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Sample records for rapid detection assays

  1. Detection of Streptococcus pyogenes using rapid visual molecular assay.

    Science.gov (United States)

    Zhao, Xiangna; He, Xiaoming; Li, Huan; Zhao, Jiangtao; Huang, Simo; Liu, Wei; Wei, Xiao; Ding, Yiwei; Wang, Zhaoyan; Zou, Dayang; Wang, Xuesong; Dong, Derong; Yang, Zhan; Yan, Xiabei; Huang, Liuyu; Du, Shuangkui; Yuan, Jing

    2015-09-01

    Streptococcus pyogenes is an increasingly important pathogen in many parts of the world. Rapid and accurate detection of S. pyogenes aids in the control of the infection. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for the specific detection of S. pyogenes. The assay incorporates two methods: a chromogenic analysis using a calcein/Mn(2+) complex and real-time turbidity monitoring to assess the reaction. Both methods detected the target DNA within 60 min under 64°C isothermal conditions. The assay used specifically designed primers to target spy1258, and correctly identified 111 strains of S. pyogenes and 32 non-S. pyogenes strains, including other species of the genus Streptococcus. Tests using reference strains showed that the LAMP assay was highly specific. The sensitivity of the assay, with a detection limit of 1.49 pg DNA, was 10-fold greater than that of PCR. The LAMP assay established in this study is simple, fast and sensitive, and does not rely upon any special equipment; thus, it could be employed in clinical diagnosis. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Rapid detection of EBOLA VP40 in microchip immunofiltration assay

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    Miethe, Peter; Gary, Dominik; Hlawatsch, Nadine; Gad, Anne-Marie

    2015-05-01

    In the spring of 2014, the Ebola virus (EBOV) strain Zaire caused a dramatic outbreak in several regions of West Africa. The RT-PCR and antigen capture diagnostic proved to be effective for detecting EBOV in blood and serum. In this paper, we present data of a rapid antigen capture test for the detection of VP40. The test was performed in a microfluidic chip for immunofiltration analysis. The chip integrates all necessary assay components. The analytical sensitivity of the rapid test was 8 ng/ml for recombinant VP40. In serum and whole blood samples spiked with virus culture material, the detection limit was 2.2 x 102 PFU/ml. The performance data of the rapid test (15 min) are comparable to that of the VP40 laboratory ELISA.

  3. [Rapid detection of Shigella dysenteriae by PCR assay].

    Science.gov (United States)

    Chen, Hongyuan; Zhong, Qingping; Wang, Li; Sun, Yuanming

    2010-09-01

    Based on the invasive plasmid antigen H gene (ipaH) of S. dysenteriae, one pair of specific primers was designed for PCR assays in this study. The concentrations of dNTP, Mg2+ and primer, dosage of Taq DNA polymerase, annealing temperature and circulating parameter in the PCR amplification system were optimized. In this way, a rapid and stable method of PCR assay for the detection of S. dysenteriae was established. The specificity and sensitivity of PCR were also analyzed. The detection limits of pure culture and genomic DNA in the PCR assay were 1.06 x 10(2) cfu/ml and 106.34 pg/PCR system, respectively. The detection limit for S. dysenteriae in artificially contaminated food samples was 3.21 x 10(4) cfu/ml. These results indicated that the PCR method for S. dysenteriae detection was simple, rapid, high in specificity and sensitivity and suitable for the detection of pathogens in foods caused by Shigella dysenteriae.

  4. Rapid detection of cryptococcal antigen by a flow assay

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    Graziano Bargiggia

    2017-10-01

    Full Text Available Cryptococcosis is a life-threatening infection caused by Cryptococcus neoformans and C. gattii. Tests for quick detection of the cryptococcal antigen are needed. This study compares the performance of a lateral flow assay (LFA to the latex agglutination method. Thirty-five cryopreserved positive samples (sera and cerebrospinal fluids plus three negative sera for control have been examined. LFA does not need high-temperature incubation or enzyme pre-treatment. All the results, except for one serum, agree with previous obtained with latex agglutination method. LFA has an important clinical utility for its rapidity and sensitivity, and it also can be used as a point-of-care test.

  5. Evaluation of a direct colorimetric assay for rapid detection of ...

    African Journals Online (AJOL)

    Yemane Berhane

    bromide (MTT) for a rapid detection of rifampicin resistance. Methods: Sputum was inoculated directly into 7H9 .... a loopful of the corresponding broth on nutrient agar and incubating it at 370C for 24 hours before performing the .... and Training in Tropical Diseases (TDR). This study was part of the MSc thesis of DW at Addis ...

  6. Analytical and clinical sensitivity of the 3M rapid detection influenza A+B assay.

    Science.gov (United States)

    Dale, Suzanne E; Mayer, Christine; Mayer, Marie C; Menegus, Marilyn A

    2008-11-01

    The performance of the 3M rapid detection influenza A+B (3M flu) assay was compared to the performance of other immunochromatographic assays. The clinical and analytical performance of the 3M flu assay was superior to that of BinaxNOW and Directigen EZ assays and equivalent to that of the QuickVue assay. The 3M flu assay offers an objective output and direct linkage to laboratory information systems.

  7. Analytical and Clinical Sensitivity of the 3M Rapid Detection Influenza A+B Assay

    Science.gov (United States)

    Dale, Suzanne E.; Mayer, Christine; Mayer, Marie C.; Menegus, Marilyn A.

    2008-01-01

    The performance of the 3M rapid detection influenza A+B (3M flu) assay was compared to the performance of other immunochromatographic assays. The clinical and analytical performance of the 3M flu assay was superior to that of BinaxNOW and Directigen EZ assays and equivalent to that of the QuickVue assay. The 3M flu assay offers an objective output and direct linkage to laboratory information systems. PMID:18832133

  8. Novel methods for improving rapid paper-based protein assays with gold nanoparticle detection

    OpenAIRE

    Lama, Lara

    2017-01-01

    This thesis describes methods for improving sensitivity in rapid singleplex and multiplex microarray assays. The assays utilize the optical characteristics of colloidal gold nanoparticles for the colorimetric detection of proteins. Multiplexed detection in sandwich immunoassays is limited by cross-reactivity between different detection antibodies. The cross-reactivity between antibodies can contribute to increased background noise - decreasing the Limit-of-Detection of the assay - or generate...

  9. A duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains.

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    Benacer, Douadi; Zain, Siti Nursheena Mohd; Lewis, John W; Khalid, Mohd Khairul Nizam Mohd; Thong, Kwai Lin

    2017-01-01

    This study aimed to develop a duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains. Primers were designed to target the rrs (LG1/LG2) and ligB (LP1/LP2) genes to confirm the presence of the Leptospira genus and the pathogenic species, respectively. The assay showed 100% specificity against 17 Leptospira strains with a limit of detection of 23.1pg/µl of leptospiral DNA and sensitivity of 103 leptospires/ml in both spiked urine and water. Our duplex endpoint PCR assay is suitable for rapid early detection of Leptospira with high sensitivity and specificity.

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

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    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.

  11. Rapid preclinical detection of sheeppox virus by a real-time PCR assay.

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    Balinsky, C A; Delhon, G; Smoliga, G; Prarat, M; French, R A; Geary, S J; Rock, D L; Rodriguez, L L

    2008-02-01

    Sheeppox virus (SPPV) is a member of the Capripoxvirus (CaPV) genus of the Poxviridae family. Members of this genus, which also include goatpox and lumpy skin disease viruses, cause economically significant disease in sheep, goats, and cattle. A rapid diagnostic assay for CaPV would be useful for disease surveillance as well as for detection of CaPV in clinical samples and for outbreak management. Here we describe a fluorogenic probe hydrolysis (TaqMan) PCR assay designed for rapid detection of CaPV and tested on sheep experimentally infected with a virulent strain of SPPV. This assay can detect SPPV in buffy coats, nasal swabs, oral swabs, scabs, and skin lesions as well as in lung and lymph nodes collected at necropsy. This single-tube diagnostic assay can be performed in 2 h or less and can detect viral DNA in preclinical, clinical, and postmortem samples.

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

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    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.

  13. 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.

  14. 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

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

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    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. Real-time PCR assay for rapid qualitative and quantitative detection of Entamoeba histolytica.

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    Orosz, Erika; Perkátai, Katalin; Kapusinszky, Beatrix; Farkas, Agnes; Kucsera, István

    2012-12-01

    Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

  17. Rapid molecular assays for the detection of yellow fever virus in low-resource settings.

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    Camille Escadafal

    2014-03-01

    Full Text Available BACKGROUND: Yellow fever (YF is an acute viral hemorrhagic disease transmitted by Aedes mosquitoes. The causative agent, the yellow fever virus (YFV, is found in tropical and subtropical areas of South America and Africa. Although a vaccine is available since the 1930s, YF still causes thousands of deaths and several outbreaks have recently occurred in Africa. Therefore, rapid and reliable diagnostic methods easy to perform in low-resources settings could have a major impact on early detection of outbreaks and implementation of appropriate response strategies such as vaccination and/or vector control. METHODOLOGY: The aim of this study was to develop a YFV nucleic acid detection method applicable in outbreak investigations and surveillance studies in low-resource and field settings. The method should be simple, robust, rapid and reliable. Therefore, we adopted an isothermal approach and developed a recombinase polymerase amplification (RPA assay which can be performed with a small portable instrument and easy-to-use lyophilized reagents. The assay was developed in three different formats (real-time with or without microfluidic semi-automated system and lateral-flow assay to evaluate their application for different purposes. Analytical specificity and sensitivity were evaluated with a wide panel of viruses and serial dilutions of YFV RNA. Mosquito pools and spiked human plasma samples were also tested for assay validation. Finally, real-time RPA in portable format was tested under field conditions in Senegal. CONCLUSION/SIGNIFICANCE: The assay was able to detect 20 different YFV strains and demonstrated no cross-reactions with closely related viruses. The RPA assay proved to be a robust, portable method with a low detection limit (<21 genome equivalent copies per reaction and rapid processing time (<20 min. Results from real-time RPA field testing were comparable to results obtained in the laboratory, thus confirming our method is suitable for

  18. 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.

  19. DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays

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    Kenjiro Nagamine

    2015-01-01

    Full Text Available Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani , and Staphylococcus aureus , which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5–50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents.

  20. 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.

  1. Fluorescence-based lateral flow assays for rapid oral fluid roadside detection of cannabis use.

    Science.gov (United States)

    Plouffe, Brian D; Murthy, Shashi K

    2017-02-01

    With the recent worldwide changes in the legalization of marijuana, there is a significant need for rapid, roadside screening test for driving under the influence of drugs. A robust, sensitive, lateral flow assay has been developed to detect recent use via oral-fluid testing for Δ 9 -tetrahydrocannabinol (THC). This proof-of-concept assay uses a fluorescent-based immunoassay detection of polymeric beads, conjugated to antibodies against native THC. The fluorescent technique allows for significantly lower limits of detection and higher precision determination of recent marijuana use without the use of urine or blood sampling-thus allowing for roadside identification. Detection levels of 0.01 ng/mL were distinguished from background and the lower limit of quantification was determined to approach 1 ng/mL. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. 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.

  3. A rapid qualitative assay for detection of Clostridium perfringens in canned food products.

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    Dave, Gayatri Ashwinkumar

    2017-01-01

    Clostridium perfringens (MTCC 1349) is a Gram-positive, anaerobic, endospore forming, and rod-shaped bacterium. This bacterium produces a variety of toxins under strict anaerobic environment. C. perfringens can grow at temperatures ranging between 20°C and 50°C. It is the major causetive agent for gas gangrene, cellulitis, septicemia, necrotic enteritis and food poisoning, which are common toxin induced conditions noted in human and animals. C. perfringens can produce produce four major types of toxins that are used for the classification of strains, classified under type A-E. Across the globe many countries, including the United States, are affected by C. perfringens food poisonings where it is ranked as one of the most common causes of food borne infections. To date, no direct one step assay for the detection of C. perfringens has been developed and only few methods are known for accurate detection of C. perfringens. Long detection and incubation time is the major consideration of these reporter assays. The prensent study proposes a rapid and reliable colorimetric assay for the detection of C. perfringens. In principale, this assay detects the para nitrophenyl (yellow colour end product) liberated due to the hydrolysis of paranitrophenyl phosphetidyl choline (PNPC) through phospholipase C (lecithinase). Constitutive secretion of phospholipase C is a charactristic feature of C. perfringens. This assay detects the presence of the extracellular lecithinse through the PNPC impragnated impregnated probe. The probe is impregnated with peranitrophenyl phosphotidyl choline ester, which is colourless substrate used by lecithinase. The designed assay is specific towards PNPC and detectes very small quantites of lecithinase under conditions used. The reaction is substrate specific, no cross reaction was observed upon incubation with other substrates. In addition, this assay gave negative results with other clostridium strains, no cross reactions were observed with other

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

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

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    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.

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

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    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.

  7. Development of multiplex-PCR assay for rapid detection of Candida spp.

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    Ni Made A. Tarini

    2010-05-01

    Full Text Available Aim Candida spp. infection commonly occur in immunocompromised patients. Biochemical assay for identification of Candida spp. is time-consuming and shows many undetermined results. Specific detection for antibody, antigen and metabolites of Candida spp. had low sensitivity and specificity. In this study, we developed a rapid diagnostic method, Multiplex-PCR, to identify Candida spp.Methods Five Candida spp. isolates were cultured, identifi ed with germ tube and API® 20 C AUX (BioMerieux® SA kit. Furthermore, DNA was purified by QIAamp DNA mini (Qiagen® kit for Multiplex-PCR assay.Results DNA detection limit by Multiplex-PCR assays for C. albicans, C. tropicalis, C. parapsilosis, C. krusei and C. glabrata were 4 pg, 0.98 pg, 0.98 pg, 0.5 pg and 16 pg respectively. This assay was also more sensitive than culture in that Multiplex-PCR could detect 2.6-2.9 x 100 CFU/ml, whereas culture 2.6-2.9 x 102 CFU/ml.Conclusion Multiplex-PCR is much more sensitive than culture and thus, can be recommended as a sensitive and specific assay for identification of Candida spp. (Med J Indones 2010; 19:83-7Keywords: Candida spp., multiplex-PCR

  8. 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.

  9. Rapid detection of Ceratocystis platani inoculum by quantitative real-time PCR assay.

    Science.gov (United States)

    Luchi, Nicola; Ghelardini, Luisa; Belbahri, Lassaâd; Quartier, Marion; Santini, Alberto

    2013-09-01

    Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect a C. platani airborne inoculum. Airborne inoculum traps (AITs) were placed in an urban setting in the city of Florence, Italy, where the disease was present. Primers and TaqMan minor groove binder (MGB) probes were designed to target cerato-platanin (CP) and internal transcribed spacer 2 (ITS2) genes. The detection limits of the assay were 0.05 pg/μl and 2 fg/μl of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for C. platani, detecting DNA concentrations as low as 1.2 × 10(-2) to 1.4 × 10(-2) pg/μl, corresponding to ∼10 conidia per ml. Airborne inoculum traps were able to detect the C. platani inoculum within 200 m of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of a C. platani inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping disease management.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Rapid PCR-based assay for Sclerotinia sclerotiorum detection on soybean seeds

    Directory of Open Access Journals (Sweden)

    Edilaine Mauricia Gelinski Grabicoski

    2015-02-01

    Full Text Available Caused by Sclerotinia sclerotiorum, white mold is an important seed-transmitted disease of soybean (Glycine max. Incubation-based methods available for the detection and quantification of seed-borne inoculum such as the blotter test, paper roll and Neon-S assay are time-consuming, laborious, and not always sensitive. In this study, we developed and evaluated a molecular assay for the detection of S. sclerotiorum in soybean seeds using a species-specific PCR (polymerase chain reaction primer set and seed soaking (without DNA extraction for up to 72 h. The PCR products were amplified in all the samples infected with the pathogen, but not in the other samples of plant material or the other seed-borne fungi DNA. The minimum amount of DNA detected was 10 pg, or one artificially infested seed in a 400-seed sample (0.25 % fungal incidence and one naturally infected seed in a 300-seed sample (0.33 % incidence. The PCR-based assay was rapid (< 9 h, did not require DNA extraction and was very sensitive.

  15. A Rapid Assay to Detect Toxigenic Penicillium spp. Contamination in Wine and Musts

    Directory of Open Access Journals (Sweden)

    Simona Marianna Sanzani

    2016-08-01

    Full Text Available Wine and fermenting musts are grape products widely consumed worldwide. Since the presence of mycotoxin-producing fungi may greatly compromise their quality characteristics and safety, there is an increasing need for relatively rapid “user friendly” quantitative assays to detect fungal contamination both in grapes delivered to wineries and in final products. Although other fungi are most frequently involved in grape deterioration, secondary infections by Penicillium spp. are quite common, especially in cool areas with high humidity and in wines obtained by partially dried grapes. In this work, a single-tube nested real-time PCR approach—successfully applied to hazelnut and peanut allergen detection—was tested for the first time to trace Penicillium spp. in musts and wines. The method consisted of two sets of primers specifically designed to target the β-tubulin gene, to be simultaneously applied with the aim of lowering the detection limit of conventional real-time PCR. The assay was able to detect up to 1 fg of Penicillium DNA. As confirmation, patulin content of representative samples was determined. Most of analyzed wines/musts returned contaminated results at >50 ppb and a 76% accordance with molecular assay was observed. Although further large-scale trials are needed, these results encourage the use of the newly developed method in the pre-screening of fresh and processed grapes for the presence of Penicillium DNA before the evaluation of related toxins.

  16. Multicenter evaluation of a new rapid automated human immunodeficiency virus antigen detection assay.

    Science.gov (United States)

    Weber, B; Mühlbacher, A; Michl, U; Paggi, G; Bossi, V; Sargento, C; Camacho, R; Fall, E H; Berger, A; Schmitt, U; Melchior, W

    1999-03-01

    Although human immunodeficiency virus (HIV) antigen assays are of limited value for monitoring antiretroviral therapy, they play an important role for confirmatory testing of fourth generation HIV screening enzyme immunoassay (EIA) reactive samples. In a multicenter study, a new automated rapid p24 antigen assay, Elecsys HIV Ag (Roche Diagnostics Boehringer Mannheim GmbH, Penzberg, Germany), was compared to FDA licensed tests (Abbott HIV-1 Ag monoclonal and Coulter HIV-1 p24 antigen assay). In the evaluation 27 seroconversion panels were included, sera from the acute phase of infection, single and follow-up samples from HIV antibody positive patients, dilution series of HIV antigen positive standards, sera and cell culture supernatants infected with different HIV-1 subtypes (A-H, and O) HIV-2 and recombinant HIV-1 (gag/env) isolates. To challenge the specificity of the new assay, 2565 unselected blood donors, sera from pregnant women, dialysis and hospitalized patients and 407 potentially cross-reactive samples were investigated. Acute HIV infection was detected in three to eight seroconversion panels earlier with Elecsys HIV Ag than with the alternative assays. Higher numbers of serum samples from HIV infected patients tested positive by Elecsys HIV Ag than with the comparative assays. All HIV-1 subtypes and HIV-2 isolates were recognized with Elecsys HIV Ag. Abbott HIV-1 Ag monoclonal and Coulter HIV-1 p24 antigen assay showed a variable sensitivity for the different HIV-1 subtypes. The specificity of Elecsys HIV Ag and Coulter HIV-1 p24 antigen assay were 99.8 and 99.93%, respectively. All the eight sera that were false reactive by Elecsys HIV Ag were tested negative with the Elecsys HIV Ag Neutralization Test. In conclusion, Elecsys HIV Ag was more sensitive than the alternative assays and showed a high specificity in combination with the neutralization assay. The very short incubation time of 18 min and the fully automated procedure of Elecsys HIV Ag which

  17. Rapid and Sensitive Reporter Gene Assays for Detection of Antiandrogenic and Estrogenic Effects of Environmental Chemicals

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Bonefeld-Jørgensen, Eva Cecilie; Larsen, John Christian

    1999-01-01

    Reports on increasing incidences in developmental abnormalities of the human male reproductive tract and the recent identifications of environmental chemicals with antiandrogenic activity necessitate the screening of a larger number of compounds in order to get an overview of potential...... antiandrogenic chemicals present in our environment. Thus, there is a great need for an effectivein vitroscreening method for (anti)androgenic chemicals. We have developed a rapid, sensitive, and reproducible reporter gene assay for detection of antiandrogenic chemicals. Chinese Hamster Ovary cells were......-on laboratory time. This assay is a powerful tool for the efficient and accurate determination and quantification of the effects of antiandrogens on reporter gene transcription. To extend the application of FuGene, the reagent was shown to be superior compared to Lipofectin for transfecting MCF7 human breast...

  18. Rapid and sensitive reporter gene assays for detection of antiandrogenic and estrogenic effects of environmental chemicals

    DEFF Research Database (Denmark)

    Vinggaard, Anne; Jørgensen, E.C.B.; Larsen, John Christian

    1999-01-01

    antiandrogenic chemicals present in our environment. Thus, there is a great need for an effective in vitro screening method for (anti)androgenic chemicals. We have developed a rapid, sensitive, and reproducible reporter gene assay for detection of antiandrogenic chemicals. Chinese Hamster Ovary cells were...... induction of luciferase activity. The classical antiandrogenic compounds hydroxy-flutamide, bicalutamide, spironolactone, and cyproterone acetate together with the pesticide(metabolite)s, vinclozolin, p,p'-DDE, and procymidone all potently inhibited the response to 0.1 nM R1881, Compared to the traditional...... cancer cells with an estrogen response element-luciferase vector. Thus, FuGene may prove to be valuable in diverse reporter gene assays involving transient transfections for screening of potential endocrine disrupters for (anti)androgenic and (anti)estrogenic properties....

  19. Rapid Cell-Based Assay for Detection and Quantification of Active Staphylococcal Enterotoxin Type D.

    Science.gov (United States)

    Rasooly, Reuven; Do, Paula M; Hernlem, Bradley J

    2017-03-01

    Food poisoning by Staphylococcus aureus is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by this bacterium and is a major source of foodborne illness. Staphylococcal enterotoxin D (SED) is one of the predominant enterotoxins recovered in Staphylococcal food poisoning incidences, including a recent outbreak in Guam affecting 300 children. Current immunology methods for SED detection cannot distinguish between the biologically active form of the toxin, which poses a threat, from the inactive form, which poses no threat. In vivo bioassays that measure emetic activity in kitten and monkeys have been used, but these methods rely upon expensive procedures using live animals and raising ethical concerns. A rapid (5 h) quantitative bioluminescence assay, using a genetically engineered T-cell Jurkat cell line expressing luciferase under regulation of nuclear factor of activated T cells response elements, in combination with the lymphoblastoid B-cell line Raji for antigen presentation, was developed. In this assay, the detection limit of biologically active SED is 100 ng/mL, which is 10 times more sensitive than the splenocyte proliferation assay, and 105 times more sensitive than monkey or kitten bioassay. Pasteurization or repeated freeze-thaw cycles had no effect on SED activity, but reduction in SED activity was shown with heat treatment at 100°C for 5 min. It was also shown that milk exhibits a protective effect on SED. This bioluminescence assay may also be used to rapidly evaluate antibodies to SED for potential therapeutic application as a measurement of neutralizing biological effects of SED. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  20. Portable ceria nanoparticle-based assay for rapid detection of food antioxidants (NanoCerac)

    Science.gov (United States)

    Sharpe, Erica; Frasco, Thalia; Andreescu, Daniel; Andreescu, Silvana

    2012-01-01

    With increased awareness of nutrition and the advocacy for healthier food choices, there exists a great demand for a simple, easy-to-use test that can reliably measure the antioxidant capacity of dietary products. We report development and characterization of a portable nanoparticle based-assay, similar to a small sensor patch, for rapid and sensitive detection of food antioxidants. The assay is based on the use of immobilized ceria nanoparticles, which change color after interaction with antioxidants by means of redox and surface chemistry reactions. Monitoring corresponding optical changes enables sensitive detection of antioxidants in which the nanoceria provides an optical ‘signature’ of antioxidant power, while the antioxidants act as reducing agents. The sensor has been tested for the detection of common antioxidant compounds including ascorbic acid, gallic acid, vanilic acid, quercetin, caffeic acid, and epigallocatechin gallate and its function has been successfully applied for the assessment of antioxidant activity in real samples (teas and medicinal mushrooms). The colorimetric response was concentration dependent, with detection limits ranging from 20–400 μM depending on the antioxidant involved. Steady-state color intensity was achieved within seconds upon addition of antioxidants. The results are presented in terms of Gallic Acid Equivalents (GAE). The sensor performed favorably when compared with commonly used antioxidant detection methods. This assay is particularly appealing for remote sensing applications, where specialized equipment is not available, and also for high throughput analysis of a large number of samples. Potential applications for antioxidant detection in remote locations are envisioned. PMID:23139929

  1. A gold immunochromatographic assay for the rapid and simultaneous detection of fifteen β-lactams

    Science.gov (United States)

    Chen, Yanni; Wang, Yongwei; Liu, Liqiang; Wu, Xiaoling; Xu, Liguang; Kuang, Hua; Li, Aike; Xu, Chuanlai

    2015-10-01

    A novel gold immunochromatographic assay (GICA) based on anti-β-lactam receptors was innovatively developed that successfully allowed rapid and simultaneous detection of fifteen β-lactams in milk samples in 5-10 minutes. By replacing the antibodies used in traditional GICA with anti-β-lactam receptors, the difficulty in producing broad specific antibodies against β-lactams was overcome. Conjugates of ampicillin with BSA and goat anti-mouse immunoglobulin (IgG) were immobilized onto the test and control lines on the nitrocellulose membrane, respectively. Since goat anti-mouse IgG does not combine with receptors, negative serum from mice labelled with gold nanoparticles (GNP) was mixed with GNP-labelled receptors. Results were obtained within 20 min using a paper-based sensor. The utility of the assay was confirmed by the analysis of milk samples. The limits of detection (LOD) for amoxicillin, ampicillin, penicillin G, penicillin V, cloxacillin, dicloxacillin, nafcillin, oxacillin, cefaclor, ceftezole, cefotaxime, ceftiofur, cefoperazone, cefathiamidine, and cefepime were 0.25, 0.5, 0.5, 0.5, 1, 5, 5, 10, 25, 10, 100, 10, 5, 5, and 2 ng mL-1, respectively, which satisfies the maximum residue limits (MRL) set by the European Union (EU). In conclusion, our newly developed GICA-based anti-β-lactam receptor assay provides a rapid and effective method for one-site detection of multiple β-lactams in milk samples.A novel gold immunochromatographic assay (GICA) based on anti-β-lactam receptors was innovatively developed that successfully allowed rapid and simultaneous detection of fifteen β-lactams in milk samples in 5-10 minutes. By replacing the antibodies used in traditional GICA with anti-β-lactam receptors, the difficulty in producing broad specific antibodies against β-lactams was overcome. Conjugates of ampicillin with BSA and goat anti-mouse immunoglobulin (IgG) were immobilized onto the test and control lines on the nitrocellulose membrane, respectively

  2. Development of a highly sensitive lateral immunochromatographic assay for rapid detection of Vibrio parahaemolyticus.

    Science.gov (United States)

    Liu, Xinfeng; Guan, Yuyao; Cheng, Shiliang; Huang, Yidan; Yan, Qin; Zhang, Jun; Huang, Guanjun; Zheng, Jian; Liu, Tianqiang

    2016-12-01

    Vibrio parahaemolyticus is widely present in brackish water all over the world, causing infections in certain aquatic animals. It is also a foodborne pathogen that causes diarrhea in humans. The aim of this study is to develop an immunochromatographic lateral flow assay (LFA) for rapid detection of V. parahaemolyticus in both aquatic products and human feces of diarrheal patients. Two monoclonal antibody (MAb) pairs, GA1a-IC9 and IC9-KB4c, were developed and proven to be highly specific and sensitive to V. parahaemolyticus. Based on the two MAb pairs, two types of LFA strips were prepared. Their testing limits for V. parahaemolyticus culture were both 1.2×103CFU/ml. The diagnostic sensitivities and specificities were both 100% for the 32 tested microbial species, including 6 Vibrio species. Subsequently, the LFA strips were used to test Whiteleg shrimps and human feces. The type II strip showed a higher diagnostic sensitivity. Its sensitivity and specificity for hepatopancreas and fecal samples from 13 Whiteleg shrimps and fecal samples from 146 human diarrheal patients were all 100%. In conclusion, our homemade type II LFA is a very promising testing device for rapid and convenient detection of V. parahaemolyticus infection not only in aquatic animals, but also in human diarrheal patients. This sensitive immunochromtographic LFA allows rapid detection of V. parahaemolyticus without requirement of culture enrichment. Copyright © 2016. Published by Elsevier B.V.

  3. Rapid screening test for detection of oxytetracycline residues in milk using lateral flow assay.

    Science.gov (United States)

    Naik, Laxmana; Sharma, Rajan; Mann, Bimlesh; Lata, Kiran; Rajput, Y S; Surendra Nath, B

    2017-03-15

    A rapid, semi-quantitative lateral flow assay (LFA) was developed to screen the oxytetracycline (OTC) antibiotics residues in milk samples. In this study a competitive immuno-assay format was established. Colloidal gold nano-particles (GNP) were prepared and used as labelling material in LFA. Polyclonal antibodies were generated against OTC molecule (anti-OTC), purified and the quality was assessed by enzyme linked immuno sorbet assay. For the first time membrane components required for LFA in milk system was optimized. GNP and anti-OTC stable conjugate preparation method was standardized, and then these components were placed over the conjugate pad. OTC coupled with carrier protein was placed on test line; species specific secondary antibodies were placed on the control line of the membrane matrix. Assay was validated by spiking OTC to antibiotic free milk samples and results could be accomplished within 5min. without need of any equipment. The visual detection limit was 30ppb. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. 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

  5. Development of a novel multiplex PCR assay for rapid detection of virulence associated genes of Pasteurella multocida from pigs

    National Research Council Canada - National Science Library

    Rajkhowa, S

    2015-01-01

    Significance and Impact of the Study: The study reports the development and evaluation of a novel multiplex PCR assay for the rapid detection of 11 important VAGs of Pasteurella multocida isolates from pigs...

  6. 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

  7. Rapid and quantitative detection of Brucella by up-converting phosphor technology-based lateral-flow assay.

    Science.gov (United States)

    Qu, Qing; Zhu, Ziwen; Wang, Yufei; Zhong, Zhijun; Zhao, Jin; Qiao, Feng; Du, Xinying; Wang, Zhoujia; Yang, Ruifu; Huang, Liuyu; Yu, Yaqin; Zhou, Lei; Chen, Zeliang

    2009-10-01

    A rapid and quantitative up-converting phosphor technology-based later-flow assay (UPT-LF assay) was developed for on-site detection of Brucella. Different Brucella species both in pure cultures and in spiked samples could be quantitatively detected. The detection limit for pure culture was 5 x 10(6)CFU/ml and the sensitivity for different spiked samples ranged from 2.0 x 10(3) to 3.9 x 10(5)CFU/mg. The UPT-LF assay showed high specificity, reproducibility and stability, providing great potential for Brucella on-site detection.

  8. 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

  9. Development of a real-time SYBR Green PCR assay for the rapid detection of Dermatophilus congolensis.

    Science.gov (United States)

    García, Alfredo; Martínez, Remigio; Benitez-Medina, José Manuel; Risco, David; Garcia, Waldo Luis; Rey, Joaquín; Alonso, Juan Manuel; Hermoso de Mendoza, Javier

    2013-01-01

    Methods such as real time (RT)-PCR have not been developed for the rapid detection and diagnosis of Dermatophilus (D.) congolensis infection. In the present study, a D. congolensis-specific SYBR Green RT-PCR assay was evaluated. The detection limit of the RT-PCR assay was 1 pg of DNA per PCR reaction. No cross-reaction with nucleic acids extracted from Pseudomonas aeruginosa, Mycobacterium tuberculosis, Staphylococcus aureus, or Austwickia chelonae was observed. Finally, the RT-PCR assay was used to evaluate clinical samples collected from naturally infected animals with D. congolensis. The results showed that this assay is a fast and reliable method for diagnosing dermatophilosis.

  10. 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

  11. Multidrug-resistant tuberculosis: Rapid molecular detection with MTBDRplus® assay in clinical samples

    Directory of Open Access Journals (Sweden)

    Rita Macedo

    2009-05-01

    Full Text Available Nowadays, the greatest concern of tuberculosis control programmes is the appearance of multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis. Rapid determination of drug resistance in clinical samples, with Mycobacterium tuberculosis complex (MTC, is the prerequisite for initiating effective chemotherapy, ensuring successful treatment of the patient and preventing further spread of drugresistant isolates.The aim of our study was to determine the sensitivity of the new MTBDRplus® assay in comparison to culture, identification and classic DST, directly from smear-positive clinical specimens.A total of 68 smear-positive sputum specimens were processed by both the classical mycobacteriological methods and the molecular assay, MTBDRplus®.MTBDRplus® assay allowed an accurate identification of MTC species by detection of the specific band in all samples, from which we also isolated and identified MTC strains by culture methods. In the samples from which we isolated susceptible strains (63.2%, wild type patterns were found using MTBDRplus® assay. The samples from which we isolated resistant strains (36.8% showed specific mutations associated with the correspondent resistant phenotype.Our study indicated that this assay allows rapid detection of resistance, always in agreement with classic methods. Resumo: Uma das principais problematicas no controlo da tuberculose e o aparecimento de casos de tuberculose multirresistente (TB-MR e tuberculose extensivamente resistente (TB-XDR. A deteccao precoce da resistencia a farmacos, directamente a partir de amostras respiratorias, e essencial para que se assegure o tratamento atempado, adequado e eficaz da tuberculose, bem como para prevenir a disseminacao destes casos de especial gravidade.O nosso objectivo foi avaliar a sensibilidade e comparar os resultados obtidos com um metodo de genetica molecular disponivel comercialmente – MTBDRplus® – e o isolamento

  12. A rapid, sensitive, simple plate assay for detection of microbial alginate lyase activity.

    Science.gov (United States)

    Sawant, Shailesh S; Salunke, Bipinchandra K; Kim, Beom Soo

    2015-09-01

    worked well for screening and identification of alginate lyase producers and non-producers from environmental samples on common laboratory media. They did this by clearly showing the presence or absence of clearance zones around the microbial colonies grown. This new method is rapid, efficient, and could easily be performed for screening a large number of microbial cultures. This is the first report on the use of Gram's iodine for the detection of alginate lyase production by microorganisms using plate assay. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. A One-Step, Real-Time PCR Assay for Rapid Detection of Rhinovirus

    Science.gov (United States)

    Do, Duc H.; Laus, Stella; Leber, Amy; Marcon, Mario J.; Jordan, Jeanne A.; Martin, Judith M.; Wadowsky, Robert M.

    2010-01-01

    One-step, real-time PCR assays for rhinovirus have been developed for a limited number of PCR amplification platforms and chemistries, and some exhibit cross-reactivity with genetically similar enteroviruses. We developed a one-step, real-time PCR assay for rhinovirus by using a sequence detection system (Applied Biosystems; Foster City, CA). The primers were designed to amplify a 120-base target in the noncoding region of picornavirus RNA, and a TaqMan (Applied Biosystems) degenerate probe was designed for the specific detection of rhinovirus amplicons. The PCR assay had no cross-reactivity with a panel of 76 nontarget nucleic acids, which included RNAs from 43 enterovirus strains. Excellent lower limits of detection relative to viral culture were observed for the PCR assay by using 38 of 40 rhinovirus reference strains representing different serotypes, which could reproducibly detect rhinovirus serotype 2 in viral transport medium containing 10 to 10,000 TCID50 (50% tissue culture infectious dose endpoint) units/ml of the virus. However, for rhinovirus serotypes 59 and 69, the PCR assay was less sensitive than culture. Testing of 48 clinical specimens from children with cold-like illnesses for rhinovirus by the PCR and culture assays yielded detection rates of 16.7% and 6.3%, respectively. For a batch of 10 specimens, the entire assay was completed in 4.5 hours. This real-time PCR assay enables detection of many rhinovirus serotypes with the Applied Biosystems reagent-instrument platform. PMID:19948820

  14. 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.

  15. 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.

  16. Development of a real-time SYBR Green PCR assay for the rapid detection of Dermatophilus congolensis

    OpenAIRE

    García, Alfredo; Martínez, Remigio; Benitez-Medina, José Manuel; Risco, David; García, Waldo Luis; Rey, Joaquín; Alonso, Juan Manuel; de Mendoza, Javier Hermoso

    2013-01-01

    Methods such as real time (RT)-PCR have not been developed for the rapid detection and diagnosis of Dermatophilus (D.) congolensis infection. In the present study, a D. congolensis-specific SYBR Green RT-PCR assay was evaluated. The detection limit of the RT-PCR assay was 1 pg of DNA per PCR reaction. No cross-reaction with nucleic acids extracted from Pseudomonas aeruginosa, Mycobacterium tuberculosis, Staphylococcus aureus, or Austwickia chelonae was observed. Finally, the RT-PCR assay was ...

  17. Nitrate reductase assay using sodium nitrate for rapid detection of multidrug resistant tuberculosis

    Science.gov (United States)

    Macedo, Maíra Bidart; Groll, Andrea Von; Fissette, Krista; Palomino, Juan Carlos; da Silva, Pedro Eduardo Almeida; Martin, Anandi

    2012-01-01

    We validated the nitrate reductase assay (NRA) for the detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) using sodium nitrate (NaNO3) in replacement of potassium nitrate (KNO3) as nitrate source. NaNO3 is cheaper than KNO3 and has no restriction on use which facilitates the implementation of NRA to detect MDR-TB. PMID:24031916

  18. Nitrate reductase assay using sodium nitrate for rapid detection of multidrug resistant tuberculosis

    OpenAIRE

    Macedo, Ma?ra Bidart; Groll, Andrea Von; Fissette, Krista; Palomino, Juan Carlos; da Silva, Pedro Eduardo Almeida; Martin, Anandi

    2012-01-01

    We validated the nitrate reductase assay (NRA) for the detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) using sodium nitrate (NaNO3) in replacement of potassium nitrate (KNO3) as nitrate source. NaNO3 is cheaper than KNO3 and has no restriction on use which facilitates the implementation of NRA to detect MDR-TB.

  19. Novel Multitarget Real-Time PCR Assay for Rapid Detection of Bordetella Species in Clinical Specimens ▿

    Science.gov (United States)

    Tatti, Kathleen M.; Sparks, Kansas N.; Boney, Kathryn O.; Tondella, Maria Lucia

    2011-01-01

    A novel multitarget real-time PCR (RT-PCR) assay for the rapid identification of Bordetella pertussis, B. parapertussis, and B. holmesii was developed using multicopy insertion sequences (ISs) in combination with the pertussis toxin subunit S1 (ptxS1) singleplex assay. The RT-PCR targets for the multiplex assay include IS481, commonly found in B. pertussis and B. holmesii; IS1001 of B. parapertussis; and the IS1001-like sequence of B. holmesii. Overall, 402 Bordetella species and 66 non-Bordetella species isolates were tested in the multitarget assay. Cross-reactivity was found only with 5 B. bronchiseptica isolates, which were positive with IS1001 of B. parapertussis. The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for all targets. A total of 197 human clinical specimens obtained during cough-illness outbreak investigations were used to evaluate the multitarget RT-PCR assay. The multiplex assay results from 87 clinical specimens were compared to the individual RT-PCR assay and culture results. The multitarget assay is useful as a diagnostic tool to confirm B. pertussis infections and to rapidly identify other Bordetella species. In conclusion, the use of this multitarget RT-PCR approach increases specificity, while it decreases the amount of time, reagents, and specimen necessary for RT-PCRs used for accurate diagnosis of pertussis-like illness. PMID:21940464

  20. Real-time cytotoxicity assay for rapid and sensitive detection of ricin from complex matrices.

    Directory of Open Access Journals (Sweden)

    Diana Pauly

    Full Text Available BACKGROUND: In the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits. METHODOLOGY/FINDINGS: This work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index-time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed. Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from complex matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material. CONCLUSIONS/SIGNIFICANCE: The cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex

  1. Real-time cytotoxicity assay for rapid and sensitive detection of ricin from complex matrices.

    Science.gov (United States)

    Pauly, Diana; Worbs, Sylvia; Kirchner, Sebastian; Shatohina, Olena; Dorner, Martin B; Dorner, Brigitte G

    2012-01-01

    In the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits. This work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index-time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed). Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from complex matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material. The cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex sample matrices.

  2. A rapid assay for Hendra virus IgG antibody detection and its titre estimation using magnetic nanoparticles and phycoerythrin.

    Science.gov (United States)

    Gao, Yuan; Pallister, Jackie; Lapierre, Florian; Crameri, Gary; Wang, Lin-Fa; Zhu, Yonggang

    2015-09-15

    Detection of Hendra viral IgG antibody in animal sera is useful for surveillance following a virus outbreak. The commonly used enzyme-linked immunosorbent assay and fluorescence-based Luminex assay typically consist of three steps and take at least several hours to complete. We have simplified the procedure to two steps in an effort to develop a rapid procedure for IgG antibody, but not IgM antibody, detection. This is achieved by conjugating the fluorescence label R-phycoerythrin directly onto the IgG binding protein Protein G. The use of magnetic nanoparticles, due to their large specific surface area, has helped reduce each of the binding steps to 20 min. As a result, the whole assay can be completed in 60 min. We also demonstrate a method to quickly estimate IgG antibody titres by assaying the sera at only two dilutions (i.e. 1:20 and 1:1000) and using the fluorescence ratio at these dilutions as an indicator of antibody titre. The results of this approach correlated well with the well-regarded serum neutralization test in virus antibody assays. This protocol reported here can be adopted in Luminex assays, fluorescence-linked immunosorbent assays and assays on microfluidics platforms for rapid antibody surveillance of Hendra and other viruses. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Rapid detection of unconjugated estriol in the serum via superparamagnetic lateral flow immunochromatographic assay.

    Science.gov (United States)

    Wang, Ce; Guan, Di; Chen, Chen; He, Shang; Liu, Xiaoting; Wang, Chengbin; Wu, Huijuan

    2018-01-01

    Unconjugated estriol (uE 3 ) is one of the main naturally occurring estrogens that plays an important role in growth and development of the fetus. Usually, the level of uE 3 is very low in men and non-pregnant women, but in pregnant women, the level of estriol has been found to be quite high. Therefore, the combination of uE 3 , AFP, and hCG is now widely used for Down Syndrome screening as a triple marker. Here, we developed a superparamagnetic lateral flow immunochromatographic assay to quantitatively detect uE 3 . The detection limit of this assay was 0.86 nmol/L and the linear range for the determination of uE 3 was from 1 to 100 nmol/L. The detection time was 15 min and the assay had very low cross-reactivity with estrone (E 1 ), estradiol (E 2 ), and progesterone. The coefficient of variation (CV) of intra- and inter-assay ranged from 5% to 13%. The magnetic signals were stable under 37 °C within 7 d. Moreover, the concentrations of uE 3 measured by lateral flow immunochromatographic assay in 230 serum samples collected from pregnant women at the Chinese People's Liberation Army General Hospital had a good correlation with those measured by time-resolved fluorescence immunoassay (R = 0.946).

  4. Evaluation of Rapid Molecular Detection Assays for Salmonella in Challenging Food Matrices at Low Inoculation Levels and Using Difficult-to-Detect Strains.

    Science.gov (United States)

    Ryan, Gina; Roof, Sherry; Post, Laurie; Wiedmann, Martin

    2015-09-01

    Assays for detection of foodborne pathogens are generally initially evaluated for performance in validation studies carried out according to guidelines provided by validation schemes (e.g., AOAC International or the International Organization for Standardization). End users often perform additional validation studies to evaluate the performance of assays in specific matrices (e.g., specific foods or raw material streams of interest) and with specific pathogen strains. However, these types of end-user validations are typically not well defined. This study was conducted to evaluate a secondary end user validation of four AOAC-validated commercial rapid detection assays (an isothermal nucleic acid amplification, an immunoassay, and two PCR-based assays) for their ability to detect Salmonella in two challenging matrices (dry pet food and dark chocolate). Inclusivity was evaluated with 68 diverse Salmonella strains at low population levels representing the limit of detection (LOD) for each assay. One assay detected all strains at the LOD, two assays detected multiple strains only at 10 times the LOD, and the fourth assay failed to detect two strains (Salmonella bongori and S. enterica subsp. houtenae) even at 1,000 times the LOD; this assay was not further evaluated. The three remaining assays were subsequently evaluated for their ability to detect five selected Salmonella strains in food samples contaminated at fractional levels. Unpaired comparisons revealed no significant difference between the results for each given assay and the results obtained with the reference assay. However, analysis of paired culture-confirmed results revealed assay false-negative rates of 4 to 26% for dry pet food and 12 to 16% for dark chocolate. Overall, our data indicate that rapid assays may have high false-negative rates when performance is evaluated under challenging conditions, including low-moisture matrices, strains that are difficult to detect, injured cells, and low inoculum

  5. Development of a novel multiplex PCR assay for rapid detection of virulence associated genes of Pasteurella multocida from pigs.

    Science.gov (United States)

    Rajkhowa, S

    2015-09-01

    As the pathogenicity of Pasteurella multocida is associated with various virulence factors (VFs), the aim of the study was to develop a novel multiplex PCR (m-PCR) assay for the rapid detection of important virulence associated genes (VAGs) of P. multocida isolates from pigs. The target recognized VFs used in the study were diverse adhesins (ptfA and pfhA), toxins (toxA), siderophores (tonB and hgbA), sialidases (nanB, nanH) and outer membrane proteins (ompA, ompH, oma87 and plpB). The primers for the genes encoding these VFs were designed by primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/) using gene sequences available in Genbank. The detection limit of the developed assay was 10(2)  CFU ml(-1) . The m-PCR did not produce any nonspecific amplification products when tested against Bordetella bronchiseptica which also commonly infects pigs. We applied m-PCR to the field samples, and the results obtained were the same as the single PCR results. The developed assay would be very useful for veterinary diagnostic laboratories and for others interested in the rapid virulence profiling of porcine P. multocida isolates circulating in the piggeries. The study reports the development and evaluation of a novel multiplex PCR assay for the rapid detection of 11 important VAGs of Pasteurella multocida isolates from pigs. Rapid and simultaneous detection of recognized VFs of the organism are essential to know the virulo-types of P. multocida isolates circulating in the piggeries. The developed novel assay will be very useful for the rapid detection of VAGs of P. multocida isolates from pigs. © 2015 The Society for Applied Microbiology.

  6. 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.

  7. Immunochromatographic strip assay for the rapid and sensitive detection of Salmonella Typhimurium in artificially contaminated tomato samples.

    Science.gov (United States)

    Shukla, Shruti; Leem, Hyerim; Lee, Jong-Suk; Kim, Myunghee

    2014-06-01

    This study was designed to confirm the applicability of a liposome-based immunochromatographic assay for the rapid detection of Salmonella enterica subsp. enterica serovar Typhimurium (Salmonella Typhimurium) in artificially contaminated tomato samples. To determine the detection limit and pre-enrichment incubation time (10, 12, and 18 h pre-enrichment in 1% buffered peptone water), the tests were performed with different cell numbers of Salmonella Typhimurium (3 × 10(0), 3 × 10(1), 3 × 10(2), and 3 × 10(3) CFU·mL(-1)) inoculated into 25 g of crushed tomato samples. The assay was able to detect as few as 30 Salmonella Typhimurium cells per 25 g of tomato samples (1.2 cells·g(-1)) after 12 h pre-enrichment incubation. Moreover, when the developed assay was compared with traditional morphological and biochemical culture-based methods as well as colloidal gold nanoparticle-based commercial test strips, the developed assay yielded positive results for the detection of Salmonella Typhimurium within a shorter period time. These findings confirm that the developed assay may have practical application for the sensitive detection of Salmonella Typhimurium in various food samples, including raw vegetables, with a relatively low detection limit and shorter analysis time.

  8. 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...

  9. Rapid detection and typing of pathogenic nonpneumophila Legionella spp. isolates using a multiplex real-time PCR assay.

    Science.gov (United States)

    Benitez, Alvaro J; Winchell, Jonas M

    2016-04-01

    We developed a single tube multiplex real-time PCR assay that allows for the rapid detection and typing of 9 nonpneumophila Legionella spp. isolates that are clinically relevant. The multiplex assay is capable of simultaneously detecting and discriminating L. micdadei, L. bozemanii, L. dumoffii, L. longbeachae, L. feeleii, L. anisa, L. parisiensis, L. tucsonensis serogroup (sg) 1 and 3, and L. sainthelensis sg 1 and 2 isolates. Evaluation of the assay with nucleic acid from each of these species derived from both clinical and environmental isolates and typing strains demonstrated 100% sensitivity and 100% specificity when tested against 43 other Legionella spp. Typing of L. anisa, L. parisiensis, and L. tucsonensis sg 1 and 3 isolates was accomplished by developing a real-time PCR assay followed by high-resolution melt (HRM) analysis targeting the ssrA gene. Further typing of L. bozemanii, L. longbeachae, and L. feeleii isolates to the serogroup level was accomplished by developing a real-time PCR assay followed by HRM analysis targeting the mip gene. When used in conjunction with other currently available diagnostic tests, these assays may aid in rapidly identifying specific etiologies associated with Legionella outbreaks, clusters, sporadic cases, and potential environmental sources. Published by Elsevier Inc.

  10. Colloidal Gold Probe-Based Immunochromatographic Strip Assay for the Rapid Detection of Microbial Transglutaminase in Frozen Surimi

    Directory of Open Access Journals (Sweden)

    Daming Fan

    2016-01-01

    Full Text Available Adding microbial transglutaminase (MTGase to frozen surimi to enable the surimi to be sold as a higher-grade product at a higher price defrauds surimi product manufacturers and undercuts legitimate industry prices. Therefore, it is important to develop an accurate method of detecting the presence of MTGase in surimi. In this study, an immunochromatographic strip assay with a colloidal gold antibody probe was successfully developed and used to rapidly and qualitatively detect MTGase in surimi samples. The results were obtained in less than 10 min. The limit for the qualitative detection of MTGase using the immunochromatographic strip assay was identified as 1.0 μg/mL. The results of the immunochromatographic strip analysis of frozen surimi samples were verified by comparison with the results of a sandwich enzyme-linked immunosorbent assay. The colloidal gold probe-based immunochromatographic strip assay was thus found to be a rapid, economical, and user friendly method of detecting MTGase in surimi.

  11. Rapid detection of Hendra virus antibodies: an integrated device with nanoparticle assay and chaotic micromixing.

    Science.gov (United States)

    Petkovic, K; Metcalfe, G; Chen, H; Gao, Y; Best, M; Lester, D; Zhu, Y

    2016-12-20

    Current diagnosis of infectious diseases such as Hendra virus (HeV) relies mostly on laboratory-based tests. There is an urgent demand for rapid diagnosis technology to detect and identify these diseases in humans and animals so that disease spread can be controlled. In this study, an integrated lab-on-a-chip device using a magnetic nanoparticle immunoassay is developed. The key features of the device are the chaotic fluid mixing, achieved by magnetically driven motion of nanoparticles with the optimal mixing protocol developed using chaotic transport theory, and the automatic liquid handling system for loading reagents and samples. The device has been demonstrated to detect Hendra virus antibodies in dilute horse serum samples within a short time of 15 minutes and the limit of detection is about 0.48 ng ml -1 . The device platform can potentially be used for field detection of viruses and other biological and chemical substances.

  12. Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay.

    Science.gov (United States)

    Binet, Rachel; Deer, Deanne M; Uhlfelder, Samantha J

    2014-06-01

    Faster detection of contaminated foods can prevent adulterated foods from being consumed and minimize the risk of an outbreak of foodborne illness. A sensitive molecular detection method is especially important for Shigella because ingestion of as few as 10 of these bacterial pathogens can cause disease. The objectives of this study were to compare the ability of four DNA extraction methods to detect Shigella in six types of produce, post-enrichment, and to evaluate a new and rapid conventional multiplex assay that targets the Shigella ipaH, virB and mxiC virulence genes. This assay can detect less than two Shigella cells in pure culture, even when the pathogen is mixed with background microflora, and it can also differentiate natural Shigella strains from a control strain and eliminate false positive results due to accidental laboratory contamination. The four DNA extraction methods (boiling, PrepMan Ultra [Applied Biosystems], InstaGene Matrix [Bio-Rad], DNeasy Tissue kit [Qiagen]) detected 1.6 × 10(3)Shigella CFU/ml post-enrichment, requiring ∼18 doublings to one cell in 25 g of produce pre-enrichment. Lower sensitivity was obtained, depending on produce type and extraction method. The InstaGene Matrix was the most consistent and sensitive and the multiplex assay accurately detected Shigella in less than 90 min, outperforming, to the best of our knowledge, molecular assays currently in place for this pathogen. Published by Elsevier Ltd.

  13. Lateral flow assay with pressure meter readout for rapid point-of-care detection of disease-associated protein.

    Science.gov (United States)

    Lin, Bingqian; Guan, Zhichao; Song, Yanling; Song, Eunyeong; Lu, Zifei; Liu, Dan; An, Yuan; Zhu, Zhi; Zhou, Leiji; Yang, Chaoyong

    2018-02-26

    Paper-based assays such as lateral flow assays are good candidates for portable diagnostics owing to their user-friendly format and low cost. In terms of analytical detection, lateral flow assays usually require dedicated instruments to obtain quantitative results. Here we demonstrate a lateral flow assay with handheld pressure meter readout for the rapid detection of disease-related protein with high sensitivity and selectivity. Based on the pressure change produced by the catalytic reaction of Pt nanoparticles related to the concentration of the target, a quantitative reaction platform was established. During the lateral flow assay, the Pt nanoparticles are aggregated in the test line to form a gray band by biomolecular recognition and finally convert the recognition signal into highly sensitive pressure readout for quantitative analysis. Without sophisticated instrumentation and complicated operations, the whole detection process can be completed within 20 minutes. The limit of detection for myoglobin (2.9 ng mL -1 in diluted serum samples) meets the requirements of clinical monitoring. With the advantages of low cost, ease of operation, high sensitivity and selectivity, the method represents a versatile platform for point-of-care testing of disease biomarkers.

  14. Rapid Detection/pathotyping of Newcastle disease virus isolates in clinical samples using real time polymerase chain reaction assay

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Abdul Wajid, Muhammad Wasim, Tahir Yaqub, Shafqat F Rehmani, Tasra Bibi, Nadia Mukhtar, Javed Muhammad, Umar Bacha, Suliman Qadir Afridi, Muhammad Nauman Zahid, Zia u ddin, Muhammad Zubair Shabbir, Kamran Abbas & Muneer Ahmad ### Abstract In the present protocol we describe the real time reverse transcription polymerase chain reaction (rRT-PCR) assay for the rapid detection/pathotyping of Newcastle disease virus (NDV) isoaltes in clinical samples. Fusion gene and matrix gene...

  15. Heteroduplex mobility assay for rapid, sensitive and specific detection of mycobacteria.

    Science.gov (United States)

    Waléria-Aleixo, A; Kroon, E G; Campos, M A; Margutti-Pinto, M E; Bonjardim, C A; Ferreira, P C

    2000-04-01

    We report an improved method for the detection and identification of mycobacteria using PCR and the heteroduplex mobility shift assay (HMA). The HMA for detection of mycobacteria was based on the microheterogeneity within the DNA coding sequences for 16S rRNA. A remarkable shift between single-stranded, heteroduplex and homoduplex bands in PAGE was observed among the Mycobacterium spp. tested. The Mycobacteria HMA (MHMA) of amplified PCR products from mycobacteria DNA coding for 16S rDNA derived from culture showed a specific heteroduplexes formed among different Mycobacterium species. Other bacterium species were distinguished from Mycobaterium due to slow migrating heteroduplexes mobility bands observed when M. bovis (BCG), M. avium, or M. fortuitum were used as a standard. The specific heteroduplexes were detected when as little as 1 etag of DNA template was used, although better results were obtained with 5 etag and when PCR products of sample test and mycobacterium standard were mixed at a ratio of 1.8. To correctly evaluate the feasibility of using MHMA to detect and identify mycobacteria, 15 clinical sample patients were tested. All MTB-positive clinical samples were identified by MHMA as well as the negative samples. In addition, MHMA will, in principle, be applicable to the detection and classification of any microorganism showing differences within the 16S rRNA as well as to the identification of new and unrecognized bacterial species.

  16. Rapid and selective detection of experimental snake envenomation - Use of gold nanoparticle based lateral flow assay.

    Science.gov (United States)

    Pawade, Balasaheb S; Salvi, Nitin C; Shaikh, Innus K; Waghmare, Arun B; Jadhav, Nitin D; Wagh, Vishal B; Pawade, Abhilasha S; Waykar, Indrasen G; Potnis-Lele, Mugdha

    2016-09-01

    In this study, we have developed a gold nanoparticle based simple, rapid lateral flow assay (LFA) for detection of Indian Cobra venom (CV) and Russell's viper venom (RV). Presently, there is no rapid, reliable, and field diagnostic test available in India, where snake bite cases are rampant. Therefore, this test has an immense potential from the public health point of view. The test is based on the principle of the paper immunochromatography assay for detection of two snake venom species using polyvalent antisnake venom antibodies (ASVA) raised in equines and species-specific antibodies (SSAbs) against venoms raised in rabbits for conjugation and impregnation respectively. The developed, snake envenomation detection immunoassay (SEDIA) was rapid, selective, and sensitive to detect venom concentrations up to 0.1 ng/ml. The functionality of SEDIA strips was confirmed by experimental envenomation in mice and the results obtained were specific for the corresponding venom. The SEDIA has a potential to be a field diagnostic test to detect snake envenomation and assist in saving lives of snakebite victims. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. A Rapid and Sensitive Assay for the Detection of Benzylpenicillin (PenG in Milk.

    Directory of Open Access Journals (Sweden)

    Anna Pennacchio

    Full Text Available Antibiotics, such as benzyl-penicillin (PenG and cephalosporin, are the most common compounds used in animal therapy. Their massive and illegal use in animal therapy and prophylaxis inevitably causes the presence of traces in foods of animal origin (milk and meat, which creates several problems for human health. With the aim to prevent the negative impact of β-lactam and, in particular, PenG residues present in the milk on customer health, many countries have established maximum residue limits (MRLs. To cope with this problem here, we propose an effective alternative, compared to the analytical methods actually employed, to quantify the presence of penicillin G using the surface plasmon resonance (SPR method. In particular, the PenG molecule was conjugated to a protein carrier to immunize a rabbit and produce polyclonal antibodies (anti-PenG. The produced antibodies were used as molecular recognition elements for the design of a competitive immune-assay for the detection of PenG by SPR experiments. The detection limit of the developed assay was found to be 8.0 pM, a value much lower than the MRL of the EU regulation limit that is fixed at 12 nM. Thus, our results clearly show that this system could be successfully suitable for the accurate and easy determination of PenG.

  18. A Rapid and Sensitive Assay for the Detection of Benzylpenicillin (PenG) in Milk.

    Science.gov (United States)

    Pennacchio, Anna; Varriale, Antonio; Esposito, Maria Grazia; Scala, Andrea; Marzullo, Vincenzo Manuel; Staiano, Maria; D'Auria, Sabato

    2015-01-01

    Antibiotics, such as benzyl-penicillin (PenG) and cephalosporin, are the most common compounds used in animal therapy. Their massive and illegal use in animal therapy and prophylaxis inevitably causes the presence of traces in foods of animal origin (milk and meat), which creates several problems for human health. With the aim to prevent the negative impact of β-lactam and, in particular, PenG residues present in the milk on customer health, many countries have established maximum residue limits (MRLs). To cope with this problem here, we propose an effective alternative, compared to the analytical methods actually employed, to quantify the presence of penicillin G using the surface plasmon resonance (SPR) method. In particular, the PenG molecule was conjugated to a protein carrier to immunize a rabbit and produce polyclonal antibodies (anti-PenG). The produced antibodies were used as molecular recognition elements for the design of a competitive immune-assay for the detection of PenG by SPR experiments. The detection limit of the developed assay was found to be 8.0 pM, a value much lower than the MRL of the EU regulation limit that is fixed at 12 nM. Thus, our results clearly show that this system could be successfully suitable for the accurate and easy determination of PenG.

  19. Rapid focused sequencing: a multiplexed assay for simultaneous detection and strain typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Rosemary S Turingan

    Full Text Available BACKGROUND: The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. METHODOLOGY/PRINCIPAL FINDINGS: We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed "Rapid Focused Sequencing," allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. CONCLUSIONS/SIGNIFICANCE: The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental

  20. Evaluation of the MeltPro TB/STR assay for rapid detection of streptomycin resistance in Mycobacterium tuberculosis.

    Science.gov (United States)

    Zhang, Ting; Hu, Siyu; Li, Guoli; Li, Hui; Liu, Xiaoli; Niu, Jianjun; Wang, Feng; Wen, Huixin; Xu, Ye; Li, Qingge

    2015-03-01

    Rapid and comprehensive detection of drug-resistance is essential for the control of tuberculosis, which has facilitated the development of molecular assays for the detection of drug-resistant mutations in Mycobacterium tuberculosis. We hereby assessed the analytical and clinical performance of an assay for streptomycin-resistant mutations. MeltPro TB/STR is a closed-tube, dual-color, melting curve analysis-based, real-time PCR test designed to detect 15 streptomycin-resistant mutations in rpsL 43, rpsL 88, rrs 513, rrs 514, rrs 517, and rrs 905-908 of M. tuberculosis. Analytical studies showed that the accuracy was 100%, the limit of detection was 50-500 bacilli per reaction, the reproducibility in the form of Tm variation was within 1.0 °C, and we could detect 20% STR resistance in mixed bacterial samples. The cross-platform study demonstrated that the assay could be performed on six models of real-time PCR instruments. A multicenter clinical study was conducted using 1056 clinical isolates, which were collected from three geographically different healthcare units, including 709 STR-susceptible and 347 STR-resistant isolates characterized on Löwenstein-Jensen solid medium by traditional drug susceptibility testing. The results showed that the clinical sensitivity and specificity of the MeltPro TB/STR was 88.8% and 95.8%, respectively. Sequencing analysis confirmed the accuracy of the mutation types. Among all the 8 mutation types detected, rpsL K43R (AAG → AGG), rpsL K88R (AAG → AGG) and rrs 514 A → C accounted for more than 90%. We concluded that MeltPro TB/STR represents a rapid and reliable assay for the detection of STR resistance in clinical isolates. Copyright © 2014. Published by Elsevier Ltd.

  1. 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.

  2. Evaluation of an Immunochromatographic Assay for the Rapid and Simultaneous Detection of Rotavirus and Adenovirus in Stool Samples

    Science.gov (United States)

    Kim, Jayoung; Kim, Han-Sung; Kim, Jae-Seok; Song, Wonkeun; Lee, Kyu Man; Lee, Sunhwa; Park, Kyoung Un; Lee, Woochang; Hong, Young Jun

    2014-01-01

    Background We evaluated the analytical and clinical performances of the SD BIOLINE Rota/Adeno Rapid kit (SD Rota/Adeno Rapid; Standard Diagnostics, Inc., Korea), an immunochromatographic assay (ICA), for the simultaneous detection of rotaviruses and adenoviruses in human stool samples. Methods We tested 400 clinical stool samples from patients with acute gastroenteritis and compared the ICA results with the results obtained by using ELISA, enzyme-linked fluorescent assays (ELFA), PCR, and multiplex reverse transcription-PCR (mRT-PCR). To assess the analytical performance of the SD BIOLINE Rota/Adeno Rapid kit, we determined its detection limit, reproducibility, cross-reactivity, and analytical reactivity for adenovirus subtypes, and performed interference studies. Results The overall agreement rates among the tested methods were 91.5% for rotavirus and 85.5% for adenovirus. On the basis of mRT-PCR, the overall agreement, positive agreement, and negative agreement rates of the ICA were 95.6%, 100%, and 94.9% for rotavirus, and 94.0%, 71.4%, and 94.8% for adenovirus, respectively. Using the ICA, we detected all the subtypes of adenovirus tested, but the analytical reactivities for adenovirus subtypes were different between the 4 adenovirus detection methods. The high reproducibility was confirmed, and no cross-reactivity or interference was detected. Conclusions The SD BIOLINE Rota/Adeno Rapid kit showed acceptable analytical and clinical performances. However, interpretation of adenovirus positive/negative result should be cautious because of different detectability for adenovirus subtypes among adenovirus detection methods. PMID:24790909

  3. Development and Validation of a Real-Time PCR Assay for Rapid Detection of Candida auris from Surveillance Samples.

    Science.gov (United States)

    Leach, L; Zhu, Y; Chaturvedi, S

    2018-02-01

    Candida auris is an emerging multidrug-resistant yeast causing invasive health care-associated infection with high mortality worldwide. Rapid identification of C. auris is of primary importance for the implementation of public health measures to control the spread of infection. To achieve these goals, we developed and validated a TaqMan-based real-time PCR assay targeting the internal transcribed spacer 2 ( ITS 2) region of the ribosomal gene. The assay was highly specific, reproducible, and sensitive, with the detection limit of 1 C. auris CFU/PCR. The performance of the C. auris real-time PCR assay was evaluated by using 623 surveillance samples, including 365 patient swabs and 258 environmental sponges. Real-time PCR yielded positive results from 49 swab and 58 sponge samples, with 89% and 100% clinical sensitivity with regard to their respective culture-positive results. The real-time PCR also detected C. auris DNA from 1% and 12% of swab and sponge samples with culture-negative results, indicating the presence of dead or culture-impaired C. auris The real-time PCR yielded results within 4 h of sample processing, compared to 4 to 14 days for culture, reducing turnaround time significantly. The new real-time PCR assay allows for accurate and rapid screening of C. auris and can increase effective control and prevention of this emerging multidrug-resistant fungal pathogen in health care facilities. Copyright © 2018 Leach et al.

  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. Rapid Assay for Simultaneous Detection and Differentiation of Immunoglobulin G Antibodies to Human Immunodeficiency Virus Type 1 (HIV-1) Group M, HIV-1 Group O, and HIV-2

    OpenAIRE

    Vallari, Ana S.; Hickman, Robert K.; Hackett, John R.; Brennan, Catherine A.; Varitek, Vincent A.; Devare, Sushil G.

    1998-01-01

    A rapid immunodiagnostic test that detects and discriminates human immunodeficiency virus (HIV) infections on the basis of viral type, HIV type 1 (HIV-1) group M, HIV-1 group O, or HIV-2, was developed. The rapid assay for the detection of HIV (HIV rapid assay) was designed as an instrument-free chromatographic immunoassay that detects immunoglobulin G (IgG) antibodies to HIV. To assess the performance of the HIV rapid assay, 470 HIV-positive plasma samples were tested by PCR and/or Western b...

  6. Multiplex real-time PCR assay for rapid detection of methicillin-resistant staphylococci directly from positive blood cultures.

    Science.gov (United States)

    Wang, Hye-Young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok; Uh, Young; Lee, Hyeyoung

    2014-06-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 10(3) CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  7. Multiplex Real-Time PCR Assay for Rapid Detection of Methicillin-Resistant Staphylococci Directly from Positive Blood Cultures

    Science.gov (United States)

    Wang, Hye-young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok

    2014-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 103 CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene. PMID:24648566

  8. 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...

  9. Rapid and Sensitive Detection of Phytophthora sojae in Soil and Infected Soybeans by Species-Specific Polymerase Chain Reaction Assays.

    Science.gov (United States)

    Wang, Yuanchao; Zhang, Wenli; Wang, Ying; Zheng, Xiaobo

    2006-12-01

    ABSTRACT Root and stem rot caused by Phytophthora sojae is one of the most destructive diseases of soybean (Glycine max) worldwide. P. sojae can survive as oospores in soil for many years. In order to develop a rapid and accurate method for the specific detection of P. sojae in soil, the internal transcribed spacer (ITS) regions of eight P. sojae isolates were amplified using polymerase chain reaction (PCR) with the universal primers DC6 and ITS4. The sequences of PCR products were aligned with published sequences of 50 other Phytophthora species, and a region specific to P. sojae was used to design the specific PCR primers, PS1 and PS2. More than 245 isolates representing 25 species of Phytophthora and at least 35 other species of pathogens were used to test the specificity of the primers. PCR amplification with PS primers resulted in the amplification of a product of approximately 330 bp, exclusively from isolates of P. sojae. Tests with P. sojae genomic DNA determined that the sensitivity of the PS primer set is approximately 1 fg. This PCR assay, combined with a simple soil screening method developed in this work, allowed the detection of P. sojae from soil within 6 h, with a detection sensitivity of two oospores in 20 g of soil. PCR with the PS primers could also be used to detect P. sojae from diseased soybean tissue and residues. Real-time fluorescent quantitative PCR assays were also developed to detect the pathogen directly in soil samples. The PS primer-based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in soil and infected soybean tissue.

  10. 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 ...

  11. Development of microLIPS (Luciferase Immunoprecipitation Systems): a novel microfluidic assay for rapid serum antibody detection

    Science.gov (United States)

    Chandrangsu, Matt; Burbelo, Peter D.; Iadarola, Michael J.; Smith, Paul D.; Morgan, Nicole Y.

    2012-06-01

    There is considerable interest in the development of rapid, point-of-care antibody detection for the diagnosis of infectious and auto-immune diseases. In this paper, we present work on the development of a self-contained microfluidic format for the Luciferase Immunoprecipitation Systems (LIPS) assay. Whereas the majority of immunoassays for antigen-specific antibodies employ either bacteria- or yeast-expressed proteins and require the use of secondary antibodies, the LIPS technique uses a fusion protein comprised of a Renilla luciferase reporter and the antigen of interest produced via mammalian cell culture, ensuring the addition of mammalian post-translational modifications. Patient serum is mixed with the fusion protein and passed over immobilized Protein A/G; after washing, the only remaining luciferase-tagged antigens are those retained by specific antibodies. These can be quantitatively measured using chemiluminescence upon the introduction of coelenterazine. The assay has been successfully employed for a wide variety of diseases in a microwell format. We report on a recent demonstration of rapid HSV-2 diagnosis with the LIPS assay in a microfluidic format, using one microliter of serum and obtaining results in under ten minutes. We will also discuss recent progress on two fronts, both aimed at the deployment of this technology in the field: first, simplifying assay operation through the automation of flow control using power-free means; and second, efforts to increase signal levels, primarily through strategies to increase antibody binding capacity, in order to move towards portable battery powered electronics.

  12. 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.

  13. 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.

  14. Rapid, Sensitive, Enzyme-Immunodotting Assay for Detecting Cow Milk Adulteration in Sheep Milk: A Modern Laboratory Project

    Science.gov (United States)

    Inda, Luis A.; Razquín, Pedro; Lampreave, Fermín; Alava, María A.; Calvo, Miguel

    1998-12-01

    Specificity, sensitivity, and experimental simplicity make the immunoenzymatic assay suitable for a variety of laboratories dedicated to diverse activities such as research, quality control in food analysis, or clinical biochemistry. In these assays, the antibody that specifically recognizes the antigen is covalently attached to an enzyme. Once the antigen-antibody immunocomplex is formed, the enzymatic reaction gives a colored product that allows the detection of the initial antigen. The aim of this work was the design of a new laboratory project appropriate for use in courses of biochemistry, immunochemistry, or analytical chemistry. The assay described here detects the presence of cow milk in milk of other species. The main application is the detection of cow milk in sheep milk and cheese. Specific proteins, immunoglobulins (IgG) of the fraudulent bovine milk, are specifically recognized and retained by antibodies immobilized on a membrane. The binding of a second antibody covalently attached to horseradish peroxidase (HRP) allows the development of a visible signal. Thus, students can rapidly detect milk adulterations using a specific, sensitive, and safe experimental approach. The experiment allows students to apply their theoretical knowledge, resulting in a stimulating experience of solving a real problem during a 4-hour laboratory period.

  15. Rapid detection of resistant tuberculosis by nitrate reductase assay performed in three settings in Brazil.

    Science.gov (United States)

    Shikama, Maria de Lourdes; Silva, Regina Ruivo Ferro E; Martins, Maria Conceição; Giampaglia, Carmen Maria Saraiva; Oliveira, Rosângela Siqueira; Silva, Rosmari F A M; Silva, Paula Ferro E; Telles, Maria Alice da Silva; Martin, Anandi; Palomino, Juan Carlos

    2009-10-01

    To evaluate nitrate reductase assay (NRA) efficacy for streptomycin, isoniazid, rifampicin and ethambutol susceptibility testing of Mycobacterium tuberculosis strains. Results were generated by three laboratories: the Instituto Adolfo Lutz (IAL) Mycobacteria Reference Laboratory and two IAL Regional Laboratories in Santo André and Sorocaba, São Paulo State, Brazil. One hundred and twenty M. tuberculosis strains were simultaneously tested using NRA and the proportion method (PM), while 117 strains were tested using both NRA and BACTEC MGIT 960 (M960). Repeatability analysis of NRA results showed rates of 100% for isoniazid and ethambutol and 97% for streptomycin and rifampicin susceptibility detection, representing substantial agreement. McNemar testing of the data also indicates that NRA and PM, as well as NRA and M960, do not differ significantly. On average, NRA results were available after 10 days. The data demonstrate that NRA is reliable for susceptibility testing of isoniazid and rifampicin, the two most important drugs for the treatment of tuberculosis. In addition, the reduction in the time necessary to obtain susceptibility results is of fundamental importance.

  16. 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.

  17. Development and application of a multiplex PCR assay for rapid detection of 4 major bacterial pathogens in ducks.

    Science.gov (United States)

    Wei, B; Cha, S-Y; Kang, M; Park, I-J; Moon, O-K; Park, C-K; Jang, H-K

    2013-05-01

    Infections with Pasteurella multocida, Salmonella enterica, Riemerella anatipestifer, and Escherichia coli result in high morbidity and mortality, which cause significant economic loss in the poultry industry. It can be difficult to distinguish these pathogens based on clinical signs because these pathogens can cause similar clinical signs and coinfections can occur. Thus, rapid and sensitive detection of these 4 major bacterial pathogens are important in ducks. The aim of this study was to develop a multiplex PCR (mPCR) assay for simultaneously detecting and identifying these 4 pathogenic bacteria in a single tube reaction. The target genes used were KMT1 of P. multocida, the invasion protein gene of S. enterica, 16S rDNA of R. anatipestifer, and the alkaline phosphatase gene of E. coli. The detection limit of the assay for all bacterial DNA was 10 pg. The mPCR did not produce any nonspecific amplification products when tested against other related pathogens, including Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens, Mycoplasma gallinarum, Mycoplasma synoviae, and Mycoplasma gallisepticum, which can also infect ducks. We applied mPCR to field samples, and the results were the same as the single PCR results. These results suggest that mPCR for the 4 bacteria is a useful and rapid technique to apply to field samples.

  18. Rapid detection and identification of 12 respiratory viruses using a dual priming oligonucleotide system-based multiplex PCR assay.

    Science.gov (United States)

    Kim, Suk Ran; Ki, Chang-Seok; Lee, Nam Yong

    2009-03-01

    Acute viral respiratory infections are among the most common causes of human disease. Rapid and accurate diagnosis of viral respiratory infections is important for providing timely therapeutic interventions. This study evaluated a new multiplex PCR assay (Seegene Inc., Seoul, Korea) for simultaneous detection and identification of 12 respiratory viruses using two primer mixes. The viruses included parainfluenza viruses 1, 2, and 3, human metapneumovirus, human coronavirus 229E/NL63 and OC43, adenovirus, influenza viruses A and B, human respiratory syncytial viruses A and B, and human rhinovirus A. The analytical sensitivity of the assay was 10-100 copies per reaction for each type of virus. There was no cross-reactivity with common bacterial or viral pathogens. A comparison with conventional viral culture and immunofluorescence was carried out using 101 respiratory specimens from 92 patients. Using viral culture, 57 specimens (56.4%) were positive without co-infection. The same viruses were identified in all 57 specimens using the multiplex PCR. Seven of the 57 specimens (12.3%) were found to be co-infected with other respiratory viruses, and 19 of 44 (43.2%) specimens which were negative by culture were positive by the multiplex PCR. The Seeplex Respiratory Virus Detection assay represents a significant improvement over the conventional methods for the detection of a broad spectrum of respiratory viruses.

  19. A rapid and sensitive assay for detection of replication-competent adenoviruses by a combination of microcarrier cell culture and quantitative PCR

    NARCIS (Netherlands)

    Schalk, Johanna A. C.; de Vries, Claudette G. J. C. A.; Orzechowski, Tom J. H.; Rots, Marianne G.

    2007-01-01

    The development of a rapid and sensitive assay for detection of replication-competent adenoviruses (RCAs) is described. This RCA assay consists of an incubation step of 4 days of adenoviral vectors on A549 cells in a microcarrier cell culture system followed by detection of amplified RCAs by

  20. Rapid DNA extraction for specific detection and quantitation of Mycobacterium tuberculosis DNA in sputum specimens using taqman assays

    Science.gov (United States)

    Gomez, Diana I.; Mullin, Caroline S.; Mora-Guzmán, Francisco; Crespo-Solis, J. Gonzalo; Fisher-Hoch, Susan P.; McCormick, Joseph B.; Restrepo, Blanca I.

    2011-01-01

    SUMMARY Rapid tuberculosis (TB) detection is critical for disease control, and further quantitation of Mycobacterium tuberculosis (Mtb) in sputum is valuable for epidemiological and clinical studies. We evaluated a simple, robust and cost-efficient in-house DNA extraction and downstream taqman approach for detection and quantitation of Mtb genomes from sputum of newly-diagnosed TB patients and non-TB controls. DNA was extracted using guanidine isothiocyanate and silica-based spin columns in less than 2h, stored frozen, and taqman assays were used to detect Mtb with IS6110 and quantify it targeting RD1 and IS1081. The taqmans had a sensitivity > 95% in 108 culture-confirmed TB patients and specificity of 100% in 43 non-TB controls. Genome counts were correlated with the Mycobacterial Growth Indicator Tubes’ (MGIT) time-to-detection values (1/TTD×1000; rho=0.66; p<0.001) in 91 TB patients (33 excluded with MGIT contamination). This linear relationship was nearly identical between mycobacteria isolated from sputum and H37Rv Mtb grown in-vitro to its log phase. TB treatment between 3 and 7 days was associated with lower 1/TTD×1000 values but not with genome counts. Together, our protocol provides rapid, specific, inexpensive and quantitative detection of Mtb DNA in fresh or stored sputa making it a robust tool for prompt TB diagnosis, and with potential use for clinical and epidemiologic studies. PMID:22088321

  1. Direct nitrate reductase assay versus microscopic observation drug susceptibility test for rapid detection of MDR-TB in Uganda.

    Directory of Open Access Journals (Sweden)

    Freddie Bwanga

    Full Text Available The most common method for detection of drug resistant (DR TB in resource-limited settings (RLSs is indirect susceptibility testing on Lowenstein-Jensen medium (LJ which is very time consuming with results available only after 2-3 months. Effective therapy of DR TB is therefore markedly delayed and patients can transmit resistant strains. Rapid and accurate tests suitable for RLSs in the diagnosis of DR TB are thus highly needed. In this study we compared two direct techniques--Nitrate Reductase Assay (NRA and Microscopic Observation Drug Susceptibility (MODS for rapid detection of MDR-TB in a high burden RLS. The sensitivity, specificity, and proportion of interpretable results were studied. Smear positive sputum was collected from 245 consecutive re-treatment TB patients attending a TB clinic in Kampala, Uganda. Samples were processed at the national reference laboratory and tested for susceptibility to rifampicin and isoniazid with direct NRA, direct MODS and the indirect LJ proportion method as reference. A total of 229 specimens were confirmed as M. tuberculosis, of these interpretable results were obtained in 217 (95% with either the NRA or MODS. Sensitivity, specificity and kappa agreement for MDR-TB diagnosis was 97%, 98% and 0.93 with the NRA; and 87%, 95% and 0.78 with the MODS, respectively. The median time to results was 10, 7 and 64 days with NRA, MODS and the reference technique, respectively. The cost of laboratory supplies per sample was low, around 5 USD, for the rapid tests. The direct NRA and MODS offered rapid detection of resistance almost eight weeks earlier than with the reference method. In the study settings, the direct NRA was highly sensitive and specific. We consider it to have a strong potential for timely detection of MDR-TB in RLS.

  2. A novel assay for rapid HIV-1 protease detection using optical sensors and magnetic carriers

    Science.gov (United States)

    Esseghaier, Chiheb; Ng, Andy; Zourob, Mohammed

    2012-10-01

    In this work, a very simple electrochemical HIV-1 protease biosensor useful for the development of an inexpensive lab-on-a- chip (LOC) device was constructed. The detection mechanism was designed to minimize the complexity either in the recognition receptor immobilization step or during the detection itself. The magnetic self-assembled monolayer of HIV-1 protease substrate peptide was able to detect as low as 10 pg/ml of the protease within 25 minutes with high specificity.

  3. Validity of the rapid strip assay test for detecting HBsAg in patients admitted to hospital in Uganda.

    Science.gov (United States)

    Seremba, E; Ocama, P; Opio, C K; Kagimu, M; Yuan, H J; Attar, N; Thomas, D L; Lee, W M

    2010-08-01

    Commercially available rapid strip assays (RSAs) for hepatitis B surface antigen (HBsAg) are used for most routine clinical testing in sub-Saharan Africa. This study evaluated the validity of RSA and a more sophisticated enzyme immunoassay (EIA) with confirmation by nucleic acid testing (NAT) in hospitalized patients in Uganda. Sera from 380 consecutive patients collected and tested for HBsAg and anti-HIV in Kampala, Uganda by RSA were sent frozen to Dallas for EIA including HBsAg, total anti-hepatitis B core, hepatitis B e antigen, and anti-HIV. NAT was performed on all HBsAg-positives and on a random sample of 102 patients that were HBsAg-negative by both assays. Overall, 31 (8%) were HBsAg positive by RSA while 50 (13%) were HBsAg-positive by EIA; 26 were concordant between the two assays. Of 55 HBsAg-positive patients, nearly all showed detectable serum hepatitis B virus (HBV) DNA by bDNA (46) or PCR (4) assay. The 26 patients who were HBsAg positive by both EIA and RSA had significantly higher median serum HBV DNA levels than the 24 patients who were HBsAg positive by EIA alone. An additional 12/102 (12%) HBsAg negative patients had very low serum HBV DNA levels by NAT. Several differences in expected results of serologic testing were observed in this large series of African patients. RSA HBsAg testing is less sensitive than EIA; even EIA failed to detect all HBV DNA positive sera. A more complex testing protocol than RSA alone will be needed in Africa to improve patient care. (c) 2010 Wiley-Liss, Inc.

  4. Prospective multicentre evaluation of the direct nitrate reductase assay for the rapid detection of extensively drug-resistant tuberculosis.

    Science.gov (United States)

    Martin, Anandi; Imperiale, Belen; Ravolonandriana, Pascaline; Coban, Ahmet Yilmaz; Akgunes, Alper; Ikram, Aamer; Satti, Luqman; Odoun, Mathieu; Pandey, Pooja; Mishra, Manvi; Affolabi, Dissou; Singh, Urvashi; Rasolofo, Voahangy; Morcillo, Nora; Vandamme, Peter; Palomino, Juan Carlos

    2014-02-01

    To perform a multicentre study evaluating the performance of the direct nitrate reductase assay (NRA) for the detection of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis in sputum samples. The study was conducted in six laboratories performing tuberculosis diagnosis that were located in six different countries. The NRA was performed directly on sputum samples in parallel with the reference method used at each site. Detection of resistance was performed for rifampicin, isoniazid, ofloxacin and kanamycin. Excellent agreement was obtained for all drugs tested at the majority of sites. The accuracy was 93.7%-100% for rifampicin, 88.2%-100% for isoniazid, 94.6%-100% for ofloxacin and 100% for kanamycin. The majority of NRA results were available at day 21 for sites 1, 2 and 5. Site 3 had a turnaround time of 13.9 days, at site 4 it was 18.4 days and at site 6 it was 16.2 days. The contamination rate ranged between 2.5% and 12%. Rapid detection of drug resistance by the direct NRA on sputum smear-positive samples was accurate and easy to implement in clinical diagnostic laboratories, making it a good alternative for rapid screening for MDR and XDR tuberculosis.

  5. Evaluation of a pan-serotype point-of-care rapid diagnostic assay for accurate detection of acute dengue infection.

    Science.gov (United States)

    Vivek, Rosario; Ahamed, Syed Fazil; Kotabagi, Shalini; Chandele, Anmol; Khanna, Ira; Khanna, Navin; Nayak, Kaustuv; Dias, Mary; Kaja, Murali-Krishna; Shet, Anita

    2017-03-01

    The catastrophic rise in dengue infections in India and globally has created a need for an accurate, validated low-cost rapid diagnostic test (RDT) for dengue. We prospectively evaluated the diagnostic performance of NS1/IgM RDT (dengue day 1) using 211 samples from a pediatric dengue cohort representing all 4 serotypes in southern India. The dengue-positive panel consisted of 179 dengue real-time polymerase chain reaction (RT-PCR) positive samples from symptomatic children. The dengue-negative panel consisted of 32 samples from dengue-negative febrile children and asymptomatic individuals that were negative for dengue RT-PCR/NS1 enzyme-linked immunosorbent assay/IgM/IgG. NS1/IgM RDT sensitivity was 89.4% and specificity was 93.8%. The NS1/IgM RDT showed high sensitivity throughout the acute phase of illness, in primary and secondary infections, in different severity groups, and detected all 4 dengue serotypes, including coinfections. This NS1/IgM RDT is a useful point-of-care assay for rapid and reliable diagnosis of acute dengue and an excellent surveillance tool in our battle against dengue. Copyright © 2016. Published by Elsevier Inc.

  6. Assessment of early thromboelastometric variables from extrinsically activated assays with and without aprotinin for rapid detection of fibrinolysis.

    Science.gov (United States)

    Dirkmann, Daniel; Görlinger, Klaus; Peters, Jürgen

    2014-09-01

    Although thromboelastometry (ROTEM®) and thrombelastography can be used for bedside diagnosis of fibrinolysis, the time needed for detection is often prolonged. Since untreated fibrinolysis can result in consumption of coagulation factors and bleeding, early diagnosis and decision making are desirable. Accordingly, we assessed ROTEM variables from extrinsically activated assays with (APTEM) and without (EXTEM) addition of aprotinin for their ability to rapidly identify fibrinolysis. Specifically, we tested the hypotheses that prolonged clotting time, clot formation time, low clot firmness (at 5, 10, 15, and 20 minutes, designated A5, A10, A15, and A20, respectively), low maximum clot firmness (MCF) in EXTEM assays, and differences in these variables from parallel APTEM and EXTEM assays (designated as Δvariables) predict fibrinolysis. Data from 411 thromboelastometric measurements (obtained from 352 patients) with fibrinolysis and from 2537 measurements without fibrinolysis (obtained from 1605 patients) were assessed and analyzed using receiver operating characteristics. Data were analyzed as a pooled fibrinolysis cohort, and subanalyses were performed from sets assigned to categories of fibrinolysis related to the timing of thrombus lysis (i.e., a decrease of clot firmness to fibrinolysis. AUCs were compared to identify the variable providing the best predictive association with fibrinolysis. As a secondary end point, optimum cutoff values at the point estimate corresponding to the greatest Youden index were calculated along with the respective sensitivities and specificities. In the pooled cohort, clot formation time (AUC: 0.652 [0.016]), α-angle (AUC: 0.675 [0.015]), A5 (AUC: 0.718 [0.013]), A10 (AUC: 0.734 [0.0.13]), A15 (AUC: 0.752 [0.013]), A20 (AUC: 0.771 [0.013]), and MCF (AUC: 0.799 [0.012]) predicted fibrinolysis. Fibrinolysis was also predicted by ΔA15 (AUC: 0.675 [0.016]), ΔA20 (AUC: 0.719 [0.015]), and ΔMCF (AUC: 0.812 [0.013]). AUCs increased in

  7. 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.

  8. Optimization of real-time PCR assay for rapid and sensitive detection of eubacterial 16S ribosomal DNA in platelet concentrates.

    NARCIS (Netherlands)

    Mohammadi, T.; Reesink, H.W.; Vandenbroucke-Grauls, C.M.J.E.; Savelkoul, P.H.M.

    2003-01-01

    A real-time PCR assay was developed for rapid detection of eubacterial 16S ribosomal DNA in platelet concentrates. The sensitivity of this assay can be hampered by contaminating DNA in the PCR reagents. Digestion of the PCR reagents with Sau3AI prior to PCR amplification was effective in eliminating

  9. Rapid multiplex detection of 10 foodborne pathogens with an up-converting phosphor technology-based 10-channel lateral flow assay.

    Science.gov (United States)

    Zhao, Yong; Wang, Haoran; Zhang, Pingping; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Yang, Ruifu; Wang, Chengbin; Zhou, Lei

    2016-02-17

    The rapid high-throughput detection of foodborne pathogens is essential in controlling food safety. In this study, a 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) assay was established for the rapid and simultaneous detection of 10 epidemic foodborne pathogens. Ten different single-target UPT-LF strips were developed and integrated into one TC-UPT-LF disc with optimization. Without enrichment the TC-UPT-LF assay had a detection sensitivity of 10(4) CFU mL(-1) or 10(5) CFU mL(-1) for each pathogen, and after sample enrichment it was 10 CFU/0.6 mg. The assay also showed good linearity, allowing quantitative detection, with a linear fitting coefficient of determination (R(2)) of 0.916-0.998. The 10 detection channels did not cross-react, so multiple targets could be specifically detected. When 279 real food samples were tested, the assay was highly consistent (100%) with culture-based methods. The results for 110 food samples artificially contaminated with single or multiple targets showed a high detection rate (≥ 80%) for most target bacteria. Overall, the TC-UPT-LF assay allows the rapid, quantitative, and simultaneous detection of 10 kinds of foodborne pathogens within 20 min, and is especially suitable for the rapid detection and surveillance of foodborne pathogens in food and water.

  10. Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples.

    Science.gov (United States)

    Zhou, Liqing; Jones, Claire; Gibani, Malick M; Dobinson, Hazel; Thomaides-Brears, Helena; Shrestha, Sonu; Blohmke, Christoph J; Darton, Thomas C; Pollard, Andrew J

    2016-01-01

    Enteric fever remains an important cause of morbidity in many low-income countries and Salmonella Paratyphi A has emerged as the aetiological agent in an increasing proportion of cases. Lack of adequate diagnostics hinders early diagnosis and prompt treatment of both typhoid and paratyphoid but development of assays to identify paratyphoid has been particularly neglected. Here we describe the development of a rapid and sensitive blood culture PCR method for detection of Salmonella Paratyphi A from blood, potentially allowing for appropriate diagnosis and antimicrobial treatment to be initiated on the same day. Venous blood samples from volunteers experimentally challenged orally with Salmonella Paratyphi A, who subsequently developed paratyphoid, were taken on the day of diagnosis; 10 ml for quantitative blood culture and automated blood culture, and 5 ml for blood culture PCR. In the latter assay, bacteria were grown in tryptone soy broth containing 2.4% ox bile and micrococcal nuclease for 5 hours (37°C) before bacterial DNA was isolated for PCR detection targeting the fliC-a gene of Salmonella Paratyphi A. An optimized broth containing 2.4% ox bile and micrococcal nuclease, as well as a PCR test was developed for a blood culture PCR assay of Salmonella Paratyphi A. The volunteers diagnosed with paratyphoid had a median bacterial burden of 1 (range 0.1-6.9) CFU/ml blood. All the blood culture PCR positive cases where a positive bacterial growth was shown by quantitative blood culture had a bacterial burden of ≥ 0.3 CFU/ ml blood. The blood culture PCR assay identified an equal number of positive cases as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood), but utilized only half the volume of specimens. The blood culture PCR method for detection of Salmonella Paratyphi A can be completed within 9 hours and offers the potential for same-day diagnosis of enteric fever. Using 5 ml blood, it exhibited a lower limit of detection equal to 0.3 CFU

  11. Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples.

    Directory of Open Access Journals (Sweden)

    Liqing Zhou

    Full Text Available Enteric fever remains an important cause of morbidity in many low-income countries and Salmonella Paratyphi A has emerged as the aetiological agent in an increasing proportion of cases. Lack of adequate diagnostics hinders early diagnosis and prompt treatment of both typhoid and paratyphoid but development of assays to identify paratyphoid has been particularly neglected. Here we describe the development of a rapid and sensitive blood culture PCR method for detection of Salmonella Paratyphi A from blood, potentially allowing for appropriate diagnosis and antimicrobial treatment to be initiated on the same day.Venous blood samples from volunteers experimentally challenged orally with Salmonella Paratyphi A, who subsequently developed paratyphoid, were taken on the day of diagnosis; 10 ml for quantitative blood culture and automated blood culture, and 5 ml for blood culture PCR. In the latter assay, bacteria were grown in tryptone soy broth containing 2.4% ox bile and micrococcal nuclease for 5 hours (37°C before bacterial DNA was isolated for PCR detection targeting the fliC-a gene of Salmonella Paratyphi A.An optimized broth containing 2.4% ox bile and micrococcal nuclease, as well as a PCR test was developed for a blood culture PCR assay of Salmonella Paratyphi A. The volunteers diagnosed with paratyphoid had a median bacterial burden of 1 (range 0.1-6.9 CFU/ml blood. All the blood culture PCR positive cases where a positive bacterial growth was shown by quantitative blood culture had a bacterial burden of ≥ 0.3 CFU/ ml blood. The blood culture PCR assay identified an equal number of positive cases as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood, but utilized only half the volume of specimens.The blood culture PCR method for detection of Salmonella Paratyphi A can be completed within 9 hours and offers the potential for same-day diagnosis of enteric fever. Using 5 ml blood, it exhibited a lower limit of detection

  12. Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples

    Science.gov (United States)

    Zhou, Liqing; Jones, Claire; Gibani, Malick M.; Dobinson, Hazel; Thomaides-Brears, Helena; Shrestha, Sonu; Blohmke, Christoph J.; Darton, Thomas C.; Pollard, Andrew J.

    2016-01-01

    Background Enteric fever remains an important cause of morbidity in many low-income countries and Salmonella Paratyphi A has emerged as the aetiological agent in an increasing proportion of cases. Lack of adequate diagnostics hinders early diagnosis and prompt treatment of both typhoid and paratyphoid but development of assays to identify paratyphoid has been particularly neglected. Here we describe the development of a rapid and sensitive blood culture PCR method for detection of Salmonella Paratyphi A from blood, potentially allowing for appropriate diagnosis and antimicrobial treatment to be initiated on the same day. Methods Venous blood samples from volunteers experimentally challenged orally with Salmonella Paratyphi A, who subsequently developed paratyphoid, were taken on the day of diagnosis; 10 ml for quantitative blood culture and automated blood culture, and 5 ml for blood culture PCR. In the latter assay, bacteria were grown in tryptone soy broth containing 2.4% ox bile and micrococcal nuclease for 5 hours (37°C) before bacterial DNA was isolated for PCR detection targeting the fliC-a gene of Salmonella Paratyphi A. Results An optimized broth containing 2.4% ox bile and micrococcal nuclease, as well as a PCR test was developed for a blood culture PCR assay of Salmonella Paratyphi A. The volunteers diagnosed with paratyphoid had a median bacterial burden of 1 (range 0.1–6.9) CFU/ml blood. All the blood culture PCR positive cases where a positive bacterial growth was shown by quantitative blood culture had a bacterial burden of ≥ 0.3 CFU/ ml blood. The blood culture PCR assay identified an equal number of positive cases as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood), but utilized only half the volume of specimens. Conclusions The blood culture PCR method for detection of Salmonella Paratyphi A can be completed within 9 hours and offers the potential for same-day diagnosis of enteric fever. Using 5 ml blood, it exhibited a

  13. Multicenter evaluation of crystal violet decolorization assay (CVDA) for rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis.

    Science.gov (United States)

    Coban, Ahmet Yilmaz; Akbal, Ahmet Ugur; Bicmen, Can; Albay, Ali; Sig, Ali Korhan; Uzun, Meltem; Selale, Deniz Sertel; Ozkutuk, Nuri; Surucuoglu, Suheyla; Albayrak, Nurhan; Ucarman, Nilay; Ozkutuk, Aydan; Esen, Nuran; Ceyhan, Ismail; Ozyurt, Mustafa; Bektore, Bayhan; Aslan, Gonul; Delialioğlu, Nuran; Alp, Alpaslan

    2016-12-16

    The aim of this multicenter study was to evaluate the performance of the crystal violet decolorization assay (CVDA) for detection of multidrug resistant tuberculosis (MDR-TB). This study was performed in 11 centers in two phases. A total of 156 isolates were tested for INH and RIF resistance. In the phase I, 106 clinical isolates were tested in the Center 1-7. In the phase 2, 156 clinical isolates were tested in the center 1-6, center 8-11. Eighty six of 156 tested isolates were the same in phase I. Agreements were 96.2-96.8% for INH and 98.1-98.7% for RIF in the phase I-II, respectively. Mean time to obtain the results in the phase I was 14.3 ± 5.4 days. In the phase II, mean time to obtain the results was 11.6 ± 3.5 days. Test results were obtained within 14days for 62.3% (66/106) of isolates in the phase I and 81.4% (127/156) of isolates in the phase II. In conclusion, CVDA is rapid, reliable, inexpensive, and easy to perform for rapid detection of MDR-TB isolates. In addition, it could be adapted for drug susceptibility testing with all drugs both in developed and developing countries.

  14. A sensitive and specific lateral flow assay for rapid detection of antibodies against glycoprotein B of Aujeszky's disease virus.

    Science.gov (United States)

    Vrublevskaya, Veronika V; Afanasyev, Vladimir N; Grinevich, Andrey A; Skarga, Yuri Y; Gladyshev, Pavel P; Ibragimova, Sagila A; Krylsky, Dmitry V; Dezhurov, Sergey V; Morenkov, Oleg S

    2017-11-01

    A direct double antibody lateral flow assay (DDA-gB-LFA) for the detection of antibodies against the glycoprotein B (gB) of Aujeszky's disease virus (ADV) in swine sera was developed. A native ADV gB was used for the preparation of a conjugate with colloidal gold particles and the immobilization on the strip membrane. The gB purified from ADV virions by immunoaffinity chromatography retained its native epitope structure after adsorption on the nitrocellulose membrane and the surface of colloidal gold particles. The diagnostic specificity and sensitivity of the DDA-gB-LFA were evaluated using 236 field swine sera. The diagnostic specificity and sensitivity of the DDA-gB-LFA compared to a commercially available gB-based ELISA were 98.0% and 98.6%, respectively, when determined with the use of the reader-detection mode, and 98.0% and 93.5%, respectively, when determined using visual detection. The DDA-gB-LFA provides a rapid, sensitive, and specific determination of ADV gB-directed antibodies in sera and can be used for the detection of ADV-exposed swine. Copyright © 2017. Published by Elsevier B.V.

  15. Evaluation of three rapid assays for detection of Clostridium difficile toxin A and toxin B in stool specimens.

    Science.gov (United States)

    Rüssmann, H; Panthel, K; Bader, R-C; Schmitt, C; Schaumann, R

    2007-02-01

    Diagnosis of Clostridium difficile-associated disease continues to be difficult for clinical microbiology laboratories. The aim of this study was to evaluate the performance of three enzyme immunoassays for detection of C. difficile toxins A and B: the recently marketed rapid enzyme immunoassay Ridascreen Clostridium difficile Toxin A/B (R-Biopharm, Darmstadt, Germany) and two established enzyme immunoassays, the C. difficile Tox A/B II Assay (TechLab, Blacksburg, VA, USA) and the ProSpecT C. difficile Toxin A/B Microplate Assay (Remel, Lenexa, KS, USA). Stool specimens (n = 383) from patients with a clinical diagnosis of antibiotic-associated diarrhea were examined by these three enzyme immunoassays and were additionally cultured for C. difficile on selective agar. Samples giving discordant enzyme immunoassay results underwent confirmatory testing by tissue culture cytotoxin B assay and by PCR for toxin A (tcdA) and toxin B (tcdB) genes from C. difficile. Using the criteria adopted for this study, 60 (15.7%) samples tested positive for toxins A and/or B. Sensitivity and specificity of the enzyme immunoassays were, respectively, 88.3 and 100% for the TechLab enzyme immunoassay, 91.7 and 100% for the R-Biopharm enzyme immunoassay, and 93.3 and 100% for the Remel enzyme immunoassay. The differences between these results are statistically not significant (p > 0.05). The results show that all three enzyme immunoassays are acceptable tests for the detection of C. difficile toxins A and B directly in fecal specimens or in toxigenic cultures.

  16. Rapid Detection and Classification of Salmonella enterica Shedding in Feedlot Cattle Utilizing the Roka Bioscience Atlas Salmonella Detection Assay for the Analysis of Rectoanal Mucosal Swabs.

    Science.gov (United States)

    Chaney, W Evan; Agga, Getahun E; Nguyen, Scott V; Arthur, Terrance M; Bosilevac, Joseph M; Dreyling, Erin; Rishi, Anantharama; Brichta-Harhay, Dayna

    2017-10-01

    With an increasing focus on preharvest food safety, rapid methods are required for the detection and quantification of foodborne pathogens such as Salmonella enterica in beef cattle. We validated the Atlas Salmonella Detection Assay (SEN), a nucleic acid amplification technology that targets Salmonella rRNA, for the qualitative detection of S. enterica with sample enrichment using immunomagnetic separation as a reference test, and we further evaluated its accuracy to predict pathogen load using SEN signal-to-cutoff (SCO) values from unenriched samples to classify animals as high or nonhigh shedders. Rectoanal mucosal swabs (RAMS) were collected from 238 beef cattle from five cohorts located in the Midwest or southern High Plains of the United States between July 2015 and April 2016. Unenriched RAMS samples were used for the enumeration and SEN SCO analyses. Enriched samples were tested using SEN and immunomagnetic separation methods for the detection of Salmonella. The SEN method was 100% sensitive and specific for the detection of Salmonella from the enriched RAMS samples. A SEN SCO value of 8, with a sensitivity of 93.5% and specificity of 94.3%, was found to be an optimum cutoff value for classifying animals as high or nonhigh shedders from the unenriched RAMS samples. The SEN assay is a rapid and reliable method for the qualitative detection and categorization of the shedding load of Salmonella from RAMS in feedlot cattle.

  17. Rapid assay for simultaneous detection and differentiation of immunoglobulin G antibodies to human immunodeficiency virus type 1 (HIV-1) group M, HIV-1 group O, and HIV-2.

    Science.gov (United States)

    Vallari, A S; Hickman, R K; Hackett, J R; Brennan, C A; Varitek, V A; Devare, S G

    1998-12-01

    A rapid immunodiagnostic test that detects and discriminates human immunodeficiency virus (HIV) infections on the basis of viral type, HIV type 1 (HIV-1) group M, HIV-1 group O, or HIV-2, was developed. The rapid assay for the detection of HIV (HIV rapid assay) was designed as an instrument-free chromatographic immunoassay that detects immunoglobulin G (IgG) antibodies to HIV. To assess the performance of the HIV rapid assay, 470 HIV-positive plasma samples were tested by PCR and/or Western blotting to confirm the genotype of the infecting virus. These samples were infected with strains that represented a wide variety of HIV strains including HIV-1 group M (subtypes A through G), HIV-1 group O, and HIV-2 (subtypes A and B). The results showed that the HIV genotype identity established by the rapid assay reliably (469 of 470 samples) correlates with the HIV genotype identity established by PCR or Western blotting. A total of 879 plasma samples were tested for IgG to HIV by a licensed enzyme immunoassay (EIA) (470 HIV-positive samples and 409 HIV-negative samples). When they were tested by the rapid assay, 469 samples were positive and 410 were negative (99.88% agreement). Twelve seroconversion panels were tested by both the rapid assay and a licensed EIA. For nine panels identical results were obtained by the two assays. For the remaining three panels, the rapid assay was positive one bleed later in comparison to the bleed at which the EIA was positive. One hundred three urine samples, including 93 urine samples from HIV-seropositive individuals and 10 urine samples from seronegative individuals, were tested by the rapid assay. Ninety-one of the ninety-three urine samples from HIV-seropositive individuals were found to be positive by the rapid assay. There were no false-positive results (98.05% agreement). Virus in all urine samples tested were typed as HIV-1 group M. These results suggest that a rapid assay based on the detection of IgG specific for selected

  18. 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.

  19. 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.

  20. Economic Impact of a New Rapid PCR Assay for Detecting Influenza Virus in an Emergency Department and Hospitalized Patients.

    Science.gov (United States)

    Soto, Marcelo; Sampietro-Colom, Laura; Vilella, Anna; Pantoja, Efraín; Asenjo, María; Arjona, Ruth; Hurtado, Juan Carlos; Trilla, Antoni; Alvarez-Martínez, Míriam José; Mira, Aurea; Vila, Jordi; Marcos, María Angeles

    2016-01-01

    Seasonal influenza causes significant morbidity and mortality and has a substantial economic impact on the healthcare system. The main objective of this study was to compare the cost per patient for a rapid commercial PCR assay (Xpert® Flu) with an in-house real-time PCR test for detecting influenza virus. Community patients with influenza like-illness attending the Emergency Department (ED) as well as hospitalized patients in the Hospital Clínic of Barcelona were included. Costs were evaluated from the perspective of the hospital considering the use of resources directly related to influenza testing and treatment. For the purpose of this study, 366 and 691 patients were tested in 2013 and 2014, respectively. The Xpert® Flu test reduced the mean waiting time for patients in the ED by 9.1 hours and decreased the mean isolation time of hospitalized patients by 23.7 hours. This was associated with a 103€ (or about $113) reduction in the cost per patient tested in the ED and 64€ ($70) per hospitalized patient. Sensitivity analyses showed that Xpert® Flu is likely to be cost-saving in hospitals with different contexts and prices.

  1. Development of a rapid, simple, and specific real-time PCR assay for detection of pseudorabies viral DNA in domestic swine herds.

    Science.gov (United States)

    Sayler, Katherine A; Bigelow, Troy; Koster, Leo G; Swenson, Sabrina; Bounds, Courtney; Hernández, Felipe; Wisely, Samantha M

    2017-07-01

    Despite successful eradication of pseudorabies virus (PRV) from the commercial pig industry in the United States in 2004, large populations of feral swine in certain regions act as wildlife reservoirs for the virus. Given the threat of reintroduction of the virus into domestic herds, a rapid, reliable, easily implemented assay is needed for detection of PRV. Although a real-time PCR (rtPCR) assay exists, improvements in rtPCR technology and a greater understanding of the diversity of PRV strains worldwide require an assay that would be easier to implement, more cost effective, and more specific. We developed a single-tube, rapid rtPCR that is capable of detecting 10 copies of PRV glycoprotein B ( gB) DNA per 20-µL total volume reaction. The assay did not produce a false-positive in samples known to be negative for the virus. The assay was negative for genetically similar herpesviruses and other porcine viruses. Our assay is a highly specific and sensitive assay that is also highly repeatable and reproducible. The assay should be a useful tool for early detection of PRV in pigs in the case of a suspected introduction or outbreak situation.

  2. Rapid detection of drug resistance and mutational patterns of extensively drug-resistant strains by a novel GenoType® MTBDRsl assay.

    Science.gov (United States)

    Singh, A K; Maurya, A K; Kant, S; Umrao, J; Kushwaha, R A S; Nag, V L; Dhole, T N

    2013-01-01

    The emergence of extensively drug-resistant tuberculosis (XDR-TB) is a major concern in the India. The burden of XDR-TB is increasing due to inadequate monitoring, lack of proper diagnosis, and treatment. The GenoType ® Mycobacterium tuberculosis drug resistance second line (MTBDRsl) assay is a novel line probe assay used for the rapid detection of mutational patterns conferring resistance to XDR-TB. The aim of this study was to study the rapid detection of drug resistance and mutational patterns of the XDR-TB by a novel GenoType ® MTBDRsl assay. We evaluated 98 multidrug-resistant (MDR) M. tuberculosis isolates for second line drugs susceptibility testing by 1% proportion method (BacT/ALERT 3D system) and GenoType ® MTBDRsl assay for rapid detection of conferring drug resistance to XDR-TB. A total of seven (17.4%) were identified as XDR-TB by using standard phenotypic method. The concordance between phenotypic and GenoType ® MTBDRsl assay was 91.7-100% for different antibiotics. The sensitivity and specificity of the MTBDRsl assay were 100% and 100% for aminoglycosides; 100% and 100% for fluoroquinolones; 91.7% and 100% for ethambutol. The most frequent mutations and patterns were gyrA MUT1 (A90V) in seven (41.2%) and gyrA + WT1-3 + MUT1 in four (23.5%); rrs MUT1 (A1401G) in 11 (64.7%), and rrs WT1-2 + MUT1 in eight (47.1%); and embB MUT1B (M306V) in 11 (64.7%) strains. These data suggest that the GenoType ® MTBDRsl assay is rapid, novel test for detection of resistance to second line anti-tubercular drugs. This assay provides additional information about the frequency and mutational patterns responsible for XDR-TB resistance.

  3. Modified RS-LAMP assay and use of lateral flow devices for rapid detection of Leifsonia xyli subsp. xyli.

    Science.gov (United States)

    Naidoo, N; Ghai, M; Moodley, K; Mkize, L; Martin, L; McFarlane, S; Rutherford, S

    2017-12-01

    Ratoon stunt (RS) caused by bacterium Leifsonia xyli subsp. xyli (Lxx) results in substantial yield losses in sugarcane (Saccharum sp. L. hybrid). Since RS does not produce reliable symptoms in the field, laboratory-based techniques are necessary for detection. Loop-mediated isothermal amplification (LAMP) assay overcomes the limitations of laboratory-based techniques which are costly, time consuming and cannot be used for near-field detection. A sensitive LAMP assay was developed to detect Lxx at 65°C in 30 min. However, carry-over contamination affected the reliability of the assay. In the present study, contaminants were successfully eliminated by incorporation of uracil nucleoside glycosylase (1 U μl -1 ) into the LAMP assay and incubation for 10 min at 37°C. To avoid the use of colorimetric reagents, lateral flow devices were successfully used for the detection of LAMP products and were equally sensitive to detection by agarose gel electrophoresis. The use of exudate from leaf sheath discs as an alternate template for the LAMP assay was found to be less sensitive than xylem sap. The preprepared master mix could be stored for up to 4 months at -20°C without any reduction in performance. These changes make the assay suitable for near-field detection in laboratories with basic facilities. This study presents a modified loop-mediated isothermal amplification (LAMP) assay for the detection of Leifsonia xyli subsp. xyli. Modifications include incorporation of uracil nucleoside glycosylase to eliminate carry-over contamination and substitution of colorimetric detection for the use of lateral flow devices. LAMP master mix was preprepared and was stably stored up to 4 months at -20°C. Sugarcane leaf sheaths worked well as a substitute to xylem sap as template, although the sensitivity was lower. These modifications allow the assay to be conducted without contamination concerns and reduction in set up time, making it ideal for near-field diagnosis. © 2017

  4. Rapid detection and identification of Brugia malayi, B. pahangi, and Dirofilaria immitis by high-resolution melting assay.

    Science.gov (United States)

    Wongkamchai, Sirichit; Monkong, Nuntiya; Mahannol, Pakpimom; Taweethavonsawat, Piyanan; Loymak, Sumat; Foongladda, Suporn

    2013-01-01

    Human lymphatic filariasis is caused by filarial worms such as Brugia malayi for which the major reservoir is domestic cats. However, domestic cats or dogs also carry nonhuman filaria such as Brugia pahangi and Dirofilaria immitis. We have developed a single-tube, real-time PCR with a high-resolution melting (HRM) analysis assay for detection and identification of B. malayi, B. pahangi, and D. immitis in blood samples. The designated primer pair in the PCR can amplify a 114-bp region of mitochondrial 12S rRNA genes of these filarial worms. Subsequently, the HRM assay showed a specific melting temperature for each species. The assay showed the highest sensitivity and specificity in comparison with DNA sequences after assessment with 34 cat and 14 dog blood samples. This assay could be helpful for epidemiological studies of reservoirs and vectors.

  5. Microscopic Observation Drug Susceptibility Assay for Rapid Diagnosis of Lymph Node Tuberculosis and Detection of Drug Resistance.

    Science.gov (United States)

    Kirwan, Daniela E; Ugarte-Gil, Cesar; Gilman, Robert H; Caviedes, Luz; Rizvi, Hasan; Ticona, Eduardo; Chavez, Gonzalo; Cabrera, José Luis; Matos, Eduardo D; Evans, Carlton A; Moore, David A J; Friedland, Jon S

    2016-01-01

    In this study, 132 patients with lymphadenopathy were investigated. Fifty-two (39.4%) were diagnosed with tuberculosis (TB). The microscopic observation drug susceptibility (MODS) assay provided rapid (13 days), accurate diagnosis (sensitivity, 65.4%) and reliable drug susceptibility testing (DST). Despite its lower sensitivity than that of other methods, its faster results and simultaneous DST are advantageous in resource-poor settings, supporting the incorporation of MODS into diagnostic algorithms for extrapulmonary TB. Copyright © 2015 Kirwan et al.

  6. 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...

  7. Field-Deployable Reverse Transcription-Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus.

    Science.gov (United States)

    Ambagala, A; Fisher, M; Goolia, M; Nfon, C; Furukawa-Stoffer, T; Ortega Polo, R; Lung, O

    2017-10-01

    Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, which can decimate the livestock industry and economy of countries previously free of this disease. Rapid detection of foot-and-mouth disease virus (FMDV) is critical to containing an FMD outbreak. Availability of a rapid, highly sensitive and specific, yet simple and field-deployable assay would support local decision-making during an FMDV outbreak. Here we report validation of a novel reverse transcription-insulated isothermal PCR (RT-iiPCR) assay that can be performed on a commercially available, compact and portable POCKIT™ analyser that automatically analyses data and displays '+' or '-' results. The FMDV RT-iiPCR assay targets the 3D region of the FMDV genome and was capable of detecting 9 copies of in vitro-transcribed RNA standard with 95% confidence. It accurately identified 63 FMDV strains belonging to all seven serotypes and showed no cross-reactivity with viruses causing similar clinical diseases in cloven-hoofed animals. The assay was able to identify FMDV RNA in multiple sample types including oral, nasal and lesion swabs, epithelial tissue suspensions, vesicular and oral fluid samples, even before the appearance of clinical signs. Clinical sensitivity of the assay was comparable or slightly higher than the laboratory-based real-time RT-PCR assay in use. The assay was able to detect FMDV RNA in vesicular fluid samples without nucleic acid extraction. For RNA extraction from more complex sample types, a commercially available taco™ mini transportable magnetic bead-based, automated extraction system was used. This assay provides a potentially useful field-deployable diagnostic tool for rapid detection of FMDV in an outbreak in FMD-free countries or for routine diagnostics in endemic countries with less structured laboratory systems. © 2016 Her Majesty the Queen in Right of Canada.

  8. 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.

  9. Rapid Detection of Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    David Perlin

    2005-08-14

    Pathogen identification is a crucial first defense against bioterrorism. A major emphasis of our national biodefense strategy is to establish fast, accurate and sensitive assays for diagnosis of infectious diseases agents. Such assays will ensure early and appropriate treatment of infected patients. Rapid diagnostics can also support infection control measures, which monitor and limit the spread of infectious diseases agents. Many select agents are highly transmissible in the early stages of disease, and it is critical to identify infected patients and limit the risk to the remainder of the population and to stem potential panic in the general population. Nucleic acid-based molecular approaches for identification overcome many of the deficiencies associated with conventional culture methods by exploiting both large- and small-scale genomic differences between organisms. PCR-based amplification of highly conserved ribosomal RNA (rRNA) genes, intergenic sequences, and specific toxin genes is currently the most reliable approach for bacterial, fungal and many viral pathogenic agents. When combined with fluorescence-based oligonucleotide detection systems, this approach provides real-time, quantitative, high fidelity analysis capable of single nucleotide allelic discrimination (4). These probe systems offer rapid turn around time (<2 h) and are suitable for high throughput, automated multiplex operations that are critical for clinical diagnostic laboratories. In this pilot program, we have used molecular beacon technology invented at the Public health Research Institute to develop a new generation of molecular probes to rapidly detect important agents of infectious diseases. We have also developed protocols to rapidly extract nucleic acids from a variety of clinical specimen including and blood and tissue to for detection in the molecular assays. This work represented a cooperative research development program between the Kramer-Tyagi/Perlin labs on probe development

  10. Analytical performance of the Alere™ i Influenza A&B assay for the rapid detection of influenza viruses.

    Science.gov (United States)

    Riazzo, Cristina; Pérez-Ruiz, Mercedes; Sanbonmatsu-Gámez, Sara; Pedrosa-Corral, Irene; Gutiérrez-Fernández, José; Navarro-Marí, José-María

    The analytical performance of the new Alere™ i Influenza A&B kit (AL-Flu) assay, based on isothermal nucleic acids amplification, was evaluated and compared with an antigen detection method, SD Bioline Influenza Virus Antigen Test (SDB), and an automated real-time RT-PCR, Simplexa™ Flu A/B & VRS Direct assay (SPX), for detection of influenza viruses. An "in-house" RT-PCR was used as the reference method. Sensitivity of AL-Flu, SDB, and SPX was 71.7%, 34.8%, and 100%, respectively. Specificity was 100% for all techniques. The turnaround time was 13min for AL-Flu, 15min for SDB, and 75min for SPX. The Alere™ i Influenza A&B assay is an optimal point-of-care assay for influenza diagnosis in clinical emergency settings, and is more sensitive and specific than antigen detection methods. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  11. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  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. Evaluation of genotype MTBDRplus assay for rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis clinical isolates from Pakistan

    Directory of Open Access Journals (Sweden)

    Hasnain Javed

    2016-01-01

    Conclusions: As evidenced in this study, the major concern with the GenoType MTBDRplus assay were false negative results. In comparison to conventional drug susceptibility testing, the assay was unable to detect 30 (30/100; 30% strains resistant to INH and 23 (23/100; 23% strains resistant to RMP. The GenoType MTBDRplus failed to identify 38 MDR (38/100; 38% strains. Resistance in those strains probably originate from mutations in other codons and/or genes than those covered by the test. For detecting INH and RMP resistance in TB cases, especially in high TB incidence countries, such as Pakistan, molecular approaches should still be a complement rather than areplacement to conventional drug susceptibility testing.

  15. 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.

  16. Performance of the Cobas® Influenza A/B Assay for Rapid Pcr-Based Detection of Influenza Compared to Prodesse ProFlu+ and Viral Culture

    Science.gov (United States)

    Chen, L.; Tian, Y.; Chen, S.; Liesenfeld, O.

    2015-01-01

    Rapid and accurate diagnosis of influenza is important for patient management and infection control. We determined the performance of the cobas® Influenza A/B assay, a rapid automated nucleic acid assay performed on the cobas® Liat System for qualitative detection of influenza A and influenza B from nasopharyngeal (NP) swab specimens. Retrospective frozen and prospectively collected NP swabs from patients with signs and symptoms of influenza collected in universal transport medium (UTM) were tested at multiple sites including CLIA-waived sites using the cobas® Influenza A/B assay. Results were compared to the Prodesse Pro-Flu+ assay and to viral culture. Compared to the Prodesse ProFlu+ Assay, sensitivities of the cobas® Influenza A/B assay for influenza A and B were 97.7 and 98.6%, respectively; specificity was 99.2 and 99.4%. Compared to viral culture, the cobas® Influenza A/B assay showed sensitivities of 97.5 and 96.9% for influenza virus A and B, respectively; specificities were 97.9% for both viruses. Polymerase chain reaction (PCR)/sequencing showed that the majority of viral culture negative but cobas® Influenza A/B positive results were true positive results, indicating that the cobas® Influenza A/B assay has higher sensitivity compared to viral culture. In conclusion, the excellent accuracy, rapid time to result, and remarkable ease of use make the cobas® Influenza A/B nucleic acid assay for use on the cobas® Liat System a highly suitable point-of-care solution for the management of patients with suspected influenza A and B infection. PMID:26716012

  17. Evaluation of the accuracy of the microplate Alamar Blue assay for rapid detection of MDR-TB in Peru.

    Science.gov (United States)

    Chauca, J A; Palomino, J-C; Guerra, H

    2007-07-01

    Tuberculosis control is hampered by the widespread increase in multidrug resistance. Rapid drug susceptibility testing would greatly aid in the adequate treatment of the disease. This study evaluates the usefulness of the colorimetric method using Alamar Blue for the rapid detection of resistance to rifampicin and isoniazid in 63 clinical isolates of Mycobacterium tuberculosis in Peru. Results obtained by receiver operating characteristic curve analysis and measures of gain in certainty showed greater diagnostic accuracy than with the gold standard, the proportion method on Löwenstein-Jensen medium.

  18. Evaluation of the novel DiaSorin LIAISON(®)Campylobacter assay for the rapid detection of Campylobacter spp.

    Science.gov (United States)

    Moure, Zaira; Rando-Segura, Ariadna; Gimferrer, Laura; Roig, Gloria; Pumarola, Tomas; Rodriguez-Garrido, Virginia

    2017-04-29

    Campylobacter spp. infection is one of the leading causes of foodborne diarrhoeal illness in humans worldwide. The purpose of this study was to evaluate the DiaSorin LIAISON(®)Campylobacter assay for human campylobacteriosis diagnosis. A total of 645 stool samples from 640 patients suspected of having gastrointestinal infection were included. A stool culture was simultaneously performed with the DiaSorin LIAISON(®)Campylobacter assay to detect the presence of Campylobacter spp. Taking the conventional culture to be the perfect gold standard, sensitivity and specificity rates of the DiaSorin LIAISON(®)Campylobacter assay were 100% and 97.7%, respectively; and 99.1% and 98.6%, respectively, when taking the culture to be the imperfect gold standard (Bayesian Model). This new assay might be a useful tool especially for the screening of negative results. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  19. 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.

  20. Development and evaluation of an up-converting phosphor technology-based lateral flow assay for rapid and quantitative detection of aflatoxin B1 in crops.

    Science.gov (United States)

    Zhao, Yong; Liu, Xiao; Wang, Xiaochen; Sun, Chongyun; Wang, Xinrui; Zhang, Pingping; Qiu, Jingfu; Yang, Ruifu; Zhou, Lei

    2016-12-01

    Contamination of grains and other crops by aflatoxin B1 (AFB1), a highly toxic aflatoxin produced by Aspergillus flavus and Aspergillus parasiticus, poses a serious threat to human health and is an important food safety issue. In this study, a competitive up-converting phosphor technology-based lateral flow (AFB1-UPT-LF) assay was developed for rapid detection of AFB1. Detection sensitivity of the proposed assay can reach 0.03ngmL -1 for standard AFB1 solutions, with the coefficients of variation (CV) less than 10% (from 1.0 to 9.4%). A good linearity (r=0.9889) was observed for quantification of AFB1 from 0.03 to 1000ngmL -1 . Except for aflatoxin M1, no cross-reactivity was found with the abrin, ricin, ochratoxin A, botulinum toxin, shiga toxin 1, shiga toxin 2, and staphylococcal enterotoxin B, even at high concentrations of 100 or 1000ngmL - 1 . After optimizing the extraction of AFB1, the assay showed good tolerance to various crop samples, with the detection limit (from 0.1 to 5ngg - 1 ) lower than the corresponding maximum residue level (MRL) set in China. The AFB1-UPT-LF assay provides a promising tool for rapid on-site detection of AFB1 because of its high sensitivity, specificity, and sample tolerance. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. 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.

  2. Development and evaluation of a pseudovirus-luciferase assay for rapid and quantitative detection of neutralizing antibodies against enterovirus 71.

    Directory of Open Access Journals (Sweden)

    Xing Wu

    Full Text Available The level of neutralizing antibodies (NtAb induced by vaccine inoculation is an important endpoint to evaluate the efficacy of EV71 vaccine. In order to evaluate the efficacy of EV71 vaccine, here, we reported the development of a novel pseudovirus system expression firefly luciferase (PVLA for the quantitative measurement of NtAb. We first evaluated and validated the sensitivity and specificity of the PVLA method. A total of 326 serum samples from an epidemiological survey and 144 serum specimens from 3 clinical trials of EV71 vaccines were used, and the level of each specimen's neutralizing antibodies (NtAb was measured in parallel using both the conventional CPE-based and PVLA-based assay. Against the standard neutralization assay based on the inhibition of the cytopathic effect (CPE, the sensitivity and specificity of the PVLA method are 98% and 96%, respectively. Then, we tested the potential interference of NtAb against hepatitis A virus, Polio-I, Polio-II, and Polio-III standard antisera (WHO and goat anti-G10/CA16 serum, the PVLA based assay showed no cross-reactivity with NtAb against other specific sera. Importantly, unlike CPE based method, no live replication-competent EV71 is used during the measurement. Taken together, PVLA is a rapid and specific assay with higher sensitivity and accuracy. It could serve as a valuable tool in assessing the efficacy of EV71 vaccines in clinical trials and disease surveillance in epidemiology studies.

  3. A broadly reactive one-step SYBR Green I real-time RT-PCR assay for rapid detection of murine norovirus.

    Directory of Open Access Journals (Sweden)

    Ken-Ichi Hanaki

    Full Text Available A one-step SYBR Green I real-time RT-PCR assay was developed for the detection and quantification of a broad range of murine noroviruses (MNVs. The primer design was based on the multiple sequence alignments of 101 sequences of the open reading frame (ORF1-ORF2 junction of MNV. The broad reactivity and quantitative capacity of the assay were validated using 7 MNV plasmids. The assay was completed within 1 h, and the reliable detection limit was 10 copies of MNV plasmid or 0.063 median tissue culture infective doses per milliliter of RAW264 cell culture-propagated viruses. The diagnostic performance of the assay was evaluated using 158 mouse fecal samples, 91 of which were confirmed to be positive. The melting curve analysis demonstrated the diversity of MNV in the samples. This is the first report of a broadly reactive one-step SYBR Green I real-time RT-PCR assay for detecting of MNVs. The rapid and sensitive performance of this assay makes it a powerful tool for diagnostic applications.

  4. Magnetically optimized SERS assay for rapid detection of trace drug-related biomarkers in saliva and fingerprints.

    Science.gov (United States)

    Yang, Tianxi; Guo, Xiaoyu; Wang, Hui; Fu, Shuyue; Wen, Ying; Yang, Haifeng

    2015-06-15

    New developments in the fields of human healthcare and social security call for the exploration of an easy and on-field method to detect drug-related biomarkers. In this paper, Au nanoparticles dotted magnetic nanocomposites (AMN) modified with inositol hexakisphosphate (IP6) were used as surface-enhanced Raman scattering (SERS) substrate to quickly monitor trace drug-related biomarkers in saliva and to on-site screen a trace drug biomarker in fingerprints. Due to inducing with an external magnet, such substrate presented a huge SERS activity, which has met the sensitivity requirement for assay to detect the drug biomarkers in saliva from the U.S. Substance Abuse and Mental Health Services Administration, and also the limit of detection for drug biomarker in fingerprint reached 100 nM. In addition, this AMN-based SERS assay was successfully conducted using a portable Raman spectrometer, which could be used to on-site and accurately differentiate between the smokers and drug addicts in near future. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. 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.

  6. Rapid detection of drug resistance and mutational patterns of extensively drug-resistant strains by a novel GenoType® MTBDRsl assay

    Directory of Open Access Journals (Sweden)

    A K Singh

    2013-01-01

    Full Text Available Background: The emergence of extensively drug-resistant tuberculosis (XDR-TB is a major concern in the India. The burden of XDR-TB is increasing due to inadequate monitoring, lack of proper diagnosis, and treatment. The GenoType ® Mycobacterium tuberculosis drug resistance second line (MTBDRsl assay is a novel line probe assay used for the rapid detection of mutational patterns conferring resistance to XDR-TB. Aim: The aim of this study was to study the rapid detection of drug resistance and mutational patterns of the XDR-TB by a novel GenoType ® MTBDRsl assay. Materials and Methods: We evaluated 98 multidrug-resistant (MDR M. tuberculosis isolates for second line drugs susceptibility testing by 1% proportion method (BacT/ALERT 3D system and GenoType ® MTBDRsl assay for rapid detection of conferring drug resistance to XDR-TB. Results: A total of seven (17.4% were identified as XDR-TB by using standard phenotypic method. The concordance between phenotypic and GenoType ® MTBDRsl assay was 91.7-100% for different antibiotics. The sensitivity and specificity of the MTBDRsl assay were 100% and 100% for aminoglycosides; 100% and 100% for fluoroquinolones; 91.7% and 100% for ethambutol. The most frequent mutations and patterns were gyrA MUT1 (A90V in seven (41.2% and gyrA + WT1-3 + MUT1 in four (23.5%; rrs MUT1 (A1401G in 11 (64.7%, and rrs WT1-2 + MUT1 in eight (47.1%; and embB MUT1B (M306V in 11 (64.7% strains. Conclusions: These data suggest that the GenoType ® MTBDRsl assay is rapid, novel test for detection of resistance to second line anti-tubercular drugs. This assay provides additional information about the frequency and mutational patterns responsible for XDR-TB resistance.

  7. A multiplex PCR assay for the rapid and sensitive detection of methicillin-resistant Staphylococcus aureus and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci.

    Science.gov (United States)

    Xu, Benjin; Liu, Ling; Liu, Li; Li, Xinping; Li, Xiaofang; Wang, Xin

    2012-11-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a global health concern, which had been detected in food and food production animals. Conventional testing for detection of MRSA takes 3 to 5 d to yield complete information of the organism and its antibiotic sensitivity pattern. So, a rapid method is needed to diagnose and treat the MRSA infections. The present study focused on the development of a multiplex PCR assay for the rapid and sensitive detection of MRSA. The assay simultaneously detected 4 genes, namely, 16S rRNA of the Staphylococcus genus, femA of S. aureus, mecA that encodes methicillin resistance, and one internal control. It was rapid and yielded results within 4 h. The analytical sensitivity and specificity of the multiplex PCR assay was evaluated by comparing it with the conventional method. The analytical sensitivity of the multiplex PCR assay at the DNA level was 10 ng DNA. The analytical specificity was evaluated with 10 reference staphylococci strains and was 100%. The diagnostic evaluation of MRSA was carried out using 360 foodborne staphylococci isolates, and showed 99.1% of specificity, 96.4% of sensitivity, 97.5% of positive predictive value, and 97.3% of negative predictive value compared to the conventional method. The inclusion of an internal control in the multiplex PCR assay is important to exclude false-negative cases. This test can be used as an effective diagnostic and surveillance tool to investigate the spread and emergence of MRSA. © 2012 Institute of Food Technologists®

  8. A rapid and low-cost microscopic observation drug susceptibility assay for detecting TB and MDR-TB among individuals infected by HIV in South India

    Directory of Open Access Journals (Sweden)

    S Solomon

    2013-01-01

    Full Text Available Background: The converging epidemics of HIV and tuberculosis (TB pose one of the greatest public health challenges of our time. Rapid diagnosis of TB is essential in view of its infectious nature, high burden of cases, and emergence of drug resistance. Objective: The purpose of this present study was to evaluate the feasibility of implementing the microscopic observation drug susceptibility (MODS assay, a novel assay for the diagnosis of TB and multi-drug-resistant tuberculosis (MDR-TB directly from sputum specimens, in the Indian setting. Materials and Methods: This study involved a cross-sectional, blinded assessment of the MODS assay on 1036 suspected cases of pulmonary TB in HIV-positive and HIV-negative patients against the radiometric method, BD-BACTEC TB 460 system. Results: Overall, the sensitivity, specificity, positive predictive value, and negative predictive value of the MODS assay in detecting MTB among TB suspected patients were 89.1%, 99.1%, 94.2%, 95.8%, respectively. In addition, in the diagnosis of drug-resistant TB, the MODS assay was 84.2% sensitive for those specimens reporting MDR, 87% sensitivity for those specimens reporting INH mono-resistance, and 100% sensitive for specimens reporting RIF mono-resistance. The median time to detection of TB in the MODS assay versus BACTEC was 9 versus 21 days (P < 0.001. Conclusion: Costing 5 to 10 times lesser than the automated culture methods, the MODS assay has the potential clinical utility as a simple and rapid method. It could be effectively used as an alternative method for diagnosing TB and detection of MDR-TB in a timely and affordable way in resource-limited settings.

  9. MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR*

    OpenAIRE

    Wang, Jian-yong; Gu, Yang-shun; Wang, Jing; Tong, Yi; Wang, Ying; Shao, Jun-bing; Qi, Ming

    2008-01-01

    Objective: Leber’s hereditary optic neuropathy (LHON) is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA (mtDNA). Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing. This study aims to develop a minor groove binder (MGB) probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction ...

  10. 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

  11. A Rapid Multiplex Real-Time PCR High-Resolution Melt Curve Assay for the Simultaneous Detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in Food.

    Science.gov (United States)

    Forghani, Fereidoun; Wei, Shuai; Oh, Deog-Hwan

    2016-05-01

    Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, are of great concern for food safety. They may also coexist in food matrices and, in the case of B. cereus and S. aureus, the resulting illnesses can resemble each other owing to similar symptoms. Therefore, their simultaneous detection may have advantages in terms of cost savings and rapidity. Given this context, a rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of these three pathogens in food was developed. The assay successfully detected B. cereus (gyrB), L. monocytogenes (hly), and S. aureus (nuc) in a single reaction, and the average melting temperatures were 76.23, 80.19, and 74.01°C, respectively. The application of SYTO9 dye and a slow melt curve analysis ramp rate (0.1°C/s) enabled the production of sharp, high-resolution melt curve peaks that were easily distinguishable from each other. The detection limit in food (milk, rice, and lettuce) was 3.7 × 10(3) CFU/g without an enrichment step and 3.7 × 10(1) CFU/g following the 10-h enrichment. Hence, the assay developed here is specific and sensitive, providing an efficient tool for implementation in food for the simultaneous detection of B. cereus, L. monocytogenes, and S. aureus .

  12. Diagnostics-in-a-Suitcase: Development of a portable and rapid assay for the detection of the emerging avian influenza A (H7N9) virus.

    Science.gov (United States)

    Abd El Wahed, Ahmed; Weidmann, Manfred; Hufert, Frank T

    2015-08-01

    In developing countries, equipment necessary for diagnosis is only available in few central laboratories, which are less accessible and of limited capacity to test large numbers of incoming samples. Moreover, the transport conditions of samples are inadequate, therefore leading to unreliable results. The development of a rapid, inexpensive, and simple test would allow mobile detection of viruses. A suitcase laboratory "Diagnostics-in-a-Suitcase" (56cm×45.5cm×26.5cm) containing all reagents and devices necessary for performing a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed. As an example, two RT-RPA assays were established for the detection of hemagglutinin (H) and neuraminidase (N) genes of the novel avian influenza (H7N9) virus. The sensitivities of the H7 and the N9 RT-RPA assays were 10 and 100 RNA molecules, respectively. The assays were performed at a single temperature (42°C). The results were obtained within 2-7min. The H7N9 RT-RPA assays did not show a cross-detection either of any other respiratory viruses affecting humans and/or birds or of the human or chicken genomes. All reagents were used, stored, and transported at ambient temperature, that is, cold chain independent. In addition, the Diagnostics-in-a-Suitcase was operated by a solar-powered battery. The developed assay protocol and mobile setup performed well. Moreover, it can be easily implemented to perform diagnoses at airports, quarantine stations, or farms for rapid on-site viral nucleic acid detection. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  13. A rapid and automated fiber optic-based biosensor assay for the detection of Salmonella in spent irrigation water used in the sprouting of sprout seeds.

    Science.gov (United States)

    Kramer, Marianne F; Lim, Daniel V

    2004-01-01

    Recent outbreaks of foodborne illness have been linked to the consumption of contaminated sprouts. The spent irrigation water used to irrigate sprouts can carry many microorganisms, including pathogenic strains of Escherichia coli and Salmonella enterica. These pathogens are believed to originate from the seeds. The U.S. Food and Drug Administration recommends that sprout producers conduct microbiological testing of spent irrigation water from each production lot at least 48 h after seeds have germinated. Microbial analysis for the detection of Salmonella is labor-intensive and takes days to complete. A rapid and automated fiber-optic biosensor assay for the detection of Salmonella in sprout rinse water was developed in this study. Alfalfa seeds contaminated with various concentrations of Salmonella Typhimurium were sprouted. The spent irrigation water was assayed 67 h after alfalfa seed germination with the RAPTOR (Research International, Monroe, Wash.), an automated fiber optic-based detector. Salmonella Typhimurium could be positively identified in spent irrigation water when seeds were contaminated with 50 CFU/g. Viable Salmonella Typhimurium cells were also recovered from the waveguides after the assay. This biosensor assay system has the potential to be directly connected to water lines within the sprout-processing facility and to operate automatically, requiring manual labor only for preventative maintenance. Therefore, the presence of Salmonella Typhimurium in spent irrigation water could be continuously and rapidly detected 3 to 5 days before the completion of the sprouting process.

  14. Evaluation of the line probe assay for the rapid detection of bacterial meningitis pathogens in cerebrospinal fluid samples from children.

    Science.gov (United States)

    Soysal, Ahmet; Toprak, Demet Gedikbasi; Türkoğlu, Salih; Bakir, Mustafa

    2017-01-11

    The aim of this study is to compare the diagnostic performance of the line probe assay (LPA) with conventional multiplex polymerase chain reaction (PCR) for Streptococcus pneumoniae as well as real-time PCR for Neisseria meningitidis and Haemophilus influenzae type b (Hib) in cerebrospinal fluid (CSF) samples from children during the multicenter national surveillance of bacterial meningitis between the years 2006 and 2009 in Turkey. During the study period 1460 subjects were enrolled and among them 841 (57%) met the criteria for probable bacterial meningitis. The mean age of subjects was 51 ± 47 months (range, 1-212 months). We performed the line probe assay in 751 (89%) CSF samples of 841 probable bacterial meningitis cases, of whom 431 (57%) were negative, 127 (17%) were positive for S. pneumoniae, 53 (7%) were positive for H. influenzae type b, and 41 (5%) were positive for N. meningitidis. The LPA was positive in 19 of 23 (82%) S. pneumoniae samples, 4 of 6 (67%) N. meningitidis samples and 2 of 2 (100%) Hib samples in CSF culture-positive cases. The specificity of the LPA for all of S. pneumoniae, H. influenzae type b, and N. meningitidis was 88% (95% CI: 85-91%), when using the standard PCR as a reference. The specificity of LPA for each of S. pneumoniae, H. influenzae type b, and N. meningitidis was 93% (95% CI: 89-95%), 96% (95% CI: 94-98%), and 99% (95% CI: 97-99%), respectively. For all of S. pneumoniae, H. influenzae type b and N. meningitidis the sensitivity of the LPA was 76% (95% CI: 70-82%) and for each of S. pneumoniae, H. influenzae type b and N. meningitidis was 72% (95% CI:63-79%), 88% (95% CI: 73-95%), and 81% (95% CI:67-92%), respectively. The LPA assay can be used to detect common bacterial meningitis pathogens in CSF samples, but the assay requires further improvement.

  15. 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.

  16. Rapid and sensitive detection of Mycoplasma synoviae by an insulated isothermal polymerase chain reaction-based assay on a field-deployable device.

    Science.gov (United States)

    Kuo, Hung-Chih; Lo, Dan-Yuan; Chen, Chiou-Lin; Tsai, Yun-Long; Ping, Jia-Fong; Lee, Chien-Hsien; Lee, Pei-Yu Alison; Chang, Hsiao-Fen Grace

    2017-01-01

    Mycoplasma synoviae (MS), causing respiratory diseases, arthritis, and eggshell apex abnormalities in avian species, is an important pathogen in the poultry industry. Implementation of a biosecurity plan is important in MS infection management. Working on a field-deployable POCKIT™ device, an insulated isothermal polymerase chain reaction (iiPCR) assay has a potential for timely MS detection on the farm. The MS iiPCR assay had limit of detection 95% of about 9 genome equivalents by testing serial dilutions of a standard DNA. The detection endpoint of the assay for detection of MS genomic DNA was comparable to a reference real-time PCR. The assay did not crossreact with other important avian pathogens, including avian reovirus, Mycoplasma gallisepticum, Staphylococcus aureus, Escherichia coli, Pasteurella multocida, and Salmonella Pullorum. When 92 synovial fluid and respiratory tract swab samples collected from chickens, turkeys, and geese suspected of MS infection were tested, the clinical performance of the MS iiPCR had 97.8% agreement (Cohen's kappa value, 0.95) with that of the reference real-time PCR. In conclusion, the MS iiPCR/POCKIT™ system, working with field-deployable manual or automatic nucleic acid extraction methods, has potential to serve as a rapid and sensitive on-site tool to facilitate timely detection of MS. © The Author 2016. Published by Oxford University Press on behalf of Poultry Science Association.

  17. 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.

  18. Comparison of a rapid immunochromatographic assay with an immunofluorescent antibody test for detection of Leishmania infantum antibodies in dogs.

    Science.gov (United States)

    Proverbio, Daniela; Spada, Eva; Perego, Roberta; Baggiani, Luciana; Bagnagatti De Giorgi, Giada; Migliazzo, Antonella; Vitale, Fabrizio

    2016-12-01

    Identification of Leishmania infantum-infected dogs is crucial for control of canine leishmaniosis. In particular, in areas where access to specialized laboratories is limited, the availability of reliable and rapid in-clinic serologic tests may support immediate diagnosis in suspected cases and permit detection of asymptomatic canine carriers of L infantum infection. The purpose of the study was to validate the immunochromatographic test (ICT) Anigen Rapid Leishmania Ab Test kit for detection of L infantum antibodies in naturally exposed dogs in comparison with the immunofluorescence antibody test (IFAT). Serum samples from 66 dogs, including 20 healthy control dogs and 46 dogs suspected or confirmed with canine leishmaniosis, were measured by both tests. Anti-Leishmania IgG titers ≥ 1:40 by IFAT were considered positive. Kappa statistic with a 95% CI was calculated to evaluate agreement between the 2 testing methods, and sensitivity, specificity, and positive and negative likelihood ratio were calculated. Anti-L infantum IgG antibodies were found in 35 of 66 samples using the IFAT test (titers 1:40-1:5120). Thirty-one out of 66 samples tested positive with the qualitative ICT. Four IFAT-positive (titers ICT-negative. The Kappa value of 0.853 demonstrated very good agreement between the 2 tests. The Anigen Rapid Leishmania Ab Test kit reliably identified canine sera with anti-L infantum IgG antibody titers ≥ 1:40. The ICT requires neither special preparation of the serum nor specialized equipment and can be stored at ambient temperature. The test is applicable as a field test because it is easy to use and provides rapid results. © 2016 American Society for Veterinary Clinical Pathology.

  19. Evaluation of Two Lyophilized Molecular Assays to Rapidly Detect Foot-and-Mouth Disease Virus Directly from Clinical Samples in Field Settings.

    Science.gov (United States)

    Howson, E L A; Armson, B; Madi, M; Kasanga, C J; Kandusi, S; Sallu, R; Chepkwony, E; Siddle, A; Martin, P; Wood, J; Mioulet, V; King, D P; Lembo, T; Cleaveland, S; Fowler, V L

    2017-06-01

    Accurate, timely diagnosis is essential for the control, monitoring and eradication of foot-and-mouth disease (FMD). Clinical samples from suspect cases are normally tested at reference laboratories. However, transport of samples to these centralized facilities can be a lengthy process that can impose delays on critical decision making. These concerns have motivated work to evaluate simple-to-use technologies, including molecular-based diagnostic platforms, that can be deployed closer to suspect cases of FMD. In this context, FMD virus (FMDV)-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) and real-time RT-PCR (rRT-PCR) assays, compatible with simple sample preparation methods and in situ visualization, have been developed which share equivalent analytical sensitivity with laboratory-based rRT-PCR. However, the lack of robust 'ready-to-use kits' that utilize stabilized reagents limits the deployment of these tests into field settings. To address this gap, this study describes the performance of lyophilized rRT-PCR and RT-LAMP assays to detect FMDV. Both of these assays are compatible with the use of fluorescence to monitor amplification in real-time, and for the RT-LAMP assays end point detection could also be achieved using molecular lateral flow devices. Lyophilization of reagents did not adversely affect the performance of the assays. Importantly, when these assays were deployed into challenging laboratory and field settings within East Africa they proved to be reliable in their ability to detect FMDV in a range of clinical samples from acutely infected as well as convalescent cattle. These data support the use of highly sensitive molecular assays into field settings for simple and rapid detection of FMDV. © 2015 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH.

  20. 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.

  1. 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.

  2. Enhanced rapidity for qualitative detection of Listeria monocytogenes using an enzyme-linked immunosorbent assay and immunochromatography strip test combined with immunomagnetic bead separation.

    Science.gov (United States)

    Shim, Won-Bo; Choi, Jin-Gil; Kim, Ji-Young; Yang, Zheng-You; Lee, Kyu-Ho; Kim, Min-Gon; Ha, Sang-Do; Kim, Keun-Sung; Kim, Kwang-Yup; Kim, Cheol-Ho; Eremin, Sergei A; Chung, Duck-Hwa

    2008-04-01

    An enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICG) strip test, and immunomagnetic bead separation (IMBS) system based on a monoclonal antibody were individually developed for the detection and isolation of Listeria monocytogenes in meat samples. The three methods showed a strong reaction with Listeria species and a weak reaction with Staphylococcus aureus. To increase the rapidity of L. monocytogenes detection, combinations of the ELISA and ICG strip test with the IMBS system (ELISA-IMBS and ICG-IMBS) were investigated. In comparative analyses of artificially inoculated meat and samples of processed meat, the ELISA and ICG strip test required 24 h of enrichment time to detect the inoculated meat samples with > or =1 X 10(2) CFU/10 g, whereas the ELISA-IMBS and ICG-IMBS required only 14 h of enrichment. Analyses of naturally contaminated meat samples (30 pork samples, 20 beef samples, 26 chicken samples, 20 fish samples, and 20 processed meat samples) performed by ELISA-IMBS, ICG-IMBS, and API kit produced similar results. The ELISA-IMBS and ICG-IMBS provide a more rapid assay than the individual ELISA and the ICG strip test and are appropriate for rapid and qualitative detection of L. monocytogenes (or Listeria species) in meat samples. With the ICG-IMBS, L. monocytogenes could be detected in meat samples within 15 h and the method has potential as a rapid, cost-effective on-site screening tool for the detection of L. monocytogenes in food samples and agricultural products at a minimum detection level of approximately 100 CFU/10 g.

  3. Evaluation of an automated rapid diagnostic assay for detection of Gram-negative bacteria and their drug-resistance genes in positive blood cultures.

    Directory of Open Access Journals (Sweden)

    Masayoshi Tojo

    Full Text Available We evaluated the performance of the Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN; Nanosphere, Northbrook, IL, USA, an automated multiplex assay for rapid identification of positive blood cultures caused by 9 Gram-negative bacteria (GNB and for detection of 9 genes associated with β-lactam resistance. The BC-GN assay can be performed directly from positive blood cultures with 5 minutes of hands-on and 2 hours of run time per sample. A total of 397 GNB positive blood cultures were analyzed using the BC-GN assay. Of the 397 samples, 295 were simulated samples prepared by inoculating GNB into blood culture bottles, and the remaining were clinical samples from 102 patients with positive blood cultures. Aliquots of the positive blood cultures were tested by the BC-GN assay. The results of bacterial identification between the BC-GN assay and standard laboratory methods were as follows: Acinetobacter spp. (39 isolates for the BC-GN assay/39 for the standard methods, Citrobacter spp. (7/7, Escherichia coli (87/87, Klebsiella oxytoca (13/13, and Proteus spp. (11/11; Enterobacter spp. (29/30; Klebsiella pneumoniae (62/72; Pseudomonas aeruginosa (124/125; and Serratia marcescens (18/21; respectively. From the 102 clinical samples, 104 bacterial species were identified with the BC-GN assay, whereas 110 were identified with the standard methods. The BC-GN assay also detected all β-lactam resistance genes tested (233 genes, including 54 bla(CTX-M, 119 bla(IMP, 8 bla(KPC, 16 bla(NDM, 24 bla(OXA-23, 1 bla(OXA-24/40, 1 bla(OXA-48, 4 bla(OXA-58, and 6 blaVIM. The data shows that the BC-GN assay provides rapid detection of GNB and β-lactam resistance genes in positive blood cultures and has the potential to contributing to optimal patient management by earlier detection of major antimicrobial resistance genes.

  4. Evaluation of an automated rapid diagnostic assay for detection of Gram-negative bacteria and their drug-resistance genes in positive blood cultures.

    Science.gov (United States)

    Tojo, Masayoshi; Fujita, Takahiro; Ainoda, Yusuke; Nagamatsu, Maki; Hayakawa, Kayoko; Mezaki, Kazuhisa; Sakurai, Aki; Masui, Yoshinori; Yazaki, Hirohisa; Takahashi, Hiroshi; Miyoshi-Akiyama, Tohru; Totsuka, Kyoichi; Kirikae, Teruo; Ohmagari, Norio

    2014-01-01

    We evaluated the performance of the Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN; Nanosphere, Northbrook, IL, USA), an automated multiplex assay for rapid identification of positive blood cultures caused by 9 Gram-negative bacteria (GNB) and for detection of 9 genes associated with β-lactam resistance. The BC-GN assay can be performed directly from positive blood cultures with 5 minutes of hands-on and 2 hours of run time per sample. A total of 397 GNB positive blood cultures were analyzed using the BC-GN assay. Of the 397 samples, 295 were simulated samples prepared by inoculating GNB into blood culture bottles, and the remaining were clinical samples from 102 patients with positive blood cultures. Aliquots of the positive blood cultures were tested by the BC-GN assay. The results of bacterial identification between the BC-GN assay and standard laboratory methods were as follows: Acinetobacter spp. (39 isolates for the BC-GN assay/39 for the standard methods), Citrobacter spp. (7/7), Escherichia coli (87/87), Klebsiella oxytoca (13/13), and Proteus spp. (11/11); Enterobacter spp. (29/30); Klebsiella pneumoniae (62/72); Pseudomonas aeruginosa (124/125); and Serratia marcescens (18/21); respectively. From the 102 clinical samples, 104 bacterial species were identified with the BC-GN assay, whereas 110 were identified with the standard methods. The BC-GN assay also detected all β-lactam resistance genes tested (233 genes), including 54 bla(CTX-M), 119 bla(IMP), 8 bla(KPC), 16 bla(NDM), 24 bla(OXA-23), 1 bla(OXA-24/40), 1 bla(OXA-48), 4 bla(OXA-58), and 6 blaVIM. The data shows that the BC-GN assay provides rapid detection of GNB and β-lactam resistance genes in positive blood cultures and has the potential to contributing to optimal patient management by earlier detection of major antimicrobial resistance genes.

  5. Rapid detection and identification of viral and bacterial fish pathogens using a DNA array‐based multiplex assay

    DEFF Research Database (Denmark)

    Lievens, B.; Frans, I.; Heusdens, C.

    2011-01-01

    for the simultaneous detection and identification of all cyprinid herpesviruses (CyHV‐1, CyHV‐2 and CyHV‐3) and some of the most important fish pathogenic Flavobacterium species, including F. branchiophilum, F. columnare and F. psychrophilum. For virus identification, the DNA polymerase and helicase genes were......Fish diseases can be caused by a variety of diverse organisms, including bacteria, fungi, viruses and protozoa, and pose a universal threat to the ornamental fish industry and aquaculture. The lack of rapid, accurate and reliable means by which fish pathogens can be detected and identified has been...... one of the main limitations in fish pathogen diagnosis and fish disease management and has consequently stimulated the search for alternative diagnostic techniques. Here, we describe a method based on multiplex and broad‐range PCR amplification combined with DNA array hybridization...

  6. Extraction-less, rapid assay for the direct detection of 2,4,6-trichloroanisole (TCA) in cork samples.

    Science.gov (United States)

    Apostolou, Theofylaktos; Pascual, Nuria; Marco, M-Pilar; Moschos, Anastassios; Petropoulos, Anastassios; Kaltsas, Grigoris; Kintzios, Spyridon

    2014-07-01

    2,4,6-trichloroanisole (TCA), the cork taint molecule, has been the target of several analytical approaches over the few past years. In spite of the development of highly efficient and sensitive tools for its detection, ranging from advanced chromatography to biosensor-based techniques, a practical breakthrough for routine cork screening purposes has not yet been realized, in part due to the requirement of a lengthy extraction of TCA in organic solvents, mostly 12% ethanol and the high detectability required. In the present report, we present a modification of a previously reported biosensor system based on the measurement of the electric response of cultured fibroblast cells membrane-engineered with the pAb78 TCA-specific antibody. Samples were prepared by macerating cork tissue and mixing it directly with the cellular biorecognition elements, without any intervening extraction process. By using this novel approach, we were able to detect TCA in just five minutes at extremely low concentrations (down to 0.2 ppt). The novel biosensor offers a number of practical benefits, including a very considerable reduction in the total assay time by one day, and a full portability, enabling its direct employment for on-site, high throughput screening of cork in the field and production facilities, without requiring any type of supporting infrastructure. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Diagnostic value of animal-side antibody assays for rapid detection of Mycobacterium bovis or Mycobacterium microti infection in South American camelids.

    Science.gov (United States)

    Lyashchenko, Konstantin P; Greenwald, Rena; Esfandiari, Javan; Rhodes, Shelley; Dean, Gillian; de la Rua-Domenech, Ricardo; Meylan, Mireille; Vordermeier, H Martin; Zanolari, Patrik

    2011-12-01

    Tuberculosis (TB) in South American camelids (SAC) is caused by Mycobacterium bovis or Mycobacterium microti. Two serological methods, rapid testing (RT) and the dual-path platform (DPP) assay, were evaluated using naturally infected SAC. The study population included 156 alpacas and 175 llamas in Great Britain, Switzerland, and the United States. TB due to M. bovis (n = 44) or M. microti (n = 8) in 35 alpacas and 17 llamas was diagnosed by gross pathology examination and culture. Control animals were from herds with no TB history. The RT and the DPP assay showed sensitivities of 71% and 74%, respectively, for alpacas, while the sensitivity for llamas was 77% for both assays. The specificity of the DPP assay (98%) was higher than that of RT (94%) for llamas; the specificities of the two assays were identical (98%) for alpacas. When the two antibody tests were combined, the parallel-testing interpretation (applied when either assay produced a positive result) enhanced the sensitivities of antibody detection to 89% for alpacas and 88% for llamas but at the cost of lower specificities (97% and 93%, respectively), whereas the serial-testing interpretation (applied when both assays produced a positive result) maximized the specificity to 100% for both SAC species, although the sensitivities were 57% for alpacas and 65% for llamas. Over 95% of the animals with evidence of TB failed to produce skin test reactions, thus confirming concerns about the validity of this method for testing SAC. The findings suggest that serological assays may offer a more accurate and practical alternative for antemortem detection of camelid TB.

  8. Diagnostic Value of Animal-Side Antibody Assays for Rapid Detection of Mycobacterium bovis or Mycobacterium microti Infection in South American Camelids▿

    Science.gov (United States)

    Lyashchenko, Konstantin P.; Greenwald, Rena; Esfandiari, Javan; Rhodes, Shelley; Dean, Gillian; de la Rua-Domenech, Ricardo; Meylan, Mireille; Vordermeier, HMartin; Zanolari, Patrik

    2011-01-01

    Tuberculosis (TB) in South American camelids (SAC) is caused by Mycobacterium bovis or Mycobacterium microti. Two serological methods, rapid testing (RT) and the dual-path platform (DPP) assay, were evaluated using naturally infected SAC. The study population included 156 alpacas and 175 llamas in Great Britain, Switzerland, and the United States. TB due to M. bovis (n = 44) or M. microti (n = 8) in 35 alpacas and 17 llamas was diagnosed by gross pathology examination and culture. Control animals were from herds with no TB history. The RT and the DPP assay showed sensitivities of 71% and 74%, respectively, for alpacas, while the sensitivity for llamas was 77% for both assays. The specificity of the DPP assay (98%) was higher than that of RT (94%) for llamas; the specificities of the two assays were identical (98%) for alpacas. When the two antibody tests were combined, the parallel-testing interpretation (applied when either assay produced a positive result) enhanced the sensitivities of antibody detection to 89% for alpacas and 88% for llamas but at the cost of lower specificities (97% and 93%, respectively), whereas the serial-testing interpretation (applied when both assays produced a positive result) maximized the specificity to 100% for both SAC species, although the sensitivities were 57% for alpacas and 65% for llamas. Over 95% of the animals with evidence of TB failed to produce skin test reactions, thus confirming concerns about the validity of this method for testing SAC. The findings suggest that serological assays may offer a more accurate and practical alternative for antemortem detection of camelid TB. PMID:22012976

  9. Evaluation of the GeneXpert MTB/RIF assay for rapid diagnosis of tuberculosis and detection of rifampin resistance in pulmonary and extrapulmonary specimens.

    Science.gov (United States)

    Zeka, Arzu N; Tasbakan, Sezai; Cavusoglu, Cengiz

    2011-12-01

    Mycobacterium tuberculosis remains one of the most significant causes of death from an infectious agent. The rapid diagnosis of tuberculosis and detection of rifampin (RIF) resistance are essential for early disease management. The GeneXpert MTB/RIF assay is a novel integrated diagnostic device for the diagnosis of tuberculosis and rapid detection of RIF resistance in clinical specimens. We determined the performance of the MTB/RIF assay for rapid diagnosis of tuberculosis and detection of rifampin resistance in smear-positive and smear-negative pulmonary and extrapulmonary specimens obtained from possible tuberculosis patients. Two hundred fifty-three pulmonary and 176 extrapulmonary specimens obtained from 429 patients were included in the study. One hundred ten (89 culture positive and 21 culture negative for M. tuberculosis) of the 429 patients were considered to have tuberculosis. In pulmonary specimens, sensitivities were 100% (27/27) and 68.6% (24/35) for smear-positive and smear-negative specimens, respectively. It had a lower sensitivity with extrapulmonary specimens: 100% for smear-positive specimens (4/4) and 47.7% for smear-negative specimens (21/44). The test accurately detected the absence of tuberculosis in all 319 patients without tuberculosis studied. The MTB/RIF assay also detected 1 RIF-resistant specimen and 88 RIF-susceptible specimens, and the results were confirmed by drug susceptibility testing. We concluded that the MTB/RIF test is a simple method, and routine staff with minimal training can use the system. The test appeared to be as sensitive as culture with smear-positive specimens but less sensitive with smear-negative pulmonary and extrapulmonary specimens that include low numbers of bacilli.

  10. Evaluation of GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing of multi-drug resistance tuberculosis in Northern India.

    Science.gov (United States)

    Maurya, Anand Kumar; Umrao, Jyoti; Singh, Amresh Kumar; Kant, Surya; Kushwaha, Ram Awadh Singh; Dhole, Tapan N

    2013-01-01

    The problem of multi-drug resistance tuberculosis (MDR-TB) is growing in several hotspots throughout the world. Rapid and accurate diagnosis of MDR-TB is crucial to facilitate early treatment and to reduce its spread in the community. The aim of the present study was to evaluate the new, novel GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing (DST) of MDR-TB cases in Northern India. A total of 550 specimens were collected from highly suspected drug resistant from pulmonary and extra-pulmonary TB cases. All the specimens were processed by Ziehl- Neelsen staining, culture, differentiation by the GenoType® CM assay, first line DST using BacT/ALERT 3D system and GenoType® MTBDRplus assay. The concordance of the GenoType® MTBDRplus assay was calculated in comparison with conventional DST results. Overall the sensitivity for detection of rifampicin, isoniazid and MDR-TB resistance by GenoType® MTBDRplus assay was 98.0%, 98.4% and 98.2% respectively. Out of 55 MDR-TB strains, 45 (81.8%), 52 (94.5%) and 17 (30.9%) strains showed mutation in rpoB, katG and inhA genes respectively (P < 0.05). The most prominent mutations in rpoB, katG and inhA genes were; 37 (67.3%) in S531L, 52 (94.5%) in S315T1 and 11 (20%) in C15T regions respectively (P < 0.05). Our study demonstrated a high concordance between the GenoType® MTBDRplus assay resistance patterns and those were observed by conventional DST with good sensitivity, specificity with short turnaround times and to control new cases of MDR-TB in countries with a high prevalence of MDR-TB.

  11. [Fluorescence microscopy and HPLC assay for rapid detection of distribution and content of resveratrol in Polygonum cuspidatum].

    Science.gov (United States)

    Bu, Xiao-Ying; Dong, Ai-Wen; Guan, Qiong-Yu; Wu, Feng

    2012-12-01

    To establish fluorescence microscopy combined with HPLC method for rapid detection the distribution and content of resveratrol tissues in different growth stages of Polygonum cuspidatum. Used sequential experiment to design conditions of frozen and observe of the section by fluorescence microscopy; Resveratrol was extracted by ultrasonic-assisted extraction and its content was detected by HPLC. The results showed that frozen condition for concentration of gum Arabic was from 20% (dipping time was 5 - 6 h) to 40% (2 - 5 min), the freezer temperature was -5 degrees C, and the thickness was 15 microm. Resveratrol in polygonum cuspidatum was mainly accumulated in the organs, tissues and cells of fiber and cellulose, its content in rhizomes declined as the following sequence: spinal cord > xylem > phloem > periderm; Its content declined in organ as the following sequence: buds > rhizomes > ground stem > leaves; The content of resveratrol in root increased with age. The results of fluorescence microscopic observation is in accordance with the HPLC results, indicating that the method is simple, fast and reliable, and provides a fast and reliable detection method for the determination of optimum harvesting period of Polygonum cuspidatum and acquisition of quality.

  12. MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR*

    Science.gov (United States)

    Wang, Jian-yong; Gu, Yang-shun; Wang, Jing; Tong, Yi; Wang, Ying; Shao, Jun-bing; Qi, Ming

    2008-01-01

    Objective: Leber’s hereditary optic neuropathy (LHON) is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA (mtDNA). Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing. This study aims to develop a minor groove binder (MGB) probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction (PCR). Methods: Forty-eight patients suspected of having LHON and their maternal relatives underwent a molecular genetic evaluation, with 20 normal individuals as a control group at the same time. A real-time PCR involving two MGB probes was used to detect the mtDNA11778 mutation and heteroplasmy. A linear standard curve was obtained by pUCmLHONG and pUCmLHONA clones. Results: All 48 LHON patients and their maternal relatives were positive for mtDNA11778 mutation in our assay, 27 heteroplasmic and 21 homoplasmic. Eighteen cases did not show an occurrence of the disease, while 9 developed the disease among the 27 heteroplasmic mutation cases. Eleven did not show an occurrence of the disease, while 10 cases developed the disease among 21 homoplasmic mutation cases. There was a significant difference in the incidence between the heteroplasmic and the homoplasmic mutation types. The time needed for running a real-time PCR assay was only 80 min. Conclusion: This real-time PCR assay is a rapid, reliable method for mtDNA mutation detection as well as heteroplasmy quantification. Detecting this ratio is very important for predicting phenotypic expression of unaffected carriers. PMID:18763310

  13. MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR.

    Science.gov (United States)

    Wang, Jian-yong; Gu, Yang-shun; Wang, Jing; Tong, Yi; Wang, Ying; Shao, Jun-bing; Qi, Ming

    2008-08-01

    Leber's hereditary optic neuropathy (LHON) is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA (mtDNA). Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing. This study aims to develop a minor groove binder (MGB) probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction (PCR). Forty-eight patients suspected of having LHON and their maternal relatives underwent a molecular genetic evaluation, with 20 normal individuals as a control group at the same time. A real-time PCR involving two MGB probes was used to detect the mtDNA11778 mutation and heteroplasmy. A linear standard curve was obtained by pUCmLHONG and pUCmLHONA clones. All 48 LHON patients and their maternal relatives were positive for mtDNA11778 mutation in our assay, 27 heteroplasmic and 21 homoplasmic. Eighteen cases did not show an occurrence of the disease, while 9 developed the disease among the 27 heteroplasmic mutation cases. Eleven did not show an occurrence of the disease, while 10 cases developed the disease among 21 homoplasmic mutation cases. There was a significant difference in the incidence between the heteroplasmic and the homoplasmic mutation types. The time needed for running a real-time PCR assay was only 80 min. This real-time PCR assay is a rapid, reliable method for mtDNA mutation detection as well as heteroplasmy quantification. Detecting this ratio is very important for predicting phenotypic expression of unaffected carriers.

  14. Development and Validation of a Real-Time PCR Assay for Rapid Detection of Two-Spotted Spider Mite, Tetranychus urticae (Acari: Tetranychidae.

    Directory of Open Access Journals (Sweden)

    Dongmei Li

    Full Text Available Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand's borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide.

  15. Development and Validation of a Real-Time PCR Assay for Rapid Detection of Two-Spotted Spider Mite, Tetranychus urticae (Acari: Tetranychidae).

    Science.gov (United States)

    Li, Dongmei; Fan, Qing-Hai; Waite, David W; Gunawardana, Disna; George, Sherly; Kumarasinghe, Lalith

    2015-01-01

    Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand's borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide.

  16. Detection of enteroviruses ribonucleic acid sequences in endomyocardial tissue from adult patients with chronic dilated cardiomyopathy by a rapid RT-PCR and hybridization assay.

    Science.gov (United States)

    Rey, L; Lambert, V; Wattré, P; Andréoletti, L

    2001-06-01

    A rapid reverse transcription polymerase chain reaction (RT-PCR) and microwell capture hybridisation assay with general specificity for enteroviruses was developed and compared with an improved nested RT-PCR for the detection of enteroviral RNA sequences in endomyocardial tissue from patients with chronic dilated cardiomyopathy. This method could detect as few as 20 genomic RNA copies per 100 mg of heart tissue homogenate and results could be obtained within 8 hours. Of the 55 biopsy specimens aseptically collected from the explanted hearts of 55 patients, 21 (38.2%) were positive by RT-PCR microplate assay, whereas only 19 (34.5%) were positive by nested RT-PCR assay and none were positive by classical cell culture assays. No enterovirus was detectable by RT-PCR or classical cell culture assays in any of the 55 heart biopsy specimens taken from organ donors without any known heart disease. Moreover, the nucleotide sequences of EV nested RT-PCR products showed greatest similarity to group B Coxsackieviruses [CVB3 (n = 12) or CVB5 (n = 3)], but also to group A Coxsackieviruses (CVA21 (n = 1) or CVA9 ( n= 3)]. The described RT-PCR and microwell capture hybridisation assay can be applied to the virological diagnosis of human enteroviral cardiac infections. Moreover our findings suggest that group B and group A Coxsackieviruses can persist in heart tissue from patients with end-stage chronic cardiomyopathy, supporting the hypothesis that these viruses could be implicated in the etiology of idiopathic dilated cardiomyopathy. Copyright 2001 Wiley-Liss, Inc.

  17. 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

    Equine influenza (EI) is an acute, highly contagious viral respiratory disease of equids. Currently, equine influenza virus (EIV) subtype H3N8 continues to be the most important respiratory pathogen of horses in many countries around the world. The need to achieve a rapid diagnosis and to implement effective quarantine and movement restrictions is critical in controlling the spread of EIV. In this study, a novel, inexpensive and user-friendly assay based on an insulated isothermal RT-PCR (iiRT-PCR) method on the POCKIT™, a field-deployable device, was described and validated for point-of-need detection of EIV-H3N8 in clinical samples. The newly established iiRT-PCR assay targeting the EIV HA3 gene was evaluated for its sensitivity using in vitro transcribed (IVT) RNA, as well as ten-fold serial dilutions of RNA extracted from the prototype H3N8 strain A/equine/Miami/1/63. Inclusivity and exclusivity panels were tested for specificity evaluation. Published real-time RT-PCR (rRT-PCR) assays targeting the NP and HA3 genes were used as the reference standards for comparison of RNA extracted from field strains and from nasal swab samples collected from experimentally infected horses, respectively. Limit of detection with a 95% probability (LoD95%) was estimated to be 11copies of IVT RNA. Clinical sensitivity analysis using RNA prepared from serial dilutions of a prototype EIV (Miami 1/63/H3N8) showed that the iiRT-PCR assay was about 100-fold more sensitive than the rRT-PCR assay targeting the NP gene of EIV subtype H3N8. The iiRT-PCR assay identified accurately fifteen EIV H3N8 strains and two canine influenza virus (CIV) H3N8 strains, and did not cross-react with H6N2, H7N7, H1N1 subtypes or any other equine respiratory viral pathogens. Finally, 100% agreement was found between the iiRT-PCR assay and the universal influenza virus type A rRT-PCR assay in detecting the EIV A/equine/Kentucky/7/07 strain in 56 nasal swab samples collected from experimentally inoculated

  18. Virus detection using Viro-Adembeads, a rapid capture system for viruses, and plaque assay in intentionally virus-contaminated beverages.

    Science.gov (United States)

    Hatano, Ben; Kojima, Asato; Sata, Tetsutaro; Katano, Harutaka

    2010-01-01

    Intentional contamination of beverages with microbes is one type of bioterrorist threat. While bacteria and fungus can be easily collected by a centrifuge, viruses are difficult to collect from virus-contaminated beverages. In this study, we demonstrated that Viro-Adembeads, a rapid-capture system for viruses using anionic polymer-coated magnetic beads, collected viruses from beverages contaminated intentionally with vaccinia virus and human herpesvirus 8. Real-time PCR showed that the recovery rates of the contaminated viruses in green tea and orange juice were lower than those in milk and water. Plaque assay showed that green tea and orange juice cut the efficiency of vaccinia virus infection in CV-1 cells. These results suggest that the efficiency of virus detection depends on the kind of beverage being tested. Viro-Adembeads would be a useful tool for detecting viruses rapidly in virus-contaminated beverages used in a bioterrorist attack.

  19. 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.

  20. Rapid detection of bluetongue virus in blood and organ samples using a capture enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Portanti, O; Luciani, M; Ronchi, G F

    2005-01-01

    An antigen capture ELISA for bluetongue (BT) virus was developed using tissue culture supernatant to identify different BT virus (BTV) serotypes 1, 2, 4, 9 and 16, which have been incriminated in the current epidemic in the Mediterranean Basin. To obtain a positive result and after amplification in tissue culture, the minimum amount of infecting virus required was 100 TCID(50). Results from the antigen capture ELISA were compared with conventional methods for virus isolation and identification, such as virus amplification on embryonated chicken eggs (ECEs), followed by tissue culture and the direct immunofluorescence test. The sensitivity and specificity of the ELISA in infected tissue culture supernatant using homogenates of BTV-positive ovine and bovine organs and blood, without a previous step in ECEs, were 100%. The assay was also applied to homogenates of chicken embryo tissues, which had been infected with different BTV serotypes. This method enabled detection of the virus with 100% sensitivity and specificity rates, also using amplification in ECEs. Furthermore, among the various embryo tissues tested, liver was found to be the most suitable for use with ELISA. In experimentally infected ovine blood samples, the ELISA revealed the presence of the virus. Given the high sensitivity and specificity obtained with the BTV serotypes in this trial, the method should greatly facilitate BT diagnosis.

  1. Rapid detection and grouping of porcine bocaviruses by an EvaGreen(®) based multiplex real-time PCR assay using melting curve analysis.

    Science.gov (United States)

    Zheng, Xiaowen; Liu, Gaopeng; Opriessnig, Tanja; Wang, Zining; Yang, Zongqi; Jiang, Yonghou

    2016-08-01

    Several novel porcine bocaviruses (PBoVs) have been identified in pigs in recent years and association of these viruses with respiratory signs or diarrhea has been suggested. In this study, an EvaGreen(®)-based multiplex real-time PCR (EG-mPCR) with melting curve analysis was developed for simultaneous detection and grouping of novel PBoVs into the same genogroups G1, G2 and G3. Each target produced a specific amplicon with a melting peak of 81.3 ± 0.34 °C for PBoV G1, 78.2 ± 0.37 °C for PBoV G2, and 85.0 ± 0.29 °C for PBoV G3. Non-specific reactions were not observed when other pig viruses were used to assess the EG-mPCR assay. The sensitivity of the EG-mPCR assay using purified plasmid constructs containing the specific viral target fragments was 100 copies for PBoV G1, 50 for PBoV G2 and 100 for PBoV G3. The assay is able to detect and distinguish three PBoV groups with intra-assay and inter-assay variations ranging from 0.13 to 1.59%. The newly established EG-mPCR assay was validated with 227 field samples from pigs. PBoV G1, G2 and G3 was detected in 15.0%, 25.1% and 41.9% of the investigated samples and coinfections of two or three PBoV groups were also detected in 25.1% of the cases, indicating that all PBoV groups are prevalent in Chinese pigs. The agreement of the EG-mPCR assay with an EvaGreen-based singleplex real-time PCR (EG-sPCR) assay was 99.1%. This EG-mPCR will serve as a rapid, sensitive, reliable and cost effective alternative for routine surveillance testing of multiple PBoVs in pigs and will enhance our understanding of the epidemiological features and possible also pathogenetic changes associated with these viruses in pigs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Apta-nanosensor preparation and in vitro assay for rapid Diazinon detection using a computational molecular approach.

    Science.gov (United States)

    Jokar, Mahmoud; Safaralizadeh, Mohammad Hassan; Hadizadeh, Farzin; Rahmani, Fatemeh; Kalani, Mohamad Reza

    2017-02-01

    Aptamers (ss-DNA or ss-RNA), also known as artificial antibodies, have been selected in vitro median to bind target molecules with high affinity and selectivity. Diazinon is one of the most widely used organophosphorus insecticides in developing and underdeveloped countries as insecticide and acaricide. Diazinon is readily absorbed from the gastrointestinal system and rapidly distributed throughout the body. Thus, the design of clinical and laboratory diagnostics using nanobiosensors is necessary. A computational approach allows us to screen or rank receptor structure and predict interaction outcomes with a deeper understanding, and it is much more cost effective than laboratory attempts. In this research, the best sequence (high affinity bind Diazinon-ssDNA) was ranked among 12 aptamers isolated from SELEX experimentation. Docking results, as the first virtual screening stage and static technique, selected frequent conformation of each aptamer. Then, the quantity and quality of aptamer-Diazinon interaction were simulated using molecular dynamics as a mobility technique. RMSD, RMSF, radius of gyration, and the number of hydrogen bonds formed between Diazinon-aptamer were monitored to assess the quantity and quality of interactions. G-quadruplex DNA aptamer (DF20) showed to be a reliable candidate for Diazinon biosensing. The apta-nanosensor designed using simulation results allowed with linearity detection in the range of .141-.65 nM and a LOD of 17.903 nM, and it was validated using a computational molecular approach.

  3. Rapid detection of Gram-negative bacteria and their drug resistance genes from positive blood cultures using an automated microarray assay.

    Science.gov (United States)

    Han, Eunhee; Park, Dong-Jin; Kim, Yukyoung; Yu, Jin Kyung; Park, Kang Gyun; Park, Yeon-Joon

    2015-03-01

    We evaluated the performance of the Verigene Gram-negative blood culture (BC-GN) assay (CE-IVD version) for identification of Gram-negative (GN) bacteria and detection of resistance genes. A total of 163 GN organisms (72 characterized strains and 91 clinical isolates from 86 patients) were tested; among the clinical isolates, 86 (94.5%) isolates were included in the BC-GN panel. For identification, the agreement was 98.6% (146/148, 95% confidence interval [CI], 92.1-100) and 70% (7/10, 95% CI, 53.5-100) for monomicrobial and polymicrobial cultures, respectively. Of the 48 resistance genes harbored by 43 characterized strains, all were correctly detected. Of the 19 clinical isolates harboring resistance genes, 1 CTX-M-producing Escherichia coli isolated in polymicrobial culture was not detected. Overall, BC-GN assay provides acceptable accuracy for rapid identification of Gram-negative bacteria and detection of resistance genes, compared with routine laboratory methods despite that it has limitations in the number of genus/species and resistance gene included in the panel and it shows lower sensitivity in polymicrobial cultures. Copyright © 2015. Published by Elsevier Inc.

  4. Rapid quantitative PCR assays for the simultaneous detection of herpes simplex virus, varicella zoster virus, cytomegalovirus, Epstein-Barr virus, and human herpesvirus 6 DNA in blood and other clinical specimens

    NARCIS (Netherlands)

    Engelmann, I.; Petzold, D. R.; Kosinska, A.; Hepkema, B. G.; Schulz, T. F.; Heim, A.

    Rapid diagnosis of human herpesvirus primary infections or reactivations is facilitated by quantitative PCRs. Quantitative PCR assays with a standard thermal cycling profile permitting simultaneous detection of herpes simplex virus (HSV), varicella zoster virus (VZV), cytomegalovirus (CMV),

  5. Rapid detection of DNMT3A R882 mutations in hematologic malignancies using a novel bead-based suspension assay with BNA(NC probes.

    Directory of Open Access Journals (Sweden)

    Velizar Shivarov

    Full Text Available Mutations in the human DNA methyl transferase 3A (DNMT3A gene are recurrently identified in several hematologic malignancies such as Philadelphia chromosome-negative myeloproliferative neoplasms (MPN, myelodysplastic syndromes (MDS, MPN/MDS overlap syndromes and acute myeloid leukemia (AML. They have been shown to confer worse prognosis in some of these entities. Notably, about 2/3 of these mutations are missense mutations in codon R882 of the gene. We aimed at the development and validation of a novel easily applicable in routine practice method for quantitative detection of the DNMT3A p.R882C/H/R/S mutations bead-based suspension assay. Initial testing on plasmid constructs showed excellent performance of BNA(NC-modified probes with an optimal hybridization temperature of 66°C. The method appeared to be quantitative and showed sensitivity of 2.5% for different mutant alleles, making it significantly superior to direct sequencing. The assay was further validated on plasmid standards at different ratios between wild type and mutant alleles and on clinical samples from 120 patients with known or suspected myeloid malignancies. This is the first report on the quantitative detection of DNMT3A R882 mutations using bead-based suspension assay with BNA(NC-modified probes. Our data showed that it could be successfully implemented in the diagnostic work-up for patients with myeloid malignancies, as it is rapid, easy and reliable in terms of specificity and sensitivity.

  6. 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.

  7. 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.

  8. An evaluation of potential interferences in a fluorimetric assay for the rapid detection of thermotolerant coliforms in sewage.

    Science.gov (United States)

    Davies, C M; Apte, S C

    2000-02-01

    A 1-h fluorimetric assay of beta-D-galactosidase activity was evaluated for determining thermotolerant coliforms (TTC) in sewage samples. Above TTC concentrations of 2.3 x 103 colony-forming units (cfu) 100 ml-1, the assay response was related to TTC concentration. However, below this concentration, a large background signal was observed which was independent of TTC concentration. A separation scheme involving various filtration treatments and additions of a beta-D-galactosidase inhibitor was devised and used to quantify the sources of this anomalous assay response. The interferences encountered were largely due to the presence in sewage of non-specific cell-free enzymes or other cell-free substances that were capable of hydrolysing the fluorogenic substrate. Despite this apparent limitation, the fluorimetric enzyme assay has potential as an 'early warning' indicator of treatment process failure and gross sewage contamination and leakage in situations where TTC concentrations exceed 2.3 x 103 cfu 100 ml-1

  9. Development of an Immunomagnetic Bead-Immunoliposome Fluorescence Assay for Rapid Detection of Escherichia coli O157:H7 in Aqueous Samples and Comparison of the Assay with a Standard Microbiological Method

    OpenAIRE

    DeCory, Thomas R.; Durst, Richard A.; Zimmerman, Scott J.; Garringer, Linda A.; Paluca, Gary; DeCory,Heleen H.; Montagna, Richard A.

    2005-01-01

    The objective of this study was to develop and optimize a protocol for the rapid detection of Escherichia coli O157:H7 in aqueous samples by a combined immunomagnetic bead-immunoliposome (IMB/IL) fluorescence assay. The protocol consisted of the filtration or centrifugation of 30- to 100-ml samples followed by incubation of the filter membranes or pellet with anti-E. coli O157:H7 immunomagnetic beads in growth medium specific for E. coli O157:H7. The resulting E. coli O157:H7-immunomagnetic b...

  10. Assessment by Ames test and comet assay of toxicity potential of polymer used to develop field-capable rapid-detection device to analyze environmental samples

    Science.gov (United States)

    Hebert, Amanda; Bishop, Michelle; Bhattacharyya, Dhiman; Gleason, Karen; Torosian, Stephen

    2015-08-01

    There is need for devices that decrease detection time of food-borne pathogens from days to real-time. In this study, a rapid-detection device is being developed and assessed for potential cytotoxicity. The device is comprised of melt-spun polypropylene coupons coated via oxidative chemical vapor deposition (oCVD) with 3,4-Ethylenedioxythiophene (EDOT), for conductivity and 3-Thiopheneethanol (3TE), allowing antibody attachment. The Ames test and comet assay have been used in this study to examine the toxicity potentials of EDOT, 3TE, and polymerized EDOT-co-3TE. For this study, Salmonella typhimurium strain TA1535 was used to assess the mutagenic potential of EDOT, 3TE and the copolymer. The average mutagenic potential of EDOT, 3TE and copolymer was calculated to be 0.86, 0.56, and 0.92, respectively. For mutagenic potential, on a scale from 0 to 1, close to 1 indicates low potential for toxicity, whereas a value of 0 indicates a high potential for toxicity. The comet assay is a single-cell gel electrophoresis technique that is widely used for this purpose. This assay measures toxicity based on the area or intensity of the comet-like shape that DNA fragments produce when DNA damage has occurred. Three cell lines were assessed; FRhK-4, BHK-21, and Vero cells. After averaging the results of all three strains, the tail intensity of the copolymer was 8.8 % and tail moment was 3.0, and is most similar to the untreated control, with average tail intensity of 5.7 % and tail moment of 1.7. The assays conducted in this study provide evidence that the copolymer is non-toxic to humans.

  11. Multicentre laboratory validation of the colorimetric redox indicator (CRI) assay for the rapid detection of extensively drug-resistant (XDR) Mycobacterium tuberculosis.

    Science.gov (United States)

    Martin, Anandi; Paasch, Fabienne; Docx, Sven; Fissette, Krista; Imperiale, Belen; Ribón, Wellman; González, Liliana Andrea; Werngren, Jim; Engström, Anna; Skenders, Girts; Juréen, Pontus; Hoffner, Sven; Del Portillo, Patricia; Morcillo, Nora; Palomino, Juan Carlos

    2011-04-01

    To perform a multicentre study to evaluate the performance of the colorimetric redox indicator (CRI) assay and to establish the MICs and critical concentrations of rifampicin, isoniazid, ofloxacin, kanamycin and capreomycin. The study was carried out in two phases. Phase I determined the MIC of each drug. Phase II established critical concentrations for the five drugs tested by the CRI assay compared with the conventional proportion method. Phase I: a strain was considered resistant by the CRI assay if the MIC was ≥0.5 mg/L for rifampicin, ≥0.25 mg/L for isoniazid, ≥4.0 mg/L for ofloxacin and ≥5.0 mg/L for kanamycin and capreomycin. Sensitivity was 99.1% for isoniazid and 100% for the other drugs and specificity was 97.9% for capreomycin and 100% for the other drugs. Phase II: the critical concentration was 0.5 mg/L for rifampicin, 0.25 mg/L for isoniazid, 2.0 mg/L for ofloxacin and 2.5 mg/L for kanamycin and capreomycin giving an overall accuracy of 98.4%, 96.6%, 96.7%, 98.3% and 90%, respectively. Results demonstrate that the CRI assay is an accurate method for the rapid detection of XDR Mycobacterium tuberculosis. The CRI assay is faster than the conventional drug susceptibility testing method using solid medium, has the same turnaround time as the BACTEC MGIT 960 system, but is less expensive, and could be an adequate method for low-income countries.

  12. High-sensitive and rapid detection of Mycobacterium tuberculosis infection by IFN-γ release assay among HIV-infected individuals in BCG-vaccinated area

    Directory of Open Access Journals (Sweden)

    Jiang Weimin

    2009-05-01

    Full Text Available Abstract Background An accurate test for Mycobacterium tuberculosis infection is urgently needed in immunosuppressed populations. The aim of this study was to investigate the diagnostic power of enzyme-linked immunospot (ELISPOT-based IFN-γ release assay in detecting active and latent tuberculosis in HIV-infected population in bacillus Calmette-Guerin (BCG-vaccinated area. A total of 100 HIV-infected individuals including 32 active tuberculosis patients were recruited. An ELISPOT-based IFN-γ release assay, T-SPOT.TB, was used to evaluate the M. tuberculosis ESAT-6 and CFP-10 specific IFN-γ response. Tuberculin skin test (TST was performed for all recruited subjects. Results The subjects were divided into group HIV+ATB (HIV-infected individuals with active tuberculosis, n = 32, group HIV+LTB (HIV-infected individuals with positive results of T-SPOT.TB assay, n = 46 and group HIV only (HIV-infected individuals with negative results of T-SPOT.TB assay and without evidence of tuberculosis infection, n = 22. In group HIV+ATB and HIV+LTB, T-SPOT.TB positive rate in subjects with TST P 85% in patients with TB treatment for less than 1 month and CD4+ T cells ≥200/μl, while for patients treated for more than 3 months and CD4+ T cells Conclusion ELISPOT-based IFN-γ release assay is more sensitive and rapid for the diagnosis of TB infection in Chinese HIV-infected individuals with history of BCG vaccination, and could be an effective tool for guiding preventive treatment with isoniazid in latently infected people and for TB control in China.

  13. An enzyme-linked immuno focus assay for rapid detection and enumeration, and a newborn mouse model for human non-polio enteroviruses associated with acute diarrhea.

    Science.gov (United States)

    Rao, C Durga; Reddy, Harikrishna; Naidu, Jagadish R; Raghavendra, A; Radhika, N S; Karande, Anjali

    2015-11-01

    We have recently reported significant association of non-polio enteroviruses (NPEVs) with acute and persistent diarrhea (18-21% of total diarrheal cases), and non-diarrheal Increased Frequency of Bowel Movements (IFoBM-ND) (about 29% of the NPEV infections) in children and that the NPEV-associated diarrhea was as significant as rotavirus diarrhea. However, their diarrhea-causing potential is yet to be demonstrated in an animal model system. Since the determination of virus titers by the traditional plaque assay takes 4-7 days, there is a need for development of a rapid method for virus titer determination to facilitate active clinical research on enterovirus-associated diarrhea. The goal of this study is to develop a cell-based rapid detection and enumeration method and to demonstrate the diarrhea-inducing potential of purified and characterized non-polio enteroviruses, which were isolated from diarrheic children. Here we describe generation of monoclonal and polyclonal antibodies against purified strains belonging to different serotypes, and development of an enzyme-linked immuno focus assay (ELIFA) for detection and enumeration of live NPEV particles in clinical and purified virus samples, and a newborn mouse model for NPEV diarrhea. Plaque-purified NPVEs, belonging to different serotypes, isolated from children with diarrhea, were grown in cell culture and purified by isopycnic CsCl density gradient centrifugation. By ELIFA, NPEVs could be detected and enumerated within 12h post-infection. Our results demonstrated that Coxsackievirus B1 (CVB1) and CVB5 strains, isolated from diarrheic children, induced severe diarrhea in orally-inoculated 9-12 day-old mouse pups, fulfilling Koch's postulates. The methods described here would facilitate studies on NPEV-associated gastrointestinal disease. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Development of a lateral flow immunochromatographic assay for rapid detection of Mycoplasma pneumoniae-specific IgM in human serum specimens.

    Science.gov (United States)

    Ou, Liming; Lv, Qingyu; Wu, Canjun; Hao, Huaijie; Zheng, Yuling; Jiang, Yongqiang

    2016-05-01

    Early diagnosis of Mycoplasma pneumoniae (MP) infection is crucial for prompt treatment and good patient outcome. However, serological tests to detect MP rapidly and conveniently are still lacking. This study aimed to use the fluorescent dye Alexa Fluor® 647 as the detection marker to develop a lateral flow immunochromatographic assay (LFIA) for detection of MP-specific IgM in serum specimen. Monoclonal mouse antibody against human IgM (μ-chain specific) and goat anti-rabbit IgG were labeled with Alexa Fluor® 647 (anti-IgM-AF647 and anti-IgG-AF647). A mixture of natural MP antigen and recombinant P1 antigen was coated as the test line (T line) and rabbit IgG was coated as the control line (C line) on a nitrocellulose (NC) membrane. The MP antigens captured IgM-anti-IgM-AF647 complex on the T line. Rabbit IgG captured anti-IgG-AF647 on the C line. The fluorescence intensity on the T line and C line was measured. Sartorius CN140 NC membrane showed higher sensitivity than CN95. The optimal reaction time for the LFIA was 10min. The area under the receiver operating characteristic curve based on 34 MP positive and 166 MP negative serum samples was 0.986 (pLFIA strips did not react with serum from patients infected with non-MP pathogens including influenza viruses and bacteria causing respiratory tract infection. The intra-assay and inter-assay coefficients of variation were between 3.28% and 10.14%. The shelf life was calculated to be 2years at room temperature. The LFIA strips and the commercial EUROIMMUN kit showed consistent results on 372 serum specimens. The overall consistency rate was 96.37% with a Kappa value of 0.842 (pLFIA in the current study may be a sensitive and specific approach to detect early MP infection rapidly and conveniently. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Rapid and highly accurate detection of Drosophila suzukii, spotted wing Drosophila (Diptera: Drosophilidae) by loop-mediated isothermal amplification assays

    Science.gov (United States)

    Drosophila suzukii, the spotted wing drosophila (SWD), is currently a major pest that causes severe economic losses to thin-skinned, small fruit growers in North America and Europe. The monitoring and early detection of SWD in the field is of the utmost importance for its proper management. Althou...

  16. A rapid detection of avian oncovirus group-specific antigens in feather pulp by the enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Korec, E; Hlozánek, I; Benda, V

    1984-01-01

    An ELISA procedure for the detection of avian sarcoma-leukosis gs antigen in feather pulp of adult birds is described. The test can be used for identifying gs+ and gs- chickens in leukosis-free populations. The titres of exogenous and endogenous virus-coded gs antigens overlap and the ELISA can be used only for a preliminary screening of unknown chicken populations.

  17. Rapid detection of avian influenza virus in chicken fecal samples by immunomagnetic capture reverse transcriptase–polymerase chain reaction assay

    DEFF Research Database (Denmark)

    Dhumpa, Raghuram; Handberg, Kurt; Jørgensen, Poul Henrik

    2011-01-01

    Avian influenza virus (AIV) causes great economic losses for the poultry industry worldwide and threatens the human population with a pandemic. The conventional detection method for AIV involves sample preparation of viral RNA extraction and purification from raw sample such as bird droppings...

  18. A commercial line probe assay for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis

    Directory of Open Access Journals (Sweden)

    Kalantri Shriprakash

    2005-07-01

    Full Text Available Abstract Background Mycobacterium tuberculosis is a leading cause of death worldwide. In multi-drug resistant tuberculosis (MDR-TB infectiousness is frequently prolonged, jeopardizing efforts to control TB. The conventional tuberculosis drug susceptibility tests are sensitive and specific, but they are not rapid. The INNO-LiPA Rif. TB ® (LiPA is a commercial line probe assay designed to rapidly detect rifampicin resistance, a marker of MDR-TB. Although LiPA has shown promising results, its overall accuracy has not been systematically evaluated. Methods We did a systematic review and meta-analysis to evaluate the accuracy of LiPA for the detection of rifampicin-resistant tuberculosis among culture isolates and clinical specimens. We searched Medline, Embase, Web of Science, BIOSIS, and Google Scholar, and contacted authors, experts and the manufacturer. Fifteen studies met our inclusion criteria. Of these, 11 studies used culture isolates, one used clinical specimens, and three used both. We used a summary receiver operating characteristic (SROC curve and Q* index to perform meta-analysis and summarize diagnostic accuracy. Results Twelve of 14 studies that applied LiPA to isolates had sensitivity greater than 95%, and 12 of 14 had specificity of 100%. The four studies that applied LiPA directly to clinical specimens had 100% specificity, and sensitivity that ranged between 80% and 100%. The SROC curve had an area of 0.99 and Q* of 0.97. Conclusion LiPA is a highly sensitive and specific test for the detection of rifampicin resistance in culture isolates. The test appears to have relatively lower sensitivity when used directly on clinical specimens. More evidence is needed before LiPA can be used to detect MDR-TB among populations at risk in clinical practice.

  19. Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams

    Directory of Open Access Journals (Sweden)

    Adeline Bidault

    2015-12-01

    Full Text Available The Gram-negative bacterium Vibrio tapetis is known as the causative agent of Brown Ring Disease (BRD in the Manila clam Venerupis (=Ruditapes philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification of V. tapetis strains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600T V. tapetis strain. Quantification curves of V. tapetis strain seeded in filtered seawater (FSW or extrapallial fluids (EF samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detect V. tapetis strains down to 1.125 101 bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitor V. tapetis load both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections by V. tapetis and for designing appropriate control measures for aquaculture purposes.

  20. [Development and comparative evaluation of up-converting phosphor technology based lateral flow assay for rapid detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp].

    Science.gov (United States)

    Li, Chunfeng; Zhang, Pingping; Wang, Xiaoying; Liu, Xiao; Zhao, Yong; Sun, Chongyun; Wang, Chengbin; Yang, Ruifu; Zhou, Lei

    2015-01-01

    To develop an up-converting phosphor technology based lateral flow (UPT-LF) assay for rapid and quantitative detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp.and make the comparison with BioThreat Alert (BTA) test strips (Tetracore Inc., USA). Using up-converting phosphor nano-particles (UCP-NPs) as the bio-marker, three double-antibody-sandwich model based UPT-LF strips including Plague-UPT-LF, Anthrax-UPT-LF, Brucella-UPT-LF were prepared and its sensitivity, accuracy, linearity and specificity were determined by detecting 10(10), 10(9), 10(8), 10(7), 10(6), 10(5) and 0 CFU/ml series of concentrations of Y.pestis, B.anthracis, Brucella standards and other 27 kinds of 10(9) CFU/ml series of contrations of bacteria strains.Furthermore, the speed, sensitivity and accuracy of bacteria standards and simulated sample detection were compared between UPT-LF and BTA system. The detection limit of Plague-UPT-LF, Anthrax-UPT-LF and Brucella-LF was 10(5) CFU/ml. The CV of series of bacteria concentrations was ≤ 15%, and the r between lg (T/C-cut-off) and lg (concentration) was 0.996,0.998 and 0.999 (F values were 1 647.57, 743.51 and 1 822.17. All the P values were Brucella-LF were excellent, while that of Anthrax-UPT-LF was a little bit regretful because of non-specific reaction with two isolates of B. subtilis and one B.cereus. On-site evaluation showed the detection time of UPT-LF for all Y.pestis, B.anthracis spore and Brucella spp.was 33, 36 and 37 min, while BTA was 115, 115 and 111 min, which revealed the higher detection speed and sensitivity of UPT-LF comparing with BTA. The negative rate of two methods for blank standard was both 5/5, the sensitivity of UPT-LF for Y.pestis,B.anthracis spore and Brucella spp. was all 10(5) CFU/ml, then BTA was 10(6), 10(6) and 10(5) CFU/ml, respectively. The detection rate of UPT-LF for all three bacteria analog positive samples was 16/16, while BTA for B.anthracis was 7/16 only. The good performance

  1. Development and validation of a SYBR Green real-time PCR assay for rapid and quantitative detection of goose interferons and proinflammatory cytokines.

    Science.gov (United States)

    Zhou, Hao; Chen, Shun; Qi, Yulin; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Liu, Fei; Chen, Xiaoyue; Cheng, Anchun

    2015-10-01

    Real time quantitative polymerase chain reaction (RT-qPCR) based on SYBR-Green I binding is a quick, reliable, and easy method for analyzing small amounts of mRNA. Viral pathogens are recognized at the time of infection by pattern recognition receptors; thus, the inflammatory cytokines (IL1β, IL6, and IL18) and antiviral cytokines (IFNα, IFNγ) are secreted by innate immune cells and induced to respond to the pathogens. The objective of this study was to develop an effective and sensitive RT-qPCR assay for the rapid and accurate quantification of goose cytokines: IFNα, IFNγ, IL1β, IL6, and IL18. Subsequently, the established methods were employed to detect the immune response in agonist-stimulated goose spleen cells in vitro. These data indicated that the established RT-qPCR is a reliable method for determining relative gene expression. The results revealed that Imiquimod led to the significant upregulation of goose IFNα (P < 0.01), IFNγ (P < 0.01), IL1β (P < 0.01), IL6 (P < 0.01), and IL18 (P < 0.05). The established methods are important for scientific research and clinical applications, which require rapid and accurate results in a short period of time. The technique can potentially be used in the further research of goose molecular immunology, which will help us understand the interactions between hosts and pathogens. © 2015 Poultry Science Association Inc.

  2. Rapid and sensitive detection of Sclerotium rolfsii associated with collar rot disease of Amorphophallus paeoniifolius by species-specific polymerase chain reaction assay.

    Science.gov (United States)

    Pravi, V; Jeeva, M L; Archana, P V

    2014-09-01

    Collar rot disease caused by Sclerotium rolfsii is an economically important disease prevailing in all Amorphophallus growing areas. The pathogen propagules surviving in soil and planting material are the major sources of inoculum. A nested PCR assay has been developed for specific detection of S. rolfsii in soil and planting material. The PCR detection limit was 10 pg in conventional assay whereas 0.1 pg in nested assay. The primers designed were found to be highly specific and could be used for accurate identification of pathogen up to species level. The protocol was standardized for detection of the pathogen in artificially and naturally infected field samples.

  3. Evaluation of a rapid analyte measurement platform and real-time reverse-transcriptase polymerase chain reaction assay West Nile virus detection system in mosquito pools.

    Science.gov (United States)

    Burkhalter, Kristen L; Horiuchi, Kalanthe; Biggerstaff, Brad J; Savage, Harry M; Nasci, Roger S

    2014-03-01

    We evaluated the commercially available Rapid Analyte Measurement Platform (RAMP) West Nile virus (WNV) antigen detection test for sensitivity and consistency with real-time reverse transcriptase polymerase chain reaction (RT-PCR) confirmation testing. Panels of samples consisting of WNV-spiked mosquito pools and negative control pools were sent to 20 mosquito abatement districts (MADs) that processed the pools using the RAMP assay. The samples were then sent to the reference laboratories used by the MADs for confirmation by real-time RT-PCR. Positive pools with virus titers of roughly 1-3 log10 PFU/ml had RAMP scores above the RAMP test positive cutoff score of 30 RAMP units, but these virus-positive samples could not be reliably confirmed by real-time RT-PCR testing. Pools with virus titers > or =4 log10 PFU/ml scored > or =50 RAMP units. Real-time RT-PCR results varied among the confirmation laboratories. With few exceptions, pools returning a RAMP score of > or =100 were confirmed with real-time RT-PCR, while pools returning a RAMP score of 50-99 appeared to be at the limit of real-time RT-PCR detection. Therefore, we recommend using a positive cutoff of 50 RAMP units with no real-time RT-PCR confirmation to maximize speed, efficiency, and economy of the RAMP assay. A more conservative approach would be to implement a "gray zone" range of 50-100 RAMP units. Pools scoring within the gray zone could be submitted for real-time RT-PCR confirmation with the understanding that positive pools may not confirm due to the inhibitory effect of the RAMP buffer on the real-time RT-PCR assay. We also conducted a series of experiments using laboratory-prepared mosquito pools spiked with WNV to compare mosquito homogenization buffers, pool sizes, and grinding methods in order to determine how these variables affect the RAMP and real-time RT-PCR assay results.

  4. Development of an immunomagnetic bead-immunoliposome fluorescence assay for rapid detection of Escherichia coli O157:H7 in aqueous samples and comparison of the assay with a standard microbiological method.

    Science.gov (United States)

    DeCory, Thomas R; Durst, Richard A; Zimmerman, Scott J; Garringer, Linda A; Paluca, Gary; DeCory, Heleen H; Montagna, Richard A

    2005-04-01

    The objective of this study was to develop and optimize a protocol for the rapid detection of Escherichia coli O157:H7 in aqueous samples by a combined immunomagnetic bead-immunoliposome (IMB/IL) fluorescence assay. The protocol consisted of the filtration or centrifugation of 30- to 100-ml samples followed by incubation of the filter membranes or pellet with anti-E. coli O157:H7 immunomagnetic beads in growth medium specific for E. coli O157:H7. The resulting E. coli O157:H7-immunomagnetic bead complexes were isolated by magnetic separation, washed, and incubated with sulforhodamine B-containing immunoliposomes specific for E. coli O157:H7; the final immunomagnetic bead-E. coli O157:H7-immunoliposome complexes were again isolated by magnetic separation, washed, and lysed with a n-octyl-beta-d-glucopyranoside to release sulforhodamine B. The final protocol took less than 8 h to complete and had a detection limit of less than 1 CFU of E. coli O157:H7 per ml in various aqueous matrices, including apple juice and cider. To validate the protocol at an independent facility, 100-ml samples of groundwater with and without E. coli O157:H7 (15 CFU) were analyzed by a public health laboratory using the optimized protocol and a standard microbiological method. While the IMB/IL fluorescence assay was able to identify E. coli O157:H7-containing samples with 100% accuracy, the standard microbiological method was unable to distinguish E. coli O157:H7-spiked samples from negative controls without further extensive workup. These results demonstrate the feasibility of using immunomagnetic beads in combination with sulforhodamine B-encapsulating immunoliposomes for the rapid detection of E. coli O157:H7 in aqueous samples.

  5. Evaluation of the Determine™ fourth generation HIV rapid assay.

    Science.gov (United States)

    Brauer, Marieke; De Villiers, Johanna C; Mayaphi, Simnikiwe H

    2013-04-01

    Assays that detect p24 antigen reduce the diagnostic window period of HIV testing. Most point-of-care HIV assays have poor sensitivity to diagnose acute HIV infection as they only detect antibodies against HIV-1 and HIV-2 (HIV-1/2). This was a cross-sectional laboratory-based study that evaluated the performance of the Determine™ HIV-1/2 Ag/Ab Combo fourth generation rapid strip - currently the only rapid assay that detects both HIV-1/2 antibodies and p24 antigen. A total of 79 serum specimens (29 positive for HIV antibodies only, 14 positive for HIV antibodies and p24 antigen, 20 HIV-negative, and 16 positive for p24 antigen only) were used for the evaluation. Results were compared with those from validated fourth generation HIV ELISAs. The Determine™ Combo rapid strips had a sensitivity of 90.7% and a specificity of 100% for the detection of HIV-1/2 antibodies. Its sensitivity for the detection of p24 antigen was only 10% (3 out of 30 p24 antigen positive specimens). This implies that most acute HIV infections will be missed with this assay. The need for a point-of-care assay which can detect acute HIV infection reliably still remains, particularly for use in a high prevalence setting such as South Africa. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Enzymic colorimetry-based DNA chip: a rapid and accurate assay for detecting mutations for clarithromycin resistance in the 23S rRNA gene of Helicobacter pylori.

    Science.gov (United States)

    Xuan, Shi-Hai; Zhou, Yu-Gui; Shao, Bo; Cui, Ya-Lin; Li, Jian; Yin, Hong-Bo; Song, Xiao-Ping; Cong, Hui; Jing, Feng-Xiang; Jin, Qing-Hui; Wang, Hui-Min; Zhou, Jie

    2009-11-01

    Macrolide drugs, such as clarithromycin (CAM), are a key component of many combination therapies used to eradicate Helicobacter pylori. However, resistance to CAM is increasing in H. pylori and is becoming a serious problem in H. pylori eradication therapy. CAM resistance in H. pylori is mostly due to point mutations (A2142G/C, A2143G) in the peptidyltransferase-encoding region of the 23S rRNA gene. In this study an enzymic colorimetry-based DNA chip was developed to analyse single-nucleotide polymorphisms of the 23S rRNA gene to determine the prevalence of mutations in CAM-related resistance in H. pylori-positive patients. The results of the colorimetric DNA chip were confirmed by direct DNA sequencing. In 63 samples, the incidence of the A2143G mutation was 17.46 % (11/63). The results of the colorimetric DNA chip were concordant with DNA sequencing in 96.83 % of results (61/63). The colorimetric DNA chip could detect wild-type and mutant signals at every site, even at a DNA concentration of 1.53 x 10(2) copies microl(-1). Thus, the colorimetric DNA chip is a reliable assay for rapid and accurate detection of mutations in the 23S rRNA gene of H. pylori that lead to CAM-related resistance, directly from gastric tissues.

  7. Rapid and sensitive detection of Mycoplasma synoviae by an insulated isothermal polymerase chain reaction-based assay on a field-deployable device

    OpenAIRE

    Kuo, Hung-Chih; Lo, Dan-Yuan; Chen, Chiou-Lin; Tsai, Yun-Long; Ping, Jia-Fong; Lee, Chien-Hsien; Lee, Pei-Yu Alison; Chang, Hsiao-Fen Grace

    2016-01-01

    Mycoplasma synoviae (MS), causing respiratory diseases, arthritis, and eggshell apex abnormalities in avian species, is an important pathogen in the poultry industry. Implementation of a biosecurity plan is important in MS infection management. Working on a field-deployable POCKIT? device, an insulated isothermal polymerase chain reaction (iiPCR) assay has a potential for timely MS detection on the farm. The MS iiPCR assay had limit of detection 95% of about 9 genome equivalents by testing se...

  8. Lateral flow assay for rapid detection of white spot syndrome virus (WSSV) using a phage-displayed peptide as bio-recognition probe.

    Science.gov (United States)

    Kulabhusan, Prabir Kumar; Rajwade, Jyutika M; Sahul Hameed, A S; Paknikar, Kishore M

    2017-06-01

    White spot disease caused by the white spot syndrome virus (WSSV) has a major socio-economic impact on shrimp farming in India. It has been realized that a field-usable diagnostic capable of rapid detection of WSSV can prevent huge economic losses in disease outbreaks. In this work, we explored the possibility of using a peptide as bio-recognition probe in a field-usable device for the detection of WSSV from infected shrimps and prawns. A commercially available random phage-display library was screened against rVP28 (a major structural protein of WSSV, expressed as a recombinant protein in Escherichia coli). A bacteriophage clone VP28-4L was obtained, and its binding to purified rVP28 protein as well as WSSV from infected shrimp Litopaeneus vannamei tissue was confirmed by ELISA and western blot. The apparent equilibrium dissociation constant (K d ,app) was calculated to be 810 nM. VP28-4L did not show cross-reactivity with any other shrimp viruses. A 12-mer peptide (pep28, with the sequence 'TFQAFDLSPFPS') displayed on the VP28-4L was synthesized, and its diagnostic potential was evaluated in a lateral flow assay (LFA). Visual detection of WSSV could be achieved using biotinylated-pep28 and streptavidin-conjugated gold nanoparticles. In LFA, 12.5 μg/mL of the virus could be detected from L. vannamei gill tissue homogenate within 20 min. Pep28 thus becomes an attractive candidate in bio-recognition of WSSV in field-usable diagnostic platforms benefitting the aquaculture sector.

  9. 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.

  10. Evaluation of a PCR-Based Universal Heteroduplex Generator Assay as a Tool for Rapid Detection of Multidrug-Resistant Mycobacterium tuberculosis in Peru

    Science.gov (United States)

    Mayta, Holger; Gilman, Robert H.; Arenas, Fanny; Valencia, Teresa; Caviedes, Luz; Montenegro, Sonia H.; Ticona, Eduardo; Ortiz, Jaime; Chumpitaz, Rosa; Evans, Carlton A.; Williams, Diana L.

    2003-01-01

    Multidrug-resistant tuberculosis is an increasing health problem worldwide, especially in developing countries. The PCR-UHG-Rif assay, which detects mutations within the rpoB gene associated with rifampin resistance, was evaluated for its ability and reliability to detect and identify drug-resistant Mycobacterium tuberculosis in a developing country where tuberculosis is highly endemic. PMID:14662980

  11. Evaluation of a PCR-based universal heteroduplex generator assay as a tool for rapid detection of multidrug-resistant Mycobacterium tuberculosis in Peru.

    Science.gov (United States)

    Mayta, Holger; Gilman, Robert H; Arenas, Fanny; Valencia, Teresa; Caviedes, Luz; Montenegro, Sonia H; Ticona, Eduardo; Ortiz, Jaime; Chumpitaz, Rosa; Evans, Carlton A; Williams, Diana L

    2003-12-01

    Multidrug-resistant tuberculosis is an increasing health problem worldwide, especially in developing countries. The PCR-UHG-Rif assay, which detects mutations within the rpoB gene associated with rifampin resistance, was evaluated for its ability and reliability to detect and identify drug-resistant Mycobacterium tuberculosis in a developing country where tuberculosis is highly endemic.

  12. A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine

    Directory of Open Access Journals (Sweden)

    Horváth Gábor V

    2010-01-01

    Full Text Available Abstract Background Progress in plant cell cycle research is highly dependent on reliable methods for detection of cells replicating DNA. Frequency of S-phase cells (cells in DNA synthesis phase is a basic parameter in studies on the control of cell division cycle and the developmental events of plant cells. Here we extend the microscopy and flow cytometry applications of the recently developed EdU (5-ethynyl-2'-deoxyuridine-based S-phase assay to various plant species and tissues. We demonstrate that the presented protocols insure the improved preservation of cell and tissue structure and allow significant reduction in assay duration. In comparison with the frequently used detection of bromodeoxyuridine (BrdU and tritiated-thymidine incorporation, this new methodology offers several advantages as we discuss here. Results Applications of EdU-based S-phase assay in microscopy and flow cytometry are presented by using cultured cells of alfalfa, Arabidopsis, grape, maize, rice and tobacco. We present the advantages of EdU assay as compared to BrdU-based replication assay and demonstrate that EdU assay -which does not require plant cell wall digestion or DNA denaturation steps, offers reduced assay duration and better preservation of cellular, nuclear and chromosomal morphologies. We have also shown that fast and efficient EdU assay can also be an efficient tool for dual parameter flow cytometry analysis and for quantitative assessment of replication in thick root samples of rice. Conclusions In plant cell cycle studies, EdU-based S-phase detection offers a superior alternative to the existing S-phase assays. EdU method is reliable, versatile, fast, simple and non-radioactive and it can be readily applied to many different plant systems.

  13. Evaluation of the accuracy of the microplate Alamar Blue assay for rapid detection of MDR-TB in Peru [Short Communication

    National Research Council Canada - National Science Library

    Chauca, J.A; Palomino, J-C; Guerra, H

    2007-01-01

    .... This study evaluates the usefulness of the colorimetric method using Alamar Blue for the rapid detection of resistance to rifampicin and isoniazid in 63 clinical isolates of Mycobacterium tuberculosis in Peru...

  14. Development of a TaqMan-based real-time PCR assay for rapid and specific detection of fowl aviadenovirus serotype 4.

    Science.gov (United States)

    Wang, Jianchang; Wang, Jinfeng; Chen, Ping; Liu, Libing; Yuan, Wanzhe

    2017-06-01

    Twelve serotypes of fowl aviadenovirus, namely, FAdV-(1-8a and 8b-11), have been identified, among which FAdV-4 is the aetiologic agent of hepatitis hydropericardium syndrome (HHS) in chickens. Outbreaks of HHS have been documented in many countries, causing significant economic losses. Real-time PCR methods described so far in the literature cross-detect different serotypes of FAdVs. In this study, we aimed to develop a TaqMan-based real-time PCR assay for the specific detection of FAdV-4. A pair of primers targeting the hexon gene and a TaqMan probe were designed. Using different copy numbers of plasmid DNA carrying the hexon gene as template, we showed the detection limit of this assay was 101 copies/reaction, which was 10 times higher than conventional PCR. The assay was highly specific for FAdV-4 and did not cross-detect 11 other serotypes of FAdVs, avian influenza virus, Newcastle disease virus, infectious bronchitis virus or subgroup J of the avian leukosis virus. The reproducibility of the assay was assessed by five independent reactions using different copy numbers of plasmid DNA (103 and 105) as template, and the results showed 0.56-1.15% coefficient of variation for inter-assay variability. Furthermore, the assay was validated with 80 clinical samples. Real-time PCR showed that 76 out of 80 samples were positive for FAdV-4 (95.0% positivity) while 68 out of 80 were tested positive by conventional PCR (85.0% positivity). Our data suggest this real-time PCR assay could be an attractive tool for screening, confirmatory diagnosis and specific differentiation of FAdV-4 infection.

  15. ASSAY FOR RAPID SCREENING OF PHYTOCHEMICALS AS ANTIMICROBIAL AGENTS

    OpenAIRE

    Ghosh Saurav; Indranil Mukherjee; Ashoke Ranjan Thakur; Shaon Ray Chaudhuri

    2013-01-01

    The present study aims to develop a rapid method for antibiotic sensitivity detection and screening of natural products for antimicrobial activity. The dimension of WBC in blood film was found to get altered when seeded with bacteria and monitored under light microscope. The shrinkage was prevented in response to antibiotic treatment and validated using statistical analysis (two sample one tailed Z test). Thus here is a prompt (4 h) assay system for detection of blood infection, antibiotic se...

  16. A Simple Modification to the Mosquito Homogenization Protocol Safely Inactivates West Nile Virus and Allows Virus Detection by the Rapid Analyte Measurement Platform (RAMP®) ASSAY.

    Science.gov (United States)

    Burkhalter, Kristen L; Biggerstaff, Brad J; Horiuchi, Kalanthe; Savage, Harry M

    2016-06-01

    We evaluated the ability of the Rapid Analyte Measurement Platform (RAMP(®)) mosquito-grinding buffer to inactivate West Nile virus (WNV) by subjecting WNV-positive samples ground in RAMP buffer to incubation intervals ranging from 5 min to 60 min. At each time point an aliquot was removed and serially diluted in bovine albumin (BA)-1 cell culture media to stop the inactivation process by RAMP buffer. Each BA-1 sample was tested for viable virus using Vero 6-well cell culture plaque assay and observed for plaques. We observed very limited inactivation of WNV (1-2 log10 plaque-forming units/ml) by RAMP buffer. Concerned for RAMP operators who may be using this assay in low-level biocontainment facilities, we developed an alternate sample homogenization protocol using Triton X-100 detergent that ensures complete WNV inactivation without compromising the performance of the RAMP assay.

  17. Evaluation of Different PCR-Based Assays and LAMP Method for Rapid Detection of Phytophthora infestans by Targeting the Ypt1 Gene

    Directory of Open Access Journals (Sweden)

    Mehran Khan

    2017-10-01

    Full Text Available Late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating diseases affecting potato and tomato worldwide. Early diagnosis of the P. infestans pathogen causing late blight should be the top priority for addressing disease epidemics and management. In this study, we performed a loop-mediated isothermal amplification (LAMP assay, conventional polymerase chain reaction (PCR, nested PCR, and real-time PCR to verify and compare the sensitivity and specificity of the reaction based on the Ypt1 (Ras-related protein gene of P. infestans. In comparison with the PCR-based assays, the LAMP technique led to higher specificity and sensitivity, using uncomplicated equipment with an equivalent time frame. All 43 P. infestans isolates, yielded positive detection results using LAMP assay showing no cross reaction with other Phytophthora spp., oomycetes or fungal pathogens. The LAMP assay yielded the lowest detectable DNA concentration (1.28 × 10-4 ng μL-1, being 10 times more sensitive than nested PCR (1.28 × 10-3 ng μL-1, 100 times more sensitive than real-time PCR (1.28 × 10-2 ng μL-1 and 103 times more sensitive than the conventional PCR assay (1.28 × 10-1 ng μL-1. In the field experiment, the LAMP assay outperformed the other tests by amplifying only diseased tissues (leaf and stem, and showing no positive reaction in healthy tissues. Overall, the LAMP assay developed in this study provides a specific, sensitive, simple, and effective visual method for detection of the P. infestans pathogen, and is therefore suitable for application in early prediction of the disease to reduce the risk of epidemics.

  18. Evaluation of Different PCR-Based Assays and LAMP Method for Rapid Detection of Phytophthora infestans by Targeting the Ypt1 Gene.

    Science.gov (United States)

    Khan, Mehran; Li, Benjin; Jiang, Yue; Weng, Qiyong; Chen, Qinghe

    2017-01-01

    Late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating diseases affecting potato and tomato worldwide. Early diagnosis of the P. infestans pathogen causing late blight should be the top priority for addressing disease epidemics and management. In this study, we performed a loop-mediated isothermal amplification (LAMP) assay, conventional polymerase chain reaction (PCR), nested PCR, and real-time PCR to verify and compare the sensitivity and specificity of the reaction based on the Ypt1 (Ras-related protein) gene of P. infestans. In comparison with the PCR-based assays, the LAMP technique led to higher specificity and sensitivity, using uncomplicated equipment with an equivalent time frame. All 43 P. infestans isolates, yielded positive detection results using LAMP assay showing no cross reaction with other Phytophthora spp., oomycetes or fungal pathogens. The LAMP assay yielded the lowest detectable DNA concentration (1.28 × 10-4 ng μL-1), being 10 times more sensitive than nested PCR (1.28 × 10-3 ng μL-1), 100 times more sensitive than real-time PCR (1.28 × 10-2 ng μL-1) and 103 times more sensitive than the conventional PCR assay (1.28 × 10-1 ng μL-1). In the field experiment, the LAMP assay outperformed the other tests by amplifying only diseased tissues (leaf and stem), and showing no positive reaction in healthy tissues. Overall, the LAMP assay developed in this study provides a specific, sensitive, simple, and effective visual method for detection of the P. infestans pathogen, and is therefore suitable for application in early prediction of the disease to reduce the risk of epidemics.

  19. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan PCR Assay

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    Hua-Ying Fu

    2016-01-01

    Full Text Available Ratoon stunting disease (RSD of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx. A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR and a fluorogenic probe (Pat1-QP targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7% of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174 were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174 were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

  20. Multicenter Evaluation of Anyplex Plus MTB/NTM MDR-TB Assay for Rapid Detection of Mycobacterium tuberculosis Complex and Multidrug-Resistant Isolates in Pulmonary and Extrapulmonary Specimens.

    Science.gov (United States)

    Sali, Michela; De Maio, Flavio; Caccuri, Francesca; Campilongo, Federica; Sanguinetti, Maurizio; Fiorentini, Simona; Delogu, Giovanni; Giagulli, Cinzia

    2016-01-01

    The rapid diagnosis of tuberculosis (TB) and the detection of drug-resistant Mycobacterium tuberculosis strains are critical for successful public health interventions. Therefore, TB diagnosis requires the availability of diagnostic tools that allow the rapid detection of M. tuberculosis and drug resistance in clinical samples. Here, we performed a multicenter study to evaluate the performance of the Seegene Anyplex MTB/NTM MDR-TB assay, a new molecular method based on a multiplex real-time PCR system, for detection of Mycobacterium tuberculosis complex (MTBC), nontuberculous mycobacteria (NTM), and genetic determinants of drug resistance. In total, the results for 755 samples (534 pulmonary and 221 extrapulmonary samples) were compared with the results of smears and cultures. For pulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 86.4% and 75.0%, respectively, and the specificities were 99% and 99.4%. For extrapulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 83.3% and 50.0%, respectively, and the specificities of both were 100%. The negative and positive predictive values of the Anyplex assay for pulmonary specimens were 97% and 100%, respectively, and those for extrapulmonary specimens were 84.6% and 100%. The sensitivities of the Anyplex assay for detecting isoniazid resistance in MTBC strains from pulmonary and extrapulmonary specimens were 83.3% and 50%, respectively, while the specificities were 100% for both specimen types. These results demonstrate that the Anyplex MTB/NTM MDR-TB assay is an efficient and rapid method for the diagnosis of pulmonary and extrapulmonary TB and the detection of isoniazid resistance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. 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

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    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

  2. A rapid method for the detection of representative coliforms in water samples: polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA).

    Science.gov (United States)

    Kuo, Jong-Tar; Cheng, Chiu-Yu; Huang, Hsiao-Han; Tsao, Chia-Fen; Chung, Ying-Chien

    2010-03-01

    Methods to detect the presence of coliform bacteria in drinking water usually involve a series of complex cultivating steps that are time-consuming and subject to external influences. For this reason, the new 16S rRNA probe has been developed in this study as an alternative detector PCR-ELISA technique that does not involve the culture of bacteria and that is able to detect, identify, and quantify the representative coliform species present in water samples. Our results indicate that this technique is both rapid (detection time of 4 h) and accurate (1.4% error rate). The limit of detection (LOD) was 5 CFU/100 ml for total coliforms, which meets the standards set by most countries for drinking water. Our comparative study demonstrated that this PCR-ELISA method is superior to current conventional methods in terms of detection time, LOD, and accuracy.

  3. Performance of the Real Fungus-ID kit based on multiplex RT-PCR assay for the rapid detection and identification of Trichophyton spp. and Microsporum spp. in clinical specimens with suspected dermatophyte infection.

    Science.gov (United States)

    Wang, H-Y; Kim, H; Choi, E H; Lee, H

    2016-01-01

    The aim of this study was to evaluate the performance of a commercially available multiplex RT-PCR assay for the rapid detection and identification of dermatophytes directly from clinical samples and cultures. The multiplex RT-PCR assay was used to evaluate 118 clinical isolates from various specimen types and a total of 140 known specimens were compared with both conventional methods, commercially available PCR-REBA, and ITS sequence analysis. In this study, multiplex RT-PCR assay yield significantly more positive results than culture (91·9 vs 39·5%) and conventional methods including KOH microscopy (91·9 vs 71·3%). Although the results among the multiplex RT-PCR, PCR-REBA and ITS sequence analysis were concordant (100%) in 118 clinical isolates, concordant results between multiplex RT-PCR assay and culture were at 66% (78/118). The overall positive rates for the PCR-REBA, multiplex RT-PCR assay and ITS sequence analysis were 98·8, 91·9, and 52·9% respectively. In addition, the concordance rate of multiplex RT-PCR assay and the PCR-REBA assay was 93% (95% confidence interval (CI), 89·9-96·1, P culture can take up to 2-3 weeks. The use of the multiplex RT-PCR molecular diagnostic assay was rapid and reliable for detecting pathogen infections. Even though the use of molecular diagnostic technology is more expensive than conventional methods, the clinical and economic benefit of saving time relative to expense remains to be elucidated. Therefore, the multiplex RT-PCR assay may provide the essential information to accelerate therapeutic decisions for earlier and adequate antibiotic treatment in the acute phase of fungal pathogen infections. © 2015 The Society for Applied Microbiology.

  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. 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

  6. Rapid Detection of Bloodstream Pathogens in Liver Transplantation Patients With FilmArray Multiplex Polymerase Chain Reaction Assays: Comparison With Conventional Methods.

    Science.gov (United States)

    Otlu, B; Bayindir, Y; Ozdemir, F; Ince, V; Cuglan, S; Hopoglu, M; Yakupogullari, Y; Kizilkaya, C; Kuzucu, C; Isık, B; Yilmaz, S

    2015-01-01

    Bloodstream infection (BSI) is an important concern in transplant patients. Early intervention with appropriate antimicrobial therapy is critical to better clinical outcome; however, there is significant delay when conventional identification methods are used. We aimed to determine the diagnostic performance of the FilmArray Blood Culture Identification Panel, a recently approved multiplex polymerase chain reaction assay detecting 24 BSI pathogens and 3 resistance genes, in comparison with the performances of conventional identification methods in liver transplant (LT) patients. A total of 52 defined sepsis episodes (signal-positive by blood culture systems) from 45 LT patients were prospectively studied. The FilmArray successfully identified 37 of 39 (94.8%) bacterial and 3 of 3 (100%) yeast pathogens in a total of 42 samples with microbial growth, failing to detect only 2 of 39 (5.1%) bacterial pathogens that were not covered by the test panel. The FilmArray could also detect additional pathogens in 3 samples that had been reported as having monomicrobial growth, and it could detect Acinetobacter baumannii in 2 samples suspected of skin flora contamination. The remaining 8 blood cultures showing a positive signal but yielding no growth were also negative by this assay. Results of MecA, KPC, and VanA/B gene detection were in high accordance. The FilmArray produced results with significantly shorter turnaround times (1.33 versus 36.2, 23.6, and 19.5 h; P FilmArray appeared as a reliable alternative diagnostic method with the potential to mitigate problems with protracted diagnosis of the BSI pathogens in LT patients. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. 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.

  8. Development and evaluation of an up-converting phosphor technology-based lateral flow assay for rapid detection of Francisella tularensis.

    Science.gov (United States)

    Hua, Fei; Zhang, Pingping; Zhang, Fuli; Zhao, Yong; Li, Chunfeng; Sun, Chongyun; Wang, Xiaochen; Yang, Ruifu; Wang, Chengbin; Yu, Ailian; Zhou, Lei

    2015-11-26

    Francisella tularensis is a potential biowarfare/bioterrorism agent and zoonotic pathogen that causes tularemia; thus, surveillance of F. tularensis and first-level emergency response using point-of-care testing (POCT) are essential. The UPT-LF POCT assay was established to quantitatively detect F. tularensis within 15 min, and the sensitivity of the assay was 10(4) CFU · mL(-1) (100 CFU/test). The linear quantitative range covered five orders of magnitude, and the coefficients of variation were less than 10%. Except Shigella dysenteriae, UPT-LF showed excellent specificity to four strains that are also potential biowarfare/bioterrorism agents and 13 food-borne pathogenic strains. Samples with pH 2-13, high ion strengths (≥ 2 mol · L(-1) solution of KCl and NaCl), high viscosities (≤ 50 mg · mL(-1) PEG20000 or ≥ 20% glycerol), and high concentrations of biomacromolecules (≥ 400 mg · mL(-1) bovine serum albumin or ≥ 80 mg · mL(-1) casein) showed little influence on the assay. For practical utilization, the tolerance limits for seven powders and eight viscera were determined, and operation errors of liquid measurement demonstrated a minor influence on the strip. Ftu-UPT-LF is a candidate POCT method because of its excellent sensitivity, specificity, and stability in complex samples, as well as low operation error.

  9. Development and application of a quantitative real-time PCR assay for rapid detection of the multifaceted yeast Kazachstania servazzii in food.

    Science.gov (United States)

    Spanoghe, Martin; Godoy Jara, Mario; Rivière, John; Lanterbecq, Deborah; Gadenne, Martine; Marique, Thierry

    2017-04-01

    The beneficial contributions of Kazachstania servazzii are well-established in various food processes. This yeast also contributes in the spoilage of finished packaged food due to abundant gas production. In particular, an occurrence of K. servazzii was recently positively correlated with the formation of severe package swelling of some prepared fresh pizzas. To circumscribe this concern, a quantitative SYBR green real-time PCR assay based on a newly designed specific primer pair targeting the ribosomal ITS1-5.8S-ITS2 region of K. servazzii was developed. The quantification was enabled using a standard curve created from serially diluted plasmids containing the target sequence of the K. servazzii strain. A validation of the assay was achieved by enumeration of K. servazzii DNA copies from artificially infected culture broths containing non-contaminated pizza substrates. The newly developed method was then tested on total DNA extracted from packaged fresh pizzas, in which certain lots were swollen and thus suspected of containing K. servazzii. This study highlights that this newly developed quantitative assay is not only sufficiently sensitive, specific and reliable to be functionally used in food control as a routine method of detection, but also promising in specific studies that seek to further characterize the dynamic of this yeast in some increasingly popular food processes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Use of a commercial enzyme-linked immunosorbent assay for rapid detection of Giardia duodenalis in dog stools in the environment: a Bayesian evaluation.

    Science.gov (United States)

    Papini, Roberto; Carreras, Giulia; Marangi, Marianna; Mancianti, Francesca; Giangaspero, Annunziata

    2013-05-01

    Giardia duodenalis is considered a potentially zoonotic protozoan. Some animal species, including infected dogs, may play an important role in the spread of Giardia cysts through environmental contamination with their feces. In the present study, a commercial enzyme-linked immunosorbent assay (ELISA) was used to examine 143 samples of dog feces collected in urban areas as an indicator of the risk of field contamination. Using a Bayesian statistical approach, the ELISA showed a sensitivity of 88.9% and a specificity of 95.8% with positive and negative predictive values of 89.6% and 95.4%, respectively. The test affords the advantage of rapid processing of fecal samples without a complex technical structure and extensive costly labor. Moreover, the present results show that the assay provides public health veterinarians with a practical tool that can be used in screening programs, as a valid alternative or as an adjunct to other tests, in order to assess the biological risk of exposure to G. duodenalis cysts from dogs in human settlements. However, the test may not be completely accurate for human health risk evaluation, as it does not discriminate between zoonotic and non-zoonotic isolates.

  11. 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

  12. Development and evaluation of an up-converting phosphor technology-based lateral flow assay for the rapid, simultaneous detection of Vibrio cholerae serogroups O1 and O139.

    Science.gov (United States)

    Hao, Min; Zhang, Pingping; Li, Baisheng; Liu, Xiao; Zhao, Yong; Tan, Hailing; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Qiu, Haiyan; Wang, Duochun; Diao, Baowei; Jing, Huaiqi; Yang, Ruifu; Kan, Biao; Zhou, Lei

    2017-01-01

    Vibrio cholerae serogroups O1 and O139 are etiological agents of cholera, a serious and acute diarrheal disease, and rapid detection of V. cholerae is a key method for preventing and controlling cholera epidemics. Here, a point of care testing (POCT) method called Vch-UPT-LF, which is an up-converting phosphor technology-based lateral flow (UPT-LF) assay with a dual-target detection mode, was developed to detect V. cholerae O1 and O139 simultaneously from one sample loading. Although applying an independent reaction pair made both detection results for the two Vch-UPT-LF detection channels more stable, the sensitivity slightly declined from 104 to 105 colony-forming units (CFU) mL-1 compared with that of the single-target assay, while the quantification ranges covering four orders of magnitude were maintained. The strip showed excellent specificity for seven Vibrio species that are highly related genetically, and nine food-borne species whose transmission routes are similar to those of V. cholerae. The legitimate arrangement of the two adjacent test lines lessened the mutual impact of the quantitation results between the two targets, and the quantification values did not differ by more than one order of magnitude when the samples contained high concentrations of both V. cholerae O1 and O139. Under pre-incubation conditions, 1×101 CFU mL-1 of V. cholerae O1 or O139 could be detected in fewer than 7 h, while the Vch-UPT-LF assay exhibited sensitivity as high as a real-time fluorescent polymerase chain reaction with fewer false-positive results. Therefore, successful development of Vch-UPT-LF as a dual-target assay for quantitative detection makes this assay a good candidate POCT method for the detection and surveillance of epidemic cholera.

  13. Short communication: A novel method using immunomagnetic separation with a fluorescent nanobeads lateral flow assay for the rapid detection of low-concentration Escherichia coli O157:H7 in raw milk.

    Science.gov (United States)

    Huang, Zhen; Cui, Xi; Xie, Quan-Yuan; Liu, Dao-Feng; Lai, Wei-Hua

    2016-12-01

    Escherichia coli O157:H7 is an important serotype of enterohemorrhagic E. coli that was first identified as a human pathogen in 1982. This pathogen causes several serious diseases. In this study, immunomagnetic separation was coupled with a fluorescent nanobeads lateral flow assay to establish a sensitive and rapid detection method for Escherichia coli O157:H7 in raw milk. The pathogen was captured from raw milk by immunomagnetic separation with immunomagnetic nanobeads and then detected using a fluorescent nanobeads lateral flow assay. A fluorescent line was formed in the test line of the test strip and quantitatively detected using a fluorescent reader. Screening times, which included immunomagnetic separation and the fluorescent nanobeads lateral flow assay, were 8, 7, 6, and 5h when 1, 5, 25, and 125 cfu of E. coli O157:H7, respectively, were inoculated into 25mL of raw milk. The established method could be widely applied to the rapid onsite detection of other pathogens to ensure food safety. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  14. Comparison of multiplex real-time PCR and PCR-reverse blot hybridization assay for the direct and rapid detection of bacteria and antibiotic resistance determinants in positive culture bottles.

    Science.gov (United States)

    Wang, Hye-Young; Kim, Seoyong; Kim, Jungho; Park, Soon Deok; Kim, Hyo Youl; Uh, Young; Lee, Hyeyoung

    2016-09-01

    The aim of this study was to evaluate the performance of a commercially available multiplex real-time PCR assay and a PCR-reverse blot hybridization assay (PCR-REBA) for the rapid detection of bacteria and identification of antibiotic resistance genes directly from blood culture bottles and to compare the results of these molecular assays with conventional culture methods. The molecular diagnostic methods were used to evaluate 593 blood culture bottles from patients with bloodstream infections. The detection positivity of multiplex real-time PCR assay for Gram-positive bacteria, Gram-negative bacteria and Candida spp. was equivalent to PCR-REBA as 99.6 %, 99.1 % and 100 %, respectively. Using conventional bacterial cultures as the gold standard, the sensitivity, specificity, positive predictive value and negative predictive value of these two molecular methods were 99.5 % [95 % confidence interval (CI), 0.980-1.000; PReal-methicillin-resistant Staphylococcusaureus multiplex real-time PCR assay targeting the mecA gene to detect methicillin resistance was lower than that of the PCR-REBA method, detecting an overall positivity of 98.4 % (n=182; 95 % CI, 0.964-1.000; P<0.009) and 99.5 % (n=184; 95 % CI, 0.985-1.000; P<0.0001), respectively. The entire two methods take about 3 h, while results from culture can take up to 48-72 h. Therefore, the use of these two molecular methods was rapid and reliable for the characterization of causative pathogens in bloodstream infections.

  15. An experimental study for rapid detection and quantification of endodontic microbiota following photo-activated disinfection via new multiplex real-time PCR assay.

    Science.gov (United States)

    Pourhajibagher, Maryam; Raoofian, Reza; Ghorbanzadeh, Roghayeh; Bahador, Abbas

    2018-01-11

    The infected root canal system harbors one of the highest accumulations of polymicrobial infections. Since the eradication of endopathogenic microbiota is a major goal in endodontic infection therapy, photo-activated disinfection (PAD) can be used as an alternative therapeutic method in endodontic treatment. Compared to cultivation-based approaches, molecular techniques are more reliable for identifying microbial agents associated with endodontic infections. The purpose of this study was to evaluate the ability of designed multiplex real-time PCR protocol for the rapid detection and quantification of six common microorganisms involved in endodontic infection before and after the PAD. Samples were taken from the root canals of 50 patients with primary and secondary/persistent endodontic infections using sterile paper points. PAD with toluidine blue O (TBO) plus diode laser was performed on root canals. Resampling was then performed, and the samples were transferred to transport medium. Then, six target microorganisms were detected using multiplex real-time PCR before and after the PAD. Veillonella parvula was found using multiplex real-time PCR to have the highest frequency among samples collected before the PAD (29.4%), followed by Porphyromonas. gingivalis (23.1%), Aggregatibacter actinomycetemcomitans (13.6%), Actinomyces naeslundii (13.0%), Enterococcus faecalis (11.5%), and Lactobacillus rhamnosus (9.4%). After TBO-mediated PAD, P. gingivalis strains, the most resistance microorganisms, were recovered in 41.7% of the samples using molecular approach (P > 0.05). As the results shown, multiplex real-time PCR as an accurate detection approach with high-throughput and TBO-mediated PAD as an efficient antimicrobial strategy due to the significant reduction of the endopathogenic count can be used for detection and treatment of microbiota involved in infected root canals, respectively. Copyright © 2018. Published by Elsevier B.V.

  16. A Real-Time PCR Assay Based on 5.8S rRNA Gene (5.8S rDNA) for Rapid Detection of Candida from Whole Blood Samples.

    Science.gov (United States)

    Guo, Yi; Yang, Jing-Xian; Liang, Guo-Wei

    2016-06-01

    The prevalence of Candida in bloodstream infections (BSIs) has increased. To date, the identification of Candida in BSIs still mainly relies on blood culture and serological tests, but they have various limitations. Therefore, a real-time PCR assay for the detection of Candida from whole blood is presented. The unique primers/probe system was designed on 5.8S rRNA gene (5.8S rDNA) of Candida genus. The analytical sensitivity was determined by numbers of positive PCRs in 12 repetitions. At the concentration of 10(1) CFU/ml blood, positive PCR rates of 100 % were obtained for C. albicans, C. parapsilosis, C. tropicalis, and C. krusei. The detection rate for C. glabrata was 75 % at 10(1) CFU/ml blood. The reaction specificity was 100 % when evaluating the assay using DNA samples from clinical isolates and human blood. The maximum CVs of intra-assay and inter-assay for the detection limit were 1.22 and 2.22 %, respectively. To assess the clinical applicability, 328 blood samples from 82 patients were prospectively tested and real-time PCR results were compared with results from blood culture. Diagnostic sensitivity of the PCR was 100 % using as gold standard blood culture, and specificity was 98.4 %. Our data suggest that the developed assay can be used in clinical laboratories as an accurate and rapid screening test for the Candida from whole blood. Although further evaluation is warranted, our assay holds promise for earlier diagnosis of candidemia.

  17. Benzodiazepine Synthesis and Rapid Toxicity Assay

    Science.gov (United States)

    Fletcher, James T.; Boriraj, Grit

    2010-01-01

    A second-year organic chemistry laboratory experiment to introduce students to general concepts of medicinal chemistry is described. Within a single three-hour time window, students experience the synthesis of a biologically active small molecule and the assaying of its biological toxicity. Benzodiazepine rings are commonly found in antidepressant…

  18. Evaluation of a rapid and simple fourth-generation HIV screening assay for qualitative detection of HIV p24 antigen and/or antibodies to HIV-1 and HIV-2.

    Science.gov (United States)

    Beelaert, G; Fransen, K

    2010-09-01

    The performance was assessed of a new, rapid, visual and qualitative immunoassay for the detection of HIV p24 antigen (Ag) and antibodies (Ab) to HIV-1 and HIV-2. Characterised serum or plasma specimens from patients diagnosed with HIV infection were tested: 179 samples of known Ab-positive patients harbouring different subtypes of HIV-1 (n=154) and HIV-2 (n=25) and 200 samples from individuals not infected with HIV. The assay's Ag sensitivity was assessed by testing HIV seroconversion panels (n=10) and primary HIV infection specimens (n=57). In addition, the influence of the genetic variability of HIV-1 on Ag detection was evaluated using dilutions of culture supernatants infected with different subtypes (n=50). The performance of the rapid test was compared to a "gold standard" testing algorithm with the use of a single Ag ELISA and with the Vironostika((R)) HIV Uni-Form II Ag/Ab test, a fourth-generation ELISA. The new assay, the Determine HIV-1/2 Combo demonstrated 100% (98.2-100.0) Ab specificity (200/200) and 100% (98.0-100.0) Ab sensitivity (179/179). In these samples, the observed Ag sensitivity was 86.6% (58/67) with the Determine HIV-1/2 Combo test and 92.5% (62/67) with the Vironostika compared to the reference single Ag ELISA. The assay could not detect Ag in one group O, one subtype F and two subtype H cell supernatant isolates. None of the HIV-2 Ag could be detected. Copyright 2010 Elsevier B.V. All rights reserved.

  19. Performance evaluation of a rapid molecular diagnostic, MultiCode based, sample-to-answer assay for the simultaneous detection of Influenza A, B and respiratory syncytial viruses

    NARCIS (Netherlands)

    J. Voermans (Jolanda); S. Seven-Deniz; P.L.A. Fraaij (Pieter); A.A. Eijck (Annemiek); M.P.G. Koopmans D.V.M. (Marion); S.D. Pas (Suzan)

    2016-01-01

    textabstractBackground Clinical signs and symptoms of different airway pathogens are generally indistinguishable, making laboratory tests essential for clinical decisions regarding isolation and antiviral therapy. Immunochromatographic tests (ICT) and direct immunofluorescence assays (DFA) have

  20. Detection of Shiga Toxins by Lateral Flow Assay

    OpenAIRE

    Ching, Kathryn H.; He, Xiaohua; Stanker, Larry H.; Lin, Alice V.; McGarvey, Jeffery A.; Hnasko, Robert

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) produce shiga toxins (Stxs) that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA) for the rapid detection of Stxs in <10 min using a pair of monoclonal antibodies that bind epitopes common to Stx1 and six Stx2 variants...

  1. 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

  2. Rapid Automated Sample Preparation for Biological Assays

    Energy Technology Data Exchange (ETDEWEB)

    Shusteff, M

    2011-03-04

    Our technology utilizes acoustic, thermal, and electric fields to separate out contaminants such as debris or pollen from environmental samples, lyse open cells, and extract the DNA from the lysate. The objective of the project is to optimize the system described for a forensic sample, and demonstrate its performance for integration with downstream assay platforms (e.g. MIT-LL's ANDE). We intend to increase the quantity of DNA recovered from the sample beyond the current {approx}80% achieved using solid phase extraction methods. Task 1: Develop and test an acoustic filter for cell extraction. Task 2: Develop and test lysis chip. Task 3: Develop and test DNA extraction chip. All chips have been fabricated based on the designs laid out in last month's report.

  3. Pyrosequencing-Based Assays for Rapid Detection of HER2 and HER3 Mutations in Clinical Samples Uncover an E332E Mutation Affecting HER3 in Retroperitoneal Leiomyosarcoma.

    Science.gov (United States)

    González-Alonso, Paula; Chamizo, Cristina; Moreno, Víctor; Madoz-Gúrpide, Juan; Carvajal, Nerea; Daoud, Lina; Zazo, Sandra; Martín-Aparicio, Ester; Cristóbal, Ion; Rincón, Raúl; García-Foncillas, Jesús; Rojo, Federico

    2015-08-17

    Mutations in Human Epidermal Growth Factor Receptors (HER) are associated with poor prognosis of several types of solid tumors. Although HER-mutation detection methods are currently available, such as Next-Generation Sequencing (NGS), alternative pyrosequencing allow the rapid characterization of specific mutations. We developed specific PCR-based pyrosequencing assays for identification of most prevalent HER2 and HER3 mutations, including S310F/Y, R678Q, L755M/P/S/W, V777A/L/M, 774-776 insertion, and V842I mutations in HER2, as well as M91I, V104M/L, D297N/V/Y, and E332E/K mutations in HER3. We tested 85 Formalin Fixed and Paraffin Embbeded (FFPE) samples and we detected three HER2-V842I mutations in colorectal carcinoma (CRC), ovarian carcinoma, and pancreatic carcinoma patients, respectively, and a HER2-L755M mutation in a CRC specimen. We also determined the presence of a HER3-E332K mutation in an urothelial carcinoma sample, and two HER3-D297Y mutations, in both gastric adenocarcinoma and CRC specimens. The D297Y mutation was previously detected in breast and gastric tumors, but not in CRC. Moreover, we found a not-previously-described HER3-E332E synonymous mutation in a retroperitoneal leiomyosarcoma patient. The pyrosequencing assays presented here allow the detection and characterization of specific HER2 and HER3 mutations. These pyrosequencing assays might be implemented in routine diagnosis for molecular characterization of HER2/HER3 receptors as an alternative to complex NGS approaches.

  4. Prospective and retrospective evaluation of the Cepheid Xpert® Flu/RSV XC assay for rapid detection of influenza A, influenza B, and respiratory syncytial virus.

    Science.gov (United States)

    Salez, Nicolas; Nougairede, Antoine; Ninove, Laetitia; Zandotti, Christine; de Lamballerie, Xavier; Charrel, Remi N

    2015-04-01

    A total of 281 clinical specimens (nasal swabs and nasopharyngeal aspirates) were tested with the Xpert® Flu/RSV XC. The results were compared to those obtained with the real-time retro transcriptase-polymerase chain reaction assays routinely used in our laboratory. The Xpert® Flu/RSV XC showed sensitivity/specificity of 97.8%/100% and 97.9%/100% for flu and respiratory syncytial virus, respectively. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Prospective evaluation of nonstructural 1 enzyme-linked immunosorbent assay and rapid immunochromatographic tests to detect dengue virus in patients with acute febrile illness.

    Science.gov (United States)

    Najioullah, Fatiha; Combet, Emilie; Paturel, Laure; Martial, Jenny; Koulmann, Laurence; Thomas, Laurent; Hatchuel, Yves; Cabié, André; Cesaire, Raymond

    2011-02-01

    We prospectively evaluated the Bio-Rad nonstructural 1 (NS1) enzyme-linked immunosorbent assay (ELISA) and lateral flow immunochromatographic assay (LFIA) in comparison to an in-place reverse transcription-polymerase chain reaction for dengue diagnosis. Among 537 consecutive samples from patients with acute febrile disease, 264 (49.2%) tested positive in reverse transcription-polymerase chain reaction (RT-PCR), 156 (29.1%) in NS1-antigen (Ag) ELISA, and 125 (23.3%) in NS1-Ag LFIA. Compared to the RT-PCR status, the specificity was 100% for the NS1-Ag ELISA and LFIA, but their respective sensitivities were 61.2% [95% confidence interval (CI), 55.2-67.2] and 49.4% (95% CI, 43.2-55.6), with nadirs of 37.9% and 24.1% on day 6 of illness. The NS1-Ag ELISA and LFIA were positive, respectively, for 48.0% and 40.7% of the secondary infections versus 85.0% and 66.7% of the primary infections. For patients LFIA reached respective sensitivities of 100% and 90.5%. Reports of results of dengue NS1-Ag assays should specify that negativity does not preclude DENV infection, and require further investigations in the case of severe disease. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Colloidal gold probe based rapid immunochromatographic strip assay for cortisol

    Energy Technology Data Exchange (ETDEWEB)

    Nara, Seema, E-mail: seemanara@mnnit.ac.in [Department of Applied Mechanics (Biotechnology), Motilal Nehru National Institute of Technology, Allahabad 211004 (India); Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi 110067 (India); Center for BioMedical Engineering, Indian Institute of Technology, New Delhi 110016 (India); Tripathi, Vinay [Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi 110067 (India); Center for BioMedical Engineering, Indian Institute of Technology, New Delhi 110016 (India); Singh, Harpal [Center for BioMedical Engineering, Indian Institute of Technology, New Delhi 110016 (India); Shrivastav, Tulsidas G. [Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi 110067 (India)

    2010-12-03

    A rapid and semi-quantitative immunochromatographic strip (ICS) test for cortisol analysis in serum was developed. The test strip was based on a competitive assay format. Colloidal gold nanoparticles were synthesized and coupled with cortisol-3-carboxymethyloxime-adipic acid dihydrazide-bovine serum albumin (F-3-CMO-ADH-BSA) antigen to directly compete with cortisol in human serum samples. F-3-CMO-ADH-BSA-gold label and uncoupled colloidal gold nanoparticles were appropriately characterized using UV-vis spectroscopy, transmission electron microscopy and atomic force microscopy. Anticortisol antibody raised against F-3-CMO-BSA immunogen in New Zealand white rabbits was coated on the NC membrane as test line. Anti-BSA antibody was used as control line. The lower detection limit of the ICS test was 30 ng mL{sup -1} with visual detection and was completed in 10 min. About 30 human serum samples were also analyzed by the developed strip test and their range of cortisol concentration was established. The developed ICS test is rapid, economic and user friendly.

  7. A new method for the rapid detection of Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus), Alaska pollock (Gadus chalcogrammus) and ling (Molva molva) using a lateral flow dipstick assay.

    Science.gov (United States)

    Taboada, Ledicia; Sánchez, Ana; Pérez-Martín, Ricardo I; Sotelo, Carmen G

    2017-10-15

    Species-specific lateral flow dipstick (LFD) assays for the identification of Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus), Alaska pollock (Gadus chalcogrammus) and ling (Molva molva) in food products were developed. The method comprises a PCR system with four sets of specific primers, for each target species. This step was also devised to dual-labeling of PCR products with biotin and 6-FAM, which are then easily read on a lateral flow dipstick, upon which these products are immobilized by a fixed biotin-ligand and visualized with anti-FAM antibody-coated gold nanoparticles. Sensitivity and selectivity were determined for each of the developed assays. Validation of the assays was performed with DNA extracted from commercial fish products, the identification of all samples by PCR-LFD was coherent with the results found with DNA sequencing. Target species were successfully detected in analyzed commercial samples, demonstrating the applicability of this method to the rapid analysis of food products. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. A homogeneous biochemiluminescent assay for detection of influenza

    Science.gov (United States)

    Hui, Kwok Min; Li, Xiao Jing; Pan, Lu; Li, X. J.

    2015-05-01

    Current methods of rapid detection of influenza are based on detection of the nucleic acids or antigens of influenza viruses. Since influenza viruses constantly mutate leading to appearance of new strains or variants of viruses, these detection methods are susceptible to genetic changes in influenza viruses. Type A and B influenza viruses contain neuraminidase, an essential enzyme for virus replication which enables progeny influenza viruses leave the host cells to infect new cells. Here we describe an assay method, the homogeneous biochemiluminescent assay (HBA), for rapid detection of influenza by detecting viral neuraminidase activity. The assay mimics the light production process of a firefly: a viral neuraminidase specific substrate containing a luciferin moiety is cleaved in the presence of influenza virus to release luciferin, which becomes a substrate to firefly luciferase in a light production system. All reagents can be formulated in a single reaction mix so that the assay involves only one manual step, i.e., sample addition. Presence of Type A or B influenza virus in the sample leads to production of strong, stable and easily detectable light signal, which lasts for hours. Thus, this influenza virus assay is suitable for use in point-of-care settings.

  9. Evaluation of two line probe assays for rapid detection of Mycobacterium tuberculosis, tuberculosis (TB) drug resistance, and non-TB Mycobacteria in HIV-infected individuals with suspected TB.

    Science.gov (United States)

    Luetkemeyer, Anne F; Kendall, Michelle A; Wu, Xingye; Lourenço, Maria Cristina; Jentsch, Ute; Swindells, Susan; Qasba, Sarojini S; Sanchez, Jorge; Havlir, Diane V; Grinsztejn, Beatriz; Sanne, Ian M; Firnhaber, Cynthia

    2014-04-01

    Limited performance data from line probe assays (LPAs), nucleic acid tests used for the rapid diagnosis of tuberculosis (TB), nontuberculosis mycobacteria (NTM), and Mycobacterium tuberculosis drug resistance are available for HIV-infected individuals, in whom paucibacillary TB is common. In this study, the strategy of testing sputum with GenoType MTBDRplus (MTBDR-Plus) and GenoType Direct LPA (Direct LPA) was compared to a gold standard of one mycobacterial growth indicator tube (MGIT) liquid culture. HIV-positive (HIV(+)) individuals with suspected TB from southern Africa and South America with tuberculosis culture positive, of which 276 (72.8%) were acid-fast bacillus (AFB) smear positive. MTBDR-Plus had a sensitivity of 81.0% and a specificity of 100%, with sensitivities of 44.1% in AFB smear-negative versus 94.6% in AFB smear-positive specimens. For specimens that were positive for M. tuberculosis by MTBDR-Plus, the sensitivity and specificity for rifampin resistance were 91.7% and 96.6%, respectively, and for isoniazid (INH) they were 70.6% and 99.1%. The Direct LPA had a sensitivity of 88.4% and a specificity of 94.6% for M. tuberculosis detection, with a sensitivity of 72.5% in smear-negative specimens. Ten of 639 MGIT cultures grew Mycobacterium avium complex or Mycobacterium kansasii, half of which were detected by Direct LPA. Both LPA assays performed well in specimens from HIV-infected individuals, including in AFB smear-negative specimens, with 72.5% sensitivity for M. tuberculosis identification with the Direct LPA and 44.1% sensitivity with MTBDR-Plus. LPAs have a continued role for use in settings where rapid identification of INH resistance and clinically relevant NTM are priorities.

  10. Detection of Shiga Toxins by Lateral Flow Assay

    Directory of Open Access Journals (Sweden)

    Kathryn H. Ching

    2015-04-01

    Full Text Available Shiga toxin-producing Escherichia coli (STEC produce shiga toxins (Stxs that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA for the rapid detection of Stxs in <10 min using a pair of monoclonal antibodies that bind epitopes common to Stx1 and six Stx2 variants. This LFA provides a rapid and sensitive test for the detection of Stxs directly from STEC culture supernatants or at risk food samples with a 0.1 ng/mL limit of detection (LOD for Stx2a. This Stx LFA is applicable for use in the rapid evaluation of Stx production from cultured E. coli strains or as a tool to augment current methods as part of food safety testing.

  11. Clostridium difficile infection diagnostics - evaluation of the C. DIFF Quik Chek Complete assay, a rapid enzyme immunoassay for detection of toxigenic C. difficile in clinical stool samples.

    Science.gov (United States)

    Johansson, Karin; Karlsson, Hanna; Norén, Torbjörn

    2016-11-01

    Diagnostic testing for Clostridium difficile infection (CDI) has, in recent years, seen the introduction of rapid dual-EIA (enzyme immunoassay) tests combining species-specific glutamate dehydrogenase (GDH) with toxin A/B. In a prospective study, we compared the C. DIFF Quik Chek Complete test to a combination of selective culture (SC) and loop-mediated isothermal amplification (LAMP) of the toxin A gene. Of 419 specimens, 68 were positive in SC including 62 positive in LAMP (14.7%). The combined EIA yielded 82 GDH positives of which 47 were confirmed toxin A/B positive (11%) corresponding to a sensitivity and specificity of 94% for GDH EIA compared to SC and for toxin A/B EIA a sensitivity of 71% and a specificity of 99% compared to LAMP. Twenty different PCR ribotypes were evenly distributed except for UK 081 where only 25% were toxin A/B positive compared to LAMP. We propose a primary use of a combined GDH toxin A/B EIA permitting a sensitive 1-h result of 379 of 419 (90%, all negatives plus GDH and toxin EIA positives) referred specimens. The remaining 10% being GDH positive should be tested for toxin A/B gene on the same day and positive results left to a final decision by the physician. © 2016 APMIS. Published by John Wiley & Sons Ltd.

  12. Simple latex agglutination assay for rapid serodiagnosis of human leptospirosis

    NARCIS (Netherlands)

    Smits, H. L.; van der Hoorn, M. A.; Goris, M. G.; Gussenhoven, G. C.; Yersin, C.; Sasaki, D. M.; Terpstra, W. J.; Hartskeerl, R. A.

    2000-01-01

    A newly developed latex agglutination assay for the detection of genus-specific Leptospira antibodies in human sera was evaluated. The assay is performed by mixing, on an agglutination card, serum with equal volumes of stabilized antigen-coated, dyed test and control latex beads and is read within 2

  13. Detection of Shiga Toxins by Lateral Flow Assay

    Science.gov (United States)

    Ching, Kathryn H.; He, Xiaohua; Stanker, Larry H.; Lin, Alice V.; McGarvey, Jeffery A.; Hnasko, Robert

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) produce shiga toxins (Stxs) that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA) for the rapid detection of Stxs in detection of Stxs directly from STEC culture supernatants or at risk food samples with a 0.1 ng/mL limit of detection (LOD) for Stx2a. This Stx LFA is applicable for use in the rapid evaluation of Stx production from cultured E. coli strains or as a tool to augment current methods as part of food safety testing. PMID:25855129

  14. A simple and rapid plate assay for screening of inulindegrading ...

    African Journals Online (AJOL)

    In this report, a simple and rapid agar plate assay was established for screening of halophilic, inulindegrading microorganisms. Two strains considered inulinolytic with this method were chosen and the inulinolytic activities in their culture supernatant were measured with the Somogyi-Nelson method, while their hydrolysis ...

  15. LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples

    OpenAIRE

    Deb, R.; Sengar, G. S.; Singh, U.; Kumar, S.; Raja, T. V.; Alex, R.; Alyethodi, R. R.; Prakash, B.

    2017-01-01

    Animal species detection is one of the crucial steps for consumer’s food analysis. In the present study we developed an in-house built loop-mediated isothermal amplification (LAMP) assay for rapid detection of adulterated cow DNA in goat milk/meat samples. The cow milk/tissue DNA in goat milk/meat samples were identified in the developed LAMP assay by either naked eye visualizing with SYBR Green I dyes or by detecting the typical ladder pattern on gel electrophoresis. This test can detect up ...

  16. Comparison of the NIDS® rapid assay with ELISA methods in immunogenicity testing of two biotherapeutics.

    Science.gov (United States)

    Pan, Jing; Small, Thomas; Qin, Dujie; Li, Shawn; Wang, Li; Chen, Dave; Pauley, Cindy; Verch, Thorsten; Kaplanski, Catherine; Bakhtiar, Ray; Vallejo, Yli Remo; Yin, Ray

    2011-01-01

    Rapid lateral flow immunogenicity assays for the detection of anti-drug antibodies (ADAs) to two biotherapeutic antibodies, an anti-HER2 antibody and an anti-TNF-α antibody, were developed using ANP Technologies, Inc.'s proprietary Nano-Intelligent Detection System (NIDS®) and compared to their ELISA counterparts. Biotin and hapten-labeled drugs are incubated with the patient serum sample to allow ADA to form a bridge complex with each drug conjugate. The reaction mixture is then added to a test strip with an anti-hapten capture zone which captures the mixed bridge complex. The bridge-complexed biotinylated drug then reacts with streptavidin-labeled gold particles in situ. The signal developed at the capture zone, which is directly proportional to ADA in the sample, is then quantitatively measured with a handheld reader. The counterpart ELISAs were run using the same reagents. Dose-response, specificity/free drug depletion, and screening cut-point assays were performed using both methods. The rapid assays' performance compare very closely to their ELISA counterparts'. Both types of assays identified the same positive samples in screening a limited population of 50 normal serum samples for the anti-HER2 antibody. In the case of anti-TNF-α, both assays identified the same positive samples out of 50 normal and 20 rheumatoid arthritis patient serum samples but differed in the assessment of two others. The rapid assay correctly identified as negative an ELISA false positive sample, and correctly tested as positive an ELISA false negative sample. Positive results were verified with a specificity/free drug depletion assay. The NIDS® rapid immunogenicity assay offers distinct advantages over current methods in simplicity, low cost, and short time to result. More importantly, the method requires no sample dilution and no washing steps which can perturb fragile complexes formed by low-affinity ADAs. Thus, the assay can potentially detect ADAs with various affinities

  17. Reliable, rapid and simple voltammetric detection of urea nitrate explosive.

    Science.gov (United States)

    Cagan, Avi; Lu, Donglai; Cizek, Karel; La Belle, Jeff; Wang, Joseph

    2008-05-01

    A highly selective and rapid electrochemical assay of the improvised explosive urea nitrate (UN) is reported. The method involves a short ( approximately 10 s) acid-catalyzed reaction of UN with 4-nitrotoluene (NT) followed by a rapid ( approximately 2 s) square-wave voltammetric (SWV) detection of the 2,4-dinitrotoluene (DNT) product. The new protocol offers great promise for a reliable field detection of UN, with significant advantages of speed, sensitivity, portability, simplicity, and cost.

  18. Rapid genetic detection of ingested Amanita phalloides.

    Science.gov (United States)

    Gausterer, Christian; Penker, Martina; Krisai-Greilhuber, Irmgard; Stein, Christina; Stimpfl, Thomas

    2014-03-01

    Mushrooms are often poorly digested by humans. Thus, their remains (tissues, spores) may persist in the gastrointestinal tract and can be detected in feces several days after mushroom consumption. In this report, we present protocols for the rapid PCR-based detection of fungal traces in a variety of complex samples. Novel primers were designed to amplify portions of ribosomal DNA from deadly poisonous European members of the genus Amanita, namely the death cap (A. phalloides), the destroying angel (A. virosa) and the fool's mushroom (A. verna), respectively. Assay sensitivity was sufficient to discover diluted DNA traces in amounts below the genomic content of a single target mushroom cell. Specificity testing was performed with DNA extracts from a variety of mushroom species. Template amplification was exclusively observed with intended targets and it was not compromised by a vast excess of non-target DNA (i.e. DNA from human and human fecal origin, respectively). A series of experiments was conducted with prepared specimens in order to follow the course of mushroom food processing and digestion. Amplification by direct PCR was successful with raw, fried and digested mixed mushrooms. To improve assay performance with fecal samples, a rapid protocol for sample pre-processing (including water-ether sedimentation and bead beating) and a modified PCR reaction mix were applied. Thereby, it was possible to detect the presence of A. phalloides DNA in spiked feces as well as in clinical samples (vomit, stool) from two independent cases of suspected mushroom poisoning. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  19. LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples.

    Science.gov (United States)

    Deb, R; Sengar, G S; Singh, U; Kumar, S; Raja, T V; Alex, R; Alyethodi, R R; Prakash, B

    2017-01-01

    Animal species detection is one of the crucial steps for consumer's food analysis. In the present study we developed an in-house built loop-mediated isothermal amplification (LAMP) assay for rapid detection of adulterated cow DNA in goat milk/meat samples. The cow milk/tissue DNA in goat milk/meat samples were identified in the developed LAMP assay by either naked eye visualizing with SYBR Green I dyes or by detecting the typical ladder pattern on gel electrophoresis. This test can detect up to minimum 5% level of cow components admixed in goat milk/meat samples and can be completed within 1 h 40 min starting from DNA extraction from milk/meat samples and can be performed in a water bath. Developed LAMP methodology is simple; rapid and sensitive techniques that can detect adulterant like cow components in goat milk/meat are more accurate than other existing DNA based technologies.

  20. Earthworm dispersal assay for rapidly evaluating soil quality.

    Science.gov (United States)

    Kim, Shin Woong; Kim, Dokyung; Moon, Jongmin; Chae, Yooeun; Kwak, Jin Il; Park, Younsu; Jeong, Seung-Woo; An, Youn-Joo

    2017-10-01

    Earthworms enhance soil functioning and are therefore key species in the soil. Their presence is generally a positive sign for a terrestrial ecosystem, because these species serve as important biomarkers in soil quality evaluations. We describe a novel bioassay, the "dispersal assay," that is a simple and rapid technique for field-based soil quality evaluations. It is based on the premise that earthworms prefer optimal soils if given the choice. Thus, assay tubes containing a reference soil were inserted in target sites, and earthworms were placed into these tubes. According to their soil preference, the earthworms dispersed into the surrounding soil, remained in the initial soil within the tubes, avoided both by crawling up the tube, or died. Furthermore, sensitivity responses to metal concentrations, electrical conductivity, and soil pH were observed in field tests. Although the dispersal assay did not completely match traditional toxicity endpoints such as earthworm survival, we found that it can serve as an in situ screening test for assessing soil quality. Overall, our dispersal assay was relatively rapid (within 24 h), had low levels of variation, and showed high correlations between earthworm behavior and soil physicochemical properties. Environ Toxicol Chem 2017;36:2766-2772. © 2017 SETAC. © 2017 SETAC.

  1. Immunochromatographic Brucella-specific immunoglobulin M and G lateral flow assays for rapid serodiagnosis of human brucellosis

    NARCIS (Netherlands)

    Smits, Henk L.; Abdoel, Theresia H.; Solera, Javier; Clavijo, Encarnacion; Diaz, Ramon

    2003-01-01

    To fulfill the need for a simple and rapid diagnostic test for human brucellosis, we used the immunochromatographic lateral flow assay format to develop two assays, one for the detection of Brucella-specific immunoglobulin M (IgM) antibodies and one for the detection of Brucella-specific IgG

  2. Evaluation of a Rapid Immunochromatographic Treponemal Antibody Test Comparing the Treponema Pallidum Particle Agglutination Assay.

    Science.gov (United States)

    Lee, Jong-Han; Lim, Chae Seung; Lee, Min-Geol; Kim, Hyon-Suk

    2015-09-01

    In addition to conventional tests, several methods for detection of treponema-specific antibodies in clinical settings have been recently introduced. We aim to comparatively evaluate a rapid immunochromatographic test (ICT) for Treponema pallidum specific antibody (SD Bioline Syphilis 3.0) and the T. pallidum particle agglutination (TPPA) assay. In all, 132 serum samples from 78 syphilis patients and 54 syphilis-negative controls were analyzed. SD Bioline Syphilis 3.0 test (Standard Diagnostic, Inc., Yongin, Korea) was evaluated and compared to Serodia TPPA assay (Fujirebio, Inc., Tokyo, Japan). All discrepant results between the two assays were repeatedly tested and evaluated by the fluorescent treponemal antibody-absorption (FTA-ABS) assay. Test reproducibility and 95% limit of detection of SD Bioline Syphilis 3.0 were determined across three different lots for seven consecutive days in triplicate. Interference due to autoantibodies and pregnancy was also tested. Percent agreement between SD Bioline Syphilis 3.0 and TPPA assays was 99.2%. Sensitivity and specificity were 100%, respectively. In TPPA assay, test-to-test, day-to-day, and lot-to-lot variations were not identified until 1:320 titer (eightfold dilutions). There was no interference due to the presence of antinuclear antibodies or samples or pregnancy. Percent agreement of SD Syphilis 3.0 and TPPA was very good. Sensitivity and specificity were appropriate for T. pallidum antibody detection. Thus, a rapid ICT could be suitable for syphilis antibody detection. © 2014 Wiley Periodicals, Inc.

  3. Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of Citrus tristeza virus, Citrus psorosis virus, and Citrus leaf blotch virus.

    Science.gov (United States)

    Osman, Fatima; Hodzic, Emir; Kwon, Sun-Jung; Wang, Jinbo; Vidalakis, Georgios

    2015-08-01

    A single real-time multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) was developed and validated using three different fluorescently labeled minor groove binding qPCR probes. To increase the detection reliability, coat protein (CP) genes from large number of different isolates of CTV, CPsV and CLBV were sequenced and a multiple sequence alignment was generated with corresponding CP sequences from the GenBank and a robust multiplex RT-qPCR assay was designed. The capacity of the multiplex RT-qPCR assay in detecting the viruses was compared to singleplex RT-qPCR designed specifically for each virus and was assessed using multiple virus isolates from diverse geographical regions and citrus species as well as graft-inoculated citrus plants infected with various combination of the three viruses. No significant difference in detection limits was found and specificity was not affected by the inclusion of the three assays in a multiplex RT-qPCR reaction. Comparison of the viral load for each virus using singleplex and multiplex RT-qPCR assays, revealed no significant differences between the two assays in virus detection. No significant difference in Cq values was detected when using one-step and two-step multiplex RT-qPCR detection formats. Optimizing the RNA extraction technique for citrus tissues and testing the quality of the extracted RNA using RT-qPCR targeting the cytochrome oxidase citrus gene as an RNA specific internal control proved to generate better diagnostic assays. Results showed that the developed multiplex RT-qPCR can streamline viruses testing of citrus nursery stock by replacing three separate singleplex assays, thus reducing time and labor while retaining the same sensitivity and specificity. The three targeted RNA viruses are regulated pathogens for California's mandatory "Section 3701

  4. Rapid and Sensitive Detection of BLAD in Cattle Population

    Directory of Open Access Journals (Sweden)

    Daniela Elena Ilie

    2014-05-01

    Full Text Available Bovine leukocyte adhesion deficiency (BLAD is an autosomal recessive disorder with negative impact on dairy cattle breeding. The molecular basis of BLAD is a single point mutation (A→G, resulting in a single amino acid substitution (aspartic acid → glycine at amino acid 128 in the adhesion molecule CD18. The object of this study was to establish a fast and sensitive molecular genotyping assay to detect BLAD carriers using high-resolution melting (HRM curve analysis. We tested animals with known genotypes for BLAD that were previously confirmed by PCR-RFLP method, and then examined the sensitivity of mutation detection using PCR followed by HRM curve analysis. BLAD carriers were readily detectable using HRM assay. Thus, the PCR-HRM genotyping method is a rapid, easily interpretable, reliable and cost-effective assay for BLAD mutant allele detection. This assay can be useful in cattle genotyping and genetic selection.

  5. Rapid Detection of the Varicella Zoster Virus

    Science.gov (United States)

    Lewis, Michelle P.; Harding, Robert

    2011-01-01

    1.Technology Description-Researchers discovered that when the Varicella Zoster Virus (VZV) reactivates from latency in the body, the virus is consistently present in saliva before the appearance of skin lesions. A small saliva sample is mixed with a specialized reagent in a test kit. If the virus is present in the saliva sample, the mixture turns a red color. The sensitivity and specificity emanates from an antibody-antigen reaction. This technology is a rapid, non-invasive, point of-of-care testing kit for detecting the virus from a saliva sample. The device is easy to use and can be used in clinics and in remote locations to quickly detect VZV and begin treatment with antiviral drugs. 2.Market Opportunity- RST Bioscience will be the first and only company to market a rapid, same day test kit for the detection of VZV in saliva. The RST detection test kit will have several advantages over existing, competitive technology. The test kit is self contained and laboratory equipment is not required for analysis of the sample. Only a single saliva sample is required to be taken instead of blood or cerebral spinal fluid. The test kit is portable, sterile and disposable after use. RST detection test kits require no electrical power or expensive storage equipment and can be used in remote locations. 3.Market Analysis- According to the CDC, it is estimated that 1 million cases of shingles occur each year in the U.S. with more than half over the age of sixty. There is a high demand for rapid diagnostics by the public. The point-of-care testing (POCT) market is growing faster than other segments of in vitro diagnostics. According to a July 2007 InteLab Corporation industry report the overall market for POCT was forecast to increase from $10.3 billion in 2005 to $18.7 billion by 2011. The market value of this test kit has not been determined. 4.Competition- The VZV vaccine prevents 50% of cases and reduces neuralgia by 66%. The most popular test detects VZV-specific IgM antibody

  6. Universal fieldable assay with unassisted visual detection

    Science.gov (United States)

    Chelyapov, Nicolas (Inventor)

    2012-01-01

    A universal detection system based on allosteric aptamers, signal amplification cascade, and eye-detectable phrase transition. A broadly applicable homogeneous detection system is provided. It utilizes components of the blood coagulation cascade in the presence of polystyrene microspheres (MS) as a signal amplifier. Russell's viper venom factor X activator (RVV-X) triggers the cascade, which results in an eye-visible phase transition--precipitation of MS bound to clotted fibrin. An allosteric RNA aptamer, RNA132, with affinity for RVV-X and human vascular endothelial growth factor (VEGF.sub.165) was created. RNA132 inhibits enzymatic activity of RVV-X. The effector molecule, VEGF.sub.165, reverses the inhibitory activity of RNA132 on RVV-X and restores its enzymatic activity, thus triggering the cascade and enabling the phase transition. Similar results were obtained for another allosteric aptamer modulated by a protein tyrosine phosphatase. The assay is instrumentation-free for both processing and readout.

  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▿

    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

  8. 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.

  9. A Rapid Zika Diagnostic Assay to Measure Neutralizing Antibodies in Patients

    Directory of Open Access Journals (Sweden)

    Chao Shan

    2017-03-01

    Full Text Available The potential association of microcephaly and other congenital abnormalities with Zika virus (ZIKV infection during pregnancy underlines the critical need for a rapid and accurate diagnosis. Due to the short duration of ZIKV viremia in infected patients, a serologic assay that detects antibody responses to viral infection plays an essential role in diagnosing patient specimens. The current serologic diagnosis of ZIKV infection relies heavily on the labor-intensive Plaque Reduction Neutralization Test (PRNT that requires more than one-week turnaround time and represents a major bottleneck for patient diagnosis. To overcome this limitation, we have developed a high-throughput assay for ZIKV and dengue virus (DENV diagnosis that can attain the “gold standard” of the current PRNT assay. The new assay is homogeneous and utilizes luciferase viruses to quantify the neutralizing antibody titers in a 96-well format. Using 91 human specimens, we showed that the reporter diagnostic assay has a higher dynamic range and maintains the relative specificity of the traditional PRNT assay. Besides the improvement of assay throughput, the reporter virus technology has also shortened the turnaround time to less than two days. Collectively, our results suggest that, along with the viral RT-PCR assay, the reporter virus-based serologic assay could be potentially used as the first-line test for clinical diagnosis of ZIKV infection as well as for vaccine clinical trials.

  10. Rapid methods for detection of bacteria

    DEFF Research Database (Denmark)

    Corfitzen, Charlotte B.; Andersen, B.Ø.; Miller, M.

    2006-01-01

    Traditional methods for detection of bacteria in drinking water e.g. Heterotrophic Plate Counts (HPC) or Most Probable Number (MNP) take 48-72 hours to give the result. New rapid methods for detection of bacteria are needed to protect the consumers against contaminations. Two rapid methods...

  11. Detection and identification of aquatic birnaviruses by PCR assay.

    Science.gov (United States)

    Blake, S L; Schill, W B; McAllister, P E; Lee, M K; Singer, J T; Nicholson, B L

    1995-04-01

    A reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for the detection and identification of aquatic birnaviruses. The four sets of primers (PrA, PrB, PrC, and PrD) that we used are specific for regions of cDNA coded by genome segment A of aquatic birnaviruses. PrA identifies a large fragment (1,180 bp) within the pVP2-coding region, and PrB identifies a 524-bp fragment within the sequence amplified by PrA. Primer set PrC frames a genome fragment (339 bp) within the NS-VP3-coding region, and PrD identifies a 174-bp sequence within the fragment identified by PrC. PrB and PrD amplified cDNAs from all nine recognized serotypes of aquatic birnavirus serogroup A as well as the N1 isolate that may represent a 10th serotype. These results indicate that these three primer sequences are highly conserved and can be used in PCR assays for group identification of these viruses. PrA routinely produced amplification products from eight serotypes but exhibited variable results with one serotype, and primer PrC identified 6 of the 11 virus isolates tested. The qualitative sensitivity of the RT-PCR assay was evaluated by comparison of the results with those of cell culture isolation assays. With the exception of one sample, the RT-PCR assay with primer PrD was as accurate as cell culture isolation for detecting virus in kidney and spleen tissues from naturally infected, asymptomatic carrier fish. These results indicate that the RT-PCR assay can be a rapid and reliable substitute for cell culture methods for the detection of aquatic birnaviruses.

  12. Rapid extraction and assay of uranium from environmental surface samples

    Energy Technology Data Exchange (ETDEWEB)

    Barrett, Christopher A.; Chouyyok, Wilaiwan; Speakman, Robert J.; Olsen, Khris B.; Addleman, Raymond Shane

    2017-10-01

    Extraction methods enabling faster removal and concentration of uranium compounds for improved trace and low-level assay are demonstrated for standard surface sampling material in support of nuclear safeguards efforts, health monitoring, and other nuclear analysis applications. A key problem with the existing surface sampling swipes is the requirement for complete digestion of sample and sampling matrix. This is a time-consuming and labour-intensive process that limits laboratory throughput, elevates costs, and increases background levels. Various extraction methods are explored for their potential to quickly and efficiently remove different chemical forms of uranium from standard surface sampling material. A combination of carbonate and peroxide solutions is shown to give the most rapid and complete form of uranyl compound extraction and dissolution. This rapid extraction process is demonstrated to be compatible with standard inductive coupled plasma mass spectrometry methods for uranium isotopic assay as well as screening techniques such as x-ray fluorescence. The general approach described has application beyond uranium to other analytes of nuclear forensic interest (e.g., rare earth elements and plutonium) as well as heavy metals for environmental and industrial hygiene monitoring.

  13. Bioluminescence inhibition assay for the detection of hydroxylated polychlorinated biphenyls.

    Science.gov (United States)

    Teasley Hamorsky, Krystal; Ensor, C Mark; Dikici, Emre; Pasini, Patrizia; Bachas, Leonidas; Daunert, Sylvia

    2012-09-18

    Hydroxylated polychlorinated biphenyls (OH-PCBs) are an important class of contaminants that mainly originate from polychlorinated biphenyl metabolism. They may conceivably be as dangerous and persistent as the parent compounds; most prominently, OH-PCBs are endocrine disruptors. Due to increasing evidence of the presence of OH-PCBs in the environment and in living organisms, including humans, and of their toxicity, methods of detection for OH-PCBs are needed in the environmental and medical fields. Herein, we describe the development and optimization of a protein-based inhibition assay for the quantification of OH-PCBs. Specifically, the photoprotein aequorin was utilized for the detection of OH-PCBs. We hypothesized that OH-PCBs interact with aequorin, and we established that OH-PCBs actually inhibit the bioluminescence of aequorin in a dose-dependent manner. We took advantage of this phenomenon to develop an assay that is capable of detecting a wide variety of OH-PCBs with a range of detection limits, the best detection limit being 11 nM for the compound 2-hydroxy-2',3,4',5',6-pentachorobiphenyl. The viability of this system for the screening of OH-PCBs in spiked biological and environmental samples was also established. We envision the implementation of this novel bioluminescence inhibition assay as a rapid, sensitive, and cost-effective method for monitoring OH-PCBs. Furthermore, to the best of our knowledge, this is the first time aequorin has been employed to detect an analyte by the inhibition of its bioluminescence reaction. Hence, this strategy may prove to be a general approach for the development of a new generation of protein-based inhibition assays.

  14. A rapid mitochondrial toxicity assay utilizing rapidly changing cell energy metabolism.

    Science.gov (United States)

    Sanuki, Yosuke; Araki, Tetsuro; Nakazono, Osamu; Tsurui, Kazuyuki

    2017-01-01

    Drug-induced liver injury is a major cause of safety-related drug-marketing withdrawals. Several drugs have been reported to disrupt mitochondrial function, resulting in hepatotoxicity. The development of a simple and effective in vitro assay to identify the potential for mitochondrial toxicity is thus desired to minimize the risk of causing hepatotoxicity and subsequent drug withdrawal. An in vitro test method called the "glucose-galactose" assay is often used in drug development but requires prior-culture of cells over several passages for mitochondrial adaptation, thereby restricting use of the assay. Here, we report a rapid version of this method with the same predictability as the original method. We found that replacing the glucose in the medium with galactose resulted in HepG2 cells immediately shifting their energy metabolism from glycolysis to oxidative phosphorylation due to drastic energy starvation; in addition, the intracellular concentration of ATP was reduced by mitotoxicants when glucose in the medium was replaced with galactose. Using our proposed rapid method, mitochondrial dysfunction in HepG2 cells can be evaluated by drug exposure for one hour without a pre-culture step. This rapid assay for mitochondrial toxicity may be more suitable for high-throughput screening than the original method at an early stage of drug development.

  15. 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...

  16. Multicenter evaluation of the BD max enteric bacterial panel PCR assay for rapid detection of Salmonella spp., Shigella spp., Campylobacter spp. (C. jejuni and C. coli), and Shiga toxin 1 and 2 genes.

    Science.gov (United States)

    Harrington, S M; Buchan, B W; Doern, C; Fader, R; Ferraro, M J; Pillai, D R; Rychert, J; Doyle, L; Lainesse, A; Karchmer, T; Mortensen, J E

    2015-05-01

    Diarrhea due to enteric bacterial pathogens causes significant morbidity and mortality in the United States and worldwide. However, bacterial pathogens may be infrequently identified. Currently, culture and enzyme immunoassays (EIAs) are the primary methods used by clinical laboratories to detect enteric bacterial pathogens. We conducted a multicenter evaluation of the BD Max enteric bacterial panel (EBP) PCR assay in comparison to culture for the detection of Salmonella spp., Shigella spp., Campylobacter jejuni, and Campylobacter coli and an EIA for Shiga toxins 1 and 2. A total of 4,242 preserved or unpreserved stool specimens, including 3,457 specimens collected prospectively and 785 frozen, retrospective samples, were evaluated. Compared to culture or EIA, the positive percent agreement (PPA) and negative percent agreement (NPA) values for the BD Max EBP assay for all specimens combined were as follows: 97.1% and 99.2% for Salmonella spp., 99.1% and 99.7% for Shigella spp., 97.2% and 98.4% for C. jejuni and C. coli, and 97.4% and 99.3% for Shiga toxins, respectively. Discrepant results for prospective samples were resolved with alternate PCR assays and bidirectional sequencing of amplicons. Following discrepant analysis, PPA and NPA values were as follows: 97.3% and 99.8% for Salmonella spp., 99.2% and 100% for Shigella spp., 97.5% and 99.0% for C. jejuni and C. coli, and 100% and 99.7% for Shiga toxins, respectively. No differences in detection were observed for samples preserved in Cary-Blair medium and unpreserved samples. In this large, multicenter study, the BD Max EBP assay showed superior sensitivity compared to conventional methods and excellent specificity for the detection of enteric bacterial pathogens in stool specimens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Plasmodium species antigen detection assays. 866... Plasmodium species antigen detection assays. (a) Identification. A Plasmodium species antigen detection assay... malaria caused by the four malaria species capable of infecting humans: Plasmodium falciparum, Plasmodium...

  18. Development of a slide agglutination assay for detection of blastomycosis.

    Science.gov (United States)

    Hatch, Wayne O; Scalarone, Gene M

    2013-11-01

    Blastomycosis, caused by the thermally dimorphic fungus Blastomyces dermatitides, which is endemic to eastern regions of the USA, is commonly misdiagnosed as a viral or bacterial infection and therefore treated improperly. Over the years, many immunodiagnostic assays to aid in the diagnosis of blastomycosis have been developed; however, a reliable assay for use in local clinics still remains elusive. Procedures for a slide agglutination assay for detection of antibody in serum from rabbits immunized with B. dermatitidis were evaluated with antigenic preparations from B. dermatitidis adsorbed to polystyrene microparticles. Yeast-phase lysates from five isolates of B. dermatitides: namely ER-593 (Eagle River, WI, USA), ER-598 (Eagle River, WI, USA), 48938 (India), B5896 (Mt. Iron, MN, USA), and T-58 (TN, USA) were evaluated for their sensitivity and specificity. Sensitivities of the lysates ranged from 29% to 83% whereas specificities ranged from 13% to 100%. Lysate ER-593 provided the most promising results with a sensitivity of 82% and specificity of 100%. This study provides suggests that a simple rapid slide agglutination assay for detecting blastomycosis may be used for screening patients with suspected B. dermatitidis infection. © 2013 The Societies and Wiley Publishing Asia Pty Ltd.

  19. Detection of pseudorabies virus by duplex droplet digital PCR assay.

    Science.gov (United States)

    Ren, Meishen; Lin, Hua; Chen, Shijie; Yang, Miao; An, Wei; Wang, Yin; Xue, Changhua; Sun, Yinjie; Yan, Yubao; Hu, Juan

    2018-01-01

    Aujeszky's disease, caused by pseudorabies virus (PRV), has damaged the economy of the Chinese swine industry. A large number of PRV gene-deleted vaccines have been constructed based on deletion of the glycoprotein E ( gE) gene combined with other virulence-related gene deletions, such as thymidine kinase ( TK), whereas PRV wild-type strains contain an intact gE gene. We developed a sensitive duplex droplet digital PCR (ddPCR) assay to rapidly detect PRV wild-type isolates and gE gene-deleted viral vaccines. We compared this assay with a TaqMan real-time PCR (qPCR) using the same primers and probes. Both assays exhibited good linearity and repeatability; however, ddPCR maintained linearity at extremely low concentrations, whereas qPCR did not. Based on positive results for both gE and gB, the detection limit of ddPCR was found to be 4.75 copies/µL in contrast of 76 copies/µL for qPCR, showing that ddPCR provided a 16-fold improvement in sensitivity. In addition, no nonspecific amplification was shown in specificity testing, and the PRV wild-type was distinguished from a gE-deleted strain. The ddPCR was more sensitive when analyzing clinical serum samples. Thus, ddPCR may become an appropriate detection platform for PRV.

  20. Comparison of two rapid assays for Clostridium difficile Common antigen and a C difficile toxin A/B assay with the cell culture neutralization assay.

    Science.gov (United States)

    Reller, Megan E; Alcabasa, Romina C; Lema, Clara A; Carroll, Karen C

    2010-01-01

    We compared 3 rapid assays for Clostridium difficile with a cell culture cytotoxicity neutralization assay (CCNA). Of 600 stool samples, 46 were positive for toxigenic C difficile. Both rapid common antigen assays were highly sensitive (91.3%-100%) and, therefore, were appropriate screening tests. The rapid toxin assay had poor sensitivity (61%) but excellent specificity (99.3%). Testing stools for glutamate dehydrogenase (step 1) and those positive with a rapid toxin assay (step 2) would correctly classify 81% of submitted specimens within 2 hours, including during periods of limited staffing (evenings, nights, and weekends). CCNA could then be used as a third step to test rapid toxin-negative samples, thereby providing a final result for the remaining 19% of samples by 48 to 72 hours. The use of rapid assays as outlined could enhance timely diagnosis of C difficile.

  1. Real-Time PCR Assay To Detect Smallpox Virus

    Science.gov (United States)

    Sofi Ibrahim, M.; Kulesh, David A.; Saleh, Sharron S.; Damon, Inger K.; Esposito, Joseph J.; Schmaljohn, Alan L.; Jahrling, Peter B.

    2003-01-01

    We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA from 48 different isolates of variola virus; 25 different strains and isolates of camelpox, cowpox, ectromelia, gerbilpox, herpes, monkeypox, myxoma, rabbitpox, raccoonpox, skunkpox, vaccinia, and varicella-zoster viruses; and two rickettsial species at concentrations mostly ranging from 100 fg/μl to 1 ng/μl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/μl to 1 ng/μl. On the Smart Cycler platform, 2 samples with false-positive results were detected among the 116 samples not containing variola virus tested; i.e., the overall specificity of the assay was 98.3%. On the LightCycler platform, five samples with false-positive results were detected (overall specificity, 95.7%). Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/μl to 1 ng/μl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform. Thus, the clinical sensitivities were 96.1% for the Smart Cycler instrument and 99.5% for the LightCycler instrument. The vast majority of these samples were derived from virus-infected cell cultures and variola virus-infected tissues; thus, the DNA material contained both viral DNA and cellular DNA. Of the 43 samples that contained purified variola virus DNA ranging in concentration from 1 fg/μl to 1 ng/μl, the assay correctly detected the virus in all 43 samples on both the Smart Cycler

  2. Rapid detection of threshold VEPs.

    Science.gov (United States)

    Mackay, Alison M; Bradnam, Michael S; Hamilton, Ruth

    2003-06-01

    To determine whether a one-dimensional (1D) Laplacian analysis detects steady-state visual evoked potentials (ssVEPs) faster than the standard O(z)-F(z) montage and to establish the optimum position of Laplacian reference electrodes. Twenty-two normal adults were shown reversing checks ranging from 1.5' to 60'. Three electrode montages were investigated: O(z)-F(z), LO-F(z) and a 1D Laplacian analysis of 3 occipital electrodes (2O(z)-(RO+LO)). RO and LO were placed symmetrically and horizontally about O(z). Five different locations for RO and LO were investigated. Recordings were analysed in the frequency domain and the presence (and detection time, DT) or absence of a ssVEP defined statistically. Effects of individual, reference electrode site and check size on DT and phase differences between recording montages were investigated. Laplacian analysis detected ssVEPs to small (3') checks faster than O(z)-F(z), by 12.3 and 4.1s on average with Laplacian reference electrodes at 15 and 20% of half-head circumference, respectively. The optimum position of reference electrodes was governed by the instantaneous spatial spread of the response and the noise coherence between midline and lateral electrodes. A 1D Laplacian analysis can reduce the time to statistical detection of ssVEPs compared to the traditional O(z)-F(z) recording for stimuli near the normal acuity threshold of adults. This in turn could be used to minimise the length of a VEP acuity assessment.

  3. Development of a rapid dipstick with latex immunochromatographic assay (DLIA for diagnosis of schistosomiasis japonica

    Directory of Open Access Journals (Sweden)

    Lu Shao-Hong

    2011-08-01

    Full Text Available Abstract Background Schistosomiasis japonica (schistosomiasis is a zoonosis that can seriously affect human health. At present, the immunodiagnostic assays for schistosomiasis detection are time-consuming and require well-trained personnel and special instruments, which can limit their use in the field. Thus, there is a pressing need for a simple and rapid immunoassay to screen patients on a large scale. In this study, we developed a novel rapid dipstick with latex immunochromatographic assay (DLIA to detect anti-Schisaosoma japonicum antibodies in human serum. Results Using latex microspheres as a color probe, DLIA was established to test standard positive and negative sera, in comparison with the classical enzyme-linked immunosorbent assay (ELISA. The sensitivity and specificity of DLIA were 95.10% (97/102 and 94.91% (261/275, respectively. The cross-reaction rates with clonorchiosis, intestinal nematodes, Angiostrongylus cantonensis and paragonimiasis were 0, 0, 0 and 42.11% respectively. All the results showed no significant difference to the ELISA. In field tests, 333 human serum samples from an endemic area were tested with DLIA, and compared with ELISA and Kato-Katz method. There was no significant difference between DLIA and ELISA on positive and negative rates of detection; however, significant differences existed between DLIA and Kato-Katz method, and between ELISA and Kato-Katz method. The kappa value between DLIA and ELISA was 0.90. Conclusions This is the first study in which DLIA was used to detect anti-Schistosoma japonicum antibody. The results show that DLIA is a simple, rapid, convenient, sensitive and specific assay for the diagnosis of schistosomiasis and is therefore very suitable for large-scale field applications and clinical detection.

  4. Assay optimization for molecular detection of Zika virus

    NARCIS (Netherlands)

    Corman, Victor M.; Rasche, Andrea; Baronti, Cecile; Aldabbagh, Souhaib; Cadar, Daniel; Reusken, Chantal Bem; Pas, Suzan D.; Goorhuis, Abraham; Schinkel, Janke; Molenkamp, Richard; Kümmerer, Beate M.; Bleicker, Tobias; Brünink, Sebastian; Eschbach-Bludau, Monika; Eis-Hübinger, Anna M.; Koopmans, Marion P.; Schmidt-Chanasit, Jonas; Grobusch, Martin P.; de Lamballerie, Xavier; Drosten, Christian; Drexler, Jan Felix

    2016-01-01

    To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection. We compared seven published real-time RT-PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we

  5. Highly specific and rapid immuno-fluorescent visualization and detection of E. coli O104:H4 with protein-A coated magnetic beads based LST-MUG assay.

    Science.gov (United States)

    Barizuddin, Syed; Balakrishnan, Baskar; Stringer, R Cody; Dweik, Majed

    2015-08-01

    A method combining immunomagnetic separation and fluorescent sensing was developed to detect Escherichia coli (E. coli) O104:H4. The antibody specific to E. coli O104:H4 was immobilized on protein A-coated magnetic beads. This protein-A-anti E. coli O104:H4 complex was used to bind Fluorescein IsoThioCyanate (FITC) labeled E. coli O104:H4 antigen (whole cell) on it. The goal was to achieve a fluorescently detectable protein-A-anti E. coli O104:H4-E. coli O104:H4 complex on the magnetic beads. Fluorescent microscopy was used to image the magnetic beads. The resulting fluorescence on the beads was due to the FITC labeled antigen binding on the protein-A-anti E. coli O104:H4 immobilized magnetic beads. This visually proves the antigen-antibody binding. The fluorescent imaging results were obtained in 2 h if the minimum available bacteria in the sample were at least 10(5) CFU/ml. If no fluorescence was observed on the magnetic beads during fluorescent imaging, it indicates the bacterial concentration in the sample to be too low for it to have bound to the magnetic beads and hence no detection was possible. To detect bacterial concentration less than 10(5) CFU/ml in the sample, an additional step was required for detection. The magnetic bead complex was added to the LST-MUG (lauryl sulfate tryptose-4-methylumbelliferyl-β-D-glucuronide), a signaling reporter. The E. coli O104:H4 grows in LST-MUG and releases β-glucuronidase enzyme. This enzyme cleaves the MUG substrate that produces 4-methylumbelliferone, a highly fluorescent species. This fluorescence was detected using a spectrofluorometer. The emission peak in the fluorescent spectrum was found to be at 450 nm. The lower and upper detection range for this LST-MUG assay was found to be 2.05×10(5)-4.09×10(8) CFU/ml. The results for the LST-MUG assay for concentrations below 10(5) CFU/ml were ascertained in 8h. The advantages of this technique include the specific detection of bacteria without an enrichment step and

  6. RAPID MONITORING OF INDICATOR COLIFORMS IN DRINKING WATER BY AN ENZYMATIC ASSAY

    Directory of Open Access Journals (Sweden)

    M. Nikaeen ، A. Pejhan ، M. Jalali

    2009-01-01

    Full Text Available Coliform group has been extensively used as an indicator of drinking water quality and historically led to the public health protection concept. Multiple tube fermentation technique has been currently used for assessment of the microbial quality of drinking water. This method, however, has limitations. Enzymatic assay constitute an alternative approach for detecting indicator bacteria, namely total coliforms and E.coli in various aquatic environments. This study compared the performance of LMX® broth as an enzymatic assay with the standard methods multiple tube fermentation technique and presence–absence test, for the detection of indicator coliforms in drinking water samples. In addition, the potential effect of water quality on the microbial detection method was assayed through measurement of some physicochemical parameters. From the 50 drinking water samples tested, 8 (16% and 7 (14% contained total coliforms and E.coli as indicated by all three techniques. Although on average the LMX recovered more total coliforms and E.Coli numbers comparing to multiple tube fermentation, but there was no significant difference. A significant difference existed between the level of residual chlorine for positive and negative samples. In conclusion, enzymatic assay showed a rapid and less labor method, allowing the simultaneous detection of total coliforms and E.coli. The method is particularly useful in the early warning of fecal pollution of drinking water.

  7. 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.

  8. A novel capillary electrophoresis-based multiplex PCR assay for detection of respiratory pathogens.

    Science.gov (United States)

    Stevenson, Jeffery B; Hymas, Weston C; Hillyard, David R

    2011-01-01

    The field of infectious disease testing has recently experienced rapid expansion in the number of multiplexed PCR-based assays available for detecting respiratory pathogens. This study provides a preliminary evaluation of a multiplex assay from Seegene that uses capillary electrophoresis as the detection platform for viral and bacterial respiratory pathogens. We compared this technology to a real-time PCR assay for 3 viral targets. Thirty respiratory samples were collected that had previously tested positive for either Flu A, Flu B, or RSV (ten of each). The Seegene assay detected 9/10 Flu A samples, 9/10 Flu B, and 10/10 RSV, for a total detection rate of 93%. The two samples that were undetected by the Seegene assay both generated late-crossing thresholds on the real-time platform, consistent with low viral loads. The Seeplex assay provides a promising alternative for multiplex respiratory testing.

  9. A modified MS2 bacteriophage plaque reduction assay for the rapid screening of antiviral plant extracts.

    Science.gov (United States)

    Cock, Ian; Kalt, F R

    2010-07-01

    Traditional methods of screening plant extracts and purified components for antiviral activity require up to a week to perform, prompting the need to develop more rapid quantitative methods to measure the ability of plant based preparations to block viral replication. We describe an adaption of an MS2 plaque reduction assay for use in S. aureus. MS2 bacteriophage was capable of infecting and replicating in B. cereus, S. aureus and F + E. coli but not F- E. coli. Indeed, both B. cereus and S. aureus were more sensitive to MS2 induced lysis than F+ E. coli. When MS2 bacteriophage was mixed with Camellia sinensis extract (1 mg/ml), Scaevola spinescens extract (1 mg/ml) or Aloe barbadensis juice and the mixtures inoculated into S. aureus, the formation of plaques was reduced to 8.9 ± 3.8%, 5.4 ± 2.4% and 72.7 ± 20.9% of the untreated MS2 control values respectively. The ability of the MS2 plaque reduction assay to detect antiviral activity in these known antiviral plant preparations indicates its suitability as an antiviral screening tool. An advantage of this assay compared with traditionally used cytopathic effect reduction assays and replicon based assays is the more rapid acquisition of results. Antiviral activity was detected within 24 h of the start of testing. The MS2 assay is also inexpensive and non-pathogenic to humans making it ideal for initial screening studies or as a simulant for pathogenic viruses.

  10. A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays

    Science.gov (United States)

    Pooley, Hannah B.; de Silva, Kumudika; Purdie, Auriol C.; Begg, Douglas J.; Whittington, Richard J.

    2016-01-01

    ABSTRACT Determining the viability of bacteria is a key outcome of in vitro cellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such as Mycobacterium avium subsp. paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viable M. avium subsp. paratuberculosis cells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and dead M. avium subsp. paratuberculosis organisms and their accuracy at low bacterial concentrations. Using the culture-based method, M. avium subsp. paratuberculosis growth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viable M. avium subsp. paratuberculosis cells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples from in vitro cellular infection assays. IMPORTANCE Rapid quantification of the viability of Mycobacterium avium subsp. paratuberculosis in samples from in vitro cellular infection assays is important, as it allows these assays to be carried out on a large scale. In vitro cellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regarding M. avium subsp. paratuberculosis viability after an in vitro infection assay; however, with the quantitative PCR and liquid culture method

  11. A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells.

    Science.gov (United States)

    O'Brien, Martha; Moehring, Danielle; Muñoz-Planillo, Raúl; Núñez, Gabriel; Callaway, Justin; Ting, Jenny; Scurria, Mike; Ugo, Tim; Bernad, Laurent; Cali, James; Lazar, Dan

    2017-08-01

    Inflammasomes are protein complexes induced by diverse inflammatory stimuli that activate caspase-1, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent, plate-based assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized lytic reagent added directly to cultured cells. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor in the lytic reagent and by use of a caspase-1 inhibitor to confirm activity. This approach enables a specific and rapid determination of caspase-1 activation. Caspase-1 activity is stable in the reagent thereby providing assay convenience and flexibility. Using this assay system, caspase-1 activation has been determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, gramicidin, MSU, R848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages (BMDMs) from mice, as well as in human primary monocytes. Caspase-1 activity was not detected in treated BMDMs derived from Casp1-/- mice, further confirming the specificity of the assay. Caspase-1 activity can be measured directly in cultured cells using the lytic reagent, or caspase-1 activity released into medium can be monitored by assay of transferred supernatant. The caspase-1 assay can be multiplexed with other assays to monitor additional parameters from the same cells, such as IL-1β release or cell death. The caspase-1 assay in combination with a sensitive real-time monitor of cell death allows one to accurately establish pyroptosis. This assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective

  12. Assay optimization for molecular detection of Zika virus

    NARCIS (Netherlands)

    Corman, Victor M.; Rasche, Andrea; Baronti, Cecile; Aldabbagh, Souhaib; Cadar, Daniel; Reusken, Chantal Bem; Pas, Suzan D.; Goorhuis, Abraham; Schinkel, Janke; Molenkamp, Richard; Kümmerer, Beate M.; Bleicker, Tobias; Brünink, Sebastian; Eschbach-Bludau, Monika; Eis-Hübinger, Anna M.; Koopmans, Marion P.; Schmidt-Chanasit, Jonas; Grobusch, Martin P.; de Lamballerie, Xavier; Drosten, Christian; Drexler, Jan Felix

    2016-01-01

    Objective To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection. Methods We compared seven published real-time RT PCR assays and two new assays that we have developed. To determine the analytical sensitivity of

  13. Is the Comet Assay a Sensitive Procedure for Detecting Genotoxicity?

    Science.gov (United States)

    Kawaguchi, Satomi; Nakamura, Takanori; Yamamoto, Ayumi; Honda, Gisho; Sasaki, Yu F.

    2010-01-01

    Although the Comet assay, a procedure for quantitating DNA damage in mammalian cells, is considered sensitive, it has never been ascertained that its sensitivity is higher than the sensitivity of other genotoxicity assays in mammalian cells. To determine whether the power of the Comet assay to detect a low level of genotoxic potential is superior to those of other genotoxicity assays in mammalian cells, we compared the results of Comet assay with those of micronucleus test (MN test). WTK1 human lymphoblastoid cells were exposed to methyl nitrosourea (MNU), ethyl nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), bleomycin (BLM), or UVC. In Comet assay, cells were exposed to each mutagen with (Comet assay/araC) and without (Comet assay) DNA repair inhibitors (araC and hydroxyurea). Furthermore, acellular Comet assay (acellular assay) was performed to determine how single-strand breaks (SSBs) as the initial damage contributes to DNA migration and/or to micronucleus formation. The lowest genotoxic dose (LGD), which is defined as the lowest dose at which each mutagen causes a positive response on each genotoxicity assay, was used to compare the power of the Comet assay to detect a low level of genotoxic potential and that of MN test; that is, a low LGD indicates a high power. Results are summarized as follows: (1) for all mutagens studied, LGDs were MN test ≦ Comet assay; (2) except for BLM, LGDs were Comet assay/araC ≦ MN test; (3) except for UVC and MNU, LGDs were acellular assay ≦ Comet assay/araC ≦ MN test ≦ Comet assay. The following is suggested by the present findings: (1) LGD in the Comet assay is higher than that in MN test, which suggests that the power of the MN test to detect a low level of genotoxic potential is superior to that of the Comet assay; (2) for the studied mutagens, all assays were able to detect all mutagens correctly, which suggests that the sensitivity of the Comet assay and that of the MN test were

  14. Evaluation and improvement of LAMP assays for detection of

    African Journals Online (AJOL)

    Abstract: Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157 are the causative agents of human diseases, and LAMP assays have been developed for detection of the seven leading STEC serogroups. Objective: To evaluate existing LAMP assays for detection of the seven STEC serogroups, ...

  15. Evaluation and improvement of LAMP assays for detection of ...

    African Journals Online (AJOL)

    Abstract: Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157 are the causative agents of human diseas- es, and LAMP assays have been developed for detection of the seven leading STEC serogroups. Objective: To evaluate existing LAMP assays for detection of the seven STEC serogroups, ...

  16. A rapid assay for on-site monitoring of infliximab trough levels: a feasibility study.

    Science.gov (United States)

    Corstjens, Paul L A M; Fidder, Herma H; Wiesmeijer, Karien C; de Dood, Claudia J; Rispens, Theo; Wolbink, Gert-Jan; Hommes, Daniel W; Tanke, Hans J

    2013-09-01

    Monitoring levels of biologicals against tumor necrosis factor (TNF) has been suggested to improve therapeutic outcomes in inflammatory bowel diseases (IBDs). This pilot study describes a rapid lateral flow (LF)-based assay for on-site monitoring of serum trough levels of humanized monoclonal antibody infliximab (IFX). The applied chromatographic method utilizes sequential flows of diluted serum, wash buffer, and an immunoglobulin generic label on LF strips with a Test line comprised of TNF-α. The successive flows permitted enrichment of IFX at the Test line before the label was applied. The label, luminescent upconverting phosphor (UCP) particles coated with protein-A, emits a 550-nm visible light upon excitation with 980-nm infrared light. IFX concentrations were determined through measurement of UCP fluorescence at the Test line. The assay was optimized to detect IFX levels as low as 0.17 μg/mL in serum. For patients with IBD, this limit is appropriate to detect levels associated with loss of response (0.5 μg IFX/mL). The assay was evaluated with clinical samples from patients with Crohn's disease and correlated well within the physiologically relevant range from 0.17 to 10 μg/mL with an IFX-specific ELISA. Performance of the assay was further successfully validated with samples from blood donors, IFX negative IBD patients, and rheumatoid arthritis patients that had developed anti-IFX antibodies. Because of its generic nature, the assay is suited for detecting most therapeutic anti-TNF-α monoclonal antibodies.

  17. Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

    DEFF Research Database (Denmark)

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

    2002-01-01

    ) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect ... axenic cultivation system for P. carinii and confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease in P. carinii DNA that exceeded the expected decrease due to dilution of the inoculum upon transfer....... In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical...

  18. A rapid assay for the biological evaluation of helicase activity.

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Dimitrios Vlachakis, Andrea Brancale, Colin Berry & Sophia Kossida ### Abstract A new assay for the measurement of helicase enzyme activity was developed for the evaluation of the potency of potential inhibitors. This assay involves the use of a DNA or RNA duplex substrate and recombinant purified helicase. The DNA duplex consists of a pair of oligonucleotides, one of which is biotinylated and the other is digoxygenin (DIG)-labelled, both at their respective 5’ termini. This ...

  19. A simple and rapid liquid chromatographic assay for evaluation of potentially counterfeit Tamiflu.

    Science.gov (United States)

    Lindegårdh, N; Hien, T T; Farrar, J; Singhasivanon, P; White, N J; Day, N P J

    2006-10-11

    A simple and rapid liquid chromatographic assay for the evaluation of potentially counterfeit oseltamivir (Tamiflu has been developed and assessed. The assay uses approximately 1mg Tamiflu powder when used for authentication and content estimate. The procedure was validated using 50 replicates analysed during five independent series with a total R.S.D. of 11.2%. The assay can also be used to monitor the exact content of oseltamivir in Tamiflu capsules. One Tamiflu capsule was transferred to a 250mL volumetric flask and 150mL water was added. The flask was placed in an ultrasonic bath at 40 degrees C for 20min to dissolve the capsule. The solution was allowed to cool to room temperature before the flask was filled up to the mark (250mL). A small aliquot was centrifuged and then directly injected into the LC-system for quantification. Oseltamivir was analysed by liquid chromatography with UV detection on a Hypersil Gold column (150mmx4.6mm) using a mobile phase containing methanol-phosphate buffer (pH 2.5; 0.1M) (50:50, v/v) at a flow rate of 1.0mL/min. The assay was implemented for the analysis of Tamiflu purchased over the Internet and at local pharmacies in Thailand and Vietnam.

  20. Microelectronic DNA assay for the detection of BRCA1 gene mutations

    Science.gov (United States)

    Chen, Hua; Han, Jie; Li, Jun; Meyyappan, Meyya

    2004-01-01

    Mutations in BRCA1 are characterized by predisposition to breast cancer, ovarian cancer and prostate cancer as well as colon cancer. Prognosis for this cancer survival depends upon the stage at which cancer is diagnosed. Reliable and rapid mutation detection is crucial for the early diagnosis and treatment. We developed an electronic assay for the detection of a representative single nucleotide polymorphism (SNP), deletion and insertion in BRCA1 gene by the microelectronics microarray instrumentation. The assay is rapid, and it takes 30 minutes for the immobilization of target DNA samples, hybridization, washing and readout. The assay is multiplexing since it is carried out at the same temperature and buffer conditions for each step. The assay is also highly specific, as the signal-to-noise ratio is much larger than recommended value (72.86 to 321.05 vs. 5) for homozygotes genotyping, and signal ratio close to the perfect value 1 for heterozygotes genotyping (1.04).

  1. 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.

  2. 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

  3. Assay optimization for molecular detection of Zika virus

    Science.gov (United States)

    Corman, Victor M; Rasche, Andrea; Baronti, Cecile; Aldabbagh, Souhaib; Cadar, Daniel; Reusken, Chantal BEM; Pas, Suzan D; Goorhuis, Abraham; Schinkel, Janke; Molenkamp, Richard; Kümmerer, Beate M; Bleicker, Tobias; Brünink, Sebastian; Eschbach-Bludau, Monika; Eis-Hübinger, Anna M; Koopmans, Marion P; Schmidt-Chanasit, Jonas; Grobusch, Martin P; de Lamballerie, Xavier; Drosten, Christian

    2016-01-01

    Abstract Objective To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection. Methods We compared seven published real-time RT–PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we constructed a synthetic universal control ribonucleic acid (uncRNA) containing all of the assays’ target regions on one RNA strand and spiked human blood or urine with known quantities of African or Asian Zika virus strains. Viral loads in 33 samples from Zika virus-infected patients were determined by using one of the new assays. Findings Oligonucleotides of the published real-time RT–PCR assays, showed up to 10 potential mismatches with the Asian lineage causing the current outbreak, compared with 0 to 4 mismatches for the new assays. The 95% lower detection limit of the seven most sensitive assays ranged from 2.1 to 12.1 uncRNA copies/reaction. Two assays had lower sensitivities of 17.0 and 1373.3 uncRNA copies/reaction and showed a similar sensitivity when using spiked samples. The mean viral loads in samples from Zika virus-infected patients were 5 × 104 RNA copies/mL of blood and 2 × 104 RNA copies/mL of urine. Conclusion We provide reagents and updated protocols for Zika virus detection suitable for the current outbreak strains. Some published assays might be unsuitable for Zika virus detection, due to the limited sensitivity and potential incompatibility with some strains. Viral concentrations in the clinical samples were close to the technical detection limit, suggesting that the use of insensitive assays will cause false-negative results. PMID:27994281

  4. Indigenous people's detection of rapid ecological change.

    Science.gov (United States)

    Aswani, Shankar; Lauer, Matthew

    2014-06-01

    When sudden catastrophic events occur, it becomes critical for coastal communities to detect and respond to environmental transformations because failure to do so may undermine overall ecosystem resilience and threaten people's livelihoods. We therefore asked how capable of detecting rapid ecological change following massive environmental disruptions local, indigenous people are. We assessed the direction and periodicity of experimental learning of people in the Western Solomon Islands after a tsunami in 2007. We compared the results of marine science surveys with local ecological knowledge of the benthos across 3 affected villages and 3 periods before and after the tsunami. We sought to determine how people recognize biophysical changes in the environment before and after catastrophic events such as earthquakes and tsunamis and whether people have the ability to detect ecological changes over short time scales or need longer time scales to recognize changes. Indigenous people were able to detect changes in the benthos over time. Detection levels differed between marine science surveys and local ecological knowledge sources over time, but overall patterns of statistically significant detection of change were evident for various habitats. Our findings have implications for marine conservation, coastal management policies, and disaster-relief efforts because when people are able to detect ecological changes, this, in turn, affects how they exploit and manage their marine resources. © 2014 Society for Conservation Biology.

  5. 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.

  6. Lateral flow assay for simultaneous detection of cellular- and humoral immune responses.

    Science.gov (United States)

    Corstjens, Paul L A M; de Dood, Claudia J; van der Ploeg-van Schip, Jolien J; Wiesmeijer, Karien C; Riuttamäki, Terhi; van Meijgaarden, Krista E; Spencer, John S; Tanke, Hans J; Ottenhoff, Tom H M; Geluk, Annemieke

    2011-10-01

    The development of a cytokine detection assay suitable for detection of multiple biomarkers for improved diagnosis of mycobacterial diseases. A lateral flow (LF) assay to detect IL-10 was developed utilizing the up-converting phosphor (UCP) reporter-technology. The assay was evaluated using blood samples of leprosy patients. Multiplex applications were explored targeting: 1) IL-10 and IFN-γ in assay buffer; 2) IL-10 and anti-phenolic glycolipid (PGL-I) antibodies in serum from leprosy patients. Detection of IL-10 below the targeted level of 100pg/mL in serum was shown. Comparison with ELISA showed a quantitative correlation with R(2) value of 0.92. Multiplexing of cytokines and simultaneous detection of cytokine and antibody was demonstrated. The UCP-LF IL-10 assay is a user-friendly, rapid alternative for IL-10 ELISAs, suitable for multiplex detection of different cytokines and can be merged with antibody-detection assays to simultaneously detect cellular- and humoral immunity. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Rapid detection of anti-Vaccinia virus neutralizing antibodies

    Directory of Open Access Journals (Sweden)

    Lichtfuss Gregor F

    2011-03-01

    Full Text Available Abstract Increasing infections with Monkeypox and Cowpox viruses pose a continuous and growing threat to human health. The standard method for detecting poxvirus neutralizing antibodies is the plaque-reduction neutralization test that is specific but also time-consuming and laborious. Therefore, a rapid and reliable method was developed to determine neutralizing antibody titers within twelve hours. The new assay measures viral mRNA transcription as a marker for actively replicating virus after incomplete neutralization using real-time PCR.

  8. Rapid and Reliable Diagnostic Algorithm for Detection of Clostridium difficile▿

    Science.gov (United States)

    Fenner, Lukas; Widmer, Andreas F.; Goy, Gisela; Rudin, Sonja; Frei, Reno

    2008-01-01

    We evaluated a two-step algorithm for detection of Clostridium difficile in 1,468 stool specimens. First, specimens were screened by an immunoassay for C. difficile glutamate dehydrogenase antigen (C.DIFF CHEK-60). Second, screen-positive specimens underwent toxin testing by a rapid toxin A/B assay (TOX A/B QUIK CHEK); toxin-negative specimens were subjected to stool culture. This algorithm allowed final results for 92% of specimens with a turnaround time of 4 h. PMID:18032627

  9. Potential rapid and simple lateral flow assay for Escherichia coli O111.

    Science.gov (United States)

    Terao, Yoshitaka; Yonekita, Taro; Morishita, Naoki; Fujimura, Tatsuya; Matsumoto, Takashi; Morimatsu, Fumiki

    2013-05-01

    We developed and evaluated a lateral flow assay (LFA) as a simple and rapid method for direct detection of Escherichia coli O111 in food after enrichment. When cell suspensions of 8 E. coli O111 strains and 77 non-E. coli O111 strains were tested with the LFA, the former all yielded positive results and the latter all yielded negative results. The minimum detection limits for the E. coli O111 strains were 1.8 × 10(3) to 5.6 × 10(5) CFU/ml of cell suspension, and the LFA was able to detect live cultures or those killed by autoclaving at nearly the same level of sensitivity. To evaluate the ability of LFA to detect its target in food, enrichment cultures of meat samples inoculated with 10-fold serial dilutions of E. coli O111 were tested with the LFA and PCR. Even when there were very few E. coli O111 cells in the meat samples (1.6 × 10(0) to 1.6 × 10(1) CFU/25 g of food), when they were cultured in modified E. coli broth with novobiocin for 22 h at 42°C, the LFA yielded positive results that corresponded to the PCR results. Although the LFA requires further evaluation and field study, these results suggest that this assay has sufficient sensitivity and specificity. This procedure can be completed with a one-step incubation after the test strip has been inserted into the sample after 22 h of culture, whereas the standard culture method requires multiple cultures, skilled personnel, a well-equipped laboratory, and 4 or 5 days. The speed and simplicity of this LFA make it suitable for use as part of routine screening assays in the food industry.

  10. Rapid diagnosis of tuberculosis using Xpert MTB/RIF assay - Report from a developing country.

    Science.gov (United States)

    Iram, Shagufta; Zeenat, Asyia; Hussain, Shahida; Wasim Yusuf, Noshin; Aslam, Maleeha

    2015-01-01

    To evaluate the diagnostic accuracy of the Xpert MTB/RIF assay for the detection of M. tuberculosis in pulmonary and extrapulmonary specimens and to compare it with conventional techniques. During a period of 10 months from December 2012 through September 2013, two hundred and forty five clinically TB suspects were enrolled for Xpert MTB\\RIF assay. The cohort comprised of 205 suspects of pulmonary TB and 40 of extrapulmonary TB (EPTB). The 40 EPTB samples included pus aspirated from different sites of the body (n=19), pleural fluid (n=11), ascitic fluid (n=7), pericardial fluid, CSF and urine one each. Ziehl-Neelsen (ZN) Stained smear microscopy, culture on LJ media and Xpert MTB/RIF assay was performed on samples from these patients. M. tuberculosis (MTB) were detected by Xpert MTB/RIF test in 111 (45.3%) out of 245 samples. Of these, 85 (34.7%) were smear positive on ZN staining and 102 (41.6%) were positive on LJ cultures. Rifampicin resistance was detected in 16 (6.5%) patients. Nine out of 19 pus samples (47.3%) were positive for MTB by Gene Xpert, 03 (15.8%) on ZN staining and 04 (21%) on LJ culture. MTB could not be detected in any other extrapulmonary sample. Xpert MTB/RIF is a sensitive method for rapid diagnosis of Tuberculosis, especially in smear negative cases and in EPTB as compared to the conventional ZN staining. Among EPTB cases the highest yield of positivity was shown in Pus samples. For countries endemic for TB GeneXpert can serve as a sensitive and time saving diagnostic modality for pulmonary and EPTB.

  11. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    National Research Council Canada - National Science Library

    Choi, Hoseok; Choi, Bomi; Seo, Ju Tae; Lee, Kyung Jin; Gye, Myung Chan; Kim, Young-Pil

    2016-01-01

    .... The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.

  12. Multiplexed lateral flow microarray assay for detection of citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis pv citri

    Science.gov (United States)

    Cary,; Bruce, R [Santa Fe, NM; Stubben, Christopher J [Los Alamos, NM

    2011-03-22

    The invention provides highly sensitive and specific assays for the major citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis, including a field deployable multiplexed assay capable of rapidly assaying for both pathogens simultaneously. The assays are directed at particular gene targets derived from pathogenic strains that specifically cause the major citrus diseases of citrus variegated chlorosis (Xylella fastidiosa 9a5c) and citrus canker (Xanthomonas axonopodis pv citri). The citrus pathogen assays of the invention offer femtomole sensitivity, excellent linear dynamic range, and rapid and specific detection.

  13. Simple and rapid spectrophotometric assay of levocetirizine in ...

    African Journals Online (AJOL)

    Simple, rapid, selective and fairly sensitive method is described for the determination of levocetirizine (LCTZ) in pure form and in its dosage forms. The method is based on the formation of intensely colored charge-transfer (CT) complexes between LCTZ as donor with two π acceptors, chloranilic acid (CAA) and 2 ...

  14. Detection of enterovirus RNA in cerebrospinal fluid : Comparison of two molecular assays

    NARCIS (Netherlands)

    de Crom, S. C. M.; Obihara, C. C.; van Loon, A. M.; Argilagos-Alvarez, A. A.; Peeters, M. F.; van Furth, A. M.; Rossen, J. W. A.

    Enterovirus (EV) and human parechovirus (HPeV) are a major cause of infection in childhood. A rapid diagnostic test may improve the management of patients with EV and HPeV infection. The aim of this study is to evaluate the performance of the GeneXpert enterovirus assay (GXEA) for detection of EV

  15. Development of a lateral flow immunochromatographic assay for the rapid diagnosis of Orf virus infections.

    Science.gov (United States)

    Zhao, Kui; He, Wenqi; Bi, Jingying; Zhang, Ximu; Zhang, Di; Huang, Houshuang; Zhang, Yuexiang; Song, Deguang; Gao, Feng

    2016-10-01

    A rapid and simple lateral-flow immunochromatographic assay (LFIA) was developed for the specific detection of Orf virus (ORFV) using two distinct monoclonal antibodies (MAbs: 5A5 and 6F2) against the ORFV ORF011 protein. The MAb 5A5 was conjugated with colloidal gold, and the MAb 6F2 and goat anti-mouse IgG were sprayed onto a nitrocellulose membrane in strips at positions designated test (T) and control (C), respectively. The results showed that samples of ORFV complexed with colloidal gold-conjugated MAb 5A5, were captured by MAb 6F2 at the T line resulting in the appearance of a purple band. When samples did not contain ORFV or when they contained a quantity of ORFV below the detection limit of the test, only the C line was visible. The analysis of sensitivity of the test demonstrated that the lowest detected quantity of ORFV was 2.03×10(3.0) TCID50/ml. Storage at room temperature for 6 months did not result in the loss of performance of the LFIA test. Using loop-mediated isothermal amplification (LAMP) as a reference test, the relative specificity and sensitivity of the LFIA test were determined to be 100% and 92.1%, respectively. Based on these results, the LFIA test developed may be a suitable tool for rapid on-site testing for ORFV infection. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Real-time PCR TaqMan assay for rapid screening of bloodstream infection

    Science.gov (United States)

    2014-01-01

    Background Sepsis is one of the main causes of mortality and morbidity. The rapid detection of pathogens in blood of septic patients is essential for adequate antimicrobial therapy and better prognosis. This study aimed to accelerate the detection and discrimination of Gram-positive (GP) and Gram-negative (GN) bacteria and Candida species in blood culture samples by molecular methods. Methods The Real-GP®, -GN®, and -CAN® real-time PCR kit (M&D, Wonju, Republic of Korea) assays use the TaqMan probes for detecting pan-GP, pan-GN, and pan-Candida species, respectively. The diagnostic performances of the real-time PCR kits were evaluated with 115 clinical isolates, 256 positive and 200 negative blood culture bottle samples, and the data were compared to results obtained from conventional blood culture. Results Eighty-seven reference strains and 115 clinical isolates were correctly identified with specific probes corresponding to GP-bacteria, GN-bacteria and Candida, respectively. The overall sensitivity and specificity of the real-time PCR kit with blood culture samples were 99.6% and 89.5%, respectively. Conclusions The Real-GP®, -GN®, and -CAN® real-time PCR kits could be useful tools for the rapid and accurate screening of bloodstream infections (BSIs). PMID:24393579

  17. 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

  18. Screening test for rapid food safety evaluation by menadione-catalysed chemiluminescent assay.

    Science.gov (United States)

    Yamashoji, Shiro; Yoshikawa, Naoko; Kirihara, Masayuki; Tsuneyoshi, Toshihiro

    2013-06-15

    The chemiluminescent assay of menadione-catalysed H2O2 production by living mammalian cells was proposed to be useful for rapid food safety evaluation. The tested foods were extracted with water, ethanol and dimethylsulfoxide, and each extract was incubated with NIH3T3, Neuro-2a and HepG2 cells for 4h. Menadione-catalysed H2O2 production by living mammalian cells exposed to each extract was determined by the chemiluminescent assay requiring only 10 min, and the viability of the cells was estimated as percentage based on H2O2 production by intact cells. In this study the cytotoxicity of food was rated in order of inhibitory effect on H2O2 production by intact cells. The well known natural toxins such as Fusarium mycotoxin, tomato toxin tomatine, potato toxin solanine and marine toxins terodotoxin and brevetoxin could be detected by the above chemiluminescent assay. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. [Rapid centrifugation assay standarization for dengue virus isolation].

    Science.gov (United States)

    Palomino, Miryam; Gutierrez, Victoria; Salas, Ramses

    2010-03-01

    The plate centrifugation assay was standardized for dengue virus isolation from serum samples. C6/36-HT cells were used determining the optimal values for centrifugation spin speed, inoculum, sera dilution, and incubation time. Then, 22 positive serum samples with viral isolation and viral strains of the four reference dengue virus serotypes were tested simultaneously by the standardized plate centrifugation method and the conventional tube culture. The isolations were typified by indirect immunofluorescent test using monoclonal antibodies. The plate centrifugation method was optimized to 200 μL of inoculum, dilution of sera 1/20, centrifugation speed at 1600 rpm/30 min, and sensitivity of 95,5% after 5 days post-inoculation. We concluded that the plate centrifugation method increased dengue virus isolation, with a significant reduction of the time of isolation for dengue virus.

  20. Rapid Antemortem Detection of CWD Prions in Deer Saliva

    Science.gov (United States)

    Haley, Nicholas J.; Denkers, Nathaniel D.; Nalls, Amy V.; Mathiason, Candace K.; Caughey, Byron; Hoover, Edward A.

    2013-01-01

    Chronic wasting disease (CWD) is an efficiently transmitted prion disease of cervids, now identified in 22 United States, 2 Canadian provinces and Korea. One hallmark of CWD is the shedding of infectious prions in saliva, as demonstrated by bioassay in deer. It is also clear that the concentration of prions in saliva, blood, urine and feces is much lower than in the nervous system or lymphoid tissues. Rapid in vitro detection of CWD (and other) prions in body fluids and excreta has been problematic due to the sensitivity limits of direct assays (western blotting, ELISA) and the presence of inhibitors in these complex biological materials that hamper detection. Here we use real-time quaking induced conversion (RT-QuIC) to demonstrate CWD prions in both diluted and prion-enriched saliva samples from asymptomatic and symptomatic white-tailed deer. CWD prions were detected in 14 of 24 (58.3%) diluted saliva samples from CWD-exposed white-tailed deer, including 9 of 14 asymptomatic animals (64.2%). In addition, a phosphotungstic acid enrichment enhanced the RT-QuIC assay sensitivity, enabling detection in 19 of 24 (79.1%) of the above saliva samples. Bioassay in Tg[CerPrP] mice confirmed the presence of infectious prions in 2 of 2 RT-QuIC-positive saliva samples so examined. The modified RT-QuIC analysis described represents a non-invasive, rapid ante-mortem detection of prions in complex biologic fluids, excreta, or environmental samples as well as a tool for exploring prion trafficking, peripheralization, and dissemination. PMID:24040235

  1. Rapid antemortem detection of CWD prions in deer saliva.

    Directory of Open Access Journals (Sweden)

    Davin M Henderson

    Full Text Available Chronic wasting disease (CWD is an efficiently transmitted prion disease of cervids, now identified in 22 United States, 2 Canadian provinces and Korea. One hallmark of CWD is the shedding of infectious prions in saliva, as demonstrated by bioassay in deer. It is also clear that the concentration of prions in saliva, blood, urine and feces is much lower than in the nervous system or lymphoid tissues. Rapid in vitro detection of CWD (and other prions in body fluids and excreta has been problematic due to the sensitivity limits of direct assays (western blotting, ELISA and the presence of inhibitors in these complex biological materials that hamper detection. Here we use real-time quaking induced conversion (RT-QuIC to demonstrate CWD prions in both diluted and prion-enriched saliva samples from asymptomatic and symptomatic white-tailed deer. CWD prions were detected in 14 of 24 (58.3% diluted saliva samples from CWD-exposed white-tailed deer, including 9 of 14 asymptomatic animals (64.2%. In addition, a phosphotungstic acid enrichment enhanced the RT-QuIC assay sensitivity, enabling detection in 19 of 24 (79.1% of the above saliva samples. Bioassay in Tg[CerPrP] mice confirmed the presence of infectious prions in 2 of 2 RT-QuIC-positive saliva samples so examined. The modified RT-QuIC analysis described represents a non-invasive, rapid ante-mortem detection of prions in complex biologic fluids, excreta, or environmental samples as well as a tool for exploring prion trafficking, peripheralization, and dissemination.

  2. 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

  3. A Rapid and Simple TLC-Densitometric Method for Assay of Clobetasol Propionate in Topical Solution.

    Science.gov (United States)

    Dolowy, Malgorzata; Kozik, Violetta; Bak, Andrzej; Jampilek, Josef; Barbusinski, Krzysztof; Thomas, Maciej; Pyka-Pajak, Alina

    2017-11-03

    A rapid, simple to use and low-cost thin-layer chromatographic procedure in normal phase system with densitometric detection at 246 nm was carefully validated according to the International Conference on Harmonisation (ICH) guidelines for assay of clobetasol propionate in topical solution containing clobetasol propionate in quantity 0.50 mg/mL. The adopted thin-layer chromatographic (TLC)-densitometric procedure could effectively separate clobetasol propionate from its related compound, namely clobetasol. It is linear for clobetasol propionate in the range of 0.188 ÷ 5 µg/spot. The limit of detection (LOD) and limit of quantification (LOQ) value is 0.061 and 0.186 µg/spot, respectively. Accuracy of proposed procedure was evaluated by recovery test. The mean recovery of studied clobetasol propionate ranges from 98.7 to 101.0%. The coefficient of variation (CV, %) obtained during intra-day and inter-day studies, which was less than 2% (0.40 ÷ 1.17%), confirms the precision of described method. The assay value of clobetasol propionate is consistent with the pharmacopoeial requirements. In conclusion, it can be suitable as a simple and economic procedure for routine quality control laboratories of clobetasol propionate in topical solution.

  4. Rapid Preclinical Detection of Sheeppox Virus by a Real-Time PCR Assay▿

    Science.gov (United States)

    Balinsky, C. A.; Delhon, G.; Smoliga, G.; Prarat, M.; French, R. A.; Geary, S. J.; Rock, D. L.; Rodriguez, L. L.

    2008-01-01

    Sheeppox virus (SPPV) is a member of the Capripoxvirus (CaPV) genus of the Poxviridae family. Members of this genus, which also include goatpox and lumpy skin disease viruses, cause economically significant disease in sheep, goats, and cattle. A rapid diagnostic assay for CaPV would be useful for disease surveillance as well as for detection of CaPV in clinical samples and for outbreak management. Here we describe a fluorogenic probe hydrolysis (TaqMan) PCR assay designed for rapid detection of CaPV and tested on sheep experimentally infected with a virulent strain of SPPV. This assay can detect SPPV in buffy coats, nasal swabs, oral swabs, scabs, and skin lesions as well as in lung and lymph nodes collected at necropsy. This single-tube diagnostic assay can be performed in 2 h or less and can detect viral DNA in preclinical, clinical, and postmortem samples. PMID:18032617

  5. Rapid and specific detection of Asian- and African-lineage Zika viruses

    Science.gov (United States)

    Chotiwan, Nunya; Brewster, Connie D.; Magalhaes, Tereza; Weger-Lucarelli, James; Duggal, Nisha K.; Rückert, Claudia; Nguyen, Chilinh; Garcia Luna, Selene M.; Fauver, Joseph R.; Andre, Barb; Gray, Meg; Black, William C.; Kading, Rebekah C.; Ebel, Gregory D.; Kuan, Guillermina; Balmaseda, Angel; Jaenisch, Thomas; Marques, Ernesto T. A.; Brault, Aaron C.; Harris, Eva; Foy, Brian D.; Quackenbush, Sandra L.; Perera, Rushika; Rovnak, Joel

    2017-01-01

    Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require precise diagnostic tools that are also appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that distinguishes Zika viruses of Asian and African lineages. The assay does not detect chikungunya virus or flaviviruses such as dengue, yellow fever, or West Nile viruses. The assay conditions allowed direct detection of Zika virus RNA in cultured infected cells; in mosquitoes; in virus-spiked samples of human blood, plasma, saliva, urine, and semen; and in infected patient serum, plasma, and semen samples without the need for RNA isolation or reverse transcription. The assay offers rapid, specific, sensitive, and inexpensive detection of the Asian-lineage Zika virus strain that is currently circulating in the Western hemisphere, and can also detect the African-lineage Zika virus strain using separate, specific primers. PMID:28469032

  6. 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.

  7. 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

    2016-01-01

    Background The AmpliVue™ Trichomonas Assay (Quidel) is a new FDA 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 (HDA) isothermal technology in conjunction with a disposable lateral-flow detection device, with a total turn-around time of approximately 45 minutes. Objective The objective of this study was to compare the performance of this new assay to wet preparation and culture, as well as to another FDA cleared nucleic acid amplification assay. Methods Four clinician collected vaginal swabs were obtained from women attending STD, family planning, and OB/GYN clinics and tested by AmpliVue™ Trichomonas Assay and comparator tests: saline microscopy, TV culture (InPouch™), and Aptima® TV (ATV). AmpliVue™ Trichomonas Assay results were compared to 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. Results A total of 992 patients, 342 symptomatic and 650 asymptomatic patients, were included in the study. Results for AmpliVue for all women combined compared to saline microscopy and culture as a composite positive comparator 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 to Aptima® was 90.7% %, while specificity was 98.9%. Conclusions The rapid AmpliVue™ Trichomonas Assay performed as well as microscopy and culture, and had comparable sensitivity and specificity to another NAAT 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. PMID:27196258

  8. Validation Study of Rapid Assays of Bioburden, Endotoxins and Other Contamination.

    Science.gov (United States)

    Shintani, Hideharu

    2016-01-01

    Microbial testing performed in support of pharmaceutical and biopharmaceutical production falls into three main categories: detection (qualitative), enumeration (quantitative), and characterization/identification. Traditional microbiological methods are listed in the compendia and discussed by using the conventional growth-based techniques, which are labor intensive and time consuming. In general, such tests require several days of incubation for microbial contamination (bioburden) to be detected, and therefore management seldom is able to take proactive corrective measures. In addition, microbial growth is limited by the growth medium used and incubation conditions, thus impacting testing sensitivity, accuracy, and reproducibility.  For more than 20 years various technology platforms for rapid microbiological methods (RMM) have been developed, and many have been readily adopted by the food industry and clinical microbiology laboratories. Their use would certainly offer drug companies faster test turnaround times to accommodate the aggressive deadlines for manufacturing processes and product release. Some rapid methods also offer the possibility for real-time microbial analyses, enabling management to respond to microbial contamination events in a more timely fashion, and can provide cost savings and higher efficiencies in quality control testing laboratories. Despite the many proven business and quality benefits and the fact that the FDA's initiative to promote the use of process analytical technology (PAT) includes rapid microbial methods, pharmaceutical and biopharmaceutical industries have been somewhat slow to embrace alternative microbial methodologies for several reasons. The major reason is that the bioburden counts detected by the incubation method and rapid assay are greatly divergent.  The use of rapid methods is a dynamic field in applied microbiology and one that has gained increased attention nationally and internationally over time. This topic

  9. Gold nanoparticle immunochromatographic assay for quantitative detection of urinary RBP

    Directory of Open Access Journals (Sweden)

    XU Kuan

    2013-04-01

    Full Text Available A rapid quantitative detection of urinary RBP was established by using nano-gold immunochromatography (sandwich method and trisodium citrate reduction method and a rapid immunochromatographic test strip was developed. Theimmunochromatographic test strip can quantitatively detect RBP within 15 minutes. The detection limit was 150ng/mL and detection range was from 150 to 5000 ng/mL. There were no cross-reactions with others kidney disease markers,such as urinary albumin (ALB,transferrin protein (TRF,β2-microglobulin (β2-MG,urinary fiber connecting protein (FN,and lysozyme (LZM. The results indicate that it is a quick and simple method with strong specificity,high sensitivity,and wide detection range. The rapid detection method will have extensive clinical applications in the early diagnosis of proximal tubular damage,kidney disease,diabetic nephropathy,and process monitoring.

  10. Integrated Biosensor Assay for Rapid Uropathogen Identification and Phenotypic Antimicrobial Susceptibility Testing.

    Science.gov (United States)

    Altobelli, Emanuela; Mohan, Ruchika; Mach, Kathleen E; Sin, Mandy Lai Yi; Anikst, Victoria; Buscarini, Maurizio; Wong, Pak Kin; Gau, Vincent; Banaei, Niaz; Liao, Joseph C

    2017-04-01

    Standard diagnosis of urinary tract infection (UTI) via urine culture for pathogen identification (ID) and antimicrobial susceptibility testing (AST) takes 2-3 d. This delay results in empiric treatment and contributes to the misuse of antibiotics and the rise of resistant pathogens. A rapid diagnostic test for UTI may improve patient care and antibiotic stewardship. To develop and validate an integrated biosensor assay for UTI diagnosis, including pathogen ID and AST, with determination of the minimum inhibitory concentration (MIC) for ciprofloxacin. Urine samples positive for Enterobacteriaceae (n=84) or culture-negative (n=23) were obtained from the Stanford Clinical Microbiology Laboratory between November 2013 and September 2014. Each sample was diluted and cultured for 5h with and without ciprofloxacin, followed by quantitative detection of bacterial 16S rRNA using a single electrochemical biosensor array functionalized with a panel of complementary DNA probes. Pathogen ID was determined using universal bacterial, Enterobacteriaceae (EB), and pathogen-specific probes. Phenotypic AST with ciprofloxacin MIC was determined using an EB probe to measure 16S rRNA levels as a function of bacterial growth. Electrochemical signals for pathogen ID at 6 SD over background were considered positive. An MIC signal of 0.4 log units lower than the no-antibiotic control indicated sensitivity. Results were compared to clinical microbiology reports. For pathogen ID, the assay had 98.5% sensitivity, 96.6% specificity, 93.0% positive predictive value, and 99.3% negative predictive value. For ciprofloxacin MIC the categorical and essential agreement was 97.6%. Further automation, testing of additional pathogens and antibiotics, and a full prospective study will be necessary for translation to clinical use. The integrated biosensor platform achieved microbiological results including MIC comparable to standard culture in a significantly shorter assay time. Further assay automation

  11. Plastic-adherent DNA aptamer-magnetic bead and quantum dot sandwich assay for Campylobacter detection.

    Science.gov (United States)

    Bruno, John G; Phillips, Taylor; Carrillo, Maria P; Crowell, Randy

    2009-05-01

    DNA aptamers were developed against MgCl(2)-extracted surface proteins from Campylobacter jejuni. The two highest affinity aptamers were selected for use in a magnetic bead (MB) and red quantum dot (QD)-based sandwich assay scheme. The assay was evaluated using both heat-killed and live C. jejuni and exhibits detection limits as low as an average of 2.5 colony forming unit (cfu) equivalents in buffer and 10-250 cfu in various food matrices. The assay exhibits low cross-reactivity with bacterial species outside the Campylobacter genus, but exhibits substantial cross-reactivity with C. coli and C. lari. The assay was evaluated with a spectrofluorometer and a commercially available handheld fluorometer, which yielded comparable detection limits and ranges. Remarkably, the sandwich assay components adhere to the inside face of polystyrene cuvettes even in food matrices near neutral pH, thereby enabling a rapid homogeneous assay, because fluorescence is concentrated to a small, thin planar area and background fluorescence from the bulk solution is minimized. The plastic cuvette-adherent technology coupled to a sensitive handheld fluorometer may enable rapid (15-20 min), portable detection of foodborne pathogens from "farm-to-fork" by obviating the slow enrichment culture phase used by other food safety tests.

  12. Rapid diagnostic tests duo as alternative to conventional serological assays for conclusive Chagas disease diagnosis.

    Science.gov (United States)

    Egüez, Karina E; Alonso-Padilla, Julio; Terán, Carolina; Chipana, Zenobia; García, Wilson; Torrico, Faustino; Gascon, Joaquim; Lozano-Beltran, Daniel-Franz; Pinazo, María-Jesús

    2017-04-01

    Chagas disease is caused by the parasite Trypanosoma cruzi. It affects several million people, mainly in Latin America, and severe cardiac and/or digestive complications occur in ~30% of the chronically infected patients. Disease acute stage is mostly asymptomatic and infection goes undiagnosed. In the chronic phase direct parasite detection is hampered due to its concealed presence and diagnosis is achieved by serological methods, like ELISA or indirect hemagglutination assays. Agreement in at least two tests must be obtained due to parasite wide antigenic variability. These techniques require equipped labs and trained personnel and are not available in distant regions. As a result, many infected people often remain undiagnosed until it is too late, as the two available chemotherapies show diminished efficacy in the advanced chronic stage. Easy-to-use rapid diagnostic tests have been developed to be implemented in remote areas as an alternative to conventional tests. They do not need electricity, nor cold chain, they can return results within an hour and some even work with whole blood as sample, like Chagas Stat-Pak (ChemBio Inc.) and Chagas Detect Plus (InBIOS Inc.). Nonetheless, in order to qualify a rapidly diagnosed positive patient for treatment, conventional serological confirmation is obligatory, which might risk its start. In this study two rapid tests based on distinct antigen sets were used in parallel as a way to obtain a fast and conclusive Chagas disease diagnosis using whole blood samples. Chagas Stat-Pak and Chagas Detect Plus were validated by comparison with three conventional tests yielding 100% sensitivity and 99.3% specificity over 342 patients seeking Chagas disease diagnosis in a reference centre in Sucre (Bolivia). Combined used of RDTs in distant regions could substitute laborious conventional serology, allowing immediate treatment and favouring better adhesion to it.

  13. A novel multiplex assay for simultaneously analysing 13 rapidly mutating Y-STRs

    NARCIS (Netherlands)

    R. Alghafri (Rashed); W. Goodwin (Will); A. Ralf (Arwin); M.H. Kayser (Manfred); S. Hadi (Sibte)

    2015-01-01

    textabstractAbstract A multiplex polymerase chain reaction (PCR) assay (RM-Yplex) was developed which is capable of simultaneously amplifying 13 recently introduced rapidly mutating Y-STR markers (RM Y-STRs). This multiplex assay is expected to aid human identity testing in forensic and other

  14. Universal primers for rapid detection of hytrosaviruses.

    Science.gov (United States)

    Abd-Alla, Adly M M; Salem, Tamer Z; Parker, Andrew G; Wang, Yongjie; Jehle, Johannes A; Vreysen, Marc J B; Boucias, Drion

    2011-01-01

    Hytrosaviridae is a proposed virus family encompassing viruses that cause salivary gland hypertrophy (SGH) syndrome in infected insects and reduce the fertility in their dipteran insect hosts. They contain a large, double stranded DNA genome of 120-190 kbp. To date, these viruses have been detected only in adult Diptera. These include hytrosaviruses detected in various tsetse fly species (Glossina spp.), the narcissus bulb fly Merodon equestris and the house fly Musca domestica. The limited number of hytrosaviruses reported to date may be a reflection of the frequent absence of external symptoms in infected adult flies and the fact that the virus does not cause rapid mortality. Based on the complete genome sequence of Glossinia pallidipes (GpSGHV) and Musca domestica (MdSGHV) salivary gland hypertrophy viruses, a PCR based methodology was developed to detect the viruses in these species. To be able to detect hytrosaviruses in other Diptera, five degenerate primer pairs were designed and tested on GpSGHV and MdSGHV DNA using gradient PCR with annealing temperatures from 37 to 61°C. Two pairs of primers were selected from p74, two pairs from PIF-1 and one pair from ODV-e66 homologous proteins. Four primer pairs generated a virus specific PCR product on both MdSGHV and GpSGHV at all tested annealing temperatures, while the ODV-e66 based primers did not generate a virus specific product with annealing temperatures higher that 47°C. No non-specific PCR product was found when using genomic DNA of infected flies as template DNA. These results offer new sets of primers that could be used to detect hytrosaviruses in other insects. Copyright © 2010 Elsevier B.V. All rights reserved.

  15. The DNA 'comet assay' as a rapid screening technique to control irradiated food.

    Science.gov (United States)

    Cerda, H; Delincée, H; Haine, H; Rupp, H

    1997-04-29

    The exposure of food to ionizing radiation is being progressively used in many countries to inactivate food pathogens, to eradicate pests, and to extend shelf-life, thereby contributing to a safer and more plentiful food supply. To ensure free consumer choice, irradiated food will be labelled as such, and to enforce labelling, analytical methods to detect the irradiation treatment in the food product itself are desirable. In particular, there is a need for simple and rapid screening methods for the control of irradiated food. The DNA comet assay offers great potential as a rapid tool to detect whether a wide variety of foodstuffs have been radiation processed. In order to simplify the test, the agarose single-layer set-up has been chosen, using a neutral protocol. Interlaboratory blind trials have been successfully carried out with a number of food products, both of animal and plant origin. This paper presents an overview of the hitherto obtained results and in addition the results of an intercomparison test with seeds, dried fruits and spices are described. In this intercomparison, an identification rate of 95% was achieved. Thus, using this novel technique, an effective screening of radiation-induced DNA fragmentation is obtained. Since other food treatments also may cause DNA fragmentation, samples with fragmented DNA suspected to have been irradiated should be analyzed by other validated methods for irradiated food, if such treatments which damage DNA cannot be excluded.

  16. The DNA `comet assay` as a rapid screening technique to control irradiated food

    Energy Technology Data Exchange (ETDEWEB)

    Cerda, H. [Department of Radioecology, The Swedish University of Agricultural Sciences, Uppsala (Sweden); Delincee, H. [Institute of Nutritional Physiology, Federal Research Centre for Nutrition, Karlsruhe (Germany); Haine, H. [Campden and Chorleywood Food Research Association, Chipping Campden, Gloucestershire (United Kingdom); Rupp, H. [Swiss Federal Office of Public Health, Section of Food Chemistry, Berne (Switzerland)

    1997-04-29

    The exposure of food to ionizing radiation is being progressively used in many countries to inactivate food pathogens, to eradicate pests, and to extend shelf-life, thereby contributing to a safer and more plentiful food supply. To ensure free consumer choice, irradiated food will be labelled as such, and to enforce labelling, analytical methods to detect the irradiation treatment in the food product itself are desirable. In particular, there is a need for simple and rapid screening methods for the control of irradiated food. The DNA comet assay offers great potential as a rapid tool to detect whether a wide variety of foodstuffs have been radiation processed. In order to simplify the test, the agarose single-layer set-up has been chosen, using a neutral protocol. Interlaboratory blind trials have been successfully carried out with a number of food products, both of animal and plant origin. This paper presents an overview of the hitherto obtained results and in addition the results of an intercomparison test with seeds, dried fruits and spices are described. In this intercomparison, an identification rate of 95% was achieved. Thus, using this novel technique, an effective screening of radiation-induced DNA fragmentation is obtained. Since other food treatments also may cause DNA fragmentation, samples with fragmented DNA suspected to have been irradiated should be analyzed by other validated methods for irradiated food, if such treatments which damage DNA cannot be excluded.

  17. Nanostructured bioluminescent sensor for rapidly detecting thrombin.

    Science.gov (United States)

    Chen, Longyan; Bao, Yige; Denstedt, John; Zhang, Jin

    2016-03-15

    Thrombin plays a key role in thrombosis and hemostasis. The abnormal level of thrombin in body fluids may lead to different diseases, such as rheumatoid arthritis, glomerulonephritis, etc. Detection of thrombin level in blood and/or urine is one of important methods for medical diagnosis. Here, a bioluminescent sensor is developed for non-invasively and rapidly detecting thrombin in urine. The sensor is assembled through conjugating gold nanoparticles (Au NPs) and a recombinant protein containing Renilla luciferase (pRluc) by a peptide, which is thrombin specific substrate. The luciferase-catalyzed bioluminescence can be quenched by peptide-conjugating Au NPs. In the presence of thrombin, the short peptide conjugating luciferase and Au NPs is digested and cut off, which results in the recovery of bioluminescence due to the release of luciferase from Au NPs. The bioluminescence intensity at 470 nm is observed, and increases with increasing concentration of thrombin. The bioluminescence intensity of this designed sensor is significantly recovered when the thrombin digestion time lasts for 10 min. In addition, a similar linear relationship between luminescence intensity and the concentration of thrombin is found in the range of 8 nM to 8 μM in both buffer and human urine spiked samples. The limit of detection is as low as 80 pM. It is anticipated that our nanosensor could be a promising tool for clinical diagnosis of thrombin in human urine. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Detection of human papillomavirus DNA by the hybrid capture assay

    Directory of Open Access Journals (Sweden)

    Carvalho Maria Odete O.

    2003-01-01

    Full Text Available Human Papillomavirus (HPV infection is the main cause of cervical cancers and cervical intraepithelial neoplasias (CIN worldwide. Consequently, it would be useful to evaluate HPV testing to screen for cervical cancer. Recently developed, the second-generation Hybrid Capture (HCA II test is a non-radioactive, relatively rapid, liquid hybridization assay designed to detect 18 HPV types, divided into high and low-risk groups. We evaluated 1055 women for HPV infection with the HCA II test. Five hundred and ten (48.3% of these women had HPV infection; 60 (11.8% had low cancer-risk HPV DNA; 269 (52.7% had high-risk HPV types and 181 (35.5% had both groups. Hence, 450 women (88.2% in this HPV-infected group had at least one high risk HPV type, and were therefore considered to be at high risk for cancer. Among the group with Papanicolaou (Pap test results, the overall prevalence of HPV DNA was 58.4%. Significant differences in HPV infection of the cervix were detected between Pap I (normal smears and Pap IV (carcinomas (p<0.0001. Values of HPV viral load obtained for Pap I and SILs were significantly different, with an upward trend (p<0.0001, suggesting a positive correlation between high viral load values and risk of SIL. Because of the high costs of the HCA II test, its use for routine cervical mass screening cannot be recommended in poor countries. Nevertheless, it is a useful tool when combined with cytology, diagnosing high-risk infections in apparently normal tissues. Use of this technique could help reduce the risk of cancer.

  19. A multiplex ligation detection assay for the characterization of Salmonella enterica strains

    DEFF Research Database (Denmark)

    Aarts, Henk J.M.; Vos, Pieter; Larsson, Jonas T.

    2011-01-01

    of four serovars each serovar was characterized by a unique virulence associated gene repertoire. The LDR microarray platform proved to be a convenient, rapid and easy to use tool with potential in tracing a Salmonella contamination in the food chain, for outbreak studies, and to provide data for risk....... The feasibility of the developed assay was verified in a method comparison study with conventional PCR using 16 Salmonella ‘test’ strains comprising eight serovars. Subsequently, the feasibility of the LDR microarray assay was also tested by analyzing 41 strains belonging to 23 serovars. With the exception......A proof of principle of a multi-target assay for genotyping Salmonella has been developed targeting 62 genomic marker sequences of Salmonella related to pathogenicity. The assay is based on multiplex ligation detection reaction (LDR) followed by customized ArrayTube® microarray detection...

  20. The comet assay: a sensitive method for detecting DNA damage in individual cells.

    Science.gov (United States)

    Liao, Wenjuan; McNutt, Michael A; Zhu, Wei-Guo

    2009-05-01

    The comet assay is a sensitive and rapid method for DNA strand break detection in individual cells, and the year 2009 represents the 25th anniversary of the first description of this methodology in 1984. Over time this method has been improved, but is still not completely standardized, and variations are currently in widespread use with emphasis on applications in research and genetic toxicology. Here we review the principles of the comet assay and cite key studies that have focused on this assay in the past 25 years. In addition, we present an example of how the comet assay was used in our laboratory for studying the induction of DNA damage in human lung cancer cells after differing doses of the cytosine analog 5-aza-2'-deoxycytidine (5-aza-CdR). Finally, some insights into the potential of this assay in cancer research and work in related fields are offered.

  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. Reliable Rapid Assay for Gonorrhea and Chlamydia in the Emergency Department.

    Science.gov (United States)

    Wilson, Sean P; Vohra, Taher; Goldberg, Jared; Price, Christopher; Calo, Sean; Mahan, Meredith; Miller, Joseph

    2017-12-01

    Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are common sexually transmitted infections seen in the emergency department (ED). Due to an inability to reliably make accurate diagnosis by physical examination, concern for unreliable follow-up, and current delays in diagnostic nucleic acid amplification testing (NAAT), presumptive treatment active against CT and NG, as described by Centers for Disease Control clinical practice guidelines, is often performed. The purpose of this study was to determine whether a rapid, urine NAAT performed in the ED is noninferior in its diagnostic sensitivity compared with a traditional, swab NAAT assay. We performed a prospective, noninferiority study comparing two U.S. Food and Drug Administration-approved NAAT assays for CT and NG: a 90-min rapid assay, the Xpert CT/NG Assay (Cepheid, Sunnyvale, CA) using a urine sample vs. a traditional assay, the Aptima Combo 2 Assay (Gen-Probe Incorporated, San Diego, CA) using a swab sample. This study was registered on Clinicaltrials.gov (NCT02386514). A total of 1162 patient samples were included in the primary analysis. We observed excellent kappa agreement between assays: NG for men, 1.00 (95% confidence interval [CI] 1.00-1.00); NG for women, 0.87 (95% CI 0.79-0.94); CT for men, 0.81 (95% CI 0.59-1.00); and CT for women: 0.85 (95% CI 0.80-0.90), as well as excellent negative and positive predictive values for the rapid assay. Although the rapid Xpert CT/NG assay's diagnostic sensitivity did not meet our prespecified threshold for noninferiority, the diagnostic characteristics are robust enough to fit into a management pathway that may reduce unnecessary antibiotic use. There may be an opportunity to utilize the rapid Xpert CT/NG assay to improve accuracy of treatment in the ED. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Rapid identification of pathogens in blood cultures with a modified fluorescence in situ hybridization assay

    NARCIS (Netherlands)

    Peters, Remco P. H.; van Agtmael, Michiel A.; Simoons-Smit, Alberdina M.; Danner, Sven A.; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.

    2006-01-01

    We evaluated a modified fluorescence in situ hybridization (FISH) assay for rapid ( <1 h) identification of microorganisms in growth-positive blood cultures. The results were compared to those of the standard FISH technique and conventional culturing. The rapid identification of microorganisms with

  4. Comparison of two multiplex PCR assays for the detection of respiratory viral infections.

    Science.gov (United States)

    Kim, Hanah; Hur, Mina; Moon, Hee-Won; Yun, Yeo-Min; Cho, Hyun Chan

    2014-10-01

    Respiratory viruses are the main causes of upper and lower respiratory tract diseases. Rapid and accurate detection of respiratory viruses is crucial for appropriate patient treatment and prevention of endemic spread. We compared two multiplex polymerase chain reaction (PCR) assays for the detection of respiratory viral pathogens. A total of 245 respiratory specimens (229 sputum samples, 14 bronchoalveolar lavage samples, 6 nasal swabs, 3 throat swabs, 7 unknown) were analyzed using two multiplex assays: One-step RV real-time PCR (BioSewoom, Seoul, Korea) and Seeplex RV 12 Detection kit (Seegene, Seoul, Korea). The results were further confirmed using sequencing as a reference. Among 245 samples (265 identifications including co-infections), the identification of respiratory viruses was 44.9% (119/265), 44.2% (117/265) and 45.3% (120/265) by One-step RV assay, Seeplex RV assay and sequencing, respectively. The concordance rate between One-step RV assay and sequencing was 95.5% (253/265), and that between Seeplex RV assay and sequencing was 89.8% (238/265) (P = 0.0189). The sensitivities of One-step RV and Seeplex RV assays were 94.1% [95% confidential interval (CI), 88.3%-97.6%] and 83.3% (95% CI, 75.4%-89.5%), respectively (P = 0.0002). The specificities of One-step RV and Seeplex RV assays were 96.6% (95% CI, 92.2%-98.9%) and 95.2% (95% CI, 90.3%-98.0%), respectively. Although the performances of One-step RV and Seeplex RV assays were overall comparable, One-step RV assay showed better sensitivity and concordance with sequencing. One-step RV assay can be a useful option for respiratory virus testing in clinical laboratories. © 2013 John Wiley & Sons Ltd.

  5. A rapid PCR-SSP assay for the hemochromatosis-associated Tyr250Stop mutation in the TFR2 gene.

    Science.gov (United States)

    Rivers, C A; Barton, J C; Acton, R T

    2001-01-01

    Several genes associated with hemochromatosis and primary iron overload have been identified. Mutations in the HFE gene have been detected in 60-100% of hemochromatosis patients of northern, central, and western European descent, although the frequencies of these mutations vary among racial and ethnic groups. Recently, a mutation in the gene encoding transferrin receptor-2 (exon 6, nucleotide 750 C --> G; Y250X) was detected by a PCR-restriction fragment length polymorphism (RFLP) method in Sicilians with hemochromatosis. We describe a modification of the original assay in which the sequence-specific priming PCR assay does not require the use of restriction endonuclease. The modified assay is robust and cost-efficient, and may be more useful for large-scale population studies because it can be performed rapidly on DNA extracted from buccal swabs.

  6. Enhanced Microbial Detection Capabilities by a Rapid Portable Instrument

    Science.gov (United States)

    Morris, Heather; Monaco, Lisa; Wainwright, Norm; Steele, Andrew; Damon, Michael; Schenk, Alison; Stimpson, Eric; Maule, Jake; Effinger, Michael

    2010-01-01

    We present data describing a progression of continuing technology development - from expanding the detection capabilities of the current PTS unit to re-outfitting the instrument with a protein microarray increasing the number of detectable compounds. To illustrate the adaptability of the cartridge format, on-orbit operations data from the ISS demonstrate the detection of the fungal cell wall compound beta-glucan using applicable LOCAD-PTS cartridges. LOCAD-PTS is a handheld device consisting of a spectrophotometer, an onboard pumping mechanism, and data storage capabilities. A suite of interchangeable cartridges lined with four distinct capillaries allow a hydrated sample to mix with necessary reagents in the channels before being pumped to the optical well for spectrophotometric analysis. The reagents housed in one type of cartridge trigger a reaction based on the Limulus Amebocyte Lysate (LAL) assay, which results in the release of paranitroaniline dye. The dye is measured using a 395 nm filter. The LAL assay detects the Gram-negative bacterial cell wall molecule, endotoxin or lipopolysaccharide (LPS). The more dye released, the greater the concentration of endotoxin in the sample. Sampling, quantitative analysis, and data retrieval require less than 20 minutes. This is significantly faster than standard culture-based methods, which require at least a 24 hour incubation period.Using modified cartridges, we demonstrate the detection of Gram negative bacteria with protein microarray technology. Additionally, we provide data from multiple field tests where both standard and advanced PTS technologies were used. These tests investigate the transfer of target microbial molecules from one surface to another. Collectively, these data demonstrate that the new cartridges expand the number of compounds detected by LOCAD-PTS, while maintaining the rapid, in situ analysis characteristic of the instrument. The unit provides relevant data for verifying sterile sample collection

  7. Henipavirus microsphere immuno-assays for detection of antibodies against Hendra virus.

    Science.gov (United States)

    McNabb, Leanne; Barr, J; Crameri, G; Juzva, S; Riddell, S; Colling, A; Boyd, V; Broder, C; Wang, L-F; Lunt, R

    2014-05-01

    Hendra and Nipah viruses (HeV and NiV) are closely related zoonotic pathogens of the Paramyxoviridae family. Both viruses belong to the Henipavirus genus and cause fatal disease in animals and humans, though only HeV is endemic in Australia. In general and due to the acute nature of the disease, agent detection by PCR and virus isolation are the primary tools for diagnostic investigations. Assays for the detection of antibodies against HeV are fit more readily for the purpose of surveillance testing in disease epidemiology and to meet certification requirements in the international movement of horses. The first generation indirect ELISA has been affected by non-specific reactions which must be resolved using virus neutralisation serology conducted at laboratory bio-safety level 4 containment (PC4). Recent developments have enabled improvements in the available serology assays. The production of an expressed recombinant truncated HeV G protein has been utilised in ELISA and in Luminex-based multiplexed microsphere assays. In the latter format, two Luminex assays have been developed for use in henipavirus serology: a binding assay (designed for antibody detection and differentiation) and a blocking assay (designed as a surrogate for virus neutralisation). Equine and canine field sera were used to evaluate the two Luminex assays relative to ELISA and virus neutralisation serology. Results showed that Luminex assays can be effective as rapid, sensitive and specific tests for the detection of HeV antibody in horse and dog sera. The tests do not require PC4 containment and are appropriate for high throughput applications as might be required for disease investigations and other epidemiological surveillance. Also, the results show that the Luminex assays detect effectively HeV vaccine-induced antibodies. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. 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.

  9. Development, validation and evaluation of a rapid PCR-nucleic acid lateral flow immuno-assay for the detection of Plasmodium and the differentiation between Plasmodium falciparum and Plasmodium vivax

    NARCIS (Netherlands)

    Mens, P.F.; Moers, A.P.H.A.; Bes, de L.M.; Flint, J.; Sak, J.R.S.; Keereecharoen, L.; Overmeir, C.; Verweij, J.J.; Hallett, R.L.; Wihokhoen, B.; Proux, S.; Schallig, H.D.F.H.; Amerongen, van A.

    2012-01-01

    Background: Molecular tools are very sensitive and specific and could be an alternative for the diagnosis of malaria. The complexity and need for expensive equipment may hamper implementation and, therefore, simplifications to current protocols are warranted. Methods: A PCR detecting the different

  10. 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.

  11. A rapid DNA extraction method suitable for human papillomavirus detection.

    Science.gov (United States)

    Brestovac, Brian; Wong, Michelle E; Costantino, Paul S; Groth, David

    2014-04-01

    Infection with oncogenic human papillomavirus (HPV) genotypes is necessary for the development of cervical cancer. Testing for HPV DNA from liquid based cervical samples can be used as an adjunct to traditional cytological screening. In addition there are ongoing viral load, genotyping, and prevalence studies. Therefore, a sensitive DNA extraction method is needed to maximize the efficiency of HPV DNA detection. The XytXtract Tissue kit is a DNA extraction kit that is rapid and so could be useful for HPV testing, particularly in screening protocols. This study was undertaken to determine the suitability of this method for HPV detection. DNA extraction from HeLa and Caski cell lines containing HPV 18 and 16 respectively together with DNA from five liquid based cervical samples were used in a HPV PCR assay. DNA was also extracted using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) as a comparison. DNA extracts were serially diluted and assayed. HPV DNA was successfully detected in cell lines and cervical samples using the XytXtract Tissue kit. In addition, the XytXtract method was found to be more sensitive than the QIAmp method as determined by a dilution series of the extracted DNA. While the XytXtract method is a closed, the QIAamp method uses a spin column with possible loss of DNA through DNA binding competition of the matrix, which could impact on the final extraction efficiency. The XytXtract is a cheap, rapid and efficient method for extracting HPV DNA from both cell lines and liquid based cervical samples. © 2014 Wiley Periodicals, Inc.

  12. One-Step Immunochromatography Assay Kit for Detecting Antibodies to Canine Parvovirus

    Science.gov (United States)

    Oh, Jin-Sik; Ha, Gun-Woo; Cho, Young-Shik; Kim, Min-Jae; An, Dong-Jun; Hwang, Kyu-Kye; Lim, Yoon-Kyu; Park, Bong-Kyun; Kang, BoKyu; Song, Dae-Sub

    2006-01-01

    This study was performed to determine the feasibility of using whole serum to detect antibodies to canine parvovirus (CPV) under nonlaboratory conditions and to evaluate the performance characteristics of an immunochromatography assay kit. Precise detection of levels of antibody against CPV in puppies can be used to determine a vaccination schedule, because maternal antibodies frequently result in the failure of protective vaccination, and can also be used to determine the antibody levels of infected puppies. Several methods for the titration of CPV antibodies have been reported, including the hemagglutination inhibition (HI) assay, which is considered the “gold standard.” These methods, however, require intricate and time-consuming procedures. In this study, a total of 386 serum specimens were tested. Compared to the HI assay, the rapid assay had a 97.1% sensitivity and a 76.6% specificity (with a cutoff HI titer of 1:80). This single-step assay could be performed rapidly and easily without special equipment. The kit provides a reliable method for detection of anti-CPV antibody where laboratory support and personnel are limited. PMID:16603622

  13. Rapid detection of equine coronavirus by reverse transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Nemoto, Manabu; Morita, Yoshinori; Niwa, Hidekazu; Bannai, Hiroshi; Tsujimura, Koji; Yamanaka, Takashi; Kondo, Takashi

    2015-04-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid detection of equine coronavirus (ECoV). This assay was conducted at 60 °C for 40 min. Specificity of the RT-LAMP assay was confirmed using several equine intestinal and respiratory pathogens in addition to ECoV. The novel assay failed to cross-react with the other pathogens tested, suggesting it is highly specific for ECoV. Using artificially synthesized ECoV RNA, the 50% detection limit of the RT-LAMP assay was 10(1.8)copies/reaction. This is a 50-fold greater sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR) assays, but a 4-fold lower sensitivity than quantitative RT-PCR (qRT-PCR) assays. Eighty-two fecal samples collected during ECoV outbreaks were analyzed. ECoV was detected in 59 samples using the RT-LAMP assay, and in 30 and 65 samples using RT-PCR or qRT-PCR assays, respectively. Although the RT-LAMP assay is less sensitive than qRT-PCR techniques, it can be performed without the need for expensive equipment. Thus, the RT-LAMP assay might be suitable for large-scale surveillance and diagnosis of ECoV infection in laboratories with limited resources. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. An immunochemical assay to detect DNA damage in bovine sperm

    NARCIS (Netherlands)

    Schans, G.P. van der; Haring, R.; Dijk- Knijnenburg, H.C.M. van; Bruijnzeel, P.L.B.; Daas, N.H.G. den

    2000-01-01

    An immunochemical assay has been developed to detect oxidative damage in bovine sperm DNA. Sperm DNA contains a large amount of oxidative damage as a result of exposure to exogenous agents, but damage also can caused by normal metabolic processes and the absence of DNA repair in the later stages of

  15. Development of a rapid and sensitive method combining a cellulose ester microfilter and a real-time quantitative PCR assay to detect Campylobacter jejuni and Campylobacter coli in 20 liters of drinking water or low-turbidity waters.

    Science.gov (United States)

    Tissier, Adeline; Denis, Martine; Hartemann, Philippe; Gassilloud, Benoît

    2012-02-01

    Investigations of Campylobacter jejuni and Campylobacter coli in samples of drinking water suspected of being at the origin of an outbreak very often lead to negative results. One of the reasons for this failure is the small volume of water typically used for detecting these pathogens (10 to 1,000 ml). The efficiencies of three microfilters and different elution procedures were determined using real-time quantitative PCR to propose a procedure allowing detection of Campylobacter in 20 liters of drinking water or low-turbidity water samples. The results showed that more than 80% of the bacteria inoculated in 1 liter of drinking water were retained on each microfilter. An elution with a solution containing 3% beef extract, 0.05 M glycine at pH 9, combined with direct extraction of the bacterial genomes retained on the cellulose ester microfilter, allowed recovery of 87.3% (±22% [standard deviation]) of Campylobacter per 1 liter of tap water. Recoveries obtained from 20-liter volumes of tap water spiked with a C. coli strain were 69.5% (±10.3%) and 78.5% (±15.1%) for 91 CFU and 36 CFU, respectively. Finally, tests performed on eight samples of 20 liters of groundwater collected from an alluvial well used for the production of drinking water revealed the presence of C. jejuni and C. coli genomes, whereas no bacteria were detected with the normative culture method in volumes ranging from 10 to 1,000 ml. In the absence of available epidemiological data and information on bacterial viability, these last results indicate only that the water resource is not protected from contamination by Campylobacter.

  16. Development of a SERS-Based Rapid Vertical Flow Assay for Point-of-Care Diagnostics.

    Science.gov (United States)

    Clarke, O J R; Goodall, B L; Hui, H P; Vats, N; Brosseau, C L

    2017-02-07

    Point-of-care (POC) diagnostic testing platforms are a growing sector of the healthcare industry as they offer the advantages of rapid provision of results, ease of use, reduced cost, and the ability to link patients to care. While many POC tests are based on chromatographic flow assay technology, this technology suffers from a lack of sensitivity along with limited capacity for multiplexing and quantitative analysis. Several recent reports have begun to investigate the feasibility of coupling chromatographic flow platforms to more advanced read-out technologies which in turn enable on-site acquisition, storage, and transmission of important healthcare metrics. One such technology being explored is surface-enhanced Raman spectroscopy or SERS. In this work, SERS is coupled for the first time to a rapid vertical flow (RVF) immunotechnology for detection of anti-HCV antibodies in an effort to extend the capabilities of this commercially available diagnostic platform. High-quality and reproducible SERS spectra were obtained using reporter-modified gold nanoparticles (AuNPs). Serial dilution studies indicate that the coupling of SERS with RVF technology shows enormous potential for next-generation POC diagnostics.

  17. Automation of plasma protein binding assay using rapid equilibrium dialysis device and Tecan workstation.

    Science.gov (United States)

    Ye, Zhengqi; Zetterberg, Craig; Gao, Hong

    2017-06-05

    Binding of drug molecules to plasma proteins is an important parameter in assessing drug ADME properties. Plasma protein binding (PPB) assays are routinely performed during drug discovery and development. A fully automated PPB assay was developed using rapid equilibrium dialysis (RED) device and Tecan workstation coupled to an automated incubator. The PPB assay was carried out in unsealed RED plates which allowed the assay to be fully automated. The plasma pH was maintained at 7.4 during the 6-h dialysis under 2% CO2 condition. The samples were extracted with acetonitrile and analyzed by liquid chromatography tandem mass spectrometry. The percent bound results of 10 commercial drugs in plasma protein binding were very similar between the automated and manual assays, and were comparable to literature values. The automated assay increases laboratory productivity and is applicable to high-throughput screening of drug protein binding in drug discovery. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Development of a quantitative real-time detection assay for hepatitis B virus DNA and comparison with two commercial assays

    NARCIS (Netherlands)

    Pas, S D; Fries, E; De Man, R A; Osterhaus, A D; Niesters, H G

    A highly reproducible and sensitive real-time detection assay based on TaqMan technology was developed for the detection of hepatitis B virus (HBV) DNA and compared with two commercially available assays. The assay was validated with the Viral Quality Control panel, which also includes EUROHEP HBV

  19. Development of a quantitative real-time detection assay for hepatitis B virus DNA and comparison with two commercial assays

    NARCIS (Netherlands)

    S.D. Pas (Suzan); E. Fries; H.G.M. Niesters (Bert); R.A. de Man (Robert); A.D.M.E. Osterhaus (Albert)

    2000-01-01

    textabstractA highly reproducible and sensitive real-time detection assay based on TaqMan technology was developed for the detection of hepatitis B virus (HBV) DNA and compared with two commercially available assays. The assay was validated with the Viral Quality Control panel,

  20. 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

  1. Development of a rapid serological assay for the diagnosis of strongyloidiasis using a novel diffraction-based biosensor technology.

    Directory of Open Access Journals (Sweden)

    Brian J Pak

    2014-08-01

    Full Text Available Strongyloidiasis is a persistent human parasitic infection caused by the intestinal nematode, Strongyloides stercoralis. The parasite has a world-wide distribution, particularly in tropical and subtropical regions with poor sanitary conditions. Since individuals with strongyloidiasis are typically asymptomatic, the infection can persist for decades without detection. Problems arise when individuals with unrecognized S. stercoralis infection are immunosuppressed, which can lead to hyper-infection syndrome and disseminated disease with an associated high mortality if untreated. Therefore a rapid, sensitive and easy to use method of diagnosing Strongyloides infection may improve the clinical management of this disease.An immunological assay for diagnosing strongyloidiasis was developed on a novel diffraction-based optical bionsensor technology. The test employs a 31-kDa recombinant antigen called NIE derived from Strongyloides stercoralis L3-stage larvae. Assay performance was tested using retrospectively collected sera from patients with parasitologically confirmed strongyloidiasis and control sera from healthy individuals or those with other parasitoses including schistosomiasis, trichinosis, echinococcosis or amebiasis who were seronegative using the NIE ELISA assay. If we consider the control group as the true negative group, the assay readily differentiated S. stercoralis-infected patients from controls detecting 96.3% of the positive cases, and with no cross reactivity observed in the control group These results were in excellent agreement (κ = 0.98 with results obtained by an NIE-based enzyme-linked immunosorbent assay (ELISA. A further 44 sera from patients with suspected S. stercoralis infection were analyzed and showed 91% agreement with the NIE ELISA.In summary, this test provides high sensitivity detection of serum IgG against the NIE Strongyloides antigen. The assay is easy to perform and provides results in less than 30 minutes

  2. Development of a competitive immunochromatographic assay for the sensitive detection of amantadine in chicken muscle.

    Science.gov (United States)

    Wu, Songsong; Zhu, Fangfei; Hu, Liming; Xia, Jun; Xu, Guomao; Liu, Daofeng; Guo, Qi; Luo, Kai; Lai, Weihua

    2017-10-01

    Amantadine (AMD) is a prohibitive veterinary medicine in the entire world. In this study, a sensitive colloidal gold immunochromatographic assay (CGICA) was established for the rapid semi-quantitative detection of AMD in chicken muscle. Under optimal conditions, the detection results were obtained in 12min with a limit of detection for 1.80ng/mL. CGICA presented a good linear range from 2.5ng/mL to 25ng/mL, with only 11.5% cross-reactivity with rimantadine. The recovery rates for the fortified samples were ranged from 81% to 120%. The coefficient of variation of the intra-assay and inter-assay was less than 15%. The accuracy of CGICA was confirmed by systematically comparing the result of the proposed method with enzyme-linked immunosorbent assay and ultra-high-performance liquid chromatography-tandem mass spectrometry. Given the advantages of its simplicity, convenience, and speediness, the proposed CGICA is suitable for the on-site rapid detection of AMD in chicken muscle. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Rapid 2,2'-bicinchoninic-based xylanase assay compatible with high throughput screening

    Science.gov (United States)

    William R. Kenealy; Thomas W. Jeffries

    2003-01-01

    High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2'-bicinchoninic-based protein reagent. Endo-1,4-â-D-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml−1. The assay is linear for sugar...

  4. Single Cell Gel Electrophoresis in DNA Damage Detection (Comet Assay

    Directory of Open Access Journals (Sweden)

    Aysen Durmaz

    2010-08-01

    Full Text Available “Single cell gel electrophoresis (SCGE”, also called “Comet Assay”, is a sensitive, reliable and rapid technique for quantifying and analyzing DNA damage in individual cells. The comet assay is widely used in living cells, researches and the applications on comet assay is becoming broader day by day. To date, the comet assay has been used for a variety of applications, including genotoxic and cytotoxic agent analyses, environmental toxicology, cancer research, and radiation biology. Briefly, in comet assay, fully frosted microscope slides were first covered with 0.5% normal melting point agarose (NMA and air-dried at the room temperature then cells were mixed with 0.5% low melting point agarose (LMA at 37oC to form a cell suspension which was spread onto the slide surface and let it solidified. A third layer of 0.5% low melting point agarose was then added and again allowed to solidify. After preparing the three layer agarose the slides were immersed in lysing solution at least for an hour. The slides were then placed in an electrophoresis tank which contained neutral or alkaline buffer solution and kept in there for a short while prior to electric field application. After electrophoresis, the slides were washed with neutralization solution or PBS and stained with a DNA-specific fluorescent dye and analyzed using a fluorescent microscope. [Archives Medical Review Journal 2010; 19(4.000: 236-

  5. Development of an enzyme linked immunosorbent assay and an immunochromatographic assay for detection of organophosphorus pesticides in different agricultural products.

    Directory of Open Access Journals (Sweden)

    Xiude Hua

    Full Text Available OBJECTIVE: Organophosphorus (OP pesticides are considered hazardous substances because of their high toxicity to nontarget species and their persistence in the environment and agricultural products. Therefore, it is important to develop a rapid, sensitive, and economical method for detecting OP pesticides and their residues in food and the environment. METHODS: A broad, selective monoclonal antibody (MAb for organophosphorus pesticides was produced. Based on the MAb, an enzyme linked immunosorbent assay (ELISA and an immunochromatography assay (ICA for detecting OP pesticides in different agricultural products were developed using a binding inhibition format on microtiter plates and a membrane strip, respectively. RESULTS: Under the optimized conditions, the IC(50 values of the ELISA ranged from 3.7 to 162.2 ng mL(-1 for the 8 OP pesticides. The matrix interferences of Apple, Chinese cabbage, and greengrocery were removed by 40-fold dilution, the recoveries from spiked samples ranged from 79.1% to 118.1%. The IC(50 values of ICA for the 8 OP pesticides ranged from 11.8 to 470.4 ng mL(-1. The matrix interference was removed from the Chinese cabbage and Apple samples with 5-fold dilution, and the interference was removed from the greengrocery samples with 20-fold dilution. The recoveries from the spiked samples ranged between 70.6 and 131.9%. The established ELISA and ICA were specific selectivity for the 8 OP pesticides. CONCLUSIONS: The established ELISA is a sensitive screening method for the detection of OP pesticides, but the ELISA detection method depends on a laboratory platform and requires a relative long assay time and several steps operation. The established ICA is very useful as a screening method for the quantitative, semi-quantitative or qualitative detection of OP pesticides in agricultural products, and it has advantages over ELISA methods with regard to factors such as the testing procedure, testing time, and matrix interferences

  6. 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.

  7. Development of real-time PCR assays for detection of megalocytiviruses in imported ornamental fish.

    Science.gov (United States)

    Gias, E; Johnston, C; Keeling, S; Spence, R P; McDonald, W L

    2011-08-01

    Megalocytiviruses have been associated globally with severe systemic disease and economic loss in farmed food fish and ornamental fish. The viruses have been spread internationally by translocation of live fish. In New Zealand, megalocytiviruses are regarded as exotic. A potential pathway for introduction has been identified, namely imported ornamental fish. In the present study, real-time PCR assays were developed for detection of megalocytiviruses using a conserved major capsid protein gene. A SYBR green assay was developed to target all known megalocytiviruses. A second real-time PCR assay using a molecular beacon was developed to specifically target gourami, Trichogaster trichopterus, iridovirus, a species of iridovirus previously linked to ornamental fish imports in Australia. The analytical sensitivity for the SYBR green and molecular beacon assays were 10 and 100 fg, respectively. The analytical specificity of the real-time PCR assays determined using genomic DNA templates from three target viruses, 12 non-target viruses and 25 aquatic bacterial species were 100%. The intra-run and inter-run coefficients of variation of both assays were <5%. The real-time PCR assays developed in this study provide rapid, sensitive, and specific detection of megalocytiviruses and gourami iridovirus. © 2011 Blackwell Publishing Ltd.

  8. Miniaturized Aptamer-Based Assays for Protein Detection

    Directory of Open Access Journals (Sweden)

    Alessandro Bosco

    2016-09-01

    Full Text Available The availability of devices for cancer biomarker detection at early stages of the disease is one of the most critical issues in biomedicine. Towards this goal, to increase the assay sensitivity, device miniaturization strategies empowered by the employment of high affinity protein binders constitute a valuable approach. In this work we propose two different surface-based miniaturized platforms for biomarker detection in body fluids: the first platform is an atomic force microscopy (AFM-based nanoarray, where AFM is used to generate functional nanoscale areas and to detect biorecognition through careful topographic measurements; the second platform consists of a miniaturized electrochemical cell to detect biomarkers through electrochemical impedance spectroscopy (EIS analysis. Both devices rely on robust and highly-specific protein binders as aptamers, and were tested for thrombin detection. An active layer of DNA-aptamer conjugates was immobilized via DNA directed immobilization on complementary single-stranded DNA self-assembled monolayers confined on a nano/micro area of a gold surface. Results obtained with these devices were compared with the output of surface plasmon resonance (SPR assays used as reference. We succeeded in capturing antigens in concentrations as low as a few nM. We put forward ideas to push the sensitivity further to the pM range, assuring low biosample volume (μL range assay conditions.

  9. Development of rapid, specific and sensitive detection of Cucumber ...

    African Journals Online (AJOL)

    detection of the CMV in infected plants using a monoclonal and polyclonal antibodies. Dotimmunobinding assays (DIBA) are useful alternatives to microtitre plate enzyme-linked immunosorbent assay (ELISA). Nine monoclonal antibodies were readily used for detected CMV by TAS-ELISA and DIBA of infected plants.

  10. Development and evaluation of a rapid dipstick assay for serodiagnosis of acute human brucellosis

    NARCIS (Netherlands)

    Smits, H. L.; Basahi, M. A.; Díaz, R.; Marrodan, T.; Douglas, J. T.; Rocha, A.; Veerman, J.; Zheludkov, M. M.; Witte, O. W.; de Jong, J.; Gussenhoven, G. C.; Goris, M. G.; van der Hoorn, M. A.

    1999-01-01

    A dipstick assay for the detection of brucella-specific immunoglobulin M antibodies was evaluated with 707 sera from 247 laboratory-confirmed brucellosis patients and 342 control sera from brucellosis-free individuals. These sera were collected from six different countries. The assay was found to be

  11. Rapid Detection of Cell-Free Mycobacterium tuberculosis DNA in Tuberculous Pleural Effusion.

    Science.gov (United States)

    Che, Nanying; Yang, Xinting; Liu, Zichen; Li, Kun; Chen, Xiaoyou

    2017-05-01

    Tuberculous pleurisy is one of the most common types of extrapulmonary tuberculosis, but its diagnosis remains difficult. In this study, we report for the first time on the detection of cell-free Mycobacterium tuberculosis DNA in pleural effusion and an evaluation of a newly developed molecular assay for the detection of cell-free Mycobacterium tuberculosis DNA. A total of 78 patients with pleural effusion, 60 patients with tuberculous pleurisy, and 18 patients with alternative diseases were included in this study. Mycobacterial culture, the Xpert MTB/RIF assay, the adenosine deaminase assay, the T-SPOT.TB assay, and the cell-free Mycobacterium tuberculosis DNA assay were performed on all the pleural effusion samples. The cell-free Mycobacterium tuberculosis DNA assay and adenosine deaminase assay showed significantly higher sensitivities of 75.0% and 68.3%, respectively, than mycobacterial culture and the Xpert MTB/RIF assay, which had sensitivities of 26.7% and 20.0%, respectively (P Mycobacterium tuberculosis DNA assay detected as few as 1.25 copies of IS6110 per ml of pleural effusion and showed good accordance of the results between repeated tests (r = 0.978, P = 2.84 × 10-10). These data suggest that the cell-free Mycobacterium tuberculosis DNA assay is a rapid and accurate molecular test which provides direct evidence of Mycobacterium tuberculosis etiology. Copyright © 2017 American Society for Microbiology.

  12. 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.

  13. 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.

  14. Rapid detection, characterization, and enumeration of foodborne pathogens.

    Science.gov (United States)

    Hoorfar, J

    2011-11-01

    enough to test for many pathogens but also many pathogens can be detected with one test. The review is mainly based on the author's scientific work that has contributed with the following new developments to this field: (i) serologic tests for large-scale screening, surveillance, or eradication programs, (ii) same-day detection of Salmonella that otherwise was considered as difficult to achieve, (iii) pathogen enumeration following a short log-phase enrichment, (iv) detection of foodborne pathogens in air samples, and finally (v) biotracing of pathogens based on mathematical modeling, even in the absence of isolate. Rapid methods are discussed in a broad global health perspective, international food supply, and for improvement of quantitative microbial risk assessments. The need for quantitative sample preparation techniques, culture-independent, metagenomic-based detection, online monitoring, a global validation infrastructure has been emphasized. The cost and ease of use of rapid assays remain challenging obstacles to surmount. © 2011 The Author. APMIS © 2011 APMIS.

  15. Rapid method for protein quantitation by Bradford assay after elimination of the interference of polysorbate 80.

    Science.gov (United States)

    Cheng, Yongfeng; Wei, Haiming; Sun, Rui; Tian, Zhigang; Zheng, Xiaodong

    2016-02-01

    Bradford assay is one of the most common methods for measuring protein concentrations. However, some pharmaceutical excipients, such as detergents, interfere with Bradford assay even at low concentrations. Protein precipitation can be used to overcome sample incompatibility with protein quantitation. But the rate of protein recovery caused by acetone precipitation is only about 70%. In this study, we found that sucrose not only could increase the rate of protein recovery after 1 h acetone precipitation, but also did not interfere with Bradford assay. So we developed a method for rapid protein quantitation in protein drugs even if they contained interfering substances. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Evaluation of simple rapid HIV assays and development of national rapid HIV test algorithms in Dar es Salaam, Tanzania

    Directory of Open Access Journals (Sweden)

    Mbwana Judica

    2009-02-01

    Full Text Available Abstract Background Suitable algorithms based on a combination of two or more simple rapid HIV assays have been shown to have a diagnostic accuracy comparable to double enzyme-linked immunosorbent assay (ELISA or double ELISA with Western Blot strategies. The aims of this study were to evaluate the performance of five simple rapid HIV assays using whole blood samples from HIV-infected patients, pregnant women, voluntary counseling and testing attendees and blood donors, and to formulate an alternative confirmatory strategy based on rapid HIV testing algorithms suitable for use in Tanzania. Methods Five rapid HIV assays: Determine™ HIV-1/2 (Inverness Medical, SD Bioline HIV 1/2 3.0 (Standard Diagnostics Inc., First Response HIV Card 1–2.0 (PMC Medical India Pvt Ltd, HIV1/2 Stat-Pak Dipstick (Chembio Diagnostic System, Inc and Uni-Gold™ HIV-1/2 (Trinity Biotech were evaluated between June and September 2006 using 1433 whole blood samples from hospital patients, pregnant women, voluntary counseling and testing attendees and blood donors. All samples that were reactive on all or any of the five rapid assays and 10% of non-reactive samples were tested on a confirmatory Inno-Lia HIV I/II immunoblot assay (Immunogenetics. Results Three hundred and ninety samples were confirmed HIV-1 antibody positive, while 1043 were HIV negative. The sensitivity at initial testing of Determine, SD Bioline and Uni-Gold™ was 100% (95% CI; 99.1–100 while First Response and Stat-Pak had sensitivity of 99.5% (95% CI; 98.2–99.9 and 97.7% (95% CI; 95.7–98.9, respectively, which increased to 100% (95% CI; 99.1–100 on repeat testing. The initial specificity of the Uni-Gold™ assay was 100% (95% CI; 99.6–100 while specificities were 99.6% (95% CI; 99–99.9, 99.4% (95% CI; 98.8–99.7, 99.6% (95% CI; 99–99.9 and 99.8% (95% CI; 99.3–99.9 for Determine, SD Bioline, First Response and Stat-Pak assays, respectively. There was no any sample which was

  17. Detection of shiga toxins by lateral flow assay

    Science.gov (United States)

    Shiga toxin-producing Escherichia coli (STEC) produce Shiga toxins (Stxs) that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript we report ...

  18. A rapid Raman detection of deoxynivalenol in agricultural products.

    Science.gov (United States)

    Yuan, Jing; Sun, Chuanwen; Guo, Xiaoyu; Yang, Tianxi; Wang, Hui; Fu, Shuyue; Li, Chuanchuan; Yang, Haifeng

    2017-04-15

    Mycotoxin results in financial damage and considerable safety risks. In this paper, the possibility of portable Raman system-based surface-enhanced Raman scattering (SERS) for a rapid detection of deoxynivalenol (DON) a mycotoxin in cereals was investigated. Under an optimized condition, SERS analysis for pure DON solution has a wide dynamic concentration range from 10-7M to 10-2M with the limit of detection (LOD) down to 100nM. Density functional theory (DFT) analysis at the level of B3LYP/6-311++G(d, p) was also preformed for vibrational assignment. For practical application, the LOD of the proposed Raman method for both DON-contaminated corns and kidney beans were validated as 10-6M and the LOD for DON-contaminated oats was 10-4M. As a perspective, the SERS-based technology could be developed into an alternatively promising assay for on-field detection of DON residues at various cereals due to it high sensitivity and selectivity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Fundamentals of rapid injection molding for microfluidic cell-based assays.

    Science.gov (United States)

    Lee, Ulri N; Su, Xiaojing; Guckenberger, David J; Dostie, Ashley M; Zhang, Tianzi; Berthier, Erwin; Theberge, Ashleigh B

    2018-01-30

    Microscale cell-based assays have demonstrated unique capabilities in reproducing important cellular behaviors for diagnostics and basic biological research. As these assays move beyond the prototyping stage and into biological and clinical research environments, there is a need to produce microscale culture platforms more rapidly, cost-effectively, and reproducibly. 'Rapid' injection molding is poised to meet this need as it enables some of the benefits of traditional high volume injection molding at a fraction of the cost. However, rapid injection molding has limitations due to the material and methods used for mold fabrication. Here, we characterize advantages and limitations of rapid injection molding for microfluidic device fabrication through measurement of key features for cell culture applications including channel geometry, feature consistency, floor thickness, and surface polishing. We demonstrate phase contrast and fluorescence imaging of cells grown in rapid injection molded devices and provide design recommendations to successfully utilize rapid injection molding methods for microscale cell-based assay development in academic laboratory settings.

  20. Immunochromatographic colloidal carbon-based assay for detection of methiocarb in surface water.

    Science.gov (United States)

    Blazková, Martina; Micková-Holubová, Barbora; Rauch, Pavel; Fukal, Ladislav

    2009-12-15

    A simple and rapid immunochromatographic assay for a sensitive and inexpensive monitoring of methiocarb in surface water was developed using a binding inhibition format on a membrane strip. In the assay, detection reagent consisted of anti-methiocarb antibody and colloidal carbon-labelled secondary antibody. Methiocarb-ovalbumin conjugate was immobilized in a test line of the strip as a capture reagent. Colour intensity of the test line in methiocarb-positive assay was visually distinguishable from that of negative sample within 10min. The optimized semi-quantitative method provided a visual detection limit of 0.5ngmL(-1). Cross-reactions with other carbamate pesticides were not found (<1%). Only a negligible matrix effect of surface water was recognized. In parallel analyses of spiked water samples, the assay results were in a good agreement with those of ELISA. The stability test indicated the strips could be used at least 2 months without change in performance. All characteristics of the visually evaluated assay mentioned above were verified by instrumental quantification of colour intensity in test lines. The developed immunochromatographic assay offers potential as a useful on-site screening tool for environmental analysis.

  1. Development and evaluation of a blocking enzyme-linked immunosorbent assay and virus neutralization assay to detect antibodies to viral hemorrhagic septicemia virus

    Science.gov (United States)

    Wilson, Anna; Goldberg, Tony; Marcquenski, Susan; Olson, Wendy; Goetz, Frederick; Hershberger, Paul; Hart, Lucas M.; Toohey-Kurth, Kathy

    2014-01-01

    Viral hemorrhagic septicemia virus (VHSV) is a target of surveillance by many state and federal agencies in the United States. Currently, the detection of VHSV relies on virus isolation, which is lethal to fish and indicates only the current infection status. A serological method is required to ascertain prior exposure. Here, we report two serologic tests for VHSV that are nonlethal, rapid, and species independent, a virus neutralization (VN) assay and a blocking enzyme-linked immunosorbent assay (ELISA). The results show that the VN assay had a specificity of 100% and sensitivity of 42.9%; the anti-nucleocapsid-blocking ELISA detected nonneutralizing VHSV antibodies at a specificity of 88.2% and a sensitivity of 96.4%. The VN assay and ELISA are valuable tools for assessing exposure to VHSV.

  2. Amorphous carbon nanoparticles: a versatile label for rapid diagnostic (immuno)assays

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Wichers, J.H.; Koets, M.; Berendsen, L.B.J.M.; Amerongen, van A.

    2012-01-01

    Carbon nanoparticles (CNPs) labeled with reporter molecules can serve as signaling labels in rapid diagnostic assays as an alternative to gold, colored latex, silica, quantum dots, or up-converting phosphor nanoparticles. Detailed here is the preparation of biomolecule-labeled CNPs and examples of

  3. A rapid assay for on-site monitoring of infliximab trough levels: a feasibility study

    NARCIS (Netherlands)

    Corstjens, Paul L. A. M.; Fidder, Herma H.; Wiesmeijer, Karien C.; de Dood, Claudia J.; Rispens, Theo; Wolbink, Gert-Jan; Hommes, Daniel W.; Tanke, Hans J.

    2013-01-01

    Monitoring levels of biologicals against tumor necrosis factor (TNF) has been suggested to improve therapeutic outcomes in inflammatory bowel diseases (IBDs). This pilot study describes a rapid lateral flow (LF)-based assay for on-site monitoring of serum trough levels of humanized monoclonal

  4. A rapid colorimetric assay for mold spore germination using XTT tetrazolium salt

    Science.gov (United States)

    Carol A. Clausen; Vina W. Yang

    2011-01-01

    Current laboratory test methods to measure efficacy of new mold inhibitors are time consuming, some require specialized test equipment and ratings are subjective. Rapid, simple quantitative assays to measure the efficacy of mold inhibitors are needed. A quantitative, colorimetric microassay was developed using XTT tetrazolium salt to metabolically assess mold spore...

  5. Sensitive detection of norovirus using phage nanoparticle reporters in lateral-flow assay.

    Directory of Open Access Journals (Sweden)

    Anna E V Hagström

    Full Text Available Noroviruses are recognized worldwide as the principal cause of acute, non-bacterial gastroenteritis, resulting in 19-21 million cases of disease every year in the United States. Noroviruses have a very low infectious dose, a short incubation period, high resistance to traditional disinfection techniques and multiple modes of transmission, making early, point-of-care detection essential for controlling the spread of the disease. The traditional diagnostic tools, electron microscopy, RT-PCR and ELISA require sophisticated and expensive instrumentation, and are considered too laborious and slow to be useful during severe outbreaks. In this paper we describe the development of a new, rapid and sensitive lateral-flow assay using labeled phage particles for the detection of the prototypical norovirus GI.1 (Norwalk, with a limit of detection of 107 virus-like particles per mL, one hundred-fold lower than a conventional gold nanoparticle lateral-flow assay using the same antibody pair.

  6. Sensitive Detection of Norovirus Using Phage Nanoparticle Reporters in Lateral-Flow Assay

    Science.gov (United States)

    Hagström, Anna E. V.; Garvey, Gavin; Paterson, Andrew S.; Dhamane, Sagar; Adhikari, Meena; Estes, Mary K.; Strych, Ulrich; Kourentzi, Katerina; Atmar, Robert L.; Willson, Richard C.

    2015-01-01

    Noroviruses are recognized worldwide as the principal cause of acute, non-bacterial gastroenteritis, resulting in 19-21 million cases of disease every year in the United States. Noroviruses have a very low infectious dose, a short incubation period, high resistance to traditional disinfection techniques and multiple modes of transmission, making early, point-of-care detection essential for controlling the spread of the disease. The traditional diagnostic tools, electron microscopy, RT-PCR and ELISA require sophisticated and expensive instrumentation, and are considered too laborious and slow to be useful during severe outbreaks. In this paper we describe the development of a new, rapid and sensitive lateral-flow assay using labeled phage particles for the detection of the prototypical norovirus GI.1 (Norwalk), with a limit of detection of 107 virus-like particles per mL, one hundred-fold lower than a conventional gold nanoparticle lateral-flow assay using the same antibody pair. PMID:25978622

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

    Directory of Open Access Journals (Sweden)

    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

  8. 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

  9. HIV-Selectest enzyme immunoassay and rapid test: ability to detect seroconversion following HIV-1 infection.

    Science.gov (United States)

    Khurana, Surender; Norris, Philip J; Busch, Michael P; Haynes, Barton F; Park, Susan; Sasono, Pretty; Mlisana, Koleka; Salim, Abdool Karim; Hecht, Frederick M; Mulenga, Joseph; Chomba, Elwyn; Hunter, Eric; Allen, Susan; Nemo, George; Rodriguez-Chavez, Isaac R; Margolick, Joseph B; Golding, Hana

    2010-01-01

    HIV-Selectest is a serodiagnostic enzyme immunoassay (EIA), containing p6 and gp41 peptides, designed to differentiate between vaccine-induced antibodies and true infections. A rapid test version of the HIV-Selectest was developed. Both assays detected HIV antibodies in men and women within 2 to 4 weeks of infection, with sensitivity similar to third-generation EIAs.

  10. Development of specific PCR assays for the detection of Cryptocaryon irritans.

    Science.gov (United States)

    Chen, W; Sun, H Y; Xie, M Q; Bai, J S; Zhu, X Q; Li, A X

    2008-07-01

    Cryptocaryon irritans is one of the most important protozoan pathogens of marine fish, causing the "white spot" disease and posing a significant problem to marine aquaculture. In the present study, a C. irritans-specific reverse primer (S15) was designed based on the published sequence of the second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) of C. irritans and used together with the conserved forward primer P1 to develop a specific polymerase chain reaction (PCR) assay for direct, rapid, and specific detection of C. irritans. The specificity of these primers was tested with both closely and distantly related ciliates (Pseudokeroronpsis rubra, Pseudokeroronpsis carnae, Euplotes sp. 1, Ichthyophthirius multifiliis, Pseudourostyla cristata, and Paramecium caudaium), and only C. irritans was detected and no product was amplified from any other ciliates examined in this study using the specific primer set P1-S15. The specific PCR assay was able to detect as low as 45 pg of C. irritans DNA and a nested PCR assay using two primer sets (P1/NC2, P1/S15) increased the sensitivity, allowing the detection of a single C. irritans. The species-specific PCR assays should provide useful tools for the diagnosis, prevention, and molecular epidemiological investigations of C. irritans infection in marine fish.

  11. A multiplex RT-PCR assay for the detection of fish picornaviruses.

    Science.gov (United States)

    Mor, Sunil K; Phelps, Nicholas B D; Barbknecht, Marisa; Hoffman, Michael A; Goyal, Sagar M

    2015-09-01

    With the emergence of high profile fish diseases in the Great Lakes region, surveillance and regulatory inspections of fish populations have increased. This has resulted in a better understanding of known pathogens and isolation of many new pathogens of fish. In this study, a multiplex RT-PCR assay was developed for the detection of three newly discovered fish picornaviruses: bluegill picornavirus-1 (BGPV-1), fathead minnow picornavirus (FHMPV), and eel picornavirus-1 (EPV-1). This assay was found to be very sensitive with a detection limit of 81.9pg/μl of extracted RNA from a pool of FHMPV and BGPV-1 and was able to detect 501 and 224 gene copies/μl of BGPV-1 and FHMPV, respectively. The assay was highly reproducible and did not cross react with other closely related pathogens. We believe that this new assay provides a rapid and cost effective tool for confirming cell culture isolates and conducting prevalence studies of these newly detected fish picornaviruses. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Evaluation of a proximity extension assay for the detection of H1 2009 pandemic influenza viruses.

    Science.gov (United States)

    Van Wesenbeeck, Liesbeth; Meeuws, Hanne; De Wolf, Hans; Stuyver, Lieven

    2013-10-01

    The rapid influenza diagnostic tests (RIDTs) are widely distributed, simple to use, but often lack sensitivity as compared to gold standard methods (viral culture and nucleic acid detection technologies). Applying RIDTs outside of epidemic or pandemic infections results in large numbers of false negatives. Hence, a sensitive RIDT that would reduce the number of false negatives would result in an increased clinical value. We evaluated the potential of a proximity extension assay (PEA) for the detection of influenza A H1 viruses. This technology makes use of antibodies to capture the pathogen, followed by molecular detection. Forty-seven nasopharyngeal swab samples, all confirmed infections of the H1 2009 pandemic influenza virus, were evaluated. The performance of PEA was compared to the RIDT Quickvue Influenza A+B assay. The success rate of the comparative assays was modeled by means of a binary logistic response model. Both assays performed equally well within the current range of viral particles, expressed as log10 copies/ml. When the actual input of viral particles was taken into account, the 95% hitrate of PEA lies within the range of 4.60-7.02 log10 copies/reaction, which is an almost 2 log10 sensitivity improvement over the 95% hitrate of the Quickvue RIDT, ranging from 6.86 to 9.37 log10 copies/reaction. The PEA method holds promise to improve sensitive detection of influenza viruses in clinical samples. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. A rapid membrane potential assay to monitor CFTR function and inhibition.

    Science.gov (United States)

    Maitra, Rangan; Sivashanmugam, Perumal; Warner, Keith

    2013-10-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) protein is an important regulator of ion transport and fluid secretion in humans. Mutations to CFTR cause cystic fibrosis, which is a common recessive genetic disorder in Caucasians. Involvement of CFTR has been noted in other important diseases, such as secretory diarrhea and polycystic kidney disease. The assays to monitor CFTR function that have been described to date either are complicated or require specialized instrumentation and training for execution. In this report, we describe a rapid FlexStation-based membrane potential assay to monitor CFTR function. In this assay, agonist-mediated activation of CFTR results in membrane depolarization that can be monitored using a fluorescent membrane potential probe. Availability of a simple mix-and-read assay to monitor the function of this important protein might accelerate the discovery of CFTR ligands to study a variety of conditions.

  14. Data visualizations to detect systematic errors in laboratory assay results.

    Science.gov (United States)

    Lötsch, Jörn

    2017-12-01

    The measurement of concentrations of drugs and endogenous substances is widely used in basic and clinical pharmacology research and service tasks. Using data science-derived visualizations of laboratory data, it is demonstrated on a real-life example that basic statistical exploration of laboratory assay results or advised standard visual methods of data inspection may fall short in detecting systematic laboratory errors. For example, data pathologies such as generating always the same value in all probes of a particular assay run may pass undetected when using standard methods of data quality check. It is shown that the use of different data visualizations that emphasize different views of the data may enhance the detection of systematic laboratory errors. A dotplot of single data in the order of assay is proposed that provides an overview on the data range, outliers and a particular type of systematic errors where similar values are wrongly measured in all probes. © 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

  15. Comparison of an enzyme-linked immunosorbent assay, an immunofluorescence assay and a hemagglutination inhibition assay for detection of antibodies to K-papovavirus in mice.

    NARCIS (Netherlands)

    J. Groen (Jan); A.D.M.E. Osterhaus (Albert); H.W.J. Broeders; H.E.M. Spijkers (Ine)

    1989-01-01

    textabstractThe sensitivity of a newly developed enzyme-linked immunosorbent assay (ELISA) for detection of antibody to K virus was compared with the sensitivities of an immunofluorescence assay (IFA) and a hemagglutination inhibition assay (HIA). Specific pathogen-free BALB/c RIVM mice, 5 weeks

  16. Rapid antigen detection test for respiratory syncytial virus diagnosis as a diagnostic tool,

    Directory of Open Access Journals (Sweden)

    Flávio da Silva Mesquita

    Full Text Available Abstract Objective: The aim of this study was to evaluate the QuickVue® RSV Test Kit (QUIDEL Corp, CA, USA as a screening tool for respiratory syncytial virus in children with acute respiratory disease in comparison with the indirect immunofluorescence assay as gold standard. In Brazil, rapid antigen detection tests for respiratory syncytial virus are not routinely utilized as a diagnostic tool, except for the diagnosis of dengue and influenza. Methods: The authors retrospectively analyzed 486 nasopharyngeal aspirate samples from children under age 5 with acute respiratory infection, between December 2013 and August 2014, the samples were analyzed by indirect immunofluorescence assay and QuickVue® RSV Test kit. Samples with discordant results were analyzed by real time PCR and nucleotide sequencing. Results: From 313 positive samples by immunofluorescence assays, 282 (90% were also positive by the rapid antigen detection test, two were positive only by rapid antigen detection test, 33 were positive only by immunofluorescence assays, and 171 were positive by both methods. The 35 samples with discordant results were analyzed by real time PCR; the two samples positive only by rapid antigen detection test and the five positive only by immunofluorescence assays were also positive by real time PCR. There was no relation between the negativity by QuickVue® RSV Test and viral load or specific strain. The QuickVue® RSV Test showed sensitivity of 90%, specificity of 98.8%, predictive positive value of 99.3%, and negative predictive value of 94.6%, with accuracy of 93.2% and agreement κ index of 0.85 in comparison to immunofluorescence assay. Conclusions: This study demonstrated that the QuickVue® RSV Test Kit can be effective in early detection of Respiratory syncytial virus in nasopharyngeal aspirate and is reliable for use as a diagnostic tool in pediatrics.

  17. Rapid antigen detection test for respiratory syncytial virus diagnosis as a diagnostic tool.

    Science.gov (United States)

    Mesquita, Flávio da Silva; Oliveira, Danielle Bruna Leal de; Crema, Daniela; Pinez, Célia Miranda Nunes; Colmanetti, Thaís Cristina; Thomazelli, Luciano Matsumia; Gilio, Alfredo Elias; Vieira, Sandra Elisabeth; Martinez, Marina Baquerizo; Botosso, Viviane Fongaro; Durigon, Edison Luiz

    The aim of this study was to evaluate the QuickVue® RSV Test Kit (QUIDEL Corp, CA, USA) as a screening tool for respiratory syncytial virus in children with acute respiratory disease in comparison with the indirect immunofluorescence assay as gold standard. In Brazil, rapid antigen detection tests for respiratory syncytial virus are not routinely utilized as a diagnostic tool, except for the diagnosis of dengue and influenza. The authors retrospectively analyzed 486 nasopharyngeal aspirate samples from children under age 5 with acute respiratory infection, between December 2013 and August 2014, the samples were analyzed by indirect immunofluorescence assay and QuickVue® RSV Test kit. Samples with discordant results were analyzed by real time PCR and nucleotide sequencing. From 313 positive samples by immunofluorescence assays, 282 (90%) were also positive by the rapid antigen detection test, two were positive only by rapid antigen detection test, 33 were positive only by immunofluorescence assays, and 171 were positive by both methods. The 35 samples with discordant results were analyzed by real time PCR; the two samples positive only by rapid antigen detection test and the five positive only by immunofluorescence assays were also positive by real time PCR. There was no relation between the negativity by QuickVue® RSV Test and viral load or specific strain. The QuickVue® RSV Test showed sensitivity of 90%, specificity of 98.8%, predictive positive value of 99.3%, and negative predictive value of 94.6%, with accuracy of 93.2% and agreement κ index of 0.85 in comparison to immunofluorescence assay. This study demonstrated that the QuickVue® RSV Test Kit can be effective in early detection of Respiratory syncytial virus in nasopharyngeal aspirate and is reliable for use as a diagnostic tool in pediatrics. Copyright © 2016 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  18. Evaluating the use of dedicated swab for rapid antigen detection ...

    African Journals Online (AJOL)

    Background: Group A streptococcus (GAS) is the most common and fearful bacterial cause in pediatric acute pharyngitis due to its serious complications. Several generations of rapid antigen detection tests (RADTs) have been developed to facilitate rapid detection of GAS pharyngitis. We assessed the value of using a ...

  19. Rapid assessment of assignments using plagiarism detection software.

    Science.gov (United States)

    Bischoff, Whitney R; Abrego, Patricia C

    2011-01-01

    Faculty members most often use plagiarism detection software to detect portions of students' written work that have been copied and/or not attributed to their authors. The rise in plagiarism has led to a parallel rise in software products designed to detect plagiarism. Some of these products are configurable for rapid assessment and teaching, as well as for plagiarism detection.

  20. 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.

  1. First direct fluorescence polarization assay for the detection and quantification of spirolides in mussel samples

    Energy Technology Data Exchange (ETDEWEB)

    Otero, Paz; Alfonso, Amparo [Departamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario s/n, 27002 Lugo (Spain); Alfonso, Carmen [CIFGA Laboratorio, Plaza de Santo Domingo, 1, 27001 Lugo (Spain); Araoz, Romulo; Molgo, Jordi [CNRS, Institut de Neurobiologie Alfred Fessard - FRC2118, Laboratoire de Neurobiologie et Developpement UPR3294, 1 Avenue de la Terrasse, 91198 Gif sur Yvette Cedex (France); Vieytes, Mercedes R. [Departamento de Fisiologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo (Spain); Botana, Luis M., E-mail: luis.botana@usc.es [Departamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario s/n, 27002 Lugo (Spain)

    2011-09-09

    Highlights: {yields} A direct assay based in the binding of nAChR to spirolide toxins by FP is described. {yields} A direct relationship between FP and 13-desMeC in the range of 10-500 nM is obtained. {yields} FP is dependent on the 13, 19-didesMeC in a higher concentration range than 13-desMeC. {yields} FP assay is a sensitive method to detect and quantify 13-desMeC in mussel samples. - Abstract: In 2009, we achieve the first inhibition FP assay to detect imine cyclic toxins. In the present paper we propose a new FP assay for direct quantify spirolides. This new method has resulted in significant improvement of sensitivity, rapidity and accessibility. In the method design, nicotinic acetylcholine receptor from Torpedo marmorata membranes labelled with a derivative of fluorescein was used. Spirolides, 13-desmethyl spirolide C (13-desMeC) and 13,19-didesmethyl spirolide C (13,19-didesMeC) were extracted and purified from cultures of the Alexandrium ostenfeldii dinoflagellate. Data showed the decrease of FP when toxin concentration was increased. Thus, a relationship between the FP units and the spirolides amount present in a sample was obtained. This direct assay is a reproducible, simple and very sensitive method with a detection limit about 25 nM for 13-desMeC and 150 nM for 13,19-didesMeC. The procedure was used to measure spirolides in mussel samples using an extraction and clean up protocol suitable for the FP assay. Results obtained show that this method is able to quantify 13-desMeC in the range of 50-350 {mu}g kg{sup -1} meat. Other liposoluble toxins did not interfere with the assay, proving a specific method. Moreover, the matrix do not affect in the range of toxin concentrations that involving risk of spirolides intoxication.

  2. Rapid and sensitive point-of-care detection of Orthopoxviruses by ABICAP immunofiltration.

    Science.gov (United States)

    Stern, Daniel; Olson, Victoria A; Smith, Scott K; Pietraszczyk, Marko; Miller, Lilija; Miethe, Peter; Dorner, Brigitte G; Nitsche, Andreas

    2016-12-09

    The rapid and reliable detection of infectious agents is one of the most challenging tasks in scenarios lacking well-equipped laboratory infrastructure, like diagnostics in rural areas of developing countries. Commercially available point-of-care diagnostic tests for emerging and rare diseases are particularly scarce. In this work we present a point-of-care test for the detection of Orthopoxviruses (OPV). The OPV ABICAP assay detects down to 1 × 104 plaque forming units/mL of OPV particles within 45 min. It can be applied to clinical material like skin crusts and detects all zoonotic OPV infecting humans, including Vaccinia, Cowpox, Monkeypox, and most importantly Variola virus. Given the high sensitivity and the ease of handling, the novel assay could be highly useful for on-site diagnostics of suspected Monkeypox virus infections in areas lacking proper laboratory infrastructure as well as rapid on-site testing of suspected bioterrorism samples.

  3. Development of a liposome-based immunochromatographic strip assay for the detection of Salmonella.

    Science.gov (United States)

    Shukla, Shruti; Leem, Hyerim; Kim, Myunghee

    2011-11-01

    Salmonella species are ubiquitous human pathogens which pose a dangerous threat to the elderly and children worldwide. In this study, to develop a more efficient assay procedure for the rapid detection of Salmonella Typhimurium, an immunochromatographic strip assay was developed using immunoliposome (anti-Salmonella IgG-tagged) encapsulated with sulforhodamine B (SRB). The detection sensitivity of the developed immunochromatographic assay was compared with a commercial immunochromatographic test strip using colloidal gold nanoparticles. The liposomes were prepared through a reverse-phase evaporation method by using a lipid and phospholipid mixture and SRB, a fluorescence dye, which was encapsulated in the phospholipid bilayer. Furthermore, the outer surface of the SRB-encapsulated liposome was conjugated with antibody (affinity-purified polyclonal goat anti-Salmonella IgG) to form an immunoliposome (size, 223 nm), used as the analytical reagent in the developed immunoassay. For this strip assay, a plastic-backed nitrocellulose strip was immobilized with two antibody zones. The lower zone of the strip referred to Salmonella antigen capture zone (test line), while the other zone served as a positive control (control line). The lower zone was coated with affinity-purified polyclonal goat anti-Salmonella IgG, while the upper zone was coated with rabbit anti-goat IgG. During capillary migration of the wicking solution (diluted liposome and Salmonella culture, each 50 μl), Salmonella was captured with surface-bound immunoliposomes at the antigen capture zone, while the unbound liposomes migrated upward and bound to another zone. The color density of the antigen capture zone was directly proportional to the amount of S. Typhimurium in the test sample. As a result, the detection limit of the immunochromatographic strip assay developed in this study against S. Typhimurium was found to be 10(2) CFU/ml, which was significantly higher than the detection limit (10(7) CFU

  4. Detection of irradiated onion by means of the comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Moreno Alvarez, Damaris L.; Prieto Miranda, Enrique Fco.; Carro Palacio, Sandra [Centro de Aplicaciones Tecnologicas y Desarrollo Nuclear. (CEADEN), Ciudad de La Habana (Cuba)]. E-mail: damaris@ceaden.edu.cu; efprieto@ceaden.edu.cu; Iglesia Enriquez, Isora [Instituto de Investigacion para la Industria Alimenticia (IIIA), Ciudad de La Habana (Cuba)

    2007-07-01

    The ionizing radiations are used as a harmless alternative treatment that it substitutes the employment of chemical treatments, which after their application in the food products can remain residuals not desired that they come to be carcinogenic. With the food irradiation is eliminated microorganisms and the storage time is prolonged, which produces benefits for the Food Industry and the consumers. In many countries the search of sensitive detecting methods of irradiated foods is promoted by the necessity of the assurance of the consumption of foods with nutritional quality and to test directly the radiation processing, for which several techniques have been developed, these are based on the changes that induce the ionizing radiations in the food products. A recommended method is the Comet Assay of DNA, it is approved by the European Committee of Standardization (EN 13784). The DNA molecule is very sensitive to gamma radiations even at low radiation dose, where the modifications produced in the molecule can be monitored for this analytical technique well-known as Comet Assay of DNA or Single Cell Gel Electrophoresis. The objective of the present paper was to evaluate the modifications of the DNA molecule of irradiated onions with the Comet Assay for several dose values, the onions were conserved at environment and refrigeration temperatures. The samples were irradiated in a self-shielding irradiator with {sup 60}Co source, dose rate of 20.45 Gy/min and absorbed dose values of 0.5; 0.6; 0.8 and 1.0 kGy. This detection method demonstrates to be one sensitive and quick technique for the qualitative detection of irradiated onions. (author)

  5. Novel PCR Assays Complement Laser Biosensor-Based Method and Facilitate Listeria Species Detection from Food.

    Science.gov (United States)

    Kim, Kwang-Pyo; Singh, Atul K; Bai, Xingjian; Leprun, Lena; Bhunia, Arun K

    2015-09-08

    The goal of this study was to develop the Listeria species-specific PCR assays based on a house-keeping gene (lmo1634) encoding alcohol acetaldehyde dehydrogenase (Aad), previously designated as Listeria adhesion protein (LAP), and compare results with a label-free light scattering sensor, BARDOT (bacterial rapid detection using optical scattering technology). PCR primer sets targeting the lap genes from the species of Listeria sensu stricto were designed and tested with 47 Listeria and 8 non-Listeria strains. The resulting PCR primer sets detected either all species of Listeria sensu stricto or individual L. innocua, L. ivanovii and L. seeligeri, L. welshimeri, and L. marthii without producing any amplified products from other bacteria tested. The PCR assays with Listeria sensu stricto-specific primers also successfully detected all species of Listeria sensu stricto and/or Listeria innocua from mixed culture-inoculated food samples, and each bacterium in food was verified by using the light scattering sensor that generated unique scatter signature for each species of Listeria tested. The PCR assays based on the house-keeping gene aad (lap) can be used for detection of either all species of Listeria sensu stricto or certain individual Listeria species in a mixture from food with a detection limit of about 10⁴ CFU/mL.

  6. Evaluation of a rapid and completely automated real-time reverse transcriptase PCR assay for diagnosis of enteroviral meningitis.

    Science.gov (United States)

    Nolte, Frederick S; Rogers, Beverly B; Tang, Yi-Wei; Oberste, M Steven; Robinson, Christine C; Kehl, K Sue; Rand, Kenneth A; Rotbart, Harley A; Romero, Jose R; Nyquist, Ann-Christine; Persing, David H

    2011-02-01

    Nucleic acid amplification tests (NAATs) for enterovirus RNA in cerebrospinal fluid (CSF) have emerged as the new gold standard for diagnosis of enteroviral meningitis, and their use can improve the management and decrease the costs for caring for children with enteroviral meningitis. The Xpert EV assay (Cepheid, Sunnyvale, CA) is a rapid, fully automated real-time PCR test for the detection of enterovirus RNA that was approved by the U.S. Food and Drug Administration for in vitro diagnostic use in March 2007. In this multicenter trial we established the clinical performance characteristics of the Xpert EV assay in patients presenting with meningitis symptoms relative to clinical truth. Clinical truth for enteroviral meningitis was defined as clinical evidence of meningitis, the absence of another detectable pathogen in CSF, and detection of enterovirus in CSF either by two reference NAATs or by viral culture. A total of 199 prospectively and 235 retrospectively collected specimens were eligible for inclusion in this study. The overall prevalence of enteroviral meningitis was 26.04%. The Xpert EV assay had a sensitivity of 94.69% (90% confidence interval [CI] = 89.79 to 97.66%), specificity of 100% (90% CI = 99.07 to 100%), positive predictive value of 100%, negative predictive value of 98.17, and an accuracy of 98.62% relative to clinical truth. The Xpert EV assay demonstrated a high degree of accuracy for diagnosis of enteroviral meningitis. The simplicity and on-demand capability of the Xpert EV assay should prove to be a valuable adjunct to the evaluation of suspected meningitis cases.

  7. Evaluation of a Rapid and Completely Automated Real-Time Reverse Transcriptase PCR Assay for Diagnosis of Enteroviral Meningitis▿

    Science.gov (United States)

    Nolte, Frederick S.; Rogers, Beverly B.; Tang, Yi-Wei; Oberste, M. Steven; Robinson, Christine C.; Kehl, K. Sue; Rand, Kenneth A.; Rotbart, Harley A.; Romero, Jose R.; Nyquist, Ann-Christine; Persing, David H.

    2011-01-01

    Nucleic acid amplification tests (NAATs) for enterovirus RNA in cerebrospinal fluid (CSF) have emerged as the new gold standard for diagnosis of enteroviral meningitis, and their use can improve the management and decrease the costs for caring for children with enteroviral meningitis. The Xpert EV assay (Cepheid, Sunnyvale, CA) is a rapid, fully automated real-time PCR test for the detection of enterovirus RNA that was approved by the U.S. Food and Drug Administration for in vitro diagnostic use in March 2007. In this multicenter trial we established the clinical performance characteristics of the Xpert EV assay in patients presenting with meningitis symptoms relative to clinical truth. Clinical truth for enteroviral meningitis was defined as clinical evidence of meningitis, the absence of another detectable pathogen in CSF, and detection of enterovirus in CSF either by two reference NAATs or by viral culture. A total of 199 prospectively and 235 retrospectively collected specimens were eligible for inclusion in this study. The overall prevalence of enteroviral meningitis was 26.04%. The Xpert EV assay had a sensitivity of 94.69% (90% confidence interval [CI] = 89.79 to 97.66%), specificity of 100% (90% CI = 99.07 to 100%), positive predictive value of 100%, negative predictive value of 98.17, and an accuracy of 98.62% relative to clinical truth. The Xpert EV assay demonstrated a high degree of accuracy for diagnosis of enteroviral meningitis. The simplicity and on-demand capability of the Xpert EV assay should prove to be a valuable adjunct to the evaluation of suspected meningitis cases. PMID:21159942

  8. Rapid detection of Panton-Valentine leukocidin from clinical isolates of Staphylococcus aureus strains by real-time PCR

    NARCIS (Netherlands)

    Deurenberg, Ruud H; Vink, Cornelis; Driessen, Christel; Bes, Michèle; London, Nancy; Etienne, Jerome; Stobberingh, Ellen E

    2004-01-01

    To allow rapid identification of Panton-Valentine leukocidin (PVL)-producing Staphylococcus aureus strains, a real-time PCR assay for detection of PVL was developed. This assay is convenient, since it can be applied directly on bacterial suspensions and does not require previous DNA purification.

  9. 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.

  10. A New Lab Developed Real Time PCR Assay for Direct Detection of C. Difficle from Stool Sample without DNA Extraction

    OpenAIRE

    Li, Brandon

    2016-01-01

    Clostridium difficile is a major cause of nosocomial antibiotic-associated infectious diarrhea and pseudomembranous colitis. Detection of C. difficile by anaerobic bacterial culture and/or cytotoxicity assays has been largely replaced by rapid enzyme immunoassays (EIA). However, due to the lack of sensitivity of stool EIA, we developed a multiplex real-time PCR assay targeting the C. difficile toxin genes tcdB. stool samples from hospitalized pediatric patients suspected of having C. difficil...

  11. Evaluation of the multiplex PCR Allplex-GI assay in the detection of bacterial pathogens in diarrheic stool samples.

    Science.gov (United States)

    Martín, Ariadna; Pérez-Ayala, Ana; Chaves, Fernando; Lora, David; Orellana, M Ángeles

    2017-10-31

    Rapid and accurate detection of the pathogens that cause gastrointestinal infection is important for appropriate therapy and proper infection control. This study assesses the performance of a new molecular assay for simultaneous detection of 13 different gastrointestinal bacteria in stool specimens. Using the Allplex GI-Bacteria (AGI-BI/AGI-BII) assay, a total of 394 stool samples were tested and the results were compared with culturing on selective differential followed by identification by mass spectroscopy. Discordant results were analyzed by a different multiplex PCR method, the Fast-Track Diagnostics Bacterial gastroenteritis (FTD-BG). The routine method (RM) detected 109 (27.7%) positive samples and the Allplex-GI assay, 261 (66.2%). Analysis of discordant results revealed that the molecular assay detected 44 pathogens that were not detected by the RM, including 23 Campylobacter spp., 11 Salmonella spp, 3 Y. enterocolitica, 2 EIEC/Shigella spp, 2 E. coli 0157, 2 C. difficile and 1 Aeromonas spp. Five cases not detected by the molecular method were detected by the RM (3 Aeromonas spp, 1 Salmonella spp and 1 Y. enterocolitica). For all targets, the percentages of sensitivity and specificity were >95%, except for Aeromonas spp., which were 81% and 99% respectively. This study suggests that Allplex-GI multiplex PCR is a sensitive and specific assay that enables a rapid and accurate diagnosis of bacterial gastrointestinal infections. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. A bispecific antibody based assay shows potential for detecting tuberculosis in resource constrained laboratory settings.

    Directory of Open Access Journals (Sweden)

    Susmita Sarkar

    Full Text Available The re-emergence of tuberculosis (TB as a global public health threat highlights the necessity of rapid, simple and inexpensive point-of-care detection of the disease. Early diagnosis of TB is vital not only for preventing the spread of the disease but also for timely initiation of treatment. The later in turn will reduce the possible emergence of multi-drug resistant strains of Mycobacterium tuberculosis. Lipoarabinomannan (LAM is an important non-protein antigen of the bacterial cell wall, which is found to be present in different body fluids of infected patients including blood, urine and sputum. We have developed a bispecific monoclonal antibody with predetermined specificities towards the LAM antigen and a reporter molecule horseradish peroxidase (HRPO. The developed antibody was subsequently used to design a simple low cost immunoswab based assay to detect LAM antigen. The limit of detection for spiked synthetic LAM was found to be 5.0 ng/ml (bovine urine, 0.5 ng/ml (rabbit serum and 0.005 ng/ml (saline and that for bacterial LAM from M. tuberculosis H37Rv was found to be 0.5 ng/ml (rabbit serum. The assay was evaluated with 21 stored clinical serum samples (14 were positive and 7 were negative in terms of anti-LAM titer. In addition, all 14 positive samples were culture positive. The assay showed 100% specificity and 64% sensitivity (95% confidence interval. In addition to good specificity, the end point could be read visually within two hours of sample collection. The reported assay might be used as a rapid tool for detecting TB in resource constrained laboratory settings.

  13. A bispecific antibody based assay shows potential for detecting tuberculosis in resource constrained laboratory settings.

    Science.gov (United States)

    Sarkar, Susmita; Tang, Xinli L; Das, Dipankar; Spencer, John S; Lowary, Todd L; Suresh, Mavanur R

    2012-01-01

    The re-emergence of tuberculosis (TB) as a global public health threat highlights the necessity of rapid, simple and inexpensive point-of-care detection of the disease. Early diagnosis of TB is vital not only for preventing the spread of the disease but also for timely initiation of treatment. The later in turn will reduce the possible emergence of multi-drug resistant strains of Mycobacterium tuberculosis. Lipoarabinomannan (LAM) is an important non-protein antigen of the bacterial cell wall, which is found to be present in different body fluids of infected patients including blood, urine and sputum. We have developed a bispecific monoclonal antibody with predetermined specificities towards the LAM antigen and a reporter molecule horseradish peroxidase (HRPO). The developed antibody was subsequently used to design a simple low cost immunoswab based assay to detect LAM antigen. The limit of detection for spiked synthetic LAM was found to be 5.0 ng/ml (bovine urine), 0.5 ng/ml (rabbit serum) and 0.005 ng/ml (saline) and that for bacterial LAM from M. tuberculosis H37Rv was found to be 0.5 ng/ml (rabbit serum). The assay was evaluated with 21 stored clinical serum samples (14 were positive and 7 were negative in terms of anti-LAM titer). In addition, all 14 positive samples were culture positive. The assay showed 100% specificity and 64% sensitivity (95% confidence interval). In addition to good specificity, the end point could be read visually within two hours of sample collection. The reported assay might be used as a rapid tool for detecting TB in resource constrained laboratory settings.

  14. Classical and new assays for detecting drug resistance in tuberculosis.

    Science.gov (United States)

    Palomino, Juan Carlos; Vandamme, Peter; Martin, Anandi

    2014-01-01

    Tuberculosis is a public health concern worldwide. Particularly worrying is the emergence of severe forms of drug resistance, such as extensively drug resistant and totally drug resistant tuberculosis, with few treatment options for the afflicted patients. To avoid further spread of drug resistance, its early detection is extremely important. Conventional phenotypic procedures to detect drug resistance depended on the in vitro slow growth of the bacteria. More recent molecular approaches such as reverse-hybridization assays and real-time PCR tests have been introduced. Newer options proposed include, faster culture-based methods and whole-genome sequencing and nanotechnology. Not yet available is a real point-of-care test, applied directly in clinical samples and reliable enough for guiding a treatment option.

  15. Detection of Mycoplasma bovis with an improved pcr assay.

    Science.gov (United States)

    Tenk, M; Bálint, A; Stipkovits, L; Biró, Judit; Dencso, L

    2006-12-01

    A Mycoplasma bovis species-specific PCR assay has been developed with improvement of a previously described method (Ghadersohi et al., 1997). This test and its semi-nested version (Hayman and Hirst, 2003) did not function at all in our hands. A new reverse primer (Mbr2) was designed using previously published sequence data. For testing specificity, DNA was extracted from the most frequently occurring mycoplasma species and bacteria of bovine origin. The new PCR detected only Mycoplasma bovis. Moreover, no cross-reaction was observed with the genetically closest relative species, M. agalactiae. The target organism could be detected in a dose as low as 150 CFU ml(-1) in broth cultures using ethidium-bromide-stained agarose gels.

  16. Rapid diagnosis of childhood pulmonary tuberculosis by Xpert MTB/RIF assay using bronchoalveolar lavage fluid.

    Science.gov (United States)

    Yin, Qing-Qin; Jiao, Wei-Wei; Han, Rui; Jiao, An-Xia; Sun, Lin; Tian, Jian-Ling; Ma, Yu-Yan; Rao, Xiao-Chun; Shen, Chen; Li, Qin-Jing; Shen, A-Dong

    2014-01-01

    In order to evaluate the diagnostic accuracy of the Xpert MTB/RIF assay on childhood pulmonary tuberculosis (PTB) using bronchoalveolar lavage fluid (BALF), we evaluated the sensitivity, specificity, positive predictive value, and negative predictive value of Xpert MTB/RIF assay using BALF in comparison with acid-fast bacilli (AFB) microscopy and Mycobacterium tuberculosis (MTB) culture for diagnosing childhood PTB using Chinese "composite clinical reference standard" (CCRS) as reference standard. Two hundred fifty-five children with suspected PTB were enrolled at Beijing Children's Hospital from September 2010 to July 2013. Compared with Chinese CCRS, the sensitivity of AFB microscopy, MTB culture, and Xpert MTB/RIF assay was 8.4%, 28.9%, and 53.0%, respectively. The specificity of three assays was all 100%. Xpert MTB/RIF assay could detect 33.9% of cases with negative MTB culture, and 48.7% of cases with negative AFB microscopy. Younger age (MTB/RIF assay. In conclusion, Xpert MTB/RIF assay using BALF can assist in diagnosing childhood PTB much faster when fiberoptic bronchoscopy is necessary according to the chest radiograph.

  17. Duplex real-time PCR assay for rapid identification of Staphylococcus aureus isolates from dairy cow milk.

    Science.gov (United States)

    Pilla, Rachel; Snel, Gustavo G M; Malvisi, Michela; Piccinini, Renata

    2013-05-01

    Staphylococcus aureus isolates from dairy cow mastitis are not always consistent with the characteristic morphology described, and molecular investigation is often needed. The aim of the study was to develop a duplex real-time PCR assay for rapid identification of Staph. aureus isolates, targeting both nuc and Sa442. Overall, 140 isolates collected from dairy cow mastitis in 90 different herds, were tested. All strains had been identified using morphological and biochemical characteristics. DNA from each strain was amplified in real-time PCR assay, to detect nuc or Sa442. Thereafter, a duplex real-time PCR assay was performed, and specificity of the amplified products was assessed by high resolution melting curve analysis. Out of 124 Staph. aureus isolates, 33 did not show the typical morphology or enzymic activity; in 118 strains, the two melt-curve peaks consistent with nuc and Sa442 were revealed, while 2 isolates showed only the peak consistent with Sa442. Four isolates bacteriologically identified as Staph. aureus, were PCR-negative and were further identified as Staph. pseudintermedius by sequencing. Staph. pseudintermedius and coagulase-negative staphylococci did not carry nuc or Sa442. The results showed the correct identification of all isolates, comprehending also coagulase-or nuc-negative Staph. aureus, while other coagulase-positive Staphylococci were correctly identified as non-Staph. aureus. Both sensitivity and specificity were 100%. High resolution melting analysis allowed easy detection of unspecific products. Finally, the duplex real-time PCR was applied directly to 40 milk samples, to detect infected mammary quarters. The assay confirmed the results of bacteriological analysis, on Staph. aureus-positive or-negative samples. Therefore, the proposed duplex real-time PCR could be used in laboratory routine as a cost-effective and powerful tool for high-throughput identification of atypical Staph. aureus isolates causing dairy cow mastitis. Also, it

  18. Rapid detection of Mycobacterium avium subsp. paratuberculosis ...

    African Journals Online (AJOL)

    Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, a suspect causative agent of Crohns disease in man, is an emerging disease of international proportions affecting all ruminants. Early stage detection of Mycobacterium avium subsp. paratuberculosis infection would accelerate progress in control ...

  19. RAPID DETECTION OF MICROBIAL CONTAMINATION IN GHANA ...

    African Journals Online (AJOL)

    2014-06-01

    Jun 1, 2014 ... pathogens in herbal medicines from Ghana. Methods: We employed different DNA extraction ... kits yielded significant amounts of DNA. PCR was able to detect pathogens present in the samples directly. ..... safety of dried spices and herbs from production and retail premises in the United Kingdom. Food.

  20. Chemiluminescence assay for the detection of biological warfare agents

    Energy Technology Data Exchange (ETDEWEB)

    Langry, K; Horn, J

    1999-11-05

    A chemiluminescent homogeneous immunoassay and a hand-size multiassay reader are described that could be used for detecting biological materials. The special feature of the assay is that it employs two different antibodies that each bind to a unique epitope on the same antigen. Each group of epitope-specific antibodies has linked to it an enzyme of a proximal-enzyme pair. One enzyme of the pair utilizes a substrate in high concentration to produce a second substrate required by the second enzyme. This new substrate enables the second enzyme to function. The reaction of the second enzyme is configured to produce light. This chemiluminescence is detected with a charge-coupled device (CCD) camera. The proximal pair enzymes must be in close proximity to one another to allow the second enzyme to react with the product of the first enzyme. This only occurs when the enzyme-linked antibodies are attached to the antigen, whether antigen is a single protein with multiple epitopes or the surface of a cell with a variety of different antigens. As a result of their juxtaposition, the enzymes produce light only in the presence of the biological material. A brief description is given as to how this assay could be utilized in a personal bio-agent detector system.

  1. Rapid and sensitive single-step radiochemical assay for catechol-O-methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Zuercher, G.; Da Prada, M. (Hoffmann-La Roche (F.) and Co., Basel (Switzerland))

    1982-01-01

    A simple, rapid and reliable radiometric assay for the determination of catechol-O-methyltransferase activity is described. The method is based on the conversion of catechol to (/sup 3/H)guaiacol by catechol-O-methyltransferase in the presence of Mg/sup 2 +/, adenosine deaminase and S-adenosyl L-(methyl-/sup 3/H)methionine. Incubation and direct extraction of (/sup 3/H)guaiacol into organic scintillation fluid, as well as counting, are performed in the same standard scintillation vial. The assay is easy to perform and more sensitive than previous analogous procedures. The method has been applied to the assay of catechol-O-methyltransferase activity in discrete brain areas and also peripheral organs of rat and in human erythrocytes.

  2. A multiplex PCR assay for the detection and quantification of Sclerotinia sclerotiorum and Botrytis cinerea.

    Science.gov (United States)

    Reich, J D; Alexander, T W; Chatterton, S

    2016-05-01

    Traditional culture methods for identifying the plant fungal pathogens Sclerotinia sclerotiorum (Lib.) de Bary and Botrytis cinerea Pers.:Fr. are slow and laborious. The goal of this study was to develop a multiplex real-time PCR (qPCR) assay to detect and quantify DNA from S. sclerotiorum and B. cinerea. A primer set (SsIGS_5) for S. sclerotiorum was designed that targeted the intergenic spacer (IGS) regions of the ribosomal DNA. Addition of a probe to the assay increased its specificity: when the primer/probe set was tested against 21 fungal species (35 strains), amplification was detected from all S. sclerotiorum strains and no other species. For qPCR, the SsIGS_5 primer and probe set exhibited a linear range from 7·0 ng to 0·07 pg target DNA (R(2)  = 0·99). SsIGS_5 was then multiplexed with a previously published primer/probe set for B. cinerea to develop a high-throughput method for the detection and quantification of DNA from both pathogens. When multiplexed, the sensitivity and specificity of both assays were not different from individual qPCR reactions. The multiplex assay is currently being used to detect and quantify S. sclerotiorum and B. cinerea DNA from aerosol samples collected in commercial seed alfalfa fields. A primer and probe set for the quantification of Sclerotinia sclerotiorum DNA in a PCR assay was developed. The probe-based nature of this assay signifies an improvement over previous assays for this species by allowing multiplex reactions while maintaining high sensitivity. The primer/probe set was used in a multiplex real-time PCR assay for the quantification of S. sclerotiorum and Botrytis cinerea DNA, enabling rapid analysis of environmental samples. In crops susceptible to both pathogens, this multiplex assay can be used to quickly quantify the presence of each pathogen. © 2016 Her Majesty the Queen in Right of Canada © 2016 The Society for Applied Microbiology. Reproduced with the permission of the Office of the

  3. Rapid detection of methicillin-resistant staphylococci by multiplex PCR

    African Journals Online (AJOL)

    Administrator

    2010-11-08

    Nov 8, 2010 ... Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 16: 11141-11156. Fang H, Hedin G (2003). Rapid screening and identification of methicillin-resistant Staphylococcus aureus from clinical samples by selective-broth and real-time PCR assay.

  4. Application of Titration-Based Screening for the Rapid Pilot Testing of High-Throughput Assays.

    Science.gov (United States)

    Zhang, Ji-Hu; Kang, Zhao B; Ardayfio, Ophelia; Ho, Pei-i; Smith, Thomas; Wallace, Iain; Bowes, Scott; Hill, W Adam; Auld, Douglas S

    2014-06-01

    Pilot testing of an assay intended for high-throughput screening (HTS) with small compound sets is a necessary but often time-consuming step in the validation of an assay protocol. When the initial testing concentration is less than optimal, this can involve iterative testing at different concentrations to further evaluate the pilot outcome, which can be even more time-consuming. Quantitative HTS (qHTS) enables flexible and rapid collection of assay performance statistics, hits at different concentrations, and concentration-response curves in a single experiment. Here we describe the qHTS process for pilot testing in which eight-point concentration-response curves are produced using an interplate asymmetric dilution protocol in which the first four concentrations are used to represent the range of typical HTS screening concentrations and the last four concentrations are added for robust curve fitting to determine potency/efficacy values. We also describe how these data can be analyzed to predict the frequency of false-positives, false-negatives, hit rates, and confirmation rates for the HTS process as a function of screening concentration. By taking into account the compound pharmacology, this pilot-testing paradigm enables rapid assessment of the assay performance and choosing the optimal concentration for the large-scale HTS in one experiment. © 2013 Society for Laboratory Automation and Screening.

  5. Quantitative methylene blue decolourisation assays as rapid screening tools for assessing the efficiency of catalytic reactions.

    Science.gov (United States)

    Kruid, Jan; Fogel, Ronen; Limson, Janice Leigh

    2017-05-01

    Identifying the most efficient oxidation process to achieve maximum removal of a target pollutant compound forms the subject of much research. There exists a need to develop rapid screening tools to support research in this area. In this work we report on the development of a quantitative assay as a means for identifying catalysts capable of decolourising methylene blue through the generation of oxidising species from hydrogen peroxide. Here, a previously described methylene blue test strip method was repurposed as a quantitative, aqueous-based spectrophotometric assay. From amongst a selection of metal salts and metallophthalocyanine complexes, monitoring of the decolourisation of the cationic dye methylene blue (via Fenton-like and non-Fenton oxidation reactions) by the assay identified the following to be suitable oxidation catalysts: CuSO 4 (a Fenton-like catalyst), iron(II)phthalocyanine (a non-Fenton oxidation catalyst), as well as manganese(II) phthalocyanine. The applicability of the method was examined for the removal of bisphenol A (BPA), as measured by HPLC, during parallel oxidation experiments. The order of catalytic activity was identified as FePc > MnPc > CuSO 4 for both BPA and MB. The quantitative MB decolourisation assay may offer a rapid method for screening a wide range of potential catalysts for oxidation processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Rapid in situ assessment for predicting soil quality using an algae-soaked disc seeding assay.

    Science.gov (United States)

    Nam, Sun-Hwa; Moon, Jongmin; Kim, Shin Woong; Kim, Hakyeong; Jeong, Seung-Woo; An, Youn-Joo

    2017-11-16

    The soil quality of remediated land is altered and this land consequently exerts unexpected biological effects on terrestrial organisms. Therefore, field evaluation of such land should be conducted using biological indicators. Algae are a promising new biological indicator since they are a food source for organisms in higher soil trophic levels and easily sampled from the soil. Field evaluation of soil characteristics is preferred to be testing in laboratory conditions because many biological effects cannot be duplicated during laboratory evaluations. Herein, we describe a convenient and rapid algae-soaked disc seeding assay for assessing soil quality in the field based on soil algae. The collection of algae is easy and rapid and the method predicts the short-term quality of contaminated, remediated, and amended farm and paddy soils. The algae-soaked disc seeding assay is yet to be extensively evaluated, and the method cannot be applied to loamy sand soil in in situ evaluations. The algae-soaked disc seeding assay is recommended for prediction of soil quality in in situ evaluations because it reflects all variations in the environment. The algae-soaked disc seeding assay will help to develop management strategies for in situ evaluation.

  7. A Fluorescence Polarization Assay To Detect Steroid Hormone Traces in Milk.

    Science.gov (United States)

    Varriale, Antonio; Pennacchio, Anna; Pinto, Gabriella; Oliviero, Giorgia; D'Errico, Stefano; Majoli, Adelia; Scala, Andrea; Capo, Alessandro; Pennacchio, Angela; Di Giovanni, Stefano; Staiano, Maria; D'Auria, Sabato

    2015-10-21

    Steroids are a class of hormones improperly used in livestock as growth-promoting agents. Due to their high risk for human health, the European Union (EU) has strictly forbidden the administration of all natural and synthetic steroid hormones to food-producing animals, and the development of new rapid detection methods are greatly encouraged. This work reports a novel fluorescence polarization assay, ready to use, capable of detecting 17β-estradiol directly in milk samples with a low limit of detection of <10 pmol. It is based on the coupling of monospecific antibodies against 17β-estradiol and fluorophores, capable of modulating the fluorescence polarization emission on the basis of the specific binding of antibodies to fluorescence-labeled 17β-estradiol derivative. The successful detection of 17β-estradiol has disclosed the development of an efficient method, easily extensible to any food matrix and having the potential to become a milestone in food quality and safety.

  8. 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.

  9. Laser-based microbiological assay for detection and quantification of bioactive compounds

    Science.gov (United States)

    Felkner, Ira C.

    1992-05-01

    New approaches have been developed for the detection and quantification of low levels of bioactive compounds that may be introduced into the environment. There is a challenge to scientists to develop analytical methods which are rapid, inexpensive, highly sensitive (e.g., ppb and ppt), have a capacity for high throughput, indicate very few false positives or negatives, and can be applied to any sample matrix relevant for the material to be detected. The basis of the system is the selective responses of sensitive biodetectors whose chemical and molecular interactions with a wide variety of toxic chemicals have been previously determined and reported. The test system assays for the differential growth rate of mutants and wild type Bacillus subtilis strains which will respond to toxic or nutrient substances according to the chemical species and bioavailability. The extent of bacterial growth is determined by the differential light scattering of a laser beam at multiple angles. The intensities at multiple angles and input to photodetection are integrated with a computerized system that collects and analyzes data. Preliminary fmdings indicate that bioactive compounds can be assayed in water, soil, or vegetation matrices; therefore, the laser-bacterial assay appears to be a rapid and inexpensive analytical tool for screening chemicals in a complex matrix.

  10. Development of a Filtration-Based Bioluminescence Assay for Detection of Microorganisms in Tea Beverages.

    Science.gov (United States)

    Shinozaki, Yohei; Igarashi, Toshinori; Harada, Yasuhiro

    2016-03-01

    The market for tea drinks as healthy beverages has been steadily expanding, and ready-to-drink beverages in polyethylene terephthalate bottles have been popular. To more rapidly and accurately test tea beverages bottled in polyethylene terephthalate for microbial contamination, a newly developed filtration device and a washing method with a commercial bioluminescence assay were combined to detect low numbers of bacterial spores, fungal conidia, and ascospores. Washing buffers were formulated with nonionic detergents from the Tween series. Commercially available tea beverages were used to evaluate the filtration capacity of the filtration device, the effect of washing buffers, and the performance of the assay. The assay was tested with serially diluted suspensions of colonies of two bacterial strains, spores of three Bacillus strains, conidia of five fungal strains, and ascospores of four fungal strains. The filtration device enabled filtration of a large sample volume (100 to 500 ml), and the washing buffer significantly decreased the background bioluminescence intensity of tea samples when compared with the no-washing method. Low numbers (1 to 10 CFU/100 ml) of the tested strains of bacteria were detected within 8 to 18 h of cultivation, and fungi were detected within 24 to 48 h. Furthermore, a whole bottle (500 ml) of mixed tea was filtered through the filtration device and microbes were detected. This method could be used for quality control of bottled beverages without preincubation.

  11. 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.

  12. Fast detection of Noroviruses using a real-time PCR assay and automated sample preparation

    Directory of Open Access Journals (Sweden)

    Schmid Michael

    2004-06-01

    Full Text Available Abstract Background Noroviruses (NoV have become one of the most commonly reported causative agents of large outbreaks of non-bacterial acute gastroenteritis worldwide as well as sporadic gastroenteritis in the community. Currently, reverse transcriptase polymerase chain reaction (RT-PCR assays have been implemented in NoV diagnosis, but improvements that simplify and standardize sample preparation, amplification, and detection will be further needed. The combination of automated sample preparation and real-time PCR offers such refinements. Methods We have designed a new real-time RT-PCR assay on the LightCycler (LC with SYBR Green detection and melting curve analysis (Tm to detect NoV RNA in patient stool samples. The performance of the real-time PCR assay was compared with that obtained in parallel with a commercially available enzyme immunoassay (ELISA for antigen detection by testing a panel of 52 stool samples. Additionally, in a collaborative study with the Baden-Wuerttemberg State Health office, Stuttgart (Germany the real-time PCR results were blindly assessed using a previously well-established nested PCR (nPCR as the reference method, since PCR-based techniques are now considered as the "gold standard" for NoV detection in stool specimens. Results Analysis of 52 clinical stool samples by real-time PCR yielded results that were consistent with reference nPCR results, while marked differences between the two PCR-based methods and antigen ELISA were observed. Our results indicate that PCR-based procedures are more sensitive and specific than antigen ELISA for detecting NoV in stool specimens. Conclusions The combination of automated sample preparation and real-time PCR provided reliable diagnostic results in less time than conventional RT-PCR assays. These benefits make it a valuable tool for routine laboratory practice especially in terms of rapid and appropriate outbreak-control measures in health-care facilities and other settings.

  13. A replaceable liposomal aptamer for the ultrasensitive and rapid detection of biotin

    Science.gov (United States)

    Sung, Tzu-Cheng; Chen, Wen-Yih; Shah, Pramod; Chen, Chien-Sheng

    2016-02-01

    Biotin is an essential vitamin which plays an important role for maintaining normal physiological function. A rapid, sensitive, and simple method is necessary to monitor the biotin level. Here, we reported a replacement assay for the detection of biotin using a replaceable liposomal aptamer. Replacement assay is a competitive assay where a sample analyte replaces the labeled competitor of analyte out of its biorecognition element on a surface. It is user friendly and time-saving because of washing free. We used aptamer as a competitor, not a biorecognition element as tradition. To label aptamers, we used cholesterol-conjugated aptamers to tag signal-amplifying-liposomes. Without the need of conjugation procedure, aptamers can be easily incorporated into the surface of dye-encapsulating liposomes. Two aptamers as competitors of biotin, ST-21 and ST-21M with different affinities to streptavidin, were studied in parallel for the detection of biotin using replacement assays. ST-21 and ST-21M aptamers reached to limits of detection of 1.32 pg/80 μl and 0.47 pg/80 μl, respectively. The dynamic ranges of our assays using ST-21 and ST-21M aptamers were seven and four orders of magnitude, respectively. This assay can be completed in 20 minutes without washing steps. These results were overall better than previous reported assays.

  14. Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations

    Science.gov (United States)

    Law, Jodi Woan-Fei; Ab Mutalib, Nurul-Syakima; Chan, Kok-Gan; Lee, Learn-Han

    2015-01-01

    The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally. Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required in many food analyses. Rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods. This review emphasizes on the principles and application of recent rapid methods for the detection of foodborne bacterial pathogens. Detection methods included are simple polymerase chain reaction (PCR), multiplex PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP) and oligonucleotide DNA microarray which classified as nucleic acid-based methods; optical, electrochemical and mass-based biosensors which classified as biosensor-based methods; enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay which classified as immunological-based methods. In general, rapid detection methods are generally time-efficient, sensitive, specific and labor-saving. The developments of rapid detection methods are vital in prevention and treatment of foodborne diseases. PMID:25628612

  15. Rapid Methods for the Detection of Foodborne Bacterial Pathogens: Principles, Applications, Advantages and Limitations

    Directory of Open Access Journals (Sweden)

    Law eJodi Woan-Fei

    2015-01-01

    Full Text Available The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally. Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required in many food analyses. Rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods. This review emphasizes on the principles and application of recent rapid methods for the detection of foodborne bacterial pathogens. Detection methods included are simple polymerase chain reaction (PCR, multiplex PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA, loop-mediated isothermal amplification (LAMP and oligonucleotide DNA microarray which classified as nucleic acid-based methods; optical, electrochemical and mass-based biosensors which classified as biosensor-based methods; enzyme-linked immunosorbent assay (ELISA and lateral flow immunoassay which classified as immunological-based methods. In general, rapid detection methods are generally time-efficient, sensitive, specific and labor-saving. The developments of rapid detection methods are vital in prevention and treatment of foodborne diseases.

  16. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

    Directory of Open Access Journals (Sweden)

    D Ransom Hardison

    Full Text Available Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs. One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R. However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R in certain labs. A fluorescence based receptor binding assay (RBA(F was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2 for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1. Fish (N = 61 of six different species were screened using the RBA(F. Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a correlated well (R2 = 0.71 with those of the RBA(F, given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F, which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F advantages include the long-term (> 5 years stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R. The RBA(F is cost-effective, allows high sample

  17. Detection of Salmonella enterica serovar Enteritidis (SE) Antibodies in Serum Using A Polystyrene Bead/SE Flagella Agglutination Assay

    Science.gov (United States)

    Serologic screening of flocks can be an important method to detect Salmonella enteritidis (SE) infections but can be labor intensive or lack specificity. Our goal was to develop a rapid agglutination assay using SE flagella adsorbed to polystyrene beads as a simple, relatively specific test to dete...

  18. 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.

  19. Evaluation of the BD Max StaphSR Assay for Rapid Identification of Staphylococcus aureus and Methicillin-Resistant S. aureus in Positive Blood Culture Broths.

    Science.gov (United States)

    Dalpke, Alexander H; Hofko, Marjeta; Hamilton, Fiona; Mackenzie, Laura; Zimmermann, Stefan; Templeton, Kate

    2015-11-01

    We evaluated the performance of the BD Max StaphSR assay for the direct detection of Staphylococcus aureus from blood culture medium. In a two-center trial, 155 blood cultures from the BD Bactec FX system and 212 from the bioMérieux BacT/Alert system were tested; 170 bottles yielded S. aureus, and all were identified correctly by the BD Max StaphSR assay. The assay required approximately 2.5 h, thus allowing rapid identification of blood cultures flagged positive. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. Dual color fluorescence in situ hybridization (FISH) assays for detecting Mycobacterium tuberculosis and Mycobacterium avium complexes and related pathogens in cultures

    OpenAIRE

    Shah, Jyotsna; Weltman, Helena; Narciso, Patricia; Murphy, Christina; Poruri, Akhila; Baliga, Shrikala; Sharon, Leesha; York, Mary; Cunningham, Gail; Miller, Steve; Caviedes, Luz; Gilman, Robert; Desmond, Edward; Ramasamy, Ranjan

    2017-01-01

    Two rapid dual color fluorescence in situ hybridization (FISH) assays were evaluated for detecting M. tuberculosis and related pathogens in cultures. The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the Mycobacterium tuberculosis complex (MTBC) and a green fluorescent probe specific for the Mycobacterium and Nocardia genera (MN Genus) to detect and distinguish MTBC from other Mycobacteria and Nocardia. A complementary MTBC-MAC FISH assay uses green and orange fluores...

  1. Whole-blood agglutination assay for on-site detection of human immunodeficiency virus infection.

    Science.gov (United States)

    Gupta, Amita; Chaudhary, Vijay K

    2003-07-01

    Simple and rapid diagnostic tests are needed to curtail human immunodeficiency virus (HIV) infection, especially in the developing and underdeveloped nations of the world. The visible-agglutination assay for the detection of HIV with the naked eye (NEVA HIV, which represents naked eye visible-agglutination assay for HIV) is a hemagglutination-based test for the detection of antibodies to HIV in whole blood. The NEVA HIV reagent is a cocktail of highly stable recombinant bifunctional antibody fusion proteins with HIV antigens which can be produced in large quantities with a high degree of purity. The test procedure involves mixing of one drop of the NEVA HIV reagent with one drop of blood sample on a glass slide. The presence of anti-HIV antibodies in the blood sample leads to clumping of erythrocytes (agglutination) that can be seen with the naked eye. Evaluation with commercially available panels of sera and clinical samples has shown that the performance of NEVA HIV is comparable to those of U.S. and European Food and Drug Administration-approved rapid as well as enzyme-linked immunosorbent assay kits. The test detects antibodies to both HIV type 1 (HIV-1) and HIV-2 in a single spot and gives results in less than 5 min. The test was developed by keeping in mind the practical constraints of testing in less developed countries and thus is completely instrument-free, requiring no infrastructure or even electricity. Because the test is extremely rapid, requires no sample preparation, and is simple enough to be performed by a semiskilled technician in any remote area, NEVA HIV is a test for the hard-to-reach populations of the world.

  2. Rapid Detection of Cellular Response to Biological Agents

    National Research Council Canada - National Science Library

    Williams, Bryan R

    2005-01-01

    Our program objective is to develop simple and rapid methods for detecting at a cellular level, individual responses to environmental stresses elaborated by exposure to infectious agents such as bacteria and viruses...

  3. Rapid Detection of Cellular Responses to Biological Agents

    National Research Council Canada - National Science Library

    Williams, Bryan

    2004-01-01

    Our program objective is to develop simple and rapid methods for detecting, at a cellular level, individual responses to environmental stresses elaborated by exposure to infectious agents such as bacteria and viruses...

  4. Rapid Detection of Cellular Responses to Biological Agents

    National Research Council Canada - National Science Library

    Williams, Bryan

    2003-01-01

    Our program objective is to develop simple and rapid methods for detecting, at a cellular level, individual responses to environmental stresses elaborated by exposure to infectious agents such as bacteria and viruses...

  5. ETV Tech Brief: Rapid Fungi and Bacteria Detection Technologies

    Science.gov (United States)

    Technical brief that summarizes the results for Mycometer, Inc. Mycometer®-test and Bactiquant®-test, which are rapid detection technologies for fungi and bacteria. The brief summarizes the results of the verification report and statement.

  6. Rapid method for detection of salmonella in meat

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention relates to a rapid method for the detection of Salmonella in meat as well as to a kit for performing said method. The method provides a time-to-result of less than 8 hours.......The present invention relates to a rapid method for the detection of Salmonella in meat as well as to a kit for performing said method. The method provides a time-to-result of less than 8 hours....

  7. Use of enzyme-linked immunosorbent assay and dipstick assay for detection of Strongyloides stercoralis infection in humans

    NARCIS (Netherlands)

    van Doorn, H. Rogier; Koelewijn, Rob; Hofwegen, Henk; Gilis, Henk; Wetsteyn, Jose C. F. M.; Wismans, Pieter J.; Sarfati, Claudine; Vervoort, Tony; van Gool, Tom

    2007-01-01

    A homemade enzyme-linked immunosorbent assay (ELISA) (Academic Medical Center ELISA [AMC-ELISA]) and a dipstick assay for the detection of anti-Strongyloides stercoralis antibodies in serum were developed and evaluated together with two commercially available ELISAs (IVD-ELISA [IVD Research, Inc.

  8. Rapid identification of ST131 Escherichia coli by a novel multiplex real-time allelic discrimination assay.

    Science.gov (United States)

    François, Patrice; Bonetti, Eve-Julie; Fankhauser, Carolina; Baud, Damien; Cherkaoui, Abdessalam; Schrenzel, Jacques; Harbarth, Stephan

    2017-09-01

    Escherichia coli sequence type 131 is increasingly described in severe hospital infections. We developed a rapid real-time allelic discrimination assay for the rapid identification of E. coli ST131 isolates. This rapid assay represents an affordable alternative to sequence-based strategies before completing characterization of potentially highly virulent isolates of E. coli. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Detection of antiphospholipid antibodies by automated chemiluminescence assay.

    Science.gov (United States)

    Capozzi, Antonella; Lococo, Emanuela; Grasso, Maria; Longo, Agostina; Garofalo, Tina; Misasi, Roberta; Sorice, Maurizio

    2012-05-31

    The laboratory diagnosis of antiphospholipid antibody syndrome (APS) requires the demonstration of antiphospholipid antibodies (aPL) by lupus anticoagulant (LAC) measured through coagulation assays, anticardiolipin IgG or IgM antibodies (aCL) and/or anti-β2-glycoprotein I IgG or IgM antibodies (anti-β2-GPI), usually detected by ELISA. In this study we tested aCL by a new automated system using the chemiluminescence principle. Our results showed that, while almost all the sera from APS patients, positive for IgG aCL and anti-β2-GPI by ELISA, were also positive for IgG aCl by chemiluminescence, only 30.13% of patients without clinical manifestations of APS, but positive for aCL and persistently negative for anti-β2-GPI (by ELISA) and LA, confirmed the positive test by chemiluminescence. This difference was highly significant (paCL and anti-β2-GPI IgG (ELISA). Thus, the new technology of automated chemiluminescence assay for measuring aPL may represent an useful tool to identify "true" APS patients. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Detection of Legionella Contamination in Tabriz Hospitals by PCR Assay

    Science.gov (United States)

    Ghotaslou, Reza; Yeganeh Sefidan, Fatemeh; Akhi, Mohammad Taghi; Soroush, Mohammad Hussein; Hejazi, Mohammad Saeid

    2013-01-01

    Purpose: The present study was designed to evaluate the occurrence of Legionella contamination in the tap water of Tabriz hospitals, Azerbaijan, Iran. Methods: One hundred and forty water samples from diverse water supply systems of 17 hospitals were collected and analyzed for the presence of Legionella spp. by PCR assay. Results: In this study, 10 of 140 (7.1%) samples were positive for Legionella which L. pneumophila was detected in 4 (2.85%) water samples. Conclusion: In conclusion, hospital potable systems are the primary reservoirs for Legionnaires’ disease. This study concludes that Legionella spp. are present in aquatic hospitals environment of Tabriz. Due to the serious risk of infections, it is better to make efforts to eliminate Legionella spp. in water supplies. PMID:24312825

  11. Detection of Legionella Contamination in Tabriz Hospitals by PCR Assay

    Directory of Open Access Journals (Sweden)

    Mohammad Saeid Hejazi

    2013-02-01

    Full Text Available Purpose: The present study was designed to evaluate the occurrence of Legionella contamination in the tap water of Tabriz hospitals, Azerbaijan, Iran. Methods: One hundred and forty water samples from diverse water supply systems of 17 hospitals were collected and analyzed for the presence of Legionella spp. by PCR assay. Results: In this study, 10 of 140 (7.1% samples were positive for Legionella which L. pneumophila was detected in 4 (2.85% water samples. Conclusion: In conclusion, hospital potable systems are the primary reservoirs for Legionnaires’ disease. This study concludes that Legionella spp. are present in aquatic hospitals environment of Tabriz. Due to the serious risk of infections, it is better to make efforts to eliminate Legionella spp. in water supplies.

  12. Detection of garlic gamma-irradiated by assay comet

    Energy Technology Data Exchange (ETDEWEB)

    Moreno Alvarez, Damaris L.; Miranda, Enrique F. Prieto; Carro, Sandra; Iglesias Enrique, Isora; Matos, Wilberto [Centro de Aplicaciones Tecnologicas y Desarrollo Nuclear (CEADEN), Ciudad de La Habana (Cuba)], e-mail: damaris@ceaden.edu.cu

    2009-07-01

    The garlic samples were irradiated in a facility with {sup 60}Co sources, at absorbed dose values of 0-0,15 kGy. The detection method utilized for the identification of the irradiated garlic was biological comet assay. The samples were classified post-irradiation several times. The irradiated samples showed high strand breaks of DNA exhibiting comets of several forms, while the not irradiated and lower dose samples showed a behavior like round shape and light comets. Significant differences were found for higher absorbed dose values at 0.06 kGy, this absorbed dose value is corresponding with the applied dose value at this food in order to avoid the germination. (author)

  13. Rapid Detection and Identification of Yersinia pestis from Food Using Immunomagnetic Separation and Pyrosequencing

    Directory of Open Access Journals (Sweden)

    Kingsley K. Amoako

    2012-01-01

    Full Text Available Interest has recently been renewed in the possible use of Y. pestis, the causative agent of plague, as a biological weapon by terrorists. The vulnerability of food to intentional contamination coupled with reports of humans having acquired plague through eating infected animals that were not adequately cooked or handling of meat from infected animals makes the possible use of Y. pestis in a foodborne bioterrorism attack a reality. Rapid, efficient food sample preparation and detection systems that will help overcome the problem associated with the complexity of the different matrices and also remove any ambiguity in results will enable rapid informed decisions to be made regarding contamination of food with biothreat agents. We have developed a rapid detection assay that combines the use of immunomagnetic separation and pyrosequencing in generating results for the unambiguous identification of Y. pestis from milk (0.9 CFU/mL, bagged salad (1.6 CFU/g, and processed meat (10 CFU/g. The low detection limits demonstrated in this assay provide a novel tool for the rapid detection and confirmation of Y. pestis in food without the need for enrichment. The combined use of the iCropTheBug system and pyrosequencing for efficient capture and detection of Y. pestis is novel and has potential applications in food biodefence.

  14. Detection of pathogens in food using a SERS-based assay in just a few hours

    Science.gov (United States)

    Shende, Chetan; Sengupta, Atanu; Huang, Hermes; Farquharson, Stuart

    2014-05-01

    In 2011 Escherichia, Listeria, and Salmonella species infected over 1.2 million people in the United States, resulting in over 23,000 hospitalizations and 650 deaths. In January 2013 President Obama signed into law the Food and Drug Administration (FDA) Food Safety Modernization Act (FSMA), which requires constant microbial testing of food processing equipment and food to minimize contamination and distribution of food tainted with pathogens. The challenge to preventing distribution and consumption of contaminated foods lies in the fact that just a few bacterial cells can rapidly multiply to millions, reaching infectious doses within a few days. Unfortunately, current methods used to detect these few cells rely on similar growth steps to multiply the cells to the point of detection, which also takes a few days. Consequently, there is a critical need for an analyzer that can rapidly extract and detect foodborne pathogens at 1000 colony forming units per gram of food in 1-2 hours (not days), and with a specificity that differentiates from indigenous microflora, so that false alarms are eliminated. In an effort to meet this need, we have been developing an assay that extracts such pathogens from food, selectively binds these pathogens, and produces surface-enhanced Raman spectra (SERS) when read by a Raman analyzer. Here we present SERS measurements of these pathogens in actual food samples using this assay.

  15. Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins.

    Science.gov (United States)

    Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M; Zakarian, Armen; Molgó, Jordi

    2012-12-04

    Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.

  16. Rapid susceptibility testing of Mycobacterium tuberculosis by bioluminescence assay of mycobacterial ATP

    Energy Technology Data Exchange (ETDEWEB)

    Nilsson, L.E.; Hoffner, S.E.; Ansehn, S.

    1988-08-01

    Mycobacterial growth was monitored by bioluminescence assay of mycobacterial ATP. Cultures of Mycobacterium tuberculosis H37Rv and of 25 clinical isolates of the same species were exposed to serial dilutions of ethambutol, isoniazid, rifampin, and streptomycin. A suppression of ATP, indicating growth inhibition, occurred for susceptible but not resistant strains within 5 to 7 days of incubation. Breakpoint concentrations between susceptibility and resistance were determined by comparing these results with those obtained by reference techniques. Full agreement was found in 99% of the assays with the resistance ratio method on Lowenstein-Jensen medium, and 98% of the assays were in full agreement with the radiometric system (BACTEC). A main advantage of the bioluminescence method is its rapidity, with results available as fast as with the radiometric system but at a lower cost and without the need for radioactive culture medium. The method provides kinetic data concerning drug effects within available in vivo drug concentrations and has great potential for both