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Sample records for acid amplification assays

  1. Enzyme-Free Nucleic Acid Amplification Assay Using a Cellphone-Based Well Plate Fluorescence Reader.

    Science.gov (United States)

    Kim, Donghyuk; Wei, Qingshan; Kim, Dong Hyeok; Tseng, Derek; Zhang, Jingzi; Pan, Eric; Garner, Omai; Ozcan, Aydogan; Di Carlo, Dino

    2018-01-02

    Nucleic acids, DNA and RNA, provide important fingerprint information for various pathogens and have significant diagnostic value; however, improved approaches are urgently needed to enable rapid detection of nucleic acids in simple point-of-care formats with high sensitivity and specificity. Here, we present a system that utilizes a series of toehold-triggered hybridization/displacement reactions that are designed to convert a given amount of RNA molecules (i.e., the analyte) into an amplified amount of signaling molecules without any washing steps or thermocycling. Fluorescent probes for signal generation were designed to consume products of the catalytic reaction in order to push the equilibrium and enhance the assay fold amplification for improved sensitivity and reaction speed. The system of toehold-assisted reactions is also modeled to better understand its performance and capabilities, and we empirically demonstrate the success of this approach with two analytes of diagnostic importance, i.e., influenza viral RNA and a micro RNA (miR-31). We also show that the amplified signal permits using a compact and cost-effective smartphone-based fluorescence reader, an important requirement toward a nucleic-acid-based point-of-care diagnostic system.

  2. Nuclease-resistant double-stranded DNA controls or standards for hepatitis B virus nucleic acid amplification assays

    Directory of Open Access Journals (Sweden)

    Zhan Sien

    2009-12-01

    Full Text Available Abstract Background Identical blood samples tested using different kits can give markedly different hepatitis B virus (HBV DNA levels, which can cause difficulty in the interpretation of viral load. A universal double-stranded DNA control or standard that can be used in all commercial HBV DNA nucleic acid amplification assay kits is urgently needed. By aligning all HBV genotypes (A-H, we found that the surface antigen gene and precore-core gene regions of HBV are the most conserved regions among the different HBV genotypes. We constructed a chimeric fragment by overlapping extension polymerase chain reaction and obtained a 1,349-bp HBVC+S fragment. We then packaged the fragment into lambda phages using a traditional lambda phage cloning procedure. Results The obtained armored DNA was resistant to DNase I digestion and was stable, noninfectious to humans, and could be easily extracted using commercial kits. More importantly, the armored DNA may be used with all HBV DNA nucleic acid amplification assay kits. Conclusions The lambda phage packaging system can be used as an excellent expression platform for armored DNA. The obtained armored DNA possessed all characteristics of an excellent positive control or standard. In addition, this armored DNA is likely to be appropriate for all commercial HBV DNA nucleic acid amplification detection kits. Thus, the constructed armored DNA can probably be used as a universal positive control or standard in HBV DNA assays.

  3. A sensitive colorimetric assay system for nucleic acid detection based on isothermal signal amplification technology.

    Science.gov (United States)

    Hu, Bo; Guo, Jing; Xu, Ying; Wei, Hua; Zhao, Guojie; Guan, Yifu

    2017-08-01

    Rapid and accurate detection of microRNAs in biological systems is of great importance. Here, we report the development of a visual colorimetric assay which possesses the high amplification capabilities and high selectivity of the rolling circle amplification (RCA) method and the simplicity and convenience of gold nanoparticles used as a signal indicator. The designed padlock probe recognizes the target miRNA and is circularized, and then acts as the template to extend the target miRNA into a long single-stranded nucleotide chain of many tandem repeats of nucleotide sequences. Next, the RCA product is hybridized with oligonucleotides tagged onto gold nanoparticles. This interaction leads to the aggregation of gold nanoparticles, and the color of the system changes from wine red to dark blue according to the abundance of miRNA. A linear correlation between fluorescence and target oligonucleotide content was obtained in the range 0.3-300 pM, along with a detection limit of 0.13 pM (n = 7) and a RSD of 3.9% (30 pM, n = 9). The present approach provides a simple, rapid, and accurate visual colorimetric assay that allows sensitive biodetection and bioanalysis of DNA and RNA nucleotides of interest in biologically important samples. Graphical abstract The colorimetric assay system for analyzing target oligonucleotides.

  4. Rapid and sensitive detection of norovirus genomes in oysters by a two-step isothermal amplification assay system combining nucleic acid sequence-based amplification and reverse transcription-loop-mediated isothermal amplification assays.

    Science.gov (United States)

    Fukuda, Shinji; Sasaki, Yukie; Seno, Masato

    2008-06-01

    We developed a two-step isothermal amplification assay system, which achieved the detection of norovirus (NoV) genomes in oysters with a sensitivity similar to that of reverse transcription-seminested PCR. The time taken for the amplification of NoV genomes from RNA extracts was shortened to about 3 h.

  5. Multicenter evaluation of the Quidel Lyra Direct C. difficile nucleic acid amplification assay.

    Science.gov (United States)

    Beck, Eric T; Buchan, Blake W; Riebe, Katherine M; Alkins, Brenda R; Pancholi, Preeti; Granato, Paul A; Ledeboer, Nathan A

    2014-06-01

    Clostridium difficile is a Gram-positive bacterium commonly found in health care and long-term-care facilities and is the most common cause of antibiotic-associated diarrhea. Rapid detection of this bacterium can assist physicians in implementing contact precautions and appropriate antibiotic therapy in a timely manner. The purpose of this study was to compare the clinical performance of the Quidel Lyra Direct C. difficile assay (Lyra assay) (Quidel, San Diego, CA) to that of a direct cell culture cytotoxicity neutralization assay (CCNA) and enhanced toxigenic culture. This study was performed at three geographically diverse laboratories within the United States using residual stool specimens submitted for routine C. difficile testing. Residual samples were tested using the Lyra assay on three real-time PCR platforms, and results were compared to those for direct CCNA and enhanced toxigenic culture. The test results for all platforms were consistent across all three test sites. The sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500 Fast DX, and ABI QuantStudio DX instruments compared to CCNA were 90.0% and 93.3%, 95.0% and 94.2%, and 93.8% and 95.0%, respectively. Compared to enhanced toxigenic culture, the sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500, and QuantStudio instruments were 82.1% and 96.9%, 89.3% and 98.8%, and 85.7% and 99.0%, respectively. Overall, the Lyra assay is easy to use and versatile and compares well to C. difficile culture methods. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  6. Rapid and Sensitive Detection of Norovirus Genomes in Oysters by a Two-Step Isothermal Amplification Assay System Combining Nucleic Acid Sequence-Based Amplification and Reverse Transcription-Loop-Mediated Isothermal Amplification Assays▿

    Science.gov (United States)

    Fukuda, Shinji; Sasaki, Yukie; Seno, Masato

    2008-01-01

    We developed a two-step isothermal amplification assay system, which achieved the detection of norovirus (NoV) genomes in oysters with a sensitivity similar to that of reverse transcription-seminested PCR. The time taken for the amplification of NoV genomes from RNA extracts was shortened to about 3 h. PMID:18456857

  7. Rapid and Sensitive Detection of Norovirus Genomes in Oysters by a Two-Step Isothermal Amplification Assay System Combining Nucleic Acid Sequence-Based Amplification and Reverse Transcription-Loop-Mediated Isothermal Amplification Assays▿

    OpenAIRE

    Fukuda, Shinji; Sasaki, Yukie; Seno, Masato

    2008-01-01

    We developed a two-step isothermal amplification assay system, which achieved the detection of norovirus (NoV) genomes in oysters with a sensitivity similar to that of reverse transcription-seminested PCR. The time taken for the amplification of NoV genomes from RNA extracts was shortened to about 3 h.

  8. Detection of virus mRNA within infected host cells using an isothermal nucleic acid amplification assay: marine cyanophage gene expression within Synechococcus sp

    Directory of Open Access Journals (Sweden)

    Hall Matthew J

    2007-06-01

    Full Text Available Abstract Background Signal-Mediated Amplification of RNA Technology (SMART is an isothermal nucleic acid amplification technology, developed for the detection of specific target sequences, either RNA (for expression or DNA. Cyanophages are viruses that infect cyanobacteria. Marine cyanophages are ubiquitous in the surface layers of the ocean where they infect members of the globally important genus Synechococcus. Results Here we report that the SMART assay allowed us to differentiate between infected and non-infected host cultures. Expression of the cyanophage strain S-PM2 portal vertex gene (g20 was detected from infected host Synechococcus sp. WH7803 cells. Using the SMART assay, we demonstrated that g20 mRNA peaked 240 – 360 minutes post-infection, allowing us to characterise this as a mid to late transcript. g20 DNA was also detected, peaking 10 hours post-infection, coinciding with the onset of host lysis. Conclusion The SMART assay is based on isothermal nucleic acid amplification, allowing the detection of specific sequences of DNA or RNA. It was shown to be suitable for differentiating between virus-infected and non-infected host cultures and for the detection of virus gene expression: the first reported use of this technology for such applications.

  9. nucleic acid amplification as used in the diagnosis and management ...

    African Journals Online (AJOL)

    DR. AMINU

    2014-06-01

    Jun 1, 2014 ... thus, amplification based methods offer superior performance ... self-sustaining sequence replication), Amplification of nucleic acid probe (e.g., ligase chain reaction and Q-beta replicase) and Signal amplification (e.g., branched-probe DNA assay). PCR ... advantages, limitations and clinical utility (Fredricks.

  10. False-positive results and contamination in nucleic acid amplification assays : Suggestions for a prevent and destroy strategy

    NARCIS (Netherlands)

    Borst, A; Box, ATA; Fluit, AC

    Contamination of samples with DNA is still a major problem in microbiology laboratories, despite the wide acceptance of PCR and other amplification techniques for the detection of frequently low amounts of target DNA. This review focuses on the implications of contamination in the diagnosis and

  11. Real-Time Detection of Noroviruses in Surface Water by Use of a Broadly Reactive Nucleic Acid Sequence-Based Amplification Assay

    Science.gov (United States)

    Rutjes, Saskia A.; van den Berg, Harold H. J. L.; Lodder, Willemijn J.; de Roda Husman, Ana Maria

    2006-01-01

    Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection assays for viruses in water are often repressed by inhibitory factors present in the environment, like humic acids and heavy metals. To study the effect of environmental inhibitors on the performance of nucleic acid sequence-based amplification (NASBA), we developed a real-time norovirus NASBA targeting part of the RNA-dependent RNA polymerase (RdRp) gene. Specificity of the assay was studied with 33 divergent clones that contained part of the targeted RdRp gene of noroviruses from 15 different genogroups. Viral RNA originated from commercial oysters, surface waters, and sewage treatment plants in The Netherlands. Ninety-seven percent of the clones derived from human noroviruses were detected by real-time NASBA. Two clones containing animal noroviruses were not detected by NASBA. We compared the norovirus detection by real-time NASBA with that by conventional reverse transcriptase PCR (RT-PCR) with large-volume river water samples and found that inhibitory factors of RT-PCR had little or no effect on the performance of the norovirus NASBA. This consequently resulted in a higher sensitivity of the NASBA assay than of the RT-PCR. We show that by combining an efficient RNA extraction method with real-time NASBA the sensitivity of norovirus detection in water samples increased at least 100 times, which consequently has implications for the outcome of the infectious risk assessment. PMID:16885286

  12. Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection.

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    Ahmed Abd El Wahed

    Full Text Available Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF. Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR are the standard method for molecular detection of the dengue virus (DENV. Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA assays were developed to detect DENV1-4.Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4 to 241 (DENV1-3 RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal and in Bangkok (Thailand. In Kedougou, the RT-RPA was operated at an ambient temperature of 38 °C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31 and 100% (n=23, respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90 and 100%(n=41, respectively.During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations.

  13. Development and evaluation of loop-mediated isothermal amplification assay for rapid detection of Capripoxvirus

    OpenAIRE

    Kanisht Batra; Aman Kumar; Vinay Kumar; Trilok Nanda; Maan, Narender S.; Sushila Maan

    2015-01-01

    Aim: The present study was undertaken to develop a nucleic acid-based diagnostic assay loop-mediated isothermal amplification assay (LAMP) targeting highly conserved genomic regions of Capripoxvirus (CaPVs) and its comparative evaluation with real-time polymerase chain reaction (PCR). Material and Methods: Lyophilized vaccine strain of sheeppox virus (SPPV) was used for optimization of LAMP assay. The LAMP assay was designed using envelope immunogenic protein (P32) coding gene targeting highl...

  14. Total tumor load assessed by one-step nucleic acid amplification assay as an intraoperative predictor for non-sentinel lymph node metastasis in breast cancer.

    Science.gov (United States)

    Nabais, Celso; Figueiredo, Joana; Lopes, Paulina; Martins, Manuela; Araújo, António

    2017-04-01

    This study aimed to determine the relationship between CK19 mRNA copy number in sentinel lymph nodes (SLN) assessed by one-step nucleic acid amplification (OSNA) technique, and non-sentinel lymph nodes (NSLN) metastization in invasive breast cancer. A model using total tumor load (TTL) obtained by OSNA technique was also constructed to evaluate its predictability. We conducted an observational retrospective study including 598 patients with clinically T1-T3 and node negative invasive breast cancer. Of the 88 patients with positive SLN, 58 patients fulfill the inclusion criteria. In the analyzed group 25.86% had at least one positive NSLN in axillary lymph node dissection. Univariate analysis showed that tumor size, TTL and number of SLN macrometastases were predictive factors for NSLN metastases. In multivariate analysis just the TTL was predictive for positive NSLN (OR 2.67; 95% CI 1.06-6.70; P = 0.036). The ROC curve for the model using TTL alone was obtained and an AUC of 0.805 (95% CI 0.69-0.92) was achieved. For TTL >1.9 × 10 5 copies/μL we got 73.3% sensitivity, 74.4% specificity and 88.9% negative predictive value to predict NSLN metastases. When using OSNA technique to evaluate SLN, NSLN metastases can be predicted intraoperatively. This prediction tool could help in decision for axillary lymph node dissection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Hyaluronic Acid Assays

    DEFF Research Database (Denmark)

    Itenov, Theis S; Kirkby, Nikolai S; Bestle, Morten H

    2015-01-01

    BACKGROUD: Hyaluronic acid (HA) is proposed as a marker of functional liver capacity. The aim of the present study was to compare a new turbidimetric assay for measuring HA with the current standard method. METHODS: HA was measured by a particle-enhanced turbidimetric immunoassay (PETIA) and enzyme...

  16. GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

    Science.gov (United States)

    Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao

    2015-06-01

    Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples.

  17. Digital Microfluidics for Nucleic Acid Amplification.

    Science.gov (United States)

    Coelho, Beatriz; Veigas, Bruno; Fortunato, Elvira; Martins, Rodrigo; Águas, Hugo; Igreja, Rui; Baptista, Pedro V

    2017-06-25

    Digital Microfluidics (DMF) has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings.

  18. Digital Microfluidics for Nucleic Acid Amplification

    Directory of Open Access Journals (Sweden)

    Beatriz Coelho

    2017-06-01

    Full Text Available Digital Microfluidics (DMF has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings.

  19. Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay.

    Science.gov (United States)

    Prithiviraj, Jothikumar; Hill, Vincent; Jothikumar, Narayanan

    2012-04-20

    In this study we report the development of a simple target-specific isothermal nucleic acid amplification technique, termed genome exponential amplification reaction (GEAR). Escherichia coli was selected as the microbial target to demonstrate the GEAR technique as a proof of concept. The GEAR technique uses a set of four primers; in the present study these primers targeted 5 regions on the 16S rRNA gene of E. coli. The outer forward and reverse Tab primer sequences are complementary to each other at their 5' end, whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The GEAR assay was performed at a constant temperature 60 °C and monitored continuously in a real-time PCR instrument in the presence of an intercalating dye (SYTO 9). The GEAR assay enabled amplification of as few as one colony forming units of E. coli per reaction within 30 min. We also evaluated the GEAR assay for rapid identification of bacterial colonies cultured on agar media directly in the reaction without DNA extraction. Cells from E. coli colonies were picked and added directly to GEAR assay mastermix without prior DNA extraction. DNA in the cells could be amplified, yielding positive results within 15 min. Published by Elsevier Inc.

  20. Prevalence of Trichomonas vaginalis and Coinfection with Chlamydia trachomatis and Neisseria gonorrhoeae in the United States as Determined by the Aptima Trichomonas vaginalis Nucleic Acid Amplification Assay

    Science.gov (United States)

    Chapin, K.; Smith, J. S.; Aslanzadeh, J.; Snook, J.; Hill, C. S.; Gaydos, C. A.

    2012-01-01

    Our aim was to determine Trichomonas vaginalis prevalence using the Aptima Trichomonas vaginalis assay (ATV; Gen-Probe) and the prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae coinfections in U.S. women undergoing screening for C. trachomatis/N. gonorrhoeae. Discarded urogenital samples from 7,593 women (18 to 89 years old) undergoing C. trachomatis/N. gonorrhoeae screening using the Aptima Combo 2 assay (Gen-Probe) in various clinical settings were tested with ATV. Overall, T. vaginalis, C. trachomatis, and N. gonorrhoeae prevalences were 8.7%, 6.7%, and 1.7%, respectively. T. vaginalis was more prevalent than C. trachomatis or N. gonorrhoeae in all age groups except the 18- to 19-year-old group. The highest T. vaginalis prevalence was in women ≥40 years old (>11%), while the highest C. trachomatis prevalence (9.2%) and N. gonorrhoeae prevalence (2.2%) were in women vaginalis, 0.61% for C. trachomatis/N. gonorrhoeae and N. gonorrhoeae/T. vaginalis, and 0.24% for C. trachomatis/N. gonorrhoeae/T. vaginalis and highest in women vaginalis prevalence differed by race/ethnicity, with the highest prevalence in black women (20.2%). T. vaginalis prevalence ranged from 5.4% in family planning clinics to 22.3% in jails. Multivariate analysis determined that ages of ≥40 years, black race, and patient locations were significantly associated with T. vaginalis infection. T. vaginalis is the most common sexually transmitted infection (STI) in women of >40 years, while C. trachomatis and N. gonorrhoeae prevalence is lowest in that age group. Higher T. vaginalis prevalence in women of >40 years is probably attributed to the reason for testing, i.e., symptomatic status versus routine screening in younger women. Coinfections were relatively low. High T. vaginalis prevalence in all age groups suggests that women screened for C. trachomatis/N. gonorrhoeae, whether asymptomatic or symptomatic, should be screened for T. vaginalis. PMID:22622447

  1. Performance of self-collected penile-meatal swabs compared to clinician-collected urethral swabs for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium by nucleic acid amplification assays.

    Science.gov (United States)

    Dize, Laura; Barnes, Perry; Barnes, Mathilda; Hsieh, Yu-Hsiang; Marsiglia, Vincent; Duncan, Della; Hardick, Justin; Gaydos, Charlotte A

    2016-10-01

    Men were enrolled in a study to assess the performance and acceptability of self-collected penile meatal swabs as compared to clinician-collected urethral swabs for sexually transmitted infections (STIs). We expected penile-meatal swabs to perform favorably to urethral swabs for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), and Mycoplasma genitalium (MG) detection by nucleic acid amplification assays (NAATs). Of 203 swab pairs tested; for CT, penile-meatal swab sensitivity was 96.8% and specificity was 98.8%. NG sensitivity and specificity were 100% and 98.9%, respectively. For TV, sensitivity was 85.0% and specificity was 96.7%. For MG sensitivity and specificity were 79.3% and 99.4%, respectively. No significant statistical differences between sample type accuracy (CT: P=0.625; NG: P=0.248; TV: P=0.344; and MG: P=0.070) existed. Most men, 90.1%, reported self-collection of penile-meatal swabs as "Very Easy" or "Easy". Self-collected penile-meatal swabs appeared acceptable for NAAT STI detection and an acceptable collection method by men. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Nucleic acid amplification of HIV-1 integrase sequence subtypes CRF01_AE and B for development of HIV anti-integrase drug resistance genotyping assay

    Science.gov (United States)

    Adlar, F. R.; Bela, B.

    2017-08-01

    To anticipate the potential use of anti-integrase drugs in Indonesia for treatment of HIV-1 infection, the development of a drug resistance genotyping assay for anti-integrase is crucial in identifying the genetic drug resistance profile of Indonesian HIV-1 strains. This experiment aimed to amplify a target region in the integrase gene of Indonesian HIV-1 subtypes CRF01_AE and B that contain genetic mutations known to confer resistance to anti-integrase drug. Eleven archived plasma samples from individuals living with HIV-1 were obtained from the Virology and Cancer Pathobiology Research Center for Health Service (VCPRC FKUI-RSCM) laboratory. One of the plasma samples contained HIV-1 subtype B, and the remaining plasma samples contained subtype CRF01_AE. The target regions for all samples were amplified through RT-PCR, with an annealing temperature of 55 °C, using the primer pair AE_POL 4086F and AE_POL 5232R that were designed by VCPRC FKUI-RSCM. The results of this experiment show that 18.2% (2/11) of the samples were successfully amplified using the one-step RT-PCR. While the primer pair was effective in amplifying the target region in the integrase gene sequence for subtype B (100%; 1/1), it had a low efficacy (10%, 1/10) for subtype CRF01_AE. In conclusion, the primer pair can be used to amplify the target region in Indonesian HIV-1 strain subtypes CRF01_AE and B. However, optimization of the PCR condition and an increased number of samples would help to determine an accurate representation of the efficacy of the primer pair.

  3. Establishment of recombinase polymerase amplification assay for five hemorrhagic fever-related viruses

    Directory of Open Access Journals (Sweden)

    Xue-feng CAO

    2017-08-01

    Full Text Available Objective To establish a one-step recombinase polymerase amplification (RPA method for pathogen screening and rapid detection in the field targeting for five hemorrhagic fever related viruses (Zaire ebola virus, Sudan ebola virus, Marburg virus, Lassa virus and Yellow fever virus. Methods The specific nucleic acid (NA fragments of each virus were selected as target genes by genome sequence analysis, and the primers and probes for RPA assays were designed according to the sequence. A series of diluted template genes were used for RPA detection to determine the sensitivity. The hemorrhagic fever-related viral nucleic acids were used for RPA detection to determine the specificity. The amplification experiments were carried out at different temperature ranging from 37℃ to 42℃ to validate the reaction temperature range. Results The RPA reaction systems of the five hemorrhagic fever viruses could effectively amplify the target genes, the sensitivities were between 1.5×102 and 1.5×103 copies. No cross reactions existed with the other hemorrhagic fever-related viral genes. Meanwhile, RPA assay could effectively amplify the target genes at 37-42℃. Conclusion The isothermal RPA assays of five hemorrhagic fever viruses are established, which may amply target genes fast and react at a wide temperature range, and be potentially useful for in field pathogens detection. DOI: 10.11855/j.issn.0577-7402.2017.06.09

  4. Nucleic Acid Amplification Testing in Suspected Child Sexual Abuse

    Science.gov (United States)

    Esernio-Jenssen, Debra; Barnes, Marilyn

    2011-01-01

    The American Academy of Pediatrics recommends that site-specific cultures be obtained, when indicated, for sexually victimized children. Nucleic acid amplification testing is a highly sensitive and specific methodology for identifying sexually transmitted infections. Nucleic acid amplification tests are also less invasive than culture, and this…

  5. Novel bioluminescent quantitative detection of nucleic acid amplification in real-time.

    Science.gov (United States)

    Gandelman, Olga A; Church, Vicki L; Moore, Cathy A; Kiddle, Guy; Carne, Christopher A; Parmar, Surendra; Jalal, Hamid; Tisi, Laurence C; Murray, James A H

    2010-11-30

    The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal

  6. Novel bioluminescent quantitative detection of nucleic acid amplification in real-time.

    Directory of Open Access Journals (Sweden)

    Olga A Gandelman

    Full Text Available BACKGROUND: The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. PRINCIPAL FINDINGS: Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. CONCLUSIONS: The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a

  7. Development and evaluation of loop-mediated isothermal amplification assay for rapid detection of Capripoxvirus

    Directory of Open Access Journals (Sweden)

    Kanisht Batra

    2015-11-01

    Full Text Available Aim: The present study was undertaken to develop a nucleic acid-based diagnostic assay loop-mediated isothermal amplification assay (LAMP targeting highly conserved genomic regions of Capripoxvirus (CaPVs and its comparative evaluation with real-time polymerase chain reaction (PCR. Material and Methods: Lyophilized vaccine strain of sheeppox virus (SPPV was used for optimization of LAMP assay. The LAMP assay was designed using envelope immunogenic protein (P32 coding gene targeting highly conserved genomic regions of CaPV responsible for causing sheep pox, goat pox, and lumpy skin disease in sheep, goat and cattle respectively. Serial tenfold dilution of SPPV recombinant plasmid DNA was used for a calculating limit of detection. Analytical sensitivity and specificity were performed. Results: The test described is quick (30 min, sensitive and specific for detection of CaPVs. The described assay did not show any cross-reactivity to other related viruses that cause apparently similar clinical signs. It was found to be ten times more sensitive than conventional PCR however, 100 times less sensitive than quantitative PCR (qPCR. LAMP assay results were monitored by color change method using picogreen dye and agarose gel electrophoresis. Conclusion: LAMP assay can be a very good alternative for CaPV detection to other molecular techniques requiring sophisticated equipments.

  8. Development and evaluation of loop-mediated isothermal amplification assay for rapid detection of Capripoxvirus.

    Science.gov (United States)

    Batra, Kanisht; Kumar, Aman; Kumar, Vinay; Nanda, Trilok; Maan, Narender S; Maan, Sushila

    2015-11-01

    The present study was undertaken to develop a nucleic acid-based diagnostic assay loop-mediated isothermal amplification assay (LAMP) targeting highly conserved genomic regions of Capripoxvirus (CaPVs) and its comparative evaluation with real-time polymerase chain reaction (PCR). Lyophilized vaccine strain of sheeppox virus (SPPV) was used for optimization of LAMP assay. The LAMP assay was designed using envelope immunogenic protein (P32) coding gene targeting highly conserved genomic regions of CaPV responsible for causing sheep pox, goat pox, and lumpy skin disease in sheep, goat and cattle respectively. Serial tenfold dilution of SPPV recombinant plasmid DNA was used for a calculating limit of detection. Analytical sensitivity and specificity were performed. The test described is quick (30 min), sensitive and specific for detection of CaPVs. The described assay did not show any cross-reactivity to other related viruses that cause apparently similar clinical signs. It was found to be ten times more sensitive than conventional PCR however, 100 times less sensitive than quantitative PCR (qPCR). LAMP assay results were monitored by color change method using picogreen dye and agarose gel electrophoresis. LAMP assay can be a very good alternative for CaPV detection to other molecular techniques requiring sophisticated equipments.

  9. Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR

    DEFF Research Database (Denmark)

    Hummelshoj, Lone; Ryder, Lars P; Madsen, Hans O

    2005-01-01

    Locked nucleic acid (LNA) is a modified DNA with increased binding affinityfor complementary DNA sequences. Our strategy was to use this property of LNA to inhibit undesired PCR amplification (e.g.,from contaminating genomic DNA) in a cDNA-based assay. By placing a short complementary LNA sequence...

  10. Instrument for Real-Time Digital Nucleic Acid Amplification on Custom Microfluidic Devices.

    Directory of Open Access Journals (Sweden)

    David A Selck

    Full Text Available Nucleic acid amplification tests that are coupled with a digital readout enable the absolute quantification of single molecules, even at ultralow concentrations. Digital methods are robust, versatile and compatible with many amplification chemistries including isothermal amplification, making them particularly invaluable to assays that require sensitive detection, such as the quantification of viral load in occult infections or detection of sparse amounts of DNA from forensic samples. A number of microfluidic platforms are being developed for carrying out digital amplification. However, the mechanistic investigation and optimization of digital assays has been limited by the lack of real-time kinetic information about which factors affect the digital efficiency and analytical sensitivity of a reaction. Commercially available instruments that are capable of tracking digital reactions in real-time are restricted to only a small number of device types and sample-preparation strategies. Thus, most researchers who wish to develop, study, or optimize digital assays rely on the rate of the amplification reaction when performed in a bulk experiment, which is now recognized as an unreliable predictor of digital efficiency. To expand our ability to study how digital reactions proceed in real-time and enable us to optimize both the digital efficiency and analytical sensitivity of digital assays, we built a custom large-format digital real-time amplification instrument that can accommodate a wide variety of devices, amplification chemistries and sample-handling conditions. Herein, we validate this instrument, we provide detailed schematics that will enable others to build their own custom instruments, and we include a complete custom software suite to collect and analyze the data retrieved from the instrument. We believe assay optimizations enabled by this instrument will improve the current limits of nucleic acid detection and quantification, improving our

  11. Instrument for Real-Time Digital Nucleic Acid Amplification on Custom Microfluidic Devices.

    Science.gov (United States)

    Selck, David A; Ismagilov, Rustem F

    2016-01-01

    Nucleic acid amplification tests that are coupled with a digital readout enable the absolute quantification of single molecules, even at ultralow concentrations. Digital methods are robust, versatile and compatible with many amplification chemistries including isothermal amplification, making them particularly invaluable to assays that require sensitive detection, such as the quantification of viral load in occult infections or detection of sparse amounts of DNA from forensic samples. A number of microfluidic platforms are being developed for carrying out digital amplification. However, the mechanistic investigation and optimization of digital assays has been limited by the lack of real-time kinetic information about which factors affect the digital efficiency and analytical sensitivity of a reaction. Commercially available instruments that are capable of tracking digital reactions in real-time are restricted to only a small number of device types and sample-preparation strategies. Thus, most researchers who wish to develop, study, or optimize digital assays rely on the rate of the amplification reaction when performed in a bulk experiment, which is now recognized as an unreliable predictor of digital efficiency. To expand our ability to study how digital reactions proceed in real-time and enable us to optimize both the digital efficiency and analytical sensitivity of digital assays, we built a custom large-format digital real-time amplification instrument that can accommodate a wide variety of devices, amplification chemistries and sample-handling conditions. Herein, we validate this instrument, we provide detailed schematics that will enable others to build their own custom instruments, and we include a complete custom software suite to collect and analyze the data retrieved from the instrument. We believe assay optimizations enabled by this instrument will improve the current limits of nucleic acid detection and quantification, improving our fundamental

  12. Quantitative reverse transcription strand displacement amplification: quantitation of nucleic acids using an isothermal amplification technique.

    Science.gov (United States)

    Nycz, C M; Dean, C H; Haaland, P D; Spargo, C A; Walker, G T

    1998-06-01

    Recent advances in nucleic acid amplification techniques have allowed for quantitation of viral nucleic acid levels in clinical specimens. The most prevalent testing is carried out for HIV viral load. Strand displacement amplification (SDA) is an isothermal DNA amplification system utilizing a restriction enzyme and a DNA polymerase with strand displacement properties. SDA was adapted for quantitative RNA amplification (QRT-SDA) of an HIV gag sequence by including AMV reverse transcriptase, a quantitative control sequence, and 32P-labeled detector oligonucleotides for the HIV and the control sequences. We have also improved the amplification efficiency by including the single-strand binding protein from gene 32 of T4 bacteriophage (T4gp32) to enhance strand displacement replication. In a preliminary analytical demonstration of the technique, RT-SDA was quantitative to within twofold over a range of 500-500,000 transcripts that were generated from a plasmid bearing an HIV gag sequence. QRT-SDA potentially represents a convenient alternative for viral load testing in a clinical setting. Copyright 1998 Academic Press.

  13. Mobile suitcase laboratory for rapid detection of Leishmania donovani using recombinase polymerase amplification assay.

    Science.gov (United States)

    Mondal, Dinesh; Ghosh, Prakash; Khan, Md Anik Ashfaq; Hossain, Faria; Böhlken-Fascher, Susanne; Matlashewski, Greg; Kroeger, Axel; Olliaro, Piero; Abd El Wahed, Ahmed

    2016-05-13

    Leishmania donovani (LD) is a protozoan parasite transmitted to humans from sand flies, which causes Visceral Leishmaniasis (VL). Currently, the diagnosis is based on presence of the anti-LD antibodies and clinical symptoms. Molecular diagnosis would require real-time PCR, which is not easy to implement at field settings. In this study, we report on the development and testing of a recombinase polymerase amplification (RPA) assay for the detection of LD. A genomic DNA sample was applied to determine the assay analytical sensitivity. The cross-reactivity of the assay was tested by DNA of Leishmania spp. and of pathogens considered for differential diagnosis. The clinical performance of the assay was evaluated on LD positive and negative samples. All results were compared with real-time PCR. To allow the use of the assay at field settings, a mobile suitcase laboratory (56 × 45.5 × 26.5 cm) was developed and operated at the local hospital in Mymensingh, Bangladesh. The LD RPA assay detected equivalent to one LD genomic DNA. The assay was performed at constant temperature (42 °C) in 15 min. The RPA assay also detected other Leishmania species (L. major, L. aethiopica and L. infantum), but did not identify nucleic acid of other pathogens. Forty-eight samples from VL, asymptomatic and post-kala-azar dermal leishmaniasis subjects were detected positive and 48 LD-negative samples were negative by both LD RPA and real-time PCR assays, which indicates 100 % agreement. The suitcase laboratory was successfully operated at the local hospital by using a solar-powered battery. DNA extraction was performed by a novel magnetic bead based method (SpeedXtract), in which a simple fast lysis protocol was applied. Moreover, All reagents were cold-chain independent. The mobile suitcase laboratory using RPA is ideal for rapid sensitive and specific detection of LD especially at low resource settings and could contribute to VL control and elimination programmes.

  14. Evaluation of a commercial loop-mediated isothermal amplification assay for diagnosis of Bordetella pertussis infection.

    Science.gov (United States)

    Kamachi, Kazunari; Moriuchi, Takumi; Hiramatsu, Yukihiro; Otsuka, Nao; Shibayama, Keigo

    2017-02-01

    We evaluated a commercial loop-mediated isothermal amplification (LAMP) assay kit for Bordetella pertussis detection. The LAMP primers were designed to target the ptxP1 allele of the pertussis toxin promoter, but the assay could detect B. pertussis ptxP3 and ptxP8 strains in addition to ptxP1 strains, with high analytical sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Multi-chamber nucleic acid amplification and detection device

    Energy Technology Data Exchange (ETDEWEB)

    Dugan, Lawrence

    2017-10-25

    A nucleic acid amplification and detection device includes an amplification cartridge with a plurality of reaction chambers for containing an amplification reagent and a visual detection reagent, and a plurality of optically transparent view ports for viewing inside the reaction chambers. The cartridge also includes a sample receiving port which is adapted to receive a fluid sample and fluidically connected to distribute the fluid sample to the reaction chamber, and in one embodiment, a plunger is carried by the cartridge for occluding fluidic communication to the reaction chambers. The device also includes a heating apparatus having a heating element which is activated by controller to generate heat when a trigger event is detected. The heating apparatus includes a cartridge-mounting section which positioned a cartridge in thermal communication with the heating element so that visual changes to the contents of the reaction chambers are viewable through the view ports.

  16. Highly Rapid Amplification-Free and Quantitative DNA Imaging Assay

    Science.gov (United States)

    Klamp, Tobias; Camps, Marta; Nieto, Benjamin; Guasch, Francesc; Ranasinghe, Rohan T.; Wiedemann, Jens; Petrášek, Zdeněk; Schwille, Petra; Klenerman, David; Sauer, Markus

    2013-01-01

    There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20 μL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of ~5 pM and a linear detection range spanning three orders of magnitude. PMID:23677392

  17. Development of Rapid Isothermal Amplification Assays for Detection of Phytophthora spp. in Plant Tissue.

    Science.gov (United States)

    Miles, Timothy D; Martin, Frank N; Coffey, Michael D

    2015-02-01

    Several isothermal amplification techniques recently have been developed that are tolerant of inhibitors present in many plant extracts, which can reduce the need for obtaining purified DNA for running diagnostic assays. One such commercially available technique that has similarities with real-time polymerase chain reaction (PCR) for designing primers and a labeled probe is recombinase polymerase amplification (RPA). This technology was used to develop two simple and rapid approaches for detection of Phytophthora spp.: one genus-specific assay multiplexed with a plant internal control and the other species-specific assays for Phytophthora ramorum and P. kernoviae. All assays were tested for sensitivity (ranging from 3 ng to 1 fg of DNA) and specificity using DNA extracted from more than 136 Phytophthora taxa, 21 Pythium spp., 1 Phytopythium sp., and a wide range of plant species. The lower limit of linear detection using purified DNA was 200 to 300 fg of DNA in all pathogen RPA assays. Six different extraction buffers were tested for use during plant tissue maceration and the assays were validated in the field by collecting 222 symptomatic plant samples from over 50 different hosts. Only 56 samples were culture positive for Phytophthora spp. whereas 91 were positive using the Phytophthora genus-specific RPA test and a TaqMan real-time PCR assay. A technique for the generation of sequencing templates from positive RPA amplifications to confirm species identification was also developed. These RPA assays have added benefits over traditional technologies because they are rapid (results can be obtained in as little as 15 min), do not require DNA extraction or extensive training to complete, use less expensive portable equipment than PCR-based assays, and are significantly more specific than current immunologically based methods. This should provide a rapid, field-deployable capability for pathogen detection that will facilitate point-of-sample collection processing

  18. Amplicon Competition Enables End-Point Quantitation of Nucleic Acids Following Isothermal Amplification.

    Science.gov (United States)

    Jiang, Yu Sherry; Stacy, Apollo; Whiteley, Marvin; Ellington, Andrew D; Bhadra, Sanchita

    2017-09-05

    It is inherently difficult to quantitate nucleic acid analytes with most isothermal amplification assays. We developed loop-mediated isothermal amplification (LAMP) reactions in which competition between defined numbers of "false" and "true" amplicons leads to order of magnitude quantitation by a single endpoint determination. These thresholded LAMP reactions were successfully used to directly and quantitatively estimate the numbers of nucleic acids in complex biospecimens, including directly from cells and in sewage, with the values obtained closely correlating with qPCR quantitations. Thresholded LAMP reactions are amenable to endpoint readout by cell phone, unlike other methods that require continuous monitoring, and should therefore prove extremely useful in developing one-pot reactions for point-of-care diagnostics without needing sophisticated material or informatics infrastructure. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Detection of infections with hepatitis B virus, hepatitis C virus, and human immunodeficiency virus by analyses of dried blood spots - performance characteristics of the ARCHITECT system and two commercial assays for nucleic acid amplification

    Science.gov (United States)

    2013-01-01

    Background Nowadays, dried blood spots (DBS) are primarily used to obtain diagnostic access to risk collectives such as intravenous drug users, who are prone to infections with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Before DBS analyses can be used in this diagnostic context, however, a comprehensive evaluation of its performance characteristics must be conducted. To the best of our knowledge, the current study presents for the first time such essential data for the Abbott ARCHITECT system, which is currently the worldwide leading platform in this field of infection diagnostics. Methods The investigation comprised 1,762 paired serum/DBS samples and a total of 3,524 determinations with the Abbott ARCHITECT HBsAg, anti-HBc, anti-HBs, anti-HCV and HIV-1-p24-antigen/anti-HIV 1/2 assays as well as with the artus HBV LC PCR and VERSANT HCV RNA qualitative (TMA) tests. Results In the context of DBS testing, a specificity of 100% was recorded for the seven serological and molecular biological assays. The analytical sensitivity of HBsAg, anti-HBc, anti-HBs, anti-HCV, HIV-1-p24-antigen/anti-HIV 1/2, HBV DNA, and HCV RNA detections in DBS eluates was 98.6%, 97.1%, 97.5%, 97.8%, 100%, 93%, and 100%, respectively. Discussion/conclusions The results obtained indicate that it is today possible to reliably detect HBsAg, anti-HBc, anti-HBs, anti-HCV and HIV-1-p24 antigen/anti-HIV 1/2 with state-of-the-art analytical systems such as the Abbott ARCHITECT in DBS eluates even when a comparatively high elution volume of 1,000 μl is used. They also provide evidence for the inherent analytical limits of DBS testing, which primarily concern the anti-HBc/anti-HBs system for individuals with HIV infections and nucleic acid tests with relatively low analytical sensitivity. PMID:23497102

  20. Detection of infections with hepatitis B virus, hepatitis C virus, and human immunodeficiency virus by analyses of dried blood spots--performance characteristics of the ARCHITECT system and two commercial assays for nucleic acid amplification.

    Science.gov (United States)

    Ross, R Stefan; Stambouli, Oumaima; Grüner, Nico; Marcus, Ulrich; Cai, Wei; Zhang, Weidong; Zimmermann, Ruth; Roggendorf, Michael

    2013-03-05

    Nowadays, dried blood spots (DBS) are primarily used to obtain diagnostic access to risk collectives such as intravenous drug users, who are prone to infections with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Before DBS analyses can be used in this diagnostic context, however, a comprehensive evaluation of its performance characteristics must be conducted. To the best of our knowledge, the current study presents for the first time such essential data for the Abbott ARCHITECT system, which is currently the worldwide leading platform in this field of infection diagnostics. The investigation comprised 1,762 paired serum/DBS samples and a total of 3,524 determinations with the Abbott ARCHITECT HBsAg, anti-HBc, anti-HBs, anti-HCV and HIV-1-p24-antigen/anti-HIV 1/2 assays as well as with the artus HBV LC PCR and VERSANT HCV RNA qualitative (TMA) tests. In the context of DBS testing, a specificity of 100% was recorded for the seven serological and molecular biological assays. The analytical sensitivity of HBsAg, anti-HBc, anti-HBs, anti-HCV, HIV-1-p24-antigen/anti-HIV 1/2, HBV DNA, and HCV RNA detections in DBS eluates was 98.6%, 97.1%, 97.5%, 97.8%, 100%, 93%, and 100%, respectively. The results obtained indicate that it is today possible to reliably detect HBsAg, anti-HBc, anti-HBs, anti-HCV and HIV-1-p24 antigen/anti-HIV 1/2 with state-of-the-art analytical systems such as the Abbott ARCHITECT in DBS eluates even when a comparatively high elution volume of 1,000 μl is used. They also provide evidence for the inherent analytical limits of DBS testing, which primarily concern the anti-HBc/anti-HBs system for individuals with HIV infections and nucleic acid tests with relatively low analytical sensitivity.

  1. Extraction, amplification and detection of DNA in microfluidic chip-based assays

    KAUST Repository

    Wu, Jinbo

    2013-12-20

    This review covers three aspects of PCR-based microfluidic chip assays: sample preparation, target amplification, and product detection. We also discuss the challenges related to the miniaturization and integration of each assay and make a comparison between conventional and microfluidic schemes. In order to accomplish these essential assays without human intervention between individual steps, the micro-components for fluid manipulation become critical. We therefore summarize and discuss components such as microvalves (for fluid regulation), pumps (for fluid driving) and mixers (for blending fluids). By combining the above assays and microcomponents, DNA testing of multi-step bio-reactions in microfluidic chips may be achieved with minimal external control. The combination of assay schemes with the use of micro-components also leads to rapid methods for DNA testing via multi-step bioreactions. Contains 259 references.

  2. A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection.

    Science.gov (United States)

    Liu, Wei; Liu, Hui-Xin; Zhang, Lin; Hou, Xue-Xia; Wan, Kang-Lin; Hao, Qin

    2016-08-03

    A novel isothermal detection for recombinase polymerase amplification with lateral flow (LF-RPA) was established for Borrelia burgdorferi (B. burgdorferi) detection in this study. This assay with high sensitivity and specificity can get a visible result without any additional equipment in 30 min. We designed a pair of primers according to recA gene of B. burgdorferi strains and a methodology evaluation was performed. The results showed that the RPA assay based on the recA gene was successfully applied in B. burgdorferi detection, and its specific amplification was only achieved from the genomic DNA of B. burgdorferi. The detection limit of the new assay was about 25 copies of the B. burgdorferi genomic DNA. Twenty Lyme borreliosis patients' serum samples were detected by LF-RPA assay, real-time qPCR and nested-PCR. Results showed the LF-RPA assay is more effective than nested-PCR for its shorter reaction time and considerably higher detection rate. This method is of great value in clinical rapid detection for Lyme borreliosis. Using the RPA assay might be a megatrend for DNA detection in clinics and endemic regions.

  3. Robustness of Salmonella loop-mediated isothermal amplification assays for food applications.

    Science.gov (United States)

    Yang, Q; Wang, F; Prinyawiwatkul, W; Ge, B

    2014-01-01

    Loop-mediated isothermal amplification (LAMP) assays have been developed recently for Salmonella detection. This study aimed at evaluating the robustness of two Salmonella LAMP assays in comparison with PCR and real-time quantitative PCR for food applications. Performance of the assays was examined under abusive preparation conditions, running temperatures and pH, and with the addition of various inhibitors and food rinses. LAMP achieved robust detection under abusive assay preparation conditions (holding at 22 and 37°C for up to 30 min) and running temperatures (57-68°C). With a hot-start DNA polymerase, PCR obtained comparable results under these temperature ranges. However, PCR performed markedly poorer under abusive pH. LAMP also showed greater tolerance to potential inhibitors than PCR. When food rinses including meat juice, chicken rinse, egg homogenate and produce homogenate were added at 20% of the reaction mix, PCR amplifications were completely inhibited, but LAMP reactions were not. Our results demonstrated that LAMP is a robust alternative to PCR in Salmonella detection for food applications. This study filled important knowledge gaps regarding the robustness of Salmonella LAMP assays. The findings will help bring Salmonella LAMP assays closer to wider applications in food testing. © 2013 The Society for Applied Microbiology.

  4. Mobile suitcase laboratory for rapid detection of Leishmania donovani using recombinase polymerase amplification assay

    OpenAIRE

    Mondal, Dinesh; Ghosh, Prakash; Khan, Md. Anik; Hossain, Faria; Böhlken-Fascher, Susanne; Matlashewski, Greg; Kroeger, Axel; Olliaro, Piero; Abd El Wahed, Ahmed

    2016-01-01

    Background Leishmania donovani (LD) is a protozoan parasite transmitted to humans from sand flies, which causes Visceral Leishmaniasis (VL). Currently, the diagnosis is based on presence of the anti-LD antibodies and clinical symptoms. Molecular diagnosis would require real-time PCR, which is not easy to implement at field settings. In this study, we report on the development and testing of a recombinase polymerase amplification (RPA) assay for the detection of LD. Methods A genomic DNA sampl...

  5. Highly Stable and Sensitive Nucleic Acid Amplification and Cell-Phone-Based Readout.

    Science.gov (United States)

    Kong, Janay E; Wei, Qingshan; Tseng, Derek; Zhang, Jingzi; Pan, Eric; Lewinski, Michael; Garner, Omai B; Ozcan, Aydogan; Di Carlo, Dino

    2017-03-28

    Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/μL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.

  6. Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the Mycobacterium avium subsp. paratuberculosis.

    Directory of Open Access Journals (Sweden)

    Sören Hansen

    Full Text Available The detection of Mycobacterium avium subsp. paratuberculosis (MAP infections in ruminants is crucial to control spread among animals and to humans. Cultivation of MAP is seen as the gold standard for detection, although it is very time consuming and labour intensive. In addition, several PCR assays have been developed to detect MAP in around 90 minutes, but these assays required highly sophisticated equipment as well as lengthy and complicated procedure.In this study, we have developed a rapid assay for the detection of MAP based on the recombinase polymerase amplification (RPA assay targeting a MAP specific region, the IS900 gene. The detection limit was 16 DNA molecules in 15 minutes as determined by the probit analysis on eight runs of the plasmid standard. Cross reactivity with other mycobacterial and environmentally associated bacterial strains was not observed. The clinical performance of the MAP RPA assay was tested using 48 MAP-positive and 20 MAP-negative blood, sperm, faecal and tissue samples. All results were compared with reads of a highly sensitive real-time PCR assay. The specificity of the MAP RPA assay was 100%, while the sensitivity was 89.5%.The RPA assay is quicker and much easier to handle than real-time PCR. All RPA reagents were cold-chain independent. Moreover, combining RPA assay with a simple extraction protocol will maximize its use at point of need for rapid detection of MAP.

  7. An aptamer assay using rolling circle amplification coupled with thrombin catalysis for protein detection.

    Science.gov (United States)

    Guo, Limin; Hao, Lihua; Zhao, Qiang

    2016-07-01

    We describe a sensitive aptamer-based sandwich assay for protein detection on microplate by using rolling circle amplification (RCA) coupled with thrombin catalysis. This assay takes advantage of RCA generating long DNA oligonucleotides with repeat thrombin-binding aptamer sequence, specific aptamer affinity binding to achieve multiple thrombin labeling, and enzyme activity of thrombin for signal generation. Protein target is specifically captured by antibody-coated microplate. Then, an oligonucleotide containing an aptamer for protein and a primer sequence is added to form a typical sandwich structure. Following a template encoded with complementary sequence of aptamer for thrombin, RCA reaction extends the primer sequence into a long oligonucleotide. Many thrombin molecules bind with the RCA product. Thrombin catalyzes the conversion of its chromogenic or fluorogenic peptide substrates into detectable products for final quantification of protein targets. We applied this strategy to the detection of a model protein target, platelet-derived growth factor-BB (PDGF-BB). Due to double signal amplifications from RCA and thrombin catalysis, this assay enabled the detection of PDGF-BB as low as 3.1 pM when a fluorogenic peptide substrate was used. This assay provides a new way for signal generation in RCA-involved assay through direct thrombin labeling, circumventing time-consuming preparation of enzyme-conjugate and affinity probes. This method has promise for a variety of analytical applications.

  8. Nucleic acid amplification of individual molecules in a microfluidic device.

    Science.gov (United States)

    Dettloff, Roger; Yang, Esther; Rulison, Aaron; Chow, Andrea; Farinas, Javier

    2008-06-01

    A microfluidic device was developed that enabled rapid polymerase chain reaction (PCR) analysis of individual DNA molecules. The device combined a means for accessing samples serially from a microtiter plate, channels for assembling eight parallel PCR reactions, and integrated resistive heaters for rapid thermocycling (>5 degrees C/s heating, >7 degrees C/s cooling) of samples as they flowed continuously through PCR channels. Amplification was monitored by fluorescence detection of Taqman probes. The long, narrow channels (10 microm x 180 microm x 40 mm) allowed sufficient separation between neighboring DNA templates to enable amplification of discreet DNA molecules. The functionality of the device was demonstrated by reproducibly amplifying a 2D6.6 CYP450 template and distinguishing between wild-type and mutant sequences using Taqman probes. A comparison of the rate of individual amplification events to the expected Poisson distribution confirmed that the device could reliably analyze individual DNA molecules. This work establishes the feasibility of rapid, single-molecule interrogation of nucleic acids.

  9. Meat Species Identification using Loop-mediated Isothermal Amplification Assay Targeting Species-specific Mitochondrial DNA.

    Science.gov (United States)

    Cho, Ae-Ri; Dong, Hee-Jin; Cho, Seongbeom

    2014-01-01

    Meat source fraud and adulteration scandals have led to consumer demands for accurate meat identification methods. Nucleotide amplification assays have been proposed as an alternative method to protein-based assays for meat identification. In this study, we designed Loop-mediated isothermal amplification (LAMP) assays targeting species-specific mitochondrial DNA to identify and discriminate eight meat species; cattle, pig, horse, goat, sheep, chicken, duck, and turkey. The LAMP primer sets were designed and the target genes were discriminated according to their unique annealing temperature generated by annealing curve analysis. Their unique annealing temperatures were found to be 85.56±0.07℃ for cattle, 84.96±0.08℃ for pig, and 85.99±0.05℃ for horse in the BSE-LAMP set (Bos taurus, Sus scrofa domesticus and Equus caballus); 84.91±0.11℃ for goat and 83.90±0.11℃ for sheep in the CO-LAMP set (Capra hircus and Ovis aries); and 86.31±0.23℃ for chicken, 88.66±0.12℃ for duck, and 84.49±0.08℃ for turkey in the GAM-LAMP set (Gallus gallus, Anas platyrhynchos and Meleagris gallopavo). No cross-reactivity was observed in each set. The limits of detection (LODs) of the LAMP assays in raw and cooked meat were determined from 10 pg/μL to 100 fg/μL levels, and LODs in raw and cooked meat admixtures were determined from 0.01% to 0.0001% levels. The assays were performed within 30 min and showed greater sensitivity than that of the PCR assays. These novel LAMP assays provide a simple, rapid, accurate, and sensitive technology for discrimination of eight meat species.

  10. Aptamer Lateral Flow Assays for Ultrasensitive Detection of β-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers.

    Science.gov (United States)

    Jauset-Rubio, Miriam; Svobodová, Markéta; Mairal, Teresa; McNeil, Calum; Keegan, Neil; El-Shahawi, Mohammad S; Bashammakh, Abdulaziz S; Alyoubi, Abdulrahman O; O'Sullivan, Ciara K

    2016-11-01

    In this work, different methodologies were evaluated in search of robust, simple, rapid, ultrasensitive, and user-friendly lateral flow aptamer assays. In one approach, we developed a competitive based lateral flow aptamer assay, in which β-conglutin immobilized on the test line of a nitrocellulose membrane and β-conglutin in the test sample compete for binding to AuNP labeled aptamer. The control line exploits an immobilized DNA probe complementary to the labeled aptamer, forcing displacement of the aptamer from the β-conglutin-aptamer complex. In a second approach, the competition for aptamer binding takes place off-strip, and following competition, aptamer bound to the immobilized β-conglutin is eluted and used as a template for isothermal recombinase polymerase amplification, exploiting tailed primers, resulting in an amplicon of a duplex flanked by single stranded DNA tails. The amplicon is rapidly and quantitatively detected using a nucleic acid lateral flow with an immobilized capture probe and a gold nanoparticle labeled reporter probe. The competitive lateral flow is completed in just 5 min, achieving a detection limit of 55 pM (1.1 fmol), and the combined competitive-amplification lateral flow requires just 30 min, with a detection limit of 9 fM (0.17 amol).

  11. Development of Chemiluminescent Lateral Flow Assay for the Detection of Nucleic Acids

    OpenAIRE

    Sam R. Nugen; Catherine Fill; Yuhong Wang

    2012-01-01

    Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety. Herein we report the development of a nucleic acid sequence-based lateral flow assay which achieves a low limit of detection using chemiluminescence. On-membrane enzymatic signal amplification is used to reduce the limit of detection to the sub-femtomol level. To demonstrate this assay, we detected synthetic nucleic acid sequences representative of Trypanosoma mRNA, th...

  12. Temperature switch PCR (TSP): Robust assay design for reliable amplification and genotyping of SNPs.

    Science.gov (United States)

    Tabone, Tania; Mather, Diane E; Hayden, Matthew J

    2009-12-03

    Many research and diagnostic applications rely upon the assay of individual single nucleotide polymorphisms (SNPs). Thus, methods to improve the speed and efficiency for single-marker SNP genotyping are highly desirable. Here, we describe the method of temperature-switch PCR (TSP), a biphasic four-primer PCR system with a universal primer design that permits amplification of the target locus in the first phase of thermal cycling before switching to the detection of the alleles. TSP can simplify assay design for a range of commonly used single-marker SNP genotyping methods, and reduce the requirement for individual assay optimization and operator expertise in the deployment of SNP assays. We demonstrate the utility of TSP for the rapid construction of robust and convenient endpoint SNP genotyping assays based on allele-specific PCR and high resolution melt analysis by generating a total of 11,232 data points. The TSP assays were performed under standardised reaction conditions, requiring minimal optimization of individual assays. High genotyping accuracy was verified by 100% concordance of TSP genotypes in a blinded study with an independent genotyping method. Theoretically, TSP can be directly incorporated into the design of assays for most current single-marker SNP genotyping methods. TSP provides several technological advances for single-marker SNP genotyping including simplified assay design and development, increased assay specificity and genotyping accuracy, and opportunities for assay automation. By reducing the requirement for operator expertise, TSP provides opportunities to deploy a wider range of single-marker SNP genotyping methods in the laboratory. TSP has broad applications and can be deployed in any animal and plant species.

  13. Temperature Switch PCR (TSP: Robust assay design for reliable amplification and genotyping of SNPs

    Directory of Open Access Journals (Sweden)

    Mather Diane E

    2009-12-01

    Full Text Available Abstract Background Many research and diagnostic applications rely upon the assay of individual single nucleotide polymorphisms (SNPs. Thus, methods to improve the speed and efficiency for single-marker SNP genotyping are highly desirable. Here, we describe the method of temperature-switch PCR (TSP, a biphasic four-primer PCR system with a universal primer design that permits amplification of the target locus in the first phase of thermal cycling before switching to the detection of the alleles. TSP can simplify assay design for a range of commonly used single-marker SNP genotyping methods, and reduce the requirement for individual assay optimization and operator expertise in the deployment of SNP assays. Results We demonstrate the utility of TSP for the rapid construction of robust and convenient endpoint SNP genotyping assays based on allele-specific PCR and high resolution melt analysis by generating a total of 11,232 data points. The TSP assays were performed under standardised reaction conditions, requiring minimal optimization of individual assays. High genotyping accuracy was verified by 100% concordance of TSP genotypes in a blinded study with an independent genotyping method. Conclusion Theoretically, TSP can be directly incorporated into the design of assays for most current single-marker SNP genotyping methods. TSP provides several technological advances for single-marker SNP genotyping including simplified assay design and development, increased assay specificity and genotyping accuracy, and opportunities for assay automation. By reducing the requirement for operator expertise, TSP provides opportunities to deploy a wider range of single-marker SNP genotyping methods in the laboratory. TSP has broad applications and can be deployed in any animal and plant species.

  14. Development of Isothermal Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Circovirus Type 2

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    Yang Yang

    2017-01-01

    Full Text Available Porcine circovirus virus type II (PCV2 is the etiology of postweaning multisystemic wasting syndrome (PMWS, porcine dermatitis, nephropathy syndrome (PDNS, and necrotizing pneumonia. Rapid diagnosis tool for detection of PCV2 plays an important role in the disease control and eradication program. Recombinase polymerase amplification (RPA assays using a real-time fluorescent detection (PCV2 real-time RPA assay and RPA combined with lateral flow dipstick (PCV2 RPA LFD assay were developed targeting the PCV2 ORF2 gene. The results showed that the sensitivity of the PCV2 real-time RPA assay was 102 copies per reaction within 20 min at 37°C and the PCV2 RPA LFD assay had a detection limit of 102 copies per reaction in less than 20 min at 37°C. Both assays were highly specific for PCV2, with no cross-reactions with porcine circovirus virus type 1, foot-and-mouth disease virus, pseudorabies virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, and classical swine fever virus. Therefore, the RPA assays provide a novel alternative for simple, sensitive, and specific identification of PCV2.

  15. Development of a loop-mediated isothermal amplification assay for rapid detection of Burkholderia mallei.

    Science.gov (United States)

    Mirzai, S; Safi, S; Mossavari, N; Afshar, D; Bolourchian, M

    2016-08-31

    The present study was conducted to establish a Loop-mediated isothermal amplification (LAMP) technique for the rapid detection of B. mallei the etiologic agent of glanders, a highly contagious disease of equines. A set of six specific primers targeting integrase gene cluster were designed for the LAMP test. The reaction was optimized using different temperatures and time intervals. The specificity of the assay was evaluated using DNA from B.pseudomallei and Pseudomonas aeruginosa. The LAMP products were analyzed both visually and under UV light after electrophoresis. The optimized conditions were found to be at 63ºC for 60 min. The assay showed high specificity and sensitivity. It was concluded that the established LAMP assay is a rapid, sensitive and practical tool for detection of B. mallei and early diagnosis of glanders.

  16. Development of an isothermal amplification-based assay for the rapid visual detection of Salmonella bacteria.

    Science.gov (United States)

    Liu, Hai-Bin; Zang, Yu-Xuan; Du, Xin-Jun; Li, Ping; Wang, Shuo

    2017-09-01

    The efficient and timely detection of pathogens is a major concern worldwide. The aim of this study was to establish a rapid detection method for Salmonella bacteria in food samples to facilitate timely treatment. Widely used detection methods currently include culture-based methods and PCR-based methods. The former are time consuming, requiring 2 to 3 d, whereas the latter have higher accuracy but are typically complicated, requiring expertise and expensive instruments. In this study, a sensitive and rapid approach for the visual and point-of-use detection of Salmonella bacteria based on recombinase polymerase amplification (RPA) and a lateral-flow (LF) nucleic acid strip was established. We designed a pair of primers according to the invA gene of Salmonella bacteria: one was modified with digoxin, and the other was modified with biotin. In the presence of the biotin- and digoxin-modified primers and target DNA, the RPA produced a substantial amount of duplex DNA attached to biotin and digoxin. The products were detected using LF strips through immunoreaction: anti-digoxin antibodies on the gold nanoparticles, digoxin on the duplex, streptavidin on the LF test line, and biotin on the duplex. The developed RPA-LF assay allowed detection of Salmonella genomic DNA in less than 20 min with simple water bath equipment or portable thermal equipment. In addition, the RPA-LF assay was highly sensitive, with a detection limit as low as 20 fg of target DNA or 1.05 × 101 cfu of bacteria in pure culture, and highly specific, exhibiting no cross-reaction with Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Shigella, Enterobacter aerogenes, or Campylobacter jejuni. Importantly, Salmonella could be detected in milk and chicken breast at concentrations as low as 1.05 × 100 cfu/mL or 1.05 × 100 cfu/g after enrichment for 2 h and in eggs at 1.05 × 100 cfu/g after enrichment for 4 h. Furthermore, RPA was more sensitive than PCR, which requires a thermal cycling

  17. Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    King Ting Lim

    2013-01-01

    Full Text Available Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA, is an important human pathogen that produces a variety of toxins and causes a wide range of infections, including soft-tissue infections, bacteremia, and staphylococcal food poisoning. A loop-mediated isothermal amplification (LAMP assay targeting the arcC gene of S. aureus was developed and evaluated with 119 S. aureus and 25 non-S. aureus strains. The usefulness of the assay was compared with the PCR method that targets spa and arcC genes. The optimal temperature for the LAMP assay was 58.5°C with a detection limit of 2.5 ng/μL and 102 CFU/mL when compared to 12.5 ng/μL and 103 CFU/mL for PCR (spa and arcC. Both LAMP and PCR assays were 100% specific, 100% sensitive, 100% positive predictive value (PPV, and 100% negative predictive value (NPV. When tested on 30 spiked blood specimens (21 MRSA, eight non-S. aureus and one negative control, the performance of LAMP and PCR was comparable: 100% specific, 100% sensitive, 100% PPV, and 100% NPV. In conclusion, the LAMP assay was equally specific with a shorter detection time when compared to PCR in the identification of S. aureus. The LAMP assay is a promising alternative method for the rapid identification of S. aureus and could be used in resource-limited laboratories and fields.

  18. Development of loop-mediated isothermal amplification (LAMP assay for rapid and sensitive identification of ostrich meat.

    Directory of Open Access Journals (Sweden)

    Amir Abdulmawjood

    Full Text Available Animal species identification is one of the primary duties of official food control. Since ostrich meat is difficult to be differentiated macroscopically from beef, therefore new analytical methods are needed. To enforce labeling regulations for the authentication of ostrich meat, it might be of importance to develop and evaluate a rapid and reliable assay. In the present study, a loop-mediated isothermal amplification (LAMP assay based on the cytochrome b gene of the mitochondrial DNA of the species Struthio camelus was developed. The LAMP assay was used in combination with a real-time fluorometer. The developed system allowed the detection of 0.01% ostrich meat products. In parallel, a direct swab method without nucleic acid extraction using the HYPLEX LPTV buffer was also evaluated. This rapid processing method allowed detection of ostrich meat without major incubation steps. In summary, the LAMP assay had excellent sensitivity and specificity for detecting ostrich meat and could provide a sampling-to-result identification-time of 15 to 20 minutes.

  19. Detection of capripoxvirus DNA using a novel loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Murray, Lee; Edwards, Lorraine; Tuppurainen, Eeva S M; Bachanek-Bankowska, Katarzyna; Oura, Chris A L; Mioulet, Valerie; King, Donald P

    2013-05-01

    Sheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are the most serious poxviruses of ruminants. They are double stranded DNA viruses of the genus Capripoxvirus, (subfamily Chordopoxvirinae) within the family Poxviridae. The aim of this study was to develop a Loop-mediated isothermal AMPlification (LAMP) assay for the detection of Capripoxvirus (CaPV) DNA. A single LAMP assay targeting a conserved region of the CaPV P32 gene was selected from 3 pilot LAMP assays and optimised by adding loop primers to accelerate the reaction time. This LAMP assay successfully detected DNA prepared from representative CaPV isolates (SPPV, GTPV and LSDV), and did not cross-react with DNA extracted from other mammalian poxviruses. The analytical sensitivity of the LAMP assay was determined to be at least 163 DNA copies/μl which is equivalent to the performance reported for diagnostic real-time PCR currently used for the detection of CaPV. LAMP reactions were monitored with an intercalating dye using a real-time PCR machine, or by agarose-gel electrophoresis. Furthermore, dual labelled LAMP products (generated using internal LAMP primers that were conjugated with either biotin or fluorescein) could be readily visualised using a lateral-flow device. This study provides a simple and rapid approach to detect CaPV DNA that may have utility for use in the field, or in non-specialised laboratories where expensive equipment is not available.

  20. A recombinase polymerase amplification assay for rapid detection of Crimean-Congo Haemorrhagic fever Virus infection.

    Directory of Open Access Journals (Sweden)

    Laura C Bonney

    2017-10-01

    Full Text Available Crimean-Congo Haemorrhagic fever Virus (CCHFV is a rapidly emerging vector-borne pathogen and the cause of a virulent haemorrhagic fever affecting large parts of Europe, Africa, the Middle East and Asia.An isothermal recombinase polymerase amplification (RPA assay was successfully developed for molecular detection of CCHFV. The assay showed rapid (under 10 minutes detection of viral extracts/synthetic virus RNA of all 7 S-segment clades of CCHFV, with high target specificity. The assay was shown to tolerate the presence of inhibitors in crude preparations of mock field samples, indicating that this assay may be suitable for use in the field with minimal sample preparation. The CCHFV RPA was successfully used to screen and detect CCHFV positives from a panel of clinical samples from Tajikistan.The assay is a rapid, isothermal, simple-to-perform molecular diagnostic, which can be performed on a light, portable real-time detection device. It is ideally placed therefore for use as a field-diagnostic or in-low resource laboratories, for monitoring of CCHF outbreaks at the point-of-need, such as in remote rural regions in affected countries.

  1. A recombinase polymerase amplification assay for rapid detection of Crimean-Congo Haemorrhagic fever Virus infection.

    Science.gov (United States)

    Bonney, Laura C; Watson, Robert J; Afrough, Babak; Mullojonova, Manija; Dzhuraeva, Viktoriya; Tishkova, Farida; Hewson, Roger

    2017-10-01

    Crimean-Congo Haemorrhagic fever Virus (CCHFV) is a rapidly emerging vector-borne pathogen and the cause of a virulent haemorrhagic fever affecting large parts of Europe, Africa, the Middle East and Asia. An isothermal recombinase polymerase amplification (RPA) assay was successfully developed for molecular detection of CCHFV. The assay showed rapid (under 10 minutes) detection of viral extracts/synthetic virus RNA of all 7 S-segment clades of CCHFV, with high target specificity. The assay was shown to tolerate the presence of inhibitors in crude preparations of mock field samples, indicating that this assay may be suitable for use in the field with minimal sample preparation. The CCHFV RPA was successfully used to screen and detect CCHFV positives from a panel of clinical samples from Tajikistan. The assay is a rapid, isothermal, simple-to-perform molecular diagnostic, which can be performed on a light, portable real-time detection device. It is ideally placed therefore for use as a field-diagnostic or in-low resource laboratories, for monitoring of CCHF outbreaks at the point-of-need, such as in remote rural regions in affected countries.

  2. A real-time loop-mediated isothermal amplification assay for rapid detection of Shigella species.

    Science.gov (United States)

    Liew, P S; Teh, C S J; Lau, Y L; Thong, K L

    2014-12-01

    Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 10(5) CFU/ml, while PCR displayed a limit of 5.9 x 10(7) CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 10(4) CFU/g, whereas PCR was 3.6 x 10(5) CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.

  3. Bacteriophage amplification assay for detection of Listeria spp. using virucidal laser treatment

    Directory of Open Access Journals (Sweden)

    I.C. Oliveira

    2012-09-01

    Full Text Available A protocol for the bacteriophage amplification technique was developed for quantitative detection of viable Listeria monocytogenes cells using the A511 listeriophage with plaque formation as the end-point assay. Laser and toluidine blue O (TBO were employed as selective virucidal treatment for destruction of exogenous bacteriophage. Laser and TBO can bring a total reduction in titer phage (ca. 10(8 pfu/mL without affecting the viability of L. monocytogenes cells. Artificially inoculated skimmed milk revealed mean populations of the bacteria as low as between 13 cfu/mL (1.11 log cfu/mL, after a 10-h assay duration. Virucidal laser treatment demonstrated better protection of Listeria cells than the other agents previously tested. The protocol was faster and easier to perform than standard procedures. This protocol constitutes an alternative for rapid, sensitive and quantitative detection of L. monocytogenes.

  4. Rapid identification of drug-type strains in Cannabis sativa using loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Kitamura, Masashi; Aragane, Masako; Nakamura, Kou; Watanabe, Kazuhito; Sasaki, Yohei

    2017-01-01

    In Cannabis sativa L., tetrahydrocannabinol (THC) is the primary psychoactive compound and exists as the carboxylated form, tetrahydrocannabinolic acid (THCA). C. sativa is divided into two strains based on THCA content-THCA-rich (drug-type) strains and THCA-poor (fiber-type) strains. Both strains are prohibited by law in many countries including Japan, whereas the drug-type strains are regulated in Canada and some European countries. As the two strains cannot be discriminated by morphological analysis, a simple method for identifying the drug-type strains is required for quality control in legal cultivation and forensic investigation. We have developed a novel loop-mediated isothermal amplification (LAMP) assay for identifying the drug-type strains of C. sativa. We designed two selective LAMP primer sets for on-site or laboratory use, which target the drug-type THCA synthase gene. The LAMP assay was accomplished within approximately 40 min. The assay showed high specificity for the drug-type strains and its sensitivity was the same as or higher than that of conventional polymerase chain reaction. We also showed the effectiveness of melting curve analysis that was conducted after the LAMP assay. The melting temperature values of the drug-type strains corresponded to those of the cloned drug-type THCA synthase gene, and were clearly different from those of the cloned fiber-type THCA synthase gene. Moreover, the LAMP assay with simple sample preparation could be accomplished within 1 h from sample treatment to identification without the need for special devices or techniques. Our rapid, sensitive, specific, and simple assay is expected to be applicable to laboratory and on-site detection.

  5. New closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination.

    Science.gov (United States)

    Karthik, K; Rathore, Rajesh; Thomas, Prasad; Arun, T R; Viswas, K N; Dhama, Kuldeep; Agarwal, R K

    2014-01-01

    Loop mediated isothermal amplification (LAMP) assay, a promising diagnostic test, has been developed for detection of different pathogens of human as well as animals. Various positive points support its use as a field level test but the major problem is product cross contamination leading to false positive results. Different methods were adopted by various researchers to control this false positive amplification due to cross contamination but all have their own advantages and disadvantages. A new closed tube LAMP assay based on agar dye capsule was developed in the present study and this technique has some advantages over the other closed tube technique.•Agar at the concentration of 1.5% was used to sandwich SYBR green dye I with the aid of intradermal syringe. This agar dye capsule was placed over the LAMP reaction mixture before it was amplified.•To eliminate the hazardous nature of Ultra Violet (UV) light during result visualization of LAMP products, the present study demonstrates the use of Light Emitting Diode (LED) lights for result visualization.•LAMP was carried out for Brucella species detection using this modified techniques yielding good results without any cross contamination and LED showed similar fluorescence compared to UV.

  6. Isothermal Amplification and Lateral-Flow Assay for Detecting Crown-Gall-Causing Agrobacterium spp.

    Science.gov (United States)

    Fuller, Skylar L; Savory, Elizabeth A; Weisberg, Alexandra J; Buser, Jessica Z; Gordon, Michael I; Putnam, Melodie L; Chang, Jeff H

    2017-09-01

    Agrobacterium is a genus of soilborne gram-negative bacteria. Members carrying oncogenic plasmids can cause crown gall disease, which has significant economic costs, especially for the orchard and nursery industries. Early and rapid detection of pathogenic Agrobacterium spp. is key to the management of crown gall disease. To this end, we designed oligonucleotide primers and probes to target virD2 for use in a molecular diagnostic tool that relies on isothermal amplification and lateral-flow-based detection. The oligonucleotide tools were tested in the assay and evaluated for detection limit and specificity in detecting alleles of virD2. One set of primers that successfully amplified virD2 when used with an isothermal recombinase was selected. Both tested probes had detection limits in picogram amounts of DNA. Probe 1 could detect all tested pathogenic isolates that represented most of the diversity of virD2. Finally, the coupling of lateral-flow detection to the use of these oligonucleotide primers in isothermal amplification helped to reduce the onerousness of the process, and alleviated reliance on specialized tools necessary for molecular diagnostics. The assay is an advancement for the rapid molecular detection of pathogenic Agrobacterium spp.

  7. Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay.

    Science.gov (United States)

    Zhou, Dinggang; Wang, Chunfeng; Li, Zhu; Chen, Yun; Gao, Shiwu; Guo, Jinlong; Lu, Wenying; Su, Yachun; Xu, Liping; Que, Youxiong

    2016-01-01

    Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg(2+), 6:1 ratio of inner vs. outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was 10-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100%) by LAMP and 97/100 cases (97%) by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable, and cost-effective for detection of the bar specific transgenic sugarcane.

  8. Detection of bar transgenic sugarcane with a rapid and visual loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Dinggang eZhou

    2016-03-01

    Full Text Available Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg2+, 6:1 ratio of inner vs outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was ten-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100% by LAMP and 97/100 cases (97% by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable and cost-effective for detection of the bar specific transgenic sugarcane.

  9. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification

    Directory of Open Access Journals (Sweden)

    Michael G. Mauk

    2015-10-01

    Full Text Available Microfluidic components and systems for rapid (<60 min, low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs are described. A microfluidic point-of-care (POC diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1 nucleic acids (NAs are extracted from relatively large (~mL volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase (“membrane” to capture sample NAs in a flow-through, filtration mode; (2 NAs captured on the membrane are isothermally (~65 °C amplified; (3 amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4 paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD better than 103 virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed.

  10. Development of loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Penicillium nordicum in dry-cured meat products.

    Science.gov (United States)

    Ferrara, M; Perrone, G; Gallo, A; Epifani, F; Visconti, A; Susca, A

    2015-06-02

    The need of powerful diagnostic tools for rapid, simple, and cost-effective detection of food-borne fungi has become very important in the area of food safety. Currently, several isothermal nucleic acid amplification methods have been developed as an alternative to PCR-based analyses. Loop-mediated isothermal amplification (LAMP) is one of these innovative methods; it requires neither gel electrophoresis to separate and visualize the products nor expensive laboratory equipment and it has been applied already for detection of pathogenic organisms. In the current study, we developed a LAMP assay for the specific detection of Penicillium nordicum, the major causative agent of ochratoxin A contamination in protein-rich food, especially dry-cured meat products. The assay was based on targeting otapksPN gene, a key gene in the biosynthesis of ochratoxin A (OTA) in P. nordicum. Amplification of DNA during the reaction was detected directly in-tube by color transition of hydroxynaphthol blue from violet to sky blue, visible to the naked eye, avoiding further post amplification analyses. Only DNAs isolated from several P. nordicum strains led to positive results and no amplification was observed from non-target OTA and non OTA-producing strains. The assay was able to detect down to 100 fg of purified targeted genomic DNA or 10(2) conidia/reaction within 60 min. The LAMP assay for detection and identification of P. nordicum was combined with a rapid DNA extraction method set up on serially diluted conidia, providing an alternative rapid, specific and sensitive DNA-based method suitable for application directly "on-site", notably in key steps of dry-cured meat production. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes.

    Science.gov (United States)

    Marras, Salvatore A E; Tyagi, Sanjay; Kramer, Fred Russell

    2006-01-01

    A number of formats for nucleic acid hybridization have been developed to identify DNA and RNA sequences that are involved in cellular processes and that aid in the diagnosis of genetic and infectious diseases. The introduction of hybridization probes with interactive fluorophore pairs has enabled the development of homogeneous hybridization assays for the direct identification of nucleic acids. A change in the fluorescence of these probes indicates the presence of a target nucleic acid, and there is no need to separate unbound probes from hybridized probes. The advantages of homogeneous hybridization assays are their speed and simplicity. In addition, homogeneous assays can be combined with nucleic acid amplification, enabling the detection of rare target nucleic acids. These assays can be followed in real time, providing quantitative determination of target nucleic acids over a broad range of concentrations.

  12. Filter-based assay for Escherichia coli in aqueous samples using bacteriophage-based amplification.

    Science.gov (United States)

    Derda, Ratmir; Lockett, Matthew R; Tang, Sindy K Y; Fuller, Renee C; Maxwell, E Jane; Breiten, Benjamin; Cuddemi, Christine A; Ozdogan, Aysegul; Whitesides, George M

    2013-08-06

    This paper describes a method to detect the presence of bacteria in aqueous samples, based on the capture of bacteria on a syringe filter, and the infection of targeted bacterial species with a bacteriophage (phage). The use of phage as a reagent provides two opportunities for signal amplification: (i) the replication of phage inside a live bacterial host and (ii) the delivery and expression of the complementing gene that turns on enzymatic activity and produces a colored or fluorescent product. Here we demonstrate a phage-based amplification scheme with an M13KE phage that delivers a small peptide motif to an F(+), α-complementing strain of Escherichia coli K12, which expresses the ω-domain of β-galactosidase (β-gal). The result of this complementation-an active form of β-gal-was detected colorimetrically, and the high level of expression of the ω-domain of β-gal in the model K12 strains allowed us to detect, on average, five colony-forming units (CFUs) of this strain in 1 L of water with an overnight culture-based assay. We also detected 50 CFUs of the model K12 strain in 1 L of water (or 10 mL of orange juice, or 10 mL of skim milk) in less than 4 h with a solution-based assay with visual readout. The solution-based assay does not require specialized equipment or access to a laboratory, and is more rapid than existing tests that are suitable for use at the point of access. This method could potentially be extended to detect many different bacteria with bacteriophages that deliver genes encoding a full-length enzyme that is not natively expressed in the target bacteria.

  13. Rapid amplification/detection of nucleic acid targets utilizing a HDA/thin film biosensor.

    Science.gov (United States)

    Jenison, Robert; Jaeckel, Heidi; Klonoski, Joshua; Latorra, David; Wiens, Jacinta

    2014-08-07

    Thin film biosensors exploit a flat, optically coated silicon-based surface whereupon formation of nucleic acid hybrids are enzymatically transduced in a molecular thin film that can be detected by the unaided human eye under white light. While the limit of sensitivity for detection of nucleic acid targets is at sub-attomole levels (60 000 copies) many clinical specimens containing bacterial pathogens have much lower levels of analyte present. Herein, we describe a platform, termed HDA/thin film biosensor, which performs helicase-dependant nucleic acid amplification on a thin film biosensor surface to improve the limit of sensitivity to 10 copies of the mecA gene present in methicillin-resistant strains of Staphylococcus. As double-stranded DNA is unwound by helicase it was either bound by solution-phase DNA primers to be copied by DNA polymerase or hybridized to surface immobilized probe on the thin film biosensor surface to be detected. Herein, we show that amplification reactions on the thin film biosensor are equivalent to in standard thin wall tubes, with detection at the limit of sensitivity of the assay occurring after 30 minutes of incubation time. Further we validate the approach by detecting the presence of the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) from positive blood culture aliquots with high specificity (signal/noise ratio of 105).

  14. Development of a loop-mediated isothermal amplification assay for rapid detection of capripoxviruses.

    Science.gov (United States)

    Das, Amaresh; Babiuk, Shawn; McIntosh, Michael T

    2012-05-01

    Sheep pox (SP), goat pox (GP), and lumpy skin disease (LSD), caused by capripoxviruses (CaPVs), are economically important diseases of sheep, goats, and cattle, respectively. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of CaPVs. LAMP primers were designed to target a conserved gene encoding the poly(A) polymerase small subunit (VP39) of CaPVs. Hydroxynaphthol blue (HNB) was incorporated to monitor assay progress by color change from violet when negative to sky blue when positive, and results were verified by agarose gel electrophoresis. The LAMP assay was shown to be highly specific for CaPVs, with no apparent cross-reactivity to other related viruses (near neighbors) or viruses that cause similar clinical signs (look-a-like viruses). The performance of LAMP was compared to that of a highly sensitive quantitative real-time PCR (qPCR) assay. LAMP and qPCR exhibited similar analytical sensitivities, with limits of detection of 3 and 8 viral genome copies, respectively. Diagnostic specificity was assessed on 36 negative specimens, including swabs and EDTA blood from control sheep, goats, and cattle. Diagnostic sensitivity was assessed on 275 specimens, including EDTA blood, swabs, and tissues from experimentally infected sheep, goats, and cattle. Overall agreement on diagnostic test results between the two assays was 90 to 95% for specificity and 89 to 100% for sensitivity. The LAMP assay described in this report is simple to use, inexpensive, highly sensitive, and particularly well suited for the diagnosis of capripox in less well equipped laboratories and in rural settings where resources are limited.

  15. Retinoic acid receptor alpha amplifications and retinoic acid sensitivity in breast cancers.

    Science.gov (United States)

    Alsafadi, Samar; Even, Caroline; Falet, Coralie; Goubar, Aicha; Commo, Frédéric; Scott, Véronique; Quidville, Virginie; Albiges, Laurence; Dieci, Maria-Vittoria; Guegan, Justine; Lazar, Vladimir; Ahomadegbe, Jean-Charles; Delaloge, Suzette; André, Fabrice

    2013-10-01

    Molecular segmentation of breast cancer allows identification of small groups of patients who present high sensitivity to targeted agents. A patient, with chemo- and trastuzumab-resistant HER2-overexpressing breast cancer, who presented concomitant acute promyelocytic leukemia, showed a response in her breast lesions to retinoic acid, arsenic, and aracytin. We therefore investigated whether RARA gene amplification could be associated with sensitivity to retinoic acid derivatives in breast cancers. Array comparative genomic hybridization and gene expression arrays were used to characterize RARA amplifications and expression in 103 breast cancer samples. In vitro activity of ATRA was characterized in T47D, SKBR3, and BT474 cell lines. Retinoic acid receptor alpha was gained or amplified in 27% of HER2-positive and 13% of HER2-negative breast cancer samples. Retinoic acid receptor alpha can be coamplified with HER2. Retinoic acid receptor alpha copy number changes could be correlated with messenger RNA expression. All-trans-retinoic acid reduced cell viability of RARA-amplified, but not RARA-normal, cell lines through apoptosis. Gene expression arrays showed that ATRA-induced apoptosis in RARA-amplified cell lines was related to an increase in CASP1 and IRF1. The results of this study suggest that breast cancers exhibiting RARA amplifications could be sensitive to retinoic acid. A phase II trial will evaluate this hypothesis in the clinical setting. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. A signal amplification assay for HSV type 1 viral DNA detection using nanoparticles and direct acoustic profiling

    Directory of Open Access Journals (Sweden)

    Hammond Richard

    2010-02-01

    Full Text Available Abstract Background Nucleic acid based recognition of viral sequences can be used together with label-free biosensors to provide rapid, accurate confirmation of viral infection. To enhance detection sensitivity, gold nanoparticles can be employed with mass-sensitive acoustic biosensors (such as a quartz crystal microbalance by either hybridising nanoparticle-oligonucleotide conjugates to complimentary surface-immobilised ssDNA probes on the sensor, or by using biotin-tagged target oligonucleotides bound to avidin-modified nanoparticles on the sensor. We have evaluated and refined these signal amplification assays for the detection from specific DNA sequences of Herpes Simplex Virus (HSV type 1 and defined detection limits with a 16.5 MHz fundamental frequency thickness shear mode acoustic biosensor. Results In the study the performance of semi-homogeneous and homogeneous assay formats (suited to rapid, single step tests were evaluated utilising different diameter gold nanoparticles at varying DNA concentrations. Mathematical models were built to understand the effects of mass transport in the flow cell, the binding kinetics of targets to nanoparticles in solution, the packing geometries of targets on the nanoparticle, the packing of nanoparticles on the sensor surface and the effect of surface shear stiffness on the response of the acoustic sensor. This lead to the selection of optimised 15 nm nanoparticles that could be used with a 6 minute total assay time to achieve a limit of detection sensitivity of 5.2 × 10-12 M. Larger diameter nanoparticles gave poorer limits of detection than smaller particles. The limit of detection was three orders of magnitude lower than that observed using a hybridisation assay without nanoparticle signal amplification. Conclusions An analytical model was developed to determine optimal nanoparticle diameter, concentration and probe density, which allowed efficient and rapid optimisation of assay parameters

  17. Development of a loop-mediated isothermal amplification assay for detection of Trichomonas vaginalis.

    Science.gov (United States)

    Reyes, John Carlo B; Solon, Juan Antonio A; Rivera, Windell L

    2014-07-01

    A loop-mediated isothermal amplification (LAMP) assay targeting the 2-kbp repeated DNA species-specific sequence was developed for detection of Trichomonas vaginalis, the causative agent of trichomoniasis. The analytical sensitivity and specificity of the LAMP assay were evaluated using pooled genital swab and urine specimens, respectively, spiked with T. vaginalis trophozoites. Genital secretion and urine did not inhibit the detection of the parasite. The sensitivity of the LAMP was 10-1000 times higher than the PCR performed. The detection limit of LAMP was 1 trichomonad for both spiked genital swab and urine specimens. Also, LAMP did not exhibit cross-reactivity with closely-related trichomonads, Trichomonas tenax and Pentatrichomonas hominis, and other enteric and urogenital microorganisms, Entamoeba histolytica, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. This is the first report of a LAMP assay for the detection of T. vaginalis and has prospective application for rapid diagnosis and control of trichomoniasis. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Development of cross-priming amplification assays for rapid and sensitive detection of Aeromonas hydrophila.

    Science.gov (United States)

    Meng, S; Wang, Y; Wang, Y; Liu, D; Ye, C

    2015-08-01

    Aeromonas hydrophila has been increasingly implicated as the aetiologic agent of various human diseases. Therefore, reliable laboratory detection and identification of this bacterium has become clinically and epidemiologically desirable. We developed a nearly instrument-free, simple molecular method for rapid detection of Aer. hydrophila using a cross-priming amplification (CPA) assay with the desA gene as the target. The desA gene is crucial for the survival and growth of Aer. hydrophila under iron starvation. The results can be visualized as colour changes without opening the reaction tubes. No false-positive results were observed for the 33 non-Aer. hydrophila strains tested to evaluate assay specificity. The limit of detection for Aer. hydrophila was approximately 200 copies of desA per reaction (on reference plasmids) and 5 × 10(3)  CFU g(-1) Aer. hydrophila in simulated human stool, which is the same sensitivity as a qPCR assay. The performance of the CPA assay was also evaluated with 100 stool specimens from diarrhoea patients and 40 environmental water samples. In conclusion, the simplicity, cost-effectiveness and nearly instrument-free platform of the CPA assay make it practical for use in primary care facilities and smaller clinical laboratories. Aeromonas hydrophila is a human pathogen that infects via exposed wounds or ingestion of contaminated water and food. In this study, a CPA-based PCR method was developed for specific, rapid, cost-effective detection of Aer. hydrophila, and the test results could be visualized without opening the reaction tubes. This is the first report on the application of the CPA method for the detection of Aer. hydrophila. This novel method could be practical for use in primary care facilities and smaller clinical laboratories. © 2015 The Society for Applied Microbiology.

  19. Reverse transcription genome exponential amplification reaction assay for rapid and universal detection of human rhinoviruses.

    Science.gov (United States)

    Guan, Li; Zhao, Lin-Qing; Zhou, Hang-Yu; Nie, Kai; Li, Xin-Na; Zhang, Dan; Song, Juan; Qian, Yuan; Ma, Xue-Jun

    2016-07-01

    Human rhinoviruses (HRVs) have long been recognized as the cause of more than one-half of acute viral upper respiratory illnesses, and they are associated with more-serious diseases in children, such as asthma, acute otitis media and pneumonia. A rapid and universal test for of HRV infection is in high demand. In this study, a reverse transcription genome exponential amplification reaction (RT-GEAR) assay targeting the HRV 5' untranslated region (UTR) was developed for pan-HRV detection. The reaction was performed in a single tube in one step at 65 °C for 60 min using a real-time fluorometer (Genie(®)II; Optigene). The RT-GEAR assay showed no cross-reactivity with common human enteroviruses, including HEV71, CVA16, CVA6, CVA10, CVA24, CVB5, Echo30, and PV1-3 or with other common respiratory viruses including FluA H3, FluB, PIV1-4, ADV3, RSVA, RSVB and HMPV. With in vitro-transcribed RNA containing the amplified regions of HRV-A60, HRV-B06 and HRV-C07 as templates, the sensitivity of the RT-GEAR assay was 5, 50 and 5 copies/reaction, respectively. Experiments to evaluate the clinical performance of the RT-GEAR assay were also carried out with a panel of 143 previously verified samples, and the results were compared with those obtained using a published semi-nested PCR assay followed by sequencing. The tested panel comprised 91 HRV-negative samples and 52 HRV-positive samples (18 HRV-A-positive samples, 3 HRV-B-positive samples and 31 HRV-C-positive samples). The sensitivity and specificity of the pan-HRVs RT-GEAR assay was 98.08 % and 100 %, respectively. The kappa correlation between the two methods was 0.985. The RT-GEAR assay based on a portable Genie(®)II fluorometer is a sensitive, specific and rapid assay for the universal detection of HRV infection.

  20. A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

    Directory of Open Access Journals (Sweden)

    Xia Li

    2016-10-01

    Full Text Available Bisphenol A (BPA detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA/Exonuclease III (Exo III-combined cascade amplification strategy. First, the duplex DNA probe (RP with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of “G-quadruplex” in lantern-like structures. Finally, the continuously enriched “G-quadruplex lanterns” were lightened by zinc(II-protoporphyrin IX (ZnPPIX generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10−17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

  1. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification

    Science.gov (United States)

    Mauk, Michael G.; Liu, Changchun; Song, Jinzhao; Bau, Haim H.

    2015-01-01

    Microfluidic components and systems for rapid (nucleic acids (NAs) are extracted from relatively large (~mL) volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase (“membrane”) to capture sample NAs in a flow-through, filtration mode; (2) NAs captured on the membrane are isothermally (~65 °C) amplified; (3) amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4) paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD) better than 103 virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed. PMID:27600235

  2. Non-instrumented incubation of a recombinase polymerase amplification assay for the rapid and sensitive detection of proviral HIV-1 DNA.

    Directory of Open Access Journals (Sweden)

    Lorraine Lillis

    Full Text Available Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs. However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS, diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25-43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10°C and rarely above 43°C. All 24 of 24 (100% HIV-1 RPA reactions amplified when incubated for 20 minutes between 31°C and 43°C. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30°C. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10°C and 30°C. All 12/12 (100% reactions amplified with chemical heat incubation from ambient temperatures of 15°C, 20°C, 25°C and 30°C. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose

  3. Evaluation of loop-mediated isothermal amplification assay for rapid diagnosis of Acanthamoeba keratitis

    Directory of Open Access Journals (Sweden)

    Abhishek Mewara

    2017-01-01

    Full Text Available Background: The clinical features of Acanthamoeba keratitis (AK are non-specific and closely resemble bacterial, viral and fungal keratitis. Materials and Methods: We compared loop-mediated isothermal amplification (LAMP with microscopy, non-nutrient agar (NNA culture and polymerase chain reaction (PCR in clinical suspects of AK. Results: Of 52 clinical samples (42 AK suspects and 10 proven bacterial, viral or fungal keratitis, 3 were positive by direct microscopy (sensitivity 60%, confidence interval [CI]: 17%–92.7%, and 5 by NNA culture, 18S rDNA PCR and LAMP (sensitivity 100%, CI: 46.3%–100%. The limit of detection of Acanthamoeba DNA was 1 pg/μl by both LAMP and PCR. Conclusion: PCR and LAMP assays targeting 18S rDNA gene were found particularly suitable for a rapid and accurate diagnosis of AK. LAMP assay takes 2–3 h lesser than PCR, and thus offers a rapid, highly sensitive and specific, simple and affordable diagnostic modality for patients suspected of AK, especially in resource limited settings

  4. Rapid, sensitive, and specific detection of Clostridium tetani by loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Jiang, Dongneng; Pu, Xiaoyun; Wu, Jiehong; Li, Meng; Liu, Ping

    2013-01-01

    Tetanus is a specific infectious disease, which is often associated with catastrophic events such as earthquakes, traumas, and war wounds. The obligate anaerobe Clostridium tetani is the pathogen that causes tetanus. Once the infection of tetanus progresses to an advanced stage within the wounds of limbs, the rates of amputation and mortality increase manifold. Therefore, it is necessary to devise a rapid and sensitive point-of-care detection method for C. tetani so as to ensure an early diagnosis and clinical treatment of tetanus. In this study, we developed a detection method for C. tetani using loop-mediated isothermal amplification (LAMP) assay, wherein the C. tetani tetanus toxin gene was used as the target gene. The method was highly specific and sensitive, with a detection limit of 10 colony forming units (CFU)/ml, and allowed quantitative analysis. While detecting C. tetani in clinical samples, it was found that the LAMP results completely agreed with those of the traditional API 20A anaerobic bacteria identification test. As compared with the traditional API test and PCR assay, LAMP detection of C. tetani is simple and rapid, and the results can be identified through naked-eye observation. Therefore, it is an ideal and rapid point-of-care testing method for tetanus.

  5. Multiplex ligation-dependent probe amplification (MLPA) assay for blood group genotyping, copy number quantification, and analysis of RH variants

    NARCIS (Netherlands)

    Veldhuisen, Barbera; van der Schoot, C. E.; de Haas, Masja

    2015-01-01

    The blood group multiplex ligation-dependent probe amplification (MLPA) is a comprehensive assay, developed for genotyping the majority of clinically relevant blood group antigens in both patients and donors. The MLPA is an easy method to apply and only requires a thermal cycler and capillary

  6. A loop-mediated isothermal amplification assay and sample preparation procedure for sensitive detection of Xanthomonas fragariae in strawberry

    Science.gov (United States)

    Xanthomonas fragariae is a bacterium that causes angular leaf spot of strawberry. Asymptomatic infections are common and contribute to the difficulties in disease management. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay with a bacterial enrichment proced...

  7. Development of Recombinase Polymerase Amplification Assays for Detection of Orientia tsutsugamushi or Rickettsia typhi.

    Directory of Open Access Journals (Sweden)

    Chien-Chung Chao

    Full Text Available Sensitive, specific and rapid diagnostic tests for the detection of Orientia tsutsugamushi (O. tsutsugamushi and Rickettsia typhi (R. typhi, the causative agents of scrub typhus and murine typhus, respectively, are necessary to accurately and promptly diagnose patients and ensure that they receive proper treatment. Recombinase polymerase amplification (RPA assays using a lateral flow test (RPA-nfo and real-time fluorescent detection (RPA-exo were developed targeting the 47-kDa gene of O. tsutsugamushi or 17 kDa gene of R. typhi. The RPA assay was capable of detecting O. tsutsugamushi or R. typhi at levels comparable to that of the quantitative PCR method. Both the RPA-nfo and RPA-exo methods performed similarly with regards to sensitivity when detecting the 17 kDa gene of R. typhi. On the contrary, RPA-exo performed better than RPA-nfo in detecting the 47 kDa gene of O. tsutsugamushi. The clinical performance of the O. tsutsugamushi RPA assay was evaluated using either human patient samples or infected mouse samples. Eight out of ten PCR confirmed positives were determined positive by RPA, and all PCR confirmed negative samples were negative by RPA. Similar results were obtained for R. typhi spiked patient sera. The assays were able to differentiate O. tsutsugamushi and R. typhi from other phylogenetically related bacteria as well as mouse and human DNA. Furthermore, the RPA-nfo reaction was completed in 20 minutes at 37°C followed by a 10 minute incubation at room temperature for development of an immunochromatographic strip. The RPA-exo reaction was completed in 20 minutes at 39°C. The implementation of a cross contamination proof cassette to detect the RPA-nfo fluorescent amplicons provided an alternative to regular lateral flow detection strips, which are more prone to cross contamination. The RPA assays provide a highly time-efficient, sensitive and specific alternative to other methods for diagnosing scrub typhus or murine typhus.

  8. Improved sensitivity of nucleic acid amplification for rapid diagnosis of tuberculous meningitis

    DEFF Research Database (Denmark)

    Johansen, Isik Somuncu; Lundgren, Bettina; Tabak, Fehmi

    2004-01-01

    Early diagnosis of tuberculous meningitis (TBM) is essential for a positive outcome; but present microbiological diagnostic techniques are insensitive, slow, or laborious. We evaluated the standard BDProbeTec ET strand displacement amplification method (the standard ProbeTec method) for the detec......Early diagnosis of tuberculous meningitis (TBM) is essential for a positive outcome; but present microbiological diagnostic techniques are insensitive, slow, or laborious. We evaluated the standard BDProbeTec ET strand displacement amplification method (the standard ProbeTec method...... diagnosis was attained by culture. Thirteen specimens from 12 patients were culture positive for M. tuberculosis complex organisms; three specimens (23%) were microscopy positive for acid-fast bacilli. Among the culture-positive specimens, the standard ProbeTec method was positive for 8 (61.......5%) and the modified assay was positive for 10 (76.9%). The overall specificity by both procedures was 98.8% compared to the results of culture. After discrepancy analysis, conducted by reviewing the patients' previous laboratory data, the specificity increased to 100%. If the cutoff value for respiratory specimens...

  9. Nucleic acid tool enzymes-aided signal amplification strategy for biochemical analysis: status and challenges.

    Science.gov (United States)

    Qing, Taiping; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Wen, Li; Shangguan, Jingfang; Mao, Zhengui; Lei, Yanli

    2016-04-01

    Owing to their highly efficient catalytic effects and substrate specificity, the nucleic acid tool enzymes are applied as 'nano-tools' for manipulating different nucleic acid substrates both in the test-tube and in living organisms. In addition to the function as molecular scissors and molecular glue in genetic engineering, the application of nucleic acid tool enzymes in biochemical analysis has also been extensively developed in the past few decades. Used as amplifying labels for biorecognition events, the nucleic acid tool enzymes are mainly applied in nucleic acids amplification sensing, as well as the amplification sensing of biorelated variations of nucleic acids. With the introduction of aptamers, which can bind different target molecules, the nucleic acid tool enzymes-aided signal amplification strategies can also be used to sense non-nucleic targets (e.g., ions, small molecules, proteins, and cells). This review describes and discusses the amplification strategies of nucleic acid tool enzymes-aided biosensors for biochemical analysis applications. Various analytes, including nucleic acids, ions, small molecules, proteins, and cells, are reviewed briefly. This work also addresses the future trends and outlooks for signal amplification in nucleic acid tool enzymes-aided biosensors.

  10. Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP Assay for the Detection of Bacterial Meningitis Pathogens

    Directory of Open Access Journals (Sweden)

    Owen Higgins

    2018-02-01

    Full Text Available Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae, N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology.

  11. Loop-mediated isothermal amplification as a reliable assay for Toxocara canis infection in pet dogs.

    Science.gov (United States)

    Khoshakhlagh, Paria; Spotin, Adel; Mahami-Oskouei, Mahmoud; Shahbazi, Abbas; Ozlati, Maryam

    2017-07-09

    Keeping of infected dogs as pet results in the potential transmission risk factors for shedding helminthic infections such as toxocariasis. Lack of accurate identification of Toxocara canis eggs in non-dewormed infected pet dogs remains a diagnostic concern among researchers. In this study, dog owners were asked to fill up a questionnaire regarding their pets and their attitude towards the deworming regimen. One hundred faecal samples were collected from pet dogs (Northwest Iran) and were subsequently identified by the ZnSo4 flotation technique, PCR and loop-mediated isothermal amplification (LAMP) assays. The DNA of the recovered T. canis eggs was then extracted and amplified by LAMP and PCR. Furthermore, ITS2 amplicons were sequenced for appraisal of the phylogenetic analysis. Nine, 5 and 11% of T. canis infections were identified by microscopy, PCR and LAMP, respectively. It was detected that LAMP was 10 times (10(-10)to 10(-13) g/μl) more sensitive than PCR (10(-10)to 10(-12) g/μl). The kappa value between LAMP and PCR indicated a faint concurrence (0.463). The kappa coefficient between LAMP and flotation technique indicated a strong agreement (0.667). The highest infection rate (n = 11) was detected in non-dewormed pet dogs, particularly those less than 3 months old (P dogs had a history of walking and kennelled behaviours in public places. The LAMP assay can address as a simple, rapid and highly sensitive technique for detecting low burden of T. canis eggs in infected pet dogs. It was proposed that the dog holder's awareness is insufficient to implement regular deworming schedules. Additionally, regional policymakers should broadly revise anthelmintic treatment guidelines.

  12. A rapid Salmonella detection method involving thermophilic helicase-dependent amplification and a lateral flow assay.

    Science.gov (United States)

    Du, Xin-Jun; Zhou, Tian-Jiao; Li, Ping; Wang, Shuo

    2017-08-01

    Salmonella is a major foodborne pathogen that is widespread in the environment and can cause serious human and animal disease. Since conventional culture methods to detect Salmonella are time-consuming and laborious, rapid and accurate techniques to detect this pathogen are critically important for food safety and diagnosing foodborne illness. In this study, we developed a rapid, simple and portable Salmonella detection strategy that combines thermophilic helicase-dependent amplification (tHDA) with a lateral flow assay to provide a detection result based on visual signals within 90 min. Performance analyses indicated that the method had detection limits for DNA and pure cultured bacteria of 73.4-80.7 fg and 35-40 CFU, respectively. Specificity analyses showed no cross reactions with Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Enterobacter aerogenes, Shigella and Campylobacter jejuni. The results for detection in real food samples showed that 1.3-1.9 CFU/g or 1.3-1.9 CFU/mL of Salmonella in contaminated chicken products and infant nutritional cereal could be detected after 2 h of enrichment. The same amount of Salmonella in contaminated milk could be detected after 4 h of enrichment. This tHDA-strip can be used for the rapid detection of Salmonella in food samples and is particularly suitable for use in areas with limited equipment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Assay for Listeria monocytogenes cells in whole blood using isotachophoresis and recombinase polymerase amplification.

    Science.gov (United States)

    Eid, Charbel; Santiago, Juan G

    2016-12-19

    We present a new approach which enables lysis, extraction, and detection of inactivated Listeria monocytogenes cells from blood using isotachophoresis (ITP) and recombinase polymerase amplification (RPA). We use an ITP-compatible alkaline and proteinase K approach for rapid and effective lysis. We then perform ITP purification to separate bacterial DNA from whole blood contaminants using a microfluidic device that processes 25 μL sample volume. Lysis, mixing, dispensing, and on-chip ITP purification are completed in a total of less than 50 min. We transfer extracted DNA directly into RPA master mix for isothermal incubation and detection, an additional 25 min. We first validate our assay in the detection of purified genomic DNA spiked into whole blood, and demonstrate a limit of detection of 16.7 fg μL -1 genomic DNA, the equivalent of 5 × 10 3 cells per mL. We then show detection of chemically-inactivated L. monocytogenes cells spiked into whole blood, and demonstrate a limit of detection of 2 × 10 4 cells per mL. Lastly, we show preliminary experimental data demonstrating the feasibility of the integration of ITP purification with RPA detection on a microfluidic chip. Our results suggest that ITP purification is compatible with RPA detection, and has potential to extend the applicability of RPA to whole blood.

  14. Loop-Mediated Isothermal Amplification Assay for Identification of Five Human Plasmodium Species in Malaysia.

    Science.gov (United States)

    Lau, Yee-Ling; Lai, Meng-Yee; Fong, Mun-Yik; Jelip, Jenarun; Mahmud, Rohela

    2016-02-01

    The lack of rapid, affordable, and accurate diagnostic tests represents the primary hurdle affecting malaria surveillance in resource- and expertise-limited areas. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cheap diagnostic method. Five species-specific LAMP assays were developed based on 18S rRNA gene. Sensitivity and specificity of LAMP results were calculated as compared with microscopic examination and nested polymerase chain reaction. LAMP reactions were highly sensitive with the detection limit of one copy for Plasmodium vivax, Plasmodium falciparum, and Plasmodium malariae and 10 copies for Plasmodium knowlesi and Plasmodium ovale. LAMP positively detected all human malaria species in all positive samples (N = 134; sensitivity = 100%) within 35 minutes. All negative samples were not amplified by LAMP (N = 67; specificity = 100%). LAMP successfully detected two samples with very low parasitemia. LAMP may offer a rapid, simple, and reliable test for the diagnosis of malaria in areas where malaria is prevalent. © The American Society of Tropical Medicine and Hygiene.

  15. Nucleic acid amplification tests for diagnosis of Neisseria gonorrhoeae oropharyngeal infections.

    Science.gov (United States)

    Bachmann, Laura H; Johnson, Robert E; Cheng, Hong; Markowitz, Lauri E; Papp, John R; Hook, Edward W

    2009-04-01

    The optimal methods for the diagnosis of pharyngeal Neisseria gonorrhoeae infection are uncertain. The objective of this study was to define the performance of culture and nucleic acid amplification tests (NAATs) for the diagnosis of pharyngeal N. gonorrhoeae. In this cross-sectional study, males and females >15 years old who acknowledged performing fellatio or cunnilingus (in the previous 2 months) were recruited from three clinics (two human immunodeficiency virus clinics and one sexually transmitted diseases clinic) located in Birmingham, AL. The test performance of culture for N. gonorrhoeae, the Gen-Probe Aptima Combo 2 transcription-mediated amplification assay (TMA), the BD ProbeTec ET amplified DNA strand displacement assay (SDA), and the Roche Cobas Amplicor PCR was defined by using a rotating "gold standard" of any positive results by two or three of the three tests that excluded the test being evaluated. A total of 961 evaluable test sets were collected. On the basis of a rotating gold standard of positive results by two of three comparator tests, the sensitivity and the specificity were as follows: culture for N. gonorrhoeae, 50.0% and 99.4%, respectively; PCR, 80.3% and 73.0%, respectively; TMA, 83.6% and 98.6%, respectively; and SDA, 93.2% and 96.3%, respectively. On the basis of a rotating gold standard of positive results by three of three comparator tests, the sensitivity and specificity were as follows: culture for N. gonorrhoeae, 65.4% and 99.0%, respectively; PCR, 91.9% and 71.8%, respectively; TMA, 100% and 96.2%, respectively; and SDA, 97.1% and 94.2%, respectively. In conclusion, currently available NAATs are more sensitive than culture for the detection of pharyngeal gonorrhea in at-risk patients. PCR is substantially less specific than culture, TMA, or SDA and should not be used for the detection of pharyngeal gonorrhea.

  16. Nucleic Acid Amplification Tests for Diagnosis of Neisseria gonorrhoeae Oropharyngeal Infections▿

    Science.gov (United States)

    Bachmann, Laura H.; Johnson, Robert E.; Cheng, Hong; Markowitz, Lauri E.; Papp, John R.; Hook, Edward W.

    2009-01-01

    The optimal methods for the diagnosis of pharyngeal Neisseria gonorrhoeae infection are uncertain. The objective of this study was to define the performance of culture and nucleic acid amplification tests (NAATs) for the diagnosis of pharyngeal N. gonorrhoeae. In this cross-sectional study, males and females >15 years old who acknowledged performing fellatio or cunnilingus (in the previous 2 months) were recruited from three clinics (two human immunodeficiency virus clinics and one sexually transmitted diseases clinic) located in Birmingham, AL. The test performance of culture for N. gonorrhoeae, the Gen-Probe Aptima Combo 2 transcription-mediated amplification assay (TMA), the BD ProbeTec ET amplified DNA strand displacement assay (SDA), and the Roche Cobas Amplicor PCR was defined by using a rotating “gold standard” of any positive results by two or three of the three tests that excluded the test being evaluated. A total of 961 evaluable test sets were collected. On the basis of a rotating gold standard of positive results by two of three comparator tests, the sensitivity and the specificity were as follows: culture for N. gonorrhoeae, 50.0% and 99.4%, respectively; PCR, 80.3% and 73.0%, respectively; TMA, 83.6% and 98.6%, respectively; and SDA, 93.2% and 96.3%, respectively. On the basis of a rotating gold standard of positive results by three of three comparator tests, the sensitivity and specificity were as follows: culture for N. gonorrhoeae, 65.4% and 99.0%, respectively; PCR, 91.9% and 71.8%, respectively; TMA, 100% and 96.2%, respectively; and SDA, 97.1% and 94.2%, respectively. In conclusion, currently available NAATs are more sensitive than culture for the detection of pharyngeal gonorrhea in at-risk patients. PCR is substantially less specific than culture, TMA, or SDA and should not be used for the detection of pharyngeal gonorrhea. PMID:19193848

  17. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 16 2010-07-01 2010-07-01 false Glial fibrillary acidic protein assay... Glial fibrillary acidic protein assay. (a) Purpose. Chemical-induced injury of the nervous system, i.e... paragraph (e)(3) in this section). Assays of glial fibrillary acidic protein (GFAP), the major intermediate...

  18. Validation of a Salmonella loop-mediated isothermal amplification assay in animal food.

    Science.gov (United States)

    Domesle, Kelly J; Yang, Qianru; Hammack, Thomas S; Ge, Beilei

    2018-01-02

    Loop-mediated isothermal amplification (LAMP) has emerged as a promising alternative to PCR for pathogen detection in food testing and clinical diagnostics. This study aimed to validate a Salmonella LAMP method run on both turbidimetry (LAMP I) and fluorescence (LAMP II) platforms in representative animal food commodities. The U.S. Food and Drug Administration (FDA)'s culture-based Bacteriological Analytical Manual (BAM) method was used as the reference method and a real-time quantitative PCR (qPCR) assay was also performed. The method comparison study followed the FDA's microbiological methods validation guidelines, which align well with those from the AOAC International and ISO. Both LAMP assays were 100% specific among 300 strains (247 Salmonella of 185 serovars and 53 non-Salmonella) tested. The detection limits ranged from 1.3 to 28 cells for six Salmonella strains of various serovars. Six commodities consisting of four animal feed items (cattle feed, chicken feed, horse feed, and swine feed) and two pet food items (dry cat food and dry dog food) all yielded satisfactory results. Compared to the BAM method, the relative levels of detection (RLODs) for LAMP I ranged from 0.317 to 1 with a combined value of 0.610, while those for LAMP II ranged from 0.394 to 1.152 with a combined value of 0.783, which all fell within the acceptability limit (2.5) for an unpaired study. This also suggests that LAMP was more sensitive than the BAM method at detecting low-level Salmonella contamination in animal food and results were available 3days sooner. The performance of LAMP on both platforms was comparable to that of qPCR but notably faster, particularly LAMP II. Given the importance of Salmonella in animal food safety, the LAMP assays validated in this study holds great promise as a rapid, reliable, and robust method for routine screening of Salmonella in these commodities. Published by Elsevier B.V.

  19. Development of a loop-mediated Isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus

    Directory of Open Access Journals (Sweden)

    Kawahara Ryuji

    2008-09-01

    Full Text Available Abstract Background Vibrio parahaemolyticus is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH and TDH-related hemolysin (TRH are known as major virulence determinants of V. parahaemolyticus. Most V. parahaemolyticus isolates from the environment do not produce TDH or TRH. Total V. parahaemolyticus has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total V. parahaemolyticus using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP assay for the sensitive and rapid detection of Vibrio parahaemolyticus. Results The assay provided markedly more sensitive and rapid detection of V. parahaemolyticus strains than conventional biochemical and PCR assays. The assay correctly identified 143 V. parahaemolyticus strains, but did not detect 33 non-parahaemolyticus Vibrio and 56 non-Vibrio strains. Sensitivity of the LAMP assay for direct detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples was 5.3 × 102 CFU per ml/g (2.0 CFU per reaction. The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay was markedly faster, requiring for amplification 13–22 min in a single colony on TCBS agar from each of 143 V. parahaemolyticus strains and less than 35 min in spiked shrimp samples. The LAMP assay for detection of V. parahaemolyticus required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination. Conclusion The LAMP assay is a sensitive, rapid and simple tool for the detection of V. parahaemolyticus and will facilitate the surveillance for control of contamination of V

  20. Amplification of the Gp41 gene for detection of mutations conferring resistance to HIV-1 fusion inhibitors on genotypic assay

    Science.gov (United States)

    Tanumihardja, J.; Bela, B.

    2017-08-01

    Fusion inhibitors have potential for future use in HIV control programs in Indonesia, so the capacity to test resistance to such drugs needs to be developed. Resistance-detection with a genotypic assay began with amplification of the target gene, gp41. Based on the sequence of the two most common HIV subtypes in Indonesia, AE and B, a primer pair was designed. Plasma samples containing both subtypes were extracted to obtain HIV RNA. Using PCR, the primer pair was used to produce the amplification product, the identity of which was checked based on length under electrophoresis. Eleven plasma samples were included in this study. One-step PCR using the primer pair was able to amplify gp41 from 54.5% of the samples, and an unspecific amplification product was seen in 1.1% of the samples. Amplification failed in 36.4% of the samples, which may be due to an inappropriate primer sequence. It was also found that the optimal annealing temperature for producing the single expected band was 57.2 °C. With one-step PCR, the designed primer pair amplified the HIV-1 gp41 gene from subtypes AE and B. However, further research should be done to determine the conditions that will increase the sensitivity and specificity of the amplification process.

  1. Development and deployment of a rapid recombinase polymerase amplification Ebola virus detection assay in Guinea in 2015

    OpenAIRE

    Faye, Oumar; Faye, Ousmane; Soropogui, B.; Patel, Pranav; Abd El Wahed, Ahmed; Loucoubar, C.; Fall, G.; Kiory, D.; Magassouba, N.; Keita, S.; Kondé, M. K.; Diallo, A.; Koivogui, L.; Karlberg, H.; Mirazimi, Ali

    2015-01-01

    In the absence of a vaccine or specific treatments for Ebola virus disease (EVD), early identification of cases is crucial for the control of EVD epidemics. We evaluated a new extraction kit (SpeedXtract (SE), Qiagen) on sera and swabs in combination with an improved diagnostic reverse transcription recombinase polymerase amplification assay for the detection of Ebola virus (EBOV-RT-RPA). The performance of combined extraction and detection was best for swabs. Sensitivity and specificity of t...

  2. Development of Chemiluminescent Lateral Flow Assay for the Detection of Nucleic Acids

    Directory of Open Access Journals (Sweden)

    Sam R. Nugen

    2012-01-01

    Full Text Available Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety. Herein we report the development of a nucleic acid sequence-based lateral flow assay which achieves a low limit of detection using chemiluminescence. On-membrane enzymatic signal amplification is used to reduce the limit of detection to the sub-femtomol level. To demonstrate this assay, we detected synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, with relevance in human and animal health in sub-Saharan Africa. The intensity of the chemiluminescent signal was evaluated by using a charge-coupled device as well as a microtiter plate reader. We demonstrated that our lateral flow chemiluminescent assay has a very low limit of detection and is easy to use. The limit of detection was determined to be 0.5 fmols of nucleic acid target.

  3. Development of Chemiluminescent Lateral Flow Assay for the Detection of Nucleic Acids

    Science.gov (United States)

    Wang, Yuhong; Fill, Catherine; Nugen, Sam R.

    2012-01-01

    Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety. Herein we report the development of a nucleic acid sequence-based lateral flow assay which achieves a low limit of detection using chemiluminescence. On-membrane enzymatic signal amplification is used to reduce the limit of detection to the sub-femtomol level. To demonstrate this assay, we detected synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, with relevance in human and animal health in sub-Saharan Africa. The intensity of the chemiluminescent signal was evaluated by using a charge-coupled device as well as a microtiter plate reader. We demonstrated that our lateral flow chemiluminescent assay has a very low limit of detection and is easy to use. The limit of detection was determined to be 0.5 fmols of nucleic acid target. PMID:25585630

  4. Diagnostic accuracy of the real-time PCR cobas(®) Liat(®) Influenza A/B assay and the Alere i Influenza A&B NEAR isothermal nucleic acid amplification assay for the detection of influenza using adult nasopharyngeal specimens.

    Science.gov (United States)

    Young, Stephen; Illescas, Patrick; Nicasio, Joclin; Sickler, Joanna Jackson

    2017-09-01

    Accurate detection of influenza requires diagnostic testing; however, methods such as RADTs and central laboratory-based tests are limited by low sensitivity and time constraints, respectively. To compare the performances of the cobas(®) Liat(®) Influenza A/B and Alere™ i Influenza A&B point-of-care (POC) assays for detecting influenza A and B viruses using fresh nasopharyngeal specimens with the GenMark Dx(®) Respiratory Viral Panel as the reference method, a FDA cleared IVD PCR test. A total of 87 samples collected in viral transport medium from adults ≥18 years of age were re-tested on both POC assays (based on the reference PCR method, 29 were influenza A and 18 were influenza B virus positive). The overall sensitivity and specificity of the cobas Influenza A/B for the detection of influenza A and B relative to reference PCR was 97.9% (95% confidence interval [CI] 88.9%, 99.6%) and 97.5% (95% CI: 87.1%, 99.6%), respectively, while the sensitivity of the Alere i Influenza A&B assay relative to the reference PCR method was 63.8% (95% CI: 49.5%, 76.0%) and the specificity was 97.5% (95% CI: 87.1%, 99.6%). The individual sensitivities and specificities of the cobas Influenza A/B assay for influenza A alone and influenza B alone were comparable to those of the reference PCR method (influenza A: sensitivity of 100% [95% CI: 88.3%, 100.0%] and specificity of 98.3% [95% CI: 90.9%, 99.7%]; influenza B: sensitivity of 94.4% [95% CI: 74.2%, 99.0%] and specificity of 100% [95% CI: 94.7%, 100.0%]). For the Alere i Influenza A&B assay, the individual specificities for influenza A and B were comparable to those of the reference PCR method (98.3% [95% CI: 90.9%, 99.7%] and 97.1% [95% CI: 90.0%, 99.2%], respectively), while the individual sensitivities were low relative to reference PCR (55.2% [95% CI: 37.5%, 71.6%] and 72.2% [95% CI: 49.1%, 87.5%], respectively). The cobas Influenza A/B assay demonstrated performance equivalent to laboratory-based PCR, and could replace

  5. Evaluation of Loop-Mediated Isothermal Amplification Assay for the Detection of Pneumocystis jirovecii in Immunocompromised Patients

    Directory of Open Access Journals (Sweden)

    Preeti Singh

    2015-01-01

    Full Text Available Pneumocystis pneumonia (PCP is one of the common opportunistic infection among HIV and non-HIV immunocompromised patients. The lack of a rapid and specific diagnostic test necessitates a more reliable laboratory diagnostic test for PCP. In the present study, the loop-mediated isothermal amplification (LAMP assay was evaluated for the detection of Pneumocystis jirovecii. 185 clinical respiratory samples, including both BALF and IS, were subjected to GMS staining, nested PCR, and LAMP assay. Of 185 respiratory samples, 12/185 (6.5%, 41/185 (22.2%, and 49/185 (26.5% samples were positive by GMS staining, nested PCR, and LAMP assay, respectively. As compared to nested PCR, additional 8 samples were positive by LAMP assay and found to be statistically significant (p<0.05 with the detection limit of 1 pg. Thus, the LAMP assay may serve as a better diagnostic tool for the detection of P. jirovecii with high sensitivity and specificity, less turn-around time, operational simplicity, single-step amplification, and immediate visual detection.

  6. Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Trichosporon asahii in Experimental and Clinical Samples

    Science.gov (United States)

    Zhou, Jianfeng; Liao, Yong; Li, Haitao; Lu, Xuelian; Han, Xiufeng; Tian, Yanli; Chen, Shanshan; Yang, Rongya

    2015-01-01

    Invasive trichosporonosis is a deep mycosis found mainly in immunocompromised hosts, and the major pathogen is Trichosporon asahii. We detected the species-specific intergenic spacers (IGS) of rRNA gene of T. asahii using a loop-mediated isothermal amplification (LAMP) assay in 15 isolates with 3 different visualization methods, including SYBR green detection, gel electrophoresis, and turbidimetric methods. The LAMP assay displayed superior rapidity to other traditional methods in the detection time; that is, only 1 h was needed for detection and identification of the pathogen DNA. Furthermore, the detection limit of the LAMP assay was more sensitive than the PCR assay. We also successfully detect the presence of T. asahii in samples from experimentally infected mice and samples from patients with invasive trichosporonosis caused by T. asahii, suggesting that this method may become useful in clinical applications in the near future. PMID:25692144

  7. Microfluidic devices for nucleic acid (NA) isolation, isothermal NA amplification, and real-time detection.

    Science.gov (United States)

    Mauk, Michael G; Liu, Changchun; Sadik, Mohamed; Bau, Haim H

    2015-01-01

    Molecular (nucleic acid)-based diagnostics tests have many advantages over immunoassays, particularly with regard to sensitivity and specificity. Most on-site diagnostic tests, however, are immunoassay-based because conventional nucleic acid-based tests (NATs) require extensive sample processing, trained operators, and specialized equipment. To make NATs more convenient, especially for point-of-care diagnostics and on-site testing, a simple plastic microfluidic cassette ("chip") has been developed for nucleic acid-based testing of blood, other clinical specimens, food, water, and environmental samples. The chip combines nucleic acid isolation by solid-phase extraction; isothermal enzymatic amplification such as LAMP (Loop-mediated AMPlification), NASBA (Nucleic Acid Sequence Based Amplification), and RPA (Recombinase Polymerase Amplification); and real-time optical detection of DNA or RNA analytes. The microfluidic cassette incorporates an embedded nucleic acid binding membrane in the amplification reaction chamber. Target nucleic acids extracted from a lysate are captured on the membrane and amplified at a constant incubation temperature. The amplification product, labeled with a fluorophore reporter, is excited with a LED light source and monitored in situ in real time with a photodiode or a CCD detector (such as available in a smartphone). For blood analysis, a companion filtration device that separates plasma from whole blood to provide cell-free samples for virus and bacterial lysis and nucleic acid testing in the microfluidic chip has also been developed. For HIV virus detection in blood, the microfluidic NAT chip achieves a sensitivity and specificity that are nearly comparable to conventional benchtop protocols using spin columns and thermal cyclers.

  8. Development of a recombinase polymerase amplification combined with lateral-flow dipstick assay for detection of bovine ephemeral fever virus.

    Science.gov (United States)

    Hou, Peili; Zhao, Guimin; Wang, Hongmei; He, Chengqiang; Huan, Yanjun; He, Hongbin

    2017-12-26

    Bovine ephemeral fever virus (BEFV), identified as the causative pathogen of bovine ephemeral fever (BEF), is responsible for increasing numbers of epidemics/outbreaks and has a significant harmful effect on the livestock industry. Therefore, a rapid detection assay is imperative for BEFV diagnosis. In this study, we described the development of lateral-flow dipstick isothermal recombinase polymerase amplification (LFD-RPA) assays for detection of BEFV. RPA primers and LF probes were designed by targeting the specific G gene, and the amplification product can be visualized on a simple lateral flow dipstick with the naked eyes. The amplification reaction was performed at 38 °C for 20 min and LFD incubation time within 5 min. The detection limit of this assay was 8 copies per reaction, and there was no cross-reactivity with other bovine infectious viruses such as bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine vesicular stomatitis virus. In addition, the assay was performed with total 128 clinical specimens and the diagnostic results were compared with conventional RT-PCR, real-time quantative(q) PCR. The result showed that the coincidence rate of BEFV LFD-RPA and real-time qPCR was 96.09% (123/128), which was higher than conventional RT-PCR. The RPA combined with LFD assay probably provides a rapid and sensitive alternative for diagnosis of BEFV infections outbreak. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. A Loop-Mediated Isothermal Amplification Assay and Sample Preparation Procedure for Sensitive Detection of Xanthomonas fragariae in Strawberry

    Science.gov (United States)

    Wang, Hehe; Turechek, William W.

    2016-01-01

    Xanthomonas fragariae is a bacterium that causes angular leaf spot of strawberry. Asymptomatic infection is common and contributes to the difficulties in disease management. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay as an efficient method for detection of asymptomatic infections of X. fragariae. In addition, a new method of sample preparation was developed that allows sampling of a larger amount of plant tissue, hence increasing the detection rate in real-life samples. The sample preparation procedure includes an overnight incubation of strawberry tissues in phosphate-buffered saline (PBS), followed by a quick sample concentration and a boiling step to extract DNA for amplification. The detection limit of the LAMP assay was approximately 2×103 CFU/mL for pure bacteria culture and 300 CFU/mL for bacteria spiked strawberry leaf and petiole samples. LAMP provided a 2–3 fold lower detection limit than the standard qPCR assay but was faster, and more user-friendly. The LAMP assay should serve as a rapid, sensitive and cost-effective tool for detecting asymptomatic infections of X. fragariae in strawberry nursery stock and contribute to improved disease management. PMID:26766068

  10. Detection of DNA sequences refractory to PCR amplification using a biophysical SERRS assay (Surface Enhanced Resonant Raman Spectroscopy.

    Directory of Open Access Journals (Sweden)

    Cécile Feuillie

    Full Text Available The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction - based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS. It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage.

  11. Detection of DNA sequences refractory to PCR amplification using a biophysical SERRS assay (Surface Enhanced Resonant Raman Spectroscopy).

    Science.gov (United States)

    Feuillie, Cécile; Merheb, Maxime M; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

    2014-01-01

    The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction - based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage.

  12. Ultrasensitive paper based nucleic acid detection realized by three-dimensional DNA-AuNPs network amplification.

    Science.gov (United States)

    Gao, Ya; Deng, Xilei; Wen, Wei; Zhang, Xiuhua; Wang, Shengfu

    2017-06-15

    A novel three-dimensional DNA-AuNPs network structure amplification strategy was employed to design a lateral flow biosensor by introducing streptavidin coated gold nanoparticles (Au-SA) in this paper. They act as amplification probes which aggregate numerous gold nanoparticles (AuNPs) on test line by forming a three-dimensional DNA-AuNPs network structure in the presence of target. Sensitive detection of nucleic acid with point-of-care analysis is significant for infectious agent, early diagnosis and treatment of genetic diseases. The use of these particles in rapid ultrasensitive point of care (POC) lateral flow assays lead to a linear range from 0.1pM to 250nM with a limit of detection of 0.01 pM without polymerase chain reaction (PCR). The proposed method could increase the sensitivity by 4 orders of magnitudes than traditional sandwich assays labeled with AuNPs. Furthermore, the assay owns good reproducibility and stability, which will prove practical diagnostic applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. A versatile size-coded flow cytometric bead assay for simultaneous detection of multiple microRNAs coupled with a two-step cascading signal amplification.

    Science.gov (United States)

    Qiu, Liying; Zhang, Yuecheng; Liu, Chenghui; Li, Zhengping

    2017-03-07

    A versatile flow cytometric bead assay is developed for the sensitive and simultaneous detection of multiple microRNAs that combines an elegant enzyme-mediated cascading signal amplification on magnetic beads with a novel bead size-encoding mechanism.

  14. Rapid detection of newly isolated Tembusu-related Flavivirus by reverse-transcription loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Wang Youling

    2011-12-01

    Full Text Available Abstract Background From April 2010 to January 2011, a severe new viral disease had devastated most duck-farming regions in China. This disease affected not only laying ducks but also meat ducks, causing huge economic losses for the poultry industry. The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP assay for the detection of the new virus related to Tembusu-related Flavivirus. Results The RT-LAMP assay is very simple and rapid, and the amplification can be completed within 50 min under isothermal conditions at 63°C by a set of 6 primers targeting the E gene based on the sequences analysis of the newly isolated viruses and other closely related Flavivirus.The monitoring of gene amplification can also be visualized by using SYBR green I fluorescent dye. In addition, the RT-LAMP assay for newly isolated Tembusu-related Flavivirus showed higher sensitivity with an RNA detection-limit of 2 copies/μL compared with 190 copies/μL of the conventional RT-PCR method. The specificity was identified without cross reaction to other common avian pathogens. By screening a panel of clinical samples this method was more feasible in clinical settings and there was higher positive coincidence rate than conventional RT-PCR and virus isolation. Conclusion The RT-LAMP assay for newly isolated Tembusu-related Flavivirus is a valuable tool for the rapid and real-time detection not only in well-equipped laboratories but also in general conditions.

  15. Development and evaluation of a novel and rapid detection assay for Botrytis cinerea based on loop-mediated isothermal amplification.

    Directory of Open Access Journals (Sweden)

    Ya-Bing Duan

    Full Text Available Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP with hydroxynaphthol blue dye (HNB. The LAMP reaction was optimal at 63 °C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10(-3 ng µL(-1 of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10(-2 ng µL(-1. Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2% were confirmed as positive by LAMP, 172 (90.1% positive by the tissue separation, while 147 (77.0% positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables.

  16. Development and evaluation of a novel and rapid detection assay for Botrytis cinerea based on loop-mediated isothermal amplification.

    Science.gov (United States)

    Duan, Ya-Bing; Ge, Chang-Yan; Zhang, Xiao-Ke; Wang, Jian-Xin; Zhou, Ming-Guo

    2014-01-01

    Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB). The LAMP reaction was optimal at 63 °C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10(-3) ng µL(-1) of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10(-2) ng µL(-1)). Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2%) were confirmed as positive by LAMP, 172 (90.1%) positive by the tissue separation, while 147 (77.0%) positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables.

  17. Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection.

    Science.gov (United States)

    Verma, Sandeep; Singh, Ruchi; Sharma, Vanila; Bumb, Ram Avtar; Negi, Narendra Singh; Ramesh, V; Salotra, Poonam

    2017-03-23

    Leishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. A robust, rapid and cost effective diagnostic test is warranted for on-time diagnosis and field application. We have developed a loop mediated isothermal amplification (LAMP) assay with primers (n = 6) based on Leishmania donovani kDNA for detection of Leishmania infection, using a closed tube to prevent cross-contamination. The assay was used to detect Leishmania infection in biological samples obtained from patients of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL). The assay was positive for L. donovani, L. tropica and L. major parasites, with the highest sensitivity towards L. donovani (1 fg DNA). The high sensitivity of the assay for detection of L. donovani was reflected in its ability to detect parasite DNA within 30 min of amplification time with a threshold detection limit of ≥25 copies per reaction. The assay detected parasite in 64 of 66 VL blood samples (sensitivity, 96.9%; 95% CI: 89.6-99.2%), 15 of 15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI:79.6-100%), 65 of 67 PKDL tissue biopsy samples (sensitivity, 97%; 95% CI:89.7-99.2%). The assay was evaluated in a few cases of CL wherein it was found positive in 8 of 10 tissue biopsies (sensitivity, 80%; 95% CI: 49-94.3%). The assay was negative in all control blood (n = 76) and tissue biopsy (n = 24) samples (specificity, 100%; 95% CI: 96.3-100%). Further, the assay was evaluated for its utility in assessment of cure in treated VL and PKDL patients. The assay detected parasite DNA in 2 of 20VL blood samples and 2 of 21 PKDL tissue samples. Out of 4 cases that were positive for parasite DNA at post treatment stage, 2 patients (1VL and 1 PKDL) returned with relapse. The study demonstrated a Leishmania genus specific closed tube LAMP assay for reliable and rapid

  18. Loop-mediated isothermal amplification (LAMP assays for the species-specific detection of Eimeria that infect chickens

    Directory of Open Access Journals (Sweden)

    Blake Damer P

    2011-11-01

    Full Text Available Abstract Background Eimeria parasites can cause the disease coccidiosis in poultry and even subclinical infection can incur economic loss. Diagnosis of infection predominantly relies on traditional techniques including lesion scoring and faecal microscopy despite the availability of sensitive molecular assays, largely due to cost and the requirement for specialist equipment. Despite longstanding proven efficacy these traditional techniques demand time and expertise, can be highly subjective and may under-diagnose subclinical disease. Recognition of the tight economic margins prevailing in modern poultry production and the impact of avian coccidiosis on poverty in many parts of the world has highlighted a requirement for a panel of straightforward and sensitive, but cost-effective, Eimeria species-specific diagnostic assays. Results Loop-mediated isothermal amplification (LAMP is an uncomplicated, quick and relatively inexpensive diagnostic tool. In this study we have developed a panel of species-specific LAMP assays targeting the seven Eimeria species that infect the chicken. Each assay has been shown to be genuinely species-specific with the capacity to detect between one and ten eimerian genomes, equivalent to less than a single mature schizont. Development of a simple protocol for template DNA preparation from tissue collected post mortem with no requirement for specialist laboratory equipment supports the use of these assays in routine diagnosis of eimerian infection. Preliminary field testing supports this hypothesis. Conclusions Development of a panel of sensitive species-specific LAMP assays introduces a valuable new cost-effective tool for use in poultry husbandry.

  19. Prospective evaluation of the Alere i Influenza A&B nucleic acid amplification versus Xpert Flu/RSV.

    Science.gov (United States)

    Nguyen Van, J C; Caméléna, F; Dahoun, M; Pilmis, B; Mizrahi, A; Lourtet, J; Behillil, S; Enouf, V; Le Monnier, A

    2016-05-01

    The rapid and accurate detection of influenza virus in respiratory specimens is required for optimal management of patients with acute respiratory infections. Because of the variability of the symptoms and the numerous other causes of influenza-like illness, the diagnosis of influenza cannot be made on the basis of clinical criteria alone. Thus, rapid influenza diagnostic tests have been developed such as the Alere i Influenza A&B isothermal nucleic acid assay. We prospectively evaluated the performance of the Alere i Influenza A&B assay in comparison with our routine Xpert Flu/RSV assay. Positive samples were subtyped according to the protocol from the National Influenza Center (Paris, France). A total of 96 respiratory nasal swab samples were analyzed: with both methods, 38 were positive and 56 were negative. Samples were prospectively collected from January 20 to April 8, 2015, from patient (86 adult and 10 pediatric patients) presenting with an influenza-like illness through the French influenza season. In comparison with the Xpert Flu/RSV assay, the overall sensitivity and specificity of the Alere i Influenza A&B assay were 95% and 100%, respectively. Our results indicate that the Alere i Influenza A&B assay has a good overall analytical performance and a high degree of concordance with the PCR-based Xpert Flu/RSV assay. The Alere i Influenza A&B isothermal nucleic acid amplification test is a powerful tool for influenza detection due to its high sensitivity and specificity as well as its ability to generate results within 15min. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Rapid detection of Salmonella in food and feed by coupling loop-mediated isothermal amplification with bioluminescent assay in real-time

    OpenAIRE

    Yang, Qianru; Domesle, Kelly J; Wang, Fei; Ge, Beilei

    2016-01-01

    Background Salmonella is among the most significant pathogens causing food and feed safety concerns. This study examined the rapid detection of Salmonella in various types of food and feed samples by coupling loop-mediated isothermal amplification (LAMP) with a novel reporter, bioluminescent assay in real-time (BART). Performance of the LAMP-BART assay was compared to a conventional LAMP and the commercially available 3M Molecular Detection Assay (MDA) Salmonella. Results The LAMP-BART assay ...

  1. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis

    Energy Technology Data Exchange (ETDEWEB)

    Santiago-Felipe, S.; Tortajada-Genaro, L.A.; Puchades, R.; Maquieira, A., E-mail: amaquieira@qim.upv.es

    2014-02-06

    Graphical abstract: -- Highlights: •Recombinase polymerase amplification is a powerful DNA method operating at 40 °C. •The combination RPA–ELISA gives excellent performances for high-throughput analysis. •Screening of food safety threats has been done using standard laboratory equipment. •Allergens, GMOs, bacteria, and fungi have been successfully determined. -- Abstract: Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR–ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA–ELISA combination is proposed for amplification at a low, constant temperature (40 °C) in a short time (40 min), for the hybridisation of labelled products to specific 5′-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA–ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA–ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings.

  2. On-Chip, Amplification-Free Quantification of Nucleic Acid for Point-of-Care Diagnosis

    Science.gov (United States)

    Yen, Tony Minghung

    This dissertation demonstrates three physical device concepts to overcome limitations in point-of-care quantification of nucleic acids. Enabling sensitive, high throughput nucleic acid quantification on a chip, outside of hospital and centralized laboratory setting, is crucial for improving pathogen detection and cancer diagnosis and prognosis. Among existing platforms, microarray have the advantages of being amplification free, low instrument cost, and high throughput, but are generally less sensitive compared to sequencing and PCR assays. To bridge this performance gap, this dissertation presents theoretical and experimental progress to develop a platform nucleic acid quantification technology that is drastically more sensitive than current microarrays while compatible with microarray architecture. The first device concept explores on-chip nucleic acid enrichment by natural evaporation of nucleic acid solution droplet. Using a micro-patterned super-hydrophobic black silicon array device, evaporative enrichment is coupled with nano-liter droplet self-assembly workflow to produce a 50 aM concentration sensitivity, 6 orders of dynamic range, and rapid hybridization time at under 5 minutes. The second device concept focuses on improving target copy number sensitivity, instead of concentration sensitivity. A comprehensive microarray physical model taking into account of molecular transport, electrostatic intermolecular interactions, and reaction kinetics is considered to guide device optimization. Device pattern size and target copy number are optimized based on model prediction to achieve maximal hybridization efficiency. At a 100-mum pattern size, a quantum leap in detection limit of 570 copies is achieved using black silicon array device with self-assembled pico-liter droplet workflow. Despite its merits, evaporative enrichment on black silicon device suffers from coffee-ring effect at 100-mum pattern size, and thus not compatible with clinical patient samples. The

  3. Development of a loop-mediated isothermal amplification assay for rapid, sensitive detection of Campylobacter jejuni in cattle farm samples.

    Science.gov (United States)

    Dong, Hee-Jin; Cho, Ae-Ri; Hahn, Tae-Wook; Cho, Seongbeom

    2014-09-01

    Campylobacter jejuni is a leading cause of bacterial foodborne disease worldwide. The detection of this organism in cattle and their environment is important for the control of C. jejuni transmission and the prevention of campylobacteriosis. Here, we describe the development of a rapid and sensitive method for the detection of C. jejuni in naturally contaminated cattle farm samples, based on real-time loop-mediated isothermal amplification (LAMP) of the hipO gene. The LAMP assay was specific (100% inclusivity and exclusivity for 84 C. jejuni and 41 non-C. jejuni strains, respectively), sensitive (detection limit of 100 fg/μl), and quantifiable (R(2) = 0.9133). The sensitivity of the LAMP assay was then evaluated for its application to the naturally contaminated cattle farm samples. C. jejuni strains were isolated from 51 (20.7%) of 246 cattle farm samples, and the presence of the hipO gene was tested using the LAMP assay. Amplification of the hipO gene by LAMP within 30 min (mean ~10.8 min) in all C. jejuni isolates (n = 51) demonstrated its rapidity and accuracy. Next, template DNA was prepared from a total of 186 enrichment broth cultures of cattle farm samples either by boiling or using a commercial kit, and the sensitivity of detection of C. jejuni was compared between the LAMP and PCR assays. In DNA samples prepared by boiling, the higher sensitivity of the LAMP assay (84.4%) compared with the PCR assay (35.5%) indicates that it is less susceptible to the existence of inhibitors in sample material. In DNA samples prepared using a commercial kit, both the LAMP and PCR assays showed 100% sensitivity. We anticipate that the use of this rapid, sensitive, and simple LAMP assay, which is the first of its kind for the identification and screening of C. jejuni in cattle farm samples, may play an important role in the prevention of C. jejuni contamination in the food chain, thereby reducing the risk of human campylobacteriosis.

  4. Design of a New Type of Compact Chemical Heater for Isothermal Nucleic Acid Amplification.

    Directory of Open Access Journals (Sweden)

    Kamal G Shah

    Full Text Available Previous chemical heater designs for isothermal nucleic acid amplification have been based on solid-liquid phase transition, but using this approach, developers have identified design challenges en route to developing a low-cost, disposable device. Here, we demonstrate the feasibility of a new heater configuration suitable for isothermal amplification in which one reactant of an exothermic reaction is a liquid-gas phase-change material, thereby eliminating the need for a separate phase-change compartment. This design offers potentially enhanced performance and energy density compared to other chemical and electric heaters.

  5. Evaluation of colorimetric loop-mediated isothermal amplification assay for visual detection of Streptococcus agalactiae and Streptococcus iniae in tilapia.

    Science.gov (United States)

    Suebsing, R; Kampeera, J; Tookdee, B; Withyachumnarnkul, B; Turner, W; Kiatpathomchai, W

    2013-10-01

    Streptococcus agalactiae and Strep. iniae are bacterial pathogens that cause streptococcosis in many fish species. An accelerated colorimetric loop-mediated isothermal amplification (LAMP) assay with pre-addition of calcein was established, and the transmission and detection of Strep. agalactiae and Strep. iniae in tilapia under natural aquatic environment were investigated. A positive reaction was observed by a colour change from orange to green through the naked eyes after completion at 63°C for 30 min with 10 times higher sensitivity than that of nested PCR assays and without cross-amplification with other fish bacterial pathogens. All sample types of Nile and red tilapia (broodstock, fertilized egg, fry) were Strep. agalactiae- and Strep. iniae positive by this new method, implying that they could be vertically transmitted. With its application for screening broodstock and fry before stocking and for monitoring fish health in grow-out ponds, the method would become very useful in fish farming industry. The application of colorimetric LAMP with pre-addition of calcein offers simple, rapid and sensitive technique with applicability for small field laboratories. This technique explored the possible vertical transmission mode of Strep. agalactiae and Strep. iniae under natural aquatic environment. It could be such preliminary data provided for the screening broodstock before breeding and/or the specific-pathogen-free production. © 2013 The Society for Applied Microbiology.

  6. A comparative study of three different nucleic acid amplification techniques combined with microchip electrophoresis for HPV16 E6/E7 mRNA detection.

    Science.gov (United States)

    Liu, Quanli; Lin, Xuexia; Lin, Luyao; Yi, Linglu; Li, Haifang; Lin, Jin-Ming

    2015-10-07

    Research towards nucleic acid amplification technologies for detection of human papillomavirus (HPV) 16 E6/E7 mRNA was carried out in combination with microchip electrophoresis (MCE). The approaches of nucleic acid sequence based amplification (NASBA), one-step RT-PCR and two-step RT-PCR were successfully developed. NASBA was a simple enzymatic reaction, which directly amplified HPV16 mRNA by isothermal amplification, leaving out the complex and tedious operation. One-step RT-PCR simplified the amplification step, while two-step RT-PCR was more sensitive and less vulnerable to the interference. Furthermore, instead of gel electrophoresis, microchip electrophoresis (MCE) for RNA assay was employed to realize high-throughput and rapid analysis. Finally, the results show that PCR-based or NASBA-based mRNA tests are valuable for HPV mRNA assay, which can be potentially applied for clinical diagnosis and prognosis of cervical and other anogenital carcinoma.

  7. NAIL: Nucleic Acid detection using Isotachophoresis and Loop-mediated isothermal amplification.

    Science.gov (United States)

    Borysiak, Mark D; Kimura, Kevin W; Posner, Jonathan D

    2015-04-07

    Nucleic acid amplification tests are the gold standard for many infectious disease diagnoses due to high sensitivity and specificity, rapid operation, and low limits of detection. Despite the advantages of nucleic acid amplification tests, they currently offer limited point-of-care (POC) utility due to the need for complex instruments and laborious sample preparation. We report the development of the Nucleic Acid Isotachophoresis LAMP (NAIL) diagnostic device. NAIL uses isotachophoresis (ITP) and loop-mediated isothermal amplification (LAMP) to extract and amplify nucleic acids from complex matrices in less than one hour inside of an integrated chip. ITP is an electrokinetic separation technique that uses an electric field and two buffers to extract and purify nucleic acids in a single step. LAMP amplifies nucleic acids at constant temperature and produces large amounts of DNA that can be easily detected. A mobile phone images the amplification results to eliminate the need for laser fluorescent detection. The device requires minimal user intervention because capillary valves and heated air chambers act as passive valves and pumps for automated fluid actuation. In this paper, we describe NAIL device design and operation, and demonstrate the extraction and detection of pathogenic E. coli O157:H7 cells from whole milk samples. We use the Clinical and Laboratory Standards Institute (CLSI) limit of detection (LoD) definitions that take into account the variance from both positive and negative samples to determine the diagnostic LoD. According to the CLSI definition, the NAIL device has a limit of detection (LoD) of 1000 CFU mL(-1) for E. coli cells artificially inoculated into whole milk, which is two orders of magnitude improvement to standard tube-LAMP reactions with diluted milk samples and comparable to lab-based methods. The NAIL device potentially offers significant reductions in the complexity and cost of traditional nucleic acid diagnostics for POC applications.

  8. Information Limited Oligonucleotide Amplification Assay for Affinity-Based, Parallel Detection Studies.

    Directory of Open Access Journals (Sweden)

    Harish Bokkasam

    Full Text Available Molecular communication systems encounter similar constraints as telecommunications. In either case, channel crosstalk at the receiver end will result in information loss that statistical analysis cannot compensate. This is because in any communication channel there is a physical limit to the amount of information that can be transmitted. We present a novel and simple modified end amplification (MEA technique to generate reduced and defined amounts of specific information in form of short fragments from an oligonucleotide source that also contains unrelated and redundant information. Our method can be a valuable tool to investigate information overflow and channel capacity in biomolecular recognition systems.

  9. Loop-mediated Isothermal Amplification Assay to Rapidly Detect Wheat Streak Mosaic Virus in Quarantined Plants

    Directory of Open Access Journals (Sweden)

    Siwon Lee

    2015-12-01

    Full Text Available We developed a loop-mediated isothermal amplification (LAMP method to rapidly diagnose Wheat streak mosaic virus (WSMV during quarantine inspections of imported wheat, corn, oats, and millet. The LAMP method was developed as a plant quarantine inspection method for the first time, and its simplicity, quickness, specificity and sensitivity were verified compared to current reverse transcription-polymerase chain reaction (RT-PCR and nested PCR quarantine methods. We were able to quickly screen for WSMV at quarantine sites with many test samples; thus, this method is expected to contribute to plant quarantine inspections.

  10. A novel CMOS image sensor system for quantitative loop-mediated isothermal amplification assays to detect food-borne pathogens.

    Science.gov (United States)

    Wang, Tiantian; Kim, Sanghyo; An, Jeong Ho

    2017-02-01

    Loop-mediated isothermal amplification (LAMP) is considered as one of the alternatives to the conventional PCR and it is an inexpensive portable diagnostic system with minimal power consumption. The present work describes the application of LAMP in real-time photon detection and quantitative analysis of nucleic acids integrated with a disposable complementary-metal-oxide semiconductor (CMOS) image sensor. This novel system works as an amplification-coupled detection platform, relying on a CMOS image sensor, with the aid of a computerized circuitry controller for the temperature and light sources. The CMOS image sensor captures the light which is passing through the sensor surface and converts into digital units using an analog-to-digital converter (ADC). This new system monitors the real-time photon variation, caused by the color changes during amplification. Escherichia coli O157 was used as a proof-of-concept target for quantitative analysis, and compared with the results for Staphylococcus aureus and Salmonella enterica to confirm the efficiency of the system. The system detected various DNA concentrations of E. coli O157 in a short time (45min), with a detection limit of 10fg/μL. The low-cost, simple, and compact design, with low power consumption, represents a significant advance in the development of a portable, sensitive, user-friendly, real-time, and quantitative analytic tools for point-of-care diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Melt analysis of mismatch amplification mutation assays (Melt-MAMA: a functional study of a cost-effective SNP genotyping assay in bacterial models.

    Directory of Open Access Journals (Sweden)

    Dawn N Birdsell

    Full Text Available Single nucleotide polymorphisms (SNPs are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA, is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ~50% to ~80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (~100 ng to ~0.1 pg. Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of

  12. Sublimation of amino acids with enantiomeric excess amplification

    Science.gov (United States)

    Guillemin, Jean-Claude; Guillemin, Jean-Claude; Bellec, Aurelien

    The notion of chirality was first reported in 1848 by Pasteur, when he mechanically separated the two enantiomers of tartrate salts.[1] Amino acids are considered as the most important building blocks of life with sugars. On the Earth, the living systems are only composed of L- amino acids and D-sugars. Nowadays, the origin of homochirality on Earth is still unknown, and there are many theories trying to explain this phenomenon. Recently Cooks [2] and Feringa [3] reported that the sublimation of small amounts of L and D amino acid mixtures containing an excess of one of them leads to a huge enantiomeric excess (ee) enhancement of the sublimate. We reinvestigated these experiments to determine the rules leading to this enhancement. Starting from mixtures of L- and DL leucine we observed increasing and decreasing of the ee in function of the starting ratios. By the use of 13C derivatives, the origin of the sublimed enantiomers has been precised. Various parameters (L and D, or L and DL mixtures, dissolution in water before sublimation, . . . ) were studied. We also took into consideration the recently proposed hypothesis of the role played by the eutectic ee in the sublimation. [4] The application of these results to find an explanation of the enantiomeric excess in meteorites or in the Primitive Earth scenarios will be discussed. 1 Pasteur, L. Ann. Phys., 1848, 24, 442. 2 R. H. Perry, C. Wu, M. Nefliu, R. G. Cooks, Chem. Commun., 2007, 1071-1073. 3 S. P. Fletcher, R. B. C. Jagt, B. L. Feringa, Chem. Commun., 2007, 2578-2580. 4 D. G. Blackmond, M. Klussmannb Chem. Commun., 2007, 3990-3996.

  13. Development and Assessment of Loop-Mediated Isothermal Amplification (LAMP Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran.

    Directory of Open Access Journals (Sweden)

    Mehrdad Ghasemian

    2014-03-01

    Full Text Available Parasitological methods for the diagnosis of Visceral leishmaniasis (VL require invasive procedures, so serological and molecular approaches have been developed but are not generally applicable in the field. We evaluated a loop mediated isothermal amplification (LAMP assay using blood from VL patients and compared it to nested PCR.Forty-seven subjects with clinical features (fever, hepatosplenomegaly and anemia were confirmed positive for VL by the direct agglutination test (DAT at titers >3200. Forty DAT negative individuals from non-endemic areas with no clinical signs or symptoms of VL served as controls. A LAMP assay was performed using a set of six primers targeting Leishmania infantum kinetoplast DNA (kDNA minicircle gene under isothermal (64 °C conditions. For nested PCR we used primers targeting the kDNA minicircle gene.The LAMP assay provided a detection limit of 1 parasite in 1 ml of peripheral blood and detected L. infantum DNA in 44 of 47 DAT-confirmed VL cases, with diagnostic sensitivity of 93.6% (95% CI. No L. infantum DNA was amplified in controls, indicating a specificity of 100%. The nested PCR yielded sensitivity of 96% (95% CI and a specificity of 100% (95% CI.The LAMP assay gave results similar to those of nested PCR but in a shorter time. The LAMP method is simple; requires no sophisticated equipment; has a short reaction time; and results, indicated by turbidity of the reaction mixture, are observable with the naked eye.

  14. Loop-mediated isothermal amplification (LAMP) assay for the rapid detection of the sexually-transmitted parasite, Trichomonas vaginalis.

    Science.gov (United States)

    Adao, Davin Edric V; Rivera, Windell L

    2016-01-01

    A loop-mediated isothermal amplification (LAMP) assay was developed to detect the sexually-transmitted parasite, Trichomonas vaginalis in vaginal swabs. The presence of T. vaginalis was detected from 121 female sex workers attending a social hygiene clinic in Balibago, Angeles City, Pampanga, Philippines using culture, polymerase chain reaction (PCR), and the developed LAMP assay. The high analytical sensitivity of LAMP detected a higher prevalence of T. vaginalis (42.06%) compared to culture (8.26%) and PCR (7.44%). Additionally, this assay did not cross-react with DNAs of other trichomonads that can infect humans such as Trichomonas tenax and Pentatrichomonas hominis as well as the pathogens, Candida albicans and Staphylococcus aureus. The LAMP assay developed had a limit of detection (0.036 ng/μl) lower than that of PCR using the primers TvK3 and TvK7 (0.36 ng/μl). Prevalence of T. vaginalis in female sex workers in this area of the Philippines may be higher than previously estimated. Discordant results of PCR and LAMP may be due to different reactions to different kinds of inhibitors in the vaginal swabs.

  15. Development of a real-time loop-mediated isothermal amplification assay for detection of Burkholderia mallei.

    Science.gov (United States)

    Pal, V; Saxena, A; Singh, S; Goel, A K; Kumar, J S; Parida, M M; Rai, G P

    2017-06-25

    Burkholderia mallei is the aetiological agent of glanders, a highly contagious and re-emerging zoonotic disease. Early diagnosis of glanders is critically important to ensure timely treatment with appropriate antibiotics in humans, and to prevent spread of infection in animals. Molecular detection of B. mallei has always been troublesome because of its genetic similarity with Burkholderia pseudomallei, the causative agent of melioidosis. In present investigation, a set of six B. mallei-specific primers were designed and a simple, rapid, specific and sensitive real-time loop-mediated isothermal amplification (LAMP) assay was developed for detection of B. mallei. The LAMP assay could detect as low as 1 pg of B. mallei genomic DNA and 5.5 × 10(3)  CFU/ml of B. mallei in spiked human blood. The assay was highly specific for B. mallei as it did not cross-react with other bacterial strains used in the study. The established LAMP assay is field adaptable and can be a better and viable alternative to PCR-based techniques for detection of B. mallei in glanders endemic areas with resource-limited settings. © 2017 Blackwell Verlag GmbH.

  16. Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Triggered Isothermal Amplification for Site-Specific Nucleic Acid Detection.

    Science.gov (United States)

    Huang, Mengqi; Zhou, Xiaoming; Wang, Huiying; Xing, Da

    2018-02-06

    A novel CRISPR/Cas9 triggered isothermal exponential amplification reaction (CAS-EXPAR) strategy based on CRISPR/Cas9 cleavage and nicking endonuclease (NEase) mediated nucleic acids amplification was developed for rapid and site-specific nucleic acid detection. CAS-EXPAR was primed by the target DNA fragment produced by cleavage of CRISPR/Cas9, and the amplification reaction performed cyclically to generate a large number of DNA replicates which were detected using a real-time fluorescence monitoring method. This strategy that combines the advantages of CRISPR/Cas9 and exponential amplification showed high specificity as well as rapid amplification kinetics. Unlike conventional nucleic acids amplification reactions, CAS-EXPAR does not require exogenous primers, which often cause target-independent amplification. Instead, primers are first generated by Cas9/sgRNA directed site-specific cleavage of target and accumulated during the reaction. It was demonstrated this strategy gave a detection limit of 0.82 amol and showed excellent specificity in discriminating single-base mismatch. Moreover, the applicability of this method to detect DNA methylation and L. monocytogenes total RNA was also verified. Therefore, CAS-EXPAR may provide a new paradigm for efficient nucleic acid amplification and hold the potential for molecular diagnostic applications.

  17. Development and deployment of a rapid recombinase polymerase amplification Ebola virus detection assay in Guinea in 2015.

    Science.gov (United States)

    Faye, Oumar; Faye, Ousmane; Soropogui, Barré; Patel, Pranav; El Wahed, Ahmed Abd; Loucoubar, Cheikh; Fall, Gamou; Kiory, Davy; Magassouba, N'Faly; Keita, Sakoba; Kondé, Mandy Kader; Diallo, Alpha Amadou; Koivogui, Lamine; Karlberg, Helen; Mirazimi, Ali; Nentwich, Oliver; Piepenburg, Olaf; Niedrig, Matthias; Weidmann, Manfred; Sall, Amadou Alpha

    2015-01-01

    In the absence of a vaccine or specific treatments for Ebola virus disease (EVD), early identification of cases is crucial for the control of EVD epidemics. We evaluated a new extraction kit (SpeedXtract (SE), Qiagen) on sera and swabs in combination with an improved diagnostic reverse transcription recombinase polymerase amplification assay for the detection of Ebola virus (EBOV-RT-RPA). The performance of combined extraction and detection was best for swabs. Sensitivity and specificity of the combined SE and EBOV-RT-RPA were tested in a mobile laboratory consisting of a mobile glovebox and a Diagnostics-in-a-Suitcase powered by a battery and solar panel, deployed to Matoto Conakry, Guinea as part of the reinforced surveillance strategy in April 2015 to reach the goal of zero cases. The EBOV-RT-RPA was evaluated in comparison to two real-time PCR assays. Of 928 post-mortem swabs, 120 tested positive, and the combined SE and EBOV-RT-RPA yielded a sensitivity and specificity of 100% in reference to one real-time RT-PCR assay. Another widely used real-time RT-PCR was much less sensitive than expected. Results were provided very fast within 30 to 60 min, and the field deployment of the mobile laboratory helped improve burial management and community engagement.

  18. On-nylon membrane detection of nucleic acid molecules by rolling circle amplification.

    Science.gov (United States)

    Xu, Xinhui; Zhang, Beibei; Gan, Ping; Wu, Jian; Dai, Wei; Zhang, Ling; Wang, Jinke

    2017-09-15

    Positively-charged nylon membrane (NM) is a general solid-phase support for nucleic acid detection due to its convenient immobilization of nucleic acid materials by direct electrostatic adherence and simple UV crosslinking. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification technique for nucleic acid detection. Near-infrared fluorescence (NIRF) is a new fluorescence technique with high sensitivity due to low background. This study developed a simple method for detecting nucleic acid molecules by combining the advantages of NM, RCA and NIRF, named NIRF-based solid phase RCA on nylon membrane (NM-NIRF-sRCA). The detection system of this method only need two kinds of nucleic acid molecules: target-specific probes with a RCA primer (P) at their 3' end and a rolling circle (RC). The detection procedure consists of four steps: (1) immobilizing detected nucleic acids on NM by UV crosslinking; (2) hybridizing NM with specific probes and RC; (3) amplifying by a RCA reaction containing biotin-dUTP; (4) incubating NM with NIRF-labeled streptavidin and imaging with a NIRF imager. The method was fully testified by detecting oligonucleotides, L1 fragments of various HPV subtypes cloned in plasmid, and E.coli genomic DNA. This study thus provides a new facile method for detecting nucleic acid molecules. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Development of loop-mediated isothermal amplification assays for genotyping of Type III Secretion System in Pseudomonas aeruginosa.

    Science.gov (United States)

    Shi, H; Chen, Z; Kan, J

    2015-10-01

    Pseudomonas aeruginosa is a well-known environmental bacterium capable of causing a variety of life-threatening human infections, with a Type III Secretion System (T3SS) as the most significant virulence determinant. P. aeruginosa strains exhibit unique T3SS virulence genotypes defined by the presence of either exoS or exoU. In this study, loop-mediated isothermal amplification (LAMP) assays for rapid detection of exoS and exoU in P. aeruginosa have been developed and evaluated. Set of four primers were designed for LAMP-based amplification of exoS and exoU respectively. The LAMP reactions were performed at 63°C for 40 min, with detection limits of 100 fg purified DNA. In 107 river water isolates, exoS and exoU were detected in 10 (9%) and 89 (83%) isolates, respectively, and in 38 soil isolates, they were detected in 7 (18%) and 31 (82%) cases respectively. In conclusion, the LAMP assays are rapid, simple and cost-effective tools for detection of the exoU- and exoS-types of P. aeruginosa strains. This method can be used for the rapid, sensitive and low-cost detection of genes (exoS and exoU) encoding proteins that are part of Type III Secretion System of Pseudomonas aeruginosa. It can serve as an efficient method in outbreak situations or in routine surveillance studies to judge virulence potential and to investigate pathogenesis of P. aeruginosa. © 2015 The Society for Applied Microbiology.

  20. Development of reverse transcription loop-mediated isothermal amplification assay as a simple detection method of Chrysanthemum stem necrosis virus in chrysanthemum and tomato.

    Science.gov (United States)

    Suzuki, Ryoji; Fukuta, Shiro; Matsumoto, Yuho; Hasegawa, Toru; Kojima, Hiroko; Hotta, Makiko; Miyake, Noriyuki

    2016-10-01

    For a simple and rapid detection of Chrysanthemum stem necrosis virus (CSNV) from chrysanthemum and tomato, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed. A primer set designed to the genome sequences of CSNV worked most efficiently at 63°C and could detect CSNV RNA within 12min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. The result of a specificity test using seven other viruses and one viroid-infectable chrysanthemum or tomato showed that the assay could amplify CSNV specifically, and a sensitivity comparison showed that the RT-LAMP assay was as sensitive as the reverse transcriptase polymerase chain reaction. The RT-LAMP assay using crude RNA, extracted simply, could detect CSNV. Overall, the RT-LAMP assay was found to be a simple, specific, convenient, and time-saving method for CSNV detection. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Simple enzymatic means to neutralize DNA contamination in nucleic acid amplification.

    Science.gov (United States)

    Ashkenas, John; Dennis, James W; Ho, Chi Yip

    2005-07-01

    Reverse transcription PCR (RT-PCR) is prone to false positives when contaminating DNA molecules are present at the start of a reaction. Contaminants that derive from earlier work using a given primer pair (carryover PCR products) are of particular concern when those primers are used routinely, as in clinical diagnostics or environmental monitoring. In addition, contamination by genomic DNA can significantly interfere with quantitative and qualitative analysis of RNAs by RT-PCR. Here we describe contaminant restriction (ConR), a method that can be used to neutralize carryover and genomic DNA contamination in RT-PCR studies. Restriction enzymes (REs) added to the amplification cocktail cleave contaminant DNA molecules while sparing the intended target nucleic acid. Restriction, reverse transcription, and amplification steps all take place in the same sealed vessel, thus avoiding any danger of recontamination. ConR eliminates carryover contamination in PCR without compromising target sequence amplification. Because the method is effective against both genomic and carryover contamination, it can be employed routinely in one-step RT-PCR, whatever the RNA target or the nature of the potential DNA contaminant. A variation of this decontamination method, amplicon primer site restriction (APSR), is effective specifically against carryover contamination. APSR, unlike ConR, can be applied during PCR-based amplification of DNA target molecules.

  2. CdTe amplification nanoplatforms capped with thioglycolic acid for electrochemical aptasensing of ultra-traces of ATP.

    Science.gov (United States)

    Shamsipur, Mojtaba; Farzin, Leila; Tabrizi, Mahmoud Amouzadeh; Shanehsaz, Maryam

    2016-12-01

    A "signal off" voltammetric aptasensor was developed for the sensitive and selective detection of ultra-low levels of adenosine triphosphate (ATP). For this purpose, a new strategy based on the principle of recognition-induced switching of aptamers from DNA/DNA duplex to DNA/target complex was designed using thioglycolic acid (TGA)-capped CdTe quantum dots (QDs) as the signal amplifying nano-platforms. Owing to the small size, high surface-to-volume ratio and good conductivity, quantum dots were immobilized on the electrode surface for signal amplification. In this work, methylene blue (MB) adsorbed to DNA was used as a sensitive redox reporter. The intensity of voltammetric signal of MB was found to decrease linearly upon ATP addition over a concentration range of 0.1nM to 1.6μM with a correlation coefficient of 0.9924. Under optimized conditions, the aptasensor was able to selectively detect ATP with a limit of detection of 45pM at 3σ. The results also demonstrated that the QDs-based amplification strategy could be feasible for ATP assay and presented a potential universal method for other small biomolecular aptasensors. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Nucleic acid amplification tests (NAATs for gonorrhoea diagnosis in women: Experience of a tertiary care hospital in north India

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    Seema Sood

    2014-01-01

    Full Text Available Background & objectives: Gonorrhoea is among the most frequent of the estimated bacterial sexually transmitted infections (STIs and has significant health implications in women. The use of nucleic acid amplification tests (NAATs has been shown to provide enhanced diagnosis of gonorrhoea in female patients. However, it is recommended that an on-going assessment of the test assays should be performed to check for any probable sequence variation occurring in the targeted region. In this study, an in-house PCR targeting opa-gene of Neisseria gonorrhoeae was used in conjunction with 16S ribosomal PCR to determine the presence of gonorrhoea in female patients attending the tertiary care hospitals. Methods: Endocervical samples collected from 250 female patients with complaints of vaginal or cervical discharge or pain in lower abdomen were tested using opa and 16S ribosomal assay. The samples were also processed by conventional methods. Results: Of the 250 female patients included in the study, only one was positive by conventional methods (microscopy and culture whereas 17 patients were found to be positive based on PCR results. Interpretation & conclusions: The clinical sensitivity of conventional methods for the detection of N. gonorrhoeae in female patients was low. The gonococcal detection rates increased when molecular method was used giving 16 additional positives. Studies should be done to find out other gene targets that may be used in the screening assays to detect the presence of gonorrhoea.

  4. A rapid assay for detection of Rose rosette virus using reverse transcription-recombinase polymerase amplification using multiple gene targets.

    Science.gov (United States)

    Babu, Binoy; Washburn, Brian K; Miller, Steven H; Poduch, Kristina; Sarigul, Tulin; Knox, Gary W; Ochoa-Corona, Francisco M; Paret, Mathews L

    2017-02-01

    Rose rosette disease caused by Rose rosette virus (RRV; genus Emaravirus) is the most economically relevant disease of Knock Out® series roses in the U.S. As there are no effective chemical control options for the disease, the most critical disease management strategies include the use of virus free clean plants for propagation and early detection and destruction of infected plants. The current diagnostic techniques for RRV including end-point reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR (RT-qPCR) are highly sensitive, but limited to diagnostic labs with the equipment and expertise; and is time consuming. To address this limitation, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay based on multiple gene targets for specific detection of RRV was developed. The assay is highly specific and did not cross react with other viruses belonging to the inclusive and exclusive genus. Dilution assays using the in vitro transcripts showed that the primer sets designed (RPA-267, RPA-131, and RPA-321) are highly sensitive, consistently detecting RRV with a detection limit of 1fg/μL. Testing of the infected plants using the primer sets indicated that the virus could be detected from leaves, stems and petals of roses. The primer pair RPA-267 produced 100% positive detection of the virus from infected leaf tissues, while primer set RPA-131 produced 100% detection from stems and petals. The primer set RPA-321 produced 83%, 87.5% and 75% positive detection from leaves, petals and stem tissues, respectively. In addition, the assay has been efficiently used in the detection of RRV infecting Knock Out® roses, collected from different states in the U.S. The assay can be completed in 20min as compared to the end-point RT-PCR assay (3-4h) and RT-qPCR (1.5h). The RT-RPA assay is reliable, rapid, highly sensitive, and can be easily used in diagnostic laboratories for detection of RRV with no need for any special equipment

  5. Clinical evaluation of a loop-mediated isothermal amplification (LAMP assay for rapid detection of Neisseria meningitidis in cerebrospinal fluid.

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    DoKyung Lee

    Full Text Available Neisseria meningitidis (Nm is a leading causative agent of bacterial meningitis in humans. Traditionally, meningococcal meningitis has been diagnosed by bacterial culture. However, isolation of bacteria from patients' cerebrospinal fluid (CSF is time consuming and sometimes yields negative results. Recently, polymerase chain reaction (PCR-based diagnostic methods of detecting Nm have been considered the gold standard because of their superior sensitivity and specificity compared with culture. In this study, we developed a loop-mediated isothermal amplification (LAMP method and evaluated its ability to detect Nm in cerebrospinal fluid (CSF.We developed a meningococcal LAMP assay (Nm LAMP that targets the ctrA gene. The primer specificity was validated using 16 strains of N. meningitidis (serogroup A, B, C, D, 29-E, W-135, X, Y, and Z and 19 non-N. meningitidis species. Within 60 min, the Nm LAMP detected down to ten copies per reaction with sensitivity 1000-fold more than that of conventional PCR. The LAMP assays were evaluated using a set of 1574 randomly selected CSF specimens from children with suspected meningitis collected between 1998 and 2002 in Vietnam, China, and Korea. The LAMP method was shown to be more sensitive than PCR methods for CSF samples (31 CSF samples were positive by LAMP vs. 25 by PCR. The detection rate of the LAMP method was substantially higher than that of the PCR method. In a comparative analysis of the PCR and LAMP assays, the clinical sensitivity, specificity, positive predictive value, and negative predictive value of the LAMP assay were 100%, 99.6%, 80.6%, and 100%, respectively.Compared to PCR, LAMP detected Nm with higher analytical and clinical sensitivity. This sensitive and specific LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings.

  6. Practical identification of eight medically important Trichosporon species by reverse line blot hybridization (RLB) assay and rolling circle amplification (RCA).

    Science.gov (United States)

    Xiao, Meng; Guo, Li-Na; Kong, Fanrong; Wang, He; Sorrell, Tania C; Li, Ruo-Yu; Jiang, Wei; Chen, Sharon C-A; Xu, Ying-Chun

    2013-04-01

    We developed a reverse line blot (RLB) hybridization-, and rolling circle amplification (RCA)-based assays for the identification of Trichoporon species and evaluated them with 48 isolates that had been previously recognized as belonging to eight species (Trichosporon asahii, T. cutaneum, T. dermatis, T. domesticum, T. inkin, T. japonicum, T. jirovecii, and T. laibachii). Results were compared to those obtained with DNA sequencing of three rRNA gene loci, i.e., the internal transcribed spacer (ITS) region, D1/D2 domain of the 28S rRNA gene and intergenic spacer 1 (IGS1) region. Using species-specific, or group-specific probes targeted at the ITS region and the D1/D2 domain, the RLB assay permitted accurate species identification of all 48 isolates with 100% specificity. Species-specific RLB probes correctly assigned 45/48 (94%) of the isolates (six species) with the exception of T. dermatis and T. japonicum isolates which were not targeted by the assay. Identification of T. dermatis relied on a positive hybridization result with the group-specific probe hybridizing with T. dermatis and T. jirovecii and the absence of a signal with the T. jirovecii-specific probe. T. japonicum strains were first assigned to the T. asahii-T. japonicum group by hybridization with the two species group-specific probe and then as T. japonicum by the absence of signal with a T. asahii-specific probe. Twelve species-specific RCA probes targeting the eight species studied detected templates of all 48 Trichosporon isolates and an artificial template of T. asteroides, all with good specificity. Both RLB and RCA are potential alternatives to DNA sequencing for the identification of Trichosporon species. The RLB approach is suited for the batched simultaneous analysis of large numbers of isolates, while RCA is more appropriate for the immediate study of single isolates. Comparative costs are US$7 and US$2 per assay for the RLB and RCA methods, respectively.

  7. Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.

    Science.gov (United States)

    Abbasi, Ibrahim; Kirstein, Oscar D; Hailu, Asrat; Warburg, Alon

    2016-10-01

    Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  8. A Simple, Inexpensive Device for Nucleic Acid Amplification without Electricity—Toward Instrument-Free Molecular Diagnostics in Low-Resource Settings

    Science.gov (United States)

    LaBarre, Paul; Hawkins, Kenneth R.; Gerlach, Jay; Wilmoth, Jared; Beddoe, Andrew; Singleton, Jered; Boyle, David; Weigl, Bernhard

    2011-01-01

    Background Molecular assays targeted to nucleic acid (NA) markers are becoming increasingly important to medical diagnostics. However, these are typically confined to wealthy, developed countries; or, to the national reference laboratories of developing-world countries. There are many infectious diseases that are endemic in low-resource settings (LRS) where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to timely treatment. One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity and power requirements of polymerase chain reaction (PCR) instrumentation (another is sample preparation). Methodology/Principal Findings In this article, we investigate the hypothesis that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays. We assess the heater's equivalence to commercially available PCR instruments through the characterization of the temperature profiles produced, and a minimal method comparison. Versions of the prototype for several different isothermal techniques are presented. Conclusions/Significance We demonstrate that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays, and that the results of those assays are not significantly different from ones incubated in parallel in commercially available PCR instruments. These results clearly suggest the potential of the non-instrumented nucleic acid amplification (NINA) heater for molecular diagnostics in LRS. When combined with other innovations in development that eliminate power requirements for sample preparation, cold reagent storage, and readout, the NINA heater will comprise part of a kit that should enable electricity-free NA testing for many important analytes. PMID:21573065

  9. A simple, inexpensive device for nucleic acid amplification without electricity-toward instrument-free molecular diagnostics in low-resource settings.

    Directory of Open Access Journals (Sweden)

    Paul LaBarre

    2011-05-01

    Full Text Available Molecular assays targeted to nucleic acid (NA markers are becoming increasingly important to medical diagnostics. However, these are typically confined to wealthy, developed countries; or, to the national reference laboratories of developing-world countries. There are many infectious diseases that are endemic in low-resource settings (LRS where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to timely treatment. One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity and power requirements of polymerase chain reaction (PCR instrumentation (another is sample preparation.In this article, we investigate the hypothesis that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays. We assess the heater's equivalence to commercially available PCR instruments through the characterization of the temperature profiles produced, and a minimal method comparison. Versions of the prototype for several different isothermal techniques are presented.We demonstrate that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays, and that the results of those assays are not significantly different from ones incubated in parallel in commercially available PCR instruments. These results clearly suggest the potential of the non-instrumented nucleic acid amplification (NINA heater for molecular diagnostics in LRS. When combined with other innovations in development that eliminate power requirements for sample preparation, cold reagent storage, and readout, the NINA heater will comprise part of a kit that should enable electricity-free NA testing for many important analytes.

  10. Development and evaluation of rapid novel isothermal amplification assays for important veterinary pathogens: Chlamydia psittaci and Chlamydia pecorum

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    Martina Jelocnik

    2017-09-01

    Full Text Available Background Chlamydia psittaci and Chlamydia pecorum are important veterinary pathogens, with the former also being responsible for zoonoses, and the latter adversely affecting koala populations in Australia and livestock globally. The rapid detection of these organisms is still challenging, particularly at the point-of-care (POC. In the present study, we developed and evaluated rapid, sensitive and robust C. psittaci-specific and C. pecorum-specific Loop Mediated Isothermal Amplification (LAMP assays for detection of these pathogens. Methods and Materials The LAMP assays, performed in a Genie III real-time fluorometer, targeted a 263 bp region of the C. psittaci-specific Cps_0607 gene or a 209 bp region of a C. pecorum-specific conserved gene CpecG_0573, and were evaluated using a range of samples previously screened using species-specific quantitative PCRs (qPCRs. Species-specificity for C. psittaci and C. pecorum LAMP targets was tested against DNA samples from related chlamydial species and a range of other bacteria. In order to evaluate pathogen detection in clinical samples, C. psittaci LAMP was evaluated using a total of 26 DNA extracts from clinical samples from equine and avian hosts, while for C. pecorum LAMP, we tested a total of 63 DNA extracts from clinical samples from koala, sheep and cattle hosts. A subset of 36 C. pecorum samples was also tested in a thermal cycler (instead of a real-time fluorometer using newly developed LAMP and results were determined as an end point detection. We also evaluated rapid swab processing (without DNA extraction to assess the robustness of these assays. Results Both LAMP assays were demonstrated to species-specific, highly reproducible and to be able to detect as little as 10 genome copy number/reaction, with a mean amplification time of 14 and 24 min for C. psittaci and C. pecorum, respectively. When testing clinical samples, the overall congruence between the newly developed LAMP assays and q

  11. Development and evaluation of rapid novel isothermal amplification assays for important veterinary pathogens: Chlamydia psittaci and Chlamydia pecorum.

    Science.gov (United States)

    Jelocnik, Martina; Islam, Md Mominul; Madden, Danielle; Jenkins, Cheryl; Branley, James; Carver, Scott; Polkinghorne, Adam

    2017-01-01

    Chlamydia psittaci and Chlamydia pecorum are important veterinary pathogens, with the former also being responsible for zoonoses, and the latter adversely affecting koala populations in Australia and livestock globally. The rapid detection of these organisms is still challenging, particularly at the point-of-care (POC). In the present study, we developed and evaluated rapid, sensitive and robust C. psittaci-specific and C. pecorum-specific Loop Mediated Isothermal Amplification (LAMP) assays for detection of these pathogens. The LAMP assays, performed in a Genie III real-time fluorometer, targeted a 263 bp region of the C. psittaci-specific Cps_0607 gene or a 209 bp region of a C. pecorum-specific conserved gene CpecG_0573, and were evaluated using a range of samples previously screened using species-specific quantitative PCRs (qPCRs). Species-specificity for C. psittaci and C. pecorum LAMP targets was tested against DNA samples from related chlamydial species and a range of other bacteria. In order to evaluate pathogen detection in clinical samples, C. psittaci LAMP was evaluated using a total of 26 DNA extracts from clinical samples from equine and avian hosts, while for C. pecorum LAMP, we tested a total of 63 DNA extracts from clinical samples from koala, sheep and cattle hosts. A subset of 36 C. pecorum samples was also tested in a thermal cycler (instead of a real-time fluorometer) using newly developed LAMP and results were determined as an end point detection. We also evaluated rapid swab processing (without DNA extraction) to assess the robustness of these assays. Both LAMP assays were demonstrated to species-specific, highly reproducible and to be able to detect as little as 10 genome copy number/reaction, with a mean amplification time of 14 and 24 min for C. psittaci and C. pecorum, respectively. When testing clinical samples, the overall congruence between the newly developed LAMP assays and qPCR was 92.3% for C. psittaci (91.7% sensitivity and 92

  12. Isolation of eicosapentaenoic acid-producing fungi from soil based on polymerase chain reaction amplification.

    Science.gov (United States)

    Hu, Jing-Rong; Zhou, Peng-Peng; Zhu, Yuan-Min; Ren, Liang; Yu, Long-Jiang

    2011-01-01

    A method was developed for rapid screening and isolation of eicosapentaenoic acid (EPA)-producing soil fungi through polymerase chain reaction (PCR) amplification. Genes coding for delta6 fatty acid desaturase and delta5 fatty acid desaturase were used as molecular markers for screening these EPA-producing fungi from soil. Three out of 65 soil fungi gave positive results through PCR amplification. Two out of these three strains were found to produce EPA when they had grown in 80 ml potato/dextrose liquid medium at (25 +/- 1) degrees C for 144 h. The EPA yields were 215.81 mg 1(-1) and 263.80 mg 1(-1), respectively. The other positive strain was detected to produce arachidonic acid (AA). This study indicates that molecular detection of genes encoding delta6 and delta5 desaturases is an efficient method for primary screening of EPA- or its related polyunsaturated fatty acids (PuFAs)-producing fungi, which can improve the screening efficiency prominently.

  13. Loop-mediated isothermal amplification (LAMP) assays for rapid detection and differentiation of Nosema apis and N. ceranae in honeybees.

    Science.gov (United States)

    Ptaszyńska, Aneta A; Borsuk, Grzegorz; Woźniakowski, Grzegorz; Gnat, Sebastian; Małek, Wanda

    2014-08-01

    Nosemosis is a contagious disease of honeybees (Apis mellifera) manifested by increased winter mortality, poor spring build-up and even the total extinction of infected bee colonies. In this paper, loop-mediated isothermal amplifications (LAMP) were used for the first time to identify and differentiate N. apis and N. ceranae, the causative agents of nosemosis. LAMP assays were performed at a constant temperature of 60 °C using two sets of six species-specific primers, recognising eight distinct fragments of 16S rDNA gene and GspSSD polymerase with strand displacement activity. The optimal time for LAMP and its Nosema species sensitivity and specificity were assessed. LAMP only required 30 min for robust identification of the amplicons. Ten-fold serial dilutions of total DNA isolated from bees infected with microsporidia were used to determine the detection limit of N. apis and N. ceranae DNAs by LAMP and standard PCR assays. LAMP appeared to be 10(3) -fold more sensitive than a standard PCR in detecting N. apis and N. ceranae. LAMP methods developed by us are highly Nosema species specific and allow to identify and differentiate N. apis and N. ceranae. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  14. Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen

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    Dong Huahuang

    2012-08-01

    Full Text Available Abstract Background HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1 which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA assays combined with polymerase chain reaction (PCR and gel electrophoresis to quantify HIV-1 p24 antigen. Method A pair of anti-p24 monoclonal antibodies (mAbs were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified. Results The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml. Conclusions When combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3–4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected

  15. Development of a one-tube multiplex reverse transcriptase-polymerase chain reaction assay for the simultaneous amplification of HIV type 1 group M gag and env heteroduplex mobility assay fragments

    OpenAIRE

    Cham, F.; Heyndrickx, L; Janssens, W; Vereecken, K.; Houwer, K.; Coppens, S.; Van Der Auwera, G.; Whittle, H; van der Groen, G

    2000-01-01

    The emergence of intersubtype recombinant HIV-1 isolates has made it imperative to analyze different regions of HIV-1 genomes. For this purpose a one-tube multiplex RT-PCR, coamplifying first-round amplicons that allow amplification of gag and env heteroduplex mobility assay (HMA) fragments from different HIV-1 group M isolates, was developed, starting with plasma samples. The multiplex RT-PCR assay is sensitive: 115 of 136 (84.5%) samples were positive for both gag and env, positive amplific...

  16. Development of a reverse transcriptase loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples

    NARCIS (Netherlands)

    Adams, Emily R.; Schoone, Gerard J.; Ageed, Al Farazdag; El Safi, Sayda; Schallig, Henk D. F. H.

    2010-01-01

    Here we describe a generic, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) assay, for the identification of Leishmania species from clinical samples. LAMP is an isothermal reaction recently developed as a point-of-care diagnostic tool. Primers were designed in the conserved

  17. Real-time sequence-validated loop-mediated isothermal amplification assays for detection of Middle East respiratory syndrome coronavirus (MERS-CoV.

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    Sanchita Bhadra

    Full Text Available The Middle East respiratory syndrome coronavirus (MERS-CoV, an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU (5 to 50 PFU/ml of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.

  18. Loop mediated isothermal amplification assay using hydroxy naphthol blue, conventional polymerase chain reaction and real-time PCR in the diagnosis of intraocular tuberculosis

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    P K Balne

    2015-01-01

    Full Text Available This study is a comparative evaluation (Chi-square test of a closed tube loop mediated isothermal amplification assay using hydroxy naphthol blue dye (HNB-LAMP, real-time polymerase chain reaction (PCR and conventional PCR in the diagnosis of intraocular tuberculosis. Considering clinical presentation as the gold standard in 33 patients, the sensitivity of HNB-LAMP assay (75.8% was higher (not significant, P value 0.2 than conventional PCR (57.6% and lower than real-time PCR (90.9%. Specificity was 100% by all three methods. No amplification was observed in negative controls (n = 20 by all three methods. The cost of the HNB-LAMP assay was Rs. 500.00 and it does not require thermocycler, therefore, it can be used as an alternative to conventional PCR in resource-poor settings.

  19. Comparison of loop-mediated isothermal amplification assay and smear microscopy with culture for the diagnostic accuracy of tuberculosis.

    Science.gov (United States)

    Gelaw, Baye; Shiferaw, Yitayal; Alemayehu, Marta; Bashaw, Abate Assefa

    2017-01-17

    Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading causes of death from infectious diseases worldwide. Sputum smear microscopy remains the most widely available pulmonary TB diagnostic tool particularly in resource limited settings. A highly sensitive diagnostic with minimal infrastructure, cost and training is required. Hence, we assessed the diagnostic performance of Loop-mediated isothermal amplification (LAMP) assay in detecting M.tuberculosis infection in sputum sample compared to LED fluorescent smear microscopy and culture. A cross-sectional study was conducted at the University of Gondar Hospital from June 01, 2015 to August 30, 2015. Pulmonary TB diagnosis using sputum LED fluorescence smear microscopy, TB-LAMP assay and culture were done. A descriptive analysis was used to determine demographic characteristics of the study participants. Analysis of sensitivity and specificity for smear microscopy and TB-LAMP compared with culture as a reference test was performed. Cohen's kappa was calculated as a measure of agreement between the tests. A total of 78 pulmonary presumptive TB patients sputum sample were analyzed. The overall sensitivity and specificity of LAMP were 75 and 98%, respectively. Among smear negative sputum samples, 33.3% sensitivity and 100% specificity of LAMP were observed. Smear microscopy showed 78.6% sensitivity and 98% specificity. LAMP and smear in series had sensitivity of 67.8% and specificity of 100%. LAMP and smear in parallel had sensitivity of 85.7% and specificity of 96%. The agreement between LAMP and fluorescent smear microscopy tests was very good (κ = 0.83, P-value ≤0.0001). TB-LAMP showed similar specificity but a slightly lower sensitivity with LED fluorescence microscopy. The specificity of LAMP and smear microscopy in series was high. The sensitivity of LAMP was insufficient for smear negative sputum samples.

  20. Rapid pathogen detection by lateral-flow immunochromatographic assay with gold nanoparticle-assisted enzyme signal amplification.

    Science.gov (United States)

    Cho, Il-Hoon; Bhunia, Arun; Irudayaraj, Joseph

    2015-08-03

    To date most LF-ICA format for pathogen detection is based on generating color signals from gold nanoparticle (AuNP) tracers that are perceivable by naked eye but often these methods exhibit sensitivity lower than those associated with the conventional enzyme-based immunological methods or mandated by the regulatory guidelines. By developing AuNP avidin-biotin constructs in which a number of enzymes can be labeled we report on an enhanced LF-ICA system to detect pathogens at very low levels. With this approach we show that as low as 100 CFU/mL of Escherichia coli O157:H7 can be detected, indicating that the limit of detection can be increased by about 1000-fold due to our signal amplification approach. In addition, extensive cross-reactivity experiments were conducted (19 different organisms were used) to test and successfully validate the specificity of the assay. Semi-quantitative analysis can be performed using signal intensities which were correlated with the target pathogen concentrations for calibration by image processing. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of common genetically modified organisms (GMOs).

    Science.gov (United States)

    Feng, Jiawang; Tang, Shiming; Liu, Lideng; Kuang, Xiaoshan; Wang, Xiaoyu; Hu, Songnan; You, Shuzhu

    2015-03-01

    Here, we developed a loop-mediated isothermal amplification (LAMP) assay for 11 common transgenic target DNA in GMOs. Six sets of LAMP primer candidates for each target were designed and their specificity, sensitivity, and reproductivity were evaluated. With the optimized LAMP primers, this LAMP assay was simply run within 45-60 min to detect all these targets in GMOs tested. The sensitivity, specificity, and reproductivity of the LAMP assay were further analyzed in comparison with those of Real-Time PCR. In consistent with real-time PCR, detection of 0.5% GMOs in equivalent background DNA was possible using this LAMP assay for all targets. In comparison with real-time PCR, the LAMP assay showed the same results with simple instruments. Hence, the LAMP assay developed can provide a rapid and simple approach for routine screening as well as specific events detection of many GMOs.

  2. A loop-mediated isothermal amplification (LAMP) assay for Strongyloides stercoralis in stool that uses a visual detection method with SYTO-82 fluorescent dye.

    Science.gov (United States)

    Watts, Matthew R; James, Gregory; Sultana, Yasmin; Ginn, Andrew N; Outhred, Alexander C; Kong, Fanrong; Verweij, Jaco J; Iredell, Jonathan R; Chen, Sharon C-A; Lee, Rogan

    2014-02-01

    An assay to detect Strongyloides stercoralis in stool specimens was developed using the loop-mediated isothermal amplification (LAMP) method. Primers were based on the 28S ribosomal subunit gene. The reaction conditions were optimized and SYTO-82 fluorescent dye was used to allow real-time and visual detection of the product. The product identity was confirmed with restriction enzyme digestion, cloning, and sequence analysis. The assay was specific when tested against DNA from bacteria, fungi and parasites, and 30 normal stool samples. Analytical sensitivity was to LAMP method. On the basis of these findings, the assay warrants further clinical validation.

  3. A comparison of the reliability of two gene targets in loop-mediated isothermal amplification assays for detecting leptospiral DNA in canine urine.

    Science.gov (United States)

    Gentilini, Fabio; Zanoni, Renato Giulio; Zambon, Elisa; Turba, Maria Elena

    2017-01-01

    We compared 2 novel loop-mediated isothermal amplification (LAMP) assays that target either the 16S ribosomal RNA ( rrs) gene or the gene encoding a 32-kDa leptospiral lipoprotein ( lipL32) in order to assess the effect of the target on the accuracy of the LAMP assays. The most sensitive assay was the rrs assay with a limit of detection (LOD) of 1.2 × 101 genome equivalents per reaction. The novel lipL32 assay showed an LOD of 1.2 × 102 genome equivalents per reaction. Both assays showed adequate specificity when tested against a collection of bacteria commonly found in voided canine urine. However, when field samples were assayed, the rrs assays gave many false-positive results and a poor positive predictive value of 8.33%. In conclusion, even if the LAMP assay is used in low prevalence areas, the lipL32 assay would be preferable. Conversely, the higher analytical sensitivity of the rrs assay could be effectively used as a screening test in endemic areas with high disease prevalence, followed by confirmation of the positive results using the lipL32 assay.

  4. Highly parallel and short-acting amplification with locus-specific primers to detect single nucleotide polymorphisms by the DigiTag2 assay.

    Directory of Open Access Journals (Sweden)

    Nao Nishida

    Full Text Available The DigiTag2 assay enables analysis of a set of 96 SNPs using Kapa 2GFast HotStart DNA polymerase with a new protocol that has a total running time of about 7 hours, which is 6 hours shorter than the previous protocol. Quality parameters (conversion rate, call rate, reproducibility and concordance were at the same levels as when genotype calls were acquired using the previous protocol. Multiplex PCR with 192 pairs of locus-specific primers was available for target preparation in the DigiTag2 assay without the optimization of reaction conditions, and quality parameters had the same levels as those acquired with 96-plex PCR. The locus-specific primers were able to achieve sufficient (concentration of target amplicon ≥5 nM and specific (concentration of unexpected amplicons <2 nM amplification within 2 hours, were also able to achieve detectable amplifications even when working in a 96-plex or 192-plex form. The improved DigiTag2 assay will be an efficient platform for screening an intermediate number of SNPs (tens to hundreds of sites in the replication analysis after genome-wide association study. Moreover, highly parallel and short-acting amplification with locus-specific primers may thus facilitate widespread application to other PCR-based assays.

  5. Colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (NINA-LAMP.

    Directory of Open Access Journals (Sweden)

    Catherine B Poole

    Full Text Available Accurate detection of filarial parasites in humans is essential for the implementation and evaluation of mass drug administration programs to control onchocerciasis and lymphatic filariasis. Determining the infection levels in vector populations is also important for assessing transmission, deciding when drug treatments may be terminated and for monitoring recrudescence. Immunological methods to detect infection in humans are available, however, cross-reactivity issues have been reported. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity, and can be used to detect infection in both humans and vectors. In this study we developed loop-mediated isothermal amplification (LAMP tests to detect three different filarial DNAs in human and insect samples using pH sensitive dyes for enhanced visual detection of amplification. Furthermore, reactions were performed in a portable, non-instrumented nucleic acid amplification (NINA device that provides a stable heat source for LAMP. The efficacy of several strand displacing DNA polymerases were evaluated in combination with neutral red or phenol red dyes. Colorimetric NINA-LAMP assays targeting Brugia Hha I repeat, Onchocerca volvulus GST1a and Wuchereria bancrofti LDR each exhibit species-specificity and are also highly sensitive, detecting DNA equivalent to 1/10-1/5000th of one microfilaria. Reaction times varied depending on whether a single copy gene (70 minutes, O. volvulus or repetitive DNA (40 min, B. malayi and W. bancrofti was employed as a biomarker. The NINA heater can be used to detect multiple infections simultaneously. The accuracy, simplicity and versatility of the technology suggests that colorimetric NINA-LAMP assays are ideally suited for monitoring the success of filariasis control programs.

  6. Evaluation of Six Commercial Nucleic Acid Amplification Tests for Detection of Neisseria gonorrhoeae and Other Neisseria Species▿

    Science.gov (United States)

    Tabrizi, Sepehr N.; Unemo, Magnus; Limnios, Athena E.; Hogan, Tiffany R.; Hjelmevoll, Stig-Ove; Garland, Susanne M.; Tapsall, John

    2011-01-01

    Molecular detection of Neisseria gonorrhoeae in extragenital samples may result in false-positive results due to cross-reaction with commensal Neisseria species or Neisseria meningitidis. This study examined 450 characterized clinical culture isolates, comprising 216 N. gonorrhoeae isolates and 234 isolates of nongonococcal Neisseria species (n = 218) and 16 isolates of other closely related bacteria, with six commercial nucleic acid amplification tests (NAATs). The six NAATs tested were Gen-Probe APTIMA COMBO 2 and APTIMA GC, Roche COBAS Amplicor CT/NG and COBAS 4800 CT/NG tests, BD ProbeTec GC Qx amplified DNA assay, and Abbott RealTime CT/NG test. All assays except COBAS Amplicor CT/NG test where four (1.9%) isolates were not detected showed a positive result with all N. gonorrhoeae isolates (n = 216). Among the 234 nongonococcal isolates examined, initial results from all assays displayed some false-positive results due to cross-reactions. Specifically, the COBAS Amplicor and ProbeTec tests showed the highest number of false-positive results, detecting 33 (14.1%) and 26 (11%) nongonococcal Neisseria isolates, respectively. On the first testing, APTIMA COMBO 2, APTIMA GC, Abbott RealTime, and Roche COBAS 4800 showed lower level of cross-reactions with five (2.1%), four (1.7%), two (1%), and two (1%) of the isolates showing low-level positivity, respectively. Upon retesting of these nine nongonococcal isolates using freshly cultured colonies, none were positive by the APTIMA COMBO 2, Abbott RealTime, or COBAS 4800 test. In conclusion, the COBAS Amplicor and ProbeTec tests displayed high number of false-positive results, while the remaining NAATs showed only sporadic low-level false-positive results. Supplementary testing for confirmation of N. gonorrhoeae NAATs remains recommended with all samples tested, in particular those from extragenital sites. PMID:21813721

  7. Visual and Real-Time Event-Specific Loop-Mediated Isothermal Amplification Based Detection Assays for Bt Cotton Events MON531 and MON15985.

    Science.gov (United States)

    Randhawa, Gurinder Jit; Chhabra, Rashmi; Bhoge, Rajesh K; Singh, Monika

    2015-01-01

    Bt cotton events MON531 and MON15985 are authorized for commercial cultivation in more than 18 countries. In India, four Bt cotton events have been commercialized; more than 95% of total area under genetically modified (GM) cotton cultivation comprises events MON531 and MON15985. The present study reports on the development of efficient event-specific visual and real-time loop-mediated isothermal amplification (LAMP) assays for detection and identification of cotton events MON531 and MON15985. Efficiency of LAMP assays was compared with conventional and real-time PCR assays. Real-time LAMP assay was found time-efficient and most sensitive, detecting up to two target copies within 35 min. The developed real-time LAMP assays, when combined with efficient DNA extraction kit/protocol, may facilitate onsite GM detection to check authenticity of Bt cotton seeds.

  8. DNA-based hybridization chain reaction amplification for assaying the effect of environmental phenolic hormone on DNA methyltransferase activity.

    Science.gov (United States)

    Xu, Zhenning; Yin, Huanshun; Han, Yunxiang; Zhou, Yunlei; Ai, Shiyun

    2014-06-04

    In this work, a novel electrochemical protocol with signal amplification for determination of DNA methylation and methyltransferase activity using DNA-based hybridization chain reaction (HCR) was proposed. After the gold electrode was modified with dsDNA, it was treated with M.SssI MTase, HpaII endonuclease, respectively. And then the HCR was initiated by the target DNA and two hairpin helper DNAs, which lead to the formation of extended dsDNA polymers on the electrode surface. The signal was amplified by the labeled biotin on the hairpin probes. As a result, the streptavidin-alkaline phosphatase (S-ALP) conjugated on the electrode surface through the specific interaction between biotin and S-ALP. ALP could convert 1-naphthyl phosphate into 1-naphthol and the latter could be electrochemically oxidized, which was used to monitor the methylation event and MTase activity. The HCR assay presents good electrochemical responses for the determination of M.SssI MTase at a concentration as low as 0.0067 uni tmL(-1). Moreover, the effects of anti-cancer drug and environmental phenolic hormone on M.SssI MTase activity were also investigated. The results indicated that 5-fluorouracil and daunorubicin hydrochloride could inhibit the activity, and the opposite results were obtained with bisphenol A and nonylphenol. Therefore, this method can not only provide a platform to screen the inhibitors of DNA MTase and develop new anticancer drugs, but also offer a novel technique to investigate the possible carcinogenesis mechanism. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Uses of Nucleic Acid Analogues in the Inhibition of Nucleic Acid Amplification

    DEFF Research Database (Denmark)

    1999-01-01

    The present invention relates to the use of nucleic acid analogues in blocking nucleic acid ampli?cation procedures and to diagnostic and analytical techniques based thereon. Also included are kits for use in the conduct of nucleic acid ampli?cation reactions....

  10. Development of a fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification assay for rapid detection of seasonal Japanese B encephalitis outbreaks in pigs.

    Science.gov (United States)

    Tian, C J; Lin, Z X; He, X M; Luo, Q; Luo, C B; Yu, H Q; Chen, R; Wu, X W; Zhu, D Z; Ren, Z J; Bi, Y Z; Ji, J

    2012-08-01

    The standardization and validation of a one-step, single-tube, accelerated fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the NS3 gene of Japanese B encephalitis virus (JEV) is described for rapid, simple, and high-throughput detection of JEV. The amplification can be completed in 35 min under isothermal conditions at 63°C by employing a set of six primers targeting the NS3 gene of JEV. The RT-LAMP assay described demonstrated high sensitivity for detecting JEV, with a detection limit in swine samples of 8.13 PFU/ml. The specificity of the selected primer sets was established by cross-reactivity studies with pathogens that exhibit similar clinical signs and testing of samples from healthy animals. The clinical applicability of the RT-LAMP assay was validated using either spiked samples or samples from seasonal outbreaks. The comparative evaluation of the RT-LAMP assay revealed 79.59 % concordance with conventional RT-PCR targeting the E gene of JEV. The RT-LAMP assay reported here is a valuable tool for rapid real-time and high-throughput seasonal infection surveillance and quarantine after outbreak through blood sampling by using ordinary real-time PCR thermocyclers without purchasing an expensive Loopamp real-time turbidimeter.

  11. Visual detection of nucleic acids based on lateral flow biosensor and hybridization chain reaction amplification.

    Science.gov (United States)

    Ying, Na; Ju, Chuanjing; Li, Zhongyi; Liu, Wensen; Wan, Jiayu

    2017-03-01

    In this study, a new lateral flow nucleic acid biosensor (LFNAB) using hybridization chain reaction (HCR) for signal amplification was developed for visual detection of nucleic acids with high sensitivity and low cost. A "sandwich-type" detection strategy was employed in our design. The sandwich system of capture probe (CP)/target DNA/reporter probe (RP)-HCR complexes was fabricated as the sensing platform. As the initiator strand, reporter probe propagated a chain reaction of hybridization events between the two hairpin probes modified with biotin, and determined whether long nicked DNA polymers were formed. The biotin-labeled double-strand DNA polymers then introduced numerous Streptavidin (SA)-labeled gold nanoparticles (AuNPs) on the lateral flow device. The CP/target DNA/RP-HCR complexes were captured on the test zone by the specific reaction between anti-Fam monoclonal antibody (anti-Fam mAb) on the test zone and Fam of the complexes. The accumulation of AuNPs on the test zone of the biosensor enabled the visual detection of specific sequences. The detection limit of specific DNA was as low as 1.76pM, which was about 2 orders lower than that of the LFNAB without HCR amplification. And the detection limit of Salmonella was 3×10 3 cfumL -1 . In conclusion, this visual detection system, HCR-LFNAB, is suitable for non-specialist personnel and point-of-care (POC) diagnosis in low-resource settings. Copyright © 2016. Published by Elsevier B.V.

  12. Application of a loop-mediated isothermal amplification (LAMP assay targeting cox1 gene for the detection of Clonorchis sinensis in human fecal samples.

    Directory of Open Access Journals (Sweden)

    S M Mazidur Rahman

    2017-10-01

    Full Text Available Clonorchiasis is prevalent in the Far East, and a major health problem in endemic areas. Infected persons may experience, if not treated, serious complications such as bile stone formation, pyogenic cholangitis, and even cholangiocarcinoma. Early diagnosis and treatment are important to prevent serious complications and, therefore, the simple and reliable diagnostic method is necessary to control clonorchiasis in endemic areas, where resources for the diagnosis are limited.The loop-mediated isothermal amplification (LAMP assay has been applied for the detection of Clonorchis sinensis DNA. Six primers targeting eight locations on the cytochrome c oxidase subunit 1 gene of C. sinensis were designed for species-specific amplification using the LAMP assay. The LAMP assay was sensitive enough to detect as little as 100 fg of C. sinensis genomic DNA and the detection limit in 100 mg of stool was as low as one egg. The assay was highly specific because no cross-reactivity was observed with the DNA of other helminths, protozoa or Escherichia coli. Then, LAMP assay was applied to human fecal samples collected from an endemic area of clonorchiasis in Korea. Using samples showing consistent results by both Kato-Katz method and real-time PCR as reference standards, the LAMP assay showed 97.1% (95% CI, 90.1-99.2 of sensitivity and 100% (95% CI, 92.9-100 of specificity. In stool samples with more than 100 eggs per gram of feces, the sensitivity achieved 100%.To detect C. sinensis in human fecal samples, the LAMP assay was applied and achieved high sensitivity and specificity. The LAMP assay can be utilized in field laboratories as a powerful tool for diagnosis and epidemiological survey of clonorchiasis.

  13. Isothermal Method of a Recombinase Polymerase Amplification Assay for the Detection of Most Common High-Risk Human Papillomavirus Type 16 and Type 18 DNA.

    Science.gov (United States)

    Ma, Biao; Fang, Jiehong; Wang, Ye; He, Haizhen; Dai, Mingyan; Lin, Wei; Su, Wei; Zhang, Mingzhou

    2017-01-01

    Cervical cancer is a common gynecologic malignant tumor and has a great impact on women's health. Human papillomavirus (HPV) is implicated in cervical cancer and precancerous lesions and the two are possibly two stages of disease progression. With the technological development of molecular biology and epidemiology, detection and treatment of HPV has become an important means to prevent cervical cancer. Here we present a novel, rapid, sensitive and specific isothermal method of recombinase polymerase amplification (RPA), which is established to detect the two most common high-risk human papillomavirus type 16 and type 18 DNA. In this study, we evaluate the efficacy of the RPA assay, incubating clinical specimens of HPV16 and HPV18 using plasmids standard. It operates at constant low temperature without the thermal instrumentation for incubation. The products can be detected via agarose gel electrophoresis assay, reverse dot blot assay, and quantitative real-time assay with SYBR Green I. We assess the diagnostic performance of the RPA assay for detecting of HPV16 and HPV18 in 335 clinical samples from patients suspected of cervical cancer. The results revealed no cross-reaction with other HPV genotypes and the RPA assay achieve a sensitivity of 100 copies. Compared with TaqMan qPCR, the RPA technique achieves exponential amplification with no need for pretreatment of sample DNA at 37°C for 20 minutes, which reveals more satisfactory performance. The agreement between the RPA and qPCR assays was 97.6% (κ = 0.89) for HPV16 positivity and 98.5% (κ = 0.81) for HPV18 positivity, indicating very good correlation between both tests. Importantly, the RPA assay was demonstrated to be a useful and powerful method for detection of HPV virus, which therefore may serve as a valuable tool for rapid diagnosis of HPV infection in both commercial and clinical applications.

  14. Genotyping of vitamin D receptor gene polymorphisms using mismatched amplification mutation assay in neonatal sepsis patients of Odisha, eastern India.

    Science.gov (United States)

    Das, Biswadeep; Patra, Saumya; Behera, Chinmay; Suar, Mryutunjay

    2016-11-01

    Vitamin D has potent antimicrobial and anti-inflammatory properties. Vitamin D deficiency has been shown to be associated with the risk of vulnerability to different infectious diseases, such as neonatal sepsis. Polymorphisms in vitamin D receptor (VDR) gene can influence the expression of vitamin D in individuals. Hence, it is essential to study the vitamin D status and VDR gene polymorphisms for assessing neonatal sepsis risk. In this study, we assessed the serum 25(OH)D, the main circulating form of vitamin D and VDR polymorphism on 120 subjects in a case-control approach, recruiting 60 subjects in each category. We genotyped Fok1, Bsm1, Apa1 and Taq1 gene polymorphisms in VDR by developing a unique mismatch amplification mutation assay (MAMA) and studied their association in both populations. VDR-MAMA primers were designed by addition of dual mismatches (DM) near the 3' end and were selected based on high ΔCt values in comparison to single mismatch (SM) primers using SYBR-Green RT-PCR, which were eventually used for VDR genotyping. Genotyping was also performed using PCR-RFLP for further confirmation. Serum 25(OH)D ELISA revealed that cases were vitamin D insufficient (Median=12.16ng/ml, 95% CI: 3.84-22.22) and controls were vitamin D sufficient (Median=30.22ng/ml, 95% CI: 20.08-46.78; pneonatal sepsis or 25(OH)D serum levels. The distributions of the Fok1, Bsm1, and Taq1 genotypes were not consistent with Hardy-Weinberg equilibrium in the control group. Future studies in larger populations are required to establish whether the VDR polymorphisms can be potentially used as genetic markers for early screening towards predisposition to neonatal sepsis risk. In this study, we describe a simple, inexpensive and rapid screening of VDR gene polymorphisms using VDR MAMA-PCR, which can be used in both clinical and research laboratories. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Development of a stable isotope dilution assay for tenuazonic acid.

    Science.gov (United States)

    Asam, Stefan; Liu, Yang; Konitzer, Katharina; Rychlik, Michael

    2011-04-13

    A stable isotope dilution assay (SIDA) for the Alternaria mycotoxin tenuazonic acid was developed. Therefore, [(13)C(6),(15)N]-tenuazonic acid was synthesized from [(13)C(6),(15)N]-isoleucine by Dieckmann intramolecular cyclization after acetoacetylation with diketene. The synthesized [(13)C(6),(15)N]-tenuazonic acid was used as the internal standard for determination of tenuazonic acid in tomato products by liquid chromatography tandem mass spectrometry after derivatization with 2,4-dinitrophenylhydrazine. Method validation revealed a limit of detection of 0.1 μg/kg and a limit of quantitation of 0.3 μg/kg. Recovery was close to 100% in the range of 3-300 μg/kg. Determination of tenuazonic acid in two samples of different tomato ketchups (naturally contaminated) was achieved with a coefficient of variation of 2.3% and 4.7%. Different tomato products (n = 16) were analyzed for their content of tenuazonic acid using the developed SIDA. Values were between 15 and 195 μg/kg (tomato ketchup, n = 9), 363 and 909 μg/kg (tomato paste, n = 2), and 8 and 247 μg/kg (pureed tomatoes and comparable products, n = 5).

  16. Diagnosis of pediatric pulmonary tuberculosis with special reference to polymerase chain reaction based nucleic acid amplification test

    Directory of Open Access Journals (Sweden)

    Shreshtha Tiwari

    2015-01-01

    Conclusion/recommendation: The present study reinforces better case detection rate with PCR-based nucleic acid amplification test as compared with microscopy and culture in pediatric pulmonary TB. PCR showed a higher correlation with clinical diagnosis as compared with microscopy and solid culture. Hence, a molecular platform should be the test of choice for detecting PPTB.

  17. Culture confirmation of gonococcal infection by recall of subjects found to be positive by nucleic acid amplification tests in general practice

    DEFF Research Database (Denmark)

    Møller, Jens Kjølseth

    2010-01-01

    To evaluate a routine notification of general practitioners to recall nucleic acid amplification test (NAAT)-positive subjects for culture of Neisseria gonorrhoeae to confirm gonococcal infection in the community.......To evaluate a routine notification of general practitioners to recall nucleic acid amplification test (NAAT)-positive subjects for culture of Neisseria gonorrhoeae to confirm gonococcal infection in the community....

  18. A method for simultaneous detection and identification of Brazilian dog- and vampire bat-related rabies virus by reverse transcription loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Saitou, Yasumasa; Kobayashi, Yuki; Hirano, Shinji; Mochizuki, Nobuyuki; Itou, Takuya; Ito, Fumio H; Sakai, Takeo

    2010-09-01

    At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme AlwI. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RT-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RT-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field. Copyright 2010 Elsevier B.V. All rights reserved.

  19. Development of Multiplex-Mismatch Amplification Mutation-PCR Assay for Simultaneous Detection of Campylobacter jejuni and Mutation in gyrA Gene Related to Fluoroquinolone Resistance.

    Science.gov (United States)

    Cui, Mingquan; Wu, Chenbin; Zhang, Peng; Wu, Congming

    2016-11-01

    Campylobacter jejuni, a foodborne pathogen, is the major cause of enteritis in humans worldwide, however, its increasing resistance to fluoroquinolones reported recently is of a major concern. In the present study, multiplex-mismatch amplification mutation assay-polymerase chain reaction (MMAMA-PCR) was developed for the first time with the aim to quickly identify C. jejuni and to detect the single nucleotide mutation (C-257 to T) frequently observed in gyrA gene, associated with the acquisition of resistance to fluoroquinolones. In this assay, mismatch amplification mutation primers for the detection of gyrA mutation in C. jejuni were coupled with primers for the hip gene encoding for hippuricase and 16S rRNA gene of C. jejuni, respectively, in the multiplex PCR assay. The specificity and accuracy of this method were analyzed by the use of 78 C. jejuni strains with previously confirmed resistance phenotypes and the mutation (C-257 to T) in gyrA gene, as well as 107 clinical isolates of various bacterial species, including 29 C. jejuni isolates. This study indicates that MMAMA-PCR is a promising assay for the rapid identification of C. jejuni with a specific mutation in gyrA gene, responsible for the resistance to fluoroquinolones.

  20. Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.

    Science.gov (United States)

    Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

    2016-06-01

    For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. © The American Society of Tropical Medicine and Hygiene.

  1. Immunocytochemistry versus nucleic acid amplification in fine needle aspirates and tissues of extrapulmonary tuberculosis

    Directory of Open Access Journals (Sweden)

    Madhu Mati Goel

    2012-01-01

    Full Text Available Background: Immunocytochemistry (ICC is an established routine diagnostic adjunct to cytology and histology for tumor diagnosis but has received little attention for diagnosis of tuberculosis. Aims: To have an objective method of direct visualization of mycobacteria or their products in clinical extrapulmonary tuberculosis (EPTB specimens, immunocytochemical localization of M. tuberculosis antigen by staining with species specific monoclonal antibody to 38-kDa antigen of Mycobacterium tuberculosis complex. Materials and Methods: Immunostaining with specific monoclonal antibody to 38-kDa antigen of Mycobacterium tuberculosis complex was done in fresh and archival fine needle aspirates and tissue granulomata of 302 cases of extrapulmonary tuberculosis and was compared with the molecular diagnostic i.e., nucleic amplification and conventional [Cytomorphology, Ziehl Neelsen (ZN staining and culture] tests and 386 controls. Results: Diagnostic indices by Bayesian analysis for all types of archival and fresh material varied from 64 to 76% in nucleic acid amplification (NAA and 96 to 98% in ICC. There was no significant difference in the diagnostic indices of ZN staining and/ or ICC in fresh or archival material whereas the sensitivity of NAA differed significantly in fresh versus archival material both in cytology (71.4% vs 52.1% and histology (51.1% vs 38.8%. ICC can be easily used on archival smears and formalin-fixed paraffin-embedded tissue sections with almost equal sensitivity and specificity as with fresh material, in contrast to NAA which showed significant difference in test results on archival and fresh material. Conclusions: Low detection sensitivity of MTB DNA in archival material from known tuberculous cases showed the limitation of in-house NAA-based molecular diagnosis. ICC was found to be sensitive, specific and a better technique than NAA and can be used as an adjunct to conventional morphology and ZN staining for the diagnosis of

  2. Real-time, sequence-specific detection of nucleic acids during strand displacement amplification.

    Science.gov (United States)

    Nadeau, J G; Pitner, J B; Linn, C P; Schram, J L; Dean, C H; Nycz, C M

    1999-12-15

    Strand displacement amplification (SDA) is an isothermal nucleic acid amplification method based on the primer-directed nicking activity of a restriction enzyme and the strand displacement activity of an exonuclease-deficient polymerase. Here we describe fluorogenic reporter probes that permit real-time, sequence-specific detection of targets amplified during SDA. The new probes possess the single-strand half of a BsoBI recognition sequence flanked on opposite sides by a fluorophore and a quencher. The probes also contain target-binding sequences located 3' to the BsoBI site. Fluorophore and quencher are maintained in sufficiently close proximity that fluorescence is quenched in the intact single-stranded probe. If target is present during SDA, the probe is converted into a fully double-stranded form and is cleaved by the restriction enzyme BsoBI, which also serves as the nicking agent for SDA. Fluorophore and quencher diffuse apart upon probe cleavage, causing increased fluorescence. Target replication may thus be followed in real time during the SDA reaction. Probe performance may be enhanced by embedding the fluorogenic BsoBI site within the loop of a folded hairpin structure. The new probe designs permit detection of as few as 10 target copies within 30 min in a closed-tube, real-time format, eliminating the possibility of carry-over contamination. The probes may be used to detect RNA targets in SDA mixtures containing reverse transcriptase. Furthermore, a two-color competitive SDA format permits accurate quantification of target levels from the real-time fluorescence data. Copyright 1999 Academic Press.

  3. Development of loop-mediated isothermal amplification-based diagnostic assays for detection of Pasteurella multocida and hemorrhagic septicemia-associated P multocida serotype B:2.

    Science.gov (United States)

    Moustafa, Ahmed M; Bennett, Mark D

    2017-02-01

    OBJECTIVE To develop 2 rapid loop-mediated isothermal amplification (LAMP) assays for detection of Pasteurella multocida DNA (Pm-LAMP assay) and P multocida DNA from strains associated with hemorrhagic septicemia (HS) in cattle and buffalo (HS-LAMP assay). SAMPLE Solutions containing 16 P multocida strains and 9 other bacterial species at various concentrations. PROCEDURES Optimal conditions were determined for running the Pm-LAMP and HS-LAMP assays. The assays were then used to detect DNA of the test organisms. Results of LAMP assays were validated against conventional PCR assays designed for specific detection of P multocida and the B:2 serotype of HS-associated strains. RESULTS Following incubation of sample reaction mixtures for 27 minutes, specificity and sensitivity of the HS-LAMP assay at template DNA amounts as low as 5 pg were 93% and 97%, respectively. When duplicates of each sample were incubated for 28 minutes (a positive result defined as positive results for both reactions of a given sample), specificity and sensitivity of the HS-LAMP assay in the same conditions increased to 100%. The best specificity and sensitivity of Pm-LAMP single (93% and 91%) and duplicate (97% and 98%) reactions at template DNA amounts as low as 10 pg were achieved at 33 and 34 minutes, respectively. CONCLUSIONS AND CLINICAL RELEVANCE These preliminary findings suggested the developed HS-LAMP assay had high sensitivity and specificity for detection of HS-associated P multocida. Additional research is needed to determine the accuracy of the assay for use on clinical specimens obtained in HS-endemic countries such as Pakistan and Thailand.

  4. Comparison between quantitative nucleic acid sequence-based amplification, real-time reverse transcriptase PCR, and real-time PCR for quantification of Leishmania parasites

    NARCIS (Netherlands)

    van der Meide, Wendy; Guerra, Jorge; Schoone, Gerard; Farenhorst, Marit; Coelho, Leila; Faber, William; Peekel, Inge; Schallig, Henk

    2008-01-01

    DNA or RNA amplification methods for detection of Leishmania parasites have advantages regarding sensitivity and potential quantitative characteristics in comparison with conventional diagnostic methods but are often still not routinely applied. However, the use and application of molecular assays

  5. Assays for urinary biomarkers of oxidatively damaged nucleic acids

    DEFF Research Database (Denmark)

    Weimann, Allan; Broedbaek, Kasper; Henriksen, Trine

    2012-01-01

    -linked immunosorbent assay). The major analytical challenge is specificity. The best combination of selectivity and speed of analysis can be obtained by liquid chromatography coupled with tandem mass spectrometric detection. This, however, is also the most demanding technique with regard to price, complexity...... and skills requirement. The available ELISA methods present considerable specificity problems and cannot be recommended at present. The oxidized nucleic acid metabolites in urine are assumed to originate from the DNA and RNA. However, direct evidence is not available. A possible contribution from...

  6. Design of a real time quantitative PCR assay to assess global mRNA amplification of small size specimens for microarray hybridisation

    Science.gov (United States)

    Choesmel, V; Foucault, F; Thiery, J P; Blin, N

    2004-01-01

    Background: Low RNA yields from clinical samples are a limiting step for microarray technology. Aims: To design an accurate real time quantitative polymerase chain reaction (PCR) assay to assess the crucial step of global mRNA amplification performed before microarray hybridisation, using less than 1 µg total RNA. Methods: Three RNA extraction procedures were compared for small size samples. Total RNA was amplified from universal RNA or the BC-H1 breast cancer micrometastatic cell line using three different protocols. Real time quantitative PCR technology was used for accurate measurement of urokinase plasminogen activator receptor and cytokeratin 8 RNA amplification rates and ratios, using primer sets binding at various distances from the 3′ end of transcripts. A 50 mer oligomeric array targeting 87 genes potentially involved in breast cancer metastatic progression was built and hybridised with amplified RNA. Results: Eighteen nanograms of total RNA could be purified from 1000 BC-H1 micrometastatic cells. Amplification rates of 25 000 to 100 000 were achieved with as little as 10 ng of starting material. However, results were highly variable, depending on the amount of starting material, gene characteristics, sample quality, and protocols used. Oligomeric array hybridisation with 20 µg reference RNA resulted in specific and reproducible signals for 83% of the genes, whereas mRNA amplification from less than 400 ng of starting material resulted in selective detection of signals from highly expressed genes. Conclusions: Improvements in the design of global mRNA amplification procedures and oligomeric arrays are needed to extract informative gene expression data from clinical samples containing limited cell numbers. PMID:15563668

  7. Combined multiplex loop-mediated isothermal amplification with lateral flow assay to detect sea and seb genes of enterotoxic Staphylococcus aureus.

    Science.gov (United States)

    Yin, H Y; Fang, T J; Wen, H W

    2016-07-01

    Staphylococcal enterotoxins (SEs) are the most common cause of food poisoning worldwide and can induce symptoms, such as diarrhoea, vomiting and abdominal cramping. Thus, the aim of this study is to develop a multiplex loop-mediated isothermal amplification combined with a lateral flow assay (m-LAMP/LFA) to simultaneously detect the sea and seb genes of Staphylococcus aureus. The amplicons of the sea gene were labelled with digoxigenin (Dig) and biotin while those of seb gene were labelled with fluorescein isothiocyanate (FITC) and biotin. These amplicons were detected using a multiplex LFA with NeutrAvidin-tagged gold nanoparticles as the detection reagent. After optimization, the detection limit of this assay was 10(2)  CFU ml(-1) Staph. aureus, which was one tenth that of a multiplex PCR. This assay did not exhibit any cross-reactivity in detecting other enterotoxic Staph. aureus strains or other food pathogens. After 6 h of enrichment, this developed assay detected 1 CFU ml(-1) of Staph. aureus in milk, apple juice, cheese and rice. The developed m-LAMP/LFA method does not require expensive equipment and can be completely implemented within an 8-h workday. Therefore, this method can provide an effective means of quickly screening staphylococcal enterotoxin A- and/or staphylococcal enterotoxin B-producing Staph. aureus in food samples. Staphylococcus aureus is one of the major foodborne pathogens worldwide, and its staphylococcal enterotoxin A and B are strongly associated with food poisoning. This work developed a multiplex loop-mediated isothermal amplification combined with a lateral flow assay (m-LAMP/LFA) to simultaneously detect the sea and seb genes of Staph. aureus in food samples. The assay has good specificity and sensitivity with ease-of-use features, making it ideal for on-site detection. © 2016 The Society for Applied Microbiology.

  8. Nucleic Acid Amplification Testing and Sequencing Combined with Acid-Fast Staining in Needle Biopsy Lung Tissues for the Diagnosis of Smear-Negative Pulmonary Tuberculosis.

    Directory of Open Access Journals (Sweden)

    Faming Jiang

    Full Text Available Smear-negative pulmonary tuberculosis (PTB is common and difficult to diagnose. In this study, we investigated the diagnostic value of nucleic acid amplification testing and sequencing combined with acid-fast bacteria (AFB staining of needle biopsy lung tissues for patients with suspected smear-negative PTB.Patients with suspected smear-negative PTB who underwent percutaneous transthoracic needle biopsy between May 1, 2012, and June 30, 2015, were enrolled in this retrospective study. Patients with AFB in sputum smears were excluded. All lung biopsy specimens were fixed in formalin, embedded in paraffin, and subjected to acid-fast staining and tuberculous polymerase chain reaction (TB-PCR. For patients with positive AFB and negative TB-PCR results in lung tissues, probe assays and 16S rRNA sequencing were used for identification of nontuberculous mycobacteria (NTM. The sensitivity, specificity, positive predictive value (PPV, negative predictive value (NPV, and diagnostic accuracy of PCR and AFB staining were calculated separately and in combination.Among the 220 eligible patients, 133 were diagnosed with TB (men/women: 76/57; age range: 17-80 years, confirmed TB: 9, probable TB: 124. Forty-eight patients who were diagnosed with other specific diseases were assigned as negative controls, and 39 patients with indeterminate final diagnosis were excluded from statistical analysis. The sensitivity, specificity, PPV, NPV, and accuracy of histological AFB (HAFB for the diagnosis of smear-negative were 61.7% (82/133, 100% (48/48, 100% (82/82, 48.5% (48/181, and 71.8% (130/181, respectively. The sensitivity, specificity, PPV, and NPV of histological PCR were 89.5% (119/133, 95.8% (46/48, 98.3% (119/121, and 76.7% (46/60, respectively, demonstrating that histological PCR had significantly higher accuracy (91.2% [165/181] than histological acid-fast staining (71.8% [130/181], P < 0.001. Parallel testing of histological AFB staining and PCR showed the

  9. Development of real-time PCR and loop-mediated isothermal amplification (LAMP) assays for the differential detection of digital dermatitis associated treponemes.

    Science.gov (United States)

    Anklam, Kelly; Kulow, Megan; Yamazaki, Wataru; Döpfer, Dörte

    2017-01-01

    Bovine digital dermatitis (DD) is a severe infectious cause of lameness in cattle worldwide, with important economic and welfare consequences. There are three treponeme phylogroups (T. pedis, T. phagedenis, and T. medium) that are implicated in playing an important causative role in DD. This study was conducted to develop real-time PCR and loop-mediated isothermal amplification (LAMP) assays for the detection and differentiation of the three treponeme phylogroups associated with DD. The real-time PCR treponeme phylogroup assays targeted the 16S-23S rDNA intergenic space (ITS) for T. pedis and T. phagedenis, and the flagellin gene (flaB2) for T. medium. The 3 treponeme phylogroup LAMP assays targeted the flagellin gene (flaB2) and the 16S rRNA was targeted for the Treponeme ssp. LAMP assay. The real-time PCR and LAMP assays correctly detected the target sequence of all control strains examined, and no cross-reactions were observed, representing 100% specificity. The limit of detection for each of the three treponeme phylogroup real-time PCR and LAMP assays was ≤ 70 fg/μl. The detection limit for the Treponema spp. LAMP assay ranged from 7-690 fg/μl depending on phylogroup. Treponemes were isolated from 40 DD lesion biopsies using an immunomagnetic separation culture method. The treponeme isolation samples were then subjected to the real-time PCR and LAMP assays for analysis. The treponeme phylogroup real-time PCR and LAMP assay results had 100% agreement, matching on all isolation samples. These results indicate that the developed assays are a sensitive and specific test for the detection and differentiation of the three main treponeme phylogroups implicated in DD.

  10. Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification

    Directory of Open Access Journals (Sweden)

    Kager Piet A

    2006-10-01

    Full Text Available Abstract Background Decisions concerning malaria treatment depend on species identification causing disease. Microscopy is most frequently used, but at low parasitaemia (Plasmodium antigen detection do often not allow for species discrimination as microscopy does, but also become insensitive at Methods This paper reports the development of a sensitive and specific real-time Quantitative Nucleic Acid Sequence Based Amplification (real-time QT-NASBA assays, based on the small-subunit 18S rRNA gene, to identify the four human Plasmodium species. Results The lower detection limit of the assay is 100 – 1000 molecules in vitro RNA for all species, which corresponds to 0.01 – 0.1 parasite per diagnostic sample (i.e. 50 μl of processed blood. The real-time QT-NASBA was further evaluated using 79 clinical samples from malaria patients: i.e. 11 Plasmodium. falciparum, 37 Plasmodium vivax, seven Plasmodium malariae, four Plasmodium ovale and 20 mixed infections. The initial diagnosis of 69 out of the 79 samples was confirmed with the developed real-time QT-NASBA. Re-analysis of seven available original slides resolved five mismatches. Three of those were initially identified as P. malariae mono-infection, but after re-reading the slides P. falciparum was found, confirming the real-time QT-NASBA result. The other two slides were of poor quality not allowing true species identification. The remaining five discordant results could not be explained by microscopy, but may be due to extreme low numbers of parasites present in the samples. In addition, 12 Plasmodium berghei isolates from mice and 20 blood samples from healthy donors did not show any reaction in the assay. Conclusion Real-time QT-NASBA is a very sensitive and specific technique with a detection limit of 0.1 Plasmodium parasite per diagnostic sample (50 μl of blood and can be used for the detection, identification and quantitative measurement of low parasitaemia of Plasmodium species, thus

  11. Nucleic Acid Amplification Testing and Sequencing Combined with Acid-Fast Staining in Needle Biopsy Lung Tissues for the Diagnosis of Smear-Negative Pulmonary Tuberculosis.

    Science.gov (United States)

    Jiang, Faming; Huang, Weiwei; Wang, Ye; Tian, Panwen; Chen, Xuerong; Liang, Zongan

    2016-01-01

    Smear-negative pulmonary tuberculosis (PTB) is common and difficult to diagnose. In this study, we investigated the diagnostic value of nucleic acid amplification testing and sequencing combined with acid-fast bacteria (AFB) staining of needle biopsy lung tissues for patients with suspected smear-negative PTB. Patients with suspected smear-negative PTB who underwent percutaneous transthoracic needle biopsy between May 1, 2012, and June 30, 2015, were enrolled in this retrospective study. Patients with AFB in sputum smears were excluded. All lung biopsy specimens were fixed in formalin, embedded in paraffin, and subjected to acid-fast staining and tuberculous polymerase chain reaction (TB-PCR). For patients with positive AFB and negative TB-PCR results in lung tissues, probe assays and 16S rRNA sequencing were used for identification of nontuberculous mycobacteria (NTM). The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy of PCR and AFB staining were calculated separately and in combination. Among the 220 eligible patients, 133 were diagnosed with TB (men/women: 76/57; age range: 17-80 years, confirmed TB: 9, probable TB: 124). Forty-eight patients who were diagnosed with other specific diseases were assigned as negative controls, and 39 patients with indeterminate final diagnosis were excluded from statistical analysis. The sensitivity, specificity, PPV, NPV, and accuracy of histological AFB (HAFB) for the diagnosis of smear-negative were 61.7% (82/133), 100% (48/48), 100% (82/82), 48.5% (48/181), and 71.8% (130/181), respectively. The sensitivity, specificity, PPV, and NPV of histological PCR were 89.5% (119/133), 95.8% (46/48), 98.3% (119/121), and 76.7% (46/60), respectively, demonstrating that histological PCR had significantly higher accuracy (91.2% [165/181]) than histological acid-fast staining (71.8% [130/181]), P pulmonary tuberculosis. For patients with positive histological AFB and

  12. The development of loop-mediated isothermal amplification (LAMP) assays for the rapid authentication of five forbidden vegetables in strict vegetarian diets

    OpenAIRE

    Meng-Shiou Lee; Ting-Ying Su; Yi-Yang Lien; Shyang-Chwen Sheu

    2017-01-01

    Plant-based food ingredients such as garlic, Chinese leek, Chinese onion, green onion and onion are widely used in many cuisines around the world. However, these ingredients known as the ?five forbidden vegetables? (FFVs) are not allowed in some vegetarian diets. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of FFVs using five respective LAMP primer sets. The specific primers targeted the ITS1-5.8S-ITS2 nuclear ribosomal DNA sequence regi...

  13. Development of Loop-Mediated Isothermal Amplification (LAMP) assay for rapid detection of Fusarium oxysporum f. sp. ciceris - wilt pathogen of chickpea.

    Science.gov (United States)

    Ghosh, Raju; Nagavardhini, Avuthu; Sengupta, Anindita; Sharma, Mamta

    2015-02-11

    Fusarium oxysporum f. sp. ciceris (Foc), the causal agent of Fusarium wilt is a devastating pathogen of chickpea. In chickpea, various soil borne pathogens produce (s) similar symptoms, therefore cannot be distinguished easily at field level. There is real need for a rapid, inexpensive, and easy to operate and maintain genotyping tool to facilitate accurate disease diagnosis and surveillance for better management of Fusarium wilt outbreaks. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay targeting the elongation factor 1 alpha gene sequence for visual detection of Foc. The LAMP reaction was optimal at 63°C for 60 min. When hydroxynaphthol blue (HNB) was added before amplification, samples with Foc DNA developed a characteristic sky blue colour but those without DNA or with the DNA of six other plant pathogenic fungi did not. Results obtained with LAMP and HNB were confirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for Foc was 10 fg of genomic DNA per reaction, while that of conventional PCR was 100 pg. In conclusion, it was found that a LAMP assay combined with HNB is simple, rapid, sensitive, and specific. The LAMP assay does not require specialized equipment, hence can be used in the field for the rapid detection of Foc. This is the first report of the use of LAMP assay for the detection of Foc. The presented LAMP method provides a specific, sensitive and rapid diagnostic tool for the distinction of Foc, with the potential to be standardized as a detection method for Foc in endemic areas and will be very useful for monitoring the disease complex in the field further suggesting the management strategies.

  14. Reverse transcription loop-mediated isothermal amplification assays for rapid identification of eastern and western strains of bluetongue virus in India.

    Science.gov (United States)

    Maan, S; Maan, N S; Batra, K; Kumar, A; Gupta, A; Rao, Panduranga P; Hemadri, Divakar; Reddy, Yella Narasimha; Guimera, M; Belaganahalli, M N; Mertens, P P C

    2016-08-01

    Bluetongue virus (BTV) infects all ruminants, including cattle, goats and camelids, causing bluetongue disease (BT) that is often severe in naïve deer and sheep. Reverse-transcription-loop-mediated-isothermal-amplification (RT-LAMP) assays were developed to detect eastern or western topotype of BTV strains circulating in India. Each assay uses four primers recognizing six distinct sequences of BTV genome-segment 1 (Seg-1). The eastern (e)RT-LAMP and western (w)RT-LAMP assay detected BTV RNA in all positive isolates that were tested (n=52, including Indian BTV-1, -2, -3, -5, -9, -10, -16, -21 -23, and -24 strains) with high specificity and efficiency. The analytical sensitivity of the RT-LAMP assays is comparable to real-time RT-PCR, but higher than conventional RT-PCR. The accelerated eRT-LAMP and wRT-LAMP assays generated detectable levels of amplified DNA, down to 0.216 fg of BTV RNA template or 108 fg of BTV RNA template within 60-90min respectively. The assays gave negative results with RNA from foot-and-mouth-disease virus (FMDV), peste des petits ruminants virus (PPRV), or DNA from Capripox viruses and Orf virus (n=10), all of which can cause clinical signs similar to BT. Both RT-LAMP assays did not show any cross-reaction among themselves. The assays are rapid, easy to perform, could be adapted as a 'penside' test making them suitable for 'front-line' diagnosis, helping to identify and contain field outbreaks of BTV. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Tylenchulus semipenetrans Using DNA Extracted from Soil

    Directory of Open Access Journals (Sweden)

    Zhi-Qiang Song

    2017-04-01

    Full Text Available Tylenchulus semipenetrans is an important and widespread plant-parasitic nematode of citrus worldwide and can cause citrus slow decline disease leading to significant reduction in tree growth and yield. Rapid and accurate detection of T. semipenetrans in soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP assay was developed to detect T. semipenetrans using DNA extracted from soil. A set of five primers was designed from the internal transcribed spacer region (ITS1 of rDNA, and was highly specific to T. semipenetrans. The LAMP reaction was performed at 63°C for 60 min. The LAMP product was visualized directly in one reaction tube by adding SYBR Green I. The detection limit of the LAMP assay was 10−2 J2/0.5 g of soil, which was 10 times more sensitive than conventional PCR (10−1 J2/0.5 g of soil. Examination of 24 field soil samples revealed that the LAMP assay was applicable to a range of soils infested naturally with T. semipenetrans, and the total assay time was less than 2.5 h. These results indicated that the developed LAMP assay is a simple, rapid, sensitive, specific and accurate technique for detection of T. semipenetrans in field soil, and contributes to the effective management of citrus slow decline disease.

  16. Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. niveum in soil.

    Science.gov (United States)

    Peng, Jun; Zhan, Yuanfeng; Zeng, Fanyun; Long, Haibo; Pei, Yuelin; Guo, Jianrong

    2013-12-01

    Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is one of the major limiting factors for watermelon production worldwide. Rapid and accurate detection of the causal pathogen is the cornerstone of integrated disease management. In this paper, a real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay was developed for the rapid and quantitative detection of Fon in soil. Positive products were amplified only from Fon isolates and not from any other species or formae speciales of F. oxysporum tested, showing a high specificity of the primer sets. The detection limit of the RealAmp assay was 1.2 pg μL(-1) genomic DNA or 10(3) spores g(-1) of artificially inoculated soil, whereas real-time PCR could detect as low as 12 fg μL(-1) or 10(2) spores g(-1). The RealAmp assay was further applied to detect eight artificially inoculated and 85 field soil samples. No significant differences were found between the results tested by the RealAmp and real-time PCR assays. The RealAmp assay is a simple, rapid and effective technique for the quantitative detection and monitoring of Fon in soil under natural conditions. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  17. A Loop-Mediated Isothermal Amplification (LAMP) Assay for Early Detection of Schistosoma mansoni in Stool Samples: A Diagnostic Approach in a Murine Model

    Science.gov (United States)

    Fernández-Soto, Pedro; Gandasegui Arahuetes, Javier; Sánchez Hernández, Alicia; López Abán, Julio; Vicente Santiago, Belén; Muro, Antonio

    2014-01-01

    Background Human schistosomiasis, mainly due to Schistosoma mansoni species, is one of the most prevalent parasitic diseases worldwide. To overcome the drawbacks of classical parasitological and serological methods in detecting S. mansoni infections, especially in acute stage of the disease, development of cost-effective, simple and rapid molecular methods is still needed for the diagnosis of schistosomiasis. A promising approach is the loop-mediated isothermal amplification (LAMP) technology. Compared to PCR-based assays, LAMP has the advantages of reaction simplicity, rapidity, specificity, cost-effectiveness and higher amplification efficiency. Additionally, as results can be inspected by the naked eye, the technique has great potential for use in low-income countries. Methodology/Principal findings A sequence corresponding to a mitochondrial S. mansoni minisatellite DNA region was selected as a target for designing a LAMP-based method to detect S. mansoni DNA in stool samples. We used a S. mansoni murine model to obtain well defined stool and sera samples from infected mice with S. mansoni cercariae. Samples were taken weekly from week 0 to 8 post-infection and the Kato-Katz and ELISA techniques were used for monitoring the infection. Primer set designed were tested using a commercial reaction mixture for LAMP assay and an in house mixture to compare results. Specificity of LAMP was tested using 16 DNA samples from different parasites, including several Schistosoma species, and no cross-reactions were found. The detection limit of our LAMP assay (SmMIT-LAMP) was 1 fg of S. mansoni DNA. When testing stool samples from infected mice the SmMIT-LAMP detected S. mansoni DNA as soon as 1 week post-infection. Conclusions/Significance We have developed, for the first time, a cost-effective, easy to perform, specific and sensitive LAMP assay for early detection of S. mansoni in stool samples. The method is potentially and readily adaptable for field diagnosis and

  18. A loop-mediated isothermal amplification (LAMP assay for early detection of Schistosoma mansoni in stool samples: a diagnostic approach in a murine model.

    Directory of Open Access Journals (Sweden)

    Pedro Fernández-Soto

    2014-09-01

    Full Text Available Human schistosomiasis, mainly due to Schistosoma mansoni species, is one of the most prevalent parasitic diseases worldwide. To overcome the drawbacks of classical parasitological and serological methods in detecting S. mansoni infections, especially in acute stage of the disease, development of cost-effective, simple and rapid molecular methods is still needed for the diagnosis of schistosomiasis. A promising approach is the loop-mediated isothermal amplification (LAMP technology. Compared to PCR-based assays, LAMP has the advantages of reaction simplicity, rapidity, specificity, cost-effectiveness and higher amplification efficiency. Additionally, as results can be inspected by the naked eye, the technique has great potential for use in low-income countries.A sequence corresponding to a mitochondrial S. mansoni minisatellite DNA region was selected as a target for designing a LAMP-based method to detect S. mansoni DNA in stool samples. We used a S. mansoni murine model to obtain well defined stool and sera samples from infected mice with S. mansoni cercariae. Samples were taken weekly from week 0 to 8 post-infection and the Kato-Katz and ELISA techniques were used for monitoring the infection. Primer set designed were tested using a commercial reaction mixture for LAMP assay and an in house mixture to compare results. Specificity of LAMP was tested using 16 DNA samples from different parasites, including several Schistosoma species, and no cross-reactions were found. The detection limit of our LAMP assay (SmMIT-LAMP was 1 fg of S. mansoni DNA. When testing stool samples from infected mice the SmMIT-LAMP detected S. mansoni DNA as soon as 1 week post-infection.We have developed, for the first time, a cost-effective, easy to perform, specific and sensitive LAMP assay for early detection of S. mansoni in stool samples. The method is potentially and readily adaptable for field diagnosis and disease surveillance in schistosomiasis-endemic areas.

  19. A formalin-free method for stabilizing cells for nucleic acid amplification, hybridization and next-generation sequencing.

    Science.gov (United States)

    Qin, Jianbing; Sanmann, Jennifer N; Kittrell, Jeff S; Althof, Pamela A; Kaspar, Erin E; Hunsley, Bradford A

    2015-12-09

    Formalin has been widely used by pathology laboratories. Its carcinogenicity has led researchers to explore formalin substitutes. Streck Cell Preservative (SCP) is a formalin-free preservative that can preserve cellular antigens. This study was undertaken to investigate the effects of cell preservation using SCP on nucleic acid amplification, hybridization, and next-generation sequencing (NGS) as compared to control frozen cells and cells fixed in the traditional cell and tissue fixative, 10 % neutral buffered formalin (NBF). The breast cancer cell line, SKBR-3, was used as a model system. Prior to nucleic acid extraction and fluorescence in situ hybridization (FISH), cells were fixed in SCP or NBF overnight at room temperature with frozen cells in parallel. Analysis showed that similar DNA extraction yields and amplification profiles determined by PCR in SCP preserved cells and control frozen cells, whereas NBF preserved cells had decreased DNA yield and impaired PCR amplification. Molecular cytogenetic studies by FISH technique indicated that the ratios of ERBB2 (HER-2/neu) signals to the chromosome 17 centromere (CEP17) were comparable for frozen cells and SCP preserved cells. The fluorescence images of both SCP fixed and control frozen cells were also clear and comparable. On the contrary, the same analysis was unsuccessful with NBF preserved cells due to poor hybridization quality. Our data also demonstrated that SCP had negligible effect on NGS testing. We conclude that SCP can be used as an alternative to NBF as a preservative for maintaining the integrity of nucleic acids for nucleic acid amplification, sequencing and FISH analysis.

  20. Tissue donation and virus safety: more nucleic acid amplification testing is needed.

    Science.gov (United States)

    Pruss, A; Caspari, G; Krüger, D H; Blümel, J; Nübling, C M; Gürtler, L; Gerlich, W H

    2010-10-01

    In tissue and organ transplantation, it is of great importance to avoid the transmission of blood-borne viruses to the recipient. While serologic testing for anti-human immunodeficiency virus (HIV)-1 and -2, anti-hepatitis C virus (HCV), hepatitis B surface antigen (HBsAg), anti-hepatitis B core antigen (HBc), and Treponema pallidum infection is mandatory, there is until now in most countries no explicit demand for nucleic acid amplification testing (NAT) to detect HIV, hepatitis B virus (HBV), and HCV infection. After a review of reports in the literature on viral transmission events, tissue-specific issues, and manufacturing and inactivation procedures, we evaluated the significance of HIV, HCV, and HBV detection using NAT  in  donors of various types of tissues and compared our results with the experiences of blood banking organizations. There is a significant risk of HIV, HCV, and HBV transmission by musculoskeletal tissues because of their high blood content and the high donor-recipient ratio. If no effective virus inactivation procedure for musculoskeletal tissue is applied, donors should be screened using NAT  for  HIV, HCV, and HBV. Serologically screened cardiovascular tissue carries a very low risk of HIV, HCV, or HBV transmission. Nevertheless, because effective virus inactivation is impossible (retention of tissue morphology) and the donor-recipient ratio may be as high as 1:10, we concluded that NAT  should be performed for HIV, HCV, and HBV as an additional safety measure. Although cornea allografts carry the lowest risk of transmitting HIV, HCV, and HBV  owing to corneal physiology, morphology, and the epidemiology of corneal diseases, NAT  for  HCV should still be performed. If the NAT  screening of a donor for HIV, HCV, and HBV is negative, quarantine storage of the donor tissue seems dispensable. In view of numerous synergistic effects with transfusion medicine, it would be advantageous for tissue banks to cooperate with blood

  1. Trichomonas vaginalis Nucleic Acid Amplification Testing at an Urban HIV Clinic.

    Science.gov (United States)

    Muzny, Christina A; Burkholder, Greer A; Fry, Karen R; Austin, Erika L; Schwebke, Jane R

    2016-08-01

    Trichomonas vaginalis is the most common nonviral sexually transmitted infection. T. vaginalis nucleic acid amplification testing (NAAT) recently became available at the University of Alabama at Birmingham human immunodeficiency virus (HIV) clinic. The objective of this study was to determine the uptake of T. vaginalis NAAT testing among clinic providers during the first year of test availability in addition to T. vaginalis prevalence and predictors based on NAAT results. This was a retrospective review of HIV+ women and men ages ≥16 years at the University of Alabama at Birmingham HIV Clinic, including those receiving a T. vaginalis NAAT on a genitourinary specimen. Between August 2014 and August 2015, 3163 HIV+ patients were seen (768 women, 2395 men), of whom 861 (27.3%) received a T. vaginalis NAAT; 402 women (52.3%) and 459 men (19.2%). Among those with T. vaginalis NAAT results, 70 (17.4%) of 402 women and 12 (2.6%) of 459 men (9 men who have sex with women, 1 man who has sex with men, 2 unknown) tested positive. In adjusted analyses for women, age ≤40 years (odds ratio [OR], 2.93; 95% confidence interval [CI], 1.23-6.96), current cocaine use (OR, 4.86; 95% CI, 1.57-15.06), and CD4 vaginalis NAAT. For those with a positive T. vaginalis NAAT, treatment was prescribed for 65 (92.9%) of 70 women and 10 (83.3%) of 12 men. Initial uptake of T. vaginalis NAAT testing was modest at this HIV clinic yet identified a high prevalence among women tested. Emphasis on the need for testing in HIV+ women is necessary.

  2. Nucleic acid amplification testing in Indian blood banks: A review with perspectives.

    Science.gov (United States)

    Ghosh, Kanjaksha; Mishra, Kanchan

    2017-01-01

    Nucleic acid amplification testing (NAT) is restricted to a few blood banks in India since 2008. This review was directed toward understanding NAT yield in different parts of the country and prevalence in the NAT of different types of virus. English literature was searched from 1990 to 2016 in PubMed, Scopus, Ind med, and Google database using properly constructed key words. Literature was collected and finally the data were synthesized. NAT results from 11 publications and one personal communication showed that till date 389387 blood units have been NAT tested from various parts of the country. NAT yield varied from 1:476 to 1:4403 in various studies. Till date, 58/2550 (2%) blood banks of India are doing NAT testing but all of them have not published their results. Majority of the centers have used ID-NAT (Individual NAT) protocol and 21 blood banks are using minipool format of the test. One center has used in-house NAT testing system. In> 70% of the time, the NAT positivity with due to hepatitis B (Hep B). For individual infection, NAT yield from the pooled data showed HIV in 1:66,000, Hep C virus 1:5484 and Hep B in 1:1761 seronegative donors. In view of the very high NAT yield (1:1361), NAT in some from needs to be universally applied in Indian blood banks. However, the high Hep B occult infection suggests stricter donor selection and immunization of adults for Hep B may be way forward toward ensuring the viral safety of blood components in India.

  3. Development of mismatch amplification mutation assays for the differentiation of MS1 vaccine strain from wild-type Mycoplasma synoviae and MS-H vaccine strains

    Science.gov (United States)

    Kreizinger, Zsuzsa; Sulyok, Kinga Mária; Grózner, Dénes; Bekő, Katinka; Dán, Ádám; Szabó, Zoltán

    2017-01-01

    Mycoplasma synoviae is an economically significant pathogen in the poultry industry, inducing respiratory disease and infectious synovitis in chickens and turkeys, and eggshell apex abnormality in chickens. Eradication, medication and vaccination are the options for controlling M. synoviae infection. Currently there are two commercial, live, attenuated vaccines available against M. synoviae: the temperature sensitive MS-H vaccine strain and the NAD independent MS1 vaccine strain. Differentiation of vaccine strains from field isolates is essential during vaccination and eradication programs. The present study provides melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) to discriminate the MS1 vaccine strain from the MS-H vaccine strain and wild-type M. synoviae isolates. The assays are based on the A/C single nucleotide polymorphism at nt11 of a HIT family protein coding gene. The melt- and agarose-MAMAs reliably distinguish the MS1 vaccine strain genotype from the MS-H vaccine strain and wild-type M. synoviae isolate genotype from 102 template number/DNA sample. No cross-reactions with other avian Mycoplasma species were observed. The assays can be performed directly on clinical samples and they can be run simultaneously with the previously described MAMAs designed for the discrimination of the MS-H vaccine strain. The developed assays are applicable in laboratories with limited facilities and promote the rapid, simple and cost effective differentiation of the MS1 vaccine strain. PMID:28419134

  4. Novel colorimetric assay of mefenamic acid using 4-amino-3,5 ...

    African Journals Online (AJOL)

    ... assay (p>0.05) and has the advantages of speed, high sensitivity, lower limit of detection and can be automated. Conclusion: The method developed could find application in in-process quality control of mefenamic acid capsules. Keywords: Mefenamic acid assay, 4-amino-3,5-dinitrobenzoic acid, colorimetry, diazotization

  5. Rapid detection of Salmonella in food and feed by coupling loop-mediated isothermal amplification with bioluminescent assay in real-time.

    Science.gov (United States)

    Yang, Qianru; Domesle, Kelly J; Wang, Fei; Ge, Beilei

    2016-06-17

    Salmonella is among the most significant pathogens causing food and feed safety concerns. This study examined the rapid detection of Salmonella in various types of food and feed samples by coupling loop-mediated isothermal amplification (LAMP) with a novel reporter, bioluminescent assay in real-time (BART). Performance of the LAMP-BART assay was compared to a conventional LAMP and the commercially available 3M Molecular Detection Assay (MDA) Salmonella. The LAMP-BART assay was 100 % specific among 178 strains (151 Salmonella and 27 non-Salmonella) tested. The detection limits were 36 cells per reaction in pure culture and 10(4) to 10(6) CFU per 25 g in spiked food and feed samples without enrichment, which were comparable to those of the conventional LAMP and 3M MDA Salmonella but 5-10 min faster. Ground turkey showed a strong inhibition on 3M MDA Salmonella, requiring at least 10(8) CFU per 25 g for detection. The correlation between Salmonella cell numbers and LAMP-BART signals was high (R (2) = 0.941-0.962), suggesting good quantification capability. After 24 h enrichment, all three assays accurately detected 1 to 3 CFU per 25 g of Salmonella among five types of food (cantaloupe, ground beef, ground turkey, shell eggs, and tomato) and three types of feed (cattle feed, chicken feed, and dry dog food) examined. However, 10(1) CFU per 25 g was required for cattle feed when tested by 3M MDA Salmonella. The Salmonella LAMP-BART assay was rapid, specific, sensitive, quantitative, and robust. Upon further validation, it may become a valuable tool for routine screening of Salmonella in various types of food and feed samples.

  6. Development and application of loop-mediated isothermal amplification assays for rapid visual detection of cry2Ab and cry3A genes in genetically-modified crops.

    Science.gov (United States)

    Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

    2014-08-27

    The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field.

  7. Development and Application of Loop-Mediated Isothermal Amplification Assays for Rapid Visual Detection of cry2Ab and cry3A Genes in Genetically-Modified Crops

    Directory of Open Access Journals (Sweden)

    Feiwu Li

    2014-08-01

    Full Text Available The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field.

  8. Rapid detection of Bombyx mori nucleopolyhedrovirus (BmNPV) by loop-mediated isothermal amplification assay combined with a lateral flow dipstick method.

    Science.gov (United States)

    Zhou, Yang; Wu, Jiege; Lin, Feng; Chen, Naifu; Yuan, Shaofei; Ding, Lina; Gao, Li; Hang, Bangxing

    2015-12-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) is a principal pathogen of the domestic silkworm. The disease often breaks out in sericultural countries and due to its high infectivity; it is difficult to control, resulting in heavy economic loss. In order to develop a rapid, sensitive visual detection and simple-to-use novel technology for detection of BmNPV, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) method was described. In this study, a set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the BmNPV gp41 gene, and the LAMP for the detection of BmNPV was developed by isothermal amplification at 61 °C for 45 min, followed by hybridization with an FITC-labeled DNA probe for 5 min and detected by LFD within 5 min. The detection limit of LAMP-LFD was 0.2 pg DNA extracted from silkworm infected with BmNPV and was 100 times more sensitive than conventional PCR. No product was generated from silkworm infected with other viruses. Furthermore, we applied the technique to detect BmNPV in the hemolymph and feces at different intervals post infection (pi). In conclusion, the novel LAMP-LFD setup presented here is simple, rapid, reliable, and has the potential for future use in the detection of BmNPV. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Polymer-based microfluidic chips for isothermal amplification of nucleic acids

    Science.gov (United States)

    Posmitnaya, Y. S.; Rudnitskaya, G. E.; Tupik, A. N.; Lukashenko, T. A.; Bukatin, A. C.; Evstrapov, A. A.

    2017-11-01

    Creation of low-cost compact devices based on microfluidic platforms for biological and medical research depends on the degree of development and enhancement of prototyping technologies. Two designs of polymer and hybrid microfluidic devices fabricated by soft lithography and intended for isothermal amplification and polymerase chain reaction are presented in this paper. The digital helicase-dependent isothermal amplification was tested in the device containing a droplet generator. Polymerase chain reaction was carried out in the hybrid microfluidic device having ten reaction chambers. A synthesized cDNA fragment of GAPDH housekeeping gene was used as a target.

  10. Chemiluminescence resonance energy transfer biosensing platform for site-specific determination of DNA methylation and assay of DNA methyltransferase activity using exonuclease III-assisted target recycling amplification.

    Science.gov (United States)

    Chen, Chun; Li, Baoxin

    2014-04-15

    Site-specific determination of DNA methylation and assay of MTase activity can be used for determining specific cancer types, providing insights into the mechanism of gene repression, and developing novel drugs to treat methylation-related diseases. Herein, we develop a simple and highly sensitive chemiluminescence (CL) biosensing platform for site-specific determination of DNA methylation using Exonuclease III (Exo III)-assisted target recycling signal amplification. After bisulfite treatment of mixture of methylated DNA and unmethylated DNA, methylated DNA can hybridize with fluorescein (FAM)-labeled probe DNA to form double-stranded DNA (dsDNA), removing the FAM-labeled probe DNA from the surface of grapheme oxide, and the chemiluminescence resonance energy transfer (CRET) sensing signal can be observed and then amplified using Exo III-based recycling strategy. The biosensing platform exhibits excellent high sensitivity, and it can ever distinguish as low as 0.002% methylation level from the mixture, which is superior to most currently reported methods used for DNA methylation assay. In addition, the proposed method can also be used to sensitively assay MTase activity with determination limit of 0.007 U/mL. This CL biosensing offers the advantages of being facile, sensitive, rapid and cost-effective. These features make the system promising for future use for early cancer diagnosis and discover of new anticancer drugs. © 2013 Published by Elsevier B.V.

  11. Utility of loop-mediated isothermal amplification assay, polymerase chain reaction, and elisa for diagnosis of leptospirosis in South Indian patients

    Directory of Open Access Journals (Sweden)

    Mallika Sengupta

    2017-01-01

    Full Text Available Background: Leptospirosis is a zoonotic disease which requires laboratory diagnosis for confirmation. Materials and Methods: In this study serum samples from adults with acute undifferentiated fever (duration ≤15 days were tested for IgM antibodies to Leptospira by ELISA, PCR for rrs gene and loop-mediated isothermal amplification (LAMP assay for LipL32 and LipL41. Results: Among the 150 sera tested, three were positive by PCR, LAMP and IgM ELISA/modified Faines' criteria, two by only PCR; seven only by LAMP assay and forty fulfilled modified Faine's criteria (illness clinically compatible and IgM ELISA positive for leptospirosis. Clinical correlation revealed renal compromise, low platelet count and severe jaundice were significantly related to leptospirosis (P < 0.05. Conclusion: This study suggests that LAMP assay could be useful for diagnosis of leptospirosis during the 1st week of illness whereas IgM ELISA forms the mainstay of diagnosis from the 2nd week onward. Further studies especially community based, comparing ELISA, PCR, LAMP, culture and microscopic agglutination test are required to evaluate the veracity of these findings.

  12. A Loop-Mediated Isothermal Amplification (LAMP) Assay for Strongyloides stercoralis in Stool That Uses a Visual Detection Method with SYTO-82 Fluorescent Dye

    Science.gov (United States)

    Watts, Matthew R.; James, Gregory; Sultana, Yasmin; Ginn, Andrew N.; Outhred, Alexander C.; Kong, Fanrong; Verweij, Jaco J.; Iredell, Jonathan R.; Chen, Sharon C-A.; Lee, Rogan

    2014-01-01

    An assay to detect Strongyloides stercoralis in stool specimens was developed using the loop-mediated isothermal amplification (LAMP) method. Primers were based on the 28S ribosomal subunit gene. The reaction conditions were optimized and SYTO-82 fluorescent dye was used to allow real-time and visual detection of the product. The product identity was confirmed with restriction enzyme digestion, cloning, and sequence analysis. The assay was specific when tested against DNA from bacteria, fungi and parasites, and 30 normal stool samples. Analytical sensitivity was to < 10 copies of target sequence in a plasmid and up to a 10-2 dilution of DNA extracted from a Strongyloides ratti larva spiked into stool. Sensitivity was increased when further dilutions were made in water, indicative of reduced reaction inhibition. Twenty-seven of 28 stool samples microscopy and polymerase chain reaction positive for S. stercoralis were positive with the LAMP method. On the basis of these findings, the assay warrants further clinical validation. PMID:24323513

  13. A model system for pathogen detection using a two-component bacteriophage/bioluminescent signal amplification assay

    Science.gov (United States)

    Bright, Nathan G.; Carroll, Richard J.; Applegate, Bruce M.

    2004-03-01

    Microbial contamination has become a mounting concern the last decade due to an increased emphasis of minimally processed food products specifically produce, and the recognition of foodborne pathogens such as Campylobacter jejuni, Escherichia coli O157:H7, and Listeria monocytogenes. This research investigates a detection approach utilizing bacteriophage pathogen specificity coupled with a bacterial bioluminescent bioreporter utilizing the quorum sensing molecule from Vibrio fischeri, N-(3-oxohexanoyl)-homoserine lactone (3-oxo-C6-HSL). The 3-oxo-C6-HSL molecules diffuse out of the target cell after infection and induce bioluminescence from a population of 3-oxo-C6-HSL bioreporters (ROLux). E. coli phage M13, a well-characterized bacteriophage, offers a model system testing the use of bacteriophage for pathogen detection through cell-to-cell communication via a LuxR/3-oxo-C6-HSL system. Simulated temperate phage assays tested functionality of the ROLux reporter and production of 3-oxo-C6-HSL by various test strains. These assays showed detection limits of 102cfu after 24 hours in a varietry of detection formats. Assays incorporating the bacteriophage M13-luxI with the ROLux reporter and a known population of target cells were subsequently developed and have shown consistent detection limits of 105cfu target organisms. Measurable light response from high concentrations of target cells was almost immediate, suggesting an enrichment step to further improve detection limits and reduce assay time.

  14. Loop-mediated isothermal amplification (LAMP) assay-A rapid detection tool for identifying red fox (Vulpes vulpes) DNA in the carcasses of harbour porpoises (Phocoena phocoena).

    Science.gov (United States)

    Heers, Teresa; van Neer, Abbo; Becker, André; Grilo, Miguel Luca; Siebert, Ursula; Abdulmawjood, Amir

    2017-01-01

    Carcasses of wild animals are often visited by different scavengers. However, determining which scavenger caused certain types of bite marks is particularly difficult and knowledge thereof is lacking. Therefore, a loop-mediated isothermal amplification (LAMP) assay (target sequence cytochrome b) was developed to detect red fox DNA in carcasses of harbour porpoises. The MSwab™ method for direct testing without prior DNA isolation was validated. As a detection device, the portable real-time fluorometer Genie® II was used, which yields rapid results and can be used in field studies without huge laboratory equipment. In addition to in vitro evaluation and validation, a stranded and scavenged harbour porpoise carcass was successfully examined for red fox DNA residues. The developed LAMP method is a valuable diagnostic tool for confirming presumable red fox bite wounds in harbour porpoises without further DNA isolation steps.

  15. Development of a loop-mediated isothermal amplification assay for rapid detection of Nocardia salmonicida, the causative agent of nocardiosis in fish.

    Science.gov (United States)

    Xia, Liqun; Zhang, Honglian; Lu, Yishan; Cai, Jia; Wang, Bei; Jian, Jichang

    2015-03-01

    Nocardia salmonicida is one of the main pathogens of fish nocardiosis. The purpose of this study was to build a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of N. salmonicida. A set of four primers were designed from the 16S-23S rRNA intergenic spacer region of N. salmonicida, and conditions for LAMP were optimized as incubating all the reagents for 60 min at 64°C. LAMP products were judged with agar gel electrophoresis as well as with the naked eye after the addition of SYBR Green I. Results showed the sensitivity of the LAMP assay was 1.68 × 10(3) CFU/ml (16.8 CFU per reaction) and 10-fold higher than that of PCR. The LAMP method was also effectively applied to detect N. salmonicida in diseased fish samples, and it may potentially facilitate the surveillance and early diagnosis of fish nocardiosis.

  16. Improved sensitivity of nucleic acid amplification for rapid diagnosis of tuberculous meningitis

    DEFF Research Database (Denmark)

    Johansen, Isik Somuncu; Lundgren, Bettina; Tabak, Fehmi

    2004-01-01

    Early diagnosis of tuberculous meningitis (TBM) is essential for a positive outcome; but present microbiological diagnostic techniques are insensitive, slow, or laborious. We evaluated the standard BDProbeTec ET strand displacement amplification method (the standard ProbeTec method) for the detec...

  17. Clinical utility of a nested nucleic acid amplification format in comparison to viral culture for the diagnosis of mucosal herpes simplex infection in a genitourinary medicine setting

    Directory of Open Access Journals (Sweden)

    Wyatt Dorothy E

    2001-08-01

    Full Text Available Abstract Background Nested nucleic acid amplification tests are often thought too sensitive or prone to generatingfalse positive results for routine use. The current study investigated the specificity and clinicalutility of a routine multiplex nested assay for mucosal herpetic infections. Methods Ninety patients, categorised into those clinically diagnosed to (a have and (b not haveherpetic infection, were enrolled. Swabs from oral and ano-genital sites were assayed by thenested assay and culture and the results assessed against clinical evaluation for diagnosingherpetic infections; cell content was also recorded. Results Twenty-six and 64 patients were thought to (a have and (b not have mucosal herpeticinfection. Taking the clinical evaluation as indicating the presence of herpetic infection, thenested polymerase chain reaction and culture had respective sensitivities of 19/26 (73% and12/26 (46% (Χ2 p = 0.02. There was no significant difference in specificities between nPCR62/64 (97% and culture 63/64 (98% (Χ2 p = 1.0. Cell content was important for viraldetection by nPCR (Χ2 p = 0.07 but not culture. Nesting was found necessary for sensitivity anddid not reduce specificity. Assay under-performance appeared related to sub-optimal cellcontent (20% but may have reflected clinical over-diagnosis. The results suggest the need forvalidating specimen cell quality. Conclusions This study questions the value of routine laboratory confirmation of mucosal herpetic infection. The adoption of a more discriminatory usage of laboratory diagnostic facilities for genital herpetic infection, taking account of cell content, and restricting it to those cases where it actually affects patient management, may be warranted.

  18. Development of a simple and rapid reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for sensitive detection of Citrus tristeza virus.

    Science.gov (United States)

    Warghane, Ashish; Misra, Pragati; Bhose, Sumit; Biswas, Kajal Kumar; Sharma, Ashwani Kumar; Reddy, M Krishna; Ghosh, Dilip Kumar

    2017-12-01

    Tristeza is a devastating disease of citrus and reported to be present in almost all countries where it is cultivated as a commercial crop. The etiological agent of this disease is Citrus tristeza virus (CTV), a member of the genus Closterovirus with in the family Closteroviridae. The pathogen is restricted to the phloem tissue of the infected citrus plant and has a monopartite ss (+) RNA genome of ∼20kb size. Till date, there is no effective control measure available for this virus. Management of tristeza depends on destruction of CTV infected field plants, production of virus-free planting material for new orchard establishment and controlling viruliferous aphid vectors responsible for field spread of the pathogen. Availability of rapid diagnostic assay is essential for rapid and efficient detection of the pathogen. In the present investigation, RT-LAMP (reverse transcription-loop mediated isothermal amplification), a highly sensitive, robust and low cost assay has been developed for rapid detection of CTV in infected citrus plant samples. Based on conserved nucleotide sequences available in GenBank and specific to p25 gene (major coat protein gene) of predominant CTV isolates of India, four primer sets (CTV-F3, CTV-B3, CTV-FIP and CTV-BIP) ware designed and custom synthesized. The amplified LAMP products obtained after maintaining isothermal condition of 65°C for 60min duration could be visible easily with necked eyes in presence of SYBR Green I (100X). Subsequently, LAMP products were verified by electrophoresis run in 1.5% agarose gel. The RT-LAMP results obtained with known CTV isolates maintained in screen house of CCRI, Nagpur were validated using field samples and thereafter it was further confirmed by conventional RT-PCR (reveres transcription-polymerase chain reaction) assay. The sensitivity of CTV-RT-LAMP protocol standardized in the present study was 100 times more than conventional one step RT-PCR assay. It also has maximum detection limit up to 0

  19. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Quality control material for cystic fibrosis... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a) Identification. Quality control material for cystic fibrosis nucleic acid assays. A quality control material for...

  20. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Science.gov (United States)

    2010-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents... panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to...: Respiratory Viral Panel Multiplex Nucleic Acid Assay;” (2) For a device that detects and identifies Human...

  1. The development of loop-mediated isothermal amplification (LAMP) assays for the rapid authentication of five forbidden vegetables in strict vegetarian diets.

    Science.gov (United States)

    Lee, Meng-Shiou; Su, Ting-Ying; Lien, Yi-Yang; Sheu, Shyang-Chwen

    2017-03-14

    Plant-based food ingredients such as garlic, Chinese leek, Chinese onion, green onion and onion are widely used in many cuisines around the world. However, these ingredients known as the "five forbidden vegetables" (FFVs) are not allowed in some vegetarian diets. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of FFVs using five respective LAMP primer sets. The specific primers targeted the ITS1-5.8S-ITS2 nuclear ribosomal DNA sequence regions among the five vegetables. The results demonstrated that the identification of FFVs using the newly developed LAMP assay is more sensitive than the traditional PCR method. Using pepper, basil, parsley, chili and ginger as references, established LAMP primer sets showed high specificity for the identification of the FFV species. Moreover, when FFVs were mixed with other plant ingredients at different ratios (100:0, 50:50, 20:80, 10:90, 5:95, 2:98, and 1:99), no cross-reactivity was evident using LAMP. Finally, genomic DNAs extracted from boiled and steamed FFVs in processed foods were used as templates; the performance of the LAMP reaction was not influenced using validated LAMP primers. Not only can FFV ingredients be identified but commercial foods containing FFVs can also be authenticated. This LAMP method will be useful for the authentication of FFVs in practical food markets in the future.

  2. Multiplex ligation-dependent probe amplification (MLPA) assay for the detection of mitochondrial DNA deletion in chronic progressive external ophthalmoplegia (CPEO).

    Science.gov (United States)

    Kwon, Min-Jung; Ki, Chang-Seok; Kim, Ji-Youn; Lee, Seung-Tae; Kim, Jong-Won; Kang, Sa-Yoon

    2011-01-01

    Chronic progressive external ophthalmoplegia (CPEO) is a mitochondrial myopathy commonly caused by deleterious changes in the mitochondrial DNA (mtDNA). We describe a 45-year-old man who was referred to us for investigation of progressive ptosis. We performed a multiplex ligation-dependent probe amplification (MLPA) assay of mtDNA from muscle tissue and peripheral blood leukocytes, and followed up with gap-polymerase chain reaction (PCR) and direct sequence analysis. Results showed a deletion of a 4,407 bp segment in the mtDNA region, ranging from nucleotide position 8,577 in the MT-ATP6 gene to nucleotide position 12,983 in the MT-ND5 gene. To the best of our knowledge, this is the first report of a CPEO patient with a large novel deletion of mtDNA genetically confirmed by MLPA assay. MLPA can be a feasible platform for clinical laboratories to detect large deletion mutations in the mtDNA for suspected cases.

  3. Giardia and Cryptosporidium spp. dissemination during wastewater treatment and comparative detection via immunofluorescence assay (IFA), nested polymerase chain reaction (nested PCR) and loop mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Gallas-Lindemann, Carmen; Sotiriadou, Isaia; Plutzer, Judit; Noack, Michael J; Mahmoudi, Mohammad Reza; Karanis, Panagiotis

    2016-06-01

    Environmental water samples from the Lower Rhine area in Germany were investigated via immunofluorescence assays (IFAs), nested polymerase chain reaction (nested PCR) and loop-mediated isothermal amplification (LAMP) to detect the presence of Giardia spp. (n=185) and Cryptosporidium spp. (n=227). The samples were concentrated through filtration or flocculation, and oocysts were purified via centrifugation through a sucrose density gradient. For all samples, IFA was performed first, followed by DNA extraction for the nested PCR and LAMP assays. Giardia cysts were detected in 105 samples (56.8%) by IFA, 62 samples (33.5%) by nested PCR and 79 samples (42.7%) by LAMP. Cryptosporidium spp. were detected in 69 samples (30.4%) by IFA, 95 samples (41.9%) by nested PCR and 99 samples (43.6%) by LAMP. According to these results, the three detection methods are complementary for monitoring Giardia and Cryptosporidium in environmental waters. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. A prospective study of two isothermal amplification assays compared with real-time PCR, CCNA and toxigenic culture for the diagnosis of Clostridium difficile infection.

    Science.gov (United States)

    Neuendorf, Martina; Guadarrama-Gonzalez, Raquel; Lamik, Birgit; MacKenzie, Colin R

    2016-02-12

    New molecular methods of detecting Clostridium difficile infection (CDI) provide the routine lab with a sensitive random access method to produce results that are available in a shorter time than traditional methods. In this prospective study a total of 989 stool specimens were tested over a period of 16 months in parallel using two isothermal amplification assays, AmpliVue® (Quidel) and Illumigene® (Meridian) and the results compared to those from toxigenic culture. In addition all specimens were tested using a cytotoxic cell neutralisation assay (CCNA) and three different Real-time PCR targeting a C. difficile-specific 16S rDNA sequence or the toxin genes tcdA, tcdB/tcdB027 or cdtB. AmpliVue® was positive in 242 (24.5%) and Illumigene® in 228 (23.1%) specimens. 167 (16.9%) specimens were positive in toxigenic culture. Real-time-tcdA and -tcdB PCR was positive in 211 (21.3%) specimens, Real-time-cdtB PCR was positive in 101 (10.2%) specimens and C. difficile-PCR (16S rDNA) in 267 (27.0%) specimens. The respective sensitivity, specificity, positive predictive value and negative predictive value compared to toxigenic culture were 91, 89, 62 and 98% for AmpliVue® and 91, 91, 67 and 98% for Illumigene®.

  5. Absolute quantitation of Met using mass spectrometry for clinical application: assay precision, stability, and correlation with MET gene amplification in FFPE tumor tissue.

    Directory of Open Access Journals (Sweden)

    Daniel V T Catenacci

    Full Text Available Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC of formalin-fixed paraffin-embedded (FFPE tissues is currently used to select for 'high Met' expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue.After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH.Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD and quantitation (LOQ for this peptide were 150 and 200 amol/µg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (<150 amol/µg to 4669.5 amol/µg. High correlation was observed between SRM Met expression and both MET GCN and MET/CEP7 ratio as determined by FISH (n = 30; R2 = 0.898. IHC did not correlate well with SRM (n = 44; R2 = 0.537 nor FISH GCN (n = 31; R2 = 0.509. A Met SRM level of ≥1500 amol/µg was 100% sensitive (95% CI 0.69-1 and 100% specific (95% CI 0.92-1 for MET amplification.The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET tumors. These results demonstrate a novel

  6. A field based detection method for Rose rosette virus using isothermal probe-based Reverse transcription-recombinase polymerase amplification assay.

    Science.gov (United States)

    Babu, Binoy; Washburn, Brian K; Ertek, Tülin Sarigül; Miller, Steven H; Riddle, Charles B; Knox, Gary W; Ochoa-Corona, Francisco M; Olson, Jennifer; Katırcıoğlu, Yakup Zekai; Paret, Mathews L

    2017-09-01

    Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early detection and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and are inconsistent in detecting the virus from symptomatic plants. Real-time RT-qPCR assay is highly sensitive for detection of RRV, but it is expensive and requires well-equipped laboratories. Both the RT-PCR and RT-qPCR cannot be used in a field-based testing for RRV. Hence a novel probe based, isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, using primer/probe designed based on the nucleocapsid gene of the RRV has been developed. The assay is highly specific and did not give a positive reaction to other viruses infecting roses belonging to both inclusive and exclusive genus. Dilution assays using the in vitro transcript showed that the primer/probe set is highly sensitive, with a detection limit of 1 fg/μl. In addition, a rapid technique for the extraction of viral RNA (<5min) has been standardized from RRV infected tissue sources, using PBS-T buffer (pH 7.4), which facilitates the virus adsorption onto the PCR tubes at 4°C for 2min, followed by denaturation to release the RNA. RT-exoRPA analysis of the infected plants using the primer/probe indicated that the virus could be detected from leaves, stems, petals, pollen, primary roots and secondary roots. In addition, the assay was efficiently used in the diagnosis of RRV from different rose varieties, collected from different states in the U.S. The entire process, including the extraction can be completed in 25min, with less sophisticated equipments. The developed assay can be used with high efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscapes. Copyright © 2017 Elsevier B

  7. Evaluation of Nucleic Acid Isothermal Amplification Methods for Human Clinical Microbial Infection Detection.

    Science.gov (United States)

    Etchebarne, Brett E; Li, Zenggang; Stedtfeld, Robert D; Nicholas, Michael C; Williams, Maggie R; Johnson, Timothy A; Stedtfeld, Tiffany M; Kostic, Tanja; Khalife, Walid T; Tiedje, James M; Hashsham, Syed A; Hughes, Mary J

    2017-01-01

    Battling infection is a major healthcare objective. Untreated infections can rapidly evolve toward the condition of sepsis in which the body begins to fail and resuscitation becomes critical and tenuous. Identification of infection followed by rapid antimicrobial treatment are primary goals of medical care, but precise identification of offending organisms by current methods is slow and broad spectrum empirical therapy is employed to cover most potential pathogens. Current methods for identification of bacterial pathogens in a clinical setting typically require days of time, or a 4- to 8-h growth phase followed by DNA extraction, purification and PCR-based amplification. We demonstrate rapid (70-120 min) genetic diagnostics methods utilizing loop-mediated isothermal amplification (LAMP) to test for 15 common infection pathogen targets, called the Infection Diagnosis Panel (In-Dx). The method utilizes filtration to rapidly concentrate bacteria in sample matrices with lower bacterial loads and direct LAMP amplification without DNA purification from clinical blood, urine, wound, sputum and stool samples. The In-Dx panel was tested using two methods of detection: (1) real-time thermocycler fluorescent detection of LAMP amplification and (2) visual discrimination of color change in the presence of Eriochrome Black T (EBT) dye following amplification. In total, 239 duplicate samples were collected (31 blood, 122 urine, 73 mucocutaneous wound/swab, 11 sputum and two stool) from 229 prospectively enrolled hospital patients with suspected clinical infection and analyzed both at the hospital and by In-Dx. Sensitivity (Se) of the In-Dx panel targets pathogens from urine samples by In-Dx was 91.1% and specificity (Sp) was 97.3%, with a positive predictive value (PPV) of 53.7% and a negative predictive value (NPV) of 99.7% as compared to clinical microbial detection methods. Sensitivity of detection of the In-Dx panel from mucocutaneous swab samples was 65.5% with a Sp of 99

  8. Evaluation of Nucleic Acid Isothermal Amplification Methods for Human Clinical Microbial Infection Detection

    Directory of Open Access Journals (Sweden)

    Brett E. Etchebarne

    2017-12-01

    Full Text Available Battling infection is a major healthcare objective. Untreated infections can rapidly evolve toward the condition of sepsis in which the body begins to fail and resuscitation becomes critical and tenuous. Identification of infection followed by rapid antimicrobial treatment are primary goals of medical care, but precise identification of offending organisms by current methods is slow and broad spectrum empirical therapy is employed to cover most potential pathogens. Current methods for identification of bacterial pathogens in a clinical setting typically require days of time, or a 4- to 8-h growth phase followed by DNA extraction, purification and PCR-based amplification. We demonstrate rapid (70–120 min genetic diagnostics methods utilizing loop-mediated isothermal amplification (LAMP to test for 15 common infection pathogen targets, called the Infection Diagnosis Panel (In-Dx. The method utilizes filtration to rapidly concentrate bacteria in sample matrices with lower bacterial loads and direct LAMP amplification without DNA purification from clinical blood, urine, wound, sputum and stool samples. The In-Dx panel was tested using two methods of detection: (1 real-time thermocycler fluorescent detection of LAMP amplification and (2 visual discrimination of color change in the presence of Eriochrome Black T (EBT dye following amplification. In total, 239 duplicate samples were collected (31 blood, 122 urine, 73 mucocutaneous wound/swab, 11 sputum and two stool from 229 prospectively enrolled hospital patients with suspected clinical infection and analyzed both at the hospital and by In-Dx. Sensitivity (Se of the In-Dx panel targets pathogens from urine samples by In-Dx was 91.1% and specificity (Sp was 97.3%, with a positive predictive value (PPV of 53.7% and a negative predictive value (NPV of 99.7% as compared to clinical microbial detection methods. Sensitivity of detection of the In-Dx panel from mucocutaneous swab samples was 65.5% with a

  9. Mobile Platform for Multiplexed Detection and Differentiation of Disease-Specific Nucleic Acid Sequences, Using Microfluidic Loop-Mediated Isothermal Amplification and Smartphone Detection.

    Science.gov (United States)

    Chen, Weili; Yu, Hojeong; Sun, Fu; Ornob, Akid; Brisbin, Ryan; Ganguli, Anurup; Vemuri, Vinay; Strzebonski, Piotr; Cui, Guangzhe; Allen, Karen J; Desai, Smit A; Lin, Weiran; Nash, David M; Hirschberg, David L; Brooks, Ian; Bashir, Rashid; Cunningham, Brian T

    2017-11-07

    New tools are needed to enable rapid detection, identification, and reporting of infectious viral and microbial pathogens in a wide variety of point-of-care applications that impact human and animal health. We report the design, construction, and characterization of a platform for multiplexed analysis of disease-specific DNA sequences that utilizes a smartphone camera as the sensor in conjunction with a hand-held "cradle" that interfaces the phone with a silicon-based microfluidic chip embedded within a credit-card-sized cartridge. Utilizing specific nucleic acid sequences for four equine respiratory pathogens as representative examples, we demonstrated the ability of the system to utilize a single 15 μL droplet of test sample to perform selective positive/negative determination of target sequences, including integrated experimental controls, in approximately 30 min. Our approach utilizes loop-mediated isothermal amplification (LAMP) reagents predeposited into distinct lanes of the microfluidic chip, which when exposed to target nucleic acid sequences from the test sample, generates fluorescent products that when excited by appropriately selected light emitting diodes (LEDs), are visualized and automatically analyzed by a software application running on the smartphone microprocessor. The system achieves detection limits comparable to those obtained by laboratory-based methods and instruments. Assay information is combined with the information from the cartridge and the patient to populate a cloud-based database for epidemiological reporting of test results.

  10. Highly-sensitive microRNA detection based on bio-bar-code assay and catalytic hairpin assembly two-stage amplification.

    Science.gov (United States)

    Tang, Songsong; Gu, Yuan; Lu, Huiting; Dong, Haifeng; Zhang, Kai; Dai, Wenhao; Meng, Xiangdan; Yang, Fan; Zhang, Xueji

    2018-04-03

    Herein, a highly-sensitive microRNA (miRNA) detection strategy was developed by combining bio-bar-code assay (BBA) with catalytic hairpin assembly (CHA). In the proposed system, two nanoprobes of magnetic nanoparticles functionalized with DNA probes (MNPs-DNA) and gold nanoparticles with numerous barcode DNA (AuNPs-DNA) were designed. In the presence of target miRNA, the MNP-DNA and AuNP-DNA hybridized with target miRNA to form a "sandwich" structure. After "sandwich" structures were separated from the solution by the magnetic field and dehybridized by high temperature, the barcode DNA sequences were released by dissolving AuNPs. The released barcode DNA sequences triggered the toehold strand displacement assembly of two hairpin probes, leading to recycle of barcode DNA sequences and producing numerous fluorescent CHA products for miRNA detection. Under the optimal experimental conditions, the proposed two-stage amplification system could sensitively detect target miRNA ranging from 10 pM to 10 aM with a limit of detection (LOD) down to 97.9 zM. It displayed good capability to discriminate single base and three bases mismatch due to the unique sandwich structure. Notably, it presented good feasibility for selective multiplexed detection of various combinations of synthetic miRNA sequences and miRNAs extracted from different cell lysates, which were in agreement with the traditional polymerase chain reaction analysis. The two-stage amplification strategy may be significant implication in the biological detection and clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. A Modified Hydroxyproline Assay Based on Hydrochloric Acid in Ehrlich's Solution Accurately Measures Tissue Collagen Content.

    Science.gov (United States)

    Cissell, Derek D; Link, Jarrett M; Hu, Jerry C; Athanasiou, Kyriacos A

    2017-04-01

    Collagen quantification has long been relevant to biomedical research and clinical practice to characterize tissues and determine disease states. The hydroxyproline assay, while a broadly employed method of quantifying collagen, uses perchloric acid to dissolve Ehrlich's reagent. Since perchloric acid poses occupational safety hazards and high costs, in this study, a new hydroxyproline assay was developed that replaces perchloric acid with a relatively safer and cheaper alternative, hydrochloric acid (HCl). To validate this biochemical technique, first, using either acid to dissolve Ehrlich's reagent, the assays were completed for native and engineered collagenous tissues. No statistical differences were identified between the assays (p = 0.32). Subsequently, both biochemical techniques were compared to amino acid analysis, considered a proteomics gold standard. Interestingly, utilizing HCl in lieu of perchloric acid yielded greater concordance with amino acid analysis (ρc = 0.980) than did the traditional assay (ρc = 0.947); that is, the HCl-based assay more closely estimates hydroxyproline content, and, consequently, true collagen content. Thus, using Ehrlich's reagent containing HCl in the hydroxyproline assay represents an advance in both mitigating laboratory safety hazards and improving biochemical collagen quantification.

  12. Multiplex ligation-dependent probe amplification assay identifies additional copy number changes compared with R-band karyotype and provide more accuracy prognostic information in myelodysplastic syndromes.

    Science.gov (United States)

    Wang, Jingya; Ai, Xiaofei; Qin, Tiejun; Xu, Zefeng; Zhang, Yue; Liu, Jinqin; Li, Bing; Fang, Liwei; Zhang, Hongli; Pan, Lijuan; Hu, Naibo; Qu, Shiqiang; Cai, Wenyu; Ru, Kun; Jia, Yujiao; Huang, Gang; Xiao, Zhijian

    2017-01-03

    Cytogenetic analysis provides important diagnostic and prognostic information for patients with Myelodysplastic syndromes (MDS) and plays an essential role in the International Prognostic Scoring System (IPSS) and the revised International Prognostic Scoring System (IPSS-R). Multiplex ligation-dependent probe amplification (MLPA) assay is a recently developed technique to identify targeted cytogenetic aberrations in MDS patients. In the present study, we evaluated the results obtained using an MLPA assay in 437 patients with MDS to determine the efficacy of MLPA analysis. Using R-banding karyotyping, 45% (197/437) of MDS patients had chromosomal abnormalities, whereas MLPA analysis detected that 35% (153/437) of MDS cases contained at least one copy-number variations (CNVs) .2/5 individuals (40%) with R-band karyotype failures had trisomy 8 detected using only MLPA. Clonal cytogenetic abnormalities were detected in 20/235 (8.5%) MDS patients with a normal R-band karyotype, and 12/20 (60%) of those patients were reclassified into a higher-risk IPSS-R prognostic category. When sequencing and cytogenetics were combined, the fraction of patients with MDS-related oncogenic lesions increased to 87.3% (233/267 cases). MLPA analysis determined that the median OS of patients with a normal karyotype (n=218) was 65 months compared with 27 months in cases with an aberrant karyotype (P=0.002) in 240 patients with normal or failed karyotypes by R-banding karyotyping. The high-resolution MPLA assay is an efficient and reliable method that can be used in conjunction with R-band karyotyping to detect chromosomal abnormalities in patients with suspected MDS. MLPA may also provide more accurate prognostic information.

  13. Multiplex ligation-dependent probe amplification assay identifies additional copy number changes compared with R-band karyotype and provide more accuracy prognostic information in myelodysplastic syndromes

    Science.gov (United States)

    Xu, Zefeng; Zhang, Yue; Liu, Jinqin; Li, Bing; Fang, Liwei; Zhang, Hongli; Pan, Lijuan; Hu, Naibo; Qu, Shiqiang; Cai, Wenyu; Ru, Kun; Jia, Yujiao; Huang, Gang; Xiao, Zhijian

    2017-01-01

    Cytogenetic analysis provides important diagnostic and prognostic information for patients with Myelodysplastic syndromes (MDS) and plays an essential role in the International Prognostic Scoring System (IPSS) and the revised International Prognostic Scoring System (IPSS-R). Multiplex ligation-dependent probe amplification (MLPA) assay is a recently developed technique to identify targeted cytogenetic aberrations in MDS patients. In the present study, we evaluated the results obtained using an MLPA assay in 437 patients with MDS to determine the efficacy of MLPA analysis. Using R-banding karyotyping, 45% (197/437) of MDS patients had chromosomal abnormalities, whereas MLPA analysis detected that 35% (153/437) of MDS cases contained at least one copy-number variations (CNVs) .2/5 individuals (40%) with R-band karyotype failures had trisomy 8 detected using only MLPA. Clonal cytogenetic abnormalities were detected in 20/235 (8.5%) MDS patients with a normal R-band karyotype, and 12/20 (60%) of those patients were reclassified into a higher-risk IPSS-R prognostic category. When sequencing and cytogenetics were combined, the fraction of patients with MDS-related oncogenic lesions increased to 87.3% (233/267 cases). MLPA analysis determined that the median OS of patients with a normal karyotype (n=218) was 65 months compared with 27 months in cases with an aberrant karyotype (P=0.002) in 240 patients with normal or failed karyotypes by R-banding karyotyping. The high-resolution MPLA assay is an efficient and reliable method that can be used in conjunction with R-band karyotyping to detect chromosomal abnormalities in patients with suspected MDS. MLPA may also provide more accurate prognostic information. PMID:27906673

  14. One-step nucleic acid amplification: the possible value in assessing sentinel lymph node metastasis during mastectomy

    Directory of Open Access Journals (Sweden)

    Hunter-Smith AE

    2018-01-01

    Full Text Available Alison E Hunter-Smith, Zenon Rayter Breast Surgery Unit, Bristol Breast Care Centre, North Bristol NHS Trust, Southmead Hospital, Westbury-on-Trym, Bristol, UK Abstract: Breast cancer is the most common cancer in women, worldwide, and 1,400 deaths per day are attributed to it. The success of national screening programs has seen breast cancers being diagnosed at an earlier stage. With conservative surgery to the breast demonstrating equivalent long-term outcomes, the last 10 years have seen a growing interest in the safety of less invasive management for the axilla in breast cancer patients. One-step nucleic acid amplification (OSNA is a validated, reliable, and efficient tool in identifying micro- and macro-metastases intraoperatively. It is the most widely used intraoperative analysis tool within the United Kingdom, and is employed by over 320 units across Europe and Asia. Recent evidence from the AMAROS, IBCSG 23-01, and ACOSOG Z0011 trials has changed surgical practice in managing the axilla of patients with breast cancer. We propose a clinical algorithm demonstrating the role of OSNA as an intraoperative analysis tool in today’s management of breast cancer as well as prospects for the future use of OSNA. Keywords: breast cancer, sentinel lymph node, intraoperative assessment, one-stop nucleic acid amplification, mastectomy

  15. Comparison of a new visual isothermal nucleic acid amplification test with PCR and skin snip analysis for diagnosis of onchocerciasis in humans.

    Science.gov (United States)

    Alhassan, Andy; Osei-Atweneboana, Mike Y; Kyeremeh, Kwadwo F; Poole, Catherine B; Li, Zhiru; Tettevi, Edward; Tanner, Nathan A; Carlow, Clotilde K S

    Accurate, simple and affordable diagnostics are needed to detect Onchocerca volvulus infection in humans. A newly developed colorimetric loop-mediated isothermal amplification (LAMP) assay was compared to PCR and skin snip analysis for diagnosis of onchocerciasis. The robustness and simplicity of the assay indicates that it may be a useful field tool for surveillance in endemic countries. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

  16. Evaluation of an individual-donation nucleic acid amplification testing algorithm for detecting hepatitis B virus infection in Chinese blood donors.

    Science.gov (United States)

    Gou, Hongna; Pan, Yang; Ge, Hongwei; Zheng, Yourong; Wu, Yaling; Zeng, Jinfeng; Yang, Zhongsi; Pan, Tong; Cun, Wei; Zhou, Guoping; Fang, Gen; Zhang, Jiahong; Zhang, Kuo; Zhang, Rui; Sun, Yu; Xie, Jiehong; Li, Jinming; Wang, Lunan

    2015-09-01

    This multicenter study was performed to evaluate the efficiency of a multiplex individual-donation nucleic acid amplification technology (ID-NAT) and discriminatory testing algorithm for detecting hepatitis B virus (HBV) infection in Chinese blood donors. A total of 1,205,796 hepatitis B surface antigen (HBsAg)-nonreactive donations from 10 blood centers were tested by ID-NAT using the Ultrio assay. Multiplex Ultrio-reactive donations were tested in the discriminatory tests as well as in quantitative polymerase chain reaction (qPCR) and in supplemental electrochemiluminescence immunoassays for HBsAg, hepatitis B surface antibody (anti-HBs), hepatitis B e antigen, and antibody to hepatitis B core antigen (anti-HBc). Meanwhile, a control group of 4317 Ultrio-nonreactive donations was tested for anti-HBc and anti-HBs. Of all donations, 2033 (0.17%) were reactive in the multiplex Ultrio assay. Among 1776 further tested samples, 548 (30.9%) were HBV discriminatory assay (dHBV)-reactive, while 1214 (68.4%) were nonreactive. Of 472 Ultrio+ and dHBV+ samples 86.2% were qPCR positive compared to 15.0% in 1046 Ultrio+ and dHBV- samples. The proportion of anti-HBc+ and anti-HBs- (potentially infectious) donations was higher in 409 Ultrio+ and dHBV+ than in 1028 Ultrio+ and dHBV- samples (51.3% vs. 31.1%, p occult carriers. Although this has no implication for blood safety, the testing algorithm needs to be redesigned to more efficiently discriminate between true and false NAT reactivity. © 2015 AABB.

  17. Chip-based device for parallel sorting, amplification, detection, and identification of nucleic acid subsequences

    Energy Technology Data Exchange (ETDEWEB)

    Beer, Neil Reginald; Colston, Jr, Billy W.

    2016-08-09

    An apparatus for chip-based sorting, amplification, detection, and identification of a sample having a planar substrate. The planar substrate is divided into cells. The cells are arranged on the planar substrate in rows and columns. Electrodes are located in the cells. A micro-reactor maker produces micro-reactors containing the sample. The micro-reactor maker is positioned to deliver the micro-reactors to the planar substrate. A microprocessor is connected to the electrodes for manipulating the micro-reactors on the planar substrate. A detector is positioned to interrogate the sample contained in the micro-reactors.

  18. A novel non-amplification assay for the detection of Leishmania spp. in clinical samples using gold nanoparticles.

    Science.gov (United States)

    Andreadou, Margarita; Liandris, Emmanouil; Gazouli, Maria; Taka, Styliani; Antoniou, Maria; Theodoropoulos, Georgios; Tachtsidis, Ilias; Goutas, Nikolaοs; Vlachodimitropoulos, Dimitrios; Kasampalidis, Ioannis; Ikonomopoulos, John

    2014-01-01

    Leishmaniosis is a zoonose caused by protozoans of the genus Leishmania. The need for accurate diagnostic investigation of cases of leishmaniosis has rendered today the use of molecular biology techniques broadly applicable. However, the reliable application of these methods requires highly-specialised personnel, dedicated equipment and space. The aim of this study was the design and construction of functionalized gold nanoparticles (AuNPs) that would be incorporated into an easily applicable DNA detection methodology for the identification of Leishmania spp. in clinical samples. AuNPs 20nm in diameter were conjugated with four oligonucleotide probes, targeting kinetoplastid minicircle DNA of Leishmania spp. In the absence of complimentary DNA, AuNPs-probes precipitate under acid environment causing a change of color from red to purple, which can be detected by visual observation. In the presence of target DNA the color of the solution remains red. The specific methodology was applied to positive and negative control samples and whole blood collected from dogs with suspected canine leishmaniosis. The method's minimum detection limit was defined to 11.5ng of target DNA per μl of sample. Repeatability and reproducibility were 100%. Relative sensitivity and specificity referenced to PCR were calculated to 92% and 100% regarding collectively control and clinical samples. The proposed approach can be considered an appealing diagnostic solution especially for screening purposes in enzootic areas, where detection of very small amounts of the targeted analyte is not top priority. © 2013.

  19. Visual Detection of Canine Parvovirus Based on Loop-Mediated Isothermal Amplification Combined with Enzyme-Linked Immunosorbent Assay and with Lateral Flow Dipstick

    Science.gov (United States)

    SUN, Yu-Ling; YEN, Chon-Ho; TU, Ching-Fu

    2013-01-01

    ABSTRACT Loop-mediated isothermal amplification (LAMP) combined with enzyme-linked immunosorbent assay (LAMP–ELISA) and with lateral flow dipstick (LAMP–LFD) are rapid, sensitive and specific methods for the visual detection of clinical pathogens. In this study, LAMP–ELISA and LAMP–LFD were developed for the visual detection of canine parvovirus (CPV). For LAMP, a set of four primers (biotin-labeled forward inner primers) was designed to specifically amplify a region of the VP2 gene of CPV. The optimum time and temperature for LAMP were 60 min and 65°C, respectively. The specific capture oligonucleotide probes, biotin-labeled CPV probe for LAMP–ELISA and fluorescein isothiocyanate-labeled CPV probe for LAMP–LFD were also designed for hybridization with LAMP amplicons on streptavidin-coated wells and LFD strips, respectively. For the comparison of detection sensitivity, conventional PCR and LAMP for CPV detection were also performed. The CPV detection limits by PCR, PCR–ELISA, LAMP, LAMP–ELISA and LAMP–LFD were 102, 102, 10−1, 10−1 and 10−1 TCID50/ml, respectively. In tests using artificially contaminated dog fecal samples, the samples with CPV inoculation levels of ≥1 TCID50/ml gave positive results by both LAMP–ELISA and LAMP–LFD. Our data indicated that both LAMP–ELISA and LAMP–LFD are promising as rapid, sensitive and specific methods for an efficient diagnosis of CPV infection. PMID:24334855

  20. Evaluation of three enzyme immunoassays and a nucleic acid amplification test for the diagnosis of Clostridium difficile-associated diarrhea at a university hospital in Brazil

    Directory of Open Access Journals (Sweden)

    Rodrigo Otávio Silveira Silva

    2014-07-01

    Full Text Available Introduction Despite the known importance of Clostridium difficile as a nosocomial pathogen, few studies regarding Clostridium difficile infection (CDI in Brazil have been conducted. To date, the diagnostic tests that are available on the Brazilian market for the diagnosis of CDI have not been evaluated. The aim of this study was to compare the performances of four commercial methods for the diagnosis of CDI in patients from a university hospital in Brazil. Methods Three enzyme immunoassays (EIAs and one nucleic acid amplification test (NAAT were evaluated against a cytotoxicity assay (CTA and toxigenic culture (TC. Stool samples from 92 patients with suspected CDI were used in this study. Results Twenty-five (27.2% of 92 samples were positive according to the CTA, and 23 (25% were positive according to the TC. All EIAs and the NAAT test demonstrated sensitivities between 59 and 68% and specificities greater than 91%. Conclusions All four methods exhibited low sensitivities for the diagnosis of CDI, which could lead to a large number of false-negative results, an increased risk of cross-infection to other patients, and overtreatment with empirical antibiotics.

  1. Standardization of CalyculinA induced PCC assay and its advantages over Okadaic acid PCC assay in Biodosimetry applications.

    Science.gov (United States)

    Nairy, Rajesha K; Yerol, Narayana; Bhat, Nagesh N; Desai, Utkarsha; Shirsath, Kapil; Yadav, Usha; K Chaurasia, Rajesh; B K, Sapra

    2016-11-29

    In the present study an attempt was made to estimate coefficients of dose response curves for PCC aberrations induced by CalyculinA and Okadaic acid, using (60)Co gamma radiation and 8 MeV pulsed electron beam for biodosimetry application. The modified method outlined by Puig et al. 2013 was used to conduct Calyculin A and Okadaic acid induced PCC assay in human blood lymphocytes.Chemical treatment was given for the last 1 h of a 48 h culture. The study was carried out in the dose range 2.5 to 20 Gy using (60)Co gamma rays and 8 MeV pulsed electron beam. Results show a linear dose dependent increase with a slope of 0.047 ± 0.001 from Calycalin A PCC and 0.048 ± 0.002 form Okadaic acid PCC. The slope of the fragments curve was 0.327 ± 0.006 from Calyculin A and 0.328 ± 0.006 from Okadaic acid PCC. Further, dose calibration studies were carried out for 8 MeV electron using Calyculin A PCC assay and the obtained slope from ring yield was 0.054 ± 0.002 and 0.427 ± 0.009 from fragment yield.

  2. Mycobacterium tuberculosis nucleic acid amplification tests reduce nosocomial tuberculosis exposure in intensive care units: A nationwide cohort study.

    Science.gov (United States)

    Wang, Jann-Yuan; Lee, Ming-Chia; Chang, Jer-Hwa; Yu, Ming-Chih; Wu, Vin-Cent; Huang, Kuo-Liang; Su, Chiu-Ping; Chao, Kun-Mao; Lee, Chih-Hsin

    2015-11-01

    This retrospective national surveillance study investigated the burden of and risk factors for nosocomial exposure of pulmonary tuberculosis (TB) in intensive care units. Patients admitted to intensive care units were identified from the National Health Insurance Research Database. During 2004-2009, there were 1 387 707 intensive care unit admissions of 900 562 adult patients. Pulmonary tuberculosis association was considered if the patient was diagnosed with pulmonary tuberculosis during admission or within 3 months after discharge. Nosocomial transmissible period was calculated based on the length of anti-tuberculosis treatment and negative-pressure isolation during admission. Pulmonary tuberculosis was associated with 1.20% of all intensive care unit admissions and 6731 (38.9%) started anti-TB treatment during admission. For the other 10 583 admissions, the diagnosis was made after discharge and anti-TB treatment was not prescribed during admission. The probability paralleled the regional tuberculosis incidence. On average, 2794 pulmonary tuberculosis associated intensive care unit admissions contributed to 42 999-44 062 days of nosocomial exposure per year. The length of nosocomial transmissible period decreased with the gradual implementation of Mycobacterium tuberculosis nucleic acid amplification tests in intensive care practice. Multivariate linear regression analysis revealed that the length of nosocomial transmissible period was inversely associated with male gender, airway symptoms prior to admission and performing M. tuberculosis nucleic acid amplification tests and mycobacterial culture. Nosocomial tuberculosis exposure is not uncommon in intensive care units. Performing rapid molecular diagnostic tests in those suspected of tuberculosis is recommended to reduce the risk of nosocomial exposure. © 2015 The Authors. Respirology published by Wiley Publishing Asia Pty Ltd on behalf of Asian Pacific Society of Respirology.

  3. Integration of nucleic acid amplification test results into hepatitis C virus supplemental serologic testing algorithms: implications for donor counseling and revision of existing algorithms.

    Science.gov (United States)

    Kleinman, Steven H; Stramer, Susan L; Brodsky, Jaye P; Caglioti, Sally; Busch, Michael P

    2006-05-01

    The routine use of hepatitis C virus (HCV) nucleic acid amplification testing (NAT) donor screening assays has provided an opportunity for revision of the current HCV supplemental testing algorithm, which requires that recombinant immunoblot assay (RIBA) be performed on every HCV enzyme immunoassay (EIA)-repeat-reactive donation. The FDA has approved variance requests to use a new algorithm that eliminates the need to perform RIBA when HCV NAT results are reactive. Data are provided in support of this new algorithm. HCV EIA (including signal-to-cutoff optical density ratio), RIBA, and NAT data were compiled from 33.2 million donations screened over an approximately 4-year period by the American Red Cross and Blood Systems Laboratories. Further, donations having specific combinations of HCV EIA, RIBA, and minipool (MP) NAT results were evaluated, with more sensitive individual-donation (ID) NAT, to construct improved counseling messages for donors. Of 47,041 EIA-repeat-reactive donations, 49.3 percent were RIBA-positive, 17.1 percent RIBA-indeterminate, and 33.5 percent RIBA-negative. NAT-reactive rates were 79.2, 2.5, and 0.18 percent for RIBA-positive, -indeterminate, and -negative donations, respectively. The new algorithm classified an additional 1 percent of donations as HCV-infected while at the same time detecting all infections classified as HCV-infected under the current algorithm. An additional 2.4 percent of RIBA-positive, MP NAT-nonreactive donations were reactive when a frozen-thawed aliquot was retested by ID NAT. Integrating HCV NAT results with RIBA results for purposes of donor notification allows more appropriate counseling messages to be given to EIA-repeat-reactive donors. The new HCV supplemental algorithm is an acceptable alternative to the current algorithm because it provides equivalent or superior accuracy in formulating donor counseling messages and may also result in reduced costs and more timely notification of infected donors.

  4. Detection of Staphylococcus epidermidis by a Quartz Crystal Microbalance Nucleic Acid Biosensor Array Using Au Nanoparticle Signal Amplification

    Directory of Open Access Journals (Sweden)

    Weiling Fu

    2008-10-01

    Full Text Available Staphylococcus epidermidis is a critical pathogen of nosocomial blood infections, resulting in significant morbidity and mortality. A piezoelectric quartz crystal microbalance (QCM nucleic acid biosensor array using Au nanoparticle signal amplification was developed to rapidly detect S. epidermidis in clinical samples. The synthesized thiolated probes specific targeting S. epidermidis 16S rRNA gene were immobilized on the surface of QCM nucleic acid biosensor arrays. Hybridization was induced by exposing the immobilized probes to the PCR amplified fragments of S. epidermidis, resulting in a mass change and a consequent frequency shift of the QCM biosensor. To further enhance frequency shift results from above described hybridizations, streptavidin coated Au nanoparticles were conjugated to the PCR amplified fragments. The results showed that the lowest detection limit of current QCM system was 1.3×103 CFU/mL. A linear correlation was found when the concentration of S. epidermidis varied from 1.3×103 to 1.3×107 CFU/mL. In addition, 55 clinical samples were detected with both current QCM biosensor system and conventional clinical microbiological method, and the sensitivity and specificity of current QCM biosensor system were 97.14% and 100%, respectively. In conclusion, the current QCM system is a rapid, low-cost and sensitive method that can be used to identify infection of S. epidermidis in clinical samples.

  5. Melamine and Cyanuric Acid do not interfere with Bradford and Ninhydrin assays for protein determination

    Science.gov (United States)

    Field, Anjalie; Field, Jeffrey

    2010-01-01

    In the fall of 2007 pet food contaminated with melamine and cyanuric acid caused kidney stones in thousands of animals. In the summer of 2008, a more serious outbreak of adulterated dairy food caused the deaths of six infants and sickened about 290,000 children in China. In all cases, melamine was likely added to inflate the apparent protein content of the foods. To determine if we could measure protein without interference from melamine and cyanuric acid we tested these compounds in the Bradford and Ninhydrin assays, two common dye-based assays for protein, as well as by ammonia release, the most common assay used in the food industry. Neither compound was detected in the Ninhydrin and Bradford assays at concentrations of >100 μg/ml. The ammonia assay detected melamine but was inconclusive with respect to cyanuric acid. To develop an accurate test for food that would not detect either chemical as a protein, assays were run on cat food and reconstituted milk powder. The Bradford assay readily measured the protein content of each food, and importantly, the addition of melamine or cyanuric acid to reconstituted milk did not affect the readings. The protein concentrations obtained for reconstituted milk powder were as expected, but those for the cat food were 10 to 30-fold lower, due to its low solubility. We conclude that dye-binding assays can be employed to detect protein in food without interference from melamine and cyanuric acid, thus reducing the incentive to use them as additives. PMID:20228949

  6. Development of a Real-Time Fluorescence Loop-Mediated Isothermal Amplification Assay for Rapid and Quantitative Detection of Fusarium oxysporum f. sp. cubense Tropical Race 4 In Soil

    Science.gov (United States)

    Pu, Jinji; Qi, Yanxiang; Yu, Qunfang; Xie, Yixian; Peng, Jun

    2013-01-01

    Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 103 spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China. PMID:24376590

  7. Loop-mediated isothermal amplification using self-avoiding molecular recognition systems and antarctic thermal sensitive uracil-DNA-glycosylase for detection of nucleic acid with prevention of carryover contamination.

    Science.gov (United States)

    Wang, Yi; Liu, Dongxin; Deng, Jianping; Wang, Yan; Xu, Jianguo; Ye, Changyun

    2017-12-15

    Loop-mediated isothermal amplification (LAMP) is the most popular technique to amplify nucleic acid sequence without the use of temperature cycling. However, LAMP is often confounded by false-positive results, arising from interactions between (hetero-dimer) or within (self-dimerization) primers, off-target hybrids and carryover contaminants. Here, we devised a new LAMP technique that is self-avoiding molecular recognition system (SAMRS) components and antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) enzyme-assisted, termed AUDG-SAMRS-LAMP. Incorporating SAMRS components into 3'-ends of LAMP primers can improve assay's specificity, which completely prevents the non-specific amplification yielding from off-target hybrids and undesired interactions between or within primers. Adding AUDG into reaction mixtures can effectively eliminate the false-positive results arising from carryover contamination, thus the genuine positive reactions are generated from the amplification of target templates. Furthermore, AUDG-SAMRS-LAMP results are confirmed using a new analysis strategy, which is developed for detecting LAMP amplicons by lateral flow biosensor (LFB). Only a single labeled primer is required in the analysis system, thus the false positive results arising from hybridization (the labeled primer and probe, or between two labeled primers) are avoided. Hence, the SAMRS components, AUDG and LFB convert traditional LAMP from a technique suited for the research laboratory into one that has practical value in the field of diagnosis. Human Tuberculosis (TB) is caused by infection with members of Mycobacterium tuberculosis complex (MTC), which are detected by the AUDG-SAMRS-LAMP technique to demonstrate the availability of target analysis. The proof-of-concept method can be reconfigured to detect various nucleic acids by redesigning the specific primers. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Usefulness of quantitative nucleic Acid sequence-based amplification for diagnosis of malaria in an academic hospital setting.

    Science.gov (United States)

    Schallig, H D F H; Schoone, G J; Lommerse, E J M; Kroon, C C M; de Vries, P J; van Gool, T

    2003-09-01

    The aim of the present study was to evaluate the usefulness of quantitative nucleic acid sequence-based amplification (QT-NASBA) to detect Plasmodium spp. in diagnostic specimens of patients suspected of having malaria in a clinical setting in a non-endemic country. During the 4-month recruitment period, 113 patients were enrolled in the study, of which 93 were diagnosed as non-malaria and 20 as malaria cases on the basis of clinical and microscopic criteria. All microscopically positive cases had QT-NASBA counts of >0.1 parasites/ micro l and there was a significant positive correlation between the parasite counts obtained with both diagnostic methods. Of the 93 microscopically negative cases, six had a positive QT-NASBA result. Three of these cases had a recent history of malaria for which specific treatment was taken. In the other three cases there was no history of malaria and QT-NASBA results in these cases were near the cut-off level (>0.1 parasites/ micro l) of the test. The results demonstrate that QT-NASBA is a useful technology for the diagnosis of malaria in a reference laboratory, and it is very helpful in cases of low parasitemia.

  9. Detecting asymptomatic Trichomonas vaginalis in females using the BD ProbeTec™ Trichomonas vaginalis Qx nucleic acid amplification test.

    Science.gov (United States)

    Lord, Emily; Newnham, Tana; Dorrell, Lucy; Jesuthasan, Gerald; Clarke, Lorraine; Jeffery, Katie; Sherrard, Jackie

    2017-03-01

    Trichomonas vaginalis (TV) rates in women are increasing and many are asymptomatic. Nucleic acid amplification tests (NAATs) are becoming the 'gold standard' for diagnosis. We aimed to establish our asymptomatic TV rates by testing all women attending Oxfordshire's Sexual Health service, regardless of symptoms, using the BD ProbeTec™ TV Qx NAATs (BDQx). During BDQx's verification process, the sensitivity and specificity were calculated using results of 220 endocervical samples from symptomatic women, compared with culture. BDQx was subsequently implemented and prospectively evaluated over 6 months in female attendees. Wet mount microscopy was also performed in symptomatics. Demographic and clinical characteristics of those diagnosed were analysed. From 220 samples tested by BDQx and culture: 5 were positive on both and one solely using BDQx, giving a sensitivity and specificity of 100% and 99.53%, respectively. In the prospective cohort, of 5775 BDQx tests, 33 (0.57%) were positive. 11/33 (33%) patients were asymptomatic. All patients diagnosed had risk factors: age >25 years (85%), residence in a deprived area (79%) and black ethnicity (21%). Despite BDQx being highly sensitive and specific, with our low TV prevalence universal screening may not be justified. Targeted screening using local demographic data merits further investigation.

  10. Tumor acidity-activatable manganese phosphate nanoplatform for amplification of photodynamic cancer therapy and magnetic resonance imaging.

    Science.gov (United States)

    Hao, Yongwei; Zheng, Cuixia; Wang, Lei; Zhang, Jinjie; Niu, Xiuxiu; Song, Qingling; Feng, Qianhua; Zhao, Hongjuan; Li, Li; Zhang, Hongling; Zhang, Zhenzhong; Zhang, Yun

    2017-10-15

    Amorphous biodegradable metal phosphate nanomaterials are considered to possess great potential in cancer theranostic application due to their promise in providing ultra-sensitive pH-responsive therapeutic benefits and diagnostic functions simultaneously. Here we report the synthesis of photosensitising and acriflavine-carrying amorphous porous manganese phosphate (PMP) nanoparticles with ultra-sensitive pH-responsive degradability and their application for a photoactivable synergistic nanosystem that imparts reactive oxygen species (ROS) induced cytotoxicity in synchrony with hypoxia-inducible factor 1α/vascular endothelial growth factor (HIF1α/VEGF) inhibitor that suppresses tumor growth and treatment escape signalling pathway. Carboxymethyl dextran (CMD) is chemically anchored on the surface of porous manganese phosphate theranostic system through the pH-responsive boronate esters. Upon the stimulus of the tumor acid microenvironment, manganese phosphate disintegrates and releases Mn 2+ ions rapidly, which are responsible for the magnetic resonance imaging (MRI) effect. Meanwhile, the released photosensitizer chlorin e6 (Ce6) produces ROS under irradiation while acriflavine (ACF) inhibits the HIF-1α/VEGF pathway during the burst release of VEGF in tumour induced by photodynamic therapy (PDT), resulting in increased therapeutic efficacy. Considering the strong pH responsivity, MRI signal amplification and drug release profile, the PMP nanoparticles offer new prospects for tumor acidity-activatable theranostic application by amplifying the PDT through inhibiting the HIF-1α /VEGF pathway timely while enhancing the MRI effect. In this study, we report the synthesis of the tumor acidity-activatable amorphous porous manganese phosphate nanoparticles and their application for a photoactivable synergistic nanosystem that imparts reactive oxygen species (ROS) induced cytotoxicity in synchrony with hypoxia-inducible factor 1α/vascular endothelial growth factor (HIF-1

  11. Development of fluorescent nanoparticle-labeled lateral flow assay for the detection of nucleic acids.

    Science.gov (United States)

    Wang, Yuhong; Nugen, Sam R

    2013-10-01

    The rapid, specific and sensitive detection of nucleic acids is of utmost importance for the identification of infectious agents, diagnosis and treatment of genetic diseases, and the detection of pathogens related to human health and safety. Here we report the development of a simple and sensitive nucleic acid sequence-based and Ru(bpy)3 (2+)-doped silica nanoparticle-labeled lateral flow assay which achieves low limit of detection by using fluorescencent nanoparticles. The detection of the synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, was utilized to demonstrate this assay. The 30 nm spherical Ru(bpy)3 (2+)-doped silica nanoparticles were prepared in aqueous medium by a novel method recently reported. The nanoparticles were modified by 3-glycidoxypropyl trimethoxysilane in order to conjugate to amine-capped oligonucleotide reporter probes. The fluorescent intensities of the fluorescent assays were quantified on a mictrotiter plate reader using a custom holder. The experimental results showed that the lateral flow fluorescent assay developed was more sensitive compared with the traditional colloidal gold test strips. The limit of detection for the fluorescent lateral flow assay developed is approximately 0.066 fmols as compared to approximately 15 fmols for the colloidal gold. The limit of detection can further be reduced about one order of magnitude when "dipstick" format was used.

  12. Analysis of Citric Acid in Beverages: Use of an Indicator Displacement Assay

    Science.gov (United States)

    Umali, Alona P.; Anslyn, Eric V.; Wright, Aaron T.; Blieden, Clifford R.; Smith, Carolyne K.; Tian, Tian; Truong, Jennifer A.; Crumm, Caitlin E.; Garcia, Jorge E.; Lee, Soal; Mosier, Meredith; Nguyen, Chester P.

    2010-01-01

    The use of an indicator displacement assay permits the visualization of binding events between host and guest molecules. An undergraduate laboratory experiment is described to demonstrate the technique in the determination of citric acid content in commercially available beverages such as soda pop and fruit juices. Through the technique, students…

  13. Comment on "Analysis of Citric Acid in Beverages: Use of an Indicator Displacement Assay"

    Science.gov (United States)

    Konski, Krzysiek; Saw, Jessica; Torriero, Angel A. J.

    2017-01-01

    This letter comments on the paper "Analysis of Citric Acid in Beverages: Use of an Indicator Displacement Assay" ["J. Chem. Educ." 2010, 87 (8), 832-835 (EJ918557)]. Discrepancies in figures and host:indicator complex behavior are discussed and an alternative experimental protocol presented.

  14. Development of a dry-reagent-based nucleic acid-sensing platform by coupling thermostabilised LATE-PCR assay to an oligonucleotide-modified lateral flow biosensor.

    Science.gov (United States)

    Ang, Geik Yong; Yu, Choo Yee; Chan, Kok Gan; Singh, Kirnpal Kaur Banga; Chan Yean, Yean

    2015-11-01

    In this study, we report for the first time the development of a dry-reagent-based nucleic acid-sensing platform by combining a thermostabilised linear-after-the-exponential (LATE)-PCR assay with a one-step, hybridisation-based nucleic acid lateral flow biosensor. The nucleic acid-sensing platform was designed to overcome the need for stringent temperature control during transportation or storage of reagents and reduces the dependency on skilled personnel by decreasing the overall assay complexity and hands-on time. The platform was developed using toxigenic Vibrio cholerae as the model organism due to the bacterium's propensity to cause epidemic and pandemic cholera. The biosensor generates result which can be visualised with the naked eyes and the limit of detection was found to be 1pg of pure genomic DNA and 10CFU/ml of toxigenic V. cholerae. The dry-reagent-based nucleic acid-sensing platform was challenged with 95 toxigenic V. cholerae, 7 non-toxigenic V. cholerae and 66 other bacterial strains in spiked stool sample and complete agreement was observed when the results were compared to that of monosialoganglioside (GM1)-ELISA. Heat-stability of the thermostabilised LATE-PCR reaction mixes at different storage temperatures (4-56°C) was investigated for up to 90days. The dry-reagent-based genosensing platform with ready-to-use assay components provides an alternative method for sequence-specific detection of nucleic acid without any cold chain restriction that is associated with conventional molecular amplification techniques. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Comparison of dacron and nylon-flocked self-collected vaginal swabs and urine for the detection of Trichomonas vaginalis using analyte-specific reagents in a transcription-mediated amplification assay.

    Science.gov (United States)

    Jang, Dan; Gilchrist, Jodi; Portillo, Eder; Smieja, Marek; Toor, Ramandeep; Chernesky, Max

    2012-04-01

    To compare self-collected vaginal swab (SCVS) types and first-catch urine (FCU) to diagnose Trichomonas vaginalis using analyte-specific reagents designed to be used in a transcription-mediated amplification assay. A total of 241 women (group A) collected a FCU and a SCVS using a dacron swab (APTIMA collection kit). A second group of 289 women (group B) collected two SCVS using one dacron swab and one nylon-flocked swab. Of 75 young women (street youth) determined to be infected with T vaginalis only seven reported symptoms of vaginal discharge or irritation. Using a cutoff of 50,000 relative light units, the sensitivity and specificity was 97.2% and 97.6%, respectively for dacron SCVS compared with 41.7% and 100% for FCU in group A; 92.3% and 98.8% for dacron SCVS and 92.3% and 99.2% for flocked-nylon SCVS in group B. The assay tested 96 samples in 6 h. Dacron and nylon-flocked SCVS performed equally well and significantly better than FCU using analyte-specific reagents in the APTIMA transcription-mediated amplification assay. Either swab type could be used for self-collection.

  16. Assay of lysergic acid diethylamide and its passage from blood into the perfused cerebral ventricles

    Science.gov (United States)

    Dras̆koci, M.

    1960-01-01

    On the isolated rat uterus, lysergic acid diethylamide had an oxytocic action in a concentration of 2×10-8; in smaller concentrations (10-9 to 10-10), which had no stimulating effect of their own, it potentiated acetylcholine-induced contractions. This potentiating effect was made the basis for assaying minute amounts of lysergic acid diethylamide. The method was used to assay this substance in plasma of cats during its intravenous infusion at a rate of 10 μg./min./kg. During these infusions 0.4 to 2 ng./min. of lysergic acid diethylamide passed into the cerebral ventricles perfused with a salt solution of a composition resembling that of cerebrospinal fluid. PMID:13818017

  17. Quantitative real-time PCR method with internal amplification control to quantify cyclopiazonic acid producing molds in foods.

    Science.gov (United States)

    Rodríguez, Alicia; Werning, María L; Rodríguez, Mar; Bermúdez, Elena; Córdoba, Juan J

    2012-12-01

    A quantitative TaqMan real-time PCR (qPCR) method that includes an internal amplification control (IAC) to quantify cyclopiazonic acid (CPA)-producing molds in foods has been developed. A specific primer pair (dmaTF/dmaTR) and a TaqMan probe (dmaTp) were designed on the basis of dmaT gene which encodes the enzyme dimethylallyl tryptophan synthase involved in the biosynthesis of CPA. The IAC consisted of a 105 bp chimeric DNA fragment containing a region of the hly gene of Listeria monocytogenes. Thirty-two mold reference strains representing CPA producers and non-producers of different mold species were used in this study. All strains were tested for CPA production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the designed qPCR method was demonstrated by the high linear relationship of the standard curves relating to the dmaT gene copy numbers and the Ct values obtained from the different CPA producers tested. The ability of the qPCR protocol to quantify CPA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1-4 log cfu/g in the different food matrices. The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g. This qPCR protocol including an IAC showed good efficiency to quantify CPA-producing molds in naturally contaminated foods avoiding false negative results. This method could be used to monitor the CPA producers in the HACCP programs to prevent the risk of CPA formation throughout the food chain. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Ultrasensitive analysis of glucose in serum by capillary electrophoresis with LIF detection in combination with signal amplification strategies and on-column enzymatic assay.

    Science.gov (United States)

    Guan, Yueqing; Zhou, Guobin

    2016-03-01

    A highly specific and sensitive method for glucose quantification in human serum samples based on on-column enzymatic assay is described. In this method, the head of the capillary was used as a nanoliter-microreactor, the diluted samples spiked with a novel fluorogenic reagent named 2-[6-(4'-amino) phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF), and the mixed enzyme solutions of glucose oxidase (GOx) and horseradish peroxidase (HRP), were individually injected into the capillary. Hydrogen peroxide (H2 O2 ) generated in situ by catalytic reaction between GOx and glucose, activates APF in the presence of HRP to form a highly fluorescent product, which was electrophoretically separated from the unreacted APF and detected by the LIF detector. The proposed method allowed the determination of glucose down to 10 nM in real samples, with RSD values lower than 3.5%, which also has the potential for measurements of multicomponents in many other systems including measurement of α-glucosidase activity and screening for its inhibitors. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Quantification of biofilm exopolysaccharides using an in situ assay with periodic acid-Schiff reagent.

    Science.gov (United States)

    Randrianjatovo-Gbalou, I; Girbal-Neuhauser, E; Marcato-Romain, C-E

    2016-05-01

    A novel approach to the quantification of extracellular polysaccharides in miniaturized biofilms presenting a wide variety of extracellular matrices was developed. The assay used the periodic acid-Schiff reagent and was first calibrated on dextran and alginate solutions. Then it was implemented on 24-h and 48-h biofilms from three strains known to produce different exopolymeric substances (Pseudomonas aeruginosa, Bacillus licheniformis, Weissella confusa). The assay allowed quantification of the total exopolysaccharides, taking into account possible interferences due to cells or other main expolymers of the matrix (eDNA, proteins). Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Simultaneous high-performance liquid chromatography assay of acetylsalicylic acid and salicylic acid in film-coated aspirin tablets.

    Science.gov (United States)

    Fogel, J; Epstein, P; Chen, P

    1984-12-28

    A reversed-phase high-performance liquid chromatography (HPLC) method has been developed for the simultaneous assay of acetylsalicylic acid (I) and salicylic acid (II) in film-coated aspirin tablets. As little as 0.1% II (relative to I) can be quantitatively determined. Using a 5-microns octadecylsilane column with water-acetonitrile-phosphoric acid (76:24:0.5) as the mobile phase enabled the chromatographic separation to be completed in 4 min. Due to the slow rate of decomposition of I to II in the extraction solvent, acetonitrile-methanol-phosphoric acid (92:8:0.5), the analysis of many samples was routinely performed by means of automated HPLC equipment. Other compounds (non-aspirin salicylates, caffeine and acetaminophen) were also separated by the chromatographic system.

  1. Fluorescence quenching of graphene oxide combined with the site-specific cleavage of restriction endonuclease for deoxyribonucleic acid demethylase activity assay

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Lijuan; Qian, Yingdan; Wu, Ping; Zhang, Hui; Cai, Chenxin, E-mail: cxcai@njnu.edu.cn

    2015-04-15

    Highlights: • An approach for sensitive and selective DNA demethylase activity assay is reported. • This assay is based on the fluorescence quenching of GO and site-specific cleavage of endonuclease. • It can determine as low as 0.05 ng mL{sup −1} of MBD2 with a linear range of 0.2–300 ng mL{sup −1}. • It has an ability to recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. • It can avoid false signals, requiring no bisulfite conversion, PCR amplification, radioisotope-labeling. - Abstract: We report on the development of a sensitive and selective deoxyribonucleic acid (DNA) demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with the site-specific cleavage of HpaII endonuclease to improve the selectivity. This approach was developed by designing a single-stranded probe (P1) that carries a binding region to facilitate the interaction with GO, which induces fluorescence quenching of the labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region, which contains a hemi-methylated site of 5′-CmCGG-3′, to specifically recognize the target (T1, a 32-mer DNA from the promoter region of p53 gene) and hybridize with it to form a P1/T1 duplex. After demethylation with MBD2, the duplex can be specifically cleaved using HpaII, which releases the labeled FAM from the GO surface and results in the recovery of fluorescence. However, this cleavage is blocked by the hemi-methylation of this site. Thus, the magnitude of the recovered fluorescence signal is related to the MBD2 activity, which establishes the basis of the DNA demethylase activity assay. This assay can determine as low as ∼(0.05 ± 0.01) ng mL{sup −1} (at a signal/noise of 3) of MBD2 with a linear range of 0.2–300 ng mL{sup −1} and recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. The advantage of this assay is its ability to avoid false signals and no

  2. Bioprocess monitoring: minimizing sample matrix effects for total protein quantification with bicinchoninic acid assay.

    Science.gov (United States)

    Reichelt, Wieland N; Waldschitz, Daniel; Herwig, Christoph; Neutsch, Lukas

    2016-09-01

    Determining total protein content is a routine operation in many laboratories. Despite substantial work on assay optimization interferences, the widely used bicinchoninic acid (BCA) assay remains widely recognized for its robustness. Especially in the field of bioprocess engineering the inaccuracy caused by interfering substances remains hardly predictable and not well understood. Since the introduction of the assay, sample pre-treatment by trichloroacetic acid (TCA) precipitation has been indicated as necessary and sufficient to minimize interferences. However, the sample matrix in cultivation media is not only highly complex but also dynamically changing over process time in terms of qualitative and quantitative composition. A significant misestimation of the total protein concentration of bioprocess samples is often observed when following standard work-up schemes such as TCA precipitation, indicating that this step alone is not an adequate means to avoid measurement bias. Here, we propose a modification of the BCA assay, which is less influenced by sample complexity. The dynamically changing sample matrix composition of bioprocessing samples impairs the conventional approach of compensating for interfering substances via a static offset. Hence, we evaluated the use of a correction factor based on an internal spike measurement for the respective samples. Using protein spikes, the accuracy of the BCA protein quantification could be improved fivefold, taking the BCA protein quantification to a level of accuracy comparable to other, more expensive methods. This will allow reducing expensive iterations in bioprocess development to due inaccurate total protein analytics.

  3. Rolling circle amplification detection of RNA and DNA

    Science.gov (United States)

    Christian, Allen T.; Pattee, Melissa S.; Attix, Cristina M.; Tucker, James D.

    2004-08-31

    Rolling circle amplification (RCA) has been useful for detecting point mutations in isolated nucleic acids, but its application in cytological preparations has been problematic. By pretreating cells with a combination of restriction enzymes and exonucleases, we demonstrate RCA in solution and in situ to detect gene copy number and single base mutations. It can also detect and quantify transcribed RNA in individual cells, making it a versatile tool for cell-based assays.

  4. [Investigation of reference intervals of blood gas and acid-base analysis assays in China].

    Science.gov (United States)

    Zhang, Lu; Wang, Wei; Wang, Zhiguo

    2015-10-01

    To investigate and analyze the upper and lower limits and their sources of reference intervals in blood gas and acid-base analysis assays. The data of reference intervals were collected, which come from the first run of 2014 External Quality Assessment (EQA) program in blood gas and acid-base analysis assays performed by National Center for Clinical Laboratories (NCCL). All the abnormal values and errors were eliminated. Data statistics was performed by SPSS 13.0 and Excel 2007 referring to upper and lower limits of reference intervals and sources of 7 blood gas and acid-base analysis assays, i.e. pH value, partial pressure of carbon dioxide (PCO2), partial pressure of oxygen (PO2), Na+, K+, Ca2+ and Cl-. Values were further grouped based on instrument system and the difference between each group were analyzed. There were 225 laboratories submitting the information on the reference intervals they had been using. The three main sources of reference intervals were National Guide to Clinical Laboratory Procedures [37.07% (400/1 079)], instructions of instrument manufactures [31.23% (337/1 079)] and instructions of reagent manufactures [23.26% (251/1 079)]. Approximately 35.1% (79/225) of the laboratories had validated the reference intervals they used. The difference of upper and lower limits in most assays among 7 laboratories was moderate, both minimum and maximum (i.e. the upper limits of pH value was 7.00-7.45, the lower limits of Na+ was 130.00-156.00 mmol/L), and mean and median (i.e. the upper limits of K+ was 5.04 mmol/L and 5.10 mmol/L, the upper limits of PCO2 was 45.65 mmHg and 45.00 mmHg, 1 mmHg = 0.133 kPa), as well as the difference in P2.5 and P97.5 between each instrument system group. It was shown by Kruskal-Wallis method that the P values of upper and lower limits of all the parameters were lower than 0.001, expecting the lower limits of Na+ with P value 0.029. It was shown by Mann-Whitney that the statistic differences were found among instrument

  5. Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification

    DEFF Research Database (Denmark)

    Silahtaroglu, Asli; Nolting, Dorrit; Andersen, Lars Dyrskjøt

    2007-01-01

    RNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA)-modified oligonucleotide probes with FISH using the tyramide signal amplification (TSA) technology. Although both approaches have previously...... been shown to increase detection sensitivity in FISH, combining these techniques into one protocol significantly decreases the time needed for miRNA detection in cryosections, while simultaneously retaining high detection sensitivity. Starting with fixation of the tissue sections, this miRNA FISH...

  6. Determination of tenuazonic acid in human urine by means of a stable isotope dilution assay.

    Science.gov (United States)

    Asam, Stefan; Habler, Katharina; Rychlik, Michael

    2013-05-01

    The content of tenuazonic acid in human urine was determined by a stable isotope dilution assay (SIDA) that was recently developed for the analysis of food commodities and extensively re-validated for urine matrix in this study. Linearity of the response curve was proven between molar ratios n(labeled standard)/n(analyte) of 0.02-100. The limits of detection and determination were 0.2 and 0.6 μg/L, respectively. The mean recovery of the stable isotope dilution assay was 102 ± 3 % in the range between 1.0 and 100 μg/L. Interassay precision was 6.7 % (relative standard deviation of three triplicate analyses of a human urine sample during 3 weeks). The method was applied to two studies dealing with urinary excretion of tenuazonic acid: In the first study, tenuazonic acid was quantified in the 24-h urine of six volunteers from Germany (three female, three male) in a concentration range of 1.3-17.3 μg/L or 2.3-10.3 ng/mg(-1) creatinine, respectively. In the second study, two volunteers (one female, one male) ingested 30 μg tenuazonic acid by consumption of naturally contaminated whole meal sorghum infant cereals and tomato juice, respectively. The urinary excretion of the ingested tenuazonic acid was 54-81 % after 6 h, depending on matrix and volunteer. After 24 h, 87-93 % of the ingested amount of tenuazonic acid was excreted, but the fate of the remaining about 10 % is open. Thus, it is not possible to exclude potential health hazards for the consumer, completely.

  7. Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method

    Directory of Open Access Journals (Sweden)

    Wan Zhixiang

    2005-07-01

    Full Text Available Abstract Background Viral hepatitis due to hepatitis B virus and hepatitis C virus are major public health problems all over the world. Traditional detection methods including polymerase chain reaction (PCR-based assays and enzyme-linked immunosorbent assays (ELISA are expensive and time-consuming. In our assay, a protein chip assay using Nano-gold Immunological Amplification and Silver Staining (NIASS method was applied to detect HBV and HCV antibodies rapidly and simultaneously. Methods Chemically modified glass slides were used as solid supports (named chip, on which several antigens, including HBsAg, HBeAg, HBcAg and HCVAg (a mixture of NS3, NS5 and core antigens were immobilized respectively. Colloidal nano-gold labelled staphylococcal protein A (SPA was used as an indicator and immunogold silver staining enhancement technique was applied to amplify the detection signals, producing black image on array spots, which were visible with naked eyes. To determine the detection limit of the protein chip assay, a set of model arrays in which human IgG was spotted were structured and the model arrays were incubated with different concentrations of anti-IgG. A total of 305 serum samples previously characterized with commercial ELISA were divided into 4 groups and tested in this assay. Results We prepared mono-dispersed, spherical nano-gold particles with an average diameter of 15 ± 2 nm. Colloidal nano-gold-SPA particles observed by TEM were well-distributed, maintaining uniform and stable. The optimum silver enhancement time ranged from 8 to 12 minutes. In our assay, the protein chips could detect serum antibodies against HBsAg, HBeAg, HBcAg and HCVAg with the absence of the cross reaction. In the model arrays, the anti-IgG as low as 3 ng/ml could be detected. The data for comparing the protein chip assay with ELISA indicated that no distinct difference (P > 0.05 existed between the results determined by our assay and ELISA respectively. Conclusion

  8. Indirect enzyme-linked immunosorbent assay for the quantitative estimation of lysergic acid diethylamide in urine.

    Science.gov (United States)

    Kerrigan, S; Brooks, D E

    1998-05-01

    A new antibody to lysergic acid diethylamide (LSD) was used to develop a novel indirect ELISA for the quantification of drug in urine. Evaluation of the new assay with the commercially available LSD ELISA (STC Diagnostics) shows improved performance. The test requires 50 microL of urine, which is used to measure concentrations of drug in the microg/L to ng/L range. The limit of detection was 8 ng/L compared with 85 ng/L in the commercial assay, and analytical recoveries were 98-106%. Our test detected 0.1 microg/L of LSD in urine with an intraassay CV of 2.4% (n = 8) compared with 6.0% for a 0.5 microg/L sample in the commercial assay (n = 20). The upper and lower limits of quantification were estimated to be 7 microg/L and 50 ng/L, respectively. Specificity was evaluated by measuring the extent of cross-reactivity with 24 related substances. Drug determination using the new assay offers both improved sensitivity and precision compared with existing methods, thus facilitating the preliminary quantitative estimation of LSD in urine at lower concentrations with a greater degree of certainty.

  9. Influenza and respiratory syncytial virus detection in clinical specimens without nucleic acid extraction using FOCUS direct disc assay is substantially equivalent to the traditional methods and the FOCUS nucleic acid extraction-dependent RT-PCR assay.

    Science.gov (United States)

    Selvaraju, Suresh B; Tierney, David; Leber, Amy L; Patel, Anami; Earley, Amalia K; Jaiswal, Dipeshkumar; Menegus, Marilyn A

    2014-03-01

    In this study, we evaluated FOCUS diagnostic's Flu A/B & RSV direct kit (Direct Disc assay), designed to detect influenza (FLU) and respiratory syncytial viruses (RSV) directly in clinical specimens without nucleic acid extraction. This novel 'sample-to-answer', nucleic acid extraction-independent assay uses a unique disc to process, amplify, and detect viral targets in up to 8 specimens at a time. The performance of this assay for detecting FLU and RSV viruses was compared to the traditional methods (culture and/or direct florescent antibody testing) using 945 nasopharyngeal swab specimens. In addition, a total of 150 consecutive clinical specimens positive for FLU (FLU A=50, FLU B=50) or RSV (n=50) were tested in parallel using the novel Direct Disc assay and FOCUS diagnostic's nucleic acid extraction-dependent assay to assess their relative performance. Compared to the traditional methods, the overall (prospective+retrospective) positive/negative percent agreement was determined to be 96.6%/98.1% for FLU A, 98.4%/99.9% for FLU B, and 99.3%/98.8% for RSV. Compared to the nucleic acid extraction-dependent assay, the positive percent agreement was 90% (n=45/50) for FLU A, 92% (n=46/50) for FLU B, and 98% (n=49/50) for RSV. Overall, the Direct Disc assay showed good agreement with both traditional methods and nucleic acid extraction-dependent assay. Although we encountered some failures compared to the nucleic acid extraction-dependent assay, these limitations must be balanced against the substantial advantages of the extraction-free nature of this assay and rapid turnaround time. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Influence of sample preparation on assay of phenolic acids from eggplant.

    Science.gov (United States)

    Luthria, Devanand L; Mukhopadhyay, Sudarsan

    2006-01-11

    Sample preparation is often overlooked and is frequently considered as "a means to an end". This systematic study with a phenolic-enriched substrate, eggplant (Solanum melongena L.), was undertaken to evaluate the substantial variations in the extraction techniques, solvents, and parameters as described in the published literature. Direct comparison of over 10 extraction procedures or conditions was performed to show the importance and influence of sample preparation on the assay of phenolic compounds. Chlorogenic acid (CA) was the most abundant phenolic acid accounting for >75% of the total phenolic acids content extracted from the eggplant sample. Optimum extraction of CA and total phenolics (TP) from Black Bell cultivar of eggplant were obtained when extractions were performed with a mixture of MeOH/H2O at a ratio of 80:20% v/v using a pressurized liquid extractor (PLE) at 100 degrees C. The amount of CA and TP extracted from eggplant by the previously reported procedures using a wrist shaker, rotary shaker, stirring, sonication, or reflux with different extraction solvents (acetone or varying composition of MeOH/H2O solvent mixtures) varied significantly between 5 and 95% as compared to PLE. The predominant phenolic acids in the free phenolic acid fraction of Black Beauty cultivar of eggplant were CA isomers. However, caffeic acid isomers were the major phenolic acids extracted from the base-hydrolyzed fraction. The total amount of caffeic acid extracted from the Italian Neon cultivar was more that twice that of four other eggplant cultivars (Orient Express, Calliope Zebra Stripe, Orient Charm Neon, and Black Beauty).

  11. Medical devices; immunology and microbiology devices; classification of respiratory viral panel multiplex nucleic acid assay. Final rule.

    Science.gov (United States)

    2009-10-09

    The Food and Drug Administration (FDA) is announcing the classification of the respiratory viral panel multiplex nucleic acid assay into class II (special controls). The special controls that will apply to the device are three guidance documents entitled: "Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay," as applicable, "Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays," and as applicable,"Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays.'' The agency classified the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device. Elsewhere in this issue of the Federal Register, FDA is announcing the availability of the guidance documents that will serve as the special controls for this device.

  12. Integrated self-regulating resistive heating for isothermal nucleic acid amplification tests (NAAT) in Lab-on-a-Chip (LoC) devices.

    Science.gov (United States)

    Pardy, Tamas; Tulp, Indrek; Kremer, Clemens; Rang, Toomas; Stewart, Ray

    2017-01-01

    Isothermal nucleic acid amplification tests (NAAT) in a Lab-on-a-Chip (LoC) format promise to bring high-accuracy, non-instrumented rapid tests to the point of care. Reliable rapid tests for infectious diseases allow for early diagnosis and treatment, which in turn enables better containment of potential outbreaks and fewer complications. A critical component to LoC NAATs is the heating element, as all NAAT protocols require incubation at elevated temperatures. We propose a cheap, integrated, self-regulating resistive heating solution that uses 2xAAA alkaline batteries as the power source, can maintain temperatures in the 60-63°C range for at least 25 minutes, and reaches the target range from room temperature in 5 minutes. 4 heating element samples with different electrical characteristics were evaluated in a thermal mock-up for a LoC NAAT device. An optimal heating element candidate was chosen based on temperature profiling. The optimal candidate was further evaluated by thermal modelling via finite element analysis of heat transfer and demonstrated suitable for isothermal nucleic acid amplification.

  13. Integrated self-regulating resistive heating for isothermal nucleic acid amplification tests (NAAT) in Lab-on-a-Chip (LoC) devices

    Science.gov (United States)

    Tulp, Indrek; Kremer, Clemens; Rang, Toomas; Stewart, Ray

    2017-01-01

    Isothermal nucleic acid amplification tests (NAAT) in a Lab-on-a-Chip (LoC) format promise to bring high-accuracy, non-instrumented rapid tests to the point of care. Reliable rapid tests for infectious diseases allow for early diagnosis and treatment, which in turn enables better containment of potential outbreaks and fewer complications. A critical component to LoC NAATs is the heating element, as all NAAT protocols require incubation at elevated temperatures. We propose a cheap, integrated, self-regulating resistive heating solution that uses 2xAAA alkaline batteries as the power source, can maintain temperatures in the 60–63°C range for at least 25 minutes, and reaches the target range from room temperature in 5 minutes. 4 heating element samples with different electrical characteristics were evaluated in a thermal mock-up for a LoC NAAT device. An optimal heating element candidate was chosen based on temperature profiling. The optimal candidate was further evaluated by thermal modelling via finite element analysis of heat transfer and demonstrated suitable for isothermal nucleic acid amplification. PMID:29267339

  14. Fluorescence quenching of graphene oxide combined with the site-specific cleavage of restriction endonuclease for deoxyribonucleic acid demethylase activity assay.

    Science.gov (United States)

    Ji, Lijuan; Qian, Yingdan; Wu, Ping; Zhang, Hui; Cai, Chenxin

    2015-04-15

    We report on the development of a sensitive and selective deoxyribonucleic acid (DNA) demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with the site-specific cleavage of HpaII endonuclease to improve the selectivity. This approach was developed by designing a single-stranded probe (P1) that carries a binding region to facilitate the interaction with GO, which induces fluorescence quenching of the labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region, which contains a hemi-methylated site of 5'-CmCGG-3', to specifically recognize the target (T1, a 32-mer DNA from the promoter region of p53 gene) and hybridize with it to form a P1/T1 duplex. After demethylation with MBD2, the duplex can be specifically cleaved using HpaII, which releases the labeled FAM from the GO surface and results in the recovery of fluorescence. However, this cleavage is blocked by the hemi-methylation of this site. Thus, the magnitude of the recovered fluorescence signal is related to the MBD2 activity, which establishes the basis of the DNA demethylase activity assay. This assay can determine as low as ∼(0.05±0.01) ng mL(-1) (at a signal/noise of 3) of MBD2 with a linear range of 0.2-300 ng mL(-1) and recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. The advantage of this assay is its ability to avoid false signals and no requirement of bisulfite conversion, PCR amplification, radioisotope labeling, or separation. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Buckyballs conjugated with nucleic acid sequences identifies microorganisms in live cell assays.

    Science.gov (United States)

    Cheng, Qingsu; Parvin, Bahram

    2017-11-09

    Rapid identification of bacteria can play an important role at the point of care, evaluating the health of the ecosystem, and discovering spatiotemporal distributions of a bacterial community. We introduce a method for rapid identification of bacteria in live cell assays based on cargo delivery of a nucleic acid sequence and demonstrate how a mixed culture can be differentiated using a simple microfluidic system. C60 Buckyballs are functionalized with nucleic acid sequences and a fluorescent reporter to show that a diversity of microorganisms can be detected and identified in live cell assays. The nucleic acid complexes include an RNA detector, targeting a species-specific sequence in the 16S rRNA, and a complementary DNA with an attached fluorescent reporter. As a result, each bacterium can be detected and visualized at a specific emission frequency through fluorescence microscopy. The C60 probe complexes can detect and identify a diversity of microorganisms that include gram-position and negative bacteria, yeast, and fungi. More specifically, nucleic-acid probes are designed to identify mixed cultures of Bacillus subtilis and Streptococcus sanguinis, or Bacillus subtilis and Pseudomonas aeruginosa. The efficiency, cross talk, and accuracy for the C60 probe complexes are reported. Finally, to demonstrate that mixed cultures can be separated, a microfluidic system is designed that connects a single source-well to multiple sinks wells, where chemo-attractants are placed in the sink wells. The microfluidic system allows for differentiating a mixed culture. The technology allows profiling of bacteria composition, at a very low cost, for field studies and point of care.

  16. Development of a Loop-Mediated Isothermal Amplification Assay for Rapid and Specific Identification of ACT Producing Alternaria alternata, the Agent of Brown Spot Disease in Tangerine.

    Science.gov (United States)

    Moghimi, Hamid; Moradi, Amir; Hamedi, Javad; Basiri, Mina

    2016-03-01

    Rapid, accurate, and easy identification of pathogenic agents has always been important in medicine, veterinary, and agriculture. The brown spot infection is among the most common diseases in tangerine caused by Alternaria alternata. Due to the existence of seven various pathotypes of A. alternata species, it is challenging and time consuming to detect a pathotype responsible for citrus brown spot. In this study, we were seeking a rapid and specific approach to identify the tangerine pathotype within the A. alternata-pathogenic species, using the loop-mediated isothermal amplification (LAMP) method and actts2 gene as a marker molecule. Nine pathogenic samples were obtained from the region of Ramsar, Iran, and certified as A. alternata-pathogenic isolates. Specific primers were designed for regions coding for Alternaria citri toxin (ACT), and the PCR and LAMP reactions were performed. Our data showed that the primers designed for the tangerine pathotype of A. alternata were specific, and in both reactions, positive results were only observed in desired pathotypes. In the other pathotypes of this species as well as other standard fungal samples as negative controls, no positive result was observed. Therefore, our results suggest the possibility to detect the tangerine-specific A. alternata pathotype from other related species with a high accuracy and in early stages of the disease.

  17. Evaluation of the pepsin digestibility assay for predicting amino acid digestibility of meat and bone meals.

    Science.gov (United States)

    Davis, T M; Parsons, C M; Utterback, P L; Kirstein, D

    2015-05-01

    Sixteen meat and bone meal (MBM) samples were obtained and selected from various company plants to provide a wide range in pepsin nitrogen digestibility values. Pepsin digestibility was determined using either 0.02 or 0.002% pepsin. Amino acid (AA) digestibility of the 16 MBM samples was then determined using a precision-fed cecectomized rooster assay. The 0.02% pepsin digestibility values were numerically higher than the 0.002% pepsin values. The values varied from 77 to 93% for 0.02% pepsin and from 67 to 91% for 0.002% pepsin. The rooster AA digestibility results showed a wide range of values among MBM samples mostly due to the 4 samples having lowest and highest AA digestibility. A precision-fed broiler chick ileal AA digestibility assay confirmed that there were large differences in AA digestibility among the MBM samples having the lowest and highest rooster digestibility values. Correlation analyses between pepsin and AA digestibility values showed that the correlation values (r) were highly significant (P values were not included in the correlation analyses, the correlation coefficient values (r) were generally very low and not significant (P > 0.05). The results indicated that the pepsin nitrogen digestibility assay is only useful for detecting large differences in AA digestibility among MBM. There also was no advantage for using 0.02 versus 0.002% pepsin. © 2015 Poultry Science Association Inc.

  18. Oligonucleotide-linked gold nanoparticle aggregates for enhanced sensitivity in lateral flow assays.

    Science.gov (United States)

    Hu, Jie; Wang, Lin; Li, Fei; Han, Yu Long; Lin, Min; Lu, Tian Jian; Xu, Feng

    2013-11-21

    Lateral flow assays (LFAs) as rapid analytical techniques promise to be widely used in point-of-care (POC) diagnostics because of their affordability and simplicity. However, LFAs still suffer from low sensitivity in detection of various biomarkers, e.g., nucleic acids. In this study, we developed a simple and general one-step signal amplification strategy, which employed oligonucleotide-linked gold nanoparticle (AuNP) aggregates to enhance the sensitivity in nucleic acid lateral flow (NALF) assays. Using a nucleic acid sequence of human immunodeficiency virus type 1 (HIV-1) as a model analyte, we observed that the detection limit of the developed NALF assay was 0.1 nM, which was improved by 2.5-fold compared with that of a non-signal amplification approach. The methodology described here could be used to detect a broad range of nucleic acids, and the general signal amplification approach could be potentially adopted in other types of LFAs.

  19. Biomaterials in light amplification

    Science.gov (United States)

    Mysliwiec, Jaroslaw; Cyprych, Konrad; Sznitko, Lech; Miniewicz, Andrzej

    2017-03-01

    Biologically produced or inspired materials can serve as optical gain media, i.e. they can exhibit the phenomenon of light amplification. Some of these materials, under suitable dye-doping and optical pumping conditions, show lasing phenomena. The emerging branch of research focused on obtaining lasing action in highly disordered and highly light scattering materials, i.e. research on random lasing, is perfectly suited for biological materials. The use of biomaterials in light amplification has been extensively reported in the literature. In this review we attempt to report on progress in the development of biologically derived systems able to show the phenomena of light amplification and random lasing together with the contribution of our group to this field. The rich world of biopolymers modified with molecular aggregates and nanocrystals, and self-organized at the nanoscale, offers a multitude of possibilities for tailoring luminescent and light scattering properties that are not easily replicated in conventional organic or inorganic materials. Of particular importance and interest are light amplification and lasing, or random lasing studies in biological cells and tissues. In this review we will describe nucleic acids and their complexes employed as gain media due to their favorable optical properties and ease of manipulation. We will report on research conducted on various biomaterials showing structural analogy to nucleic acids such as fluorescent proteins, gelatins in which the first distributed feedback laser was realized, and also amyloids or silks, which, due to their dye-doped fiber-like structure, allow for light amplification. Other materials that were investigated in that respect include polysaccharides, like starch exhibiting favorable photostability in comparison to other biomaterials, and chitosan, which forms photonic crystals or cellulose. Light amplification and random lasing was not only observed in processed biomaterials but also in living

  20. Properties of kojic acid and curcumin: Assay on cell B16-F1

    Science.gov (United States)

    Sugiharto, Ariff, Arbakariya; Ahmad, Syahida; Hamid, Muhajir

    2016-03-01

    Ultra violet (UV) exposure and oxidative stress are casually linked to skin disorders. They can increase melanin synthesis, proliferation of melanocytes, and hyperpigmentation. It is possible that antioxidants or inhibitors may have a beneficial effect on skin health to reduce hyperpigmentation. In the last few years, a huge number of natural herbal extracts have been tested to reduce hyperpigmentation. The objective of this study was to determine and to compare of kojic acid and curcumin properties to viability cell B16-F1. In this study, our data showed that the viability of cell B16-F1 was 63.91% for kojic acid and 64.12% for curcumin at concentration 100 µg/ml. Further investigation assay of antioxidant activities, indicated that IC50 for kojic acid is 63.8 µg/ml and curcumin is 16.05 µg/ml. Based on the data, kojic acid and curcumin have potential antioxidant properties to reduce hyperpigmentation with low toxicity effect in cell B16-F1.

  1. Evaluation of a monoclonal antibody-based immunoradiometric assay for prostatic acid phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Davies, S.N.; Gochman, N.

    1983-01-01

    This report evaluates a new immunoradiometric assay for prostatic acid phosphatase in serum, based on a dual monoclonal antibody reaction system (Hybritech-TANDEM). A solidphase antibody binds the acid phosphatase molecule and a second monoclonal antibody to a different antigenic site serves as the /sup 125/I-radiolabel. The method was tested on 67 patients with various stages of prostatic carcinoma and 134 patients without the disease. It also was compared with a conventional polyclonal radioimmunoassay (NEN) and an enzymatic activity method (duPont aca). The upper limit for the TANDEM assay on nondiseased male patients was found to be 2.0 microgram/L. Based on this upper limit of normal, the diagnostic sensitivity of the method for all cases of prostatic carcinoma was 60%. Researchs could not distinguish the enzyme released in abnormal amounts due to benign prostatic hypertrophy and certain nonprostatic malignant diseases from that of prostatic carcinoma. The diagnostic specificity was calculated at 95%. For the clinically undetectable Stage 1 disease, sensitivity was 44% (four abnormal values out of nine cases). The TANDEM procedure is simple to use and reproducible.

  2. Ordering folate assays is no longer justified for investigation of anemias, in folic acid fortified countries

    Directory of Open Access Journals (Sweden)

    von Kuster Kenneth

    2010-01-01

    Full Text Available Abstract Background Since 1998, in the countries where there is mandatory fortification of grain products with folic acid, folate deficiency has become very rare. Consequently, we decided to find out whether there is any justification for ordering folate assays for investigation of anemias. Methods We reviewed serum folate (SF and red cell folate (RF data at two teaching hospitals in Canada. At the Health Sciences Centre (HSC the folate data for the year 2001 were analyzed and the medical records of those with low SF or low RF were reviewed. At St. Boniface General Hospital(SBGHall folate data between January 1996 and Dec 31,2004 were analyzed and the medical records of all who had low RF between January 1,1999 and December 31,2004 were reviewed. Results In 2001, at HSC, 11 out of 2154(0.5%SF were low( Conclusion In countries where there is mandatory fortification of grain products with folic acid, folate deficiency to the degree that could cause anemia is extremely rare. Ordering folate assays for investigation of anemias, in these countries, is waste of time and money. The result of these tests is more likely to mislead the physicians than to provide any useful information.

  3. A simple assay for the measurement of plasma antioxidant status using spontaneous autoxidation of homovanillic acid.

    Science.gov (United States)

    Gaboriau, François; Delcros, Jean Guy; Moulinoux, Jacques Philippe

    2002-01-01

    Reactive oxygen species (ROS) contribute to the development of pathophysiological processes, hence the increasing interest in modulating the antioxidant status of patient by nutritional or pharmacological intervention. Antioxidants act by preventing the formation of ROS (inhibitory effect) and/or by trapping these species (scavenger effect). We have developed a simple, sensitive, and reliable test to measure the total antioxidative efficiency of plasma or other biological fluids using microliter samples. Autoxidation of homovanillic acid (HVA) gives rise to fluorescent dimers. Antioxidants contained in the plasma (or free aqueous solutions) scavenge the ROS involved in this process and transiently stop the linear increase in fluorescence intensity during a time (delay) proportional to the total concentration of antioxidants and their scavenging efficiency. In addition to this scavenging effect, the kinetics of HVA autoxidation, restarting after the delay, reflects the ability of the plasma antioxidants to inhibit the ROS-triggered autoxidation. The rate of the HVA autoxidation depended on the temperature, the protonation of the phenolic group, and on the presence of peroxide, peroxyl radicals, and peroxidase as well as metal ions. This Fenton-like reaction was transiently stopped by various ROS scavengers including quercetin, ascorbic acid, and thiol derivatives (glutathione and N-acetylcystein) while metal chelating agents such as desferrioxamine, ethylene diamine tetracetic acid (EDTA), and polyamine only reduced its rate. The main advantages of this new assay are its versatility to investigate in a single run both the scavenging and inhibitory components of the antioxidant capacity, and its relevance to the reactive hydroxyl radical. As shown in this study, the increase in the antioxidant capacity of human plasma during pharmacological supplementation with antioxidant illustrates one of the various fields of application of this assay.

  4. Quantification of bacterial species of the vaginal microbiome in different groups of women, using nucleic acid amplification tests

    OpenAIRE

    Jespers Vicky; Menten Joris; Smet Hilde; Poradosú Sabrina; Abdellati Saïd; Verhelst Rita; Hardy Liselotte; Buvé Anne; Crucitti Tania

    2012-01-01

    Abstract Background The vaginal microbiome plays an important role in urogenital health. Quantitative real time Polymerase Chain Reaction (qPCR) assays for the most prevalent vaginal Lactobacillus species and bacterial vaginosis species G. vaginalis and A. vaginae exist, but qPCR information regarding variation over time is still very limited. We set up qPCR assays for a selection of seven species and defined the temporal variation over three menstrual cycles in a healthy Caucasian population...

  5. Comparison of Antioxidant Evaluation Assays for Investigating Antioxidative Activity of Gallic Acid and Its Alkyl Esters in Different Food Matrices.

    Science.gov (United States)

    Phonsatta, Natthaporn; Deetae, Pawinee; Luangpituksa, Pairoj; Grajeda-Iglesias, Claudia; Figueroa-Espinoza, Maria Cruz; Le Comte, Jérôme; Villeneuve, Pierre; Decker, Eric A; Visessanguan, Wonnop; Panya, Atikorn

    2017-08-30

    The addition of antioxidants is one of the strategies to inhibit lipid oxidation, a major cause of lipid deterioration in foods leading to rancidity development and nutritional losses. However, several studies have been reported that conventional antioxidant assays, e.g., TPC, ABTS, FRAP, and ORAC could not predict antioxidant performance in several foods. This study aimed to investigate the performance of two recently developed assays, e.g., the conjugated autoxidizable triene (CAT) and the apolar radical-initiated conjugated autoxidizable triene (ApoCAT) assays to predict the antioxidant effectiveness of gallic acid and its esters in selected food models in comparison with the conventional antioxidant assays. The results indicated that the polarities of the antioxidants have a strong impact on antioxidant activities. In addition, different oxidant locations demonstrated by the CAT and ApoCAT assays influenced the overall antioxidant performances of the antioxidants with different polarities. To validate the predictability of the assays, the antioxidative performance of gallic acid and its alkyl esters was investigated in oil-in-water (O/W) emulsions, bulk soybean oils, and roasted peanuts as the lipid food models. The results showed that only the ApoCAT assay could be able to predict the antioxidative performances in O/W emulsions regardless of the antioxidant polarities. This study demonstrated that the relevance of antioxidant assays to food models was strongly dependent on physical similarities between the tested assays and the food structure matrices.

  6. Abscisic acid signaling: thermal stability shift assays as tool to analyze hormone perception and signal transduction.

    Science.gov (United States)

    Soon, Fen-Fen; Suino-Powell, Kelly M; Li, Jun; Yong, Eu-Leong; Xu, H Eric; Melcher, Karsten

    2012-01-01

    Abscisic acid (ABA) is a plant hormone that plays important roles in growth and development. ABA is also the central regulator to protect plants against abiotic stresses, such as drought, high salinity, and adverse temperatures, and ABA signaling is therefore a promising biotechnological target for the generation of crops with increased stress resistance. Recently, a core signal transduction pathway has been established, in which ABA receptors, type 2C protein phosphatases, and AMPK-related protein kinases control the regulation of transcription factors, ion channels, and enzymes. Here we use a simple protein thermal stability shift assay to independently validate key aspects of this pathway and to demonstrate the usefulness of this technique to detect and characterize very weak (Kd ≥ 50 µM) interactions between receptors and physiological and synthetic agonists, to determine and analyze protein-protein interactions, and to screen small molecule inhibitors.

  7. Abscisic acid signaling: thermal stability shift assays as tool to analyze hormone perception and signal transduction.

    Directory of Open Access Journals (Sweden)

    Fen-Fen Soon

    Full Text Available Abscisic acid (ABA is a plant hormone that plays important roles in growth and development. ABA is also the central regulator to protect plants against abiotic stresses, such as drought, high salinity, and adverse temperatures, and ABA signaling is therefore a promising biotechnological target for the generation of crops with increased stress resistance. Recently, a core signal transduction pathway has been established, in which ABA receptors, type 2C protein phosphatases, and AMPK-related protein kinases control the regulation of transcription factors, ion channels, and enzymes. Here we use a simple protein thermal stability shift assay to independently validate key aspects of this pathway and to demonstrate the usefulness of this technique to detect and characterize very weak (Kd ≥ 50 µM interactions between receptors and physiological and synthetic agonists, to determine and analyze protein-protein interactions, and to screen small molecule inhibitors.

  8. A label-free fluorescence assay for thrombin based on aptamer exonuclease protection and exonuclease III-assisted recycling amplification-responsive cascade zinc(II)-protoporphyrin IX/G-quadruplex supramolecular fluorescent labels.

    Science.gov (United States)

    Lv, Yanqin; Xue, Qingwang; Gu, Xiaohong; Zhang, Shuqiu; Liu, Jifeng

    2014-05-21

    A simple, label-free and sensitive fluorescence protein assay has been developed on the basis of aptamer exonuclease protection and exonuclease III (Exo III)-assisted recycling amplification-responsive cascade ZnPPIX/G-quadruplex supramolecular fluorescent labels. In the sensing system, a special aptamer probe containing the aptamer sequence at the 3'-terminus and the DNAzyme sequence at the 5'-terminus was applied, which has the capacity to recognize a protein target with high affinity and specificity. Exonuclease I (Exo I) can efficiently catalyze the degradation of free single stranded DNA probes in the 3' to 5' direction. In the presence of the target protein, the strong binding between the target protein and its aptamer can protect aptamer probes from degradation. Subsequently, the protected aptamer probes act as catalysators to trigger hybridization with the hairpin DNA probe that contains a partially "caged" G-quadruplex sequence. Upon interaction with the protected aptamer probes, the hairpin opens to yield the active G-quadruplex structure. In the presence of exonuclease III (Exo III), Exo III-assisted recycling amplification occurs generating numerous G-quadruplex supramolecular structures. The zinc(ii)-protoporphyrin IX (ZnPPIX) fluorophore binds to the G-quadruplexes and this results in the enhanced fluorescence of the fluorophore. The resulting fluorescence of the ZnPPIX/G-quadruplex provides the readout signal for the sensing event. Thrombin is used as the model analyte in the current proof-of-concept. The developed method was demonstrated to have very high sensitivity for the detection of proteins with a limit of detection of 0.2 pM without using washes or separations. In addition, this new method for protein detection is simple and inherits all the advantages of aptamers. The mechanism, moreover, may be generalized and used for other forms of protein analysis.

  9. Simultaneous detection and differentiation of dengue virus serotypes 1-4, Japanese encephalitis virus, and West Nile virus by a combined reverse-transcription loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Yin Jianhua

    2011-07-01

    Full Text Available Abstract Background Rapid identification and differentiation of mosquito-transmitted flaviviruses in acute-phase sera of patients and field-caught vector mosquitoes are important for the prediction and prevention of large-scale epidemics. Results We developed a flexible reverse-transcription loop-mediated isothermal amplification (RT-LAMP unit for the detection and differentiation of dengue virus serotypes 1-4 (DENV1-4, Japanese encephalitis virus (JEV, and West Nile virus (WNV. The unit efficiently amplified the viral genomes specifically at wide ranges of viral template concentrations, and exhibited similar amplification curves as monitored by a real-time PCR engine. The detection limits of the RT-LAMP unit were 100-fold higher than that of RT-PCR in 5 of the six flaviviruses. The results on specificity indicated that the six viruses in the assay had no cross-reactions with each other. By examining 66 viral strains of DENV1-4 and JEV, the unit identified the viruses with 100% accuracy and did not cross-react with influenza viruses and hantaviruses. By screening a panel of specimens containing sera of 168 patients and 279 pools of field-caught blood sucked mosquitoes, results showed that this unit is high feasible in clinical settings and epidemiologic field, and it obtained results 100% correlated with real-time RT-PCR. Conclusions The RT-LAMP unit developed in this study is able to quickly detect and accurately differentiate the six kinds of flaviviruses, which makes it extremely feasible for screening these viruses in acute-phase sera of the patients and in vector mosquitoes without the need of high-precision instruments.

  10. Electrospin-coating of nitrocellulose membrane enhances sensitivity in nucleic acid-based lateral flow assay.

    Science.gov (United States)

    Yew, Chee-Hong Takahiro; Azari, Pedram; Choi, Jane Ru; Li, Fei; Pingguan-Murphy, Belinda

    2018-06-07

    Point-of-care biosensors are important tools developed to aid medical diagnosis and testing, food safety and environmental monitoring. Paper-based biosensors, especially nucleic acid-based lateral flow assays (LFA), are affordable, simple to produce and easy to use in remote settings. However, the sensitivity of such assays to infectious diseases has always been a restrictive challenge. Here, we have successfully electrospun polycaprolactone (PCL) on nitrocellulose (NC) membrane to form a hydrophobic coating to reduce the flow rate and increase the interaction rate between the targets and gold nanoparticles-detecting probes conjugates, resulting in the binding of more complexes to the capture probes. With this approach, the sensitivity of the PCL electrospin-coated test strip has been increased by approximately ten-fold as compared to the unmodified test strip. As a proof of concept, this approach holds great potential for sensitive detection of targets at point-of-care testing. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Antioxidant Capacity of Gallic Acid in vitro Assayed on Human Erythrocytes.

    Science.gov (United States)

    Suwalsky, Mario; Colina, José; Gallardo, María José; Jemiola-Rzeminska, Malgorzata; Strzalka, Kazimierz; Manrique-Moreno, Marcela; Sepúlveda, Benjamín

    2016-12-01

    Gallic acid (GA) is a polyphenol present in many plants. This study was aimed to investigate the molecular interaction of GA with the human erythrocyte membrane and to determine its antioxidant capacity. The molecular interaction with the membrane of human red cells and the antioxidant property was assayed on both human red cells and molecular models of its membrane. Observations by optical, scanning electron, and defocusing microscopy demonstrated that GA is capable to convert red cells from their normal biconcave shape to crenated echinocytes. This result indicates that GA molecules are positioned in the outer monolayer of the red cell membrane. Dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were selected as classes of phospholipids found in the outer and inner monolayers of the red cell membrane, respectively. X-ray diffraction and differential scanning calorimetry showed that GA was preferentially bound to DMPC bilayers. Experiments related to the antioxidant capacity of GA indicated that this compound offsets HClO oxidative capacity on DMPE bilayers. In addition, optical, scanning, defocusing microscopy, and hemolysis assays confirmed the protective capacity of GA against HClO deleterious effects on human red cells. As a conclusion, GA would be capable to block the access of oxidants into the lipid bilayer, and thus avoid their access into red cells.

  12. Immunochromatographic strip assay for detection of bioactive Ganoderma triterpenoid, ganoderic acid A in Ganoderma lingzhi.

    Science.gov (United States)

    Sakamoto, Seiichi; Kikkawa, Nao; Kohno, Toshitaka; Shimizu, Kuniyoshi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2016-10-01

    Ganoderic acid A (GAA) is one of the major Ganoderma triterpenes produced by medicinal mushroom belonging to the genus Ganoderma (Ganodermataceae). Due to its interesting pharmacological activities, Ganoderma species have been traditionally used in China for the treatment of various diseases. Herein, we developed a colloidal gold-based immunochromatographic strip assay (ICA) for the rapid detection of GAA using highly specific monoclonal antibody against GAA (MAb 12A) conjugated with gold nanoparticles. Using the developed ICA, the detection of GAA can be completed within 15min after dipping the test strip into an analyte solution with the limit of detection (LOD) for GAA of ~500ng/mL. In addition, this system makes it possible to perform a semi-quantitative analysis of GAA in Ganoderma lingzhi, where high reliability was evaluated by enzyme-linked immunosorbent assay (ELISA). The newly developed ICA can potentially be applied to the standardization of Ganoderma using GAA as an index because GAA is major triterpenoid present much in the mushroom. Copyright © 2016. Published by Elsevier B.V.

  13. New Fpg probe chemistry for direct detection of recombinase polymerase amplification on lateral flow strips.

    Science.gov (United States)

    Powell, Michael L; Bowler, Frank R; Martinez, Aurore J; Greenwood, Catherine J; Armes, Niall; Piepenburg, Olaf

    2018-02-15

    Rapid, cost-effective and sensitive detection of nucleic acids has the ability to improve upon current practices employed for pathogen detection in diagnosis of infectious disease and food testing. Furthermore, if assay complexity can be reduced, nucleic acid amplification tests could be deployed in resource-limited and home use scenarios. In this study, we developed a novel Fpg (Formamidopyrimidine DNA glycosylase) probe chemistry, which allows lateral flow detection of amplification in undiluted recombinase polymerase amplification (RPA) reactions. The prototype nucleic acid lateral flow chemistry was applied to a human genomic target (rs1207445), Campylobacter jejuni 16S rDNA and two genetic markers of the important food pathogen E. coli O157:H7. All four assays have an analytical sensitivity between 10 and 100 copies DNA per amplification. Furthermore, the assay is performed with fewer hands-on steps than using the current RPA Nfo lateral flow method as dilution of amplicon is not required for lateral flow analysis. Due to the simplicity of the workflow, we believe that the lateral flow chemistry for direct detection could be readily adapted to a cost-effective single-use consumable, ideal for use in non-laboratory settings. Copyright © 2017. Published by Elsevier Inc.

  14. Specific, Sensitive, and Rapid Diagnosis of Active Toxoplasmosis by a Loop-Mediated Isothermal Amplification Method Using Blood Samples from Patients ▿

    OpenAIRE

    Lau, Yee Ling; Meganathan, Puviarasi; Sonaimuthu, Parthasarathy; Thiruvengadam, Girija; Nissapatorn, Veeranoot; Chen, Yeng

    2010-01-01

    Loop-mediated isothermal amplification (LAMP), a rapid nucleic acid amplification method, was developed for the clinical diagnosis of toxoplasmosis. Three LAMP assays based on the SAG1, SAG2, and B1 genes of Toxoplasma gondii were developed. The sensitivities and specificities of the LAMP assays were evaluated by comparison with the results of conventional nested PCR. The LAMP assays were highly sensitive and had a detection limit of 0.1 tachyzoite, and no cross-reactivity with the DNA of oth...

  15. Highly simplified lateral flow-based nucleic acid sample preparation and passive fluid flow control

    Science.gov (United States)

    Cary, Robert E.

    2015-12-08

    Highly simplified lateral flow chromatographic nucleic acid sample preparation methods, devices, and integrated systems are provided for the efficient concentration of trace samples and the removal of nucleic acid amplification inhibitors. Methods for capturing and reducing inhibitors of nucleic acid amplification reactions, such as humic acid, using polyvinylpyrrolidone treated elements of the lateral flow device are also provided. Further provided are passive fluid control methods and systems for use in lateral flow assays.

  16. Medical Devices; Immunology and Microbiology Devices; Classification of the Streptococcus SPP. Nucleic Acid-Based Assay. Final order.

    Science.gov (United States)

    2017-10-30

    The Food and Drug Administration (FDA or we) is classifying the Streptococcus spp. nucleic acid-based assay into class II (special controls). The special controls that apply to the device type are identified in this order and will be part of the codified language for the Streptococcus spp. nucleic acid-based assay's classification. We are taking this action because we have determined that classifying the device into class II (special controls) will provide a reasonable assurance of safety and effectiveness of the device. We believe this action will also enhance patients' access to beneficial innovative devices, in part by reducing regulatory burdens.

  17. Nucleic acid-based assays for the detection of high-risk human papillomavirus: a technical review.

    Science.gov (United States)

    Gibson, Jane S

    2014-09-01

    Nucleic acid-based high-risk human papillomavirus (hrHPV) testing is essential to contemporary cervical cancer screening. The numbers of commercially available assays approved by the US Food and Drug Administration for HPV nucleic acid detection have increased, each offering various approaches to analysis. An understanding of the methodologies associated with HPV testing is important to the practice of laboratory medicine. An overview of instruments, chemistries, laboratory workflows, and test limitations associated with current US Food and Drug Administration-approved assays is provided. © 2014 American Cancer Society.

  18. Diagnostic Accuracy of Loop-mediated Isothermal Amplifica-tion Assay as a Field Molecular Tool for Rapid Mass Screening of Old World Leishmania Infections in Sand Flies and In Vitro Culture

    Directory of Open Access Journals (Sweden)

    Mehdi GHODRATI

    2017-12-01

    Full Text Available AbstractBackground: We employed a highly sensitive loop-mediated isothermal amplification (LAMP by targeting 18S rRNA gene to identify the rapid mass screening of Leishmania infections in captured sand flies of southwest Iran and In vitro culture. Methods: One hundred fifty sand flies were collected from 11 sites adjacent to Iraqi’s borders in southern parts of Khuzestan Province by using sticky sheets of paper and CDC miniature light traps during late May 2014 to Nov 2015. Following morphological identification of sand flies species, the DNA of infected samples was extracted and amplified by PCR and LAMP assays by targeting ITS-rDNA and 18S rRNA genes. The PCR amplicons were directly sequenced to conduct the phylogenetic analysis Results: Ten (6.6% Leishmania infections were identified by LAMP assay (detection limit 0.01 parasites DNA among infected Sergentomyia baghdadis, S. sintoni and Phlebotomus papatasi sand flies that was more sensitive than PCR (n=6.4%; (detection limit 101parasites DNA. LAMP can identify 101-106promastigotes/100 µl RPMI 1640 while PCR recognized104-106 promastigotes. The majority infection rate of sand flies was confirmed to L. major inferred by phylogenetic analysis. Conclusion: This is the first exploration characterized the Old World Leishmania infections by LAMP technique in both infected sand flies and In vitro conditions. The LAMP method because of its shorter reaction time, robustness, more sensitivity, lack of requirement of complicated equipment and visual discriminatory of positivity can be appeared a promising tool instead of PCR to identify low Leishmania loads and entomological monitoring of leishmaniasis in resource-limited endemic of the world.

  19. Amplification variable factor amplifier

    NARCIS (Netherlands)

    Akitsugu, Oshita; Nauta, Bram

    2007-01-01

    PROBLEM TO BE SOLVED: To provide an amplification factor variable amplifier capable of achieving temperature compensation of an amplification factor over a wide variable amplification factor range. ; SOLUTION: A Gilbert type amplification factor variable amplifier 11 amplifies an input signal and

  20. Amplification variable factor amplifier

    NARCIS (Netherlands)

    Akitsugu, O.; Nauta, Bram

    2006-01-01

    PROBLEM TO BE SOLVED: To provide an amplification factor variable amplifier capable of achieving temperature compensation of an amplification factor over a wide variable amplification factor range. ; SOLUTION: A Gilbert type amplification factor variable amplifier 11 amplifies an input signal and

  1. Digital Assays Part II: Digital Protein and Cell Assays.

    Science.gov (United States)

    Basu, Amar S

    2017-08-01

    A digital assay is one in which the sample is partitioned into many containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, . . .). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotype and phenotype. Where part I of this review focused on the fundamentals of partitioning and digital PCR, part II turns its attention to digital protein and cell assays. Digital enzyme assays measure the kinetics of single proteins with enzymatic activity. Digital enzyme-linked immunoassays (ELISAs) quantify antigenic proteins with 2 to 3 log lower detection limit than conventional ELISA, making them well suited for low-abundance biomarkers. Digital cell assays probe single-cell genotype and phenotype, including gene expression, intracellular and surface proteins, metabolic activity, cytotoxicity, and transcriptomes (scRNA-seq). These methods exploit partitioning to 1) isolate single cells or proteins, 2) detect their activity via enzymatic amplification, and 3) tag them individually by coencapsulating them with molecular barcodes. When scaled, digital assays reveal stochastic differences between proteins or cells within a population, a key to understanding biological heterogeneity. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows.

  2. Method and apparatus for purifying nucleic acids and performing polymerase chain reaction assays using an immiscible fluid

    Science.gov (United States)

    Koh, Chung-Yan; Light, Yooli Kim; Piccini, Matthew Ernest; Singh, Anup K.

    2017-10-31

    Embodiments of the present invention are directed toward devices, systems, and methods for purifying nucleic acids to conduct polymerase chain reaction (PCR) assays. In one example, a method includes generating complexes of silica beads and nucleic acids in a lysis buffer, transporting the complexes through an immiscible fluid to remove interfering compounds from the complexes, further transporting the complexes into a density medium containing components required for PCR where the nucleic acids disassociate from the silica beads, and thermocycling the contents of the density medium to achieve PCR. Signal may be detected from labeling agents in the components required for PCR.

  3. Quantification of bacterial species of the vaginal microbiome in different groups of women, using nucleic acid amplification tests.

    Science.gov (United States)

    Jespers, Vicky; Menten, Joris; Smet, Hilde; Poradosú, Sabrina; Abdellati, Saïd; Verhelst, Rita; Hardy, Liselotte; Buvé, Anne; Crucitti, Tania

    2012-05-30

    The vaginal microbiome plays an important role in urogenital health. Quantitative real time Polymerase Chain Reaction (qPCR) assays for the most prevalent vaginal Lactobacillus species and bacterial vaginosis species G. vaginalis and A. vaginae exist, but qPCR information regarding variation over time is still very limited. We set up qPCR assays for a selection of seven species and defined the temporal variation over three menstrual cycles in a healthy Caucasian population with a normal Nugent score. We also explored differences in qPCR data between these healthy women and an 'at risk' clinic population of Caucasian, African and Asian women with and without bacterial vaginosis (BV), as defined by the Nugent score. Temporal stability of the Lactobacillus species counts was high with L. crispatus counts of 108 copies/mL and L. vaginalis counts of 106 copies/mL. We identified 2 types of 'normal flora' and one 'BV type flora' with latent class analysis on the combined data of all women. The first group was particularly common in women with a normal Nugent score and was characterized by a high frequency of L. crispatus, L. iners, L. jensenii, and L. vaginalis and a correspondingly low frequency of L. gasseri and A. vaginae. The second group was characterized by the predominance of L. gasseri and L. vaginalis and was found most commonly in healthy Caucasian women. The third group was commonest in women with a high Nugent score but was also seen in a subset of African and Asian women with a low Nugent score and was characterized by the absence of Lactobacillus species (except for L. iners) but the presence of G. vaginalis and A. vaginae. We have shown that the quantification of specific bacteria by qPCR contributes to a better description of the non-BV vaginal microbiome, but we also demonstrated that differences in populations such as risk and ethnicity also have to be taken into account. We believe that our selection of indicator organisms represents a feasible strategy

  4. Quantification of bacterial species of the vaginal microbiome in different groups of women, using nucleic acid amplification tests

    Directory of Open Access Journals (Sweden)

    Jespers Vicky

    2012-05-01

    Full Text Available Abstract Background The vaginal microbiome plays an important role in urogenital health. Quantitative real time Polymerase Chain Reaction (qPCR assays for the most prevalent vaginal Lactobacillus species and bacterial vaginosis species G. vaginalis and A. vaginae exist, but qPCR information regarding variation over time is still very limited. We set up qPCR assays for a selection of seven species and defined the temporal variation over three menstrual cycles in a healthy Caucasian population with a normal Nugent score. We also explored differences in qPCR data between these healthy women and an ‘at risk’ clinic population of Caucasian, African and Asian women with and without bacterial vaginosis (BV, as defined by the Nugent score. Results Temporal stability of the Lactobacillus species counts was high with L. crispatus counts of 108 copies/mL and L. vaginalis counts of 106 copies/mL. We identified 2 types of ‘normal flora’ and one ‘BV type flora’ with latent class analysis on the combined data of all women. The first group was particularly common in women with a normal Nugent score and was characterized by a high frequency of L. crispatus, L. iners, L. jensenii, and L. vaginalis and a correspondingly low frequency of L. gasseri and A. vaginae. The second group was characterized by the predominance of L. gasseri and L. vaginalis and was found most commonly in healthy Caucasian women. The third group was commonest in women with a high Nugent score but was also seen in a subset of African and Asian women with a low Nugent score and was characterized by the absence of Lactobacillus species (except for L. iners but the presence of G. vaginalis and A. vaginae. Conclusions We have shown that the quantification of specific bacteria by qPCR contributes to a better description of the non-BV vaginal microbiome, but we also demonstrated that differences in populations such as risk and ethnicity also have to be taken into account. We believe

  5. Cytotoxicity test of 40, 50 and 60% citric acid as dentin conditioner by using MTT assay on culture cell line

    Directory of Open Access Journals (Sweden)

    Christian Khoswanto

    2008-09-01

    Full Text Available Background: Open dentin is always covered by smear layer, therefore before restoration is performed, cavity or tooth which has been prepared should be clean from dirt. The researchers suggested that clean dentin surface would reach effective adhesion between resin and tooth structure, therefore dentin conditioner like citric acid was used to reach the condition. Even though citric acid is not strong acid but it can be very erosive to oral mucous. Several requirements should be fulfilled for dental product such as non toxic, non irritant, biocompatible and should not have negative effect against local, systemic or biological environment. Cytotoxicity test was apart of biomaterial evaluation and needed for standard screening. Purpose: This study was to know the cytotoxicity of 40, 50, 60% citric acid as dentin conditioner using MTT assay. Method: This study is an experimental research using the Post-Test Only Control Group Design. Six samples of each 40, 50 and 60% citric acid for citotoxicity test using MTT assay. The density of optic formazan indicated the number of living cells. All data were statistically analyzed by one way ANOVA. Result: The percentage of living cells in 40, 50 and 60% citric acid were 95.14%, 93.42% and 93.14%. Conclusion: Citric acid is non toxic and safe to be used as dentine conditioner.

  6. Assessment of 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure using a modified D-glucaric acid assay

    Energy Technology Data Exchange (ETDEWEB)

    Steinberg, K.K.; MacNeil, M.L.; Karon, J.M.; Stehr, P.A.; Neese, J.W.; Needham, L.L.

    1985-01-01

    An enzyme-inhibition assay was evaluated and modified to quantify D-glucaric acid in a population exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). The modified assay combined improvements described separately in previous reports, including pH adjustment by standard addition of buffers rather than by titration, an optimum pH of 2.3 for converting D-glucaric acid to 1,4-glucarolactone, and the use of the relation reciprocal of absorbance versus concentration for calculating unknowns. Reference limits for adult males were 0.06-5.90 mmol D-glucaric acid/mol creatinine and for adult females 0.87-6.23 mmol D-glucaric acid/mol creatinine. Children under the age of 15 yr had a reference range of 0-8.34 mmol D-glucaric acid/mol creatinine. Persons on anticonvulsant therapy excreted from 3 to 10 times the upper reference limits of D-glucaric acid. Urinary concentrations of D-glucaric acid in persons identified as being at high risk for exposure to 2,3,7,8-TCDD, were not significantly different from concentrations in those identified as being at low risk for such exposure.

  7. Comparison of the Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa ProFlu+™ assay for detecting influenza and respiratory syncytial viruses.

    Science.gov (United States)

    Selvaraju, Suresh B; Bambach, Adrienne V; Leber, Amy L; Patru, Maria-Magdalena; Patel, Anami; Menegus, Marilyn A

    2014-09-01

    The relative performance of 2 widely used reverse transcription polymerase chain reaction (RT-PCR) assays, the Focus diagnostics Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa Proflu+™ assay, was evaluated using 735 prospectively and retrospectively collected nasopharyngeal swab specimens. Overall, the assays showed positive and negative agreements of 100% and 99.7% for influenza A, 98.1% and 99.9% for influenza B, and 99.3% and 99.5% for respiratory syncytial virus. The relative analytical sensitivity of the 2 assays was also similar. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

    Directory of Open Access Journals (Sweden)

    Anja Gulliksen

    2012-01-01

    Full Text Available The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n=28 from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.

  9. Chlamydia trachomatis infection positivity rates determined by nucleic acid amplification test in patients of hospitals in the northeastern region of Ukraine.

    Science.gov (United States)

    Belozorov, Alexei; Fedets, Olga; Chastii, Tatjana; Milutina, Elena; Sokol, Oksana; Grigorova, Ritsa; Unuchko, Sergey

    2017-12-01

    There are no accurate data regarding the prevalence of Chlamydia trachomatis infection in Ukraine. This study aims to estimate the prevalence in the northeastern region of the country through reviewing nucleic acid amplification test results in patients of medical institutions in the Kharkov region during 2014-2016. Samples from 6920 patients (5028 women and 1892 men) aged 12-76 years were tested. The overall positivity rate was 4.5% (95% CI 4.0-5.0): 3.9% (95% CI 3.4-4.5) in women and 6.1% (95% CI 5.1-7.3) in men. The highest prevalence was found in the 16-20 (8.5%, CI 6.3-11.4) and 21-25 (8.0%, CI 6.7-9.4) year age groups. The prevalence in men was higher than in women in all investigated groups. The results show the need for more attention to the prevention, diagnosis, and treatment of chlamydial infection in these age groups of women and men in this region.

  10. Point-Counterpoint: A Nucleic Acid Amplification Test for Streptococcus pyogenes Should Replace Antigen Detection and Culture for Detection of Bacterial Pharyngitis.

    Science.gov (United States)

    Pritt, Bobbi S; Patel, Robin; Kirn, Thomas J; Thomson, Richard B

    2016-10-01

    Nucleic acid amplification tests (NAATs) have frequently been the standard diagnostic approach when specific infectious agents are sought in a clinic specimen. They can be applied for specific agents such as S. pyogenes, or commercial multiplex NAATs for detection of a variety of pathogens in gastrointestinal, bloodstream, and respiratory infections may be used. NAATs are both rapid and sensitive. For many years, S. pyogenes testing algorithms used a rapid and specific group A streptococcal antigen test to screen throat specimens, followed, in some clinical settings, by a throat culture for S. pyogenes to increase the sensitivity of its detection. Now S. pyogenes NAATs are being used with increasing frequency. Given their accuracy, rapidity, and ease of use, should they replace antigen detection and culture for the detection of bacterial pharyngitis? Bobbi Pritt and Robin Patel of the Mayo Clinic, where S. pyogenes NAATs have been used for well over a decade with great success, will explain the advantages of this approach, while Richard (Tom) Thomson and Tom Kirn of the NorthShore University HealthSystem will discuss their concerns about this approach to diagnosing bacterial pharyngitis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  11. Ultra-fast electronic detection of antimicrobial resistance genes using isothermal amplification and Thin Film Transistor sensors.

    Science.gov (United States)

    Hu, Chunxiao; Kalsi, Sumit; Zeimpekis, Ioannis; Sun, Kai; Ashburn, Peter; Turner, Carrie; Sutton, J Mark; Morgan, Hywel

    2017-10-15

    A low cost thin-film transistor (TFT) nanoribbon (NR) sensor has been developed for rapid real-time detection of DNA amplification using an isothermal Recombinase Polymerase Amplification (RPA) method. The semiconductor chip measures DNA amplification through a pH change, rather than via fluorescence. The utility of the method was demonstrated by amplifying CTX-M and NDM, two genes that confer bacterial resistance to cephalosporins and carbapenems, respectively. It is shown that this approach provides extremely fast and sensitive detection. It can detect <10 copies of the gene in genomic DNA extracted from E. coli or K. pneumoniae clinical isolates within a few minutes. A differential readout system was developed to minimize the effect of primer-dimer amplification on the assay. The simple device has the potential for low cost, portable and real-time nucleic acid analysis as a Point of Care device. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  12. A universal colorimetry for nucleic acids and aptamer-specific ligands detection based on DNA hybridization amplification.

    Science.gov (United States)

    Li, Shuang; Shang, Xinxin; Liu, Jia; Wang, Yujie; Guo, Yingshu; You, Jinmao

    2017-07-01

    We present a universal amplified-colorimetric for detecting nucleic acid targets or aptamer-specific ligand targets based on gold nanoparticle-DNA (GNP-DNA) hybridization chain reaction (HCR). The universal arrays consisted of capture probe and hairpin DNA-GNP. First, capture probe recognized target specificity and released the initiator sequence. Then dispersed hairpin DNA modified GNPs were cross-linked to form aggregates through HCR events triggered by initiator sequence. As the aggregates accumulate, a significant red-to purple color change can be easily visualized by the naked eye. We used miRNA target sequence (miRNA-203) and aptamer-specific ligand (ATP) as target molecules for this proof-of-concept experiment. Initiator sequence (DNA2) was released from the capture probe (MNP/DNA1/2 conjugates) under the strong competitiveness of miRNA-203. Hairpin DNA (H1 and H2) can be complementary with the help of initiator DNA2 to form GNP-H1/GNP-H2 aggregates. The absorption ratio (A620/A520) values of solutions were a sensitive function of miRNA-203 concentration covering from 1.0 × 10-11 M to 9.0 × 10-10 M, and as low as 1.0 × 10-11 M could be detected. At the same time, the color changed from light wine red to purple and then to light blue have occurred in the solution. For ATP, initiator sequence (5'-end of DNA3) was released from the capture probe (DNA3) under the strong combination of aptamer-ATP. The present colorimetric for specific detection of ATP exhibited good sensitivity and 1.0 × 10-8 M ATP could be detected. The proposed strategy also showed good performances for qualitative analysis and quantitative analysis of intracellular nucleic acids and aptamer-specific ligands. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Camera-based ratiometric fluorescence transduction of nucleic acid hybridization with reagentless signal amplification on a paper-based platform using immobilized quantum dots as donors.

    Science.gov (United States)

    Noor, M Omair; Krull, Ulrich J

    2014-10-21

    Paper-based diagnostic assays are gaining increasing popularity for their potential application in resource-limited settings and for point-of-care screening. Achievement of high sensitivity with precision and accuracy can be challenging when using paper substrates. Herein, we implement the red-green-blue color palette of a digital camera for quantitative ratiometric transduction of nucleic acid hybridization on a paper-based platform using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). A nonenzymatic and reagentless means of signal enhancement for QD-FRET assays on paper substrates is based on the use of dry paper substrates for data acquisition. This approach offered at least a 10-fold higher assay sensitivity and at least a 10-fold lower limit of detection (LOD) as compared to hydrated paper substrates. The surface of paper was modified with imidazole groups to assemble a transduction interface that consisted of immobilized QD-probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as an acceptor. A hybridization event that brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs was responsible for a FRET-sensitized emission from the acceptor dye, which served as an analytical signal. A hand-held UV lamp was used as an excitation source and ratiometric analysis using an iPad camera was possible by a relative intensity analysis of the red (Cy3 photoluminescence (PL)) and green (gQD PL) color channels of the digital camera. For digital imaging using an iPad camera, the LOD of the assay in a sandwich format was 450 fmol with a dynamic range spanning 2 orders of magnitude, while an epifluorescence microscope detection platform offered a LOD of 30 fmol and a dynamic range spanning 3 orders of magnitude. The selectivity of the hybridization assay was demonstrated by detection of a single nucleotide polymorphism at a contrast ratio of 60:1. This work provides an

  14. A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods.

    Science.gov (United States)

    Nixon, Gavin J; Svenstrup, Helle F; Donald, Carol E; Carder, Caroline; Stephenson, Judith M; Morris-Jones, Stephen; Huggett, Jim F; Foy, Carole A

    2014-12-01

    Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These 'isothermal' methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

  15. A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods

    Directory of Open Access Journals (Sweden)

    Gavin J. Nixon

    2014-12-01

    Full Text Available Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR. There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These ‘isothermal’ methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT, akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

  16. Medical Devices; Immunology and Microbiology Devices; Classification of the Nucleic Acid-Based Device for the Amplification, Detection, and Identification of Microbial Pathogens Directly From Whole Blood Specimens. Final order.

    Science.gov (United States)

    2017-10-16

    The Food and Drug Administration (FDA or we) is classifying the nucleic acid-based device for the amplification, detection, and identification of microbial pathogens directly from whole blood specimens into class II (special controls). The special controls that apply to the device type are identified in this order and will be part of the codified language for the nucleic acid-based device for the amplification, detection, and identification of microbial pathogens directly from whole blood specimens' classification. We are taking this action because we have determined that classifying the device into class II (special controls) will provide a reasonable assurance of safety and effectiveness of the device. We believe this action will also enhance patients' access to beneficial innovative devices, in part by reducing regulatory burdens.

  17. Nucleic Acid Biosensor Synthesis of an All-in-One Universal Blocking Linker Recombinase Polymerase Amplification with a Peptide Nucleic Acid-Based Lateral Flow Device for Ultrasensitive Detection of Food Pathogens.

    Science.gov (United States)

    Xu, Yuancong; Wei, Yujun; Cheng, Nan; Huang, Kunlun; Wang, Weiran; Zhang, Li; Xu, Wentao; Luo, Yunbo

    2018-01-02

    In this study, a whole-course nucleic-acid-constructed biosensor that combines the all-in-one concepts of the universal blocking linker recombinase polymerase amplification (UBLRPA) and a peptide nucleic acid (PNA)-based lateral flow device (PLFD) has been developed for the ultrasensitive detection of food pathogens. Using the preamplification UBLRPA principle, a universal linker and C3 space blocker were utilized to produce the universal linker single-duplex DNA products. The developed amplification system was employed to convert duplex products to a single strand. In the signal recognition strategy, a special PNA probe was successfully employed in the portable PLFD. The UBLRPA products were identified visually using the PLFD through dual hybridization (a PNA probe on the gold nanoparticle (Au-NP) was combined with a universal linker on the end of the products; a PNA capture probe was used on the test line and a universal linker on the other end of the products). The accumulation of Au-NPs produced a characteristic red band, enabling the visual detection of a food pathogen without further testing. To demonstrate the value of the all-in-one biosensor, Salmonella enterica subsp. enterica serovar typhimurium was used as a model organism. The biosensor showed high selectivity and extraordinary repeatability using S. typhimurium, and the limit of detection was 4 CFU mL -1 . Furthermore, when milk samples artificially contaminated with S. typhimurium were analyzed, the analysis was completed within 30 min without complicated instrumentation. The results exhibited good precision and recovery. This portable all-in-one biosensor demonstrates great promise for the screening of pathogens in food and environmental samples.

  18. Toxicity of the Alternaria metabolites alternariol, alternariol methyl ether, altenuene, and tenuazonic acid in the chicken embryo assay.

    OpenAIRE

    Griffin, G F; Chu, F S

    1983-01-01

    The effects in the chicken embryo assay of four Alternaria metabolites (alternariol [AOH], alternariol methyl ether [AME], altenuene [ALT], and tenuazonic acid [TA]) were investigated. Administered to 7-day-old chicken embryos by yolk sac injection, AOH, AME, and ALT caused no mortality or teratogenic effect at doses up to 1,000, 500, and 1,000 micrograms per egg, respectively. TA exhibited a calculated 50% lethal dose of 548 micrograms per egg, with no teratogenic effect observed at either l...

  19. Increased Detection of Pharyngeal and Rectal Gonorrhea in Men Who Have Sex With Men After Transition From Culture To Nucleic Acid Amplification Testing.

    Science.gov (United States)

    Cornelisse, Vincent J; Chow, Eric P F; Huffam, Sarah; Fairley, Christopher K; Bissessor, Melanie; De Petra, Vesna; Howden, Benjamin P; Denham, Ian; Bradshaw, Catriona S; Williamson, Deborah; Chen, Marcus Y

    2017-02-01

    This before-and-after study measured the impact of a change in testing methods from culture to nucleic acid amplification testing (NAAT) on the detection of pharyngeal and rectal gonorrhea in men who have sex with men (MSM) on a sexual health service level, including the effect on subgroups anticipated to have higher rates of gonorrhea. In March 2015, Melbourne Sexual Health Centre changed its laboratory method for gonococcal testing from culture to NAAT using the Aptima Combo 2 and Aptima GC tests. We compared the proportion of tests positive for rectal and pharyngeal gonorrhea in MSM using culture in 2014 with those using NAAT in 2015. The proportion of tests positive for rectal gonorrhea by NAAT was double that obtained by culture (8% vs 3.9%; prevalence ratio [PR], 2.0; 95% confidence interval [CI], 1.8-2.4) and 5-fold for pharyngeal gonorrhea (8.3% vs 1.6%; PR, 5.2; 95% CI, 4.2-6.4). Similar increases in test positivity were observed in human immunodeficiency virus (HIV)-positive and HIV-negative men. By NAAT, test positivity for rectal gonorrhea was higher in HIV-positive compared with HIV-negative men (15.4% vs 7.3%; PR, 2.1; 95% CI, 1.7-2.6). Culture and NAAT had similar test positivity for rectal gonorrhea among men who reported contact with gonorrhea (24.9% vs 25.3%, PR 1.0, 95% CI 0.8-1.4) and men who presented with symptoms of proctitis (22.2% vs 27.9%, PR 1.3, 95% CI 0.8-2.0). A switch from culture to Aptima Combo 2 testing for extragenital gonorrhea in MSM increased detection and was most marked for pharyngeal infections.

  20. Effects of humic acid on DNA quantification with Quantifiler® Human DNA Quantification kit and short tandem repeat amplification efficiency.

    Science.gov (United States)

    Seo, Seung Bum; Lee, Hye Young; Zhang, Ai Hua; Kim, Hye Yeon; Shin, Dong Hoon; Lee, Soong Deok

    2012-11-01

    Correct DNA quantification is an essential part to obtain reliable STR typing results. Forensic DNA analysts often use commercial kits for DNA quantification; among them, real-time-based DNA quantification kits are most frequently used. Incorrect DNA quantification due to the presence of PCR inhibitors may affect experiment results. In this study, we examined the alteration degree of DNA quantification results estimated in DNA samples containing a PCR inhibitor by using a Quantifiler® Human DNA Quantification kit. For experiments, we prepared approximately 0.25 ng/μl DNA samples containing various concentrations of humic acid (HA). The quantification results were 0.194-0.303 ng/μl at 0-1.6 ng/μl HA (final concentration in the Quantifiler reaction) and 0.003-0.168 ng/μl at 2.4-4.0 ng/μl HA. Most DNA quantity was undetermined when HA concentration was higher than 4.8 ng/μl HA. The C (T) values of an internal PCR control (IPC) were 28.0-31.0, 36.5-37.1, and undetermined at 0-1.6, 2.4, and 3.2 ng/μl HA. These results indicate that underestimated DNA quantification results may be obtained in the DNA sample with high C (T) values of IPC. Thus, researchers should carefully interpret the DNA quantification results. We additionally examined the effects of HA on the STR amplification by using an Identifiler® kit and a MiniFiler™ kit. Based on the results of this study, it is thought that a better understanding of various effects of HA would help researchers recognize and manipulate samples containing HA.

  1. A touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections

    Directory of Open Access Journals (Sweden)

    Feeney Susan A

    2004-10-01

    Full Text Available Abstract Background Immunofluorescence and virus culture are the main methods used to diagnose acute respiratory virus infections. Diagnosing these infections using nucleic acid amplification presents technical challenges, one of which is facilitating the different optimal annealing temperatures needed for each virus. To overcome this problem we developed a diagnostic molecular strip which combined a generic nested touchdown protocol with in-house primer master-mixes that could recognise 12 common respiratory viruses. Results Over an 18 month period a total of 222 specimens were tested by both immunofluorescence and the molecular strip. The specimens came from 103 males (median age 3.5 y, 80 females (median age 9 y and 5 quality assurance scheme specimens. Viruses were recovered from a number of specimen types including broncho-alveolar lavage, nasopharyngeal secretions, sputa, post-mortem lung tissue and combined throat and nasal swabs. Viral detection by IF was poor in sputa and respiratory swabs. A total of 99 viruses were detected in the study from 79 patients and 4 quality control specimens: 31 by immunofluorescence and 99 using the molecular strip. The strip consistently out-performed immunofluorescence with no loss of diagnostic specificity. Conclusions The touchdown protocol with pre-dispensed primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses which were negative by immunofluorescence. Results by immunofluorescence were available after an average of 4–12 hours while molecular strip results were available within 24 hours, considerably faster than viral culture. The combined strip and touchdown protocol proved to be a convenient and reliable method of testing for multiple viruses in a routine setting.

  2. A novel method for the purification of DNA by capturing nucleic acid and magnesium complexes on non-woven fabric filters under alkaline conditions for the gene diagnosis of tuberculosis by loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Fukasawa, Tadashi; Oda, Naozumi; Wada, Yasunao; Tamaru, Aki; Fukushima, Yukari; Nakajima, Chie; Suzuki, Yasuhiko

    2010-07-01

    A novel method for purifying DNA from clinical samples based on the complex formation of DNA and magnesium ion (Mg(2+)) was developed for the detection of Mycobacterium tuberculosis. The formation of a DNA-Mg(2+) complex under alkaline conditions was observed by analyzing electrophoretic mobility reduction of DNA on agarose gel. The DNA-Mg(2+) complex increases the efficacy of DNA recovery from the sample solution on polyethylene terephthalate non-woven fabric (PNWF) filters. Among the various divalent metal cations, only Mg(2+) was associated with this effect. The applicability of DNA recovered on the PNWF filter was examined for the gene amplification method; loop-mediated isothermal amplification (LAMP). DNA on the PNWF filter could be used for the amplification of specific DNA fragments without elution from the filter. Using this method, DNA was directly purified from M. tuberculosis spiked sputum and examined by LAMP assay, showing a high sensitivity in comparison to the commercially available DNA extraction kit. These results indicated that the method developed in this study is useful for rapid gene diagnosis of tuberculosis.

  3. Molecularly imprinted titania nanoparticles for selective recognition and assay of uric acid

    Science.gov (United States)

    Mujahid, Adnan; Khan, Aimen Idrees; Afzal, Adeel; Hussain, Tajamal; Raza, Muhammad Hamid; Shah, Asma Tufail; uz Zaman, Waheed

    2015-06-01

    Molecularly imprinted titania nanoparticles are su ccessfully synthesized by sol-gel method for the selective recognition of uric acid. Atomic force microscopy is used to study the morphology of uric acid imprinted titania nanoparticles with diameter in the range of 100-150 nm. Scanning electron microscopy images of thick titania layer indicate the formation of fine network of titania nanoparticles with uniform distribution. Molecular imprinting of uric acid as well as its subsequent washing is confirmed by Fourier transformation infrared spectroscopy measurements. Uric acid rebinding studies reveal the recognition capability of imprinted particles in the range of 0.01-0.095 mmol, which is applicable in monitoring normal to elevated levels of uric acid in human blood. The optical shift (signal) of imprinted particles is six times higher in comparison with non-imprinted particles for the same concentration of uric acid. Imprinted titania particles have shown substantially reduced binding affinity toward interfering and structurally related substances, e.g. ascorbic acid and guanine. These results suggest the possible application of titania nanoparticles in uric acid recognition and quantification in blood serum.

  4. Relationship between human semen parameters and deoxyribonucleic acid damage assessed by the neutral comet assay.

    Science.gov (United States)

    Trisini, Ana T; Singh, Narendra P; Duty, Susan M; Hauser, Russ

    2004-12-01

    To explore the association between semen parameters and sperm DNA damage. Cross-sectional. Andrology clinic. Two hundred fifty-seven men undergoing infertility assessment. None. Sperm concentration and motility were measured using computer-assisted sperm analysis; morphology was scored using the strict criteria. The neutral comet assay was used to measure sperm DNA damage. Comet assay parameters included comet extent, percent DNA in the comet tail, and tail distributed moment, an integrated measure of length and intensity. We also scored cells that were too long to measure (>300 microm), which we referred to as cells with high DNA damage. Men older than 35 years had a statistically significant increase in the number of cells with high DNA damage as compared with younger men. In age-adjusted regression analyses, the most consistent associations were found between semen parameters and the number of cells with high DNA damage. For an interquartile range change in the number of cells with high DNA damage, sperm concentration declined 14.2 x 10(6)/mL, motility declined 4.3%, and morphology declined 0.5%. Comet extent and percent DNA in the comet tail were also associated with a decline in sperm concentration and motility, respectively. Although there were associations between semen and comet assay parameters, their magnitudes were weak, suggesting that the comet assay provides additional independent information on sperm function.

  5. Comet assay in evaluating deoxyribonucleic acid damage after out-of-hospital cardiac arrest

    Science.gov (United States)

    Hazuková, Radka; Řezáčová, Martina; Köhlerová, Renata; Tomek, Tomáš; Čermáková, Eva; Kočí, Jaromír; Pleskot, Miloslav

    2017-01-01

    Objective: This study aimed to investigate whether out-of-hospital cardiac arrest (OHCA) may induce severe DNA damage measured using comet assay in successfully resuscitated humans and to evaluate a short-term prognostic role. Methods: In this prospective, controlled, blinded study (1/2013–1/2014), 41 patients (age, 63±14 years) successfully resuscitated from non-traumatic OHCA and 10 healthy controls (age, 53±17 years) were enrolled. DNA damage [double-strand breaks (DSBs) and single-strand breaks (SSBs)] was measured using comet assay in peripheral lymphocytes sampled at admission. Clinical data were recorded (according to Utstein style). A good short-term prognosis was defined as survival for 30 days. Results: Among the patients, there were 71% (29/41) short-term survivors. After OHCA, DNA damage (DSBs and SSBs) was higher (11.0±7.6% and 0.79±2.41% in tail) among patients than among controls (1.96±1.63% and 0.02±0.03% in tail), and it was more apparent for DSBs (pcomet assay in patients successfully resuscitated from OHCA. Whether DNA damage measured using comet assay may be a prognostic marker remains unknown, although our data may encourage some suggestions. PMID:28639949

  6. Assay of urinary protein-bound sialic acid can differentiate steroidsensitive nephrotic syndrome from steroid-resistant cases

    Directory of Open Access Journals (Sweden)

    Niranjan Gopal

    2016-01-01

    Full Text Available The protein selectivity index as measured from the ratio of urinary immunoglobulin to albumin failed to differentiate between steroid-sensitive (SS and steroid-resistant (SR cases of nephrotic syndrome (NS. Sialic acid contributes negative charges to many plasma proteins. The negative charge is a determinant of protein excretion rate. The prognostic significance of assay of urinary excretion of protein-bound sialic acid in NS has not been evaluated. Hence, the present study was designed to evaluate whether measurement of urinary protein bound sialic acid (UPBSA can be used as a marker to differentiate SS from SR cases of NS. The urine samples of 70 (47 SS and 23 SR pediatric NS children were assayed for UPBSA by Aminoff′s method. The levels were compared and the receiver-operator curve was drawn to determine the optimum cutoff point to differentiate among the groups before starting the therapy. The excretion of UPBSA in SR cases of NS was significantly higher than that of SS cases (P<0.05. The optimum cutoff limit for UPBSA was 2.71 μg/mg of proteins with 75% sensitivity and 75.5% specificity for differentiating SS cases from SR cases (area under the plasma- concentration time curve = 0.814, P = 0.009. We conclude that UPBSA can differentiate SR cases from SS cases of NS in pediatric patients and may help in predicting the response to steroid therapy.

  7. Simultaneous determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in dried blood spots: Second-tier LC-MS/MS assay for newborn screening of propionic acidemia, methylmalonic acidemias and combined remethylation disorders.

    Directory of Open Access Journals (Sweden)

    Péter Monostori

    Full Text Available Increased propionylcarnitine levels in newborn screening are indicative for a group of potentially severe disorders including propionic acidemia (PA, methylmalonic acidemias and combined remethylation disorders (MMACBL. This alteration is relatively non-specific, resulting in the necessity of confirmation and differential diagnosis in subsequent tests. Thus, we aimed to develop a multiplex approach for concurrent determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid from the same dried blood spot (DBS as in primary screening (second-tier test. We also set out to validate the method using newborn and follow-up samples of patients with confirmed PA or MMACBL.The assay was developed using liquid chromatography-tandem mass spectrometry and clinically validated with retrospective analysis of DBS samples from PA or MMACBL patients.Reliable determination of all three analytes in DBSs was achieved following simple and fast (<20 min sample preparation without laborious derivatization or any additional pipetting steps. The method clearly distinguished the pathological and normal samples and differentiated between PA and MMACBL in all stored newborn specimens. Methylcitric acid was elevated in all PA samples; 3-hydroxypropionic acid was also high in most cases. Methylmalonic acid was increased in all MMACBL specimens; mostly together with methylcitric acid.A liquid chromatography-tandem mass spectrometry assay allowing simultaneous determination of the biomarkers 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in DBSs has been developed. The assay can use the same specimen as in primary screening (second-tier test which may reduce the need for repeated blood sampling. The presented preliminary findings suggest that this method can reliably differentiate patients with PA and MMACBL in newborn screening. The validated assay is being evaluated prospectively in a pilot project for extension of the German newborn

  8. Simultaneous determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in dried blood spots: Second-tier LC-MS/MS assay for newborn screening of propionic acidemia, methylmalonic acidemias and combined remethylation disorders.

    Science.gov (United States)

    Monostori, Péter; Klinke, Glynis; Richter, Sylvia; Baráth, Ákos; Fingerhut, Ralph; Baumgartner, Matthias R; Kölker, Stefan; Hoffmann, Georg F; Gramer, Gwendolyn; Okun, Jürgen G

    2017-01-01

    Increased propionylcarnitine levels in newborn screening are indicative for a group of potentially severe disorders including propionic acidemia (PA), methylmalonic acidemias and combined remethylation disorders (MMACBL). This alteration is relatively non-specific, resulting in the necessity of confirmation and differential diagnosis in subsequent tests. Thus, we aimed to develop a multiplex approach for concurrent determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid from the same dried blood spot (DBS) as in primary screening (second-tier test). We also set out to validate the method using newborn and follow-up samples of patients with confirmed PA or MMACBL. The assay was developed using liquid chromatography-tandem mass spectrometry and clinically validated with retrospective analysis of DBS samples from PA or MMACBL patients. Reliable determination of all three analytes in DBSs was achieved following simple and fast (<20 min) sample preparation without laborious derivatization or any additional pipetting steps. The method clearly distinguished the pathological and normal samples and differentiated between PA and MMACBL in all stored newborn specimens. Methylcitric acid was elevated in all PA samples; 3-hydroxypropionic acid was also high in most cases. Methylmalonic acid was increased in all MMACBL specimens; mostly together with methylcitric acid. A liquid chromatography-tandem mass spectrometry assay allowing simultaneous determination of the biomarkers 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in DBSs has been developed. The assay can use the same specimen as in primary screening (second-tier test) which may reduce the need for repeated blood sampling. The presented preliminary findings suggest that this method can reliably differentiate patients with PA and MMACBL in newborn screening. The validated assay is being evaluated prospectively in a pilot project for extension of the German newborn screening

  9. Dinitrophenyl derivatization of imino acids, spectral characteristics and HPLC analysis: application in urinary peptide-derived hydroxyproline and proline assay.

    Science.gov (United States)

    Badadani, Mallikarjun; Babu, S V Suresh; Shetty, K Taranath

    2007-03-01

    Assay of urinary imino acids, in particular peptide derived, is of immense utility in diagnosis of collagen-related disorders. The often-used methods for hydrolysis of urinary peptides need a long time and are cumbersome, hence the need for relatively simpler, but effective methods. The method described, based on alkaline hydrolysis by autoclaving for 60 min followed by pre-column dinitrophenyl (DNP) derivatization and high-performance liquid chromatography (HPLC) analysis, demonstrates the complete hydrolysis and stability of urinary peptide derived imino acids. DNP derivatives of both imino acids had identical lambda max (380 nm) with molar epsilon of 28.224 x 10(3) and 17.036 x 10(3), respectively, for hydroxyproline (Hyp) and proline (Pro). HPLC run, extending up to 18 min, resolved major components of collagen products, namely Hyp, Hyl, Gly, Pro and Lys, with retention times of 6.5, 9.8, 10.5, 11.2 and 12.55 min, respectively. The assay method conformed to linear response for individual amino acid concentrations of 0.5-4.0 nmol per injection, with goodness of fit (r(2) value) 0.99 for both Hyp and Pro, and detection limit of 0.05-4.0 nmol of DNP derivatives. The recovery of Pro and Hyp, when spiked with urine prior to hydrolysis, were found to be 95% and 92%, respectively. Alkaline hydrolysis by autoclaving and DNP derivatization of imino acids followed by HPLC provides a method for the analysis of peptide-derived Hyp and Pro in urine. Hence, it is of utility to study collagen disorders.

  10. Non-aqueous titrimetric assay of gabapentin in capsules using perchloric acid as titrant

    OpenAIRE

    Sameer A. M. Abdulrahman; KANAKAPURA BASAVAIAH

    2011-01-01

    Two simple, rapid, accurate and inexpensive methods using visual and potentiometric titrimetric techniques are described for the determination of gabapentin (GBP) in bulk drug as well as in capsules. The methods are based on the neutralization reaction of the primary amino group of GBP with acetous perchloric acid as titrant in anhydrous acetic acid medium. The end point was detected either visually using crystal violet as indicator or potentiometrically using a modified glass electrode SCE e...

  11. A Cross-Reactivity of Fenofibric Acid With MDMA DRI Assay.

    Science.gov (United States)

    Bugier, Sarah; Garcia-Hejl, Carine; Vest, Philippe; Plantamura, Julie; Chianea, Denis; Renard, Christophe

    2016-09-01

    Within the framework of routine fitness examinations, French Air Force military crew underwent urine testing for 3,4 methylenedioxymetamphetamine (MDMA [ecstasy]). The cross-reactivity of a dyslipidemic drug, fenofibrate, with an MDMA immunoassay was studied and confirmed on a large population sample. A 3-year retrospective study was performed on the MDMA DRI Ecstasy Assay on the Unicel DXC 600. In the event of positive test result, a confirmatory testing was carried out by gas chromatography/mass spectrometry (GC/MS) to establish the presence of MDMA. When analysis by GC/MS did not confirm the presence of MDMA, a false-positive result was suspected and the samples were analyzed by high-performance liquid chromatography-mass spectrometry to identify a potential interfering substance. A total of 15,169 urine samples, from 7,803 patients, were tested for 3 years. Of the tested samples, 22 (0.15%) were positive by DRI Ecstasy Assay. None of them were positive by GC/MS. A cross-reactivity of fenofibrate's metabolite with MDMA using this assay was systematically found. Fenofibrate's interference with MDMA immunoassay was confirmed. Fenofibrate being widely prescribed, physicians had to be alerted that this treatment could lead to false-positive results. Reprint & Copyright © 2016 Association of Military Surgeons of the U.S.

  12. Pelvic inflammatory disease risk following negative results from chlamydia nucleic acid amplification tests (NAATs) versus non-NAATs in Denmark: A retrospective cohort.

    Science.gov (United States)

    Davies, Bethan; Turner, Katy M E; Benfield, Thomas; Frølund, Maria; Andersen, Berit; Westh, Henrik; Ward, Helen

    2018-01-01

    Nucleic Acid Amplification Tests (NAATs) are the recommended test type for diagnosing Chlamydia trachomatis (chlamydia). However, less sensitive diagnostic methods-including direct immunofluorescence (IF) and enzyme-linked immunoassay (ELISA)-remain in use in lower resourced settings. We estimate the risk of pelvic inflammatory disease (PID) following undiagnosed infection in women tested with non-NAATs and estimate the health gain from using accurate diagnostic tests. We used Denmark's national Chlamydia Study dataset to extract all chlamydia tests performed in women aged 15-34 years (1998-2001). Tests were categorised as non-NAAT (IF/ELISA) or NAAT and limited to each woman's first test in the study period. We linked test data to hospital presentations for PID within 12 months from the Danish National Patient Register. The study included 272,105 women with a chlamydia test, just under half (44.78%, n = 121,857) were tested using NAATs. Overall, 6.38% (n = 17,353) tested positive for chlamydia and 0.64% (n = 1,732) were diagnosed with PID within 12 months. The risk of PID following a positive chlamydia test did not differ by test type (NAAT 0.81% [95% CI 0.61-1.00], non-NAAT 0.78% [0.59-0.96]). The risk of PID following a negative test was significantly lower in women tested with NAATs compared to non-NAATs (0.55% [0.51-0.59] compared to 0.69% [0.64-0.73]; adjusted odds ratio (AOR) 0.83 [0.75-0.93]). We estimate that 18% of chlamydia infections in women tested with a non-NAAT were undiagnosed and that the risk of progression from undiagnosed chlamydia infection to PID within 12 months was 9.52% (9.30-9.68). Using non-NAATs could lead to an excess 120 cases of PID per 100,000 women tested compared to using NAATs. The key limitations of this study are under ascertainment of PID cases, misclassification bias in chlamydia and PID exposure status, bias to the association between clinical presentation and test type and the presence of unmeasured confounders (including

  13. Toxicity of the Alternaria metabolites alternariol, alternariol methyl ether, altenuene, and tenuazonic acid in the chicken embryo assay.

    Science.gov (United States)

    Griffin, G F; Chu, F S

    1983-12-01

    The effects in the chicken embryo assay of four Alternaria metabolites (alternariol [AOH], alternariol methyl ether [AME], altenuene [ALT], and tenuazonic acid [TA]) were investigated. Administered to 7-day-old chicken embryos by yolk sac injection, AOH, AME, and ALT caused no mortality or teratogenic effect at doses up to 1,000, 500, and 1,000 micrograms per egg, respectively. TA exhibited a calculated 50% lethal dose of 548 micrograms per egg, with no teratogenic effect observed at either lethal or sublethal doses.

  14. Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.

    Directory of Open Access Journals (Sweden)

    David S Boyle

    Full Text Available Improved access to effective tests for diagnosing tuberculosis (TB has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110 and 20 fg (IS1081were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9 and 86.1% (95%CI: 78.1, 94.1 respectively (n = 71. Specificities were 100% and 88.6% (95% CI: 80.8, 96.1 respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2 and 70.8% (95%CI: 62.9, 78.7 were obtained (n = 90. Specificities were 95.4 (95% CI: 92.3,98.1 and 88% (95% CI: 83.6, 92.4 respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB

  15. Pharmacological characterization of human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 in a fluorescence-based membrane potential assay

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Bräuner-Osborne, Hans

    2004-01-01

    We have expressed the human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 stably in HEK293 cells and characterized the transporters pharmacologically in a conventional [(3) H]-d-aspartate uptake assay and in a fluorescence-based membrane potential assay, the FLIPR Membrane Potential (...

  16. Ninhydrin-sodium molybdate chromogenic analytical probe for the assay of amino acids and proteins

    Science.gov (United States)

    Anantharaman, Shivakumar; Padmarajaiah, Nagaraja; Al-Tayar, Naef Ghllab Saeed; Shrestha, Ashwinee Kumar

    2017-02-01

    A sensitive method has been proposed for the quantification of amino acids and proteins using ninhydrin and sodium molybdate as chromogenic substrates in citrate buffer of pH 5.6. A weak molybdate-hydrindantin complex plays the role in the formation of Ruhemann's purple. The linear response for the amino acid, amino acid mixture and Bovine serum albumin is between 0.999 and 66.80 μM, 1.52 and 38 μM and 5 and 100 μg/L, respectively. The molar absorptivity of the individual amino acid by the proposed reaction extends from 0.58 × 104 to 2.86 × 104 M- 1 cm- 1. The linearity equations for the proposed ninhydrin-molybdate for amino acid mixture is Abs = 0.021 × Conc (μM) - 0.002. The applicability of the proposed method has been justified in food and biological samples in conjunction with Kjeldahl method.

  17. Direct loop mediated isothermal amplification on filters for quantification of Dehalobacter in groundwater.

    Science.gov (United States)

    Stedtfeld, Robert D; Stedtfeld, Tiffany M; Samhan, Farag; Kanitkar, Yogendra H; Hatzinger, Paul B; Cupples, Alison M; Hashsham, Syed A

    2016-12-01

    Nucleic acid amplification of biomarkers is increasingly used to monitor microbial activity and assess remedial performance in contaminated aquifers. Previous studies described the use of filtration, elution, and direct isothermal amplification (i.e. no DNA extraction and purification) as a field-able means to quantify Dehalococcoides spp. in groundwater. This study expands previous work with direct loop mediated isothermal amplification (LAMP) for the detection and quantification of Dehalobacter spp. in groundwater. Experiments tested amplification of DNA with and without crude lysis and varying concentrations of humic acid. Three separate field-able methods of biomass concentration with eight aquifer samples were also tested, comparing direct LAMP with traditional DNA extraction and quantitative PCR (qPCR). A new technique was developed where filters were amplified directly within disposable Gene-Z chips. The direct filter amplification (DFA) method eliminated an elution step and provided a detection limit of 10(2)Dehalobacter cells per 100mL. LAMP with crudely lysed Dehalobacter had a negligible effect on threshold time and sensitivity compared to lysed samples. The LAMP assay was more resilient than traditional qPCR to humic acid in sample, amplifying with up to 100mg per L of humic acid per reaction compared to 1mg per L for qPCR. Of the tested field-able concentrations methods, DFA had the lowest coefficient of variation among Dehalobacter spiked groundwater samples and lowest threshold time indicating high capture efficiency and low inhibition. While demonstrated with Dehalobacter, the DFA method can potentially be used for a number of applications requiring field-able, rapid (<60min) and highly sensitive quantification of microorganisms in environmental water samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. HPLC assay of acetylsalicylic acid, paracetamol, caffeine and phenobarbital in tablets.

    Science.gov (United States)

    Franeta, J T; Agbaba, D; Eric, S; Pavkov, S; Aleksic, M; Vladimirov, S

    2002-09-01

    This paper present a HPLC method for simultaneous determination of acetylsalicylic acid, paracetamol, caffeine and phenobarbital in tablets, using chromatographic system consisting a Bio Rad 18 01 solvent pump, Rheodine 71 25 injector and Bio Rad 18 01 UV-Vis Detector. Separation was achieved using Bio SiL HL C18, 5 microm, 250 x 4.6 mm column. Mixture of acetonitrile-water (25:75 v/v) adjusted to pH 2.5 with phosphoric acid was used as a mobile phase at a flow rate of 2.0 ml min(-1). UV detection was at 207 nm range 0.01 AUFS. Under the same conditions it was possible to determine the level of salicylic acid. The chromatographic parameters such as retention times, capacity factor, peak asymmetry, selectivity factor and resolution factor was determined. The validation parameters: linearity (r > 0.998), intra-day precision (RSD: 0.36-1.89%) and inter-day precision (RSD: 0.58-2.18%), sensitivity (LOD: 9 x 10(-5)-1.7 x 10(-4) mg ml(-1) and LOQ: 2.5 x 10(-4)-5.6 x 10(-4) mg ml(-1)), accuracy (recoveries: 98.35-99.14%) and reproducibility (recovery values: 98.74-102.08% for acetylsalicylic acid, 99.93-102.11% for paracetamol, 98.25-102.12% for caffeine and 98.15-102.3% for phenobarbital) (RSD: 1.21-1.85%) were found to be satisfactory. The proposed HPLC method has been applied for the determination of acetylsalicylic acid, paracetamol, caffeine and phenobarbital in Malophenum tablets. The obtained RSD values were within 0.99-1.21%. The developed method is rapid and sensitive and therefore suitable for routine control of these drugs in dosage form.

  19. Rapid, Fully Automated Digital Immunoassay for p24 Protein with the Sensitivity of Nucleic Acid Amplification for Detecting Acute HIV Infection.

    Science.gov (United States)

    Cabrera, Carlos; Chang, Lei; Stone, Mars; Busch, Michael; Wilson, David H

    2015-11-01

    Nucleic acid testing (NAT) has become the standard for high sensitivity in detecting low levels of virus. However, adoption of NAT can be cost prohibitive in low-resource settings where access to extreme sensitivity could be clinically advantageous for early detection of infection. We report development and preliminary validation of a simple, low-cost, fully automated digital p24 antigen immunoassay with the sensitivity of quantitative NAT viral load (NAT-VL) methods for detection of acute HIV infection. We developed an investigational 69-min immunoassay for p24 capsid protein for use on a novel digital analyzer on the basis of single-molecule-array technology. We evaluated the assay for sensitivity by dilution of standardized preparations of p24, cultured HIV, and preseroconversion samples. We characterized analytical performance and concordance with 2 NAT-VL methods and 2 contemporary p24 Ag/Ab combination immunoassays with dilutions of viral isolates and samples from the earliest stages of HIV infection. Analytical sensitivity was 0.0025 ng/L p24, equivalent to 60 HIV RNA copies/mL. The limit of quantification was 0.0076 ng/L, and imprecision across 10 runs was 4000-fold greater sensitivity than contemporary immunoassays for p24 and sensitivity equivalent to that of NAT methods for early detection of HIV. The data indicate that NAT-level sensitivity for acute HIV infection is possible with a simple, low-cost digital immunoassay. © 2015 American Association for Clinical Chemistry.

  20. Comparison of EMIT II, CEDIA, and DPC RIA assays for the detection of lysergic acid diethylamide in forensic urine samples.

    Science.gov (United States)

    Wiegand, Russell F; Klette, Kevin L; Stout, Peter R; Gehlhausen, Jay M

    2002-10-01

    In an effort to determine a practical, efficient, and economical alternative for the use of a radioimmunoassay (RIA) for the detection of lysergic acid diethylamide (LSD) in human urine, the performance of two photometric immunoassays (Dade Behring EMIT II and Microgenics CEDIA) and the Diagnostics Products Corp. (DPC) RIA were compared. Precision, accuracy, and linearity of the 3 assays were determined by testing 60 replicates (10 for RIA) at 5 different concentrations below and above the 500-pg/mL LSD cut-off. The CEDIA and RIA exhibited better accuracy and precision than the EMIT II immunoassay. In contrast, the EMIT II and CEDIA demonstrated superior linearity r2 = 0.9809 and 0.9540, respectively, as compared with the RIA (r2 = 0.9062). The specificity of the three assays was assessed using compounds that have structural and chemical properties similar to LSD, common over-the-counter products, prescription drugs and some of their metabolites, and other drugs of abuse. Of the 144 compounds studied, the EMIT II cross-reacted with twice as many compounds as did the CEDIA and RIA. Specificity was also assessed in 221 forensic human urine specimens that previously screened positive for LSD by the EMIT II assay. Of these, only 11 tested positive by CEDIA, and 3 were positive by RIA. This indicated a comparable specificity performance between CEDIA and RIA. This also was consistent with a previously reported high false-positive rate of EMIT II (low specificity). Each of the immunoassays correctly identified LSD in 23 out of 24 human urine specimens that had previously been found to contain LSD by gas chromatography-mass spectrometry at a cut-off concentration of 200 pg/mL. The CEDIA exhibited superior precision, accuracy, and decreased cross-reactivity to compounds other than LSD as compared with the EMIT II assay and does not necessitate the handling of radioactive materials.

  1. Development of betulinic acid as an agonist of TGR5 receptor using a new in vitro assay

    Directory of Open Access Journals (Sweden)

    Lo SH

    2016-08-01

    Full Text Available Shih-Hsiang Lo,1,2 Kai-Chung Cheng,3 Ying-Xiao Li,3,4 Chin-Hong Chang,4,5 Juei-Tang Cheng,4,6 Kung-Shing Lee7,8 1Division of Cardiology, Department of Internal Medicine, Zhongxing Branch of Taipei City Hospital, 2Department of History and Geography, University of Taipei, Taipei, Taiwan; 3Department of Psychosomatic Internal Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan; 4Department of Medical Research, 5Department of Neurosurgery, Chi-Mei Medical Center, Yong Kang, 6Institute of Medical Science, College of Health Science, Chang Jung Christian University, Tainan, 7Department of Surgery, Pingtung Hospital, 8Division of Neurosurgery, Department of Surgery, Kaohsiung Medical University Chung-Ho Memorial Hospital, School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Background: G-protein-coupled bile acid receptor 1, also known as TGR5 is known to be involved in glucose homeostasis. In animal models, treatment with a TGR5 agonist induces incretin secretion to reduce hyperglycemia. Betulinic acid, a triterpenoid present in the leaves of white birch, has been introduced as a selective TGR5 agonist. However, direct activation of TGR5 by betulinic acid has not yet been reported. Methods: Transfection of TGR5 into cultured Chinese hamster ovary (CHO-K1 cells was performed to establish the presence of TGR5. Additionally, TGR5-specific small interfering RNA was employed to silence TGR5 in cells (NCI-H716 cells that secreted incretins. Uptake of glucose by CHO-K1 cells was evaluated using a fluorescent indicator. Amounts of cyclic adenosine monophosphate and glucagon-like peptide were quantified using enzyme-linked immunosorbent assay kits. Results: Betulinic acid dose-dependently increases glucose uptake by CHO-K1 cells transfected with TGR5 only, which can be considered an alternative method instead of radioligand binding assay. Additionally, signals coupled to TGR5 activation are also

  2. Non-aqueous titrimetric assay of gabapentin in capsules using perchloric acid as titrant

    Directory of Open Access Journals (Sweden)

    SAMEER A.M. ABDULRAHMAN

    2011-06-01

    Full Text Available Two simple, rapid, accurate and inexpensive methods using visual and potentiometric titrimetric techniques are described for the determination of gabapentin (GBP in bulk drug as well as in capsules. The methods are based on the neutralization reaction of the primary amino group of GBP with acetous perchloric acid as titrant in anhydrous acetic acid medium. The end point was detected either visually using crystal violet as indicator or potentiometrically using a modified glass electrode SCE electrode system. Both methods are applicable over the range 1.0-16.0 mg of GBP and the titration reaction follows a 1:1 stoichiometry. The methods were successfully applied to the determination of GBP in capsules. The validity of the proposed methods was further ascertained by parallel determination by a reference method and by recovery studies via standard-addition technique.

  3. Colorimetric deoxyribonucleic acid hybridization assay for rapid screening of Salmonella in foods: collaborative study.

    Science.gov (United States)

    Curiale, M S; Klatt, M J; Mozola, M A

    1990-01-01

    A collaborative study was performed in 11 laboratories to validate a colorimetric DNA hybridization (DNAH) method for rapid detection of Salmonella in foods. The method was compared to the standard culture method for detection of Salmonella in nonfat dry milk, milk chocolate, soy isolate, dried whole egg, ground black pepper, and raw ground turkey. Samples inoculated with high (0.4-2 cells/g) and low (0.04-0.2 cells/g) levels of Salmonella and uninoculated control samples were included in each food group analyzed. There was no significant difference in the proportion of samples positive by DNAH and culture procedure for any of the 6 foods. The colorimetric DNA hybridization assay screening method has been adopted official first action as a rapid screening method for detection of Salmonella in all foods.

  4. Real-time detection of noroviruses in surface water by use of a broadly reactive nucleic acid sequence-based amplification assay.

    NARCIS (Netherlands)

    Rutjes, Saskia A; Berg, Harold H J L van den; Lodder, Willemijn J; Roda Husman, Ana Maria de

    2006-01-01

    Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection

  5. Plasmon-enhanced fluorescence imaging with silicon-based silver chips for protein and nucleic acid assay.

    Science.gov (United States)

    Yuan, Bing; Jiang, Xiangxu; Yao, Chu; Bao, Meimei; Liu, Jiaojiao; Dou, Yujiang; Xu, Yinze; He, Yao; Yang, Kai; Ma, Yuqiang

    2017-02-22

    Metal-enhanced fluorescence shows great potential for improving the sensitivity of fluoroscopy, which has been widely used in protein and nucleic acid detection for biosensor and bioassay applications. In comparison with the traditional glass-supported metal nanoparticles (MNPs), the introduction of a silicon substrate has been shown to provide an increased surface-enhanced Raman scattering (SERS) effect due to the coupling between the MNPs and the semiconducting silicon substrate. In this work, we further study the fluorescence-enhanced effect of the silicon-supported silver-island (Ag@Si) plasmonic chips. In particular, we investigate their practical application of improving the traditional immunoassay such as the biotin-streptavidin-based protein assay and the protein-/nucleic acid-labeled cell and tissue samples. The protein assay shows a wavelength-dependent enhancement effect of the Ag@Si chip, with an enhancement factor ranging from 1.2 (at 532 nm) to 57.3 (at 800 nm). Moreover, for the protein- and nucleic acid-labeled cell and tissue samples, the Ag@Si chip provides a fluorescence enhancement factor of 3.0-4.1 (at 800 nm) and a significant improvement in the signal/background ratio for the microscopy images. Such a ready accommodation of the fluorescence-enhanced effect for the immunoassay samples with simple manipulations indicates broad potential for applications of the Ag@Si chip not only in biological studies but also in the clinical field. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Current state and future perspectives of loop-mediated isothermal amplification (LAMP)-based diagnosis of filamentous fungi and yeasts.

    Science.gov (United States)

    Niessen, Ludwig

    2015-01-01

    Loop-mediated isothermal amplification is a rather novel method of enzymatic deoxyribonucleic acid amplification which can be applied for the diagnosis of viruses, bacteria, and fungi. Although firmly established in viral and bacterial diagnosis, the technology has only recently been applied to a noteworthy number of species in the filamentous fungi and yeasts. The current review gives an overview of the literature so far published on the topic by discussing the different groups of fungal organisms to which the method has been applied. Moreover, the method is described in detail as well as the different possibilities available for signal detection and quantification and sample preparation. Future perspective of loop-mediated isothermal amplification-based assays is discussed in the light of applicability for fungal diagnostics.

  7. Novel Signal-Enhancing Approaches for Optical Detection of Nucleic Acids—Going beyond Target Amplification

    Directory of Open Access Journals (Sweden)

    Laura Miotke

    2015-09-01

    Full Text Available Detection of low-abundance nucleic acids is a challenging task, which over the last two decades has been solved using enzymatic target amplification. Enzymatic synthesis enhances the signal so that diverse, scientifically and clinically relevant molecules can be identified and studied, including cancer DNA, viral nucleic acids, and regulatory RNAs. However, using enzymes increases the detection time and cost, not to mention the high risk of mistakes with amplification and data alignment. These limitations have stimulated a growing interest in enzyme-free methods within researchers and industry. In this review we discuss recent advances in signal-enhancing approaches aimed at nucleic acid diagnostics that do not require target amplification. Regardless of enzyme usage, signal enhancement is crucial for the reliable detection of nucleic acids at low concentrations. We pay special attention to novel nanomaterials, fluorescence microscopy, and technical advances in detectors for optical assessment. We summarize sensitivity parameters of the currently available assays and devices which makes this review relevant to the broad spectrum of researchers working in fields from biophysics, to engineering, to synthetic biology and bioorganic chemistry.

  8. Post-Fragmentation Whole Genome Amplification-Based Method

    Science.gov (United States)

    Benardini, James; LaDuc, Myron T.; Langmore, John

    2011-01-01

    This innovation is derived from a proprietary amplification scheme that is based upon random fragmentation of the genome into a series of short, overlapping templates. The resulting shorter DNA strands (fragmentation whole genome amplification-based technology provides a robust and accurate method of amplifying femtogram levels of starting material into microgram yields with no detectable allele bias. The amplified DNA also facilitates the preservation of samples (spacecraft samples) by amplifying scarce amounts of template DNA into microgram concentrations in just a few hours. Based on further optimization of this technology, this could be a feasible technology to use in sample preservation for potential future sample return missions. The research and technology development described here can be pivotal in dealing with backward/forward biological contamination from planetary missions. Such efforts rely heavily on an increasing understanding of the burden and diversity of microorganisms present on spacecraft surfaces throughout assembly and testing. The development and implementation of these technologies could significantly improve the comprehensiveness and resolving power of spacecraft-associated microbial population censuses, and are important to the continued evolution and advancement of planetary protection capabilities. Current molecular procedures for assaying spacecraft-associated microbial burden and diversity have inherent sample loss issues at practically every step, particularly nucleic acid extraction. In engineering a molecular means of amplifying nucleic acids directly from single cells in their native state within the sample matrix, this innovation has circumvented entirely the need for DNA extraction regimes in the sample processing scheme.

  9. Colorimetric Enzymatic Assay of L-Malic Acid Using Dehydrogenase from Baker’s Yeast

    Directory of Open Access Journals (Sweden)

    Cecilia Laluce

    2008-01-01

    Full Text Available A colorimetric method has been developed and optimized to measure L-malic acid in samples of fruit juices and wine. This method is based on oxidation of the analyte, catalyzed by malate dehydrogenase (MDH from dry baker’s yeast, and in combination with the reduction of a tetrazolium salt (MTT: 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide. In the present study, the method exhibited sensitivity in the range of 500–4000 mM of L-malic acid in the reaction cuvette, with the lower detection limit of 6.7·10^-2 g/L, the upper limit of 53.6·10^-2 g/L and a maximum standard deviation of only 2.5 % for the analyzed samples. The MDH activity from baker’s yeast was also optimized, the enzyme showed a high stability at pH=8.0–9.0 and the activity was maintained completely at temperatures up to 40 °C for 1 hour. The results show that the colorimetric method using enzymatic preparations from dry baker’s yeast is a simple and low-cost method with possibility of wide application

  10. World Health Organization International Standard To Harmonize Assays for Detection of Mycoplasma DNA.

    Science.gov (United States)

    Nübling, C Micha; Baylis, Sally A; Hanschmann, Kay-Martin; Montag-Lessing, Thomas; Chudy, Michael; Kreß, Julia; Ulrych, Ursula; Czurda, Stefan; Rosengarten, Renate

    2015-09-01

    Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the detection of mycoplasma contamination of cell cultures. Assay features, like the limit of detection or quantification, vary widely between different mycoplasma NAT assays. Biological reference materials may be useful for harmonization of mycoplasma NAT assays. An international feasibility study included lyophilized preparations of four distantly related mycoplasma species (Acholeplasma laidlawii, Mycoplasma fermentans, M. orale, M. pneumoniae) at different concentrations which were analyzed by 21 laboratories using 26 NAT assays with a qualitative, semiquantitative, or quantitative design. An M. fermentans preparation was shown to decrease the interassay variation when used as a common reference material. The preparation was remanufactured and characterized in a comparability study, and its potency (in NAT-detectable units) across different NATs was determined. The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) established this preparation to be the "1st World Health Organization international standard for mycoplasma DNA for nucleic acid amplification technique-based assays designed for generic mycoplasma detection" (WHO Tech Rep Ser 987:42, 2014) with a potency of 200,000 IU/ml. This WHO international standard is now available as a reference preparation for characterization of NAT assays, e.g., for determination of analytic sensitivity, for calibration of quantitative assays in a common unitage, and for defining regulatory requirements in the field of mycoplasma testing. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. Translation of a laboratory-validated equine herpesvirus-1 specific real-time PCR assay into an insulated isothermal polymerase chain reaction (iiPCR) assay for point-of-need diagnosis using POCKIT™ nucleic acid analyzer.

    Science.gov (United States)

    Balasuriya, Udeni B R; Lee, Pei-Yu Alison; Tsai, Yun-Long; Tsai, Chuan-Fu; Shen, Yu-Han; Chang, Hsiao-Fen Grace; Skillman, Ashley; Wang, Hwa-Tang Thomas; Pronost, Stéphane; Zhang, Yan

    2017-03-01

    Equine herpesvirus myeloencephalopathy (EHM), a major problem for the equine industry in the United States, is caused by equine herpesvirus-1 (EHV-1). In addition, EHV-1 is associated with upper respiratory disease, abortion, and chorioretinal lesions in horses. Here we describe the development and evaluation of an inexpensive, user-friendly insulated isothermal PCR (iiPCR) method targeting open reading 30 (ORF30) to detect both neuropathogenic and non-neuropathogenic strains on the field-deployable POCKIT™ device for point-of-need detection of EHV-1. The analytical sensitivity of the EHV-1 iiPCR assay was 13 genome equivalents per reaction. The assay did not cross react with ten non-target equine viral pathogens. Performance of the EHV-1 iiPCR assay was compared to two previously described real-time PCR (qPCR) assays in two laboratories by using 104 archived clinical samples. All 53 qPCR-positive and 46 of the 51 qPCR-negative samples tested positive and negative, respectively, by the iiPCR. The agreement between the two assays was 95.19% (confidence interval 90.48-99.90%) with a kappa value of 0.90. In conclusion, the newly developed EHV-1 iiPCR assay is robust to provide specificity and sensitivity comparable to qPCR assays for the detection of EHV-1 nucleic acid in clinical specimens. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Single Laboratory Validation of A Ready-to-Use Phosphatase Inhibition Assay for Detection of Okadaic Acid Toxins

    Directory of Open Access Journals (Sweden)

    Luis Mata

    2012-04-01

    Full Text Available A phosphatase inhibition assay for detection of okadaic acid (OA toxins in shellfish, OkaTest, was single laboratory validated according to international recognized guidelines (AOAC, EURACHEM. Special emphasis was placed on the ruggedness of the method and stability of the components. All reagents were stable for more than 6 months and the method was highly robust under normal laboratory conditions. The limit of detection and quantification were 44 and 56 µg/kg, respectively; both below the European legal limit of 160 µg/kg. The repeatability was evaluated with 2 naturally contaminated samples. The relative standard deviation (RSD calculated was 1.4% at a level of 276 µg/kg and 3.9% at 124 µg/kg. Intermediate precision was estimated by testing 10 different samples (mussel and scallop on three different days and ranged between 2.4 and 9.5%. The IC50 values of the phosphatase used in this assay were determined for OA (1.2 nM, DTX-1 (1.6 nM and DTX-2 (1.2 nM. The accuracy of the method was estimated by recovery testing for OA (mussel, 78–101%; king scallop, 98–114%, DTX-1 (king scallop, 79–102% and DTX-2 (king scallop, 93%. Finally, the method was qualitatively compared to the mouse bioassay and LC-MS/MS.

  13. Fibrinolytic Activity and Dose-Dependent Effect of Incubating Human Blood Clots in Caffeic Acid Phenethyl Ester: In Vitro Assays

    Directory of Open Access Journals (Sweden)

    Abuzar Elnager

    2015-01-01

    Full Text Available Background. Caffeic acid phenethyl ester (CAPE has been reported to possess time-dependent fibrinolytic activity by in vitro assay. This study is aimed at investigating fibrinolytic dose-dependent activity of CAPE using in vitro assays. Methods. Standardized human whole blood (WB clots were incubated in either blank controls or different concentrations of CAPE (3.75, 7.50, 15.00, 22.50, and 30.00 mM. After 3 hours, D-dimer (DD levels and WB clot weights were measured for each concentration. Thromboelastography (TEG parameters were recorded following CAPE incubation, and fibrin morphology was examined under a confocal microscope. Results. Overall, mean DD (μg/mL levels were significantly different across samples incubated with different CAPE concentrations, and the median pre- and postincubation WB clot weights (grams were significantly decreased for each CAPE concentration. Fibrin removal was observed microscopically and indicated dose-dependent effects. Based on the TEG test, the Ly30 fibrinolytic parameter was significantly different between samples incubated with two different CAPE concentrations (15.0 and 22.50 mM. The 50% effective dose (ED50 of CAPE (based on DD was 1.99 mg/mL. Conclusions. This study suggests that CAPE possesses fibrinolytic activity following in vitro incubation and that it has dose-dependent activities. Therefore, further investigation into CAPE as a potential alternative thrombolytic agent should be conducted.

  14. Single laboratory validation of a ready-to-use phosphatase inhibition assay for detection of okadaic acid toxins.

    Science.gov (United States)

    Smienk, Henry G F; Calvo, Dolores; Razquin, Pedro; Domínguez, Elena; Mata, Luis

    2012-05-01

    A phosphatase inhibition assay for detection of okadaic acid (OA) toxins in shellfish, OkaTest, was single laboratory validated according to international recognized guidelines (AOAC, EURACHEM). Special emphasis was placed on the ruggedness of the method and stability of the components. All reagents were stable for more than 6 months and the method was highly robust under normal laboratory conditions. The limit of detection and quantification were 44 and 56 µg/kg, respectively; both below the European legal limit of 160 µg/kg. The repeatability was evaluated with 2 naturally contaminated samples. The relative standard deviation (RSD) calculated was 1.4% at a level of 276 µg/kg and 3.9% at 124 µg/kg. Intermediate precision was estimated by testing 10 different samples (mussel and scallop) on three different days and ranged between 2.4 and 9.5%. The IC(50) values of the phosphatase used in this assay were determined for OA (1.2 nM), DTX-1 (1.6 nM) and DTX-2 (1.2 nM). The accuracy of the method was estimated by recovery testing for OA (mussel, 78-101%; king scallop, 98-114%), DTX-1 (king scallop, 79-102%) and DTX-2 (king scallop, 93%). Finally, the method was qualitatively compared to the mouse bioassay and LC-MS/MS.

  15. Relationship between in vitro assays and standardized ileal amino acid digestibility of animal protein meals in broilers.

    Science.gov (United States)

    Rochell, S J; Kuhlers, D L; Dozier, W A

    2013-01-01

    Two identical trials were conducted to determine the relationship of a novel digestive enzyme assay, Poultry Complete IDEA (PC IDEA), and the pepsin digestibility assay with standardized ileal amino acid digestibility (SIAAD) of 20 animal protein meals (APM) fed to broilers from 25 to 30 d of age. Animal protein meals included 10 meat and bone meals (MBM) consisting of bovine, porcine, or mixed bovine and porcine raw materials (BP), and 10 animal protein blends containing animal proteins from various species. Treatments consisted of 20 semi-purified diets containing 1 APM as the sole source of dietary amino acids (AA), and 1 N-free diet to determine endogenous ileal AA flow. With the exception of the N-free diet, diets were formulated to contain 20% CP. In each trial, 756 Ross × Ross 708 male broilers were housed in battery cages and randomly assigned to 21 dietary treatments on d 25 (12 birds per cage; 3 replicate cages), and ileal digesta were collected on d 30 for determination of SIAAD. Pepsin digestibility and PC IDEA were determined for APM samples from each experimental diet (3 replicates per trial; 6 total replicates). Pepsin digestibility and PC IDEA were both correlated (P digestibility on SIAAD resulted in the following equations: % Lys SIAAD = [-9.65 + (0.38 × % PC IDEA predicted Lys digestibility) + (0.69 × % pepsin digestibility)], % Met SIAAD = [-35.95 + (0.62 × % PC IDEA predicted Met digestibility) + (0.75 × % pepsin digestibility)], % Thr SIAAD = [-77.5 + (0.39 × % PC IDEA predicted Thr digestibility) + (1.37 × % pepsin digestibility)]. Values of R(2) were 0.46, 0.47, and 0.55 for Lys, Met, and Thr, respectively. The relatively low R(2) values may have been due to the limited range in SIAAD observed for the 20 APM, and additional data on APM varying in SIAAD are needed.

  16. Antitumor activity of zoledronic acid in primary breast cancer cells determined by the ATP tumor chemosensitivity assay

    Directory of Open Access Journals (Sweden)

    Fehm Tanja

    2012-07-01

    Full Text Available Abstract Background The NeoAzure study has demonstrated that the use of the bisphosphonate zoledronic acid (Zol in the neoadjuvant setting increases the rate of complete response in primary breast cancer and therefore indicates direct antitumor activity. The purpose of this study was to compare the antitumor effect of Zol with standard chemotherapy in primary breast cancer cells using ATP-tumor chemosensitivity assay (ATP-TCA. Methods Breast cancer specimens were obtained from patients with breast cancer who underwent primary breast cancer surgery at the Department of Obstetrics and Gynecology, Tübingen, Germany, between 2006 through 2009. Antitumor effects of Zol, TAC (Docetaxel, Adriamycin, Cyclophosphamide and FEC (5-Fluorouracil, Epirubicin, Cyclophosphamide were tested in 116 fresh human primary breast cancer specimens using ATP-TCA. ATP-TCA results were analyzed with different cut-off levels for the half maximal inhibitory concentration (IC50, for IC90 and for the sensitivity index (IndexSUM. Each single agent or combination was tested at six doubling dilutions from 6.25, 12.5, 25, 50, 100, and 200% of test drug concentrations (TDC derived from the plasma peak concentrations determined by pharmacokinetic data. The assay was carried out in duplicate wells with positive and negative controls. Results The median IndexSUM value was lower for Zol than for the combined regimen FEC (36.8% and TAC (12.9%, respectively, indicating increased antitumor activity of Zol in primary breast cancer cells. The difference regarding Zol and FEC was significant (p  Conclusion Zoledronic acid has a strong antitumor effect on primary breast cancer cells in vitro which is equal or superior to commonly used chemotherapeutic regimens for treating breast cancer.

  17. Nucleic acid detection system and method for detecting influenza

    Science.gov (United States)

    Cai, Hong; Song, Jian

    2015-03-17

    The invention provides a rapid, sensitive and specific nucleic acid detection system which utilizes isothermal nucleic acid amplification in combination with a lateral flow chromatographic device, or DNA dipstick, for DNA-hybridization detection. The system of the invention requires no complex instrumentation or electronic hardware, and provides a low cost nucleic acid detection system suitable for highly sensitive pathogen detection. Hybridization to single-stranded DNA amplification products using the system of the invention provides a sensitive and specific means by which assays can be multiplexed for the detection of multiple target sequences.

  18. Microfluidic continuous flow digital loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Rane, Tushar D; Chen, Liben; Zec, Helena C; Wang, Tza-Huei

    2015-02-07

    Digital nucleic acid detection is rapidly becoming a popular technique for ultra-sensitive and quantitative detection of nucleic acid molecules in a wide range of biomedical studies. Digital polymerase chain reaction (PCR) remains the most popular way of conducting digital nucleic acid detection. However, due to the need for thermocycling, digital PCR is difficult to implement in a streamlined manner on a single microfluidic device, leading to complex fragmented workflows and multiple separate devices and instruments. Loop-mediated isothermal amplification (LAMP) is an excellent isothermal alternative to PCR with potentially better specificity than PCR because of the use of multiple primer sets for a nucleic acid target. Here we report a microfluidic droplet device implementing all the steps required for digital nucleic acid detection including droplet generation, incubation and in-line detection for digital LAMP. As compared to microchamber or droplet array-based digital assays, the continuous flow operation of this device eliminates the constraints on the number of total reactions imposed by the footprint of the device and the analysis throughput caused by the time for lengthy incubation and transfer of materials between instruments.

  19. Evaluation of loop mediated isothermal amplification for diagnosis of ...

    African Journals Online (AJOL)

    Tuberculosis (TB) remains an important global public health problem. The lack of rapid and accurate diagnostic testing is an important impediment to global tuberculosis control. Loop mediated isothermal amplification (LAMP) is a rapid method for nucleic acid amplification. In this study, we assessed the performance of an ...

  20. Heat induces gene amplification in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Bin, E-mail: yanbin@mercyhealth.com [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Mercy Cancer Center, Mercy Medical Center-North Iowa, Mason City, IA 50401 (United States); Ouyang, Ruoyun [Department of Respiratory Medicine, The Second Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410011 (China); Huang, Chenghui [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Department of Oncology, The Third Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410013 (China); Liu, Franklin [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Neill, Daniel [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Li, Chuanyuan [Dermatology, Duke University Medical Center, Durham, NC 27710 (United States); Dewhirst, Mark [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and

  1. Perfluorooctanoic acid (PFOA) but not perfluorooctane sulfonate (PFOS) showed DNA damage in comet assay on Paramecium caudatum.

    Science.gov (United States)

    Kawamoto, Kosuke; Oashi, Takahiro; Oami, Kazunori; Liu, Wei; Jin, Yihe; Saito, Norimitsu; Sato, Itaru; Tsuda, Shuji

    2010-12-01

    Persistent perfluorinated organic compounds such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are distributed widely in the global environment including wildlife and human. In this study, we investigated the genotoxicity of PFOS and PFOA using the novel in vivo comet assay developed for Paramecium caudatum. For the comet assay, large nuclei squeezed out of the paramecia with 0.25 M sucrose containing 0.6% Triton X-100 were embedded in a layer of agarose gel placed over the slide glass. N-methyl-N´-nitro-N-nitrosoguanidine (MNNG) and 2-aminoanthracene (2-AA) were successfully used for positive controls. Productions of 8-hydroxydeoxyguanosine (8-OH-dG) and intracellular reactive oxygen species (ROS) were also measured in paramecia. PFOS did not cause DNA damage on any conditions examined. On the other hand, 12 and 24 hr exposure to PFOA (100 µM) increased DNA migration in electrophoresis condition at pH 13, but not at pH 12.1, suggesting that the DNA damage may be alkali labile site (such as apurinic/apyrimidinic (AP) site). Exposure of paramecia to 100 µM PFOA for 1, 3 and 24 hr and to 10 µM PFOA for 24 hr significantly increased intracellular ROS. Under the same condition, however, 8-OH-dG level was not affected by PFOA. The PFOA-induced DNA damage was not abolished by the application of 100 µM GSH which completely inhibited the increase of intracellular ROS. In conclusion, the PFOA-induced in vivo DNA damage was first shown in paramecia, and the DNA damage might not be directly attributable to increase in intracellular ROS.

  2. Convenient and Efficient Synthesis of a Lanthanide3+-Coordinated, Diethylene Triamine Pentaacetic Acid Labeled Biopolymer as an Assay for the Cholecystokinin B Receptor

    Science.gov (United States)

    Gao, F.; Handl, H.; Vagner, J.; Hruby, V.; Gillies, R.

    2009-01-01

    To develop an assay for the cholecystokinin B receptor with an Eu3+-labeled cholecystokinin peptide via a diethylene triamine pentaacetic acid chelating linker, a commercial dianhydride diethylene triamine pentaacetic acid precursor was directly attached to the N-terminus of cholecystokinin peptides by a solid-phase synthesis method with a satisfactory yield and purity after reverse-phase high-performance liquid chromatography separation. Lanthanide was then coordinated to the peptide via a diethylene triamine pentaacetic acid bifunctional agent. This method is a useful approach to the large-scale synthesis of lanthanide3+-coordinated, diethylene triamine pentaacetic acid labeled biopolymers. This research provides not only a simple and convenient method for the preparation of lanthanide-based peptide ligand libraries but also possible lanthanide-based high-throughput screening of peptide receptors with a timeresolved fluorescence assay system. Five biopolymers were synthesized and characterized with high-resolution electrospray ionization in this study. PMID:19562042

  3. Sensitive real-time PCR detection of pathogenic Leptospira spp. and a comparison of nucleic acid amplification methods for the diagnosis of leptospirosis.

    Science.gov (United States)

    Waggoner, Jesse J; Balassiano, Ilana; Abeynayake, Janaki; Sahoo, Malaya K; Mohamed-Hadley, Alisha; Liu, Yuanyuan; Vital-Brazil, Juliana Magalhães; Pinsky, Benjamin A

    2014-01-01

    Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species. For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p = 0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (pLeptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay. The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests.

  4. Analytical evaluation of three enzymatic assays for measuring total bile acids in plasma using a fully-automated clinical chemistry platform.

    Science.gov (United States)

    Danese, Elisa; Salvagno, Gian Luca; Negrini, Davide; Brocco, Giorgio; Montagnana, Martina; Lippi, Giuseppe

    2017-01-01

    Although the clinical significance of measuring bile acids concentration in plasma or serum has been recognized for long in patients with hepatobiliary disease and/or bile acid malabsorption, the reference separation techniques are expensive and mostly unsuitable for early diagnosis and for measuring large volumes of samples. Therefore, this study was aimed to evaluate the analytical performance of three commercial enzymatic techniques for measuring total bile acids in plasma using a fully-automated clinical chemistry platform. Three commercial enzymatic assays (from Diazyme, Randox and Sentinel) were adapted for use on a Cobas Roche c501. We performed imprecision and linearity studies, and we compared results with those obtained using a reference liquid chromatography-mass spectrometry (LC-MS) technique on an identical set of lithium-heparin plasma samples. Total imprecision was optimal, always equal or lower than 3%. All assays had optimal linearity between 3-138 μmol/L. The comparison studies showed good correlation with LC-MS data (Spearman's correlation coefficients always >0.92), but all plasma samples values were significantly underestimated using the commercial enzymatic assays (-44% for Diazyme, -16% for Randox and -12% for Sentinel). The agreement at the 10 and 40 μmol/L diagnostic thresholds of total bile acids in plasma ranged between 86-92%. This discrepancy was found to be mainly attributable to a heterogeneous composition in terms of bile acids content of the three assay calibrators. This study suggests that the analytical performance of the three commercial enzymatic assays is excellent, thus confirming that automation of this important test by means of enzymatic assessment may be feasible, practical, reliable and supposedly cheap. Nevertheless, the underestimation of values compared to the reference LC-MS also suggests that the local definition and validation of reference ranges according to the combination between the specific enzymatic assay

  5. Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis

    Science.gov (United States)

    2014-01-01

    Background Nucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing. Methods A specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38°C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay. Results The lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n = 77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45°C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined. Conclusions Combining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in

  6. Detection of Chlamydia trachomatis and Neisseria gonorrhoeae based on cross-priming amplification.

    Science.gov (United States)

    Yu, B; An, Y; Xu, G; Shan, H

    2016-05-01

    Rapid, sensitive and specific isothermal nucleic acid amplification methods of Chlamydia trachomatis (C. trachomatis) and Neisseria gonorrhoeae (N. gonorrhoeae) have been developed based on cross-priming amplification (CPA). The amplicon of CPA can be detected by a disposable amplicon cross-contamination proof device. The whole assay takes 1-1·5 h from amplification to read out. Ten fold serial dilutions of quantified plasmids were used to test the CPA assay sensitivities, with the detection limits of 45 copies per reaction and 65 copies per reaction for C. trachomatis and N. gonorrhoeae respectively. The specificities of CPA assays for C. trachomatis and N. gonorrhoeae were tested by using total DNA extracted from nine other bacterial strains, and no cross-reactivity was detected. Eighty clinical cervical or vaginal swab specimens were tested by both CPA and real-time polymerase chain reaction (PCR) with the consistencies of 98·75% (79/80) and 97·5% (78/80) for C. trachomatis and N. gonorrhoeae respectively. Using real-time PCR as a reference standard, the clinical sensitivity (positive) and specificity (negative) of CPA was found to be 98·15% (53/54) and 100% (26/26) for C. trachomatis, and 93·75% (30/32) and 100% (48/48) for N. gonorrhoeae. Eight swab specimens tested positive for both C. trachomatis and N. gonorrhoeae simultaneously by real-time PCR and CPA assay. This study demonstrated that CPA is an affordable and accessible assay for C. trachomatis and N. gonorrhoeae detection, with high sensitivity and specificity. Rapid and specific detection of the sexually transmitted pathogens Chlamydia trachomatis (C. trachomatis) and Neisseria gonorrhoeae (N. gonorrhoeae) would enable early treatment and management of their spread. Here, for the first time, a user friendly DNA isothermal amplification method named cross-priming amplification (CPA), was used to test C. trachomatis and N. gonorrhoeae with high sensitivity and specificity. The

  7. A biotin-drug extraction and acid dissociation (BEAD) procedure to eliminate matrix and drug interference in a protein complex anti-drug antibody (ADA) isotype specific assay.

    Science.gov (United States)

    Niu, Hongmei; Klem, Thomas; Yang, Jinsong; Qiu, Yongchang; Pan, Luying

    2017-07-01

    Monitoring anti-drug antibody (ADA) responses in patients receiving protein therapeutics treatment is an important safety assessment for regulatory agencies, drug manufacturers, clinicians and patients. Recombinant human IGF-1/IGFBP-3 (rhIGF-1/rhIGFBP-3) is a 1:1 formulation of naturally occurring protein complex. The individual IGF-1 and IGFBP-3 proteins have multiple binding partners in serum matrix with high binding affinity to each other, which presents challenges in ADA assay development. We have developed a biotin-drug extraction with acid dissociation (BEAD) procedure followed by an electrochemiluminescence (ECL) direct assay to overcome matrix and drug interference. The method utilizes two step acid dissociation and excess biotin-drug to extract total ADA, which are further captured by soluble biotin-drug and detected in an ECL semi-homogeneous direct assay format. The pre-treatment method effectively eliminates interference by serum matrix and free drug, and enhances assay sensitivity. The assays passed acceptance criteria for all validation parameters, and have been used for clinical sample Ab testing. This method principle exemplifies a new approach for anti-isotype ADA assays, and could be an effective strategy for neutralizing antibody (NAb), pharmacokinetic (PK) and biomarker analysis in need of overcoming interference factors. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Content of the Alternaria mycotoxin tenuazonic acid in food commodities determined by a stable isotope dilution assay.

    Science.gov (United States)

    Asam, Stefan; Lichtenegger, Martina; Liu, Yang; Rychlik, Michael

    2012-02-01

    The Alternaria mycotoxin tenuazonic acid (TA) was quantified in fruit juices (n = 50), cereals (n = 12) and spices (n = 38) using a recently developed stable isotope dilution assay (SIDA). [(13) C6,(15) N]-TA was used as the internal standard. Method validation revealed low limits of detection (LODs) of 0.15 μg/kg (fruit juices), 1.0 μg/kg (cereals) and 17 μg/kg (spices). The respective limits of quantitation were about three times higher. Recovery was about 100% for all matrices. The precision (relative standard deviation of replicate analyses of naturally contaminated samples) was 4.2% (grape juice; 1.7 μg/kg), 3.5% (whole wheat flour; 36 μg/kg) and 0.9% (curry powder; 215 μg/kg). The median content of TA in the analyzed samples was 1.8 μg/kg (fruit juices), 16 μg/kg (cereals) and 500 μg/kg (spices). Positive samples amounted to 86% (fruit juices), 92% (cereals) and 87% (spices).

  9. Study of the population dynamics of a mixed bacterial culture able to degrade cyanuric acid in a packed bed reactor, using RAPD (Random amplification of polymorphic DNA) technique

    Energy Technology Data Exchange (ETDEWEB)

    Galindez-Najera, S.; Llamas-Martinez, M.; Ahuatzi-Chacon, D.; Ruiz-Ordaz, N.; Galindez-Mayer, C. J.

    2009-07-01

    Cyanuric acid is a biodegradation byproduct of triazinic compounds. Because of its low carbon to nitrogen ratio, a complementary carbon source is usually needed for its complete biodegradation. In this work, glucose was used as extra carbon source. Cyanuric hydrolase is the first enzyme in cyanuric acid (CA) catabolism, and is produced by a wide number of microorganisms. (Author)

  10. Quantitative analysis of caffeic and ferulic acids in oatmeal. Comparison of a conventional method with a stable isotope dilution assay.

    Science.gov (United States)

    Guth, H; Grosch, W

    1994-09-01

    [13C]Caffeic acid and [13C]ferulic acid were synthesized and then used as internal standards for the determination of these acids (free and esterified) in oatmeal. A comparative study indicated that 84% of the ferulic acid, but only 32% of the caffeic acid, which is more susceptible to oxidation than the former, could be found by a conventional analytical approach.

  11. A reliable phenotypic assay for detection of ESBLs and AmpCs in MBL-producing gram-negative bacteria with the use of aminophenylboronic acid, dipicolinic acid and cloxacillin.

    Science.gov (United States)

    Datta, Saswati; Chatterjee, Somdatta; Mitra, Shravani; Basu, Sulagna

    2015-08-01

    ESBLs and AmpCs may escape detection when they coexist with metallo-β-lactamases such as New Delhi Metallo-β-lactamases-1. In this study a combination disk assay was established using cefotaxime, cefotaxime/clavulanic acid, cefotaxime/clavulanic acid/cloxacillin, cefoxitin and cefoxitin/phenylboronic acid/cloxacillin on Mueller Hinton agar supplemented with dipicolinic acid for determination of β-lactamases in the presence of NDM-1. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Lateral flow nucleic acid biosensor for sensitive detection of microRNAs based on the dual amplification strategy of duplex-specific nuclease and hybridization chain reaction.

    Directory of Open Access Journals (Sweden)

    Na Ying

    Full Text Available MicroRNAs (miRNAs constitute novel biomarkers for various diseases. Accurate and quantitative analysis of miRNA expression is critical for biomedical research and clinical theranostics. In this study, a method was developed for sensitive and specific detection of miRNAs via dual signal amplification based on duplex specific nuclease (DSN and hybridization chain reaction (HCR. A reporter probe (RP, comprising recognition sequence (3' end modified with biotin for a target miRNA of miR-21 and capture sequence (5' end modified with Fam for HCR product, was designed and synthesized. HCR was initiated by partial sequence of initiator probe (IP, the other part of which can hybridize with capture sequence of RP, and was assembled by hairpin probes modified with biotin (H1-bio and H2-bio. A miR-21 triggered cyclical DSN cleavage of RP, which was immobilized to a streptavidin (SA coated magnetic bead (MB. The released Fam labeled capture sequence then hybridized with the HCR product to generate a detectable dsDNA. This polymer was then dropped on lateral flow strip and positive result was observed. The proposed method allowed quantitative sequence-specific detection of miR-21 (with a detection limit of 2.1 fM, S/N = 3 in a dynamic range from 100 fM to 100 pM, with an excellent ability to discriminate differences in miRNAs. The method showed acceptable testing recoveries for the determination of miRNAs in serum.

  13. Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.

    Directory of Open Access Journals (Sweden)

    Shichu Huang

    Full Text Available In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10(-2 pg of C. difficile DNA while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health.

  14. A novel functional assay for simultaneous determination of total fatty acid beta-oxidation flux and acylcarnitine profiling in human skin fibroblasts using (2)H(31)-palmitate by isotope ratio mass spectrometry and electrospray tandem mass spectrometry

    NARCIS (Netherlands)

    Law, Lap-Kay; Tang, Nelson Leung-Sang; Hui, Joannie; Ho, Chung-Shun; Ruiter, Jos; Fok, Tai-Fai; Wanders, Ronald J. A.; Lam, Christopher Wai-Kei

    2007-01-01

    BACKGROUND: Two separate and complementary assays, total mitochondrial fatty acid beta-oxidation (FAO) flux rate and acylcarnitine profiling, have been used to establish a definitive diagnosis of FAO defects (FAOD) in cultured cells. We developed a novel functional assay for total FAO rate assay by

  15. Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant-Associated Fungi.

    Science.gov (United States)

    Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao

    2016-09-29

    The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant-associated fungi due to the similar homologies of sequences in primer-annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3' end of the primer-binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant-associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant-associated fungi.

  16. Clostridium difficile testing algorithms using glutamate dehydrogenase antigen and C. difficile toxin enzyme immunoassays with C. difficile nucleic acid amplification testing increase diagnostic yield in a tertiary pediatric population.

    Science.gov (United States)

    Ota, Kaede V; McGowan, Karin L

    2012-04-01

    We evaluated the performance of the rapid C. diff Quik Chek Complete's glutamate dehydrogenase antigen (GDH) and toxin A/B (CDT) tests in two algorithmic approaches for a tertiary pediatric population: algorithm 1 entailed initial testing with GDH/CDT followed by loop-mediated isothermal amplification (LAMP), and algorithm 2 entailed GDH/CDT followed by cytotoxicity neutralization assay (CCNA) for adjudication of discrepant GDH-positive/CDT-negative results. A true positive (TP) was defined as positivity by CCNA or positivity by LAMP plus another test (GDH, CDT, or the Premier C. difficile toxin A and B enzyme immunoassay [P-EIA]). A total of 141 specimens from 141 patients yielded 27 TPs and 19% prevalence. Sensitivity, specificity, positive predictive value, and negative predictive value were 56%, 100%, 100%, and 90% for P-EIA and 81%, 100%, 100%, and 96% for both algorithm 1 and algorithm 2. In summary, GDH-based algorithms detected C. difficile infections with superior sensitivity compared to P-EIA. The algorithms allowed immediate reporting of half of all TPs, but LAMP or CCNA was required to confirm the presence or absence of toxigenic C. difficile in GDH-positive/CDT-negative specimens.

  17. Species specific identification of spore-producing microbes using the gene sequence of small acid-soluble spore coat proteins for amplification based diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    McKinney, Nancy (Decatur, GA)

    2002-01-01

    PCR (polymerase chain reaction) primers for the detection of certain Bacillus species, such as Bacillus anthracis. The primers specifically amplify only DNA found in the target species and can distinguish closely related species. Species-specific PCR primers for Bacillus anthracis, Bacillus globigii and Clostridium perfringens are disclosed. The primers are directed to unique sequences within sasp (small acid soluble protein) genes.

  18. Electrochemical quantification of some water soluble vitamins in commercial multi-vitamin using poly-amino acid caped by graphene quantum dots nanocomposite as dual signal amplification elements.

    Science.gov (United States)

    Shadjou, Nasrin; Hasanzadeh, Mohammad; Omari, Ali

    2017-12-15

    Rapid analyses of some water soluble vitamins (Vitamin B2, B9, and C) in commercial multi vitamins could be routinely performed in analytical laboratories. This study reports on the electropolymerization of a low toxic and biocompatible polymer "poly aspartic acid-graphene quantum dots" as a novel strategy for surface modification of glassy carbon electrode and preparation a new interface for measurement of selected vitamins in commercial multi vitamins. Electrochemical deposition, as a well-controlled synthesis procedure, has been used for subsequently layer-by-layer preparation of graphene quantum dots nanostructures on a poly aspartic acid using cyclic voltammetry techniques in the regime of -1.5 to 2 V. The field emission scanning electron microscopy indicated immobilization of graphene quantum dots onto poly aspartic acid film. The modified electrode possessed as an effective electroactivity for detection of water soluble vitamins by using cyclic voltammetry, chronoamperometry and differential pulse voltammetry. Enhancement of peak currents is ascribed to the fast heterogeneous electron transfer kinetics that arise from the synergistic coupling between the excellent properties of poly aspartic acid as semiconducting polymer, graphene quantum dots as high density of edge plane sites and chemical modification. Under the optimized analysis conditions, the prepared sensor for detection of VB2, VB9, and VC showed a low limit of quantification 0.22, 0.1, 0.1 μM, respectively. Copyright © 2017. Published by Elsevier Inc.

  19. Macromechanical Parametric Amplification

    DEFF Research Database (Denmark)

    Neumeyer, Stefan

    Parametric amplification is obtained by adding parametric excitation to direct (externally driven) excitation for boosting near-resonant oscillations. It is utilized for mass and force sensing, switching and signal processing, filtering, timing, signal amplification, and appears promising...... for energy harvesting. Using analytical, numerical, and experimental methods, the thesis focuses on superthreshold pumping (above the systems parametric instability threshold), nonlinear effects, frequency response backbones, and frequency detuning effects for parametric amplifiers. Part one of the thesis...... but parametrically excited frequency response backbone is proposed instead of the classic unforced and undamped backbone. With the modified and more general backbone, it is shown how the response of a superthreshold pumped amplifier is related to respectively the pure directly and pure parametrically excited...

  20. Rapid electrochemiluminescence assays of polymerase chain reaction products.

    Science.gov (United States)

    Kenten, J H; Casadei, J; Link, J; Lupold, S; Willey, J; Powell, M; Rees, A; Massey, R

    1991-09-01

    We demonstrate the first use of an electrochemiluminescent (ECL) label, [4-(N-succimidyloxycarbonylpropyl)-4'-methyl-2,2'- bipyridine]ruthenium(II) dihexafluorophosphate (Origen label; IGEN Inc.), in DNA probe assays. This label allows rapid (less than 25 min) quantification and detection of polymerase chain reaction (PCR)-amplified products from oncogenes, viruses, and cloned genes. For the PCR, we used labeled oligonucleotide primers complementary to human papiloma virus and the Ha-ras oncogene. These samples were followed by ECL analysis or hybridization with specific, Origen-labeled oligonucleotide probes. These studies demonstrate the speed, specificity, and effectiveness of the new ECL labels, compared with 32P, for nucleic acid probe applications. We describe formats involving conventional methodologies and a new format that requires no wash step, allowing simple and rapid sample analysis. These rapid assays also reduce PCR contamination, by requiring less sample handling. Improvements in ECL detectability are currently under investigation for use in DNA probe assays without amplification.

  1. Simultaneous elimination of carryover contamination and detection of DNA with uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP).

    Science.gov (United States)

    Hsieh, Kuangwen; Mage, Peter L; Csordas, Andrew T; Eisenstein, Michael; Soh, H Tom

    2014-04-11

    We report a one-pot, closed-vessel enzymatic assay that eliminates carryover contamination while preserving robust DNA amplification in loop-mediated isothermal amplification (LAMP), providing reliable and rapid detection of target DNA in contaminated samples.

  2. Superlinear Amplitude Amplification

    OpenAIRE

    Grover, Lov K.

    2008-01-01

    Quantum search/amplitude amplification algorithms are designed to be able to amplify the amplitude in the target state linearly with the number of operations. Since the probability is the square of the amplitude, this results in the success probability rising quadratically with the number of operations. This paper presents a new kind of quantum search algorithm in which the amplitude of the target state, itself increases quadratically with the number of operations. However, the domain of appl...

  3. Analytical specificity and sensitivity of the APTIMA Combo 2 and APTIMA GC assays for detection of commensal Neisseria species and Neisseria gonorrhoeae on the Gen-Probe Panther instrument.

    Science.gov (United States)

    Golparian, Daniel; Tabrizi, Sepehr N; Unemo, Magnus

    2013-02-01

    Genetic detection of Neisseria gonorrhoeae is replacing culture for increased diagnostic sensitivity. Specificity of several nucleic acid amplification tests is suboptimal. Herein, the Gen-Probe APTIMA Combo 2 and APTIMA GC assays had 100% specificity and 100% sensitivity after confirmatory testing, when testing 298 isolates of nongonococcal Neisseria and related species and 205 gonococcal isolates.

  4. Flavonoids, flavonoid metabolites, and phenolic acids inhibit oxidative stress in the neuronal cell line HT-22 monitored by ECIS and MTT assay: a comparative study.

    Science.gov (United States)

    Kling, Beata; Bücherl, Daniel; Palatzky, Peter; Matysik, Frank-Michael; Decker, Michael; Wegener, Joachim; Heilmann, Jörg

    2014-03-28

    A real-time and label-free in vitro assay based on electric cell-substrate impedance sensing (ECIS) was established, validated, and compared to an end-point MTT assay within an experimental trial addressing the cytoprotective effects of 19 different flavonoids, flavonoid metabolites, and phenolic acids and their methyl esters on the HT-22 neuronal cell line, after induction of oxidative stress with tert-butyl hydroperoxide. Among the flavonoids under study, only those with a catechol unit and an additional 4-keto group provided cytoprotection. The presence of a 2,3-double bond was not a structural prerequisite for a neuroprotective effect. In the case of the phenolics, catechol substitution was the only structural requirement for activity. The flavonoids and other phenolics with a ferulic acid substitution or a single hydroxy group showed no activity. Electrochemical characterization of all compounds via square-wave voltammetry provided a rather specific correlation between cytoprotective activity and redox potential for the active flavonoids, but not for the active phenolics with a low molecular weight. Moreover this study was used to compare label-free ECIS recordings with results of the established MTT assay. Whereas the former provides time-resolved and thus entirely unbiased information on changes of cell morphology that are unequivocally associated with cell death, the latter requires predefined exposure times and a strict causality between metabolic activity and cell death. However, MTT assays are based on standard lab equipment and provide a more economic way to higher throughput.

  5. On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer

    Energy Technology Data Exchange (ETDEWEB)

    Noor, M. Omair; Tavares, Anthony J.; Krull, Ulrich J., E-mail: ulrich.krull@utoronto.ca

    2013-07-25

    Graphical abstract: -- Highlights: •Solid-phase multiplexed QD-FRET nucleic acid assay in electrokinetic fluidic chip. •Concurrent detection of two oligonucleotides based on channel length coverage. •Selection of “turn-on” and “turn-off” signals from two acceptor dyes and two colors of immobilized QDs, respectively. •No loss in assay sensitivity when implementing multiplexed assay format. -- Abstract: A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical

  6. A streptavidin paramagnetic-particle based competition assay for the evaluation of the optical selectivity of quadruplex nucleic acid fluorescent probes.

    Science.gov (United States)

    Largy, Eric; Hamon, Florian; Teulade-Fichou, Marie-Paule

    2012-05-01

    Although quadruplex nucleic acids are thought to be involved in many biological processes, they are massively overwhelmed by duplex DNA in the cell. Small molecules, able to probe quadruplex nucleic acids with high optical selectivity, could possibly achieve the visualization of these processes. The aim of the method described herein is to evaluate quickly the optical selectivity of quadruplex nucleic acid probes, in isothermal conditions, using widely available materials, small quantities of oligonucleotides and virtually any kind and quantity of biological competitor. The assay relies on the use of streptavidin-coated paramagnetic particles and biotinylated quadruplex forming oligonucleotides, allowing a quick and easy separation of the quadruplex target from the competitor. In the present study, two quadruplex nucleic acids (the DNA and RNA human telomeric repeats) have been used as targets while a duplex DNA oligonucleotide, total DNA, total RNA, another quadruplex nucleic acid and a protein have been used as competitors. The optical selectivity of various probes, displaying different photophysical properties and binding selectivities, has been successfully examined, allowing the identification of a best candidate for further cell microscopy experiments. This assay allows a quick and reliable assessment of the labeling properties of a quadruplex binder in cellular environment conditions. It is an interesting alternative to gel electrophoresis experiments since it is performed in solution, has a well-resolved separation system and allows easy quantifications. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Deployable laboratory response to influenza pandemic; PCR assay field trials and comparison with reference methods.

    Directory of Open Access Journals (Sweden)

    Timothy J J Inglis

    Full Text Available BACKGROUND: The influenza A/H1N1/09 pandemic spread quickly during the Southern Hemisphere winter in 2009 and reached epidemic proportions within weeks of the official WHO alert. Vulnerable population groups included indigenous Australians and remote northern population centres visited by international travellers. At the height of the Australian epidemic a large number of troops converged on a training area in northern Australia for an international exercise, raising concerns about their potential exposure to the emerging influenza threat before, during and immediately after their arrival in the area. Influenza A/H1N1/09 became the dominant seasonal variant and returned to Australia during the Southern winter the following year. METHODS: A duplex nucleic acid amplification assay was developed within weeks of the first WHO influenza pandemic alert, demonstrated in northwestern Australia shortly afterwards and deployed as part of the pathology support for a field hospital during a military exercise during the initial epidemic surge in June 2009. RESULTS: The nucleic acid amplification assay was twice as sensitive as a point of care influenza immunoassay, as specific but a little less sensitive than the reference laboratory nucleic acid amplification assay. Repetition of the field assay with blinded clinical samples obtained during the 2010 winter influenza season demonstrated a 91.7% congruence with the reference laboratory method. CONCLUSIONS: Rapid in-house development of a deployable epidemic influenza assay allowed a flexible laboratory response, effective targeting of limited disease control resources in an austere military environment, and provided the public health laboratory service with a set of verification tools for resource-limited settings. The assay method was suitable for rapid deployment in time for the 2010 Northern winter.

  8. In vitro amplification of ovine prions from scrapie-infected sheep from Great Britain reveals distinct patterns of propagation

    Directory of Open Access Journals (Sweden)

    Thorne Leigh

    2012-11-01

    Full Text Available Abstract Background Protein misfolding cyclic amplification (PMCA is a method that facilitates the detection of prions from many sources of transmissible spongiform encephalopathy (TSE. Sheep scrapie represents a unique diversity of prion disease agents in a range of susceptible PRNP genotypes. In this study PMCA was assessed on a range of Great Britain (GB sheep scrapie isolates to determine the applicability to veterinary diagnosis of ovine TSE. Results PrPSc amplification by protein misfolding cyclic amplification (PMCA was assessed as a diagnostic tool for field cases of scrapie. The technique was initially applied to thirty-seven isolates of scrapie from diverse geographical locations around GB, and involved sheep of various breeds and PRNP genotypes. All samples were amplified in either VRQ and/or ARQ PrPC substrate. For PrPSc from sheep with at least one VRQ allele, all samples amplified efficiently in VRQ PrPC but only PrPSc from ARH/VRQ sheep amplified in both substrates. PrPSc from ARQ/ARQ sheep displayed two amplification patterns, one that amplified in both substrates and one that only amplified in ARQ PrPC. These amplification patterns were consistent for a further 14/15 flock/farm mates of these sheep. Furthermore experimental scrapie strains SSBP1, Dawson, CH1641 and MRI were analysed. SSBP1 and Dawson (from VRQ/VRQ sheep amplified in VRQ but not ARQ substrate. MRI scrapie (from ARQ/ARQ sheep nor CH1641 did not amplify in ARQ or VRQ substrate; these strains required an enhanced PMCA method incorporating polyadenylic acid (poly(A to achieve amplification. Conclusions PrPsc from 52 classical scrapie GB field isolates amplified in VRQ or ARQ or both substrates and supports the use of PMCA as a rapid assay for the detection of a wide range of ovine classical scrapie infections involving multiple PRNP genotypes and scrapie strains.

  9. Real-Time Fluorogenic Polymerase Chain Reaction (PCR) Assays to Nucleic Acid-Based Detection of Simulants and Biothreat Agents

    National Research Council Canada - National Science Library

    Khan, Akbar S; O'Connell, Kevin P; Bucher, Jennifer R; Anderson, Patricia E; Cao, Cheng J; Gostomski, Mark V; Valdes, James J

    2003-01-01

    .... We describe here assays for the detection of the gene encoding staphylococcal enterotoxin A (SEA), a toxin produced by the bacterium Staphylococcus aureus and an agent responsible for a significant fraction of food poisoning incidents worldwide...

  10. A paper-based resonance energy transfer nucleic acid hybridization assay using upconversion nanoparticles as donors and quantum dots as acceptors

    Energy Technology Data Exchange (ETDEWEB)

    Doughan, Samer; Uddayasankar, Uvaraj; Krull, Ulrich J., E-mail: ulrich.krull@utoronto.ca

    2015-06-09

    Highlights: • Covalent immobilization of upconversion nanoparticles on paper. • LRET-based label free DNA detection using quantum dots as acceptors. • Use of polyethylene glycol to eliminate non-specific adsorption of quantum dots. • Improved analytical performance compared to analogous assays. - Abstract: Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reduction amination to develop a luminescence resonance energy transfer (LRET)-based nucleic acid hybridization assay. This first account of covalent immobilization of UCNPs on paper for a bioassay reports an optically responsive method that is sensitive, reproducible and robust. The immobilized UCNPs were decorated with oligonucleotide probes to capture HPRT1 housekeeping gene fragments, which in turn brought reporter conjugated quantum dots (QDs) in close proximity to the UCNPs for LRET. This sandwich assay could detect unlabeled oligonucleotide target, and had a limit of detection of 13 fmol and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs, which are excellent LRET acceptors, demonstrated improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was attributed to the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The kinetics of hybridization were determined to be diffusion limited and full signal development occurred within 3 min.

  11. Carcinoembryonic antigen: assay following heat compared with perchloric acid extraction in patients with colon cancer, non-neoplastic gastrointestinal diseases, or chronic renal failure.

    Science.gov (United States)

    Witherspoon, L R; Shuler, S E; Alyea, K; Husserl, F E

    1983-10-01

    Heat inactivation has been proposed as an alternative to perchloric acid (PCA) precipitation for the extraction of carcinoembryonic antigen (CEA) from human plasma. We examined a commercial RIA kit using heat inactivation, and compared results with those obtained with PCA precipitation. Adequate sensitivity (1.5 micrograms CEA/l plasma), satisfactory analytical recovery of CEA added to plasma, and dilutional linearity of samples found to have elevated CEA concentrations, were demonstrated for the heat-inactivation assay. Between-assay precision was better with the heat inactivation than with the PCA assay. Although the absolute concentration of CEA estimated after heat inactivation was consistently lower than that estimated after PCA extraction of plasma specimens, there was excellent correlation between results obtained with the two methods in colon cancer patients free of disease, colon cancer patients with residual or recurrent disease, patients with benign gastrointestinal disease, and in patients with chronic renal failure. We conclude that the heat-inactivation assay is an excellent alternative to the PCA assay.

  12. Carcinoembryonic antigen: assay following heat compared with perchloric acid extraction in patients with colon cancer, non-neoplastic gastrointestinal diseases, or chronic renal failure

    Energy Technology Data Exchange (ETDEWEB)

    Witherspoon, L.R.; Shuler, S.E.; Alyea, K.; Husserl, F.E.

    1983-10-01

    Heat inactivation has been proposed as an alternative to perchloric acid (PCA) precipitation for the extraction of carcinoembryonic antigen (CEA) from human plasma. A commercial RIA kit using heat inactivation was examined and results compared with those obtained with PCA precipitation. Adequate sensitivity (1.5 ..mu..g CEA/I plasma), satisfactory analytical recovery of CEA added to plasma, and dilutional linearity of samples found to have elevated CEA concentrations, were demonstrated for the heat-inactivation assay. Between-assay precision was better with the heat inactivation than with the PCA assay. Although the absolute concentration of CEA estimated after heat inactivation was consistently lower than that estimated after PCA extraction of plasma specimens, there was excellent correlation between results obtained with the two methods in colon cancer patients free of disease, colon cancer patients with residual or recurrent disease, patients with benign gastrointestinal disease, and in patients with chronic renal failure. The heat-inactivation assay is an excellent alternative to the PCA assay.

  13. Liquid chromatography-tandem mass spectrometry assay for the quantification of free and total sialic acid in human cerebrospinal fluid.

    NARCIS (Netherlands)

    Ham, M. van der; Koning, T.J. de; Lefeber, D.J.; Fleer, A.; Prinsen, B.H.; Sain-van der Velden, M.G. de

    2010-01-01

    BACKGROUND: Analysis of sialic acid (SA) metabolites in cerebrospinal fluid (CSF) is important for clinical diagnosis. In the present study, a high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) method for free sialic acid (FSA) and total sialic acid (TSA) in human CSF was

  14. Liquid chromatography-tandem mass spectrometry assay for the quantification of free and total sialic acid in human cerebrospinal fluid

    NARCIS (Netherlands)

    van der Ham, Maria; de Koning, Tom J; Lefeber, Dirk; Fleer, André; Prinsen, Berthil H C M T; de Sain-van der Velden, Monique G M

    2010-01-01

    BACKGROUND: Analysis of sialic acid (SA) metabolites in cerebrospinal fluid (CSF) is important for clinical diagnosis. In the present study, a high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) method for free sialic acid (FSA) and total sialic acid (TSA) in human CSF was

  15. Thermal stability of quadruplex primers for highly versatile isothermal DNA amplification.

    Science.gov (United States)

    Mathias, Jordan; Okyere, Robert; Lomidze, Levan; Gvarjaladze, David; Musier-Forsyth, Karin; Kankia, Besik

    2014-01-01

    Quadruplex priming amplification (QPA) allows isothermal amplification of nucleic acids with improved yield and simplified detection. This assay is based on a DNA quadruplex, GGGTGGGTGGGTGGG (G3T), which in the presence of specific cations possesses unusually high thermal stability. QPA employs truncated G3T sequences as primers, which upon polymerase elongation, self-dissociate from the binding site and allow the next round of priming without thermal unfolding of amplicons. The rate of amplification strongly depends on the thermal stability of the primer/primer binding site (PBS) complex and to date QPA has been demonstrated to work over a narrow temperature range. To expand the capabilities of QPA, in the present study, we studied the fold and thermodynamic properties of the wild-type G3T and variants containing sequence modifications or extensions at the 5'-end. Circular dichroism studies demonstrate that the substitution of thymidines by other nucleotides or GC addition at the 5'-end does not change the parallel fold of G3T. Thermal unfolding experiments revealed that purine bases incorporated at loop positions and 5'-end dinucleotide extension significantly destabilize the quadruplex, while loop pyrimidines have almost no effect. Overall, the results of these studies suggest that linear isothermal QPA can be performed over a wide temperature range to accommodate both thermophilic and mesophilic DNA polymerases. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Amplification of an MFS transporter encoding gene penT significantly stimulates penicillin production and enhances the sensitivity of Penicillium chrysogenum to phenylacetic acid.

    Science.gov (United States)

    Yang, Jing; Xu, Xinxin; Liu, Gang

    2012-11-20

    Penicillin is historically important as the first discovered drug against bacterial infections in human. Although the penicillin biosynthetic pathway and regulatory mechanism have been well studied in Penicillium chrysogenum, the compartmentation and molecular transport of penicillin or its precursors are still poorly understood. In search of the genomic database, more than 830 open reading frames (ORFs) were found to encode transmembrane proteins of P. chrysogenum. In order to investigate their roles on penicillin production, one of them (penT) was selected and cloned. The deduced protein of penT belongs to the major facilitator superfamily (MFS) and contains 12 transmembrane spanning domains (TMS). During fermentation, the transcription of penT was greatly induced by penicillin precursors phenylacetic acid (PAA) and phenoxyacetic acid (POA). Knock-down of penT resulted in significant decrease of penicillin production, while over-expression of penT under the promoter of trpC enhanced the penicillin production. Introduction of an additional penT in the wild-type strain of P. chrysogenum doubled the penicillin production and enhanced the sensitivity of P. chrysogenum to the penicillin precursors PAA or POA. These results indicate that penT stimulates penicillin production probably through enhancing the translocation of penicillin precursors across fungal cellular membrane. Copyright © 2012. Published by Elsevier Ltd.

  17. Loopback rolling circle amplification for ultrasensitive detection of Kras gene.

    Science.gov (United States)

    Xu, Huo; Wu, Dong; Jiang, Yifan; Zhang, Rongbo; Wu, Qingzheng; Liu, Yiyun; Li, Feng; Wu, Zai-Sheng

    2017-03-01

    Mutations in Kras gene may be used as a diagnostic marker and a target for treatment of the broad spectrum of human cancers. In this study, we developed a new class of amplification assay, double-hairpin molecular beacon (DHMB)-based cascade rolling circle amplification (RCA), for ultrasensitive and selective detection of Kras gene in a homogenous solution. Specifically, target DNA can hybridize with DHMB and activate cyclical target strand-displacement polymerization (CTDP) and nicking-mediated strand-displacement polymerization (NMDP). The resulting nicked/displaced fragments substantially outnumber target DNA and cause the cascade rolling circle amplification (C-RCA) and nicked fragment-induced strand-displacement polymerization (NFDP). Even if four amplification processes are designed, only DHMB, padlock probe and polymerization primer are involved. Under optimized conditions, this screening system exhibits a linear range of 5 orders of magnitude (from 100fM to 20nM), and the detection limit is down to 16fM. Moreover, the developed biosensing system offers a high assay specificity for perfectly matched target DNA, and the measured data from practical samples demonstrated the potential application in the cancer diagnoses. As a proof-of-concept genetic assay, the novel signaling strategy, as well as desirable analytical capability, would significantly benefit the development of versatile amplification gene profiling platforms, revealing great promise in biological studies and medical diagnostics. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Loop-mediated isothermal amplification for the rapid detection of Chrysanthemum chlorotic mottle viroid (CChMVd).

    Science.gov (United States)

    Park, Jungan; Jung, Yuchul; Kil, Eui-Joon; Kim, Jaedeok; Thi Tran, Dung; Choi, Seung-Kook; Yoon, Ju-Yeon; Cho, Won Kyong; Lee, Sukchan

    2013-10-01

    Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method offering rapid, sensitive, and convenient diagnosis of infectious diseases. Chrysanthemum chlorotic mottle viroid (CChMVd) causes one of the most serious viral diseases in chrysanthemum in Korea. A sensitive LAMP assay was developed for rapidly detecting CChMVd infection. The assay was based on a set of four primers matching the specific region of the CChMVd genome. The CChMVd LAMP primer sets were designed using the sequences from nonsymptomatic and symptomatic CChMVd isolates in Korea. The efficiency and specificity of this method were optimized using Bst DNA polymerase, which allowed for increased viroid detection sensitivity. The reaction was carried out at 65 °C for 90 min, and was improved by adding SYBR Green I dye to the inside of the reaction tube lid prior to amplification. The results indicate that this LAMP method will be useful for chrysanthemum viroid disease monitoring and detecting CChMVd infectious disease. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  19. Rapid detection of equine influenza virus H3N8 subtype by insulated isothermal RT-PCR (iiRT-PCR) assay using the POCKIT™ Nucleic Acid Analyzer.

    Science.gov (United States)

    Balasuriya, Udeni B R; Lee, Pei-Yu Alison; Tiwari, Ashish; Skillman, Ashley; Nam, Bora; Chambers, Thomas M; Tsai, Yun-Long; Ma, Li-Juan; Yang, Pai-Chun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

    2014-10-01

    horses. Therefore, the EIV H3N8 subtype specific iiRT-PCR assay along with the portable POCKIT™ Nucleic Acid Analyzer provides a highly reliable, sensitive and specific on-site detection system of both equine and canine influenza viruses. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Hardness amplification in nondeterministic logspace

    OpenAIRE

    Gupta, Sushmita

    2007-01-01

    A hard problem is one which cannot be easily computed by efficient algorithms. Hardness amplification is a procedure which takes as input a problem of mild hardness and returns a problem of higher hardness. This is closely related to the task of decoding certain error-correcting codes. We show amplification from mild average case hardness to higher average case hardness for nondeterministic logspace and worst-to-average amplification for nondeterministic linspace. Finally we explore possible ...

  1. Paper-based solid-phase multiplexed nucleic acid hybridization assay with tunable dynamic range using immobilized quantum dots as donors in fluorescence resonance energy transfer.

    Science.gov (United States)

    Noor, M Omair; Krull, Ulrich J

    2013-08-06

    A multiplexed solid-phase nucleic acid hybridization assay on a paper-based platform is presented using multicolor immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize two types of QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) and red-emitting QDs (rQDs) served as donors with Cy3 and Alexa Fluor 647 (A647) acceptors. The gQD/Cy3 FRET pair served as an internal standard, while the rQD/A647 FRET pair served as a detection channel, combining the control and analytical test zones in one physical location. Hybridization of dye-labeled oligonucleotide targets provided the proximity for FRET sensitized emission from the acceptor dyes, which served as an analytical signal. Hybridization assays in the multicolor format provided a limit of detection of 90 fmol and an upper limit of dynamic range of 3.5 pmol. The use of an array of detection zones was designed to provide improved analytical figures of merit compared to that which could be achieved on one type of array design in terms of relative concentration of multicolor QDs. The hybridization assays showed excellent resistance to nonspecific adsorption of oligonucleotides. Selectivity of the two-plex hybridization assay was demonstrated by single nucleotide polymorphism (SNP) detection at a contrast ratio of 50:1. Additionally, it is shown that the use of preformed QD-probe oligonucleotide conjugates and consideration of the relative number density of the two types of QD-probe conjugates in the two-color assay format is advantageous to maximize assay sensitivity and the upper limit of dynamic range.

  2. A Paper-Based Sandwich Format Hybridization Assay for Unlabeled Nucleic Acid Detection Using Upconversion Nanoparticles as Energy Donors in Luminescence Resonance Energy Transfer

    Directory of Open Access Journals (Sweden)

    Feng Zhou

    2015-09-01

    Full Text Available Bioassays based on cellulose paper substrates are gaining increasing popularity for the development of field portable and low-cost diagnostic applications. Herein, we report a paper-based nucleic acid hybridization assay using immobilized upconversion nanoparticles (UCNPs as donors in luminescence resonance energy transfer (LRET. UCNPs with intense green emission served as donors with Cy3 dye as the acceptor. The avidin functionalized UCNPs were immobilized on cellulose paper and subsequently bioconjugated to biotinylated oligonucleotide probes. Introduction of unlabeled oligonucleotide targets resulted in a formation of probe-target duplexes. A subsequent hybridization of Cy3 labeled reporter with the remaining single stranded portion of target brought the Cy3 dye in close proximity to the UCNPs to trigger a LRET-sensitized emission from the acceptor dye. The hybridization assays provided a limit of detection (LOD of 146.0 fmol and exhibited selectivity for one base pair mismatch discrimination. The assay was functional even in undiluted serum samples. This work embodies important progress in developing DNA hybridization assays on paper. Detection of unlabeled targets is achieved using UCNPs as LRET donors, with minimization of background signal from paper substrates owing to the implementation of low energy near-infrared (NIR excitation.

  3. Multiple displacement amplification as an adjunct to PCR-based detection of Staphylococcus aureus in synovial fluid

    Directory of Open Access Journals (Sweden)

    Johnson Sandra

    2010-10-01

    Full Text Available Abstract Background Detection of bacterial nucleic acids in synovial fluid following total joint arthroplasty with suspected infection can be difficult; among other technical challenges, inhibitors in the specimens require extensive sample preparation and can diminish assay sensitivity even using polymerase chain reaction (PCR-based methods. To address this problem a simple protocol for prior use of multiple displacement amplification (MDA as an adjunct to PCR was established and tested on both purified S. aureus DNA as well as on clinical samples known to contain S. aureus nucleic acids. Findings A single round of MDA on purified nucleic acids resulted in a > 300 thousand-fold increase in template DNA on subsequent quantitative PCR (qPCR analysis. MDA use on clinical samples resulted in at least a 100-fold increase in sensitivity on subsequent qPCR and required no sample preparation other than a simple alkali/heat lysis step. Mixed samples of S. aureus DNA with a 103 - 104-fold excess of human genomic DNA still allowed for MDA amplification of the minor bacterial component to the threshold of detectability. Conclusion MDA is a promising technique that may serve to significantly enhance the sensitivity of molecular assays in cases of suspected joint infection while simultaneously reducing the specimen handling required.

  4. Integrated amplification microarray system in a lateral flow cell for warfarin genotyping from saliva.

    Science.gov (United States)

    Sebastian, Thomas; Cooney, Christopher G; Parker, Jennifer; Qu, Peter; Perov, Alexander; Golova, Julia B; Pozza, Lindsay; Iwasiow, Rafal M; Holmberg, Rebecca

    2014-02-15

    Genetic polymorphisms in the CYP2C9 and VKORC1 genes have been linked to sensitivity of the anticoagulant drug warfarin. The aim of this study is to demonstrate a method for warfarin sensitivity genotyping using gel element microarray technology in a simplified workflow from sample collection to analysis and detection. We developed an integrated amplification microarray system combining PCR amplification, target labeling, and microarray hybridization within a single, closed-amplicon "lateral flow cell" for genotyping three single nucleotide polymorphisms (SNPs) that influence warfarin response. We combined nucleic acid extraction of saliva using the TruTip technology together with the lateral flow cell assay and with fully automated array detection and analysis. The analytical performance of the assay was tested using 20 genotyped human genomic DNA samples and found to be sensitive down to 330 input genomic copies (1 ng). A follow-up pre-clinical evaluation was performed with 65 blinded saliva samples and the genotyping results were in agreement with those determined by bidirectional sequencing. Combined with the use of non-invasive saliva samples, rapid DNA extraction, the lateral flow cell, automatic imaging and data analysis provides a simple and fast sample-to-answer microarray test for warfarin sensitivity genotyping. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Chemiluminescence method for the assay of perphenazine in drug formulation using permanganate in sulphuric acid with flow injection technique and a chemometrical optimization approach.

    Science.gov (United States)

    Sultan, S M; Abdennabi, A M; Almuaibed, A M

    1999-08-09

    An accurate selective flow injection chemiluminescence (CL) method for the assay of perphenazine was explored. In the method 394 ppm permanganate solution was used as a chemiluminogenic reagent in 0.289 mol dm(-3) sulphuric acid media. A photomultiplier tube was used as a detector at a total flow rate of 4.94 ml/min. Perphenazine was determined by a linear calibration plot of the following equation in the range 50-350 ppm: mV=-4.488+0.1162C, with a correlation coefficient of 0.9989 for five measurements and a relative standard deviation less than 2.33. A sampling frequency not less than 110 samples h(-1) was established. Three factors namely, the flow rate, sulphuric acid and permanganate concentrations were found to have an influence on the amount of chemiluminescence intensity produced. Therefore, their interaction effects were thoroughly investigated by employing the 2(3) factorial design chemometrical approach and the results obtained revealed a higher interaction between sulphuric acid and permanganate and a less significant interaction for both reagents with the flow rate. The interaction of variables observed necessitated the conduct of the super modified simplex optimization procedure which has resulted in offering the proper optimum conditions as stated above and led to the quantitative assay of perphenazine. An interference study indicated that the method was suitable for application in pharmaceutical preparations.

  6. A smart ZnO@polydopamine-nucleic acid nanosystem for ultrasensitive live cell mRNA imaging by the target-triggered intracellular self-assembly of active DNAzyme nanostructures† †Electronic supplementary information (ESI) available: Oligonucleotide sequences, DLS and zeta potential measurements, TEM images, absorption and fluorescence spectra, cytotoxicity assay and CLSM images. See DOI: 10.1039/c6sc04633a Click here for additional data file.

    Science.gov (United States)

    He, Dinggeng; He, Xing; Yang, Xue

    2017-01-01

    Efficient strategies for the ultrasensitive imaging of gene expression in living cells are essential in chemistry and cell biology. Here, we report a novel and efficient enzyme-free dual signal amplification strategy for live cell mRNA imaging by using a smart nucleic acid hairpin-based nanosystem. This nanosystem consists of a ZnO nanoparticle core, an interlayer of polydopamine and an outer layer of four hairpin DNA (hpDNA) probes. Such a core–shell nanosystem facilitates the cellular uptake of molecular hairpin payloads, protects them from nuclease digestion, and delivers them into the cytoplasm by the acid-triggered dissolution of the ZnO core. In the presence of target mRNA, the released hpDNA probes self-assemble via HCR into wire-shaped active DNAzymes that catalyze the generation of a fluorescence signal. The target-initiated HCR events and DNAzyme cascades offer efficient dual amplification and enable the ultrasensitive detection of mRNA with a femtomolar detection limit. Live cell assays show an intense fluorescence response from a tumor-related biomarker survivin mRNA only in tumor cells untreated with a survivin expression repressor YM155, but not in normal cells. The developed nanosystem provides a potential platform for the amplified imaging of low-abundance disease-related biomarkers in live cells. PMID:28553521

  7. Efficient audio power amplification - challenges

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Michael A.E.

    2005-07-01

    For more than a decade efficient audio power amplification has evolved and today switch-mode audio power amplification in various forms are the state-of-the-art. The technical steps that lead to this evolution are described and in addition many of the challenges still to be faced and where extensive research and development are needed is covered. (au)

  8. Efficient Audio Power Amplification - Challenges

    DEFF Research Database (Denmark)

    Andersen, Michael Andreas E.

    2005-01-01

    For more than a decade efficient audio power amplification has evolved and today switch-mode audio power amplification in various forms are the state-of-the-art. The technical steps that lead to this evolution are described and in addition many of the challenges still to be faced and where...... extensive research and development are needed is covered....

  9. Detection of Ganoderic Acid A in Ganoderma lingzhi by an Indirect Competitive Enzyme-Linked Immunosorbent Assay.

    Science.gov (United States)

    Sakamoto, Seiichi; Kohno, Toshitaka; Shimizu, Kuniyoshi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2016-05-01

    Ganoderma is a genus of medicinal mushroom traditionally used for treating various diseases. Ganoderic acid A is one of the major bioactive Ganoderma triterpenoids isolated from Ganoderma species. Herein, we produced a highly specific monoclonal antibody against ganoderic acid A (MAb 12 A) and developed an indirect competitive ELISA for the highly sensitive detection of ganoderic acid A in Ganoderma lingzhi, with a limit of detection of 6.10 ng/mL. Several validation analyses support the accuracy and reliability of the developed indirect competitive ELISA for use in the quality control of Ganoderma based on ganoderic acid A content. Furthermore, quantitative analysis of ganoderic acid A in G. lingzhi revealed that the pileus exhibits the highest ganoderic acid A content compared with the stipe and spore of the fruiting body; the best extraction efficiency was found when 50 % ethanol was used, which suggests the use of a strong liquor to completely harness the potential of Ganoderma triterpenoids in daily life. Georg Thieme Verlag KG Stuttgart · New York.

  10. High-performance liquid chromatography-tandem mass spectrometry assay of fatty acid amide hydrolase (FAAH) in blood: FAAH inhibition as clinical biomarker.

    Science.gov (United States)

    Yapa, Udeni; Prusakiewicz, Jeffery J; Wrightstone, Ann D; Christine, Lori J; Palandra, Joe; Groeber, Elizabeth; Wittwer, Arthur J

    2012-02-15

    Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for the degradation of the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA). FAAH inhibitors may be useful in treating many disorders involving inflammation and pain. Although brain FAAH may be the relevant target for inhibition, rat studies show a correlation between blood and brain FAAH inhibition, allowing blood FAAH activity to be used as a target biomarker. Building on experience with a rat leukocyte FAAH activity assay using [³H]AEA, we have developed a human leukocyte assay using stably labeled [²H₄]AEA as substrate. The deuterium-labeled ethanolamine reaction product ([²H₄]EA) was analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in the positive electrospray ionization (ESI) mode. The response for [²H₄]EA was linear from 10 nM to 10 μM, and the analysis time was less than 6 min/sample. Results using the [²H₄]AEA and HPLC-MS/MS method agreed well with those obtained using the [³H]AEA radiometric assay. In addition to using a nonradioactive substrate, the HPLC-MS/MS method had increased sensitivity with lower background. Importantly, the assay preserved partial FAAH inhibition resulting from ex vivo treatment with a time-dependent irreversible inhibitor, suggesting its utility with clinical samples. The assay has been used to profile the successful inhibition of FAAH in recent clinical trials. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Assay of calcium borogluconate veterinary medicines for calcium gluconate, boric acid, phosphorus, and magnesium by using inductively coupled plasma emission spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Lyons, D.J.; Spann, K.P.

    1985-03-01

    An inductively coupled plasma spectrometric method is described for the determination of 4 elements (Ca, B, P, and Mg) in calcium borogluconate veterinary medicines. Samples are diluted, acidified, and sprayed directly into the plasma. Reproducibility relative confidence intervals for a single sample assay are +/- 1.4% (calcium), +/- 1.8% (boron), +/- 2.6% (phosphorus), and +/- 1.4% (magnesium). The total element concentrations for each of 4 elements compared favorably with concentrations determined by alternative methods. Formulation estimates of levels of calcium gluconate, boric acid, phosphorus, and magnesium salts can be made from the analytical data.

  12. Peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) assay for specific detection of Mycobacterium immunogenum and DNA-FISH assay for analysis of pseudomonads in metalworking fluids and sputum.

    Science.gov (United States)

    Selvaraju, Suresh B; Kapoor, Renuka; Yadav, Jagjit S

    2008-01-01

    Specific and rapid detection and quantification of mycobacteria in contaminated metalworking fluid (MWF) are problematic due to complexity of the matrix and heavy background co-occurring microflora. Furthermore, cross-reactivity among neighboring species of Mycobacterium makes species differentiation difficult for this genus. Here, we report for the first time a species-specific peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method for Mycobacterium immunogenum, a non-tuberculous Mycobacterium species prevalent in MWF and implicated in occupational lung disease hypersensitivity pneumonitis and pseudo-outbreaks. A novel species-specific 14-bp PNA probe was designed for M. immunogenum based on its 16S rRNA gene sequence and was validated for specificity, by testing against a panel of other phylogenetically closely related rapidly growing mycobacteria and representative species of gram-positive, gram-negative, and acid fast organisms. In addition, a DNA-FISH protocol was optimized for co-detection of Pseudomonas, the most predominantly co-occurring genus in contaminated MWF. Reliable quantification for both the test organisms was achieved at or above a cell density of 10(3)cellsml(-1), a recognized minimum limit for microscopic quantification. The mycobacterial PNA-FISH assay was successfully adapted to human sputum demonstrating its potential for clinical diagnostic applications in addition to industrial MWF monitoring, to assess MWF-associated exposures and pseudo-outbreaks.

  13. Determination of vitamin B6 vitamers and pyridoxic acid in plasma: development and evaluation of a high-performance liquid chromatographic assay

    DEFF Research Database (Denmark)

    Bisp, Marianne R; Bor, Mustafa Vakur; Heinsvig, Else-Marie

    2002-01-01

    were eluted with a gradient of acetonitrile (0.5-15%) in a potassium phosphate buffer with 1-octanesulfonic acid and triethylamine, pH 2.16. The concentration of the vitamers was determined with fluorescence detector (328 nm excitation, 393 nm emission) after postcolumn derivatization with phosphate...... vitamers pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxamine 5'-phosphate (PMP), pyridoxine (PN), and pyridoxamine (PM) and the degradation product 4-pyridoxic acid (4-PA). The separation was accomplished using a C18 (ODS) analytical column and an ion-pair reversed-phase chromatography. B6 vitamers...... buffer containing 1 g/L sodium bisulfite. The performance of the assay was evaluated by analyzing six plasma samples with interrelated concentration and two control samples (unspiked and vitamer spiked) over a 3-months period. The HPLC method was able to identify PLP, 4-PA, PM, PL, PN, and PMP from all...

  14. Preventing false-negatives in the in vitro skin sensitization testing of acid anhydrides using interleukin-8 release assays.

    Science.gov (United States)

    Narita, Kazuto; Vo, Phuc Thi Hong; Yamamoto, Kenta; Kojima, Hajime; Itagaki, Hiroshi

    2017-08-01

    In vitro safety tests may be used as replacements for animal tests owing to their accuracy and high-throughput performance. However, several in vitro skin sensitization tests produce false-negative results such as acid anhydride. Here, we investigated the relationship between false-negative results of acid anhydride and its hydrolysis by aqueous vehicle. Differences in the pattern of hydrolysis for phthalic anhydride (PAH) due to addition of 1 drop of stock solution of PAH in liquid paraffin (LP) dispersion medium and PAH in DMSO were analyzed in a cell-free system. The results showed that use of LP dispersion medium stabilized the concentration of PAH in water over 5min by sustained-release, although almost all PAH converted to phthalic acid in water within 5min using DMSO. Additionally, treatment of THP-1 cells with PAH and phthalic acid using LP dispersion medium for 5min resulted in a 32-fold increase in IL-8 release for PAH as compared with that in the vehicle control. In contrast, for PAH using aqueous vehicle and phthalic acid using LP dispersion medium, there were no significant increases in IL-8 release. Similarly, using LP dispersion medium, trimellitic anhydride significantly increased IL-8 release was observed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Design and performance testing of a DNA extraction assay for sensitive and reliable quantification of acetic acid bacteria directly in red wine using real time PCR

    Directory of Open Access Journals (Sweden)

    Cédric eLONGIN

    2016-06-01

    Full Text Available Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence there is a real need for a rapid, specific, sensitive and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR. Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP at 1% (v/v during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 mL to 10 mL. Thus the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage.

  16. In vitro amplification of PrPSc derived from the brain and blood of sheep infected with scrapie.

    Science.gov (United States)

    Thorne, Leigh; Terry, Linda A

    2008-12-01

    Scrapie is a fatal, naturally transmissible, neurodegenerative prion disease that affects sheep and goats and is characterized by the accumulation of a misfolded protein, PrPSc, converted from host-encoded PrPc, in the central nervous system of affected animals. Highly efficient in vitro conversion of host PrPc to PrPSc has been achieved in models of scrapie and in natural prion diseases by protein misfolding cyclic amplification (PMCA). Here, we demonstrate amplification, by serial PMCA, of PrPSc from individual sources of scrapie-infected sheep. Efficiency of amplification was affected by the pairing of the source of PrPSc with the control brain substrate of different genotypes of PrP. In line with previous studies, efficiency of amplification was greatly enhanced with the addition of a synthetic polyanion, polyadenylic acid (PolyA), facilitating rapid detection of low levels of PrPSc from body fluids such as blood. To this end PrPSc was amplified, in a 3 day PMCA assay, from blood leukocyte preparations from VRQ/VRQ scrapie-affected sheep at clinical end point. While PolyA-assisted PMCA resulted in spontaneous conversion of PrPc, we were able to distinguish blood samples from unaffected and affected sheep under controlled conditions. This study demonstrates that highly efficient amplification of PrPSc can be achieved for ovine scrapie from both brain and blood from naturally infected sheep and shows potential applications for improvements in current diagnostics and pre-mortem testing.

  17. Detection of Botrytis cinerea by loop-mediated isothermal amplification.

    Science.gov (United States)

    Tomlinson, J A; Dickinson, M J; Boonham, N

    2010-12-01

    To develop a sensitive, rapid and simple method for detection of Botrytis cinerea based on loop-mediated isothermal amplification (LAMP) that would be suitable for use outside a conventional laboratory setting. A LAMP assay was designed based on the intergenic spacer of the B. cinerea nuclear ribosomal DNA (rDNA). The resulting assay was characterized in terms of sensitivity and specificity using DNA extracted from cultures. The assay consistently amplified 65 pg B. cinerea DNA. No cross-reactivity was observed with a range of other fungal pathogens, with the exception of the closely related species Botrytis pelargonii. Use of a novel real-time LAMP platform (the OptiGene Genie I) allowed detection of B. cinerea in infected rose petals, with amplification occurring in cut flowers, fruit and vegetables. © 2010 British Crown Copyright. Letters in Applied Microbiology 51, 650-657 © 2010 The Society for Applied Microbiology.

  18. Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

    Science.gov (United States)

    Morandi, Luca; de Biase, Dario; Visani, Michela; Cesari, Valentina; De Maglio, Giovanna; Pizzolitto, Stefano; Pession, Annalisa; Tallini, Giovanni

    2012-01-01

    The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes. PMID:22558339

  19. Allele specific locked nucleic acid quantitative PCR (ASLNAqPCR: an accurate and cost-effective assay to diagnose and quantify KRAS and BRAF mutation.

    Directory of Open Access Journals (Sweden)

    Luca Morandi

    Full Text Available The use of tyrosine kinase inhibitors (TKIs requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR. The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes.

  20. Development of a loop-mediated isothermal amplification assay for ...

    African Journals Online (AJOL)

    Administrator

    2011-09-28

    Sep 28, 2011 ... 1National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China. 2College of ... pure cultures was 10-fold higher than that of polymerase chain reaction (PCR), with a detection limit of. 34 CFU ... multiplex PCR, and real-time PCR have been used to detect M.

  1. In vivo genotoxicity testing of the amnesic shellfish poison (domoic acid) in piscine erythrocytes using the micronucleus test and the comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Cavas, Tolga [Mersin University, Faculty of Sciences and Letters, Department of Biology, 33343 Mersin (Turkey)], E-mail: tcavas@mersin.edu.tr; Koenen, Serpil [Mersin University, Faculty of Sciences and Letters, Department of Biology, 33343 Mersin (Turkey)

    2008-11-11

    Domoic acid (DA) is a neurotoxic amino acid naturally produced in the marine environment by some diatom species belonging to the genus Pseudo-nitzschia. Although the neurotoxic properties of DA have been demonstrated, very little is known about in vivo genotoxicity of DA on aquatic organisms. In the present paper, an in vivo study on the genotoxic effects of domoic acid was carried out on a fish, Oreochromis niloticus, using the micronucleus test and the comet assay. The fish were exposed to three doses of domoic acid (1, 5 and 10 {mu}g/g body weight) by intracoelomic injections. Ethyl methane sulphonate at a single dose of 5 mg/l was used as positive control. Analysis of micronuclei, nuclear abnormalities and DNA damage were carried out on peripheral erythrocytes sampled 24, 48 and 72 h post-treatment. Our results revealed significant increases in the frequencies of micronuclei, nuclear abnormalities as well as DNA strand breaks and thus demonstrated the genotoxic potential of DA on fish.

  2. Phenolic Contents and Antioxidant Potential of Crataegus Fruits Grown in Tunisia as Determined by DPPH, FRAP, and β-Carotene/Linoleic Acid Assay

    Directory of Open Access Journals (Sweden)

    Farouk Mraihi

    2013-01-01

    Full Text Available Crataegus fruit is one of most important fruits in Tunisian flora. Some fruits of this genus are edible. This study was undertaken in order to examine the benefits of these fruits in human health and their composition of antioxidants including total polyphenol, flavonoids, proanthocyanidins content, and total anthocyanins. The antioxidative properties of the ultrasonic methanolic extract were assessed by different in vitro methods such as the FRAP, DPPH, and β-carotene/linoleic acid assay. We concluded that peel fraction of red fruits possessed relatively high antioxidant activity and might be a rich source of natural antioxidants in comparison with the pulp and seed fruit extract. The results also showed that hawthorn yellow fruit presents lower amounts of phenolic content, absence of anthocyanins, and less antioxidant capacity. Most of peel and seed fractions were stronger than the pulp fractions in antioxidant activity based on their DPPH IC50, FRAP values, and results of β-carotene/linoleic acid. The total phenolic compounds contents were also highly correlated with the DPPH method and the FRAP assay.

  3. Ethamsylate (Dicynone) interference in determination of serum creatinine, uric acid, triglycerides, and cholesterol in assays involving the Trinder reaction; in vivo and in vitro.

    Science.gov (United States)

    Dastych, Milan; Wiewiorka, Ondrej; Benovská, Miroslava

    2014-01-01

    The aim of our research was the quantification of interfering properties of the haemostatic drug Dicynone (ethamsylate) in serum creatinine, uric acid, cholesterol, and triglyceride assays using the Trinder reaction. Blood from patients was collected before and 15 minutes after administration of 500 mg Dicynone dose i.v. and the above mentioned analytes were quantified using Roche assays (Cobas 8000). In our in vitro experiment, we measured concentrations of the analytes in pooled serum aliquots with final concentrations of Dicynone additions 0, 30, 60, 150, and 300 mg/L. Aliquots with 60 mg/L Dicynone were also measured at 2, 6, and 8 hours after initial measurement when stored in 22 degrees C and 4 degrees C for comparison. Concentrations of the measured analytes in samples from patients administered with a 500 mg dose of Dicynone were lower in all cases (n = 10) when compared to values in samples taken immediately before treatment. The in vitro samples showed that considerable negative interference occurred even with the low concentrations of Dicynone additions (30 and 60 mg/L), showing the strongest negative interference in creatinine values, followed by uric acid, triglycerides, and cholesterol. Using in vitro samples, we showed strong time and temperature dependence on Dicynone interference. We found and proved significant negative interference of the drug Dicynone (ethamsylate) in the clinical analysis of blood using in vivo and in vitro experiments. Furthermore, we observed a change of this effect in serum matrix over time and at different storage temperatures.

  4. Effects of Acid Stress on Aerobic Decomposition of Algal and Aquatic Macrophyte Detritus: Direct Comparison in a Radiocarbon Assay

    Science.gov (United States)

    Schoenberg, Steven A.; Benner, Ronald; Armstrong, Anthony; Sobecky, Patricia; Hodson, Robert E.

    1990-01-01

    Radiolabeled phytoplankton and macrophyte lignocelluloses were incubated at pHs 4 and 7 in water from a naturally acidic freshwater wetland (Okefenokee Swamp; ambient pH, 3.8 to 4.2), a freshwater reservoir (L-Lake; pH 6.7 to 7.2), and a marine marsh (Sapelo Island; pH ∼7.8). The data suggest that acidity is an important factor in explaining the lower decomposition rates of algae in Okefenokee Swamp water relative to L-Lake or Sapelo Island water. The decomposition of algal substrate was less sensitive to low pH (∼5 to 35% inhibition) than was the decomposition of lignocellulose (∼30 to 70% inhibition). These substrate-dependent differences were greater and more consistent in salt marsh than in L-lake incubations. In both freshwater sites, the extent to which decomposition was suppressed by acidity was greater for green algal substrate than for mixed diatom or blue-green algal (cyanobacteria) substrates. The use of different bases to adjust pH or incubation in a defined saltwater medium had no significant effect on substrate-dependent differences. Although pH differences with lignocellulose were larger in marine incubations, amendment of lakewater with marine bacteria or with calcium, known to stabilize exoenzymes in soils, did not magnify the sensitivity of decomposition to acid stress. PMID:16348097

  5. Rapid Diagnosis of Trichomonas vaginalis by Testing Vaginal Swabs in an Isothermal Helicase-Dependent AmpliVue Assay.

    Science.gov (United States)

    Gaydos, Charlotte A; Hobbs, Marcia; Marrazzo, Jeanne; Schwebke, Jane; Coleman, Jenell S; Masek, Billie; Dize, Laura; Jang, Dan; Li, Jenny; Chernesky, Max

    2016-06-01

    The AmpliVue Trichomonas Assay (Quidel) is a new Federal Drug Administration-cleared rapid test for qualitative detection of Trichomonas vaginalis (TV) DNA in female vaginal specimens. The assay is based on BioHelix's helicase-dependent amplification isothermal technology in conjunction with a disposable lateral-flow detection device, with a total turnaround time of approximately 45 minutes. The objective of this study was to compare the performance of this new assay to wet preparation and culture as well as to another Federal Drug Administration-cleared nucleic acid amplification assay. Four clinician collected vaginal swabs were obtained from women attending sexually transmitted disease, family planning, and OB/GYN clinics and tested by AmpliVue Trichomonas Assay and comparator tests: saline microscopy, TV culture (InPouch), and Aptima TV. AmpliVue Trichomonas Assay results were compared with a composite positive comparator (CPC) as determined by the results from culture and/or wet mount microscopic examination. At least one of either the wet preparation or culture reference test results was required to be positive to establish CPC. A total of 992 patients, 342 symptomatic and 650 asymptomatic patients, were included in the study. Results for AmpliVue for all women combined compared with saline microscopy and culture as a CPC yielded a sensitivity of 100%. Specificity for all women was 98.2%. Overall percent agreement versus Aptima TV was 97.8%. Sensitivity for AmpliVue compared with Aptima was 90.7% %, whereas specificity was 98.9%. The rapid AmpliVue Trichomonas Assay performed as well as microscopy and culture, and had comparable sensitivity and specificity to another nucleic acid amplification test for the detection of TV. This study provided evidence of new diagnostic options and indicated very good performance of amplified testing for detection of TV in symptomatic and asymptomatic women.

  6. α-Eleostearic acid-containing triglycerides for a continuous assay to determine lipase sn-1 and sn-3 regio-preference.

    Science.gov (United States)

    El Alaoui, Meddy; Soulère, Laurent; Noiriel, Alexandre; Queneau, Yves; Abousalham, Abdelkarim

    2017-08-01

    Lipases are essentially described as sn-1 and sn-3 regio-selective. Actually few methods are available to measure this lipase regio-selectivity, moreover they require chiral chromatography analysis or specific derivations which are discontinuous and time consuming. In this study we describe a new, convenient, sensitive and continuous spectrophotometric method to screen lipases regio-selectivity using synthetic triglycerides (TG) containing α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) either at the sn-1 position [1-α-eleostearoyl-2,3-octadecyl-sn-glycerol (sn-EOO)] or at the sn-3 position [1,2-octadecyl-3-α-eleostearoyl-sn-glycerol (sn-OOE)] and coated onto the wells of microtiter plates. A non-hydrolysable ether bond, with a non UV-absorbing alkyl chain, was introduced at the other sn positions to prevent acyl chain migration during TG synthesis or lipolysis. The synthesis of TG containing α-eleostearic acid was performed from S-glycidol in six steps to obtain sn-EOO and in five steps to sn-OOE. The α-eleostearic acid conjugated triene constitutes an intrinsic chromophore and, consequently, confers the strong UV absorption properties of this free fatty acid as well as of the TG harboring it. The lipase activity on coated sn-EOO or sn-OOE was measured by the increase in the absorbance at 272nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. Human and porcine pancreatic lipases, guinea pig pancreatic lipase related protein 2, Thermomyces lanuginosus lipase, Candida antarctica lipase A and Candida antarctica lipase B were all used to validate the assay. This continuous high-throughput screening method could determine directly without any processes after lipolysis the regio-selectivity of various lipases. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Novel fluorescent nanoparticles for ultrasensitive identification of nucleic acids by optical methods

    DEFF Research Database (Denmark)

    Mulberg, Mads Westergaard; Taskova, Maria; Thomsen, Rasmus P.

    2017-01-01

    For decades, the detection of nucleic acids and their interactions at low abundances has been a challenging task. Present nucleic acid diagnostics are primarily based on enzymatic reactions including sequencing, polymerase-chain reaction and microarrays. However, the use of enzymatic amplification...... interferes with the initial biomolecular system, is limited to in vitro assays, often time consuming and rather expensive. Therefore, there is interest in new amplification-free detection methods. A tremendous progress has been made in fluorescence based optical detection of biomolecules. In this work, we...... aimed at developing efficient tools for amplification-free nucleic acid detection. The result of simple and inexpensive polymerization in the presence of fluorescent dyes and additional functionalization reagents was ultra-bright fluorescent nanoparticles modified with additional groups...

  8. Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay

    Science.gov (United States)

    Jauset-Rubio, Miriam; Svobodová, Markéta; Mairal, Teresa; McNeil, Calum; Keegan, Neil; Saeed, Ayman; Abbas, Mohammad Nooredeen; El-Shahawi, Mohammad S.; Bashammakh, Abdulaziz S.; Alyoubi, Abdulrahman O.; O´Sullivan, Ciara K.

    2016-01-01

    Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 × 10−11 M (190 amol), equivalent to 8.67 × 105 copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 °C. The use of the tailed primers obviates the need for hapten labelling and consequent use of capture and reporter antibodies, whilst also avoiding the need for any post-amplification processing for the generation of single stranded DNA, thus presenting an assay that can facilely find application at the point of need. PMID:27886248

  9. Qualitative detection of class IIa bacteriocinogenic lactic acid bacteria from traditional Chinese fermented food using a YGNGV-motif-based assay.

    Science.gov (United States)

    Liu, Wenli; Zhang, Lanwei; Yi, Huaxi; Shi, John; Xue, Chaohui; Li, Hongbo; Jiao, Yuehua; Shigwedha, Nditange; Du, Ming; Han, Xue

    2014-05-01

    In the present study, a YGNGV-motif-based assay was developed and applied. Given that there is an increasing demand for natural preservatives, we set out to obtain lactic acid bacteria (LAB) that produce bacteriocins against Gram-positive and Gram-negative bacteria. We here isolated 123 LAB strains from 5 types of traditional Chinese fermented food and screened them for the production of bacteriocins using the agar well diffusion assay (AWDA). Then, to acquire LAB producing class IIa bacteriocins, we used a YGNGV-motif-based assay that was based on 14 degenerate primers matching all class IIa bacteriocin-encoding genes currently deposited in NCBI. Eight of the LAB strains identified by AWDA could inhibit Gram-positive and Gram-negative bacteria; 5 of these were YGNGV-amplicon positive. Among these 5 isolates, amplicons from 2 strains (Y31 and Y33) matched class IIa bacteriocin genes. Strain Y31 demonstrated the highest inhibitory activity and the best match to a class IIa bacteriocin gene in NCBI, and was identified as Enterococcus faecium. The bacteriocin from Enterococcus avium Y33 was 100% identical to enterocin P. Both of these strains produced bacteriocins with strong antimicrobial activity against Listeria monocytogenes, Escherichia coli, and Bacillus subtilis, hence these bacteriocins hold promise as potential bio-preservatives in the food industry. These findings also indicated that the YGNGV-motif-based assay used in this study could identify novel class IIa bacteriocinogenic LAB, rapidly and specifically, saving time and labour by by-passing multiple separation and purification steps. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Liquid Chromatography-Tandem Mass Spectrometry Assay of Leukocyte Acid α-Glucosidase for Post-Newborn Screening Evaluation of Pompe Disease

    Science.gov (United States)

    Lin, Na; Huang, Jingyu; Violante, Sara; Orsini, Joseph J.; Caggana, Michele; Hughes, Erin E.; Stevens, Colleen; DiAntonio, Lisa; Liao, Hsuan Chieh; Hong, Xinying; Ghomashchi, Farideh; Kumar, Arun Babu; Zhou, Hui; Kornreich, Ruth; Wasserstein, Melissa; Gelb, Michael H.; Yu, Chunli

    2017-01-01

    BACKGROUND Pompe disease (PD) is the first lysosomal storage disorder to be added to the Recommended Uniform Screening Panel for newborn screening. This condition has a broad phenotypic spectrum, ranging from an infantile form (IOPD), with severe morbidity and mortality in infancy, to a late-onset form (LOPD) with variable onset and progressive weakness and respiratory failure. Because the prognosis and treatment options are different for IOPD and LOPD, it is important to accurately determine an individual’s phenotype. To date, no enzyme assay of acid α-glucosidase (GAA) has been described that can differentiate IOPD vs LOPD using blood samples. METHODS We incubated 10 μL leukocyte lysate and 25 μL GAA substrate and internal standard (IS) assay cocktail for 1 h. The reaction was purified by a liquid–liquid extraction. The extracts were evaporated and reconstituted in 200 μL methanol and analyzed by LC-MS/MS for GAA activity. RESULTS A 700-fold higher analytical range was observed with the LC-MS/MS assay compared to the fluorometric method. When GAA-null and GAA-containing fibroblast lysates were mixed, GAA activity could be measured accurately even in the range of 0%–1% of normal. The leukocyte GAA activity in IOPD (n = 4) and LOPD (n = 19) was 0.44–1.75 nmol · h−1 · mg−1 and 2.0–6.5 nmol · h−1 · mg−1, respectively, with no overlap. The GAA activity of pseudodeficiency patients ranged from 3.0–28.1 nmol · h−1 · mg−1, showing substantial but incomplete separation from the LOPD group. CONCLUSIONS This assay allows determination of low residual GAA activity in leukocytes. IOPD, LOPD, and pseudodeficiency patients can be partially differentiated by measuring GAA using blood samples. PMID:28196920

  11. A systematic review and economic evaluation of intraoperative tests [RD-100i one-step nucleic acid amplification (OSNA) system and Metasin test] for detecting sentinel lymph node metastases in breast cancer.

    Science.gov (United States)

    Huxley, Nicola; Jones-Hughes, Tracey; Coelho, Helen; Snowsill, Tristan; Cooper, Chris; Meng, Yang; Hyde, Chris; Mújica-Mota, Rubén

    2015-01-01

    In breast cancer patients, sentinel lymph node biopsy is carried out at the same time as the removal of the primary tumour to postoperatively test with histopathology for regional metastases in the sentinel lymph node. Those patients with positive test results are then operated on 2-4 weeks after primary surgery to remove the lymph nodes from the axilla (axillary lymph node dissection, ALND). New molecular tests RD-100i [one-step nucleic acid amplification (OSNA); based on messenger RNA amplification to identify the cytokeratin-19 (CK19) gene marker] (Sysmex, Norderstedt, Germany) and Metasin (using the CK19 and mammaglobin gene markers) (Cellular Pathology, Princess Alexandra Hospital NHS Trust, Harlow, UK) are intended to provide an intraoperative diagnosis, thereby avoiding the need for postoperative histopathology and, in positive cases, a second operation for ALND. To evaluate the clinical effectiveness and cost-effectiveness of using OSNA and Metasin in the NHS in England for the intraoperative diagnosis of sentinel lymph nodes metastases, compared with postoperative histopathology, the current standard. Electronic databases including MEDLINE, MEDLINE In-Process & Other Non-Indexed Citations, EMBASE, The Cochrane Library and the Health Economic Evaluations Database as well as clinical trial registries, grey literature and conference proceedings were searched up to July 2012. A systematic review of the evidence was carried out using standard methods. Single-gate studies were used to estimate the accuracy of OSNA with histopathology as the reference standard. The cost-effectiveness analysis adapted an existing simulation model of the long-term costs and health implications of early breast cancer diagnostic outcomes. The model accounted for the costs of an extended first operation with intraoperative testing, the loss of health-related quality of life (disutility) from waiting for postoperative test results, disutility and costs of a second operation, and long

  12. Avaliação do protocolo PCR4 de Marchetti em tecidos parafinizados para o diagnóstico da tuberculose cutânea e ganglionar Evaluation of Marchetti PCR4 amplification assay to the diagnosis of cutaneous and lymph node tuberculosis from formalin-fixed paraffin-embedded tissue

    Directory of Open Access Journals (Sweden)

    Luciana Botinelly Mendonça Fujimoto

    2007-06-01

    methods of diagnose including bacteriological and histopathological assays involve some technical difficulties, and the polymerase chain reaction - PCR arise as an alternative method allowing specific results faster than the others. In this context the accuracy of PCR4 Marchetti et al. protocol was compared with traditional methods. MATERIAL AND METHOD: Nested-PCR for IS6110 (123 pb were applied on 83 CLTb suspicious formalin fixed and paraffin embedded samples of tissues (52 cutaneous and 31 lymph node, obtained from 1997 to 2002. All cases were evaluated by bacteriological and histopathological methods. Accuracy analyses were carried out between the PCR amplification results and those related on bacteriological and histopathological methods. RESULTS AND DISCUSSIONS: Positive results of PCR4 were about 50.6% (59.6% in cutaneous samples and of 35.5% in lymph nodes samples. In both groups were observed false-negative and false-positive results. Some hypotheses that explain those results are related to the presence of IS6110 in environmental mycobacterias in the Amazon region and the absence of standardized DNA concentration to amplification assays. CONCLUSIONS: The proposed protocol was as positive as others ones available in the literature. Definitive Tb diagnostic can be obtained on lymph node paraffin embedded PCR in association with bacteriological or histopathological method. A better accuracy of an amplification assay applied to cutaneous Tb suspicious lesions has to be still under research.

  13. Development of an Interaction Assay between Single-Stranded Nucleic Acids Trapped with Silica Particles and Fluorescent Compounds

    Directory of Open Access Journals (Sweden)

    R. Maeda

    2012-09-01

    Full Text Available Biopolymers are easily denatured by heating, a change in pH or chemical substances when they are immobilized on a substrate. To prevent denaturation of biopolymers, we developed a method to trap a polynucleotide on a substrate by hydrogen bonding using silica particles with surfaces modified by aminoalkyl chains ([A-AM silane]/SiO2. [A-AM silane]/SiO2 was synthesized by silane coupling reaction of N-2-(aminoethyl-3-aminopropyltrimethoxysilane (A-AM silane with SiO2 particles with a diameter of 5 μm at 100 °C for 20 min. The surface chemical structure of [A-AM silane]/SiO2 was characterized by Fourier transform infrared spectroscopy and molecular orbital calculations. The surface of the silica particles was modified with A-AM silane and primary amine groups were formed. [A-AM silane]/SiO2 was trapped with single-stranded nucleic acids [(Poly-X; X = A (adenine, G (guanine and C (cytosine] in PBS solution at 37 °C for 1 h. The single-stranded nucleic acids were trapped on the surface of the [A-AM silane]/SiO2 by hydrogen bonding to form conjugated materials. The resulting complexes were further conjugated by derivatives of acridine orange (AO as fluorescent labels under the same conditions to form [AO:Poly-X:A-AM silane]/SiO2 complexes. Changes in the fluorescence intensity of these complexes originating from interactions between the single-stranded nucleic acid and aromatic compounds were also evaluated. The change in intensity displayed the order [AO: Poly-G: A-AM silane]/SiO2 > [AO:Poly-A:A-AM silane]/SiO2 >> [AO:Poly-C:A-AM silane]/SiO2. This suggests that the single-stranded nucleic acids conjugated with aminoalkyl chains on the surfaces of SiO2 particles and the change in fluorescence intensity reflected the molecular interaction between AO and the nucleic-acid base in a polynucleotide.

  14. Next generation Chirped Pulse Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Nees, J.; Biswal, S.; Mourou, G. [Univ. Michigan, Center for Ultrafast Optical Science, Ann Arbor, MI (United States); Nishimura, Akihiko; Takuma, Hiroshi

    1998-03-01

    The limiting factors of Chirped Pulse Amplification (CPA) are discussed and experimental results of CPA in Yb:glass regenerative amplifier are given. Scaling of Yb:glass to the petawatt level is briefly discussed. (author)

  15. A rapid and sensitive assay for determination of doxycycline using thioglycolic acid-capped cadmium telluride quantum dots

    Science.gov (United States)

    Tashkhourian, Javad; Absalan, Ghodratollah; Jafari, Marzieh; Zare, Saber

    2016-01-01

    A rapid, simple and inexpensive spectrofluorimetric sensor for determination of doxycycline based on its interaction with thioglycolic acid-capped cadmium telluride quantum dots (TGA/CdTe QDs) has been developed. Under the optimum experimental conditions, the sensor exhibited a fast response time of determination of doxycycline in a concentration range of 1.9 × 10-6-6.1 × 10-5 mol L-1 with a detection limit of 1.1 × 10-7 mol L-1. The sensor was applied for determination of doxycycline in honey and human serum samples.

  16. Development of a Medium-Throughput Targeted LCMS Assay to Detect Endogenous Cellular Levels of Malonyl-CoA to Screen Fatty Acid Synthase Inhibitors.

    Science.gov (United States)

    Hopcroft, Philip J; Fisher, David I

    2016-02-01

    The fatty acid synthase (FAS) enzyme in mammalian cells is a large multidomain protein responsible for de novo synthesis of fatty acids. The steps catalyzed by FAS involve the condensation of acetyl-CoA and malonyl-CoA moieties in the presence of NADPH until palmitate is formed. Inhibition of FAS causes an accumulation of intracellular malonyl-CoA, as this metabolite is essentially committed to fatty acid synthesis once formed. Detection of intracellular metabolites for screening can be problematic due to a lack of appropriate tools, but here we describe a targeted liquid chromatography-mass spectroscopy (LCMS) method to directly measure endogenous levels of malonyl-CoA to drive a drug development structure-activity relationship (SAR) screening cascade. Our process involves preparation of samples at 96-well scale, normalization postpermeabilization via use of a whole-well imaging platform, and the LCMS detection methodology. The assay is amenable to multiplexing cellular endpoints, has a typical Z' of >0.6, and has high reproducibility of EC50 values. © 2015 Society for Laboratory Automation and Screening.

  17. Multiplexed Molecular Assays for Rapid Rule-Out of Foot-and-Mouth Disease

    Energy Technology Data Exchange (ETDEWEB)

    Lenhoff, R; Naraghi-Arani, P; Thissen, J; Olivas, J; Carillo, C; Chinn, C; Rasmussen, M; Messenger, S; Suer, L; Smith, S M; Tammero, L; Vitalis, E; Slezak, T R; Hullinger, P J; Hindson, B J; Hietala, S; Crossley, B; Mcbride, M

    2007-06-26

    A nucleic acid-based multiplexed assay was developed that combines detection of foot-and-mouth disease virus (FMDV) with rule-out assays for two other foreign animal diseases and four domestic animal diseases that cause vesicular or ulcerative lesions indistinguishable from FMDV infection in cattle, sheep and swine. The FMDV 'look-alike' diagnostic assay panel contains five PCR and twelve reverse transcriptase PCR (RT-PCR) signatures for a total of seventeen simultaneous PCR amplifications for seven diseases plus incorporating four internal assay controls. It was developed and optimized to amplify both DNA and RNA viruses simultaneously in a single tube and employs Luminex{trademark} liquid array technology. Assay development including selection of appropriate controls, a comparison of signature performance in single and multiplex testing against target nucleic acids, as well of limits of detection for each of the individual signatures is presented. While this assay is a prototype and by no means a comprehensive test for FMDV 'look-alike' viruses, an assay of this type is envisioned to have benefit to a laboratory network in routine surveillance and possibly for post-outbreak proof of freedom from foot-and-mouth disease.

  18. Genotoxicity of 5-aminolevulinic and 4,5-dioxovaleric acids in the salmonella/microsuspension mutagenicity assay and SOS chromotest.

    Science.gov (United States)

    Onuki, Janice; Rech, Célia Maria; Medeiros, Marisa H G; de A Umbuzeiro, Gisela; Di Mascio, Paolo

    2002-01-01

    5-Aminolevulinic acid (ALA) is a heme precursor that accumulates in some porphyric disorders and in lead poisoning which can undergo metal-catalyzed oxidation producing reactive oxygen species and the keto-aldehyde, 4,5-dioxovaleric acid (DOVA). Evidence in vitro of ALA-induced DNA lesions suggests that ALA and DOVA have mutagenic potential that could possibly contribute to an increased frequency of hepatocellular carcinoma (HCC) in patients with acute intermittent porphyria (AIP). In this study, we evaluated the genotoxic potential of ALA and DOVA. In the absence of exogenous metabolic activation, ALA and DOVA were mutagenic in Salmonella typhimurium tester strain TA104. ALA was also mutagenic in S. typhimurium TA102, but not in TA98, TA100, or TA1535, indicating an oxidative mechanism. Removal of H(2)O(2) with catalase gave only partial protection, suggesting generation of other mutagenic species. Both ALA and DOVA damaged the DNA of Escherichia coli PQ37, inducing the SOS response detected by an increase in beta-galactosidase activity. These results verified the potential mutagenic activity of ALA and DOVA and reinforce the hypothesis that DNA damage induced by ALA may be associated with the development of HCC in individuals suffering from AIP. Copyright 2002 Wiley-Liss, Inc.

  19. Sensitive detection of DNA methyltransferase using the dendritic rolling circle amplification-induced fluorescence.

    Science.gov (United States)

    Song, Weiling; Luan, Yawen; Guo, Xiaoyan; He, Peng; Zhang, Xiaoru

    2017-03-01

    The analysis of DNA methylation and MTase activities is very important in the early clinical diagnosis of cancer, on purposes of providing insights into the mechanism of gene repression and developing novel drugs of treating methylation-related diseases. Combining the dendritic rolling circle amplification and Mg(2+)-dependent DNAzyme with a function of catalyzing the generation of a fluorophore-labeled nucleic acid acting as readout signal for the analyses, a new fluorescent method for DNA methyltransferase detection was reported. In the presence of DNA methyltransferases (MTase), the methylation-responsive sequence of double-stranded DNA probe was methylated and then cleaved by the methylation-sensitive restriction endonuclease DpnI. The cleaved hybrid DNA probe then functioned as a signal primer to initiate the dendritic rolling circle amplification reaction, containing a circular DNA and a structurally tailored hairpin structure. Subsequently, the circular nucleic acid template produced a complementary sequence to the Mg(2+)-dependent DNAzyme and a sequence identical to the loop region of the co-added hairpin structure. At last, a fluorescence readout signal was afforded by the DNAzyme-catalyzed cleavage of a fluorophore/quencher-modified substrate. This method enabled the analysis of the target MTase with a detection limit up to 0.36 U mL(-1), and a dynamic range was obtained from 1.0 to 10 U mL(-1). Moreover, the proposed strategy was successfully applied in real sample assay. With this assay, the inhibitors of MTase were evaluated and screened which might be helpful for the discovery of anticancer drugs. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Whole genome amplification: Use of advanced isothermal method ...

    African Journals Online (AJOL)

    Laboratory method for amplifying genomic deoxyribonucleic acid (DNA) samples aiming to generate more amounts and sufficient quantity DNA for subsequent specific analysis is named whole genome amplification (WGA). This method is only way to increase input material from few cells and limited DNA contents.

  1. Effect of okadaic acid on carpet shell clam (Ruditapes decussatus) haemocytes by in vitro exposure and harmful algal bloom simulation assays.

    Science.gov (United States)

    Prado-Alvarez, Maria; Flórez-Barrós, Fernanda; Méndez, Josefina; Fernandez-Tajes, Juan

    2013-06-01

    Okadaic acid (OA), produced by dinoflagellates during harmful algal blooms (HAB), belongs to the Diarrheic Shellfish Poisoning toxins that cause gastrointestinal symptoms in humans after consumption. In the present work, Ruditapes decussatus haemocytes were selected to evaluate the effect of OA on cell viability, enzymatic status and immune capacity through the measure by flow cytometry of apoptosis-cell death, non-specific esterase activity and phagocytosis. In order to compare different exposure conditions, two experiments were developed: in vitro exposure to OA and HAB simulation by feeding clams with the OA producer, Prorocentrum lima. Apoptosis was not OA dose-dependent and cell death increased in both assays. Phagocytosis of latex beads and esterase activity decreased in haemocytes incubated with OA. In contrast, esterases increased during the feeding with P. lima. Our results showed that OA and the simulated HAB caused damages on haemocyte functions and viability.

  2. Sialic acid and sialyl-lactose glyco-conjugates: design, synthesis and binding assays to lectins and swine influenza H1N1 virus.

    Science.gov (United States)

    Zevgiti, Stella; Zabala, Juliana Gonzalez; Darji, Ayub; Dietrich, Ursula; Panou-Pomonis, Eugenia; Sakarellos-Daitsiotis, Maria

    2012-01-01

    The terminal parts of the influenza hemagglutinin (HA) receptors α2,6- and α2,3-sialyllactoses were conjugated to an artificial carrier, named sequential oligopeptide carrier (SOC(4) ), to formulate human and avian receptor mimics, respectively. SOC(4) , formed by the tripeptide unit Lys-Aib-Gly, adopts a rigid helicoids-type conformation, which enables the conjugation of biomolecules to the Lys-N(ε) H(2) groups. By doing so, it preserves their initial conformations and functionalities of the epitopes. We report that SOC(4) -glyco-conjugate bearing two copies of the α2,6-sialyllactose is specifically recognized by the biotinylated Sambucus nigra (elderberry) bark lectin, which binds preferentially to sialic acid in an α2,6-linkage. SOC(4) -glyco-conjugate bearing two copies of the α2,3-sialyllactose was not recognized by the biotinylated Maackia amurensis lectin, despite its well-known α2,3-sialyl bond specificity. However, preliminary immune blot assays showed that H1N1 virus binds to both the SOC(4) -glyco-conjugates immobilized onto nitrocellulose membrane. It is concluded that Ac-SOC(4) [(Ac)(2) ,(3'SL-Aoa)(2) ]-NH(2) 5 and Ac-SOC(4) [(Ac)(2) ,(6'SL-Aoa)(2) ]-NH(2) 6 mimic the HA receptors. These findings could be useful for easy screening of binding and inhibition assays of virus-receptor interactions. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

  3. A facile, sensitive, and highly specific trinitrophenol assay based on target-induced synergetic effects of acid induction and electron transfer towards DNA-templated copper nanoclusters.

    Science.gov (United States)

    Li, Haiyin; Chang, Jiafu; Hou, Ting; Ge, Lei; Li, Feng

    2016-11-01

    Reliable, selective and sensitive approaches for trinitrophenol (TNP) detection are highly desirable with respect to national security and environmental protection. Herein, a simple and novel fluorescent strategy for highly sensitive and specific TNP assay has been successfully developed, which is based on the quenching of the fluorescent poly(thymine)-templated copper nanoclusters (DNA-CuNCs), through the synergetic effects of acid induction and electron transfer. Upon the addition of TNP, donor-acceptor complexes between the electron-deficient nitro-groups in TNP and the electron-donating DNA templates are formed, resulting in the close proximity between TNP and CuNCs. Moreover, the acidity of TNP contributes to the pH decrease of the system. These factors combine to dramatically quench the fluorescence of DNA-CuNCs, providing a "signal-off" strategy for TNP sensing. The as-proposed strategy demonstrates high sensitivity for TNP assay, and a detection limit of 0.03μM is obtained, which is lower than those reported by using organic fluorescent materials. More significantly, this approach shows outstanding selectivity over a number of TNP analogues, such as 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), 2,4-dinitrophenol (DNP), 3-nitrophenol (NP), nitrobenzene (NB), phenol (BP), and toluene (BT). Compared with previous studies, this method does not need complex DNA sequence design, fluorescent dye labeling, or sophisticated organic reactions, rendering the strategy with additional advantages of simplicity and cost-effectiveness. In addition, the as-proposed strategy has been adopted for the detection of TNP in natural water samples, indicating its great potential to be applied in the fields of public safety and environmental monitoring. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Cascade Invasive Reaction and Nanoparticle Hybridization for Subtyping of Influenza A Virus

    Science.gov (United States)

    Chi, Ying; Ge, Yiyue; Zhao, Kangchen; Zou, Bingjie; Liu, Bin; Qi, Xian; Bian, Qian; Shi, Zhiyang; Zhu, Fengcai; Zhou, Minghao; Cui, Lunbiao; Su, Chuan

    2017-01-01

    Considering the fatal human victims and economic loss caused by influenza virus infection every year, methodologies for rapid and on-site detection of influenza viruses are urgently needed. LAMP is the most commonly used nucleic acid isothermal amplification technology suitable for on-site use. However, for multiplex LAMP, differentiation of the amplicons derived from multiple targets is still challengeable currently. Here we developed a multiplex RT-LAMP assay for simultaneous amplification of three prominent subtypes of influenza viruses (A/H5, A/H7 and 2009A/H1). The amplicons were further identified by cascade invasive reaction and nanoparticle hybridization in separate target-specific detection tubes (referred to as mRT-LAMP-IRNH). The analytic sensitivities of the assay are 10 copies of RNA for all the three HA subtypes, and the specificity reached 100%. Clinical specimen analysis showed this assay had a combined sensitivity and specificity of 98.1% and 100%, respectively. Overall, the mRT-LAMP-IRNH assay can be used as a cost-saving method that utilizes a simple instrument to detect A/H5, A/H7, and 2009A/H1 influenza viruses, especially in resource-limited settings. PMID:28322309

  5. Principles of Whole-Genome Amplification.

    Science.gov (United States)

    Czyz, Zbigniew Tadeusz; Kirsch, Stefan; Polzer, Bernhard

    2015-01-01

    Modern molecular biology relies on large amounts of high-quality genomic DNA. However, in a number of clinical or biological applications this requirement cannot be met, as starting material is either limited (e.g., preimplantation genetic diagnosis (PGD) or analysis of minimal residual cancer) or of insufficient quality (e.g., formalin-fixed paraffin-embedded tissue samples or forensics). As a consequence, in order to obtain sufficient amounts of material to analyze these demanding samples by state-of-the-art modern molecular assays, genomic DNA has to be amplified. This chapter summarizes available technologies for whole-genome amplification (WGA), bridging the last 25 years from the first developments to currently applied methods. We will especially elaborate on research application, as well as inherent advantages and limitations of various WGA technologies.

  6. Simple and rapid spectrophotometric assay of albendazole in pharmaceuticals using iodine and picric acid as CT complexing agents

    Directory of Open Access Journals (Sweden)

    Nagaraju Swamy

    2014-12-01

    Full Text Available Two simple, rapid and inexpensive spectrophotometric methods are described for the determination of albendazole (ALB in bulk drug and in tablets. The methods are based on charge-transfer (CT complexation reaction involving ALB as n-donor and iodine as σ-acceptor (method A in dichloromethane or picric acid (PA as π-acceptor (method B in chloroform. The absorbance of CT complexes was measured at 380 nm for method A, and 415 nm for method B. The optimization of the experimental conditions is described. Under optimum conditions, Beer's law obeyed over the concentration ranges 8.0-240 and 2.4-42 μg mL-1 for method A and method B, respectively. The apparent molar absorptivity of CT complexes at the respective λmax are calculated to be 1.17×103 and 5.22×103 L mol-1cm-1 respectively, and the corresponding Sandell sensitivity values are 0.2273 and 0.0509 ng cm-2. The limits of detection (LOD and quantification (LOQ are calculated to be (0.69 and 2.08, and (0.10 and 0.30 μg mL-1 with method A, and method B, respectively. The intra-day and inter-day accuracy expressed as % RE and precision expressed as % RSD were less than 3%. The methods were applied to the determination of ALB in tablets.

  7. A FRET-based assay for characterization of alternative splicing events using peptide nucleic acid fluorescence in situ hybridization.

    Science.gov (United States)

    Blanco, Ana M; Rausell, Laura; Aguado, Begoña; Perez-Alonso, Manuel; Artero, Rubén

    2009-09-01

    We describe a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction by FRET measure. As a proof of concept we analyzed two alternative splicing events originating from lymphocyte antigen 6 (LY6) complex, locus G5B (LY6G5B) pre-mRNA. These are characterized by the removal of the first intron (Fully Spliced Isoform, FSI) or by retention of such intron (Intron-Retained Isoform, IRI). The use of PNA probe pairs labeled with donor (Cy3) and acceptor (Cy5) fluorophores, suitable to FRET, flanking FSI and IRI specific splice junctions specifically detected both mRNA isoforms in HeLa cells. We have observed that the method works efficiently with probes 5-11 nt apart. The data supports that this FRET-based PNA fluorescence in situ hybridization (FP-FISH) method offers a conceptually new approach for characterizing at the subcellular level not only splice variant isoform structure, location and dynamics but also potentially a wide variety of close range RNA-RNA interactions.

  8. Effects of two plant growth regulators, indole-3-acetic acid and β-naphthoxyacetic acid, on genotoxicity in Drosophila SMART assay and on proliferation and viability of HEK293 cells from the perspective of carcinogenesis.

    Science.gov (United States)

    Karadeniz, Asuman; Kaya, Bülent; Savaş, Burhan; Topcuoğlu, Ş Fatih

    2011-10-01

    In this study, the mutagenic and recombinogenic effects of indole-3-acetic acid (IAA), a plant growth regulator naturally synthesized in plants but produced synthetically, and β-naphthoxyacetic acid (BNOA), a synthetic plant growth regulator widely used in agricultural regions, were investigated using the somatic mutation and recombination test (SMART) in Drosophila wings. The effect of the same plant growth regulators against the proliferation and viability of a human immortalized embryonic kidney HEK293 cells which is at the early stage of carcinogenesis were also examined with MTT and trypan-blue exclusion assays. For the SMART assay, two different crosses were used: a standard and a high-bioactivation (HB) cross, involving the flare-3 and the multiple wing hairs markers. The HB cross involved flies characterized by an increased cytochrome P-450-dependent bioactivation capacity, which permits the more efficient biotransformation of promutagens and procarcinogens. In both crosses, the wings of the two types of progeny, inversion-free marker heterozygotes and balancer heterozygotes, were analyzed. The results show that IAA and BNOA are not mutagenic or recombinogenic in the wing cells of Drosophila. Furthermore, neither plant growth regulator affected the proliferation rate of HEK293 cells; however, both of them induced cell death at high concentrations.

  9. Transporter-mediated uptake of UDP-glucuronic acid by human liver microsomes: assay conditions, kinetics, and inhibition.

    Science.gov (United States)

    Rowland, Andrew; Mackenzie, Peter I; Miners, John O

    2015-01-01

    This study characterized the kinetics, variability, and factors that affect UDP-glucuronic acid (UDP-GlcUA) uptake by human liver microsomes (HLM). Biphasic kinetics were observed for UDP-GlcUA uptake by HLM. Uptake affinities (assessed as Kd) of the high- and low-affinity components differed by more than an order of magnitude (13 ± 6 vs. 374 ± 175 µM), but were comparable in terms of the maximal rate of uptake, with mean Vmax values differing less than 2.3-fold (56 ± 26 vs. 131 ± 35 pmol/min per mg). Variability in total intrinsic transporter activity (Uint) for microsomal UDP-GlcUA uptake across 12 livers was less than 4-fold. Experiments performed to optimize the conditions for microsomal UDP-GlcUA uptake demonstrated that both components were trans-stimulated by preloading (luminal addition) with an alternate UDP-sugar, and essentially abolished by the thiol-alkylating agent N-ethylmaleimide. Furthermore, interaction studies undertaken with a panel of drugs, alternate UDP-sugars, and glucuronide conjugates, at low (2.5 μM) and high (1000 μM) UDP-GlcUA concentrations, demonstrated that both components were inhibited to varying extents. Notably, the nucleoside analogs zidovudine, stavudine, lamivudine, and acyclovir inhibited both the high- and low- affinity components of microsomal UDP-GlcUA uptake by >45% at an inhibitor concentration of 100 μM. Taken together, these data demonstrate that human liver microsomal UDP-GlcUA uptake involves multiple protein-mediated components, and raises the possibility of impaired in vivo glucuronidation activity resulting from inhibition of UDP-GlcUA uptake into the endoplasmic reticulum membrane by drugs and other compounds. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  10. A fluorescence-coupled assay for gamma aminobutyric acid (GABA reveals metabolic stress-induced modulation of GABA content in neuroendocrine cancer.

    Directory of Open Access Journals (Sweden)

    Joseph E Ippolito

    Full Text Available Pathways involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA have been implicated in the pathogenesis of high grade neuroendocrine (NE neoplasms as well as neoplasms from a non-NE lineage. Using The Cancer Genome Atlas, overexpression of the GABA synthetic enzyme, glutamate decarboxylase 1 (GAD1, was found to be associated with decreased disease free-survival in prostate adenocarcinoma and decreased overall survival in clear cell renal cell carcinomas. Furthermore, GAD1 was found to be expressed in castrate-resistant prostate cancer cell lines, but not androgen-responsive cell lines. Using a novel fluorescence-coupled enzymatic microplate assay for GABA mediated through reduction of resazurin in a prostate neuroendocrine carcinoma (PNEC cell line, acid microenvironment-induced stress increased GABA levels while alkaline microenvironment-induced stress decreased GABA through modulation of GAD1 and glutamine synthetase (GLUL activities. Moreover, glutamine but not glucose deprivation decreased GABA through modulation of GLUL. Consistent with evidence in prokaryotic and eukaryotic organisms that GABA synthesis mediated through GAD1 may play a crucial role in surviving stress, GABA may be an important mediator of stress survival in neoplasms. These findings identify GABA synthesis and metabolism as a potentially important pathway for regulating cancer cell stress response as well as a potential target for therapeutic strategies.

  11. Molecular screening of wine lactic acid bacteria degrading hydroxycinnamic acids.

    Science.gov (United States)

    de las Rivas, Blanca; Rodríguez, Héctor; Curiel, José Antonio; Landete, José María; Muñoz, Rosario

    2009-01-28

    The potential to produce volatile phenols from hydroxycinnamic acids was investigated for lactic acid bacteria (LAB) isolated from Spanish grape must and wine. A PCR assay was developed for the detection of LAB that potentially produce volatile phenols. Synthetic degenerate oligonucleotides for the specific detection of the pdc gene encoding a phenolic acid decarboxylase were designed. The pdc PCR assay amplifies a 321 bp DNA fragment from phenolic acid decarboxylase. The pdc PCR method was applied to 85 strains belonging to the 6 main wine LAB species. Lactobacillus plantarum, Lactobacillus brevis, and Pediococcus pentosaceus strains produce a positive response in the pdc PCR assay, whereas Oenococcus oeni, Lactobacillus hilgardii, and Leuconostoc mesenteroides strains did not produce the expected PCR product. The production of vinyl and ethyl derivatives from hydroxycinnamic acids in culture media was determined by high-performance liquid chromatography. A relationship was found between pdc PCR amplification and volatile phenol production, so that the LAB strains that gave a positive pdc PCR response produce volatile phenols, whereas strains that did not produce a PCR amplicon did not produce volatile phenols. The proposed method could be useful for a preliminary identification of LAB strains able to produce volatile phenols in wine.

  12. Diagnosis of brugian filariasis by loop-mediated isothermal amplification.

    Directory of Open Access Journals (Sweden)

    Catherine B Poole

    Full Text Available In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis.

  13. Enzyme assays

    OpenAIRE

    Bisswanger, Hans

    2014-01-01

    The essential requirements for enzyme assays are described and frequently occurring errors and pitfalls as well as their avoidance are discussed. The main factors, which must be considered for assaying enzymes, are temperature, pH, ionic strength and the proper concentrations of the essential components like substrates and enzymes. Standardization of these parameters would be desirable, but the diversity of the features of different enzymes prevents unification of assay conditions. Neverthele...

  14. Target amplification for broad spectrum microbial diagnostics and detection.

    Science.gov (United States)

    Leski, Tomasz A; Malanoski, Anthony P; Stenger, David A; Lin, Baochuan

    2010-02-01

    Microarrays are massively parallel detection platforms that were first used extensively for gene expression studies, but have also been successfully applied to microbial detection in a number of diverse fields requiring broad-range microbial identification. This technology has enabled researchers to gain an insight into the microbial diversity of environmental samples, facilitated discovery of a number of new pathogens and enabled studies of multipathogen infections. In contrast to gene expression studies, the concentrations of targets in analyzed samples for microbial detection are usually much lower, and require the use of nucleic acid amplification techniques. The rapid advancement of manufacturing technologies has increased the content of the microarrays; thus, the required amplification is a challenging problem. The constant parallel improvements in both microarray and sample amplification techniques in the near future may lead to a radical progression in medical diagnostics and systems for efficient detection of microorganisms in the environment.

  15. Method for chemical amplification based on fluid partitioning in an immiscible liquid

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Brian L.; Colston, Bill W.; Elkin, Christopher J.

    2017-02-28

    A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

  16. Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres.

    Directory of Open Access Journals (Sweden)

    Delyan P Ivanov

    Full Text Available Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.

  17. Assay systems for screening food and natural substances that have anti-hyperuricemic activity: uric acid production in cultured hepatocytes and purine bodies-induced hyperuricemic model mice.

    Science.gov (United States)

    Adachi, Shin-Ichi; Yoshizawa, Fumiaki; Yagasaki, Kazumi

    2017-06-01

    Hyperuricemia is characterized by the high uric acid (UA) level in serum (or plasma) and has been considered to be an important risk factor for gout. In the present study, we have attempted to construct an assay system for UA production in vitro employing cultured AML12 hepatocytes. UA levels in balanced salt solution (BSS) in the presence of UA precursor nucleosides, adenosine, inosine, guanosine and xanthine, at 12.5, 25, and 100 µM were significantly higher than BSS alone and their effects were dose-dependent, while all the UA precursors did not significantly increase intracellular UA levels. Hence, UA levels in BSS were thereafter adopted as an index of UA production. UA production from nucleosides was significantly higher than that from nucleotides (GMP, IMP and AMP). UA production from guanosine and inosine in combination (GI mixture) as well as nucleosides increased time-dependently and almost linearly up to 2 h. Selecting GI mixture, effects of allopurinol, a widely used anti-hyperuricemic agent, and quercetin, a well-known polyphenol in onion and strawberry, on UA production were examined. Both allopurinol and quercetin dose-dependently (0.1, 0.3 and 1 μM for allopurinol and 10, 30, and 100 μM for quercetin) and significantly reduced UA production in the hepatocytes. They also significantly reduced hyperuricemia induced by intraperitoneal injection of UA precursor purine bodies to mice at a single oral dose of 10 (allopurinol) or 200 (quercetin) mg/kg body weight. This assay system for UA production in cultured hepatocytes is considered to be useful to search for novel anti-hyperuricemic compounds in foods and natural resources with possibility to have human health benefits.

  18. Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

    Science.gov (United States)

    Ivanov, Delyan P.; Parker, Terry L.; Walker, David A.; Alexander, Cameron; Ashford, Marianne B.; Gellert, Paul R.; Garnett, Martin C.

    2014-01-01

    Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money. PMID:25119185

  19. Detection and quantification of the toxic microalgae Karenia brevis using lab on a chip mRNA sequence-based amplification.

    Science.gov (United States)

    Loukas, Christos-Moritz; McQuillan, Jonathan S; Laouenan, Florian; Tsaloglou, Maria-Nefeli; Ruano-Lopez, Jesus M; Mowlem, Matthew C

    2017-08-01

    Now and again, the rapid proliferation of certain species of phytoplankton can give rise to Harmful Algal Blooms, which pose a serious threat to marine life and human health. Current methods of monitoring phytoplankton are limited by poor specificity or by the requirement to return samples to a highly resourced, centralised lab. The Lab Card is a small, microfluidic cassette which, when used in tandem with a portable Lab Card Reader can be used to sensitively and specifically quantify harmful algae in the field, from nucleic acid extracts using RNA amplification; a sensitive and specific method for the enumeration of potentially any species based on their unique genetic signatures. This study reports the culmination of work to develop a Lab Card-based genetic assay to quantify the harmful algae Karenia brevis using mRNA amplification by the Nucleic Acid Sequence Based Amplification (NASBA) method. K. brevis cells were quantified by amplification of the rbcL gene transcript in nucleic acid extracts of K. brevis cell samples. A novel enzyme dehydration and preservation method was combined with a pre-existing reagent Gelification method to prepare fully preserved Lab Cards with a shelf-life of at least six weeks prior to use. Using an internal control (IC), the Lab Card-based rbcL NASBA was demonstrated for the quantification of K. brevis from cell extracts containing between 50 and 5000 cells. This is the first demonstration of quantitation of K. brevis using IC-NASBA on a Lab Card. Copyright © 2017. Published by Elsevier B.V.

  20. Shining a light on LAMP assays--a comparison of LAMP visualization methods including the novel use of berberine.

    Science.gov (United States)

    Fischbach, Jens; Xander, Nina Carolin; Frohme, Marcus; Glökler, Jörn Felix

    2015-04-01

    The need for simple and effective assays for detecting nucleic acids by isothermal amplification reactions has led to a great variety of end point and real-time monitoring methods. Here we tested direct and indirect methods to visualize the amplification of potato spindle tuber viroid (PSTVd) by loop-mediated isothermal amplification (LAMP) and compared features important for one-pot in-field applications. We compared the performance of magnesium pyrophosphate, hydroxynaphthol blue (HNB), calcein, SYBR Green I, EvaGreen, and berberine. All assays could be used to distinguish between positive and negative samples in visible or UV light. Precipitation of magnesium-pyrophosphate resulted in a turbid reaction solution. The use of HNB resulted in a color change from violet to blue, whereas calcein induced a change from orange to yellow-green. We also investigated berberine as a nucleic acid-specific dye that emits a fluorescence signal under UV light after a positive LAMP reaction. It has a comparable sensitivity to SYBR Green I and EvaGreen. Based on our results, an optimal detection method can be chosen easily for isothermal real-time or end point screening applications.

  1. Genome position and gene amplification

    Czech Academy of Sciences Publication Activity Database

    Jirsová, Pavla; Snijders, A.M.; Kwek, S.; Roydasgupta, R.; Fridlyand, J.; Tokuyasu, T.; Pinkel, D.; Albertson, D. G.

    2007-01-01

    Roč. 8, č. 6 (2007), r120 ISSN 1474-760X Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : gene amplification * array comparative genomic hybridization * oncogene Subject RIV: BO - Biophysics Impact factor: 6.589, year: 2007