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Sample records for based detection assays

  1. Comet assay for rapid detection of base damage in foods

    International Nuclear Information System (INIS)

    Single cell gel electrophoresis (SCGE) or comet assay technique a sensitive, reliable and rapid method for DNA double and single strand break, alkali- labile site and delayed repair site detection in individual cells. In recent years, this method has been widely used for studies of DNA repair, genetic toxicology, and environmental biomontoring, however, this technique serves as an important tool for detection of DNA damage in living organism and is increasing being used in genetic testing of industrial chemicals, environmental agent's contaminations. This research paper helps to evaluate the oxidant agent's effects of exposure to organic pollutants by using comet assay techniques. This study used five samples of each food sample (Meat, Chicken, Rice, Fruits, Vegetables and Tea) to evaluate the genotoxic effects of exposure, to environmental agent's pollutants. The experimental data suggest that the DNA damage parameters ( Tail length, Tail width 1 ) were found higher value in exposed population when compared with the ratio of the length to width that cells exhibiting no migration having a ratio of 1. The percentage and distribution of cells in exposed population of cells also increases with the increase in values. This study demonstrates that, using sensitive techniques, it is possible to detect environmental agent's risks at an early stage. (Author)

  2. Immunological-based assays for specific detection of shrimp viruses.

    Science.gov (United States)

    Chaivisuthangkura, Parin; Longyant, Siwaporn; Sithigorngul, Paisarn

    2014-02-12

    Among shrimp viral pathogens, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal agents, causing serious problems for both the whiteleg shrimp, Penaeus (Litopenaeus) vannamei, and the black tiger shrimp, Penaeus (Penaeus) monodon. Another important virus that infects P. vannamei is infectious myonecrosis virus (IMNV), which induces the white discoloration of affected muscle. In the cases of taura syndrome virus and Penaeus stylirostris densovirus (PstDNV; formerly known as infectious hypodermal and hematopoietic necrosis virus), their impacts were greatly diminished after the introduction of tolerant stocks of P. vannamei. Less important viruses are Penaeus monodon densovirus (PmDNV; formerly called hepatopancreatic parvovirus), and Penaeus monodon nucleopolyhedrovirus (PemoNPV; previously called monodon baculovirus). For freshwater prawn, Macrobrachium rosenbergii nodavirus and extra small virus are considered important viral pathogens. Monoclonal antibodies (MAbs) specific to the shrimp viruses described above have been generated and used as an alternative tool in various immunoassays such as enzyme-linked immunosorbent assay, dot blotting, Western blotting and immunohistochemistry. Some of these MAbs were further developed into immunochromatographic strip tests for the detection of WSSV, YHV, IMNV and PemoNPV and into a dual strip test for the simultaneous detection of WSSV/YHV. The strip test has the advantages of speed, as the result can be obtained within 15 min, and simplicity, as laboratory equipment and specialized skills are not required. Therefore, strip tests can be used by shrimp farmers for the pond-side monitoring of viral infection. PMID:24567913

  3. Molecular-Based Assay for Simultaneous Detection of Four Plasmodium spp. and Wuchereria bancrofti Infections

    OpenAIRE

    MEHLOTRA, RAJEEV K.; Gray, Laurie R; Blood-Zikursh, Melinda J.; Kloos, Zachary; Henry-Halldin, Cara N.; Tisch, Daniel J.; Thomsen, Edward; Reimer, Lisa; Kastens, Will; Baea, Manasseh; Baea, Kaye; Baisor, Moses; Tarongka, Nandao; Kazura, James W; Zimmerman, Peter A

    2010-01-01

    Four major malaria-causing Plasmodium spp. and lymphatic filariasis-causing Wuchereria bancrofti are co-endemic in many tropical and sub-tropical regions. Among molecular diagnostic assays, multiplex polymerase chain reaction (PCR)–based assays for the simultaneous detection of DNAs from these parasite species are currently available only for P. falciparum and W. bancrofti or P. vivax and W. bancrofti. Using a post-PCR oligonucleotide ligation detection reaction–fluorescent microsphere assay ...

  4. Nanobeads-based assays. The case of gluten detection

    International Nuclear Information System (INIS)

    In order to verify if the use of nanobeads of poly[phenylacetylene-(co-acrylic acid)] (PPA/AA) in the ELISA test would affect the immune-activity of the antibodies (Ab) and/or the activity of the enzymes used to label the Ab anti-rabbit IGg, in this work we immobilized the horse liver peroxidase labelled Ab anti-rabbit IGg onto PPA/AA nanobeads. The gluten test was chosen as the model to demonstrate the usefulness of these nanobeads in immunoassays. The synthesis of PPA/AA nanobeads was performed by a modified emulsion polymerization. Self-assembly of nanospheres with mean diameter equal to 200 nm was achieved by casting aqueous suspensions. The materials were characterized by traditional spectroscopic techniques, while the size and dispersion of the particles were analysed by scanning electron microscopy (SEM) measurements. The obtained results show that the immobilization process of the Abs onto PPA/AA did not affect either the immune-response of the Abs or the functional activity of the peroxidase suggesting the usefulness of PPA/AA for the design of advanced nanobeads-based assays for the simultaneous screening of several analytes in complex media.

  5. Nanobeads-based assays. The case of gluten detection

    Science.gov (United States)

    Venditti, Iole; Fratoddi, Ilaria; Vittoria Russo, Maria; Bellucci, Stefano; Crescenzo, Roberta; Iozzino, Luisa; Staiano, Maria; Aurilia, Vincenzo; Varriale, Antonio; Rossi, Mosè; D'Auria, Sabato

    2008-11-01

    In order to verify if the use of nanobeads of poly[phenylacetylene-(co-acrylic acid)] (PPA/AA) in the ELISA test would affect the immune-activity of the antibodies (Ab) and/or the activity of the enzymes used to label the Ab anti-rabbit IGg, in this work we immobilized the horse liver peroxidase labelled Ab anti-rabbit IGg onto PPA/AA nanobeads. The gluten test was chosen as the model to demonstrate the usefulness of these nanobeads in immunoassays. The synthesis of PPA/AA nanobeads was performed by a modified emulsion polymerization. Self-assembly of nanospheres with mean diameter equal to 200 nm was achieved by casting aqueous suspensions. The materials were characterized by traditional spectroscopic techniques, while the size and dispersion of the particles were analysed by scanning electron microscopy (SEM) measurements. The obtained results show that the immobilization process of the Abs onto PPA/AA did not affect either the immune-response of the Abs or the functional activity of the peroxidase suggesting the usefulness of PPA/AA for the design of advanced nanobeads-based assays for the simultaneous screening of several analytes in complex media.

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

    OpenAIRE

    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 ant...

  7. Medically Relevant Assays with a Simple Smartphone and Tablet Based Fluorescence Detection System

    Directory of Open Access Journals (Sweden)

    Piotr Wargocki

    2015-05-01

    Full Text Available Cell phones and smart phones can be reconfigured as biomedical sensor devices but this requires specialized add-ons. In this paper we present a simple cell phone-based portable bioassay platform, which can be used with fluorescent assays in solution. The system consists of a tablet, a polarizer, a smart phone (camera and a box that provides dark readout conditions. The assay in a well plate is placed on the tablet screen acting as an excitation source. A polarizer on top of the well plate separates excitation light from assay fluorescence emission enabling assay readout with a smartphone camera. The assay result is obtained by analysing the intensity of image pixels in an appropriate colour channel. With this device we carried out two assays, for collagenase and trypsin using fluorescein as the detected fluorophore. The results of collagenase assay with the lowest measured concentration of 3.75 µg/mL and 0.938 µg in total in the sample were comparable to those obtained by a microplate reader. The lowest measured amount of trypsin was 930 pg, which is comparable to the low detection limit of 400 pg for this assay obtained in a microplate reader. The device is sensitive enough to be used in point-of-care medical diagnostics of clinically relevant conditions, including arthritis, cystic fibrosis and acute pancreatitis.

  8. An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification.

    Science.gov (United States)

    Wu, Xuping; Wang, Jianfang; Song, Jinyun; Li, Jiayan; Yang, Yongfeng

    2016-09-01

    Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection. PMID:27283884

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

    International Nuclear Information System (INIS)

    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. (author)

  10. 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.

  11. An aptamer-based dipstick assay for the rapid and simple detection of aflatoxin B1.

    Science.gov (United States)

    Shim, Won-Bo; Kim, Min Jin; Mun, Hyoyoung; Kim, Min-Gon

    2014-12-15

    A rapid and simple dipstick assay based on an aptamer has been developed for the determination of aflatoxin B1 (AFB1). The dipstick assay format was based on a competitive reaction of the biotin-modified aptamer specific to AFB1 between target and cy5-modified DNA probes. Streptavidin and anti-cy5 antibody as capture reagents were immobilized at test and control lines on a membrane of the dipstick assay. After optimization, the limit of detection for the dipstick assay was 0.1 ng/ml AFB1 in buffer. The method was confirmed to be specific to AFB1, and the entire process of the assay can be completed within 30 min. Aqueous methanol (20%) provided a good extraction efficiency, and the matrix influence from corn extracts was successfully reduced through 2-fold dilution. The results of AFB1 analysis for corn samples spiked with known concentration of AFB1 by the dipstick assay and ELISA showed good agreement. The cut-off value of the dipstick assay for corn samples was 0.3 ng/g AFB1. Therefore, the dipstick assay is first reported and considered as a rapid, simple, on-site and inexpensive screening tool for AFB1 determination in grains as well as a corn. PMID:25032679

  12. A biolayer interferometry-based assay for rapid and highly sensitive detection of biowarfare agents.

    Science.gov (United States)

    Mechaly, Adva; Cohen, Hila; Cohen, Ofer; Mazor, Ohad

    2016-08-01

    Biolayer interferometry (BLI) is an optical technique that uses fiber-optic biosensors for label-free real-time monitoring of protein-protein interactions. In this study, we coupled the advantages of the Octet Red BLI system (automation, fluidics-free, and on-line monitoring) with a signal enhancement step and developed a rapid and sensitive immunological-based method for detection of biowarfare agents. As a proof of concept, we chose to demonstrate the efficacy of this novel assay for the detection of agents representing two classes of biothreats, proteinaceous toxins, and bacterial pathogens: ricin, a lethal plant toxin, and the gram-negative bacterium Francisella tularensis, the causative agent of tularemia. The assay setup consisted of biotinylated antibodies immobilized to the biosensor coupled with alkaline phosphatase-labeled antibodies as the detection moiety to create nonsoluble substrate crystals that precipitate on the sensor surface, thereby inducing a significant wavelength interference. It was found that this BLI-based assay enables sensitive detection of these pathogens (detection limits of 10 pg/ml and 1 × 10(4) pfu/ml ricin and F. tularensis, respectively) within a very short time frame (17 min). Owing to its simplicity, this assay can be easily adapted to detect other analytes in general, and biowarfare agents in particular, in a rapid and sensitive manner. PMID:27156814

  13. Detection of Hepatitis B Virus DNA by Duplex Scorpion Primer-based PCR Assay

    Institute of Scientific and Technical Information of China (English)

    KONG De-Ming孔德明; SHEN Han-Xi沈含熙; MI Huai-Feng宓怀风

    2004-01-01

    The application of a new fiuorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.

  14. Electrochemical chip-based genomagnetic assay for detection of high-risk human papillomavirus DNA.

    Science.gov (United States)

    Bartosik, Martin; Durikova, Helena; Vojtesek, Borivoj; Anton, Milan; Jandakova, Eva; Hrstka, Roman

    2016-09-15

    Cervical cancer, being the fourth leading cause of cancer death in women worldwide, predominantly originates from a persistent infection with a high-risk human papillomavirus (HPV). Detection of DNA sequences from these high-risk strains, mostly HPV-16 and HPV-18, represents promising strategy for early screening, which would help to identify women with higher risk of cervical cancer. In developing countries, inadequate screening options lead to disproportionately high mortality rates, making a fast and inexpensive detection schemes highly important. Electrochemical sensors and assays offer an alternative to current methods of detection. We developed an electrochemical-chip based assay, in which target HPV DNA is captured via magnetic bead-modified DNA probes, followed by an antidigoxigenin-peroxidase detection system at screen-printed carbon electrode chips, enabling parallel measurements of eight samples simultaneously. We show sensitive detection in attomoles of HPV DNA, selective discrimination between HPV-16 and HPV-18 and good reproducibility. Most importantly, we show application of the assay into both cancer cell lines and cervical smears from patients. The electrochemical results correlated well with standard methods, making this assay potentially applicable in clinical practice. PMID:27132004

  15. Novel fabrication of immunochromatographic assay based on up conversion phosphors for sensitive detection of clenbuterol.

    Science.gov (United States)

    Wang, Peilong; Wang, Ruiguo; Zhang, Wei; Su, Xiaoou; Luo, Haifeng

    2016-03-15

    A novel and ultra sensitive immunochromatographic assay sensor (ICA) based on up conversion phosphor (UCP) for quantitative detection of clenbuterol (CL) was developed. Monoclonal antibody against CL was labeled with UCP beads. The detection strategy is based on competitive immunoreaction between CL antibodies conjugated to UCP beads and CL or CL antigen on the UCP-ICA sensor. It enables ultra sensitive detection of CL in one single test without complicated sample preparation. Sensing results can be obtained within 10 min. Under optimized conditions, visual limit of detection (vLOD) of UCP-ICA for CL was 0.1 ng/mL. Calculated LOD (cLOD) for CL, as low as 0.01 ng/mL, could be achieved with the UCP-ICA sensor. Recoveries of CL in various sample matrixes ranged from 73.0% to 92.2% and relative standard deviations (RSD) were below 12%. The assay was evaluated with spiked and real samples and the results were compared with liquid chromatography-tandem mass. The developed novel assay method based on UCP could be a potential alternative format for on site and rapid detection of CL as well as other illegal drugs. PMID:26516687

  16. A REAL-TIME PCR-BASED ASSAY FOR DETECTION OF WUCHERERIA BANCROFTI DNA IN BLOOD AND MOSQUITOES

    OpenAIRE

    Rao, Ramakrishna U.; ATKINSON, LAURA J.; Reda M R Ramzy; Helmy, Hanan; Farid, Hoda A.; Bockarie, Moses J.; Susapu, Melinda; LANEY, SANDRA J.; Williams, Steven A.; Weil, Gary J

    2006-01-01

    We developed and evaluated real-time polymerase chain reaction (PCR) assays for detecting Wuchereria bancrofti DNA in human blood and in mosquitoes. An assay based on detection of the W. bancrofti “LDR” repeat DNA sequence was more sensitive than an assay for Wolbachia 16S rDNA. The LDR-based assay was sensitive for detecting microfilarial DNA on dried membrane filters or on filter paper. We also compared real-time PCR with conventional PCR (C-PCR) for detecting W. bancrofti DNA in mosquito s...

  17. Multi-residue detection of pesticides using a sensitive immunochip assay based on nanogold enhancement.

    Science.gov (United States)

    Lan, Meijing; Guo, Yirong; Zhao, Ying; Liu, Yihua; Gui, Wenjun; Zhu, Guonian

    2016-09-28

    This paper describes the development of a new multiplex immunoassay for simultaneous detection of seven pesticides (triazophos, methyl-parathion, fenpropathrin, carbofuran, thiacloprid, chlorothalonil, and carbendazim). Sixteen pairs of pesticide antibodies and antigens were screened for reactivity and cross-reaction. A microarray chip consisting of seven antigens immobilized on a nitrocellulose membrane was then constructed. Nanogold was employed for labeling and signal amplification to obtain a sensitive colorimetric immunoassay. The direct and indirect detection formats were further compared using primary antibody-gold and secondary antibody-gold conjugates as tracers. An integrated 7-plex immunochip assay based on the indirect model was established and optimized. The detection limits for the pesticides were 0.02-6.45 ng mL(-1), which meets detection requirements for pesticide residues. Naked-eye assessment showed the visual detection limits of the assay ranged from 1 to 100 ng mL(-1). Spiked recovery results demonstrated that the immunochip assay had potential for multi-analysis of pesticide residues in vegetables and fruits. The proposed microarray methodology is a flexible and versatile tool, which can be applied to other competitive multiplex immunoassays for small molecular compounds. PMID:27619097

  18. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  19. Innovative mode of action based in vitro assays for detection of marine neurotoxins

    OpenAIRE

    Nicolas, J.A.Y.

    2015-01-01

    Innovative mode of action based in vitro assays for detection of marine neurotoxins J. Nicolas, P.J.M. Hendriksen, T.F.H. Bovee, I.M.C.M. Rietjens Marine biotoxins are naturally occurring compounds produced by particular phytoplankton species. These toxins often accumulate in seafood and thereby represent a threat to consumers. Regulatory limits have been set for lipophilic marine biotoxins (diarrhetic shellfish poisons (DSPs) and azaspiracids (AZPs)) and for most marine neurotoxins (amnesic ...

  20. Paper-based fluorescence resonance energy transfer assay for directly detecting nucleic acids and proteins.

    Science.gov (United States)

    Li, Hua; Fang, Xueen; Cao, Hongmei; Kong, Jilie

    2016-06-15

    Paper-based fluorescence resonance energy transfer assay (FRET) is gaining great interest in detecting macro-biological molecule. It is difficult to achieve conveniently and fast detection for macro-biological molecule. Herein, a graphene oxide (GO)-based paper chip (glass fiber) integrated with fluorescence labeled single-stranded DNA (ssDNA) for fast, inexpensive and direct detection of biological macromolecules (proteins and nucleic acids) has been developed. In this paper, we employed the Cy3/FAM-labeled ssDNA as the reporter and the GO as quencher and the original glass fiber paper as data acquisition substrates. The chip which was designed and fabricated by a cutting machine is a miniature biosensor that monitors fluorescence recovery from resonance energy transfer. The hybridization assays and fluorescence detection were all simplified, and the surface of the chip did not require immobilization or washing. A Nikon Eclipse was employed as excited resource and a commercial digital camera was employed for capturing digital images. This paper-based microfluidics chip has been applied in the detection of proteins and nucleic acids. The biosensing capability meets many potential requirements for disease diagnosis and biological analysis. PMID:26807518

  1. 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.

  2. A tissue biopsy-based epigenetic multiplex PCR assay for prostate cancer detection

    Directory of Open Access Journals (Sweden)

    Van Neste Leander

    2012-06-01

    Full Text Available Abstract Background PSA-directed prostate cancer screening leads to a high rate of false positive identifications and an unnecessary biopsy burden. Epigenetic biomarkers have proven useful, exhibiting frequent and abundant inactivation of tumor suppressor genes through such mechanisms. An epigenetic, multiplex PCR test for prostate cancer diagnosis could provide physicians with better tools to help their patients. Biomarkers like GSTP1, APC and RASSF1 have demonstrated involvement with prostate cancer, with the latter two genes playing prominent roles in the field effect. The epigenetic states of these genes can be used to assess the likelihood of cancer presence or absence. Results An initial test cohort of 30 prostate cancer-positive samples and 12 cancer-negative samples was used as basis for the development and optimization of an epigenetic multiplex assay based on the GSTP1, APC and RASSF1 genes, using methylation specific PCR (MSP. The effect of prostate needle core biopsy sample volume and age of formalin-fixed paraffin-embedded (FFPE samples was evaluated on an independent follow-up cohort of 51 cancer-positive patients. Multiplexing affects copy number calculations in a consistent way per assay. Methylation ratios are therefore altered compared to the respective singleplex assays, but the correlation with patient outcome remains equivalent. In addition, tissue-biopsy samples as small as 20 μm can be used to detect methylation in a reliable manner. The age of FFPE-samples does have a negative impact on DNA quality and quantity. Conclusions The developed multiplex assay appears functionally similar to individual singleplex assays, with the benefit of lower tissue requirements, lower cost and decreased signal variation. This assay can be applied to small biopsy specimens, down to 20 microns, widening clinical applicability. Increasing the sample volume can compensate the loss of DNA quality and quantity in older samples.

  3. A new scintillation proximity assay-based approach for the detection of KRAS mutations

    Energy Technology Data Exchange (ETDEWEB)

    Lee, So-Young; Lim, Jae-Cheong; Cho, Eun-Ha; Jung, Sung-Hee [Korea Atomic Energy Research Institute (KAERI), Daejeon (Korea, Republic of). Radioisotope Research Div.

    2016-04-01

    KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with {sup 125}I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

  4. A new scintillation proximity assay-based approach for the detection of KRAS mutations

    International Nuclear Information System (INIS)

    KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with 125I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

  5. Comparison of histopathology and PCR based assay for detection of experimentally induced toxoplasmosis in murine model

    Institute of Scientific and Technical Information of China (English)

    Vikrant Sudan; A K Tewari; R Singh; Harkirat Singh

    2015-01-01

    Objective:To compare histopathology and PCR based detection in diagnosis of experimentally induced toxoplasmosis of RH human strain of the parasite in murine models. Methods:A comparison of histopathology and PCR based detection was done to diagnose experimentally induced toxoplasmosis in ten inbred swiss albino mice after intraperitoneal inoculation of 100 tachyzoites of laboratory mantained human RH strain of the parasite. Tissue samples from lung, liver, spleen, brain, heart and kidney were taken and processed for histopathological examination while all the samples also were subjected to PCR, using primers directed to the multicopy of SAG 3 gene, in dublicates. Results: Histopathology revealed presence of tachyzoites only in liver while along with lung, liver, spleen and brain tissue yielded desired positive PCR amplicons. Conclusions:The SAG 3 based PCR is able to diagnose toxoplasmosis in those tissues which are declared negative by histopathological assay.

  6. Comparison of histopathology and PCR based assay for detection of experimentally induced toxoplasmosis in murine model

    Institute of Scientific and Technical Information of China (English)

    Vikrant; Sudan; A.K.Tewari; R; Singh; Harkirat; Singh

    2015-01-01

    Objective:To compare histopathology and PCR based detection in diagnosis of experimentally induced toxoplasmosis of RH human strain of the parasite in murine models.Methods:A comparison of histopathology and PCR based detection was done to diagnose experimentally induced toxoplasmosis in ten inbred swiss albino mice after intraperitoneal inoculation of 100 tachyzoites of laboratory mantained human RH strain of the parasite.Tissue samples from lung,liver,spleen,brain,heart and kidney were taken and processed for histopathological examination while all the samples also were subjected to PCR,using primers directed to the multicopy of SAG 3 gene,in dublicates.Results:Histopathology revealed presence of tachyzoites only in liver while along with lung,liver,spleen and brain tissue yielded desired positive PCR amplicons.Conclusions:The SAG 3 based PCR is able to diagnose toxoplasmosis in those tissues which are declared negative by histopathological assay.

  7. Quantum-dot submicrobead-based immunochromatographic assay for quantitative and sensitive detection of zearalenone.

    Science.gov (United States)

    Duan, Hong; Chen, Xuelan; Xu, Wei; Fu, Jinhua; Xiong, Yonghua; Wang, Andrew

    2015-01-01

    Mycotoxin pollutants are commonly related to cereal products and cause fatal threats in food safety, and therefore require simple and sensitive detection. In this work, quantum-dot (QD) submicrobeads (QBs) were synthesized by encapsulating CdSe/ZnS QDs using the microemulsion technique. The resultant QBs, with approximately 2800 times brighter luminescence than the corresponding QDs, were explored as novel fluorescent probes in the immunochromatographic assay (ICA) for sensitive and quantitative detection of zearalenone (ZEN) in corns. Various parameters that influenced the sensitivity and stability of QB-based ICA (QB-ICA) were investigated and optimized. The optimal QB-ICA exhibits good dynamic linear detection for ZEN over the range of 0.125 ng/mL to 10 ng/mL with a median inhibitory concentration of 1.01±0.09 ng/mL (n=3). The detection limits for ZEN in a standard solution and real corn sample (dilution ratio of 1:30) are 0.0625 ng/mL and 3.6 µg/kg, respectively, which is much better than that of a previously reported gold nanoparticle-based ICA method. Forty-six natural corn samples are assayed using both QB-ICA and enzyme-linked immunosorbent assay. The two methods show a highly significant correlation (R(2)=0.92). Nine ZEN-contaminated samples were further confirmed with liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the QB-ICA results also exhibited good agreement with LC-MS/MS method. In brief, this work demonstrates that QB-ICA is capable of rapid, sensitive screening of toxins in food analysis, and shows great promise for point-of-care testing of other analytes. PMID:25476288

  8. PathogenMip Assay: A Multiplex Pathogen Detection Assay

    OpenAIRE

    Akhras, Michael S.; Sreedevi Thiyagarajan; Villablanca, Andrea C.; Davis, Ronald W; Pål Nyrén; Nader Pourmand

    2007-01-01

    The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The ...

  9. Rapid and quantitative detection of C-reactive protein based on quantum dots and immunofiltration assay

    Directory of Open Access Journals (Sweden)

    Zhang PF

    2015-09-01

    Full Text Available Pengfei Zhang,1,* Yan Bao,1,* Mohamed Shehata Draz,2,3,* Huiqi Lu,1 Chang Liu,1 Huanxing Han11Center for Translational Medicine, Changzheng Hospital, Second Military Medical University, Shanghai, People’s Republic of China; 2Zhejiang-California International Nanosystems Institute, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China; 3Faculty of Science, Tanta University, Tanta, Egypt*These authors contributed equally to this workAbstract: Convenient and rapid immunofiltration assays (IFAs enable on-site “yes” or “no” determination of disease markers. However, traditional IFAs are commonly qualitative or semi-quantitative and are very limited for the efficient testing of samples in field diagnostics. Here, we overcome these limitations by developing a quantum dots (QDs-based fluorescent IFA for the quantitative detection of C-reactive proteins (CRP. CRP, the well-known diagnostic marker for acute viral and bacterial infections, was used as a model analyte to demonstrate performance and sensitivity of our developed QDs-based IFA. QDs capped with both polyethylene glycol (PEG and glutathione were used as fluorescent labels for our IFAs. The presence of the surface PEG layer, which reduced the non-specific protein interactions, in conjunction with the inherent optical properties of QDs, resulted in lower background signal, increased sensitivity, and ability to detect CRP down to 0.79 mg/L with only 5 µL serum sample. In addition, the developed assay is simple, fast and can quantitatively detect CRP with a detection limit up to 200 mg/L. Clinical test results of our QD-based IFA are well correlated with the traditional latex enhance immune-agglutination aggregation. The proposed QD-based fluorescent IFA is very promising, and potentially will be adopted for multiplexed immunoassay and in field point-of-care test.Keywords: C-reactive proteins, point-of-care test, Glutathione capped QDs, PEGylation

  10. Novel biosensor-based microarray assay for detecting rs8099917 and rs12979860 genotypes

    Institute of Scientific and Technical Information of China (English)

    Pei-Yuan Li; Xiao-Jun Zhou; Lan Yao; Xin-Hua Fang; Jiang-Nan Ren; Jia-Wu Song

    2012-01-01

    AIM:To evaluate a novel biosensor-based microarray (BBM) assay for detecting rs12979860 and rs8099917genotypes.METHODS:Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control probes were designed,constructed and arrayed on an optical biosensor to develop a microarray assay.Two sets of primers were used in a one tube polymerase chain reaction (PCR) system to amplify two target fragments simultaneously.The biosensor microarray contained probes that had been sequenced to confirm that they included the rsS099917C/T or rs12979860G/T alleles of interest and could serve as the specific assay standards.In addition to rehybridization of four probes of known sequence,a total of 40 clinical samples collected from hepatitis C seropositive patients were also tested.The target fragments of all 40 samples were amplified in a 50 μL PCR system.Ten μL of each amplicon was tested by BBM assay,and another 40 μL was used for sequencing.The agreement of the results obtained by the two methods was tested statistically using the kappa coefficient.The sensitivity of the BBM assay was evaluated using serial dilutions of ten clinical blood samples containing 103-104 white cells/lμL.RESULTS:As shown by polyacrylamide gel electrophoresis,two target segments of the interleukin 28B-associated polymorphisms (SNPs) were successfully amplified in the one-tube PCR system.The lengths of the two amplified fragments were consistent with the known length of the target sequences,137 and 159bps.After hybridization of the PCR amplicons with the probes located on the BBM array,the signals of each allele of both the rs8099917 SNPs and rs12979860 SNPs were observed simultaneously and were clearly visible by the unaided eye.The signals were distinct from each other,could be interpreted visually,and accurately recorded using an ordinary digital camera.To evaluate the specificity of the assay,both the plasmids and clinical samples were applied to the microarray

  11. On-chip, aptamer-based sandwich assay for detection of glycated hemoglobins via magnetic beads.

    Science.gov (United States)

    Li, Jinglun; Chang, Ko-Wei; Wang, Chih-Hung; Yang, Ching-Hsuan; Shiesh, Shu-Chu; Lee, Gwo-Bin

    2016-05-15

    Diabetes can be diagnosed and monitored by measurement of the cutoff ratio between glycated hemoglobins (HbA1c) and total hemoglobin (Hb), which does not require a fasting blood sample and is less influenced by biological variations. In this study, we combined the advantages of the microfluidic system and the selected low-cost, stable and specific aptamers and developed an integrated, aptamer-based microfluidic system for automatic glycated hemoglobin measurements. The detection process of human whole blood can be totally automated in this integrated microfluidic system. According to the experimental results, when compared to conventional bench-top manual assays, reagent consumption was significantly reduced by 75%, and the analysis time was reduced from 3.5h to 30min. Besides, the novelty in this research also lies in the simultaneously performed two parallel assays for detection of Hb and HbA1c in a single chip. Therefore, this sensitive and low-cost aptamer-based microfluidic system may become a promising tool for point-of -care diagnosis of diabetes. PMID:26797251

  12. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    Directory of Open Access Journals (Sweden)

    Alvaro Díaz-Badillo

    2014-04-01

    Full Text Available Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

  13. A Cell-Based Fluorescent Assay to Detect the Activity of Shiga Toxin and Other Toxins That Inhibit Protein Synthesis

    Science.gov (United States)

    Escherichia coli O157:H7, a major cause of food-borne illness, produces Shiga toxins that block protein synthesis by inactivating the ribosome. In this chapter we describe a simple cell-based fluorescent assay to detect Shiga toxins and inhibitors of toxin activity. The assay can also be used to d...

  14. Alternative Methods for the Detection of Emerging Marine Toxins: Biosensors, Biochemical Assays and Cell-Based Assays

    Directory of Open Access Journals (Sweden)

    Laia Reverté

    2014-11-01

    Full Text Available The emergence of marine toxins in water and seafood may have a considerable impact on public health. Although the tendency in Europe is to consolidate, when possible, official reference methods based on instrumental analysis, the development of alternative or complementary methods providing functional or toxicological information may provide advantages in terms of risk identification, but also low cost, simplicity, ease of use and high-throughput analysis. This article gives an overview of the immunoassays, cell-based assays, receptor-binding assays and biosensors that have been developed for the screening and quantification of emerging marine toxins: palytoxins, ciguatoxins, cyclic imines and tetrodotoxins. Their advantages and limitations are discussed, as well as their possible integration in research and monitoring programs.

  15. Alternative methods for the detection of emerging marine toxins: biosensors, biochemical assays and cell-based assays.

    Science.gov (United States)

    Reverté, Laia; Soliño, Lucía; Carnicer, Olga; Diogène, Jorge; Campàs, Mònica

    2014-12-01

    The emergence of marine toxins in water and seafood may have a considerable impact on public health. Although the tendency in Europe is to consolidate, when possible, official reference methods based on instrumental analysis, the development of alternative or complementary methods providing functional or toxicological information may provide advantages in terms of risk identification, but also low cost, simplicity, ease of use and high-throughput analysis. This article gives an overview of the immunoassays, cell-based assays, receptor-binding assays and biosensors that have been developed for the screening and quantification of emerging marine toxins: palytoxins, ciguatoxins, cyclic imines and tetrodotoxins. Their advantages and limitations are discussed, as well as their possible integration in research and monitoring programs. PMID:25431968

  16. Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design

    Science.gov (United States)

    Pilotte, Nils; Papaiakovou, Marina; Grant, Jessica R.; Bierwert, Lou Ann; Llewellyn, Stacey; McCarthy, James S.; Williams, Steven A.

    2016-01-01

    Background The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world’s most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays. Methodology/Principal Findings Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay. Conclusions/Significance The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other

  17. Template reporter bacteriophage platform and multiple bacterial detection assays based thereon

    Science.gov (United States)

    Goodridge, Lawrence (Inventor)

    2007-01-01

    The invention is a method for the development of assays for the simultaneous detection of multiple bacteria. A bacteria of interest is selected. A host bacteria containing plasmid DNA from a T even bacteriophage that infects the bacteria of interest is infected with T4 reporter bacteriophage. After infection, the progeny bacteriophage are plating onto the bacteria of interest. The invention also includes single-tube, fast and sensitive assays which utilize the novel method.

  18. Development of a Luminex-Based DIVA Assay for Serological Detection of African Horse Sickness Virus in Horses.

    Science.gov (United States)

    Sánchez-Matamoros, A; Nieto-Pelegrín, E; Beck, C; Rivera-Arroyo, B; Lecollinet, S; Sailleau, C; Zientara, S; Sánchez-Vizcaíno, J M

    2016-08-01

    African horse sickness (AHS) is considered a fatal re-emergent vector-borne disease of horses. In the absence of any effective treatment for AHS, vaccination remains the most effective form of disease control. The new generation of vaccines, such as one based on purified, inactivated AHS virus (AHSV, serotype 4), which does not induce antibodies against non-structural protein 3 (NS3), enables the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA assays). As detecting AHS in AHSV-free countries may lead to restrictions on international animal movements and thereby cause significant economic damage, these DIVA assays are crucial for reducing movement restrictions. In this article, we describe a Luminex-based multiplex assay for DIVA diagnosis of AHS, and we validate it in a duplex format to detect antibodies against structural protein 7 (VP7) and NS3 in serum samples from horses vaccinated with inactivated AHSV4 vaccine or infected with a live virus of the same serotype. Results of the Luminex-based assay for detecting anti-NS3 antibodies showed good positive correlation with results from an in-house enzyme-linked immunosorbent assay (ELISA). Thus, the Luminex-based technique described here may allow multiplex DIVA antibody detection in a single sample in less than 2 h, and it may prove adaptable for the development of robust, multiplex serological assays. PMID:27090377

  19. Heteropolymeric triplex-based genomic assay to detect pathogens or single-nucleotide polymorphisms in human genomic samples.

    Directory of Open Access Journals (Sweden)

    Jasmine I Daksis

    Full Text Available Human genomic samples are complex and are considered difficult to assay directly without denaturation or PCR amplification. We report the use of a base-specific heteropolymeric triplex, formed by native duplex genomic target and an oligonucleotide third strand probe, to assay for low copy pathogen genomes present in a sample also containing human genomic duplex DNA, or to assay human genomic duplex DNA for Single Nucleotide Polymorphisms (SNP, without PCR amplification. Wild-type and mutant probes are used to identify triplexes containing FVL G1691A, MTHFR C677T and CFTR mutations. The specific triplex structure forms rapidly at room temperature in solution and may be detected without a separation step. YOYO-1, a fluorescent bis-intercalator, promotes and signals the formation of the specific triplex. Genomic duplexes may be assayed homogeneously with single base pair resolution. The specific triple-stranded structures of the assay may approximate homologous recombination intermediates, which various models suggest may form in either the major or minor groove of the duplex. The bases of the stable duplex target are rendered specifically reactive to the bases of the probe because of the activity of intercalated YOYO-1, which is known to decondense duplex locally 1.3 fold. This may approximate the local decondensation effected by recombination proteins such as RecA in vivo. Our assay, while involving triplex formation, is sui generis, as it is not homopurine sequence-dependent, as are "canonical triplexes". Rather, the base pair-specific heteropolymeric triplex of the assay is conformation-dependent. The highly sensitive diagnostic assay we present allows for the direct detection of base sequence in genomic duplex samples, including those containing human genomic duplex DNA, thereby bypassing the inherent problems and cost associated with conventional PCR based diagnostic assays.

  20. An improved respiratory syncytial virus neutralization assay based on the detection of green fluorescent protein expression and automated plaque counting

    OpenAIRE

    van Remmerden Yvonne; Xu Fang; van Eldik Mandy; Heldens Jacco GM; Huisman Willem; Widjojoatmodjo Myra N

    2012-01-01

    Abstract Background Virus neutralizing antibodies against respiratory syncytial virus (RSV) are considered important correlates of protection for vaccine evaluation. The established plaque reduction assay is time consuming, labor intensive and highly variable. Methods Here, a neutralization assay based on a modified RSV strain expressing the green fluorescent protein in combination with automated detection and quantification of plaques is described. Results The fluorescence plaque reduction a...

  1. Development of PCR-based assays for detecting and differentiating three species of botrytis infecting broad bean

    Science.gov (United States)

    Botrytis cinerea, B. fabae and B. fabiopsis are known to cause chocolate spot on broad bean. This study was conducted to develop PCR-based assays to detect and differentiate this three species. Two sets of primers, Bc-f/Bc-r for B. cinerea and Bfab-f/Bfab-r for B. fabiopsis, were designed based on t...

  2. 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. PMID:26939661

  3. Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: Assay development and analytical validation

    DEFF Research Database (Denmark)

    Lübeck, Peter Stephensen; Wolffs, P.; On, Stephen L.W.;

    2003-01-01

    As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S...... carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions...

  4. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    OpenAIRE

    Ågren, Joakim; Hamidjaja, Raditijo A; Hansen, Trine; Ruuls, Robin; Thierry, Simon; Vigre, Håkan; Janse, Ingmar; Sundström, Anders; Segerman, Bo; Koene, Miriam; Löfström, Charlotta; van Rotterdam, Bart; Derzelle, Sylviane

    2013-01-01

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. ...

  5. Use of a Phage-Based Assay for Phenotypic Detection of Mycobacteria Directly from Sputum

    OpenAIRE

    Park, D J; Drobniewski, F. A.; Meyer, A.; Wilson, S M

    2003-01-01

    The phage amplified biologically assay is a new method for rapid and low-cost phenotypic determination of the drug sensitivities of Mycobacterium tuberculosis isolates and the detection of viable organisms in patient specimens. Infection of slowly growing mycobacteria with phage (phage D29) was followed by chemical virucide destruction of extracellular phage. Infected mycobacteria were mixed in culture with rapidly growing sensor cells, which the phage can also infect; i.e., lytic amplificati...

  6. White blood cell-based detection of asymptomatic scrapie infection by ex vivo assays.

    Directory of Open Access Journals (Sweden)

    Sophie Halliez

    Full Text Available Prion transmission can occur by blood transfusion in human variant Creutzfeldt-Jakob disease and in experimental animal models, including sheep. Screening of blood and its derivatives for the presence of prions became therefore a major public health issue. As infectious titer in blood is reportedly low, highly sensitive and robust methods are required to detect prions in blood and blood derived products. The objectives of this study were to compare different methods--in vitro, ex vivo and in vivo assays--to detect prion infectivity in cells prepared from blood samples obtained from scrapie infected sheep at different time points of the disease. Protein misfolding cyclic amplification (PMCA and bioassays in transgenic mice expressing the ovine prion protein were the most efficient methods to identify infected animals at any time of the disease (asymptomatic to terminally-ill stages. However scrapie cell and cerebellar organotypic slice culture assays designed to replicate ovine prions in culture also allowed detection of prion infectivity in blood cells from asymptomatic sheep. These findings confirm that white blood cells are appropriate targets for preclinical detection and introduce ex vivo tools to detect blood infectivity during the asymptomatic stage of the disease.

  7. Development and Evaluation of Real-Time PCR-Based Fluorescence Assays for Detection of Chlamydia pneumoniae

    OpenAIRE

    Tondella, Maria Lucia C.; Talkington, Deborah F.; Holloway, Brian P.; Scott F Dowell; Cowley, Karyn; Soriano-Gabarro, Montse; Elkind, Mitchell S.; Fields, Barry S.

    2002-01-01

    Chlamydia pneumoniae is an important respiratory pathogen recently associated with atherosclerosis and several other chronic diseases. Detection of C. pneumoniae is inconsistent, and standardized PCR assays are needed. Two real-time PCR assays specific for C. pneumoniae were developed by using the fluorescent dye-labeled TaqMan probe-based system. Oligonucleotide primers and probes were designed to target two variable domains of the ompA gene, VD2 and VD4. The limit of detection for each of t...

  8. Microbial based assay for specific detection of β-lactam group of antibiotics in milk

    OpenAIRE

    Das, Sougata; Kumar, Naresh; Vishweswaraiah, Raghu Hirikyathanahalli; Haldar, Lopamudra; Gaare, Manju; Singh, Vinai Kumar; Puniya, Anil Kumar

    2011-01-01

    The spore forming Bacillus cereus (66) was screened for the induction of β-lactamase in presence of an inducer using iodometric assay. A significant induction in marker enzyme was observed in B. cereus 66 at maximum residual limit (MRL) of penicillin, ampicillin, cloxacillin, amoxicillin, cefalexin, and cephazolin belonging to β-lactam group of antibiotics. A microbial based assay, where enzyme induction was optimized at pH 7.0, temperature 30°C, and whey powder (0.25%) after 4 h of incubatio...

  9. Nucleic acid detection assays

    Energy Technology Data Exchange (ETDEWEB)

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann; Dahlberg, James E.

    2005-04-05

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  10. Microcystin-LR detection based on indirect competitive enzyme-linked immunosorbent assay

    Institute of Scientific and Technical Information of China (English)

    SHENG Jianwu; HE Miao; YU Shaoqing; SHI Hanchang; QIAN Yi

    2007-01-01

    Microcystins (MCs) are a group of closely related toxic cyclic heptapeptides produced by common cyanobacteria,which cause lots of accidents and threatens human health.In this paper,an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established and used to detect microcystin-LR (MC-LR) in drinking and surface waters.The concentration of coating antigen was 5 μg/mL,the dilution of monoclonal antibody MC10E7 was 1:3 000,the dilution of enzyme tracer (goat anti-mouse IgG-peroxidase) was 1:3 000,the standard concentration of MC-LR ranged from 0.001 μg/L to 30 μg/L,and o-phenylenediamine was used as substrate.The assay showed high relativity with high performance liquid chromatography(HPLC) with a correlation coefficient of more than 99%.The relative standard deviation was less than 10%,the detection limit was achieved down to 0.01 μg/L and up to 5.1 μg/L.The quantitative detection range was from 0.03 μg/L to 3 μg/L,and the antibody had high specificity for [4-arginine]microcystins.It performed well in spite of the influence of the real samples.

  11. Metal enhanced fluorescence on nanoporous gold leaf-based assay platform for virus detection.

    Science.gov (United States)

    Ahmed, Syed Rahin; Hossain, Md Ashraf; Park, Jung Youn; Kim, Soo-Hyung; Lee, Dongyun; Suzuki, Tetsuro; Lee, Jaebeom; Park, Enoch Y

    2014-08-15

    In the present study, a rapid, sensitive and quantitative detection of influenza A virus targeting hemagglutinin (HA) was developed using hybrid structure of quantum dots (QDs) and nanoporous gold leaf (NPGL). NPGL film was prepared by dealloying bimetallic film where its surface morphology and roughness were fairly controlled. Anti-influenza A virus HA antibody (ab66189) was bound with NPGL and amine (-NH2) terminated QDs. These biofunctionalized NPGL and QDs formed a complex with the influenza virus A/Beijing/262/95 (H1N1) and the photoluminescence (PL) intensities of QDs were linearly correlated with the concentrations of the virus up to 1ng/mL while no PL was observed in the absence of the virus, or in bovine serum albumin (BSA, 1µg/mL) alone. In addition, it was demonstrated that this assay detected successfully influenza virus A/Yokohama/110/2009 (H3N2) that is isolated from a clinical sample, at a concentration of ca. 50 plaque forming units (PFU)/mL. This detection limit is 2-order more sensitive than a commercially available rapid influenza diagnostic test. From these results, the proposed assay may offer a new strategy to monitor influenza virus for public health. PMID:24607620

  12. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences.

    Science.gov (United States)

    Ågren, Joakim; Hamidjaja, Raditijo A; Hansen, Trine; Ruuls, Robin; Thierry, Simon; Vigre, Håkan; Janse, Ingmar; Sundström, Anders; Segerman, Bo; Koene, Miriam; Löfström, Charlotta; Van Rotterdam, Bart; Derzelle, Sylviane

    2013-11-15

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays. PMID:24005110

  13. An improved respiratory syncytial virus neutralization assay based on the detection of green fluorescent protein expression and automated plaque counting

    Directory of Open Access Journals (Sweden)

    van Remmerden Yvonne

    2012-10-01

    Full Text Available Abstract Background Virus neutralizing antibodies against respiratory syncytial virus (RSV are considered important correlates of protection for vaccine evaluation. The established plaque reduction assay is time consuming, labor intensive and highly variable. Methods Here, a neutralization assay based on a modified RSV strain expressing the green fluorescent protein in combination with automated detection and quantification of plaques is described. Results The fluorescence plaque reduction assay in microplate format requires only two days to complete and is simple and reproducible. A good correlation between visual and automated counting methods to determine RSV neutralizing serum antibody titers was observed. Conclusions The developed virus neutralization assay is suitable for high-throughput testing and can be used for both animal studies and (large scale vaccine clinical trials.

  14. An alternative nested-PCR assay for the detection of Toxoplasma gondii strains based on GRA7 gene sequences.

    Science.gov (United States)

    Costa, Maria Eduarda S M; Oliveira, Claudio Bruno S; Andrade, Joelma Maria de A; Medeiros, Thatiany A; Neto, Valter F Andrade; Lanza, Daniel C F

    2016-07-01

    Toxoplasma gondii is a widespread parasite able to infect virtually any nucleated cells of warm-blooded hosts. In some cases, T. gondii detection using already developed PCR primers can be inefficient in routine laboratory tests, especially to detect atypical strains. Here we report a new nested-PCR protocol able to detect virtually all T. gondii isolates. Analyzing 685 sequences available in GenBank, we determine that GRA7 is one of the most conserved genes of T. gondii genome. Based on an alignment of 85 GRA7 sequences new primer sets that anneal in the highly conserved regions of this gene were designed. The new GRA7 nested-PCR assay providing sensitivity and specificity equal to or greater than the gold standard PCR assays for T. gondii detection, that amplify the B1 sequence or the repetitive 529bp element. PMID:27036222

  15. Microtiterplate phosphate assay based on luminescence quenching of a terbium complex amenable to decay time detection

    International Nuclear Information System (INIS)

    We describe a terbium-ligand complex (TbL) for a microtiterplate assay for phosphate (P) in the 0.3-100 μmol L-1 range based on luminescence quenching. As the pH optimum is at neutral pH (7.4) the probe is quenched by both, primary (H2PO4-) and secondary phosphate (HPO42-). The LOD is 110 nmol L-1. A Stern-Volmer study revealed that quenching is mostly static. Due to the ms-decay time of TbL, the first luminescence lifetime assay for phosphate could also be developed. The lifetime-based calibration plot is linear between 0.5 and 5 μmol L-1 of P. The effect of various surfactants on assay performance and a study on interferents are presented. The probe was successfully applied to determination of P in commercial plant fertilizers and validated against the molybdenum blue test. The probe is the most sensitive lanthanide-based probe for phosphate.

  16. Scalable DNA-Based Magnetic Nanoparticle Agglutination Assay for Bacterial Detection in Patient Samples

    DEFF Research Database (Denmark)

    Mezger, Anja; Fock, Jeppe; Antunes, Paula Soares Martins;

    2015-01-01

    and on the introduction of a magnetic incubation scheme. This enables multiplex detection of Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa at clinically relevant concentrations, demonstrating a factor of 30 improvement in sensitivity compared to previous MNP-based detection schemes. Thanks...

  17. Multiparameter Analysis-Based Electrochemiluminescent Assay for Simultaneous Detection of Multiple Biomarker Proteins on a Single Interface.

    Science.gov (United States)

    Liang, Wenbin; Fan, Chenchen; Zhuo, Ying; Zheng, Yingning; Xiong, Chengyi; Chai, Yaqin; Yuan, Ruo

    2016-05-01

    Electrochemiluminescent (ECL) assay with high sensitivity has been considered as one of the potential strategies to simultaneously detect multiple biomarker proteins. However, it was essential, but full of challenges, to overcome the limitation caused by cross reactions among different ECL indicators. Herein, the multiparameter analysis of ECL-potential signals demonstrated by multivariate linear algebraic equations was first employed in the simultaneous ECL assay to realize multiple detection of biomarker proteins on a single interface. Additionally, owing to the exponential amplification of self-synthesized nucleotide dendrimer by hybridization chain reaction (HCR) and rolling circle amplification (RCA), the developed simultaneous ECL assay showed improved sensitivity and satisfactory accuracy for the detection of N-terminal of the prohormone brain natriuretic peptide (BNPT) and cardiac troponin I (cTnI). Furthermore, a self-designed magnetic beads-based flow system was also employed to improve the feasibility and analysis speed of the simultaneous ECL assay. Importantly, the proposed strategy enabled simultaneous detection of multiple biomarker proteins simply, which could be readily expanded for the multiplexed estimation of various kinds of proteins and nucleotide sequence also, revealing a new avenue for early disease diagnosis with higher efficiency. PMID:27064937

  18. Planar optical waveguide based sandwich assay sensors and processes for the detection of biological targets including early detection of cancers

    Science.gov (United States)

    Martinez, Jennifer S.; Swanson, Basil I.; Shively, John E.; Li, Lin

    2009-06-02

    An assay element is described including recognition ligands adapted for binding to carcinoembryonic antigen (CEA) bound to a film on a single mode planar optical waveguide, the film from the group of a membrane, a polymerized bilayer membrane, and a self-assembled monolayer containing polyethylene glycol or polypropylene glycol groups therein and an assay process for detecting the presence of CEA is described including injecting a possible CEA-containing sample into a sensor cell including the assay element, maintaining the sample within the sensor cell for time sufficient for binding to occur between CEA present within the sample and the recognition ligands, injecting a solution including a reporter ligand into the sensor cell; and, interrogating the sample within the sensor cell with excitation light from the waveguide, the excitation light provided by an evanescent field of the single mode penetrating into the biological target-containing sample to a distance of less than about 200 nanometers from the waveguide thereby exciting any bound reporter ligand within a distance of less than about 200 nanometers from the waveguide and resulting in a detectable signal.

  19. A novel immunochromatographic assay based on a time-resolved chemiluminescence strategy for the multiplexed detection of ractopamine and clenbuterol.

    Science.gov (United States)

    Wang, Wenwen; Su, Xiaoxiao; Ouyang, Hui; Wang, Lin; Fu, Zhifeng

    2016-04-21

    A novel multiplexed immunochromatographic assay (ICA) based on a time-resolved chemiluminescence (CL) strategy was developed for quantitative detection of β-agonists, by utilizing ractopamine (RAC) and clenbuterol (CLE) as the models. Different from conventional multiplexed ICA methods which usually require two or more test lines, this strategy was developed for detection of two β-agonists by using only one test line on the nitrocellulose membrane. In this study, horseradish peroxidase and alkaline phosphatase were used as the signal probes to label RAC antibody and CLE antibody, respectively. The two CL reactions with flash type and glow type kinetics characteristics were triggered simultaneously by injecting the coreactants, then the signals for RAC and CLE detections were recorded at 3 s and 300 s after coreactants injection, respectively. Owing to the utilization of CL detection, this protocol showed ideal sensitivity for quantitation. Under the optimal conditions, the detection limits for RAC and CLE were 0.17 ng mL(-1) and 0.067 ng mL(-1) (S/N = 3), respectively. The whole assay process can be accomplished within 20 min without complicated sample pretreatment. The proposed method was successfully applied for the detection of RAC and CLE in spiked swine urine. It opens up a new pathway for designing a low cost, time-efficiency and multiplexed strategy for rapid screening and field assay. PMID:27026603

  20. A Homogenous Fluorescence Quenching Based Assay for Specific and Sensitive Detection of Influenza Virus A Hemagglutinin Antigen

    Directory of Open Access Journals (Sweden)

    Longyan Chen

    2015-04-01

    Full Text Available Influenza pandemics cause millions of deaths worldwide. Effective surveillance is required to prevent their spread and facilitate the development of appropriate vaccines. In this study, we report the fabrication of a homogenous fluorescence-quenching-based assay for specific and sensitive detection of influenza virus surface antigen hemagglutinins (HAs. The core of the assay is composed of two nanoprobes namely the glycan-conjugated highly luminescent quantum dots (Gly-QDs, and the HA-specific antibody-modified gold nanoparticle (Ab-Au NPs. When exposed to strain-specific HA, a binding event between the HA and the two nanoprobes takes place, resulting in the formation of a sandwich complex which subsequently brings the two nanoprobes closer together. This causes a decrease in QDs fluorescence intensity due to a non-radiative energy transfer from QDs to Au NPs. A resulting correlation between the targets HA concentrations and fluorescence changes can be observed. Furthermore, by utilizing the specific interaction between HA and glycan with sialic acid residues, the assay is able to distinguish HAs originated from viral subtypes H1 (human and H5 (avian. The detection limits in solution are found to be low nanomolar and picomolar level for sensing H1-HA and H5-HA, respectively. Slight increase in assay sensitivity was found in terms of detection limit while exposing the assay in the HA spiked in human sera solution. We believe that the developed assay could serve as a feasible and sensitive diagnostic tool for influenza virus detection and discrimination, with further improvement on the architectures.

  1. Rapid and quantitative detection of 4(5)-methylimidazole in caramel colours: A novel fluorescent-based immunochromatographic assay.

    Science.gov (United States)

    Wu, Xinlan; Huang, Minghui; Yu, Shujuan; Kong, Fansheng

    2016-01-01

    A novel fluorescence-based immunochromatographic assay (ICA) for rapid detecting 4(5)-methylimidazole (4-MI) is presented in this study. In our work, the conjugates of fluorescent microspheres (FMs) and 4-MI monoclonal antibody were used as probe for ICA. Under optimal conditions, a standard curve of ICA-based detection of 4-MI was developed, linear detection ranged from 0.50 to 32.0 mg/L. The cross-reactivities were observed less than 3.93% by detecting 6 selected structural analogues of 4-MI. The recoveries of 4-MI in caramels detection were ranged from 82.85% to 102.31%, with the coefficient of variation (n = 3) below 9.06%. Quantitative comparison of the established fluorescence-based ICA with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) analysis of real caramel colour samples indicated a good correlation among the methods. Therefore, our developed fluorescence-based ICA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of 4-MI in food safety control. PMID:26213047

  2. A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants.

    Science.gov (United States)

    Gao, Siyue; Glasser, Jessica; He, Lili

    2016-01-01

    We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be applied to the detection of various chemical contaminants. Silver nanoparticles (Ag NPs) are synthesized via reduction of silver nitrate by sodium citrate. Then the NPs are aggregated by sodium chloride to form nanoclusters that could be trapped in the pores of the filter membrane. A syringe is connected to the filter holder, with a filter membrane inside. By loading the nanoclusters into the syringe and passing through the membrane, the liquid goes through the membrane but not the nanoclusters, forming a SERS-active membrane. When testing the analyte, the liquid sample is loaded into the syringe and flowed through the Ag NPs coated membrane. The analyte binds and concentrates on the Ag NPs coated membrane. Then the membrane is detached from the filter holder, air dried and measured by a Raman instrument. Here we present the study of the volume effect of Ag NPs and sample on the detection sensitivity as well as the detection of 10 ppb ferbam and 1 ppm ampicillin using the developed assay. PMID:26966831

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

    Directory of Open Access Journals (Sweden)

    Dong Huahuang

    2012-08-01

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

  4. Validation of the performance of a GMO multiplex screening assay based on microarray detection

    NARCIS (Netherlands)

    Leimanis, S.; Hamels, S.; Naze, F.; Mbongolo, G.; Sneyers, M.; Hochegger, R.; Broll, H.; Roth, L.; Dallmann, K.; Micsinai, A.; Dijk, van J.P.; Kok, E.J.

    2008-01-01

    A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray, has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target elements first amplified by PCR, followed by direct

  5. Preclinical detection of porcine circovirus type 2 infection using an ultrasensitive nanoparticle DNA probe-based PCR assay.

    Science.gov (United States)

    Huang, Yong; Zhang, Xiujuan; Du, Qian; Wang, Fengyu; Zhao, Xiaomin; Zhang, Wenlong; Tong, Dewen

    2014-01-01

    Porcine circovirus type 2 (PCV2) has emerged as one of the most important pathogens affecting swine production globally. Preclinical identification of PCV2 is very important for effective prophylaxis of PCV2-associated diseases. In this study, we developed an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR) for PCV2 detection. Magnetic microparticles coated with PCV2 specific DNA probes were used to enrich PCV2 DNA from samples, then gold nanoparticles coated with PCV2 specific oligonucleotides were added to form a sandwich nucleic acid-complex. After the complex was formed, the oligonucleotides were released and characterized by PCR. This assay exhibited about 500-fold more sensitive than conventional PCR, with a detection limit of 2 copies of purified PCV2 genomic DNA and 10 viral copies of PCV2 in serum. The assay has a wide detection range for all of PCV2 genotypes with reliable reproducibility. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1, porcine parvovirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus and classical swine fever virus. The positive detection rate of PCV2 specific UNDP-PCR in 40 preclinical field samples was 27.5%, which appeared greater than that by conventional and real-time PCR and appeared application potency in evaluation of the viral loads levels of preclinical infection samples. The UNDP-PCR assay reported here can reliably rule out false negative results from antibody-based assays, provide a nucleic acid extraction free, specific, ultrasensitive, economic and rapid diagnosis method for preclinical PCV2 infection in field, which may help prevent large-scale outbreaks. PMID:24842840

  6. Preclinical detection of porcine circovirus type 2 infection using an ultrasensitive nanoparticle DNA probe-based PCR assay.

    Directory of Open Access Journals (Sweden)

    Yong Huang

    Full Text Available Porcine circovirus type 2 (PCV2 has emerged as one of the most important pathogens affecting swine production globally. Preclinical identification of PCV2 is very important for effective prophylaxis of PCV2-associated diseases. In this study, we developed an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR for PCV2 detection. Magnetic microparticles coated with PCV2 specific DNA probes were used to enrich PCV2 DNA from samples, then gold nanoparticles coated with PCV2 specific oligonucleotides were added to form a sandwich nucleic acid-complex. After the complex was formed, the oligonucleotides were released and characterized by PCR. This assay exhibited about 500-fold more sensitive than conventional PCR, with a detection limit of 2 copies of purified PCV2 genomic DNA and 10 viral copies of PCV2 in serum. The assay has a wide detection range for all of PCV2 genotypes with reliable reproducibility. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1, porcine parvovirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus and classical swine fever virus. The positive detection rate of PCV2 specific UNDP-PCR in 40 preclinical field samples was 27.5%, which appeared greater than that by conventional and real-time PCR and appeared application potency in evaluation of the viral loads levels of preclinical infection samples. The UNDP-PCR assay reported here can reliably rule out false negative results from antibody-based assays, provide a nucleic acid extraction free, specific, ultrasensitive, economic and rapid diagnosis method for preclinical PCV2 infection in field, which may help prevent large-scale outbreaks.

  7. A qPCR-based Multiplex Assay for Detection of Wuchereria bancrofti, Plasmodium falciparum, and Plasmodium vivax DNA

    OpenAIRE

    Rao, Ramakrishna U.; Huang, Yuefang; Bockarie, Moses J.; Susapu, Melinda; LANEY, SANDRA J.; Weil, Gary J

    2008-01-01

    The purpose of this study was to develop multiplex qPCR assays for simultaneous detection of Wuchereria bancrofti (Wb), Plasmodium falciparum (Pf) and P. vivax (Pv) in mosquitoes. We optimized the assays with purified DNA samples and then used these assays to test DNA samples isolated from Anopheles punctulatus mosquitoes collected in villages in Papua New Guinea where these infections are co-endemic. Singleplex assays detected Wb, Pf, and Pv DNA in 32%, 19% and 15% of the mosquito pools, res...

  8. Preclinical Detection of Porcine Circovirus Type 2 Infection Using an Ultrasensitive Nanoparticle DNA Probe-Based PCR Assay

    OpenAIRE

    Huang, Yong; Zhang, Xiujuan; Du, Qian; Wang, Fengyu; Zhao, Xiaomin; Zhang, Wenlong; Tong, Dewen

    2014-01-01

    Porcine circovirus type 2 (PCV2) has emerged as one of the most important pathogens affecting swine production globally. Preclinical identification of PCV2 is very important for effective prophylaxis of PCV2-associated diseases. In this study, we developed an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR) for PCV2 detection. Magnetic microparticles coated with PCV2 specific DNA probes were used to enrich PCV2 DNA from samples, then gold nanoparticles coated with PCV2 specifi...

  9. Electrochemical Enzyme-Linked Immunosorbent Assay (ELISA) for α-Fetoprotein Based on Glucose Detection with Multienzyme-Nanoparticle Amplification

    OpenAIRE

    Ning Gan; Bo Li; Li Lin; Bo Situ; Han-Kun Zhou; Xiao-Mao Yin; Xiao-Hui Yan; Qin-Lan Liu; Lei Zheng

    2013-01-01

    Since glucose biosensors are one of the most popular and widely used point-of-care testing devices, a novel electrochemical enzyme-linked immunosorbent assay (ELISA) for protein biomarkers has been developed based on a glucose detection strategy. In this study, α-fetoprotein (AFP) was used as the target protein. An electrochemical ELISA system was constructed using anti-AFP antibodies immobilized on microwell plates as the capture antibody (Ab1) and multi-label bioconjugates as signal tracer....

  10. Comparative Analysis of Cultural Isolation and Pcr Based Assay for Detection of Campylobacter Jejuni In Food and Faecal Samples

    OpenAIRE

    Singh, Harkanwaldeep; Rathore, R. S.; Singh, Satparkash; Cheema, Pawanjit Singh

    2011-01-01

    In the present study, the efficacy of polymerase chain reaction (PCR) based on mapA gene of C. jejuni was tested for detection of Campylobacter jejuni in naturally infected as well as spiked faecal and food samples of human and animal origin. Simultaneously, all the samples were subjected to the cultural isolation of organism and biochemical characterization. The positive samples resulted in the amplification of a DNA fragment of size ~589 bp in PCR assay whereas the absence of such amplicon ...

  11. Comparative analysis of cultural isolation and PCR based assay for detection of Campylobacter jejuni in food and faecal samples

    OpenAIRE

    Harkanwaldeep Singh; Rathore, R. S.; Satparkash Singh; Pawanjit Singh Cheema

    2011-01-01

    In the present study, the efficacy of polymerase chain reaction (PCR) based on mapA gene of C. jejuni was tested for detection of Campylobacter jejuni in naturally infected as well as spiked faecal and food samples of human and animal origin. Simultaneously, all the samples were subjected to the cultural isolation of organism and biochemical characterization. The positive samples resulted in the amplification of a DNA fragment of size ~589 bp in PCR assay whereas the absence of such amplicon ...

  12. Colloidal gold probe-based immunochromatographic assay for the rapid detection of lead ions in water samples

    International Nuclear Information System (INIS)

    One-step immunochromatographic assay (ICA) has been developed using colloidal gold-labeled monoclonal antibody probe for the rapid detection of lead ions in water samples. The ICA was based on the theory of competitive reactivity, and the results can be easily judged based on the presence or absence of a red colored test line with visual detection. Under optimal conditions, this method shows high detecting sensitivity with a LOD (limit of detection) of 50 ng/ml. Stability test indicates that the immunochromatographic strips are stable for 8 weeks at room temperature. During practical application, nanometer TiO2 is used to enrich the lead ions in water samples. The ICA is successfully applied in the measurement of lead ion concentrations in local water samples, and the results are highly consistent with that of ICP-MS. Detecting lead ions with ICA can be done within 4 min and is very useful for the rapid onsite testing. - Development of immunochromatographic assay for lead ions facilitates the rapid detection and regulation of environmental pollution.

  13. Alternative Methods for the Detection of Emerging Marine Toxins: Biosensors, Biochemical Assays and Cell-Based Assays

    OpenAIRE

    Laia Reverté; Lucía Soliño; Olga Carnicer; Jorge Diogène; Mònica Campàs

    2014-01-01

    The emergence of marine toxins in water and seafood may have a considerable impact on public health. Although the tendency in Europe is to consolidate, when possible, official reference methods based on instrumental analysis, the development of alternative or complementary methods providing functional or toxicological information may provide advantages in terms of risk identification, but also low cost, simplicity, ease of use and high-throughput analysis. This article gives an overview of th...

  14. 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.

  15. Validation study of a receptor-based lateral flow assay for detection of beta-lactam antibiotics in milk.

    Science.gov (United States)

    Abouzied, Mohamed; Sarzynski, Michael; Walsh, Aaron; Wood, Heather; Mozola, Mark

    2009-01-01

    A validation study designed to meet the requirements of the AOAC Research Institute and the U.S. Food and Drug Administration (FDA), Center for Veterinary Medicine, was conducted for a receptor-based, immunochromatographic method (BetaStar US) for detection of beta-lactam antibiotic residues in raw, commingled bovine milk. The assay was found to detect amoxicillin, ampicillin, cephapirin, cloxacillin, and penicillin G at levels below the FDA tolerance/safe levels but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments. Results of the Part I (internal) and Part II (independent laboratory) dose-response studies using spiked samples were in very close agreement for all five drugs tested, with differences between the Part I and Part II 90/95% sensitivity values ranging from 0 to 1 ppb. The test was able to detect all five drugs at the approximate 90/95% sensitivity levels when present as incurred residues in milk collected from cows that had been treated with the specific drug. A sixth drug, ceftiofur, was found to be undetectable at levels of 1000 control milk samples. The assay was found to be applicable to the testing of frozen raw milk samples. Results of ruggedness experiments established the operating parameter tolerances for the BetaStar US assay. Results of cross-reactivity testing established that the assay detects certain other beta-lactam drugs (dicloxacillin and ticarcillin), but it does not cross-react with any of 30 drugs belonging to other classes. Abnormally high bacterial or somatic cell counts in raw milk produced no interference with the ability of the test to detect beta-lactams at tolerance/safe levels. PMID:19610391

  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. PMID:27377230

  17. Efficacy of a commercial polymerase chain reaction-based assay for detection of Salmonella spp. in animal feeds.

    Science.gov (United States)

    Maciorowski, K G; Pillai, S D; Ricke, S C

    2000-10-01

    Salmonellosis is a cyclic problem in the food industry, to which animal feed has been contributory. Current conventional methods of Salmonella spp. detection require 96 h for detection and confirmation. With modern and just-in-time production schedules, a 96-h hold represents a significant expense in storage and decontamination. The commercially available assay, 'BAX for Screening/Salmonella' (BAX), is based on the principle of the polymerase chain reaction and may represent a significant decrease in assay time. Seven fresh feed formulations, two fresh feed ingredients, seven stored feeds and two stored feed ingredients were artificially contaminated with a primary poultry isolate of Salmonella typhimurium and analysed by conventional and BAX methodology. The results of BAX agreed with conventional plating results for 16 of 18 samples spiked with 1200 cfu 10 g(-1) of feed and 13 of 18 samples spiked with 40 cfu 10 g(-1) of feed. Indigenous Salmonella spp. were detected in five of eight samples of poultry diets by conventional methods. With BAX, Salmonella spp. could not be detected in any of the samples after only 7 h of enrichment but could be detected in two dietary samples after 13 h of enrichment and four dietary samples after 24 h of enrichment. Specific sequences of salmonella DNA that were extracted from poultry diets could be detected with BAX. PMID:11054177

  18. A Luminex-based single DNA fragment amplification assay as a practical tool for detecting and serotyping dengue virus.

    Science.gov (United States)

    Cabral-Castro, Mauro Jorge; Peralta, Regina Helena Saramago; Cavalcanti, Marta Guimarães; Puccioni-Sohler, Marzia; Carvalho, Valéria Lima; da Costa Vasconcelos, Pedro Fernando; Peralta, José Mauro

    2016-10-01

    Dengue is a mosquito-borne viral infection that can evolve from subclinical to severe forms of disease. Early recognition during initial primary and secondary infections correlates with a reduced case-fatality rate in susceptible groups. The aim of this study was to standardize a DNA hybridization assay based on the Luminex technology for detecting and serotyping dengue virus (DENV). Reference DENVs representing the four different serotypes were used as controls to standardize the test. For validation, 16 DENV isolates obtained from a reference laboratory were analyzed in a double-blind manner to validate the test. Sixty blood samples from patients suspected of having dengue fever were used to evaluate the methodology after the validation step, and the results were compared with the reference semi-nested RT-PCR. Additionally, five human samples of each Zika and Chikungunya confirmed patients were used for specificity analysis. The Luminex-based assay correctly identified all 16 DENV isolates. In the evaluation step, the results of the RT-PCR/Luminex assay showed a concordance of 86.7% with those of the semi-nested RT-PCR. None of other virus infection samples was amplified. This is the first description of a hybridization assay that can discriminate the four DENV serotypes using probes against a single DENV sequence. The results indicated that the RT-PCR/Luminex DENV assay designed and evaluated in this study is a valuable additional tool for the early and rapid detection and serotyping of DENV, which could, in the future, be applied to new targets such as the Zika and Chikungunya viruses. PMID:27393681

  19. ELISA-based assay for IP-10 detection from filter paper samples

    DEFF Research Database (Denmark)

    Drabe, Camilla Heldbjerg; Blauenfeldt, Thomas; Ruhwald, Morten

    2014-01-01

    IP-10 is a small pro-inflammatory chemokine secreted primarily from monocytes and fibroblasts. Alterations in IP-10 levels have been associated with inflammatory conditions including viral and bacterial infections, immune dysfunction, and tumor development. IP-10 is increasingly recognized as a b...... biomarker that predicts severity of various diseases and can be used in the immunodiagnostics of Mycobacterium tuberculosis and cytomegalovirus infection. Here, we describe an ELISA-based method to detect IP-10 from dried blood and plasma spot samples....

  20. Nanoparticle-Based Histidine-Rich Protein-2 Assay for the Detection of the Malaria Parasite Plasmodium falciparum.

    Science.gov (United States)

    Castro-Sesquen, Yagahira E; Kim, Chloe; Gilman, Robert H; Sullivan, David J; Searson, Peter C

    2016-08-01

    A nanoparticle-based assay for detection and quantification of Plasmodium falciparum histidine-rich protein 2 (HRP2) in urine and serum is reported. The assay uses magnetic beads conjugated with anti-HRP2 antibody for protein capture and concentration, and antibody-conjugated quantum dots for optical detection. Western blot analysis demonstrated that magnetic beads allow the concentration of HRP2 protein in urine by 20-fold. The concentration effect was achieved because large volume of urine can be incubated with beads, and magnetic separation can be easily performed in minutes to isolate beads containing HRP2 protein. Magnetic beads and quantum dots conjugated to anti-HRP2 antibodies allows the detection of low concentrations of HRP2 protein (0.5 ng/mL), and quantification in the range of 33-2,000 ng/mL corresponding to the range associated with non-severe to severe malaria. This assay can be easily adapted to a noninvasive point-of-care test for classification of severe malaria. PMID:27185769

  1. Detection of Puccinia kuehnii Causing Sugarcane Orange Rust with a Loop-Mediated Isothermal Amplification-Based Assay.

    Science.gov (United States)

    Chandra, Amaresh; Keizerweerd, Amber T; Grisham, Michael P

    2016-03-01

    Puccinia kuehnii is a fungal pathogen that causes orange rust in sugarcane, which is now prevalent in many countries. At the early stage of disease, it is almost indistinguishable from brown rust, which is caused by Puccinia melanocephala. Although several PCR assays are available to detect these diseases, the loop-mediated isothermal amplification (LAMP)-based assay has been reported to be more economical and easier to perform. Under isothermal conditions, DNA is amplified with high specificity and rapidity. Moreover, visual judgment of color change without further post-amplification processing makes the method convenient. The present study was undertaken to detect P. kuehnii genomic DNA using four primers corresponding to a unique DNA sequence of P. kuehnii. The LAMP assay was found to be optimal when 8 mM MgSO4 was used and the reaction was incubated at 63 °C for 90 min. Positive samples showed a color change from orange to green upon SYBR Green I dye addition. Specificity of the LAMP test was checked with DNA of P. melanocephala, which showed no reaction. Sensitivity of the LAMP method was observed to be the same as real-time PCR at 0.1 ng, thus providing a rapid and more affordable option for early disease detection. PMID:26837389

  2. Fluorescent single-stranded DNA-based assay for detecting unchelated Gadolinium(III) ions in aqueous solution.

    Science.gov (United States)

    Edogun, Osafanmwen; Nguyen, Nghia Huu; Halim, Marlin

    2016-06-01

    The main concern pertaining to the safety of Gadolinium(III)-based contrast agents (GBCAs) is the toxicity caused by the unchelated ion, which may be inadvertently present in the solution due most commonly to excess unreacted starting material or dissociation of the complexes. Detecting the aqueous free ion during the synthesis and preparation of GBCA solutions is therefore instrumental in ensuring the safety of the agents. This paper reports the development of a sensitive fluorogenic sensor for aqueous unchelated Gadolinium(III) (Gd(III)). Our design utilizes single-stranded oligodeoxynucleotides with a specific sequence of 44 bases as the targeting moiety. The fluorescence-based assay may be run at ambient pH with very small amounts of samples in 384-well plates. The sensor is able to detect nanomolar concentration of Gd(III), and is relatively unresponsive toward a range of biologically relevant ions and the chelated Gd(III). Although some cross-reactivity with other trivalent lanthanide ions, such as Europium(III) and Terbium(III), is observed, these are not commonly found in biological systems and contrast agents. This convenient and rapid method may be useful in ascertaining a high purity of GBCA solutions. Graphical abstract Fluorescent aptamer-based assay for detecting unchelated Ln(III) ions in aqueous solution. PMID:27071762

  3. Development of colloidal gold-based immunochromatographic assay for rapid detection of Mycoplasma suis in porcine plasma.

    Science.gov (United States)

    Meng, Kai; Sun, Wenjing; Zhao, Peng; Zhang, Limei; Cai, Dongjie; Cheng, Ziqiang; Guo, Huijun; Liu, Jianzhu; Yang, Dubao; Wang, Shujing; Chai, Tongjie

    2014-05-15

    A one-step immunochromatographic assay using gold nanoparticles coated with polyclonal antibody (pAb) against Mycoplasma suis (M. suis) was developed in this study for the detection of M. suis in porcine plasma. The colloidal gold was prepared by the reduction of gold salt with sodium citrate coupled with pAb against M. suis. The pAb was produced by immunizing the BALB/c mice with recombinant MSG1 (rMSG1) protein from M. suis expressed in Escherichia coli. The optimal concentrations of the capture antibody and the coating antibody were 12 μg/ml and 1.5 mg/ml, respectively, and that of the blocking buffer was 1% bovine serum albumin. The lower detection limit of the immunochromatographic assay test was 100 ng/ml with visual detection under optimal conditions of analysis. Classical swine fever virus, porcine reproductive and respiratory syndrome virus, swine pneumonia mycoplasma, swine toxoplasma, and porcine parvovirus were used to evaluate the specificity of the immunochromatographic strips. No cross-reaction of the antibodies with other related swine pathogens was observed. This qualitative test based on the visual evaluation of the results did not require any equipment. The assay time for M. suis detection was less than 10 min, suitable for rapid detection at the grassroots level. The one-step colloidal gold immunochromatographic strips that we developed had high specificity and sensitivity. Therefore, this method would be feasible, convenient, rapid, and effective for detecting M. suis in porcine plasma. PMID:24434494

  4. Cy5 labeled single-stranded DNA-polydopamine nanoparticle conjugate-based FRET assay for reactive oxygen species detection

    Directory of Open Access Journals (Sweden)

    Lina Ma

    2015-03-01

    Full Text Available This work reports on a simple and feasible fluorescence resonance energy transfer (FRET assay for detecting reactive oxygen species (ROS both in solution and living cell using polydopamine nanoparticle (PDA NP as energy acceptor and Cy5 labeled single-stranded DNA (Cy5-ssDNA as energy donor. The Cy5-ssDNA and PDA NPs form self-assembled conjugates (Cy5-ssDNA-PDA NP conjugates via π-stacking interactions. In the presence of ROS, the PDA NP adsorbed Cy5-ssDNAs can be effectively cleaved, resulting in the release of Cy5 molecules into solution and recovery of fluorescence emission of Cy5. In order to obtain ROS solution, the glucose oxidase-catalyzed oxidation reaction of glucose with O2 is employed to generate hydrogen peroxide for Fenton-like reaction. The formation of ROS in Fenton-like reaction can be detected as low as glucose oxidase-catalyzed oxidation of 100 pM glucose by the Cy5-ssDNA-PDA NP conjugate-based FRET assay. The recovery ratio of Cy5 fluorescence intensity is increased linearly with logarithm of glucose concentration from 100 pM to 1 μM, demonstrating that the FRET assay has wide dynamic range. In particular, intracellular ROS has been successfully detected in chemical stimulated HepG-2 cells by the Cy5-ssDNA-PDA NP conjugate-based FRET assay with a fluorescence microscopy, indicating that this approach has great potential to monitor ROS in living cells.

  5. Electrochemical enzyme-linked immunosorbent assay (ELISA) for α-fetoprotein based on glucose detection with multienzyme-nanoparticle amplification.

    Science.gov (United States)

    Liu, Qin-Lan; Yan, Xiao-Hui; Yin, Xiao-Mao; Situ, Bo; Zhou, Han-Kun; Lin, Li; Li, Bo; Gan, Ning; Zheng, Lei

    2013-01-01

    Since glucose biosensors are one of the most popular and widely used point-of-care testing devices, a novel electrochemical enzyme-linked immunosorbent assay (ELISA) for protein biomarkers has been developed based on a glucose detection strategy. In this study, α-fetoprotein (AFP) was used as the target protein. An electrochemical ELISA system was constructed using anti-AFP antibodies immobilized on microwell plates as the capture antibody (Ab1) and multi-label bioconjugates as signal tracer. The bioconjugates were synthesized by attaching glucoamylase and the secondary anti-AFP antibodies (Ab2) to gold nanoparticles (AuNPs). After formation of the sandwich complex, the Ab2-glucoamylase-AuNPs conjugates converted starch into glucose in the presence of AFP. The concentration of AFP can be calculated based on the linear relation between AFP and glucose, the concentration of which can be detected by the glucose biosensor. When the AFP concentration ranged from 0.05 to 100 ng/mL, a linear calibration plot (i (µA) = 13.62033 - 2.86252 logCAFP (ng/mL), r = 0.99886) with a detection limit of 0.02 ng/mL was obtained under optimal conditions. The electrochemical ELISA developed in this work shows acceptable stability and reproducibility, and the assay for AFP spiked in human serum also shows good recovery (97.0%-104%). This new method could be applied for detecting any protein biomarker with the corresponding antibodies. PMID:24129276

  6. New high-performance liquid chromatography assay for glycosyltransferases based on derivatization with anthranilic acid and fluorescence detection.

    Science.gov (United States)

    Anumula, Kalyan Rao

    2012-07-01

    Assays were developed using the unique labeling chemistry of 2-aminobenzoic acid (2AA; anthranilic acid, AA) for measuring activities of both β1-4 galactosyltransferase (GalT-1) and α2-6 sialyltransferase (ST-6) by high-performance liquid chromatography (HPLC) with fluorescence detection (Anumula KR. 2006. Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal Biochem. 350:1-23). N-Acetylglucosamine (GlcNAc) and N-acetyllactosamine were used as acceptors and uridine diphosphate (UDP)-galactose and cytidine monophosphate (CMP)-N-acetylneuraminic acid (NANA) as donors for GalT-1 and ST-6, respectively. Enzymatic products were labeled in situ with AA and were separated from the substrates on TSKgel Amide 80 column using normal-phase conditions. Enzyme units were determined from the peak areas by comparison with the concomitantly derivatized standards Gal-β1-4GlcNAc and NANA-α2-6 Gal-β1-4GlcNAc. Linearity (time and enzyme concentration), precision (intra- and interassay) and reproducibility for the assays were established. The assays were found to be useful in monitoring the enzyme activities during isolation and purification. The assays were highly sensitive and performed equal to or better than the traditional radioactive sugar-based measurements. The assay format can also be used for measuring the activity of other transferases, provided that the carbohydrate acceptors contain a reducing end for labeling. An assay for glycoprotein acceptors was developed using IgG. A short HPLC profiling method was developed for the separation of IgG glycans (biantennary G0, G1, G2, mono- and disialylated), which facilitated the determination of GalT-1 and ST-6 activities in a rapid manner. Furthermore, this profiling method should prove useful for monitoring the changes in IgG glycans in clinical settings. PMID:22459802

  7. Improved detection of Tritrichomonas foetus in bovine diagnostic specimens using a novel probe-based real time PCR assay.

    Science.gov (United States)

    McMillen, Lyle; Lew, Ala E

    2006-11-01

    A Tritrichomonas foetus-specific 5' Taq nuclease assay using a 3' minor groove binder-DNA probe (TaqMan MGB) targeting conserved regions of the internal transcribed spacer-1 (ITS-1) was developed and compared to established diagnostic procedures. Specificity of the assay was evaluated using bovine venereal microflora and a range of related trichomonad species. Assay sensitivity was evaluated with log(10) dilutions of known numbers of cells, and compared to that for microscopy following culture (InPouch TF test kit) and the conventional TFR3-TFR4 PCR assay. The 5' Taq nuclease assay detected a single cell per assay from smegma or mucus which was 2500-fold or 250-fold more sensitive than microscopy following selective culture from smegma or mucus respectively, and 500-fold more sensitive than culture followed by conventional PCR assay. The sensitivity of the conventional PCR assay was comparable to the 5' Taq nuclease assay when testing purified DNA extracted from clinical specimens, whereas the 5' Taq nuclease assay sensitivity improved using crude cell lysates, which were not suitable as template for the conventional PCR assay. Urine was evaluated as a diagnostic specimen providing improved and equivalent levels of T. foetus detection in spiked urine by both microscopy following culture and direct 5' Taq nuclease detection, respectively, compared with smegma and mucus, however inconclusive results were obtained with urine samples from the field study. Diagnostic specimens (n=159) were collected from herds with culture positive animals and of the 14 animals positive by 5' Taq nuclease assay, 3 were confirmed by selective culture/microscopy detection (Fisher's exact test P<0.001). The 5' Taq nuclease assay described here demonstrated superior sensitivity to traditional culture/microscopy and offers advantages over the application of conventional PCR for the detection of T. foetus in clinical samples. PMID:16860481

  8. Minimizing antibody cross-reactivity in multiplex detection of biomarkers in paper-based point-of-care assays

    Science.gov (United States)

    Dias, J. T.; Lama, L.; Gantelius, J.; Andersson-Svahn, H.

    2016-04-01

    Highly multiplexed immunoassays could allow convenient screening of hundreds or thousands of protein biomarkers simultaneously in a clinical sample such as serum or plasma, potentially allowing improved diagnostic accuracy and clinical management of many conditions such as autoimmune disorders, infections, and several cancers. Currently, antibody microarray-based tests are limited in part due to cross reactivity from detection antibody reagents. Here we present a strategy that reduces the cross-reactivity between nanoparticle-bound reporter antibodies through the application of ultrasound energy. By this concept, it was possible to achieve a sensitivity 103-fold (5 pg mL-1) lower than when no ultrasound was applied (50 ng mL-1) for the simultaneous detection of three different antigens. The detection limits and variability achieved with this technique rival those obtained with other types of multiplex sandwich assays.Highly multiplexed immunoassays could allow convenient screening of hundreds or thousands of protein biomarkers simultaneously in a clinical sample such as serum or plasma, potentially allowing improved diagnostic accuracy and clinical management of many conditions such as autoimmune disorders, infections, and several cancers. Currently, antibody microarray-based tests are limited in part due to cross reactivity from detection antibody reagents. Here we present a strategy that reduces the cross-reactivity between nanoparticle-bound reporter antibodies through the application of ultrasound energy. By this concept, it was possible to achieve a sensitivity 103-fold (5 pg mL-1) lower than when no ultrasound was applied (50 ng mL-1) for the simultaneous detection of three different antigens. The detection limits and variability achieved with this technique rival those obtained with other types of multiplex sandwich assays. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr09207h

  9. HLA-B27 detection – comparison of genetic sequence-based method and flow cytometry assay

    OpenAIRE

    Skalska, Urszula; Kozakiewicz, Anna; Maśliński, Włodzimierz; Jurkowska, Monika

    2015-01-01

    Objectives The presence of human leukocyte antigen B27 (HLA-B27) is strongly associated with ankylosing spondylitis. HLA-B27 testing is routinely applied in the diagnosis of this disease. The aim of the present study was to compare two methods of HLA-B27 detection – a genetic sequence-based method and a flow cytometry assay. Material and methods Peripheral blood was obtained from 300 individuals with suspected spondyloarthropathy. Expression of HLA-B27 on the T cell surface was analysed by fl...

  10. An organic indicator functionalized graphene oxide nanocomposite-based colorimetric assay for the detection of sarcosine

    Science.gov (United States)

    Xue, Zhonghua; Yin, Bo; Wang, Hui; Li, Mengqian; Rao, Honghong; Liu, Xiuhui; Zhou, Xinbin; Lu, Xiaoquan

    2016-03-01

    Rapid detection of sarcosine is a key requirement for both diagnosis and treatment of disease. We report here a simple yet sensitive colorimetric nanocomposite platform for rapid detection of sarcosine in alkaline media. The approach exploited the benefits of a rapid color-producing reaction between an organic indicator, 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS), and the analyte of sarcosine species as well as the good catalytic ability of graphene oxide (GO) to the formation of highly colored products due to its good water dispersibility, extremely large surface area and facile surface modification. As a result, a NQS functionalized GO nanocomposite through π-π stacking has been demonstrated to be useful as a highly efficient catalyst system for the selective and sensitive colorimetric determination of sarcosine by providing a nanocomposite-amplified colorimetric response. Meanwhile, the strategy offered excellent selectivity toward sarcosine species against other amino acids as well as a satisfying detection limit of 0.73 μM. More importantly, by using an electrochemical method, a credible sensing mechanism of GO nanocomposite-based colorimetric platform for a special analyte determination can be easily verified and elucidated, which also provides an attractive alternative to conventional characterization strategies.Rapid detection of sarcosine is a key requirement for both diagnosis and treatment of disease. We report here a simple yet sensitive colorimetric nanocomposite platform for rapid detection of sarcosine in alkaline media. The approach exploited the benefits of a rapid color-producing reaction between an organic indicator, 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS), and the analyte of sarcosine species as well as the good catalytic ability of graphene oxide (GO) to the formation of highly colored products due to its good water dispersibility, extremely large surface area and facile surface modification. As a result, a NQS

  11. Development of a novel genetically modified bioluminescent-bacteria-based assay for detection of fluoroquinolones in animal-derived foods.

    Science.gov (United States)

    Cheng, Guyue; Dong, Xiaobing; Wang, Yulian; Peng, Dapeng; Wang, Xu; Hao, Haihong; Xie, Shuyu; Qu, Wei; Liu, Zhenli; Yuan, Zonghui

    2014-12-01

    Fluoroquinolones (FQNs) are broad-spectrum antibacterial agents widely used in animal husbandry and aquaculture. The residues and antimicrobial resistance of such antibiotics are a major public health concern. To realize multianalyte detection of FQN residues, a genetically modified bacterium, Escherichia coli pK12 harboring plasmid pRecAlux3, was constructed in this study to develop a bioluminescent-bacteria-based assay for the detection of FQNs in animal-derived foods. This assay was based on the principle of induction of an SOS response by FQNs via inducing the recA-promoter-fused luciferase reporter gene existing on the plasmid pRecAlux3. E. coli pK12 was able to recognize 11 FQNs: difloxacin, enrofloxacin, ciprofloxacin, sarafloxacin, norfloxacin, danofloxacin, ofloxacin, pefloxacin, lomefloxacin, marbofloxacin, and orbifloxacin. This method could be applied to 11 edible tissues, including milk, fish muscle, and the muscles, livers, and kidneys of cattle, chickens, and pigs, with a very simple and rapid sample extraction procedure using only phosphate-buffered saline. The limits of detection of the FQNs were between 12.5 and 100 μg kg(-1), all of which were lower than the maximum residue limits. Most of the recoveries of the FQNs were in the range from 60 to 120 %, and the interassay coefficients of variation were less than 30 %. This method, confirmed by high-performance liquid chromatography, is reliable and can be used as both a screening test and a semiquantitative assay, when the identity of a single type of FQN is known. PMID:25354889

  12. An electrochemiluminescent assay for high sensitive detection of mercury (II) based on isothermal rolling circular amplification

    Energy Technology Data Exchange (ETDEWEB)

    Zhou Xiaoming; Su Qiang [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631 (China); Xing Da, E-mail: xingda@scnu.edu.cn [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631 (China)

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer Bst DNA polymerase shows specific function on the T-Hg{sup 2+}-T biomimetic structure. Black-Right-Pointing-Pointer T-Hg{sup 2+}-T can be formed in the presence of Hg{sup 2+}, thus induces the RCA reaction. Black-Right-Pointing-Pointer Sub-nanomolar sensitivity and excellent selectivity were achieved for Hg{sup 2+} detection. - Abstract: In this study, we firstly demonstrated that Bst DNA polymerase shows specific recognition and function on the T-Hg{sup 2+}-T biomimetic structure. Based on this, a novel available electrochemiluminescence (ECL) sensor for Hg{sup 2+} has been developed. In this strategy, magnet beads tagged primer was designed to complementary to the region of the circular padlock probe but with two T-T mismatches at the 3 Prime end. The mismatched primers cannot be extended by Bst DNA polymerase in the absence of Hg{sup 2+}. Stable T-Hg{sup 2+}-T can be formed in the presence of Hg{sup 2+}, thus induces the elongation and amplification reaction by DNA polymerase with a rolling circular amplification (RCA) mechanism. Subsequently, the resulted RCA products are hybridized with the tris (bipyridine) ruthenium (TBR)-tagged probes and detected by ECL platform. Current method shows a sub-nanomolar sensitivity and excellent selectivity over a spectrum of interference metal ions.

  13. Organophosphorus pesticides detection using broad-specific single-stranded DNA based fluorescence polarization aptamer assay.

    Science.gov (United States)

    Zhang, Cunzheng; Wang, Li; Tu, Zhui; Sun, Xing; He, Qinghua; Lei, Zhaojing; Xu, Chongxin; Liu, Yuan; Zhang, Xiao; Yang, Jingyi; Liu, Xianjin; Xu, Yang

    2014-05-15

    An approach is developed to detect the organophosphorus pesticides via competitive binding to a recombinant broad-specificity DNA aptamer with a molecular beacon (MB), the binding of the MB to the aptamer results in the activation of a fluorescent signal, which can be measured for pesticide quantification. Aptamers selected via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) were structurally modified and truncated to narrow down the binding region of the target, which indicated that loops of the aptamer contributed different functions for different chemical recognition. Thereafter, a variant fused by two different minimum functional structures, was clarified with broad specificity and increased affinity. Further molecular docking and molecular dynamics simulations was conducted to understand the molecular interaction between DNA structure and chemicals. 3D modeling revealed a hot spot area formed by 3 binding sites, forces including hydrogen bonds and van der Waals interactions appear to play a significant role in enabling and stabilizing the binding of chemicals. Finally, an engineered aptamer based approach for the detection of organophosphorus pesticides was successfully applied in a test using a real sample, the limit of quantification (LOQ) for phorate, profenofos, isocarbophos, and omethoate reached 19.2, 13.4, 17.2, and 23.4 nM (0.005 mg L(-1)), respectively. PMID:24384262

  14. Novel Monoclonal Antibody-Based Immunodiagnostic Assay for Rapid Detection of Deamidated Gluten Residues.

    Science.gov (United States)

    Masiri, Jongkit; Benoit, Lora; Katepalli, Madhu; Meshgi, Mahzad; Cox, David; Nadala, Cesar; Sung, Shao-Lei; Samadpour, Mansour

    2016-05-11

    Gluten derived from wheat and related Triticeae can induce gluten sensitivity as well as celiac disease. Consequently, gluten content in foods labeled "gluten-free" is regulated. Determination of potential contamination in such foods is achieved using immunoassays based on monoclonal antibodies (mAbs) that recognize specific epitopes present in gluten. However, food-processing measures can affect epitope recognition. In particular, preparation of wheat protein isolate through deamidation of glutamine residues significantly limits the ability of commercial gluten testing kits in their ability to recognize gluten. Adding to this concern, evidence suggests that deamidated gluten imparts more pathogenic potential in celiac disease than native gluten. To address the heightened need for antibody-based tools that can recognize deamidated gluten, we have generated a novel mAb, 2B9, and subsequently developed it as a rapid lateral flow immunoassay. Herein, we report the ability of the 2B9-based lateral flow device (LFD) to detect gluten from wheat, barley, and rye and deamidated gluten down to 2 ppm in food as well as its performance in food testing. PMID:27087556

  15. MICROSPHERE-BASED FLOW CYTOMETRY PROTEASE ASSAYS FOR USE IN PROTEASE ACTIVITY DETECTION AND HIGH-THROUGHPUT SCREENING

    OpenAIRE

    Saunders, Matthew J.; Edwards, Bruce S.; Zhu, Jingshu; Sklar, Larry A.; Graves, Steven W.

    2010-01-01

    This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein specific protease of interest and results can be measured in both real time and as end point fluorescence assays on a flow cytometer. End point assays...

  16. Colloidal gold nanoparticle probe-based immunochromatographic assay for the rapid detection of chromium ions in water and serum samples

    International Nuclear Information System (INIS)

    An immunochromatographic assay (ICA) using gold nanoparticles coated with monoclonal antibody (McAb) for the detection of chromium ions (Cr) in water and serum samples was developed, optimized, and validated. Gold nanoparticles coated with affinity- purified monoclonal antibodies against isothiocyanobenzyl-EDTA (iEDTA)-chelated Cr3+ were used as the detecting reagent in this completive immunoassay-based one- step test strip. The ICA was investigated to measure chromium speciation in water samples. Chromium standard samples of 0-80 ng/mL in water were determined by the test strips. The results showed that the visual lowest detection limit (LDL) of the test strip was 50.0 ng/mL. A portable colorimetric lateral flow reader was used for the quantification of Cr. The results indicated that the linear range of the ICA with colorimetric detection was 5-80 ng/mL. The ICA was also validated for the detection of chromium ions in serum samples. The test trips showed high stability in that they could be stored at at 37 C for at least 12 weeks without significant loss of activity. The test strip also showed good selectivity for Cr detection with negligible interference from other heavy metals. Because of its low cost and short testing time (within 5 min), the test strip is especially suitable for on-site large- scale screening of Cr-polluted water samples, biomonitoring of Cr exposure, and many other field applications.

  17. Colloidal gold nanoparticle probe-based immunochromatographic assay for the rapid detection of chromium ions in water and serum samples

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xi; Xiang, Jun-Jian; Tang, Yong; Zhang, Xiao-Li; Fu, Qiang-Qiang; Zou, Jun-Hui; Lin, Yuehe

    2012-09-01

    An immunochromatographic assay (ICA) using gold nanoparticles coated with monoclonal antibody (McAb) for the detection of chromium ions (Cr) in water and serum samples was developed, optimized, and validated. Gold nanoparticles coated with affinity- purified monoclonal antibodies against isothiocyanobenzyl-EDTA (iEDTA)-chelated Cr3+ were used as the detecting reagent in this completive immunoassay-based one- step test strip. The ICA was investigated to measure chromium speciation in water samples. Chromium standard samples of 0-80 ng/mL in water were determined by the test strips. The results showed that the visual lowest detection limit (LDL) of the test strip was 50.0 ng/mL. A portable colorimetric lateral flow reader was used for the quantification of Cr. The results indicated that the linear range of the ICA with colorimetric detection was 5-80 ng/mL. The ICA was also validated for the detection of chromium ions in serum samples. The test trips showed high stability in that they could be stored at at 37 C for at least 12 weeks without significant loss of activity. The test strip also showed good selectivity for Cr detection with negligible interference from other heavy metals. Because of its low cost and short testing time (within 5 min), the test strip is especially suitable for on-site large- scale screening of Cr-polluted water samples, biomonitoring of Cr exposure, and many other field applications.

  18. Integrated microfluidic system for rapid detection of influenza H1N1 virus using a sandwich-based aptamer assay.

    Science.gov (United States)

    Tseng, Yi-Ting; Wang, Chih-Hung; Chang, Chih-Peng; Lee, Gwo-Bin

    2016-08-15

    The rapid spread of influenza-associated H1N1 viruses has caused serious concern in recent years. Therefore, there is an urgent need for the development of automatic, point-of-care devices for rapid diagnosis of the influenza virus. Conventional approaches suffer from several critical issues; notably, they are time-consuming, labor-intensive, and are characterized by relatively low sensitivity. In this work, we present a new approach for fluorescence-based detection of the influenza A H1N1 virus using a sandwich-based aptamer assay that is automatically performed on an integrated microfluidic system. The entire detection process was shortened to 30min using this chip-based system which is much faster than the conventional viral culture method. The limit of detection was significantly improved to 0.032 hemagglutination unit due to the high affinity and high specificity of the H1N1-specific aptamers. The results showed that the two-aptamer microfluidic system had about 10(3) times higher sensitivity than the conventional serological diagnosis. It was demonstrated that the developed microfluidic system may play as a powerful tool in the detection of the H1N1 virus. PMID:27054814

  19. Electrochemical Enzyme-Linked Immunosorbent Assay (ELISA for α-Fetoprotein Based on Glucose Detection with Multienzyme-Nanoparticle Amplification

    Directory of Open Access Journals (Sweden)

    Ning Gan

    2013-10-01

    Full Text Available Since glucose biosensors are one of the most popular and widely used point-of-care testing devices, a novel electrochemical enzyme-linked immunosorbent assay (ELISA for protein biomarkers has been developed based on a glucose detection strategy. In this study, α-fetoprotein (AFP was used as the target protein. An electrochemical ELISA system was constructed using anti-AFP antibodies immobilized on microwell plates as the capture antibody (Ab1 and multi-label bioconjugates as signal tracer. The bioconjugates were synthesized by attaching glucoamylase and the secondary anti-AFP antibodies (Ab2 to gold nanoparticles (AuNPs. After formation of the sandwich complex, the Ab2-glucoamylase-AuNPs conjugates converted starch into glucose in the presence of AFP. The concentration of AFP can be calculated based on the linear relation between AFP and glucose, the concentration of which can be detected by the glucose biosensor. When the AFP concentration ranged from 0.05 to 100 ng/mL, a linear calibration plot (i (µA = 13.62033 − 2.86252 logCAFP (ng/mL, r = 0.99886 with a detection limit of 0.02 ng/mL was obtained under optimal conditions. The electrochemical ELISA developed in this work shows acceptable stability and reproducibility, and the assay for AFP spiked in human serum also shows good recovery (97.0%–104%. This new method could be applied for detecting any protein biomarker with the corresponding antibodies.

  20. Development of a real-time RT-PCR assay based on primer-probe energy transfer for the detection of all serotypes of bluetongue virus

    DEFF Research Database (Denmark)

    Leblanc, N; Rasmussen, Thomas Bruun; Fernandez, J;

    2010-01-01

    A real-time RT-PCR assay based on the primer–probe energy transfer (PriProET) was developed to detect all 24 serotypes of bluetongue virus (BTV). BTV causes serious disease, primarily in sheep, but in other ruminants as well. A distinguishing characteristic of the assay is its tolerance toward...... tests showed no positive results for heterologous pathogens. The assay was tested on clinical samples from BTV 8 outbreaks in Sweden and Denmark in 2008. The lowest detection limit for that serotype, determined with PCR standards, was 57 genome copies. The assay sensitivity for some other serotypes that...... circulate currently in Europe was also determined. BTV 2, 4, 9 and 16 were tested on available cell culture samples and the detection limits were 109, 12, 13 and 24 copies, respectively. This assay provides an important tool for early and rapid detection of a wide range of BTV strains, including emerging...

  1. Toward an international standard for PCR-based detection of Escherichia coli O157 - Part 1. Assay development and multi-center validation

    DEFF Research Database (Denmark)

    Abdulmawjood, A.; Bulte, M.; Cook, N.;

    2003-01-01

    As part of a major European research project, a diagnostic PCR assay, including an internal amplification control, was developed and validated in a collaborative trial for the detection of Escherichia coli O157. The assay is based on amplification of sequences of the rJbE O157 gene. The collabora...

  2. Comparative analysis of cultural isolation and PCR based assay for detection of Campylobacter jejuni in food and faecal samples

    Directory of Open Access Journals (Sweden)

    Harkanwaldeep Singh

    2011-03-01

    Full Text Available In the present study, the efficacy of polymerase chain reaction (PCR based on mapA gene of C. jejuni was tested for detection of Campylobacter jejuni in naturally infected as well as spiked faecal and food samples of human and animal origin. Simultaneously, all the samples were subjected to the cultural isolation of organism and biochemical characterization. The positive samples resulted in the amplification of a DNA fragment of size ~589 bp in PCR assay whereas the absence of such amplicon in DNA extracted from E. coli, Listeria, Salmonella and Staphylococcus confirmed the specificity of the primers. Of randomly collected 143 faecal samples comprising human diarrheic stools (43, cattle diarrheic faeces (48 and poultry faecal swabs (52 only 4, 3 and 8, respectively, could be detected by isolation whereas 6, 3 and 10, respectively, were found positive by PCR. However, among food samples viz. beef (30, milk (35, cheese (30, only one beef sample was detected both by culture as well as PCR. Additionally, PCR was found to be more sensitive for C. jejuni detection in spiked faecal and food samples (96.1% each as relative to culture isolation which could detect the organism in 86.7% and 80% samples, respectively. The results depicted the superior efficacy of PCR for rapid screening of samples owing to its high sensitivity, specificity and automation potential.

  3. Molecular heterogeneity for bovine alpha-mannosidosis: PCR based assays for detection of breed-specific mutations.

    Science.gov (United States)

    Berg, T; Healy, P J; Tollersrud, O K; Nilssen, O

    1997-01-01

    DNA tests, based on the polymerase chain reaction (PCR), were developed for the detection of two breed-specific mutations responsible for the autosomal recessive disorder bovine alpha-mannosidosis. The tests involve separate amplification of two exons of the lysosomal alpha-mannosidase gene followed by restriction enzyme digestion of the amplicons. We demonstrate that one of the mutations, the 662G-->A transition, is responsible for alpha-mannosidosis in Galloway cattle. The other mutation, the 961T-->C transition, is uniquely associated with alpha-mannosidosis in Angus, Murray Grey and Brangus cattle from Australia. The 961T-->C mutation was also detected in Red Angus cattle exported from Canada to Australia as embryos. All 39 animals classified as heterozygotes on the basis of biochemical assays were heterozygous for one of the two mutations. None of 102 animals classified as homozygous-normal on the basis of biochemical assays possessed the mutations. Our results indicate that the two breed-specific mutations may have arisen in Scotland and by the export of animals and germplasm disseminated to America, New Zealand and Australia. PMID:9491457

  4. Niche nanoparticle-based FRET assay for bleomycin detection via DNA scission.

    Science.gov (United States)

    Pei, Haimeng; Zheng, Yiqun; Kong, Rongmei; Xia, Lian; Qu, Fengli

    2016-11-15

    We describe a highly sensitive nanoparticle-based fluorescence resonance energy transfer (FRET) probe developed without using molecular fluorophores as donors and acceptors. The success of this work relies on the strategy that DNA scission was designed to occur to the probe when target presented, which enabled the fluorescence signal "turn-on" of graphene quantum dots (GQDs) and thus quantitative analysis. In particular, amino-modified SiO2 NPs were initially coated by GQDs to form highly emitting SiO2/GQDs, followed by conjunction with DNA functionalized gold nanoparticles (Au NPs-DNA) to form SiO2/GQDs-DNA-Au NPs composite. Owing to the FRET interactions between the GQDs and Au NPs, the fluorescence of GQDs was effectively quenched by Au NPs. When bleomycin (BLM), a model analyte, was mixed with the probe, the fluorescence signal of GQDs would be restored due to the removal of Au NPs from the SiO2/GQDs surface by DNA scission treatment with BLM in the presence of Fe (II). The current FRET probe shows a good linear relationship between the fluorescence intensity and the concentration of BLM in the range from 0.5nM to 1μM with a detection limit of 0.2nM. The probe also shows satisfactory results for the analysis of clinical serum samples. This method provides versatility to the application of GQDs in FRET biosensing and could be potentially extended to other similar systems by replacing the linker between the GQDs and Au NPs. PMID:27155119

  5. Sensitive detection of novel Indian isolate of BTV 21 using ns1 gene based real-time PCR assay

    Directory of Open Access Journals (Sweden)

    Gaya Prasad

    2013-06-01

    Full Text Available Aim: The study was conducted to develop ns1 gene based sensitive real-time RT-PCR assay for diagnosis of India isolates of bluetongue virus (BTV. Materials and Methods: The BTV serotype 21 isolate (KMNO7 was isolated from Andhra Pradesh and propagated in BHK-21 cell line in our laboratory. The Nucleic acid (dsRNA of virus was extracted using Trizol method and cDNA was prepared using a standard protocol. The cDNA was allowed to ns1 gene based group specific PCR to confirm the isolate as BTV. The viral RNA was diluted 10 folds and the detection limit of ns1 gene based RT-PCR was determined. Finally the tenfold diluted viral RNA was subjected to real-time RT-PCR using ns1 gene primer and Taq man probe to standardized the reaction and determine the detection limit. Results: The ns1 gene based group specific PCR showed a single 366bp amplicon in agarose gel electrophoresis confirmed the sample as BTV. The ns1 gene RT-PCR using tenfold diluted viral RNA showed the detection limit of 70.0 fg in 1%agarose gel electrophoresis. The ns1 gene based real time RT-PCR was successfully standardized and the detection limit was found to be 7.0 fg. Conclusion: The ns1 gene based real-time RT-PCR was successfully standardized and it was found to be 10 times more sensitive than conventional RT-PCR. Key words: bluetongue, BTV21, RT-PCR, Real time RT-PCR, ns1 gene [Vet World 2013; 6(8.000: 554-557

  6. Three-in-one enzyme assay based on single molecule detection in femtoliter arrays.

    Science.gov (United States)

    Liebherr, Raphaela B; Hutterer, Albert; Mickert, Matthias J; Vogl, Franziska C; Beutner, Andrea; Lechner, Alfred; Hummel, Helmut; Gorris, Hans H

    2015-09-01

    Large arrays of femtoliter-sized chambers are important tools for single molecule research as well as bioanalytical applications. We have optimized the design and fabrication of two array types consisting of 250 × 250 (62 500) femtoliter chambers either by surface etching of fused silica slides or by polydimethylsiloxane (PDMS) molding. Highly diluted solutions of β-galactosidase were enclosed in such arrays to monitor the fluorogenic reactions of hundreds of individual enzyme molecules in parallel by wide-field fluorescence microscopy. An efficient mechanical sealing procedure was developed to prevent diffusion of the fluorescent reaction product out of the chambers. Different approaches for minimizing non-specific surface adsorption were explored. The signal acquisition was optimized to grant both a large field of view and an efficient signal acquisition from each femtoliter chamber. The optimized femtoliter array has enabled a three-in-one enzyme assay system: First, the concentration of active enzyme can be determined in a digital way by counting fluorescent chambers in the array. Second, the activity of the enzyme bulk solution is given by averaging many individual substrate turnover rates without the need for knowing the exact enzyme concentration. Third-unlike conventional enzyme assays-the distribution of individual substrate turnover rates yields insight into the conformational heterogeneity in an enzyme population. The substrate turnover rates of single β-galactosidase molecules were found to be broadly distributed and independent of the type of femtoliter array. In general, both types of femtoliter arrays are highly sensitive platforms for enzyme analysis at the single molecule level and yield consistent results. Graphical Abstract Isolation and analysis of individual enzyme molecules in large arrays of femtoliter-sized chambers. PMID:26253226

  7. Single-cell multiple gene expression analysis based on single-molecule-detection microarray assay for multi-DNA determination

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Xianwei [School of Life Sciences, Shandong University, Jinan 250100 (China); Zhang, Xiaoli [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin, Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2015-01-07

    Highlights: • A single-molecule-detection (SMD) microarray for 10 samples is fabricated. • The based-SMD microarray assay (SMA) can determine 8 DNAs for each sample. • The limit of detection of SMA is as low as 1.3 × 10{sup −16} mol L{sup −1}. • The SMA can be applied in single-cell multiple gene expression analysis. - Abstract: We report a novel ultra-sensitive and high-selective single-molecule-detection microarray assay (SMA) for multiple DNA determination. In the SMA, a capture DNA (DNAc) microarray consisting of 10 subarrays with 9 spots for each subarray is fabricated on a silanized glass coverslip as the substrate. On the subarrays, the spot-to-spot spacing is 500 μm and each spot has a diameter of ∼300 μm. The sequence of the DNAcs on the 9 spots of a subarray is different, to determine 8 types of target DNAs (DNAts). Thus, 8 types of DNAts are captured to their complementary DNAcs at 8 spots of a subarray, respectively, and then labeled with quantum dots (QDs) attached to 8 types of detection DNAs (DNAds) with different sequences. The ninth spot is used to detect the blank value. In order to determine the same 8 types of DNAts in 10 samples, the 10 DNAc-modified subarrays on the microarray are identical. Fluorescence single-molecule images of the QD-labeled DNAts on each spot of the subarray are acquired using a home-made single-molecule microarray reader. The amounts of the DNAts are quantified by counting the bright dots from the QDs. For a microarray, 8 types of DNAts in 10 samples can be quantified in parallel. The limit of detection of the SMA for DNA determination is as low as 1.3 × 10{sup −16} mol L{sup −1}. The SMA for multi-DNA determination can also be applied in single-cell multiple gene expression analysis through quantification of complementary DNAs (cDNAs) corresponding to multiple messenger RNAs (mRNAs) in single cells. To do so, total RNA in single cells is extracted and reversely transcribed into their cDNAs. Three

  8. Single-cell multiple gene expression analysis based on single-molecule-detection microarray assay for multi-DNA determination

    International Nuclear Information System (INIS)

    Highlights: • A single-molecule-detection (SMD) microarray for 10 samples is fabricated. • The based-SMD microarray assay (SMA) can determine 8 DNAs for each sample. • The limit of detection of SMA is as low as 1.3 × 10−16 mol L−1. • The SMA can be applied in single-cell multiple gene expression analysis. - Abstract: We report a novel ultra-sensitive and high-selective single-molecule-detection microarray assay (SMA) for multiple DNA determination. In the SMA, a capture DNA (DNAc) microarray consisting of 10 subarrays with 9 spots for each subarray is fabricated on a silanized glass coverslip as the substrate. On the subarrays, the spot-to-spot spacing is 500 μm and each spot has a diameter of ∼300 μm. The sequence of the DNAcs on the 9 spots of a subarray is different, to determine 8 types of target DNAs (DNAts). Thus, 8 types of DNAts are captured to their complementary DNAcs at 8 spots of a subarray, respectively, and then labeled with quantum dots (QDs) attached to 8 types of detection DNAs (DNAds) with different sequences. The ninth spot is used to detect the blank value. In order to determine the same 8 types of DNAts in 10 samples, the 10 DNAc-modified subarrays on the microarray are identical. Fluorescence single-molecule images of the QD-labeled DNAts on each spot of the subarray are acquired using a home-made single-molecule microarray reader. The amounts of the DNAts are quantified by counting the bright dots from the QDs. For a microarray, 8 types of DNAts in 10 samples can be quantified in parallel. The limit of detection of the SMA for DNA determination is as low as 1.3 × 10−16 mol L−1. The SMA for multi-DNA determination can also be applied in single-cell multiple gene expression analysis through quantification of complementary DNAs (cDNAs) corresponding to multiple messenger RNAs (mRNAs) in single cells. To do so, total RNA in single cells is extracted and reversely transcribed into their cDNAs. Three types of c

  9. Modified Indirect Porcine Circovirus (PCV) Type 2-Based and Recombinant Capsid Protein (ORF2)-Based Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to PCV

    OpenAIRE

    Nawagitgul, Porntippa; Harms, Perry A.; Morozov, Igor; Thacker, Brad J.; Sorden, Steven D.; Lekcharoensuk, Chalermpol; Paul, Prem S.

    2002-01-01

    Postweaning multisystemic wasting syndrome of swine associated with porcine circovirus (PCV) is a recently reported and economically important disease. Simple and reliable diagnostic methods are needed for detecting antibodies to PCV type 2 (PCV2) for monitoring of PCV infection. Here, we report the development of two modified indirect enzyme-linked immunosorbent assays (ELISAs): a PCV2 ELISA based on cell-culture-propagated PCV2 and an ORF2 ELISA based on recombinant major capsid protein. PC...

  10. Improvement of the Mutation-Discrimination Threshold for Rare Point Mutations by a Separation-Free Ligase Detection Reaction Assay Based on Fluorescence Resonance Energy Transfer.

    Science.gov (United States)

    Hagihara, Kenta; Tsukagoshi, Kazuhiko; Nakajima, Chinami; Esaki, Shinsuke; Hashimoto, Masahiko

    2016-01-01

    We previously developed a separation-free ligase detection reaction assay based on fluorescence resonance energy transfer from a donor quantum dot to an acceptor fluorescent dye. This assay could successfully detect one cancer mutation among 10 wild-type templates. In the current study, the mutation-discrimination threshold was improved by one order of magnitude by replacing the original acceptor dye (Alexa Fluor 647) with another fluorescent dye (Cyanine 5) that was spectrally similar but more fluorescent. PMID:26960620

  11. A recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) for lungworm detection in seals

    OpenAIRE

    Ulrich, Sophia Arlena; Lehnert, Kristina; Siebert, Ursula; Strube, Christina

    2015-01-01

    Background Pinnipeds are frequently infected by the lungworms Otostrongylus circumlitus and Parafilaroides gymnurus (Metastrongyloidea). Infections are frequently associated with secondary bacterial bronchopneumonia and are often lethal. To date, a reliable lungworm diagnosis in individual seals is only possible during necropsy as examination of faeces collected from resting places does not allow assignment to individuals. Therefore, a diagnostic tool for lungworm detection in living seals is...

  12. Antibody-based biosensor assays for the detection of zilpaterol and markers for prostate cancer

    OpenAIRE

    Dillon, Mary

    2008-01-01

    The research presented in this thesis describes the production and application of antibodies against the drug of abuse zilpaterol, and the application of antibodies against prostate-specific antigen (PSA), a cancer marker. Polyclonal antibodies were used in the development of immunoassays in a competitive ELISA format and on the Biacore (a surface plasmon resonance-based optical biosensor capable of monitoring biomolecular interactions in 'real-time'). A zilpaterol-HSA conjugate was u...

  13. A highly sensitive dual-readout assay based on gold nanoclusters for folic acid detection

    International Nuclear Information System (INIS)

    We describe a sensitive fluorometric and colorimetric dual-readout probe for folic acid (FA). It is based on the use of the gold nanoclusters (AuNCs) and cysteamine–modified gold nanoparticles (cyst-AuNPs). The bovine serum albumin stabilized AuNCs exhibit strong fluorescence emission at 652 nm. Upon addition of cyst-AuNPs, the fluorescence intensity of the AuNCs showed dramatic decrease due to the surface plasmon enhanced energy transfer process. This is due to an FA-induced aggregation of the cyst-AuNPs which shifts the absorption peaks from 530 to 670 nm. Thus, the surface plasmon enhanced energy transfer between cyst-AuNPs and AuNCs is weakened and the fluorescence intensity of AuNCs is recovered. The fluorescence intensity of the AuNCs/cyst-AuNPs system is proportional to the concentration of FA in the range from 0.11 to 2.27 μmol L−1. The dual-readout probe reported here was successfully applied to the determination of FA in spiked serum samples and folic acid tablets. (author)

  14. The development of a GeXP-based multiplex reverse transcription-PCR assay for simultaneous detection of sixteen human respiratory virus types/subtypes

    Directory of Open Access Journals (Sweden)

    Li Jin

    2012-08-01

    Full Text Available Abstract Background Existing standard non-molecular diagnostic methods such as viral culture and immunofluorescent (DFA are time-consuming, labor intensive or limited sensitivity. Several multiplex molecular assays are costly. Therefore, there is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens. Methods A GeXP-based multiplex RT-PCR assay (GeXP assay was developed to detect simultaneously sixteen different respiratory virus types/subtypes. Seventeen sets of chimeric primers were used to initiate the RT-PCR, and one pair of universal primers was used for the subsequent cycles of the RT-PCR. The specificity of the GeXP assay was examined with positive controls for each virus type/subtype. The sensitivity was evaluated by performing the assay on serial ten-fold dilutions of in vitro-transcribed RNA of all RNA viruses and the plasmids containing the Adv and HBoV target sequence. GeXP assay was further evaluated using 126 clinical specimens and compared with Luminex xTAG RVP Fast assay. Results The GeXP assay achieved a sensitivity of 20–200 copies for a single virus and 1000 copies when all of the 16 pre-mixed viral targets were present. Analyses of 126 clinical specimens using the GeXP assay demonstrated that GeXP assay and the RVP Fast assay were in complete agreement for 109/126 (88.51% of the specimens. GeXP assay was more sensitive than the RVP Fast assay for the detection of HRV and PIV3, and slightly less sensitive for the detection of HMPV, Adv, RSVB and HBoV. The whole process of the GeXP assay for the detection of 12 samples was completed within 2.5 hours. Conclusions In conclusion, the GeXP assay is a rapid, cost-effective, sensitive, specific and high throughput method for the detection of respiratory virus infections.

  15. A simple aptamer-based fluorescent assay for the detection of Aflatoxin B1 in infant rice cereal.

    Science.gov (United States)

    Chen, Lu; Wen, Fang; Li, Ming; Guo, Xiaodong; Li, Songli; Zheng, Nan; Wang, Jiaqi

    2017-01-15

    A fluorescent assay for the rapid, sensitive and specific detection of Aflatoxin B1 (AFB1) was developed in this study. Initially, a DNA/DNA duplex was formed between a fluorescein-labeled AFB1 aptamer and its partially complementary DNA strand containing a quencher moiety, resulting in fluorescence quenching due to the close proximity of fluorophore and quencher. Upon the addition of AFB1, an aptamer/AFB1 complex was generated to release the quencher-modified DNA strand, thus recovered the fluorescence of fluorescein and enabled quantitative detection for AFB1 by monitoring fluorescence enhancement. Under optimized conditions, this assay exhibited a linear response to AFB1 in the range of 5-100ng/mL with a detection limit down to 1.6ng/mL. Trials of this assay in infant rice cereal with satisfactory recovery in the range of 93.0%-106.8%, demonstrate that the new assay could be a potential sensing platform for AFB1 determination in food. PMID:27542489

  16. Sample preincubation strategy for sensitive and quantitative detection of clenbuterol in swine urine using a fluorescent microsphere-based immunochromatographic assay.

    Science.gov (United States)

    Deng, Sheng L; Shan, Shan; Xu, Chao L; Liu, Dao F; Xiong, Yong H; Wei, Hua; Lai, Wei H

    2014-11-01

    We describe an ultrasensitive and quantitative immunochromatographic assay to determine the amount of clenbuterol (CLB) in swine urine. In this study, fluorescein isothiocyanate polystyrene fluorescent microspheres were used as probes. A sample preincubation strategy was introduced to this immunochromatographic assay. Results showed that the strategy evidently improved the sensitivity and accuracy of lateral flow assay. The method was completed in 20 min, and a half-maximal inhibitory concentration of 0.13 μg liter(-1) was obtained. The limit of detection of the proposed method to determine CLB in swine urine was 0.01 μg liter(-1), which was lower than the limit of detection of immunochromatographic assays without preincubation. Intra- and interday recoveries of spiked swine urine ranged from 85.0 to 107.5%. The relative standard deviation values of the preincubated test strip ranged from 2.7 to 12.5%. Analysis of the CLB in swine urine samples showed that the result obtained from the lateral flow assay is consistent with that obtained from a commercial enzyme-linked immunosorbent assay kit. Our results suggest that the developed fluorescent microsphere-based immunochromatographic assay may be useful as a rapid screening method to detect CLB quantitatively. PMID:25364937

  17. A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus.

    Science.gov (United States)

    Su, Mingjun; Li, Chunqiu; Guo, Donghua; Wei, Shan; Wang, Xinyu; Geng, Yufei; Yao, Shuang; Gao, Jing; Wang, Enyu; Zhao, Xiwen; Wang, Zhihui; Wang, Jianfa; Wu, Rui; Feng, Li; Sun, Dongbo

    2016-05-01

    Recently, porcine deltacoronavirus (PDCoV) has been proven to be associated with enteric disease in piglets. Diagnostic tools for serological surveys of PDCoV remain in the developmental stage when compared with those for other porcine coronaviruses. In our study, an indirect enzyme-linked immunosorbent assay (ELISA) (rPDCoV-N-ELISA) was developed to detect antibodies against PDCoV using a histidine-tagged recombinant nucleocapsid (N) protein as an antigen. The rPDCoV-N-ELISA did not cross-react with antisera against porcine epidemic diarrhea virus, swine transmissible gastroenteritis virus, porcine group A rotavirus, classical swine fever virus, porcine circovirus-2, porcine pseudorabies virus, and porcine reproductive and respiratory syndrome virus; the receiver operating characteristic (ROC) curve analysis revealed 100% sensitivity and 90.4% specificity of the rPDCoV-N-ELISA based on samples of known status (n=62). Analyses of field samples (n=319) using the rPDCoV-N-ELISA indicated that 11.59% of samples were positive for antibodies against PDCoV. These data demonstrated that the rPDCoV-N-ELISA can be used for epidemiological investigations of PDCoV and that PDCoV had a low serum prevalence in pig population in Heilongjiang province, northeast China. PMID:26668175

  18. 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.

  19. Simultaneous Detection of Seven Enteric Viruses Associated with Acute Gastroenteritis by a Multiplexed Luminex-Based Assay

    OpenAIRE

    Liu, Yan; Xu, Zi-qian; Zhang, Qing; Jin, Miao; Yu, Jie-mei; Li, Jin-song; Liu, Na; Cui, Shu-Xian; Kong, Xiang-Yu; Wang, Hong; Li, Hui-Ying; Cheng, Wei-Xia; Ma, Xue-Jun; Duan, Zhao-jun

    2012-01-01

    Rapid and broad diagnostic methods are needed for the identification of viral agents of gastroenteritis. In this study, we used Luminex xMAP technology to develop a multiplexed assay for the simultaneous identification of major enteric viral pathogens, including rotavirus A (RVA), noroviruses (NoVs) (including genogroups GI and GII), sapoviruses (SaV), human astrovirus (HAstV), enteric adenoviruses (EAds), and human bocavirus 2 (HBoV2). The analytical sensitivity allowed detection of 103 (EAd...

  20. Matrix effects on a cell-based assay used for the detection of paralytic shellfish toxins in bivalve shellfish samples.

    Science.gov (United States)

    Aballay-Gonzalez, Ambbar; Ulloa, Viviana; Rivera, Alejandra; Hernández, Víctor; Silva, Macarena; Caprile, Teresa; Delgado-Rivera, Lorena; Astuya, Allisson

    2016-05-01

    Detecting marine biotoxins such as paralytic shellfish toxins (PSTs) is essential to ensuring the safety of seafood. The mouse bioassay is the internationally accepted method for monitoring PSTs, but technical and ethical issues have led to a search for new detection methods. The mouse neuroblastoma cell-based assay (Neuro-2a CBA) using ouabain and veratridine (O/V) has proven useful for the detection of PSTs. However, CBAs are sensitive to shellfish-associated matrix interferences. As the extraction method highly influences matrix interferences, this study compared three extraction protocols: Association of Official Analytical Chemists (AOAC) 2005.06, AOAC 2011.02 and an alternative liquid-liquid method. These methods were used to assess the matrix effect of extracts from four commercially important bivalve species (Chilean mussel, Magellan mussel, clam and Pacific oyster) in Neuro-2a CBA. Extracts from all three protocols caused a toxic effect in Neuro-2a cells (without O/V) when tested at a concentration of 25 mg of tissue-equivalent (TE) ml(-1). The greatest toxicity was obtained through the AOAC 2011.02 protocol, especially for the Chilean mussel and Pacific oyster extracts. Similar toxicity levels (less than 15%) were observed in all extracts at 3.1 mg TE ml(-1). When assessed in Neuro-2a CBA, AOAC 2005.06 extracts presented the lowest matrix interferences, while the highest interferences were observed for AOAC 2011.02 in Magellan mussel and clam extracts. Finally, the AOAC 2005.06 and alternative protocols were compared using Chilean mussel samples fortified with 40 and 80 µg STX per 100 g meat. The AOAC 2005.06 method demonstrated better results. In conclusion, the AOAC 2005.06 extracts exhibited the fewest interferences in the Neuro-2a CBA. Therefore, this extraction method should be considered for the implementation of Neuro-2a CBA as a high-throughput screening methodology for PST detection. PMID:27002718

  1. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    International Nuclear Information System (INIS)

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples

  2. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    Energy Technology Data Exchange (ETDEWEB)

    Huang, S-H; Tsai, M-H; Lin, C-W [Department of Biotechnology, College of Health Science, Asia University, Wufeng, Taichung, Taiwan (China); Yang, T-C; Chuang, P-H [Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan (China); Tsai, I-S; Lu, H-C [Nanotechnology Research Center, Feng Chia University, Taichung, Taiwan (China); Wan Lei; Lin, Y-J [Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung, Taiwan (China); Lai, C-H [Department of Microbiology and Immunology, China Medical University, Taichung, Taiwan (China)], E-mail: cwlin@mail.cmu.edu.tw

    2008-10-08

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

  3. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    Science.gov (United States)

    Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

    2008-10-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

  4. A versatile method to design stem-loop primer-based quantitative PCR assays for detecting small regulatory RNA molecules.

    Directory of Open Access Journals (Sweden)

    Zsolt Czimmerer

    Full Text Available Short regulatory RNA-s have been identified as key regulators of gene expression in eukaryotes. They have been involved in the regulation of both physiological and pathological processes such as embryonal development, immunoregulation and cancer. One of their relevant characteristics is their high stability, which makes them excellent candidates for use as biomarkers. Their number is constantly increasing as next generation sequencing methods reveal more and more details of their synthesis. These novel findings aim for new detection methods for the individual short regulatory RNA-s in order to be able to confirm the primary data and characterize newly identified subtypes in different biological conditions. We have developed a flexible method to design RT-qPCR assays that are very sensitive and robust. The newly designed assays were tested extensively in samples from plant, mouse and even human formalin fixed paraffin embedded tissues. Moreover, we have shown that these assays are able to quantify endogenously generated shRNA molecules. The assay design method is freely available for anyone who wishes to use a robust and flexible system for the quantitative analysis of matured regulatory RNA-s.

  5. A TaqMan-based real-time PCR assay for porcine parvovirus 4 detection and quantification in reproductive tissues of sows

    Science.gov (United States)

    Porcine parvovirus 4 (PPV4) is a DNA virus, and a member of the Parvoviridae family within the Bocavirus genera. It was recently detected in swine, but its epidemiology and pathology remain unclear. A TaqMan-based real-time polymerase chain reaction (qPCR) assay targeting a conserved region of the O...

  6. A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus

    OpenAIRE

    Su, Mingjun; Li, Chunqiu; Guo, Donghua; Wei, Shan; Wang, Xinyu; GENG, Yufei; Yao, Shuang; Gao, Jing; WANG, Enyu; ZHAO, Xiwen; Wang, Zhihui; Wang, Jianfa; Wu, Rui; Feng, Li; SUN, Dongbo

    2015-01-01

    Recently, porcine deltacoronavirus (PDCoV) has been proven to be associated with enteric disease in piglets. Diagnostic tools for serological surveys of PDCoV remain in the developmental stage when compared with those for other porcine coronaviruses. In our study, an indirect enzyme-linked immunosorbent assay (ELISA) (rPDCoV-N-ELISA) was developed to detect antibodies against PDCoV using a histidine-tagged recombinant nucleocapsid (N) protein as an antigen. The rPDCoV-N-ELISA did not cross-re...

  7. Development of a TaqMan Probe-Based Insulated Isothermal Polymerase Chain Reaction (iiPCR) Assay for Detection of Fusarium oxysporum f. sp. cubense Race 4.

    Science.gov (United States)

    Lin, Ying-Hong; Lin, Yi-Jia; Chang, Tsai-De; Hong, Li-Ling; Chen, Tzu-Yu; Chang, Pi-Fang Linda

    2016-01-01

    This study developed a novel and inexpensive detection method based on a TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) method for the rapid detection of Panama disease caused by Fusarium oxysporum f. sp. cubense (Foc) race 4, which is currently among the most serious fungal vascular diseases worldwide. By using the portable POCKIT™ device with the novel primer set iiFoc-1/iiFoc-2, the Foc race 4 iiPCR assay (including DNA amplification and signal monitoring) could be completed within one hour. The developed Foc race 4 iiPCR assay is thus a user-friendly and efficient platform designed specifically for the detection of Foc race 4. The detection limit of this optimized Foc iiPCR system was estimated to be 1 copy of the target standard DNA as well as 1 fg of the Foc genomic DNA. This approach can serve as a rapid detection method for in planta detection of Foc race 4 in field-infected banana. It was concluded that this molecular detection procedure based on iiPCR has good potential for use as an efficient detection method. PMID:27448242

  8. Development of a TaqMan Probe-Based Insulated Isothermal Polymerase Chain Reaction (iiPCR) Assay for Detection of Fusarium oxysporum f. sp. cubense Race 4

    Science.gov (United States)

    Lin, Yi-Jia; Chang, Tsai-De; Hong, Li-Ling; Chen, Tzu-Yu; Chang, Pi-Fang Linda

    2016-01-01

    This study developed a novel and inexpensive detection method based on a TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) method for the rapid detection of Panama disease caused by Fusarium oxysporum f. sp. cubense (Foc) race 4, which is currently among the most serious fungal vascular diseases worldwide. By using the portable POCKIT™ device with the novel primer set iiFoc-1/iiFoc-2, the Foc race 4 iiPCR assay (including DNA amplification and signal monitoring) could be completed within one hour. The developed Foc race 4 iiPCR assay is thus a user-friendly and efficient platform designed specifically for the detection of Foc race 4. The detection limit of this optimized Foc iiPCR system was estimated to be 1 copy of the target standard DNA as well as 1 fg of the Foc genomic DNA. This approach can serve as a rapid detection method for in planta detection of Foc race 4 in field-infected banana. It was concluded that this molecular detection procedure based on iiPCR has good potential for use as an efficient detection method. PMID:27448242

  9. A PCR-based diagnostic assay for the detection of Roseovarius crassostreae in Crassostrea virginica affected by juvenile oyster disease (JOD)

    Science.gov (United States)

    Maloy, A.P.; Barber, B.J.; Boettcher, K.J.

    2005-01-01

    We have developed a PCR-assay for the diagnosis of juvenile oyster disease (JOD) based on the detection of Roseovarius crassostreae directly from affected oysters. Species-specific primers are used to amplify the 16S-23S rDNA internal transcribed spacer (ITS) of R. crassostreae, and confirmation of product identity is accomplished by restriction enzyme analysis. No false positives were obtained with either closely related bacterial species or from other DNAs present in oyster samples. The assay has the potential to detect as few as 10 cells of R. crassostreae per oyster when samples are taken from the inner valve surfaces of the animal. Inclusion of material from soft body surfaces is not necessary, and may reduce sensitivity approximately 10-fold. In a JOD-affected population, a positive PCR result was obtained from all oysters from which these bacteria were subsequently cultured. The assay also detected the presence of R. crassostreae in 2 oysters from which no R. crassostreae isolates were recovered. No R. crassostreae was detected by either PCR or bacteriology in oysters from a population that was not exhibiting JOD-signs. This assay is expected to advance regional disease management efforts and provide valuable insights into the disease process and epizootiology of JOD. ?? Inter-Research 2005.

  10. A novel bead-based assay to detect specific antibody responses against Toxoplasma gondii and Trichinella spiralis simultaneously in sera of experimentally infected swine

    Directory of Open Access Journals (Sweden)

    Bokken Gertie CAM

    2012-03-01

    Full Text Available Abstract Background A novel, bead-based flow cytometric assay was developed for simultaneous determination of antibody responses against Toxoplasma gondii and Trichinella spiralis in pig serum. This high throughput screening assay could be an alternative for well known indirect tests like ELISA. One of the advantages of a bead-based assay over ELISA is the possibility to determine multiple specific antibody responses per single sample run facilitated by a series of antigens coupled to identifiable bead-levels. Furthermore, inclusion of a non-coupled bead-level in the same run facilitates the determination of, and correction for non-specific binding. The performance of this bead-based assay was compared to one T. spiralis and three T. gondii ELISAs. For this purpose, sera from T. gondii and T. spiralis experimentally infected pigs were used. With the experimental infection status as gold standard, the area under the curve, Youden Index, sensitivity and specificity were determined through receiver operator curve analysis. Marginal homogeneity and inter-rater agreement between bead-based assay and ELISAs were evaluated using McNemar's Test and Cohen's kappa, respectively. Results Results indicated that the areas under the curve of the bead-based assay were 0.911 and 0.885 for T. gondii and T. spiralis, respectively, while that of the T. gondii ELISAs ranged between 0.837 and 0.930 and the T. spiralis ELISA was 0.879. Bead-based T. gondii assay had a sensitivity of 86% and specificity of 96%, while the ELISAs ranged between 64-84% and 93-99%, respectively. The bead-based T. spiralis assay had a sensitivity of 68% and specificity of 100% while the ELISA scored 72% and 95%, respectively. Marginal homogeneity was found between the T. gondii bead-based test and one of the T. gondii ELISAs. Moreover, in this test combination and between T. spiralis bead-based assay and respective ELISA, an excellent inter-rater agreement was found. When results of

  11. Detection of Salmonella spp, Salmonella Enteritidis and Typhimurium in naturally infected broiler chickens by a multiplex PCR-based assay

    Science.gov (United States)

    Paião, F.G.; Arisitides, L.G.A.; Murate, L.S.; Vilas-Bôas, G.T.; Vilas-Boas, L.A.; Shimokomaki, M.

    2013-01-01

    The presence of Salmonella in the intestinal tract, on the chickens skin and among their feathers, may cause carcasses contamination during slaughtering and processing and possibly it is responsible by the introduction of this microorganism in the slaughterhouses. A rapid method to identify and monitor Salmonella and their sorovars in farm is becoming necessary. A pre-enriched multiplex polymerase chain reaction (m-PCR) assay employing specific primers was developed and used to detect Salmonella at the genus level and to identify the Salmonella enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterica serovar Typhimurium (S. Typhimurium) in broiler chicken swab samples. The method was validated by testing DNA extract from 90 fresh culture cloacal swab samples from poultry chicken cultured in phosphate buffer peptone water at 37 °C for 18 h. The final results showed the presence of Salmonella spp. in 25% of samples, S. Enteritidis was present in 12% of the Salmonella-positive samples and S. Typhimurium in 3% of the samples. The m-PCR assay developed in this study is a specific and rapid alternative method for the identification of Salmonella spp. and allowed the observation of specific serovar contamination in the field conditions within the locations where these chickens are typically raised. PMID:24159281

  12. Development of bioreporter assays for the detection of bioavailability of long-chain alkanes based on the marine bacterium Alcanivorax borkumensis strain SK2.

    Science.gov (United States)

    Kumari, Rekha; Tecon, Robin; Beggah, Siham; Rutler, Rebecca; Arey, J Samuel; van der Meer, Jan Roelof

    2011-10-01

    Long-chain alkanes are a major component of crude oil and therefore potentially good indicators of hydrocarbon spills. Here we present a set of new bacterial bioreporters and assays that allow to detect long-chain alkanes. These reporters are based on the regulatory protein AlkS and the alkB1 promoter from Alcanivorax borkumensis SK2, a widespread alkane degrader in marine habitats. Escherichia coli cells with the reporter construct reacted strongly to octane in short-term (6 h) aqueous suspension assays but very slightly only to tetradecane, in line with what is expected from its low water solubility. In contrast, long-term assays (up to 5 days) with A. borkumensis bioreporters showed strong induction with tetradecane and crude oil. Gel-immobilized A. borkumensis reporter cells were used to demonstrate tetradecane and crude oil bioavailability at a distance from a source. Alcanivorax borkumensis bioreporters induced fivefold more rapid and more strongly when allowed physical contact with the oil phase in standing flask assays, suggesting a major contribution of adhered cells to the overall reporter signal. Using the flask assays we further demonstrated the effect of oleophilic nutrients and biosurfactants on oil availability and degradation by A. borkumensis. The fluorescence signal from flask assays could easily be captured with a normal digital camera, making such tests feasible to be carried out on, e.g. marine oil responder vessels in case of oil accidents. PMID:21895911

  13. Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathryn L.; Marks, James D.; Varnum, Susan M.

    2012-11-15

    Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A-G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the present study, we have developed an ELISA-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotype A, B, C, D, E and F. With engineered high-affinity antibodies, the assays have sensitivities in buffer of 8 fM (1.2 pg/mL) for serotypes A and B, and 32 fM (4.9 pg/mL) for serotypes C, D, E, and F. Using clinical and environmental samples (serum and milk), the microarray is capable of detecting BoNT/A-F to the same levels as in standard buffer. Cross reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical or environmental samples.

  14. Simultaneous detection of eight immunosuppressive chicken viruses using a GeXP analyser-based multiplex PCR assay

    OpenAIRE

    Zeng, Tingting; Xie, Zhixun; Xie, Liji; Deng, Xianwen; Xie, Zhiqin; Luo, Sisi; Huang, Li; Huang, Jiaoling

    2015-01-01

    Background Immunosuppressive viruses are frequently found as co-infections in the chicken industry, potentially causing serious economic losses. Because traditional molecular biology methods have limited detection ability, a rapid, high-throughput method for the differential diagnosis of these viruses is needed. The objective of this study is to develop a GenomeLab Gene Expression Profiler Analyser-based multiplex PCR method (GeXP-multiplex PCR) for simultaneous detection of eight immunosuppr...

  15. Development of a Solid-Phase Receptor-Based Assay for the Detection of Cyclic Imines Using a Microsphere-Flow Cytometry System

    OpenAIRE

    Laura P. Rodríguez; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Louzao, M. Carmen; Taylor, Palmer; Talley, Todd; Botana, Luis M.

    2013-01-01

    Biologically active macrocycles containing a cyclic imine were isolated for the first time from aquaculture sites in Nova Scotia, Canada, during the 1990s. These compounds display a “fast-acting” toxicity in the traditional mouse bioassay for lipophilic marine toxins. Our work aimed at developing receptor-based detection method for spirolides using a microsphere/flow cytometry Luminex system. For the assay two alternatives were considered as binding proteins, the Torpedo marmorata nicotinic a...

  16. Comparison of monoclonal antibody-based sandwich enzyme-linked immunosorbent assay and virus isolation for detection of peste des petits ruminants virus in goat tissues and secretions.

    OpenAIRE

    Saliki, J T; House, J A; MEBUS, C.A.; Dubovi, E. J.

    1994-01-01

    A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (S-ELISA) was developed for specific detection of peste des petits ruminants virus. Compared with virus isolation in Vero cell cultures using 89 paired tissue and secretion samples from six experimentally infected goats, S-ELISA was significantly more sensitive (71.9% versus 65.2%; P < 0.05). The S-ELISA is a suitable alternative to virus isolation.

  17. Microbead agglutination based assays

    KAUST Repository

    Castro, David

    2013-11-28

    A method for detecting the presence of an analyte in a sample can include adding a plurality of microparticles of a first-type to the sample, where each microparticle of the first-type includes a first binding partner configured to interact with at least a first portion of the analyte, adding a plurality of microparticles of a second-type to the sample, where each microparticle of the second-type includes a second binding partner configured to interact with at least a second portion of the analyte, the first portion of the analyte being different from the second portion of the analyte, and identifying an aggregate including at least one microparticle of the first-type, at least one microparticle of the second-type and the analyte, where the aggregate indicates the presence of the analyte.

  18. Development and validation of fast duplex real-time PCR assays based on SYBER Green florescence for detection of bovine and poultry origins in feedstuffs.

    Science.gov (United States)

    Safdar, Muhammad; Junejo, Yasmeen

    2015-04-15

    SYBR duplex real-time polymerase chain reaction (SDRT-PCR) with melting curve analysis was developed that can unite the reward of multiplex PCR with real-time PCR to recognize animal genes rapidly in feedstuffs. The method merges the use of bovine (Bos taurus) and poultry (Gallus gallus) specific primers that amplify small fragments (amplicons melting peaks simultaneously at 79.5 °C and 87.5 °C, respectively. Multiplex analysis of the reference feed samples showed that the detection limit of the assay was 0.001% for bovine and poultry species. Based upon the assay results it has been concluded that SDRT-PCR assay might be an efficient tool for the verification of species origin in feedstuffs. PMID:25466073

  19. Simultaneous detection of eight swine reproductive and respiratory pathogens using a novel GeXP analyser-based multiplex PCR assay.

    Science.gov (United States)

    Zhang, Minxiu; Xie, Zhixun; Xie, Liji; Deng, Xianwen; Xie, Zhiqin; Luo, Sisi; Liu, Jiabo; Pang, Yaoshan; Khan, Mazhar I

    2015-11-01

    A new high-throughput GenomeLab Gene Expression Profiler (GeXP) analyser-based multiplex PCR assay was developed for the detection of eight reproductive and respiratory pathogens in swine. The reproductive and respiratory pathogens include North American porcine reproductive and respiratory syndrome virus (PRRSV-NA), classical swine fever virus (CSFV), porcine circovirus 2 (PCV-2), swine influenza virus (SIV) (including H1 and H3 subtypes), porcine parvovirus (PPV), pseudorabies virus (PRV) and Japanese encephalitis virus (JEV). Nine pairs of specific chimeric primers were designed and used to initiate PCRs, and one pair of universal primers was used for subsequent PCR cycles. The specificity of the GeXP assay was examined using positive controls for each virus. The sensitivity was evaluated using serial ten-fold dilutions of in vitro-transcribed RNA from all of the RNA viruses and plasmids from DNA viruses. The GeXP assay was further evaluated using 114 clinical specimens and was compared with real-time PCR/single RT-PCR methods. The specificity of the GeXP assay for each pathogen was examined using single cDNA/DNA template. Specific amplification peaks of the reproductive and respiratory pathogens were observed on the GeXP analyser. The minimum copies per reaction detected for each virus by the GeXP assay were as follows: 1000 copies/μl for PRV; 100 copies/μl for CSFV, JEV, PCV-2 and PPV; and 10 copies/μl for SIV-H1, SIV-H3 and PRRSV-NA. Analysis of 114 clinical samples using the GeXP assay demonstrated that the GeXP assay had comparable detection to real-time PCR/single RT-PCR. This study demonstrated that the GeXP assay is a new method with high sensitivity and specificity for the identification of these swine reproductive and respiratory pathogens. The GeXP assay may be adopted for molecular epidemiological surveys of these reproductive and respiratory pathogens in swine populations. PMID:26259690

  20. Ultrasensitive and quantitative detection of a new β-agonist phenylethanolamine A by a novel immunochromatographic assay based on surface-enhanced Raman scattering (SERS).

    Science.gov (United States)

    Li, Mingxin; Yang, Hong; Li, Shuqun; Zhao, Kang; Li, Jianguo; Jiang, Danni; Sun, Lulu; Deng, Anping

    2014-11-12

    Phenylethanolamine A (PA) is a new kind of β-agonist, which was illegally used as a feed additive for growth promotion in China. In this study, a novel immunochromatographic assay (ICA) based on surface-enhanced Raman scattering (SERS) for the ultrasensitive and quantitative detection of phenylethanolamine A is presented. The principle of this new ICA is similar to that based on colloidal gold particles, but using Au(MBA)@Ag-Ab [e.g., polyclonal antibody of PA labeled Au@Ag core-shell nanoparticles (NPs) sandwiched with a Raman reporter (4-mercaptobenzoic acid, MBA)] as a probe. After ICA procedures, the specific Raman scattering intensity of MBA on the test line was measured for quantitative detection of PA. This assay was completed within 15 min. The IC50 and limit of detection (LOD) values of the ICA for PA detection were 0.06 ng mL(-1) and 0.32 pg mL(-1), respectively, which were 1-3 orders of magnitude lower than those obtained by other immunoassays, indicating the ultrasensitivity of this ICA. There was no cross-reactivity (CR) of the assay with another three β-agonists (ractopamine, clenbuterol, and salbutamol), suggesting high specificity of the SERS-based ICA. A spiking experiment revealed that the recoveries of PA from pig urine samples were in range of 99.9- 101.2% with relative standard deviations (RSDs) of 3.6-5.8%. The results demonstrated that this SERS-based ICA was able to quantitatively detect PA in urine samples with high sensitivity, specificity, precision, and accuracy and might be a powerful method for the analysis of other target analytes in the food area. PMID:25343225

  1. A locked nucleic acid (LNA-based real-time PCR assay for the rapid detection of multiple bacterial antibiotic resistance genes directly from positive blood culture.

    Directory of Open Access Journals (Sweden)

    Lingxiang Zhu

    Full Text Available Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA-based quantitative real-time PCR (LNA-qPCR method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1-10 colony forming units (CFU per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4% were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.

  2. Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Leishmania infantum in canine leishmaniasis based on cysteine protease B genes.

    Science.gov (United States)

    Chaouch, Melek; Mhadhbi, Moez; Adams, Emily R; Schoone, Gerard J; Limam, Sassi; Gharbi, Zyneb; Darghouth, Mohamed Aziz; Guizani, Ikram; BenAbderrazak, Souha

    2013-11-15

    We developed a Leishmania infantum specific LAMP assay that was carried out using a set of, six primers targeting the cysteine protease B multi copy gene of L. infantum. Our result shows that we, successfully detect the L. infantum DNA and that amplification is specific as no cross reaction was seen, with L. major, L. tropica, L. turanica, L. aethiopica, L. tarentolae, L. gerbilii, Trypanosoma cruzi or, human genomic DNA. When compared to conventional cpb based PCR, the sensitivity of LAMP assay, was higher with a detection limit of 50 fg/μl of genomic L. infantum parasite DNA. Accurate and rapid, diagnosis of canine leishmaniasis (CanL) is an important issue that allows early treatment and, prevents transmission. Our developed LAMP assay was used to evaluate occurrences of Leishmania infantum in seventy five (75) dogs from the field. Blood samples were used to perform LAMP assay, classical PCR, IFAT and microscopy that was used as gold standard. The IFAT in addition to, microscopy, are the basic techniques used for CanL diagnosis at the School of Veterinary Medicine, where we obtained our samples. Compared to molecular methods, the serology (IFAT) test shows the, best sensitivity (88.57%) with, however, a much lower specificity (52.5%) due to a relatively high, number of false-positive results (22 animals). The PCR assay shows a low sensitivity (37.14%) and, specificity around (82.5%). Our LAMP assay shows a suitable sensitivity (54%) and a good specificity, (80%), with however, positive (70%) and negative (66%) predictive values. Furthermore, the best, positive likelihood ratio (LR+) was obtained by LAMP assay (2.7). This technique presents the highest, kappa value (with a fair agreement of 0.34). Moreover, the relative stability of the reagents indicates, that LAMP may be a good alternative to a conventional PCR, especially under field conditions. Finally in, a brief cost evaluation, the LAMP assay compares favorably with other molecular diagnostic tests. This

  3. PCR-based detection of Plasmodium in Anopheles mosquitoes: a comparison of a new high-throughput assay with existing methods

    Directory of Open Access Journals (Sweden)

    Bass Chris

    2008-09-01

    Full Text Available Abstract Background Detection of the four malaria-causing Plasmodium species (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae within their mosquito hosts is an essential component of vector control programmes. Several PCR protocols have been developed for this purpose. Many of these methods, while sensitive, require multiple PCR reactions to detect and discriminate all four Plasmodium species. In this study a new high-throughput assay was developed and compared with three previously described PCR techniques. Methods A new assay based on TaqMan SNP genotyping was developed to detect all four Plasmodium species and discriminate P. falciparum from P. vivax, P. ovale and P. malariae. The sensitivity and the specificity of the new assay was compared to three alternative PCR approaches and to microscopic dissection of salivary glands in a blind trial of 96 single insect samples that included artificially infected Anopheles stephensi mosquitoes. The performance of the assays was then compared using more than 450 field-collected specimens that had been stored on silica gel, in ethanol or in isopropanol. Results The TaqMan assay was found to be highly specific when using Plasmodium genomic DNA as template. Tests of analytical sensitivity and the results of the blind trial showed the TaqMan assay to be the most sensitive of the four methods followed by the 'gold standard' nested PCR approach and the results generated using these two methods were in good concordance. The sensitivity of the other two methods and their agreement with the nested PCR and TaqMan approaches varied considerably. In trials using field collected specimens two of the methods (including the nested protocol showed a high degree of non-specific amplification when using DNA derived from mosquitoes stored in ethanol or isopropanol. The TaqMan method appeared unaffected when using the same samples. Conclusion This study describes a new high-throughput Taq

  4. Development of an enzyme-linked-receptor assay based on Syrian hamster β2-adrenergic receptor for detection of β-agonists.

    Science.gov (United States)

    Cheng, Guyue; Li, Feng; Peng, Dapeng; Huang, Lingli; Hao, Haihong; Liu, Zhenli; Wang, Yulian; Yuan, Zonghui

    2014-08-15

    β-Adrenergic agonists (β-agonists) are illegally used in animal husbandry, threatening the health of consumers. To realize multianalyte detection of β-agonists, a β2-adrenergic receptor (β2-AR) was cloned from Syrian hamster lung and heterogeneously expressed by Spodoptera frugiperda (Sf9) cells. The recombinant β2-AR was purified from intracellular soluble proteins of infected Sf9 cells, and was utilized to establish an enzyme-linked-receptor assay (ELRA) to detect a group of β-agonists simultaneously. This assay was based on direct competitive inhibition of binding of horseradish peroxidase-labeled ractopamine to the immobilized β2-AR proteins by β-agonists. The IC50 and limit of detection values for ractopamine were 30.38μgL(-1) and 5.20μgL(-1), respectively. Clenbuterol and salbutamol showed 87.7% and 58.5% cross-reactivities with ractopamine, respectively. This assay is simple, rapid, and environmentally friendly, showing a potential application in the screening of β-agonists in animal feeds. PMID:24853343

  5. Development of a DNA Microarray-Based Assay for the Detection of Sugar Beet Root Rot Pathogens.

    Science.gov (United States)

    Liebe, Sebastian; Christ, Daniela S; Ehricht, Ralf; Varrelmann, Mark

    2016-01-01

    Sugar beet root rot diseases that occur during the cropping season or in storage are accompanied by high yield losses and a severe reduction of processing quality. The vast diversity of microorganism species involved in rot development requires molecular tools allowing simultaneous identification of many different targets. Therefore, a new microarray technology (ArrayTube) was applied in this study to improve diagnosis of sugar beet root rot diseases. Based on three marker genes (internal transcribed spacer, translation elongation factor 1 alpha, and 16S ribosomal DNA), 42 well-performing probes enabled the identification of prevalent field pathogens (e.g., Aphanomyces cochlioides), storage pathogens (e.g., Botrytis cinerea), and ubiquitous spoilage fungi (e.g., Penicillium expansum). All probes were proven for specificity with pure cultures from 73 microorganism species as well as for in planta detection of their target species using inoculated sugar beet tissue. Microarray-based identification of root rot pathogens in diseased field beets was successfully confirmed by classical detection methods. The high discriminatory potential was proven by Fusarium species differentiation based on a single nucleotide polymorphism. The results demonstrate that the ArrayTube constitute an innovative tool allowing a rapid and reliable detection of plant pathogens particularly when multiple microorganism species are present. PMID:26524545

  6. Detection of Eperythrozoon wenyoni by PCR assay

    Institute of Scientific and Technical Information of China (English)

    Jian WANG; Yutao ZHU; Jianhua QIN; Fumei ZHANG; Yuelan ZHAO

    2009-01-01

    The objective of this research was to develop a detection method for Eperythrozoon wenyoni infection using polymerase chain reaction (PCR) assay technique. A pair of primers was designed and synthesized according to the conservative sequence 16S rRNA. The PCR assay was performed with the primers. A 985-bp fragment was amplified by using PCR. The amplified fragments with the expected size were identified by EcoR I restriction digestion. The crossing-reaction, specific-reaction and duplicate-reaction indicated that the PCR method is a specific, sensitive, fast and effective method for diagnosing E. Wenyoni infection at group level.

  7. Application of fluorescence based molecular assays for improved detection and typing of Brucella strains in clinical samples

    Directory of Open Access Journals (Sweden)

    Kiril Krstevski

    2015-10-01

    Full Text Available Bacteria from the genus Brucella are causative agents of brucellosis – a zoonotic disease which affects many wild and domestic animal species and humans. Taking into account the significant socio-economic and public health impact of brucellosis, its control is of great importance for endemic areas. The chosen control strategy could be successful only if adapted to the current epidemiological situation. This implies that a choice of appropriate diagnostic procedures for detection and typing of Brucella spp. strains are of essential importance. Significant advancement of molecular techniques and their advantages compared to classical methods, give strong arguments in promotion of these techniques as a powerful tool for comprehensive diagnostics of brucellosis. Considering this, the major tasks of the study were to select and implement molecular tests for detection and genotyping Brucella spp. and evaluate their performances using DNA from cultivated brucellae (islolates and limited number of tissue samples from seropositive animals. The obtained results confirmed that implemented real time PCR for Brucella spp. detection, as well as MLVA-16 used for genotyping, have excellent analytical sensitivity (4.2 fg of Brucella DNA were successfully detected and genotyped. Furthermore, compared to bacteriological cultivation of Brucella spp., real time PCR and MLVA-16 protocols showed superior diagnostic sensitivity and detected Brucella DNA in tissues from which Brucella could not be cultivated. Based on the summarized study results, we propose a diagnostic algorithm for detection and genotyping of Brucella spp. bacteria. Routine use of proposed diagnostic algorithm will improve the effectiveness of infection confirmation and help for accurate evaluation of epidemiological situation.

  8. Aggregated silver nanoparticles based surface-enhanced Raman scattering enzyme-linked immunosorbent assay for ultrasensitive detection of protein biomarkers and small molecules.

    Science.gov (United States)

    Liang, Jiajie; Liu, Hongwu; Huang, Caihong; Yao, Cuize; Fu, Qiangqiang; Li, Xiuqing; Cao, Donglin; Luo, Zhi; Tang, Yong

    2015-06-01

    Lowering the detection limit is critical to the design of bioassays required for medical diagnostics, environmental monitoring, and food safety regulations. The current sensitivity of standard color-based analyte detection limits the further use of enzyme-linked immunosorbent assays (ELISAs) in research and clinical diagnoses. Here, we demonstrate a novel method that uses the Raman signal as the signal-generating system of an ELISA and combines surface-enhanced Raman scattering (SERS) with silver nanoparticles aggregation for ultrasensitive analyte detection. The enzyme label of the ELISA controls the dissolution of Raman reporter-labeled silver nanoparticles through hydrogen peroxide and generates a strong Raman signal when the analyte is present. Using this assay, prostate-specific antigen (PSA) and the adrenal stimulant ractopamine (Rac) were detected in whole serum and urine at the ultralow concentrations of 10(-9) and 10(-6) ng/mL, respectively. The methodology proposed here could potentially be applied to other molecules detection as well as PSA and Rac. PMID:25928837

  9. 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

    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...... 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...

  10. Visual detection and microplate assay for Staphylococcus aureus based on aptamer recognition coupled to tyramine signal amplification

    International Nuclear Information System (INIS)

    We have developed a specific method for the visual detection of Staphylococcus aureus based on aptamer recognition coupled to tyramine signal amplification technology. A biotinylated aptamer specific for S. aureus was immobilized on the surface of the wells of a microplate via biotin-avidin binding. Then, the target bacteria (S. aureus), the biotinylated-aptamer-streptavidin-HRP conjugates, biotinylated tyramine, hydrogen peroxide and streptavidin-HRP were successively placed in the wells of the microplate. After adding TMB reagent and stop solution, the intensity of the yellow reaction product can be visually inspected or measured with a plate reader. Under optimized conditions, there is a linear relationship between absorbance at 450 nm and the concentration of S. aureus in the 10 to 107 cfu mL−1 concentration range (with an R2 of 0.9976). The limit of detection is 8 cfu mL−1. (author)

  11. A TaqMan-based real time PCR assay for specific detection and quantification of Xylella fastidiosa strains causing bacterial leaf scorch in oleander

    Science.gov (United States)

    A TaqMan-based real-time PCR assay is developed for strains of X. fastidiosa causing oleander leaf scorch. The assay uses primers WG-OLS-F1 and WG-OLS-R1 and the fluorescent probe WG-OLS-P1, designed based on unique sequences present only in genomic sequence of oleander strain Ann1. The assay is spe...

  12. Array-in-well platform-based multiplex assay for the simultaneous detection of anti-HIV- and treponemal-antibodies, and Hepatitis B surface antigen.

    Science.gov (United States)

    Talha, Sheikh M; Saviranta, Petri; Hattara, Liisa; Vuorinen, Tytti; Hytönen, Jukka; Khanna, Navin; Pettersson, Kim

    2016-02-01

    Multiplex assays detecting sets of related clinical analytes simultaneously can save considerable amount of time and resources. Array-in-well (AIW) is a powerful platform for the multiplex detection of different analytes where microarrays can be printed at the bottom of microtiter wells, thus combining the potential of microarrays with the ease of handling microtiter wells. We have developed a single-step AIW assay for the simultaneous screening of HIV, Treponema pallidum subspecies pallidum (causing syphilis) and Hepatitis B virus infections targeting the specific detection of anti-HIV- and treponemal-antibodies and Hepatitis B surface antigen (HBsAg), respectively, using two different fluorescent label technologies i.e. DyLight 633 and europium nanoparticle. Double-antigen assay formats were used for anti-HIV- and treponemal-antibody detection that can simultaneously detect both IgG and IgM, and thus reduce the window period of detection. AIW assay was evaluated with well characterized serum/plasma samples (n=111), and the qualitative results were in near complete agreement with those of the reference assays. The AIW assay exhibited 100% sensitivities for all three analytes, and 100% specificities for anti-HIV antibodies and HBsAg, and 98.6% specificity for treponemal antibodies. The limit of detection of HBsAg in AIW assay was 0.18 ng/ml. This high performing AIW assay has the potential to be used as a multiplex screening test for these three infections. PMID:26711310

  13. Development and Evaluation of a Novel and Rapid Detection Assay for Botrytis cinerea Based on Loop-Mediated Isothermal Amplification

    OpenAIRE

    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 ...

  14. Design and development of PCR-free highly sensitive electrochemical assay for detection of telomerase activity using Nano-based (liposomal) signal amplification platform.

    Science.gov (United States)

    Alizadeh-Ghodsi, Mohammadreza; Zavari-Nematabad, Ali; Hamishehkar, Hamed; Akbarzadeh, Abolfazl; Mahmoudi-Badiki, Tohid; Zarghami, Faraz; Pourhassan Moghaddam, Mohammad; Alipour, Esmaeel; Zarghami, Nosratollah

    2016-06-15

    Telomerase, which has been detected in almost all kinds of cancer tissues, is considered as an important tumor marker for early cancer diagnostics. In the present study, an electrochemical method based on liposomal signal amplification platform is proposed for simple, PCR-free, and highly sensitive detection of human telomerase activity, extracted from A549 cells. In this strategy, telomerase reaction products, which immobilized on streptavidin-coated microplate, hybridized with biotinylated capture probes. Then, dopamine-loaded biotinylated liposomes are attached through streptavidin to biotinylated capture probes. Finally, liposomes are ruptured by methanol and the released-dopamine is subsequently measured using differential pulse voltammetry technique by multi-walled carbon nanotubes modified glassy carbon electrode. Using this strategy, the telomerase activity extracted from 10 cultured cancer cells could be detected. Therefore, this approach affords high sensitivity for telomerase activity detection and it can be regarded as an alternative to telomeric repeat amplification protocol assay, having the advantages of simplicity and less assay time. PMID:26874110

  15. Development and evaluation of a SYBR green-based real time RT-PCR assay for detection of the emerging avian influenza A (H7N9 virus.

    Directory of Open Access Journals (Sweden)

    Zheng Zhu

    Full Text Available Most recently a novel avian-origin influenza A (H7N9 virus emerged in China and has been associated with lots of human infection and fatal cases. Genetic analysis of the viral genome revealed that this reassortant virus might be better adapted to humans than other avian influenza viruses. Molecular diagnostic methods are thus urgently needed in public health laboratories. In this study, a SYBR green-based one-step real time reverse transcription-PCR (RT-PCR was developed to detect the novel H7N9 virus. The primer pairs on the basis of the hemagglutinin and neuraminidase gene sequences of H7N9 viruses amplified subtype-specific fragments with Tm values of 80.77±0.06°C for H7 and 81.20±0.17°C for N9 respectively. The standard curves showed a dynamic linear range across 6 log units of RNA copy number (10(6 to 10(1 copies/ µl with a detection limit of 10 copies per reaction for both H7 and N9 assays by using serial ten-fold diluted in-vitro transcribed viral RNA. In addition, no cross-reactivity was observed with seasonal H1N1, H1N1 pdm09, H3N2, H5N1 and H9N2 viruses as well as other human respiratory viruses. When the assay was further evaluated in H7N9 virus infected clinical samples, positive amplification signals were obtained in all of the specimens with the accordance between H7 and N9 assays. Therefore, the established SYBR green-based real time RT-PCR assay could provide a rapid, sensitive, specific and reliable alternative approach with lower costs for high throughput screening of suspected samples from humans, animals and environments in first line public health laboratories.

  16. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish

    OpenAIRE

    Hardison, D. Ransom; Holland, William C.; McCall, Jennifer R.; Bourdelais, Andrea J.; Baden, Daniel G.; Darius, H. Taiana; Chinain, Mireille; Tester, Patricia A.; Shea, Damian; Harold A. Flores Quintana; Morris, James A.; Litaker, R. Wayne

    2016-01-01

    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 s...

  17. Nanoparticles assembled by aptamers and crystal violet for arsenic(iii) detection in aqueous solution based on a resonance Rayleigh scattering spectral assay

    Science.gov (United States)

    Wu, Yuangen; Zhan, Shenshan; Xing, Haibo; He, Lan; Xu, Lurong; Zhou, Pei

    2012-10-01

    Aptamer-assembled nanomaterials have captured much attention from the field of analytical chemistry in recent years. Although they have been regarded as a promising tool for heavy metal monitoring, report involving aptamer-based biosensors for arsenic detection are rare. Herein we developed a highly sensitive and selective aptamer biosensor for As(iii) detection based on a Resonance Rayleigh Scattering (RRS) spectral assay. Prior to As(iii) detection, we firstly assembled a variety of nanoparticles with different sizes via controlling the concentration of arsenic-binding aptamers in crystal violet (CV) solutions. The results of photon correlation spectroscopy (PCS) and scanning probe microscope (SPM) testified that the introduction of As(iii) had indeed changed the size of nanoparticles, which caused a great variation in the RRS intensity at 310 nm. In the presence of 100 ppb As(iii), a maximum decline in the ratio of RRS intensity was achieved for large nanoparticles assembled from 200 nM of aptamers and CV molecules, where the average size of nanoparticles had decreased from 273 nm to 168 nm. In the case of small nanoparticles, the maximum increase ratio of the RRS intensity was obtained when the concentration of aptamer was over 600 nM. Combined with an RRS spectral assay, an effective biosensor has been developed for As(iii) detection, using the above large and small nanoparticles as the target recognition element. The present biosensor has a detection limit as low as 0.2 ppb, a dynamic range from 0.1 ppb to 200 ppb, and high selectivity over other metal ions. Such an efficient biosensor will play an important role in environmental detection.

  18. Development of bioreporter assays for the detection of bioavailability of long chain alkanes based on the marine bacterium Alcanivorax borkumensis strain SK2

    OpenAIRE

    Kumari, Rekha; Tecon, Robin; Beggah, Siham; Rutler, Rebecca; Arey, J. Samuel; van der Meer, Jan Roelof

    2011-01-01

    Long-chain alkanes are a major component of crude oil and therefore potentially good indicators of hydrocarbon spills. Here we present a set of new bacterial bioreporters and assays that allow to detect long-chain alkanes. These reporters are based on the regulatory protein AlkS and the alkB1 promoter from Alcanivorax borkumensis SK2, a widespread alkane degrader in marine habitats. Escherichia coli cells with the reporter construct reacted strongly to octane in short-term (6 h) aqueous suspe...

  19. Microchip based and immunochromatographic strip assays for the visual detection of interleukin-6 and of tumor necrosis factor α using gold nanoparticles as labels

    International Nuclear Information System (INIS)

    The enzyme-linked immunesorbent assay (ELISA) is most frequently used for the determination of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). However, this technique is time-consuming and requires a substantial sample volume. We have developed (a) a new kind of colloidal gold-based immunochromatographic strip, and (b) a colloidal gold-based immunochromatographic microchip for simultaneous, rapid, and visual detection of IL-6 and TNF-α. The lowest limit of detection is 62.5 ng mL−1 for IL-6, and 30 ng mL−1 for TNF-α. Both the strip method and the microchip method are highly specific, user-friendly, and can be read out without the need for instrumentation. (author)

  20. Mrassf1a-pap, a novel methylation-based assay for the detection of cell-free fetal DNA in maternal plasma.

    Directory of Open Access Journals (Sweden)

    Jessica M E van den Oever

    Full Text Available OBJECTIVES: RASSF1A has been described to be differentially methylated between fetal and maternal DNA and can therefore be used as a universal sex-independent marker to confirm the presence of fetal sequences in maternal plasma. However, this requires highly sensitive methods. We have previously shown that Pyrophosphorolysis-activated Polymerization (PAP is a highly sensitive technique that can be used in noninvasive prenatal diagnosis. In this study, we have used PAP in combination with bisulfite conversion to develop a new universal methylation-based assay for the detection of fetal methylated RASSF1A sequences in maternal plasma. METHODS: Bisulfite sequencing was performed on maternal genomic (gDNA and fetal gDNA from chorionic villi to determine differentially methylated regions in the RASSF1A gene using bisulfite specific PCR primers. Methylation specific primers for PAP were designed for the detection of fetal methylated RASSF1A sequences after bisulfite conversion and validated. RESULTS: Serial dilutions of fetal gDNA in a background of maternal gDNA show a relative percentage of ~3% can be detected using this assay. Furthermore, fetal methylated RASSF1A sequences were detected both retrospectively as well as prospectively in all maternal plasma samples tested (n = 71. No methylated RASSF1A specific bands were observed in corresponding maternal gDNA. Specificity was further determined by testing anonymized plasma from non-pregnant females (n = 24 and males (n = 21. Also, no methylated RASSF1A sequences were detected here, showing this assay is very specific for methylated fetal DNA. Combining all samples and controls, we obtain an overall sensitivity and specificity of 100% (95% CI 98.4%-100%. CONCLUSIONS: Our data demonstrate that using a combination of bisulfite conversion and PAP fetal methylated RASSF1A sequences can be detected with extreme sensitivity in a universal and sex-independent manner. Therefore, this assay could be of great

  1. Creation of a gold nanoparticle based electrochemical assay for the detection of inhibitors of bacterial cytochrome bd oxidases.

    Science.gov (United States)

    Fournier, Eugénie; Nikolaev, Anton; Nasiri, Hamid R; Hoeser, Jo; Friedrich, Thorsten; Hellwig, Petra; Melin, Frederic

    2016-10-01

    Cytochrome bd oxidases are membrane proteins expressed by bacteria including a number of pathogens, which make them an attractive target for the discovery of new antibiotics. An electrochemical assay is developed to study the activity of these proteins and inhibition by quinone binding site tool compounds. The setup relies on their immobilization at electrodes specifically modified with gold nanoparticles, which allows achieving a direct electron transfer to/from the heme cofactors of this large enzyme. After optimization of the protein coverages, the assay shows at pH7 a good reproducibility and readout stability over time, and it is thus suitable for further screening of small molecule collections. PMID:27314676

  2. A ferrofluid-based homogeneous assay for highly sensitive and selective detection of single-nucleotide polymorphisms.

    Science.gov (United States)

    Shen, Wei; Lim, Cai Le; Gao, Zhiqiang

    2013-09-21

    A simple and low-cost colorimetric assay utilizing ferrofluidic nanoparticulate probes (FNPs) and a ligase for single-nucleotide polymorphism genotyping is described. Excellent sensitivity and selectivity were accomplished through the engagement of the FNPs and a ligase chain reaction. PMID:23923128

  3. Analyte detection using an active assay

    Science.gov (United States)

    Morozov, Victor; Bailey, Charles L.; Evanskey, Melissa R.

    2010-11-02

    Analytes using an active assay may be detected by introducing an analyte solution containing a plurality of analytes to a lacquered membrane. The lacquered membrane may be a membrane having at least one surface treated with a layer of polymers. The lacquered membrane may be semi-permeable to nonanalytes. The layer of polymers may include cross-linked polymers. A plurality of probe molecules may be arrayed and immobilized on the lacquered membrane. An external force may be applied to the analyte solution to move the analytes towards the lacquered membrane. Movement may cause some or all of the analytes to bind to the lacquered membrane. In cases where probe molecules are presented, some or all of the analytes may bind to probe molecules. The direction of the external force may be reversed to remove unbound or weakly bound analytes. Bound analytes may be detected using known detection types.

  4. Detection of Salmonella in Shellfish Using SYBR Green™ I-Based Real-Time Multiplexed PCR Assay Targeting invA and spvB

    KAUST Repository

    Gangwar, Maulshree

    2012-09-23

    A SYBR Green™ I-based real-time multiplexed PCR assay was developed targeting invA and spvB for the detection of Salmonella strains in shellfish after both hns and invA genes were identified in all Salmonella strains. Simultaneously, the 16S rRNA gene was used as a PCR internal amplification control (IAC). All 89 Salmonella strains tested in this study exhibited amplification of invA, whereas only 21 (23. 6 %) were PCR positive for spvB. The sensitivity of detection of all three targeted genes was 1 ng, which is equivalent to approximately 105 colony-forming unit (CFU) of Salmonella enterica. The analysis showed specific PCR products that were identified by reproducible melt temperature profiles (invA, 84. 27 ± 1. 7 °C; spvB, 88. 76 ± 1. 0 °C; and 16S rRNA gene, 87. 16 ± 0. 8 °C). The sensitivity of detection was 10 pg purified DNA (invA) or 105 CFU in 1 mL pure culture of S. enterica ATCC 14028. The above molecular detection method for Salmonella strains was successfully applied to the oyster homogenates (food matrix). An initial inoculum of 106 and 102 CFU Salmonella in 1 ml seeded oyster tissue homogenate was detected by multiplexed PCR for all three genes after 5 and 24 h of enrichment, respectively. Natural oysters isolated from Gulf of Mexico during the winter months exhibited negative PCR amplification results suggesting the absence of Salmonella. In contrast to conventional PCR, real-time multiplex PCR assay developed in this study is rapid and sensitive and will help Interstate Shellfish Sanitation Conference undertake appropriate measures to monitor Salmonella in oysters, thereby preventing disease outbreaks and consequently protecting consumer health. © 2012 Springer Science+Business Media, LLC.

  5. Development of a Novel Fluorescence Assay Based on the Use of the Thrombin-Binding Aptamer for the Detection of O6-Alkylguanine-DNA Alkyltransferase Activity

    Directory of Open Access Journals (Sweden)

    Maria Tintoré

    2010-01-01

    Full Text Available Human O6-alkylguanine-DNA alkyltransferase (hAGT is a DNA repair protein that reverses the effects of alkylating agents by removing DNA adducts from the O6 position of guanine. Here, we developed a real-time fluorescence hAGT activity assay that is based on the detection of conformational changes of the thrombin-binding aptamer (TBA. The quadruplex structure of TBA is disrupted when a central guanine is replaced by an O6-methyl-guanine. The sequence also contains a fluorophore (fluorescein and a quencher (dabsyl attached to the opposite ends. In the unfolded structure, the fluorophore and the quencher are separated. When hAGT removes the methyl group from the central guanine of TBA, it folds back immediately into its quadruplex structure. Consequently, the fluorophore and the quencher come into close proximity, thereby resulting in decreased fluorescence intensity. Here, we developed a new method to quantify the hAGT without using radioactivity. This new fluorescence resonance energy transfer assay has been designed to detect the conformational change of TBA that is induced by the removal of the O6-methyl group.

  6. Irradiation detection of food by DNA Comet Assay

    International Nuclear Information System (INIS)

    Microgel electrophoresis of single cells or nuclei (DNA Comet Assay) has been investigated to detect irradiation treatment of more than 50 food commodities e.g. meats, seafood, cereals, pulses, nuts, fruits and vegetables, and spices. The foodstuffs have been exposed to radiation doses covering the range of potential commercial irradiation for inactivation of pathogenic and spoilage micro-organisms, for insect disinfestation and for shelf-life extension. The Comet Assay is based on detection of DNA fragments presumptive to irradiation. For most of the food items investigated, the assay can be applied successfully for irradiation detection by working out different conditions of the assay. However, with some of the foods difficulties arose due to - lack of discrimination between the irradiated and unirradiated food samples due to the presence of the same kinds of comets in both cases and the total absence of the typical intact cells in unirradiated samples. - Sufficient DNA material was not available from some of the foods. - Insufficient lysis of the cell walls in case of some plant foods. In conclusion, the DNA Comet Assay can help to detect the irradiation treatment of several varieties of foods using low-cost equipment in a short time of analysis. (orig.)

  7. IS711-based real-time PCR assay as a tool for detection of Brucella spp. in wild boars and comparison with bacterial isolation and serology

    Directory of Open Access Journals (Sweden)

    Miserez Raymond

    2009-07-01

    Full Text Available Abstract Background Control of brucellosis in livestock, wildlife and humans depends on the reliability of the methods used for detection and identification of bacteria. In the present study, we describe the evaluation of the recently established real-time PCR assay based on the Brucella-specific insertion sequence IS711 with blood samples from 199 wild boars (first group of animals and tissue samples from 53 wild boars (second group of animals collected in Switzerland. Results from IS711 real-time PCR were compared to those obtained by bacterial isolation, Rose Bengal Test (RBT, competitive ELISA (c-ELISA and indirect ELISA (i-ELISA. Results In the first group of animals, IS711 real-time PCR detected infection in 11.1% (16/144 of wild boars that were serologically negative. Serological tests showed different sensitivities [RBT 15.6%, c-ELISA 7.5% and i-ELISA 5.5%] and only 2% of blood samples were positive with all three tests, which makes interpretation of the serological results very difficult. Regarding the second group of animals, the IS711 real-time PCR detected infection in 26% of animals, while Brucella spp. could be isolated from tissues of only 9.4% of the animals. Conclusion The results presented here indicate that IS711 real-time PCR assay is a specific and sensitive tool for detection of Brucella spp. infections in wild boars. For this reason, we propose the employment of IS711 real-time PCR as a complementary tool in brucellosis screening programs and for confirmation of diagnosis in doubtful cases.

  8. Detection of Early Stage Apoptotic Cells Based on Label-Free Cytochrome c Assay Using Bioconjugated Metal Nanoclusters as Fluorescent Probes.

    Science.gov (United States)

    Shamsipur, Mojtaba; Molaabasi, Fatemeh; Hosseinkhani, Saman; Rahmati, Fereshteh

    2016-02-16

    Cytochrome c (Cyt c) is an important biomarker in cell lysates for the early stage of apoptosis or anticancer agents. Here, two novel label-free fluorescence assays based on hemoglobin-stabilized gold nanoclusters (Hb/AuNCs) and aptamer-stabilized silver nanoclusters (DNA/AgNCs) for analysis of Cyt c are presented. The heme group of the protein induces sensitive sensing platforms accompanied by the decreased fluorescence of both metal nanoclusters. The quenching processes observed found to be based on the fluorescence resonance energy transfer mechanism from Hb/AuNCs to Cyt c and photoinduced electron transfer from DNA/AgNCs to the aptamer-Cyt c complex. The linear range for Cyt c was found to be 0-10 μM for Hb/AuNCs and from 0 to 1 μM for DNA/AgNCs, with limits of detection of ∼15 nM. On the basis of strong binding affinity of DNA aptamers for their target proteins, the DNA/AgNCs probe was successfully applied to the quantitative determination of Cyt c in cell lysates, which opens a new avenue to early diagnostics and drug screening with high sensitivity. Compared to the conventional Western blot method, the presented assays are low cost, easy to prepare the fluorescent probes, and sensitive, while overall time for the detection and quantitation of Cyt c from isolated mitochondria is only 20 min. The proposed method for Cyt c detection may also be useful for the study of those materials that cause mitochondrial dysfunction and apoptotic cell death. PMID:26812937

  9. 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,...

  10. Development and application of loop-mediated isothermal amplification assays based on ITS-1 for rapid detection of Toxoplasma gondii in pork.

    Science.gov (United States)

    Zhuo, Xunhui; Huang, Bin; Luo, Jiaqing; Yu, Haijie; Yan, Baolong; Yang, Yi; Du, Aifang

    2015-03-15

    The loop-mediated isothermal amplification (LAMP) assay is a novel method that rapidly amplifies DNA with high specificity and sensitivity under isothermal conditions. In this study, we established a LAMP assay with six primers targeting a highly conserved region of Toxoplasma gondii ITS-1 sequence. The amplification protocol completes within 30 min under isothermal condition in a 65°C water bath while specificity tests confirmed no cross-reactivity with DNA templates of Neospora caninum, Eimeria tenella, Cryptosporidium parvum, Listeria monocytogenes and Streptococcus suis. The detection limit of the LAMP assay was 0.9 fg T. gondii genomic DNA, a sensitivity that was 10-fold higher than that of a conventional PCR assay. Both LAMP assay and conventional PCR were applied to detect T. gondii genomic DNA in 118 diaphragm samples obtained from pig farms in Zhejiang Province, China. Our results showed that the LAMP assay is more sensitive than conventional PCR (13.56% and 9.32%). The LAMP assay established in this study provides a simple, specific, sensitive and rapid method of T. gondii genomic DNA detection, hence is expected to plays an important role in the monitoring of T. gondii contamination in various food products. PMID:25624074

  11. The synchronous active neutron detection assay system

    International Nuclear Information System (INIS)

    We have begun to develop a novel technique for active neutron assay of fissile material in spent nuclear fuel. This approach will exploit a 14-MeV neutron generator developed by Schlumberger. The technique, termed synchronous active neutron detection (SAND), follows a method used routinely in other branches of physics to detect very small signals in presence of large backgrounds. Synchronous detection instruments are widely available commercially and are termed ''lock-in'' amplifiers. We have implemented a digital lock-in amplifier in conjunction with the Schlumberger neutron generator to explore the possibility of synchronous detection with active neutrons. The Schlumberger system can operate at up to a 50% duty factor, in effect, a square wave of neutron yield. Results are preliminary but promising. The system is capable of resolving the fissile material contained in a small fraction of the fuel rods in a cold fuel assembly; it also appears resilient to background neutron interference. The interrogating neutrons appear to be non-thermal and penetrating. Work remains to fully explore relevant physics and optimize instrument design

  12. Development of an F57 Sequence-Based Real-Time PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Milk

    OpenAIRE

    Tasara, T; Stephan, R.

    2005-01-01

    A light cycler-based real-time PCR (LC-PCR) assay that amplifies the F57 sequence of Mycobacterium avium subsp. paratuberculosis was developed. This assay also includes an internal amplification control template to monitor the amplification conditions in each reaction. The targeted F57 sequence element is unique for M.avium subsp. paratuberculosis and is not known to exist in any other bacterial species. The assay specificity was demonstrated by evaluation of 10 known M. avium subsp. paratube...

  13. Real-Time Detection and Identification of Chlamydophila Species in Veterinary Specimens by Using SYBR Green-Based PCR Assays

    DEFF Research Database (Denmark)

    Nordentoft, Steen; Kabell, Susanne; Pedersen, Karl

    2011-01-01

    Chlamydiaceae and differentiate the most prevalent veterinary Chlamydophila species: Cp. psittaci, Cp. abortus, Cp. felis, and Cp. caviae. By adding bovine serum albumin to the master mixes, target DNA could be detected directly in crude lysates of enzymatically digested conjunctival or pharyngeal swabs or...

  14. Development of a PCR Assay Based on a Single-Base Pair Substitution for the Detection of Aeromonas caviae by Targeting the gyrB Gene.

    Science.gov (United States)

    Payattikul, Penpan; Longyant, Siwaporn; Sithigorngul, Paisarn; Chaivisuthangkura, Parin

    2015-09-01

    Aeromonas caviae is a bacterial pathogen that causes various infectious diseases in both humans and animals. To facilitate its detection, we developed species-specific primer sets targeting polymorphisms in the gyrB gene for use in a PCR assay. The technique was able to detect 100% (29/29) of the A. caviae strains tested using either of two sets of primers (designated ACF1-ACR and ACF3-ACR), which produced 293-bp and 206-bp amplicons, respectively. Another set of primers (designated ACF2-ACR) yielded a 237-bp amplicon and exhibited 90% (26/29) positive results with respect to A. caviae. None of the primer sets exhibited cross-reactivity with 12 non-A. caviae isolates and 52 other non-Aeromonas bacteria. The detection limit using the ACF2-ACR and ACF3-ACR primer sets in pure culture was 1.6 × 10(3) CFU/mL, or 6 CFU per reaction, whereas that of the ACF1-ACR primer set was 1.6 × 10(4) CFU/mL, or 60 CFU per reaction. In the case of spiked Nile Tilapia Oreochromis niloticus, the sensitivity of all primer sets without enrichment was 1.8 × 10(4) CFU/g, or 30 CFU per reaction. Primer set ACF3-ACR was the best for a PCR assay targeting the gyrB gene, and the PCR technique developed was rapid, specific, and sensitive for the identification of A. caviae. PMID:26223267

  15. Specific detection of DNA and RNA targets using a novel isothermal nucleic acid amplification assay based on the formation of a three-way junction structure

    OpenAIRE

    Wharam, Susan D.; Marsh, Peter; Lloyd, John S.; Ray, Trevor D.; Mock, Graham A.; Assenberg, René; McPhee, Julie E.; Brown, Philip; Weston, Anthony; Cardy, Donald L. N.

    2001-01-01

    The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets. The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure. One probe (template for the RNA signal) contains a non-functional single-stranded T7 RNA polymerase promoter sequence. This ...

  16. Diagnostic efficacy of monoclonal antibody based sandwich enzyme linked immunosorbent assay (ELISA for detection of Fasciola gigantica excretory/secretory antigens in both serum and stool

    Directory of Open Access Journals (Sweden)

    Zoheiry Mona K

    2011-09-01

    Full Text Available Abstract Background This research was carried out to develop a reliable monoclonal antibody (MoAb-based sandwich enzyme linked immunosorbent assay (ELISA for the diagnosis of active Fasciola gigantica infection in both serum and stool for comparative purposes. Methods From a panel of MoAbs raised against F. gigantica excretory/secretory antigens (ES Ags, a pair (12B/11D/3F and 10A/9D/10G was chosen due to its high reactivity and strict specificity to F. gigantica antigen by indirect ELISA. Results The two MoAbs were of the IgG1 and IgG2a subclasses, respectively. Using SDS-PAGE and EITB, the selected MoAbs recognized 83, 64, 45 and 26 kDa bands of ES Ags. The lower detection limit of ELISA assay was 3 ng/ml. In stool, the sensitivity, specificity and diagnostic efficacy of ELISA was 96%, 98.2 and 97.1%; while in serum they were 94%, 94.6% and 94.3%, respectively. Moreover, a positive correlation was found between ova count in stool of F. gigantica infected patients and the OD readings of ELISA in both stool and serum samples (r = 0.730, p Conclusions These data showed that the use of MoAb-based sandwich ELISA for the detection of F. gigantica coproantigens in stool specimens was superior to serum samples; it provides a highly efficient, non-invasive technique for the diagnosis of active F. gigantica infection.

  17. Detection of Pseudomonas aeruginosa by a triplex polymerase chain reaction assay based on lasI/R and gyrB genes.

    Science.gov (United States)

    Aghamollaei, Hosseine; Moghaddam, Mehrdad M; Kooshki, Hamid; Heiat, Mohammad; Mirnejad, Reza; Barzi, Nastaran S

    2015-01-01

    Pseudomonas aeruginosa is a nosocomial pathogen, which, due to its inherent and acquired resistance to a wide range of antibiotics, causes high mortality rates. Therefore, rapid detection of the bacterium with high specificity and sensitivity plays a critical role in the control of the pathogenic bacterium. The aim of this study was to evaluate the accuracy and specificity of a prompt detection of the bacterium based on a triplex polymerase chain reaction that amplifies the lasI, lasR and gyrB genes. For this purpose, 30 clinical isolates of P. aeruginosa and 30 wound biopsy samples were retrieved from clinical diagnostic laboratories. After the extraction of the chromosomal DNA, the desired genes were amplified using uniplex and triplex PCR with appropriate primers. The specificity of the primers was evaluated by a comparison of the PCR results for P. aeruginosa clinical samples and non-Pseudomonas species control samples. The sensitivity of the primers was determined using a serial dilution of the genomic DNA template (100 ng to 100 fg) and by a comparison of the PCR and bacterial culture results. The results showed that the triplex PCR assay was positive for all of the samples (100%), while the PCR identifications were negative for non-Pseudomonas species. Additionally, at 10(-4) and 10(-5) diluted genomic DNA from P. aeruginosa (10 pg and 1 pg), the triplex PCR test was positive for the Las and gyrB genes in all of the samples, respectively. Based on these results, the designed primers can be used for the rapid, specific and sensitive diagnosis of P. aeruginosa in a triplex PCR assay. PMID:25863575

  18. Multifunctional Oval Shape Gold Nanoparticle Based Selective Detection of Breast Cancer Cells Using Simple Colorimetric and Highly Sensitive Two-Photon Scattering Assay

    Science.gov (United States)

    Lu, Wentong; Arumugam, Sri Ranjini; Senapati, Dulal; Singh, Anant K.; Arbneshi, Tahir; Yu, Sadia Afrin Khan Hongtao; Ray, Paresh Chandra

    2010-01-01

    Breast cancer is the most common cancer among women and it is the second leading cause of cancer deaths in women today. The key to the effective and ultimately successful treatment of diseases such as cancer is an early and accurate diagnosis. Driven by the need, in this article, we report for the first time a simple colorimetric and highly sensitive two-photon scattering assay for highly selective and sensitive detection of breast cancer SK-BR-3 cell lines in 100-cells/ml level using multifunctional (monoclonal anti-HER2/c-erb-2 antibody and S6 RNA aptamers conjugated) oval shape gold nanoparticle based nanoconjugate. When multifunctional oval shape gold nanoparticles were mixed with breast cancer SK-BR-3 cell line, a distinct color change occurs and two-photon scattering intensity increases by about 13 times. Experimental data with HaCaT non-cancerous cell line, as well as with MDA-MB-231 breast cancer cell line clearly demonstrated that our assay was highly sensitive to SK-BR-3 and it was able to distinguish from other breast cancer cell line which expresses low levels of HER-2. The mechanism of selectivity and assay’s response change, have been discussed. Our experimental results reported here open up a new possibility of rapid, easy and reliable diagnosis of cancer cell lines by monitoring the colorimetric change and measuring TPS intensity from multifunctional gold nanosystems. PMID:20155973

  19. Nucleoprotein-based indirect enzyme-linked immunosorbent assay(indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus

    Institute of Scientific and Technical Information of China (English)

    Yi; Huang; Youjie; Zhu; Mengshi; Yang; Zhenqing; Zhang; Donglin; Song; Zhiming; Yuan

    2014-01-01

    Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays(ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in antiEbola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G(Ig G) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA’s ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.

  20. A duplex real-time PCR assay based on TaqMan technology for simultaneous detection and differentiation of canine adenovirus types 1 and 2.

    Science.gov (United States)

    Dowgier, Giulia; Mari, Viviana; Losurdo, Michele; Larocca, Vittorio; Colaianni, Maria Loredana; Cirone, Francesco; Lucente, Maria Stella; Martella, Vito; Buonavoglia, Canio; Decaro, Nicola

    2016-08-01

    Canine adenoviruses are a major cause of disease in dogs, coyotes, red foxes and wolves, as well as in other carnivores and marine mammals. Canine adenovirus type 1 (CAdV-1) and canine adenovirus type 2 (CAdV-2) cause infectious canine hepatitis (ICH) and infectious tracheobronchitis (ITB), respectively. In this study, a duplex real-time PCR assay for simultaneous detection and characterisation of CAdV-1 and CAdV-2 was developed by using a single primer pair and virus-specific probes. The assay was validated testing standard DNAs produced on purpose and clinical samples of various matrices known to be positive for CAdV-1, CAdV-2 or both viruses. Precise calculation of DNA loads in samples containing a wide range of viral amounts was allowed by generating a standard curve for absolute quantification. The assay was proven to be highly specific, since no cross-reactions with the different CAdV type was observed, and sensitive, being able to detect less than 10 copies of CAdV-1/CAdV-2 DNA. The low intra-assay and interassay coefficient of variations demonstrated a high repeatability, thus confirming the potential use of this assay for quantitative detection of CAdV-1 and CAdV-2 for rapid diagnosis and epidemiological investigations. PMID:27040113

  1. WASP: a Web-based Allele-Specific PCR assay designing tool for detecting SNPs and mutations

    Directory of Open Access Journals (Sweden)

    Assawamakin Anunchai

    2007-08-01

    Full Text Available Abstract Background Allele-specific (AS Polymerase Chain Reaction is a convenient and inexpensive method for genotyping Single Nucleotide Polymorphisms (SNPs and mutations. It is applied in many recent studies including population genetics, molecular genetics and pharmacogenomics. Using known AS primer design tools to create primers leads to cumbersome process to inexperience users since information about SNP/mutation must be acquired from public databases prior to the design. Furthermore, most of these tools do not offer the mismatch enhancement to designed primers. The available web applications do not provide user-friendly graphical input interface and intuitive visualization of their primer results. Results This work presents a web-based AS primer design application called WASP. This tool can efficiently design AS primers for human SNPs as well as mutations. To assist scientists with collecting necessary information about target polymorphisms, this tool provides a local SNP database containing over 10 million SNPs of various populations from public domain databases, namely NCBI dbSNP, HapMap and JSNP respectively. This database is tightly integrated with the tool so that users can perform the design for existing SNPs without going off the site. To guarantee specificity of AS primers, the proposed system incorporates a primer specificity enhancement technique widely used in experiment protocol. In particular, WASP makes use of different destabilizing effects by introducing one deliberate 'mismatch' at the penultimate (second to last of the 3'-end base of AS primers to improve the resulting AS primers. Furthermore, WASP offers graphical user interface through scalable vector graphic (SVG draw that allow users to select SNPs and graphically visualize designed primers and their conditions. Conclusion WASP offers a tool for designing AS primers for both SNPs and mutations. By integrating the database for known SNPs (using gene ID or rs number

  2. Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples.

    Science.gov (United States)

    Brooks, B W; Devenish, J; Lutze-Wallace, C L; Milnes, D; Robertson, R H; Berlie-Surujballi, G

    2004-10-01

    A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was described and evaluated for use as a presumptive screening test for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. A total of 725 diagnostic samples collected in the field and submitted in Clark's transport enrichment medium (TEM) were analyzed. Cultural isolation of C. fetus was used as the standard for comparison. After incubation of the TEM vials for 4-5 days, fluid was removed for culture and ELISA testing. A sandwich ELISA format was used and the target antigen was C. fetus lipopolysaccharides (LPS). A rabbit anti-C. fetus polyclonal antiserum was used as the capture antibody. Murine monoclonal antibodies (MAbs) to C. fetus serotype A and B LPS core and O-polysaccharides and a goat anti-mouse horseradish peroxidase conjugate were used as detection antibodies. ELISA and culture results for the diagnostic samples were in complete agreement. Seven hundred and eight samples were negative by both tests. All 17 culture positive samples were positive by ELISA with a MAb to LPS core. The ELISA with MAbs to LPS O-polysaccharides detected all culture positive samples with the homologous C. fetus serotype. Sixty-six preputial wash samples from three known C. fetus culture positive bulls were also analyzed. Forty-nine of these samples were positive by both ELISA and culture, 16 were positive by ELISA only, and one was negative by both ELISA and culture. The results indicate that this ELISA is useful as a screening test for the detection of C. fetus in diagnostic samples. PMID:15381269

  3. Nanoparticle-based assay for the detection of virgin argan oil adulteration and its rapid quality evaluation.

    Science.gov (United States)

    Zougagh, M; Salghi, R; Dhair, S; Rios, A

    2011-03-01

    A new method, based on the formation of gold nanoparticles (AuNPs) and spectrophotometric analysis, is proposed to determine total phenolic acids in virgin argan oil samples. These compounds have reducibility due to the presence of the phenol group in their molecular structure, and a redox reaction occurs in the presence of HAuCl(4). The formation of AuNPs as a result of the redox reaction leading to colour changes can be visually observed, resulting in strong light signals that show absorption at 555 nm. As ferulic acid represents more than 95% of the total phenolic acid content of virgin argan oil, this compound was used as an adulteration marker to carry out the screening of samples for the evaluation of the authenticity of virgin argan oils. The analytical features of this screening method also allowed a low precision quantization of the quality of the product. Then, a reference HPLC-DAD/FD method was used to confirm the potential adulterated samples, as well as to provide a detailed quantitative analysis of the most representative phenolic compounds in the samples. The overall screening-confirmation strategy was validated by analysing pure virgin argan oil samples and argan oil samples adulterated with other commercial vegetable oils, demonstrating the reliability of the results. This approach is characterised by its simplicity, low cost, rapid information and responded to practical laboratories needs. PMID:21221541

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

    OpenAIRE

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

    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 hum...

  5. PCR-based detection of Plasmodium in Anopheles mosquitoes: a comparison of a new high-throughput assay with existing methods.

    OpenAIRE

    Bass Chris; Nikou Dimitra; Blagborough Andrew M; Vontas John; Sinden Robert E; Williamson Martin S; Field Linda M

    2008-01-01

    Abstract Background Detection of the four malaria-causing Plasmodium species (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) within their mosquito hosts is an essential component of vector control programmes. Several PCR protocols have been developed for this purpose. Many of these methods, while sensitive, require multiple PCR reactions to detect and discriminate all four Plasmodium species. In this study a new high-throughput assay was developed and compa...

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

    OpenAIRE

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

    2006-01-01

    Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection assays for viruses in water are often repressed by inhibitory factors present in the environment, like humic acids and heavy metals. To study the effect of environmental inhibitors on the performa...

  7. Real-time detection of noroviruses in surface water by use of a broadly reactive nucleic acid sequence-based amplification assay.

    OpenAIRE

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

    2006-01-01

    Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection assays for viruses in water are often repressed by inhibitory factors present in the environment, like humic acids and heavy metals. To study the effect of environmental inhibitors on the performa...

  8. Successful Application of a Direct Detection Slide-Based Sequential Phenotype/Genotype Assay Using Archived Bone Marrow Smears and Paraffin Embedded Tissue Sections

    OpenAIRE

    Bedell, Victoria; Forman, Stephen J.; Gaal, Karl; Pullarkat, Vinod; Weiss, Lawrence M.; Slovak, Marilyn L.

    2007-01-01

    Identification of genetic abnormalities in pathological samples is critical for accurate diagnosis, risk stratification, detection of minimal residual disease, and assessment of response to therapy. Interphase fluorescence in situ hybridization analysis is the standard cytogenetic assay used by many laboratories to detect specific clonal karyotypic aberrations in formalin-fixed, paraffin-embedded tissue. However, direct correlation with immunophenotype or morphology in individual cells is rar...

  9. 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

    2015-12-01

    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

  10. Pistachio (Pistacia vera L.) Detection and Quantification Using a Murine Monoclonal Antibody-Based Direct Sandwich Enzyme-Linked Immunosorbent Assay.

    Science.gov (United States)

    Liu, Changqi; Chhabra, Guneet S; Sathe, Shridhar K

    2015-10-21

    A commercially available direct sandwich enzyme-linked immunosorbent assay (ELISA) (BioFront Technologies, Tallahassee, FL, USA) using murine anti-pistachio monoclonal antibodies (mAbs) as capture and detection antibodies was evaluated. The assay was sensitive (limit of detection = 0.09 ± 0.02 ppm full fat pistachio, linear detection range = 0.5-36 ppm, 50% maximum signal concentration = 7.9 ± 0.7 ppm), reproducible (intra- and inter-assay variability < 24% CV), and rapid (post-extraction testing time ∼ 1.5 h). The target antigen was stable and detectable in whole pistachio seeds subjected to autoclaving (121 °C, 15 psi, 15, 30 min), blanching (100 °C, 5, 10 min), frying (191 °C, 1 min), microwaving (500, 1000 W, 3 min), and dry roasting (140 °C, 30 min; 168 °C, 12 min). No cross-reactivity was observed in 156 food matrices, each tested at 100,000 ppm, suggesting the ELISA to be pistachio specific. The pistachio recovery ranges for spiked (10 ppm) and incurred (10-50000 ppm) food matrices were 93.1-125.6% and 35.7-112.2%, respectively. The assay did not register any false-positive or -negative results among the tested commercial and laboratory prepared samples. PMID:26416205

  11. PNA-based DNA assay with attomolar detection limit based on polygalacturonic acid mediated in-situ deposition of metallic silver on a gold electrode

    International Nuclear Information System (INIS)

    An electrochemical method is described for the ultrasensitive determination of sequence-specific DNA by using polysaccharide-mediated in-situ deposition of metallic silver on a gold electrode. Specifically, a thiolated peptide nucleic acid (PNA) is immobilized on the gold electrode via formation of a self-assembled monolayer. Following hybridization between PNA and target single-stranded DNA (ssDNA), polygalacturonic acid (PGUA) is introduced to the PNA/DNA heteroduplexes via phosphate-zirconium-carboxylate coordination interaction. Next, the vicinal hydroxy groups of the polysaccharide backbone are cleaved and oxidized into aldehyde groups. These act as reductants and convert added silver ions into metallic silver which in-situ deposits on the gold electrode, and then is stripped off electrochemically into a solution of KCl where it is accurately determined by differential pulse voltammetry. Under optimal conditions, this assay exhibits a wide linear response range in that the stripping current is related to the logarithm of the concentration of target ssDNA in the 0.1 fM to 10 pM range, with a detection limit as low as 2.5 aM. The method displays excellent specificity in clearly differentiating mismatched oligonucleotide fragments. We therefore believe that this method has a large potential in terms of genotyping of single-nucleotide polymorphism. Moreover, this kind of signal amplification possesses a very large capability with respect to ultrasensitive quantitation of low-abundant biomarkers. (author)

  12. Real‑time, fast neutron detection for stimulated safeguards assay

    International Nuclear Information System (INIS)

    The advent of low‑hazard organic liquid scintillation detectors and real‑time pulse‑shape discrimination (PSD) processing has suggested a variety of modalities by which fast neutrons, as opposed to neutrons moderated prior to detection, can be used directly to benefit safeguards needs. In this paper we describe a development of a fast‑neutron based safeguards assay system designed for the assessment of 235U content in fresh fuel. The system benefits from real‑time pulse‑shape discrimination processing and auto‑calibration of the detector system parameters to ensure a rapid and effective set‑up protocol. These requirements are essential in optimising the speed and limit of detection of the fast neutron technique, whilst minimising the intervention needed to perform the assay.

  13. Development and comparison of a Primer-Probe Energy Transfer based assay and a 5' conjugated Minor Groove Binder assay for sensitive real-time PCR detection of infectious laryngotracheitis virus.

    Science.gov (United States)

    McMenamy, M J; McKillen, J; Hjertner, B; Kiss, I; Yacoub, A; Leijon, M; Duffy, C; Belák, S; Welsh, M; Allan, G

    2011-08-01

    In this study the design and development of two real-time PCR assays for the rapid, sensitive and specific detection of infectious laryngotracheitis virus (ILTV) DNA is described. A Primer-Probe Energy Transfer (PriProET) assay and 5' conjugated Minor Groove Binder (MGB) method are compared and contrasted. Both have been designed to target the thymidine kinase gene of the ILTV genome. Both PriProET and MGB assays are capable of detecting 20 copies of a DNA standard per reaction and are linear from 2×10(8) to 2×10(2)copies/μl. Neither PriProET, nor MGB reacted with heterologous herpesviruses, indicating a high specificity of the two methods as novel tools for virus detection and identification. This study demonstrates the suitability of PriProET and 5' conjugated MGB probes as real-time PCR chemistries for the diagnosis of respiratory diseases caused by ILTV. PMID:21539859

  14. A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins.

    Directory of Open Access Journals (Sweden)

    Preeti Sharma

    Full Text Available Staphylococcal and streptococcal exotoxins, also known as superantigens, mediate a range of diseases including toxic shock syndrome, and they exacerbate skin, pulmonary and systemic infections caused by these organisms. When present in food sources they can cause enteric effects commonly known as food poisoning. A rapid, sensitive assay for the toxins would enable testing of clinical samples and improve surveillance of food sources. Here we developed a bead-based, two-color flow cytometry assay using single protein domains of the beta chain of T cell receptors engineered for high-affinity for staphylococcal (SEA, SEB and TSST-1 and streptococcal (SpeA and SpeC toxins. Site-directed biotinylated forms of these high-affinity agents were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD50 values of 400 pg/ml (SEA, 3 pg/ml (SEB, 25 pg/ml (TSST-1, 6 ng/ml (SpeA, and 100 pg/ml (SpeC. These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. A multiplex format of the assay showed reduced sensitivity due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected toxins in supernatants derived from cultures of Staphylococcus aureus. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.

  15. Assay of angiotensin I-converting enzyme-inhibiting activity based on the detection of 3-hydroxybutyrate with water-soluble tetrazolium salt.

    Science.gov (United States)

    Lam, Le Hoang; Shimamura, Tomoko; Manabe, Sachiyo; Ishiyama, Munetaka; Ukeda, Hiroyuki

    2008-08-01

    A newly synthesized substrate, 3-hydroxybutyrylglycyl-glycyl-glycine (3HB-GGG), was applied to the assay of ACE-inhibiting activity to overcome the smaller selectivity and sensitivity of the conventional method. In this study, an ACE-inhibiting assay was improved by the use of a water-soluble tetrazolium salt, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt (WST-1), for the detection of 3-hydroxybutyrate, derived from 3HB-GGG. The optimized conditions were as follows: 0.333 mM NAD(+), 0.333 mM WST-1, 0.1 mM EDTA, 0.633 U ml(-1) diaphorase, and 0.700 U ml(-1) 3-hydroxybutyrate dehydrogenase. The developed assay was efficiently applicable to evaluate the ACE-inhibiting activity of practical ACE inhibitors. PMID:18689950

  16. Nonradioactive PCR-enzyme-linked immunosorbent assay method for detection of human cytomegalovirus DNA.

    OpenAIRE

    Allen, R. D.; Pellett, P E; Stewart, J A; Koopmans, M

    1995-01-01

    We developed a rapid, sensitive, and specific PCR-based assay for human cytomegalovirus (HCMV). The assay includes primer and probe sequences derived from conserved HCMV nucleotide sequences and nonradioactive hybridization-confirmation. The assay detected between 10 and 100 viral genomes. All HCMV clinical isolates tested (39 of 39) gave positive reactions.

  17. Clinical Impact of Laboratory Implementation of Verigene BC-GN Microarray-Based Assay for Detection of Gram-Negative Bacteria in Positive Blood Cultures.

    Science.gov (United States)

    Walker, Tamar; Dumadag, Sandrea; Lee, Christine Jiyoun; Lee, Seung Heon; Bender, Jeffrey M; Cupo Abbott, Jennifer; She, Rosemary C

    2016-07-01

    Gram-negative bacteremia is highly fatal, and hospitalizations due to sepsis have been increasing worldwide. Molecular tests that supplement Gram stain results from positive blood cultures provide specific organism information to potentially guide therapy, but more clinical data on their real-world impact are still needed. We retrospectively reviewed cases of Gram-negative bacteremia in hospitalized patients over a 6-month period before (n = 98) and over a 6-month period after (n = 97) the implementation of a microarray-based early identification and resistance marker detection system (Verigene BC-GN; Nanosphere) while antimicrobial stewardship practices remained constant. Patient demographics, time to organism identification, time to effective antimicrobial therapy, and other key clinical parameters were compared. The two groups did not differ statistically with regard to comorbid conditions, sources of bacteremia, or numbers of intensive care unit (ICU) admissions, active use of immunosuppressive therapy, neutropenia, or bacteremia due to multidrug-resistant organisms. The BC-GN panel yielded an identification in 87% of Gram-negative cultures and was accurate in 95/97 (98%) of the cases compared to results using conventional culture. Organism identifications were achieved more quickly post-microarray implementation (mean, 10.9 h versus 37.9 h; P < 0.001). Length of ICU stay, 30-day mortality, and mortality associated with multidrug-resistant organisms were significantly lower in the postintervention group (P < 0.05). More rapid implementation of effective therapy was statistically significant for postintervention cases of extended-spectrum beta-lactamase-producing organisms (P = 0.049) but not overall (P = 0.12). The Verigene BC-GN assay is a valuable addition for the early identification of Gram-negative organisms that cause bloodstream infections and can significantly impact patient care, particularly when resistance markers are detected. PMID:27098961

  18. Two Types of Assays for Detecting Frog Sperm Chemoattraction

    OpenAIRE

    Burnett, Lindsey A.; Tholl, Nathan; Chandler, Douglas E.

    2011-01-01

    Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assay...

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

    Directory of Open Access Journals (Sweden)

    Wan Zhixiang

    2005-07-01

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

  20. PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples.

    Science.gov (United States)

    Kox, L F; van Leeuwen, J; Knijper, S; Jansen, H M; Kolk, A H

    1995-01-01

    A PCR and a reverse cross blot hybridization assay were developed for the detection and identification of mycobacteria in clinical samples. The PCR amplifies a part of the DNA coding for 16S rRNA with a set of primers that is specific for the genus Mycobacterium and that flanks species-specific sequences within the genes coding for 16S rRNA. The PCR product is analyzed in a reverse cross blot hybridization assay with probes specific for M. tuberculosis complex (pTub1), M. avium (pAvi3), M. intracellulare (pInt5 and pInt7), M. kansasii complex-M. scrofulaceum complex (pKan1), M. xenopi (pXen1), M. fortuitum (pFor1), M. smegmatis (pSme1), and Mycobacterium spp. (pMyc5a). The PCR assay can detect 10 fg of DNA, the equivalent of two mycobacteria. The specificities of the probes were tested with 108 mycobacterial strains (33 species) and 31 nonmycobacterial strains (of 17 genera). The probes pAvi3, pInt5, pInt7, pKan1, pXen1, and pMyc5a were specific. With probes pTub1, pFor1, and pSme1, slight cross hybridization occurred. However, the mycobacterial strains from which the cross-hybridizing PCR products were derived belonged to nonpathogenic or nonopportunistic species which do not occur in clinical samples. The test was used on 31 different clinical specimens obtained from patients suspected of having mycobacterial disease, including a patient with a double mycobacterial infection. The samples included sputum, bronchoalveolar lavage, tissue biopsy samples, cerebrospinal fluid, pus, peritoneal fluid, pleural fluid, and blood. The results of the PCR assay agreed with those of conventional identification methods or with clinical data, showing that the test can be used for the direct and rapid detection and identification of mycobacteria in clinical samples. PMID:8586707

  1. Planar optical waveguide based sandwich assay sensors and processes for the detection of biological targets including protein markers, pathogens and cellular debris

    Science.gov (United States)

    Martinez, Jennifer S.; Swanson, Basil I.; Grace, Karen M.; Grace, Wynne K.; Shreve, Andrew P.

    2009-06-02

    An assay element is described including recognition ligands bound to a film on a single mode planar optical waveguide, the film from the group of a membrane, a polymerized bilayer membrane, and a self-assembled monolayer containing polyethylene glycol or polypropylene glycol groups therein and an assay process for detecting the presence of a biological target is described including injecting a biological target-containing sample into a sensor cell including the assay element, with the recognition ligands adapted for binding to selected biological targets, maintaining the sample within the sensor cell for time sufficient for binding to occur between selected biological targets within the sample and the recognition ligands, injecting a solution including a reporter ligand into the sensor cell; and, interrogating the sample within the sensor cell with excitation light from the waveguide, the excitation light provided by an evanescent field of the single mode penetrating into the biological target-containing sample to a distance of less than about 200 nanometers from the waveguide thereby exciting the fluorescent-label in any bound reporter ligand within a distance of less than about 200 nanometers from the waveguide and resulting in a detectable signal.

  2. A multi-detection assay for malaria transmitting mosquitoes.

    Science.gov (United States)

    Lee, Yoosook; Weakley, Allison M; Nieman, Catelyn C; Malvick, Julia; Lanzaro, Gregory C

    2015-01-01

    The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays. PMID:25867057

  3. 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

    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. The fea...... assessors that support bio-traceability models....

  4. Comparison of use of enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes for simultaneous detection of Entamoeba histolytica and E. dispar.

    OpenAIRE

    Mirelman, D; Nuchamowitz, Y; Stolarsky, T

    1997-01-01

    A comparison of the use of three commercially available enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes to detect and differentiate Entamoeba histolytica from E. dispar was carried out. Only the Techlab kit did not cross-react with E. dispar antigens, but it was 100 times less sensitive than PCR in detection of and differentiation between the two types of Entamoeba.

  5. Detection of radiation-induced apoptosis using the comet assay

    International Nuclear Information System (INIS)

    The electrophoresis pattern of apoptotic cells detected by the comet assay has a characteristic small head and spread tail. This image has been referred to as an apoptotic comet, but it has not been previously proven to be apoptotic cells by any direct method. In order to identify this image obtained by the comet assay as corresponding to an apoptotic cell, the frequency of appearance of apoptosis was examined using CHO-K1 and L5178Y cells which were exposed to gamma irradiation. As a method for detecting apoptosis, the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay was used. When the frequency of appearance of apoptotic cells following gamma irradiation was observed over a period of time, there was a significant increase in appearance of apoptosis when using the TUNEL assay. However, there was only a slight increase when using the comet assay. In order to verify the low frequency of appearance of apoptosis when using the comet assay, we attempted to use the TUNEL assay to satin the apoptotic comets detected in the comet assay. The apoptotic comets were TUNEL positive and the normal comets were TUNEL negative. This indicates that the apoptotic comets were formed from DNA fragments with 3'-hydroxy ends that are generated as cells undergo apoptosis. Therefore, it was understood that the characteristic pattern of apoptotic comets detected by the comet assay corresponds to cells undergoing apoptosis. (author)

  6. Determining lower limits of detection of digital PCR assays for cancer-related gene mutations

    OpenAIRE

    Milbury, Coren A.; Qun Zhong; Jesse Lin; Miguel Williams; Jeff Olson; Link, Darren R.; Brian Hutchison

    2014-01-01

    Digital PCR offers very high sensitivity compared to many other technologies for processing molecular detection assays. Herein, a process is outlined for determining the lower limit of detection (LoD) of two droplet-based digital PCR assays for point mutations of the epidermal growth factor receptor (EGFR) gene. Hydrolysis probe mutation-detection assays for EGFR p.L858R and p.T790M mutations were characterized in detail. Furthermore, sixteen additional cancer-related mutation assays were exp...

  7. Development and Validation of LNA-Based Quantitative Real-Time PCR Assays for Detection and Identification of the Root-Knot Nematode Meloidogyne enterolobii in Complex DNA Backgrounds.

    Science.gov (United States)

    Kiewnick, Sebastian; Frey, Jürg E; Braun-Kiewnick, Andrea

    2015-09-01

    Meloidogyne enterolobii is a quarantine root-knot nematode posing a major threat to agricultural production systems worldwide. It attacks many host plants, including important agricultural crops, ornamentals, and trees. M. enterolobii is a highly virulent and pathogenic root-knot nematode species, able to reproduce on plants resistant to other Meloidogyne spp. Significant crop damage has been reported in Asia, South America, Africa, the United States, France, and greenhouses in Switzerland. To identify potential introduction pathways and ensure appropriate phytosanitary measures and management strategies, accurate detection and identification tools are needed. Therefore, two real-time quantitative polymerase chain reaction (PCR) assays based on the second intergenic spacer region of the ribosomal DNA cistron and the cytochrome oxidase c subunit I (COI) gene using locked nucleic acid probes were developed and validated for fast and reliable detection and identification of M. enterolobii. Analytical specificity was confirmed with 16 M. enterolobii populations, 16 populations of eight closely related Meloidogyne spp., and four species from other nematode genera. Optimizing and testing the assays on two real-time PCR platforms revealed an analytical sensitivity of one juvenile in a background of 1,000 nematodes and the intended limit of detection of one juvenile per 100 ml of soil. Both assays performed equally well, with the COI-based assay showing a slightly better performance concerning detection of M. enterolobii target DNA in complex DNA backgrounds. PMID:25775103

  8. Magnetic Barcode Assay for Genetic Detection of Pathogens

    Science.gov (United States)

    Liong, Monty; Hoang, Anh N.; Chung, Jaehoon; Gural, Nil; Ford, Christopher B.; Min, Changwook; Shah, Rupal R.; Ahmad, Rushdy; Fernandez-Suarez, Marta; Fortune, Sarah M.; Toner, Mehmet; Lee, Hakho; Weissleder, Ralph

    2013-01-01

    The task of rapidly identifying patients infected with Mycobacterium tuberculosis (MTB) in resource-constrained environments remains a challenge. A sensitive and robust platform that does not require bacterial isolation or culture is critical in making informed diagnostic and therapeutic decisions. Here we introduce a platform for the detection of nucleic acids based on a magnetic barcoding strategy. PCR-amplified mycobacterial genes are sequence-specifically captured on microspheres, labeled by magnetic nanoprobes, and detected by nuclear magnetic resonance. All components are integrated into a single, small fluidic cartridge for streamlined on-chip operation. We use this platform to detect MTB and identify drug-resistance strains from mechanically processed sputum samples within 2.5 hours. The specificity of the assay is confirmed by a panel of clinically relevant non-MTB bacteria, and the clinical utility is demonstrated by the measurements in MTB-positive patient specimens. Combined with portable systems, the magnetic barcode assay holds promise to become a sensitive, high-throughput, and low-cost platform for point-of-care diagnostics. PMID:23612293

  9. Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease.

    Directory of Open Access Journals (Sweden)

    Panat Anuracpreeda

    Full Text Available Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden.In this study, we have produced a monoclonal antibody (MoAb against recombinant F. gigantica cathepsin L1 (rFgCatL1, and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA and immunochromatographic (IC test for rapid detection of circulating cathepsin L1 protease (CatL1 in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities.The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA, and 93%, 100% and 98.2% (for IC test, while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA, and 96.7%, 100% and 99.1% (for IC test.These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.

  10. Colloidal gold-based indirect competitive immunochromatographic assay for rapid detection of bioactive isoflavone glycosides daidzin and genistin in soy products.

    Science.gov (United States)

    Sakamoto, Seiichi; Yusakul, Gorawit; Pongkitwitoon, Benyakan; Tanaka, Hiroyuki; Morimoto, Satoshi

    2016-03-01

    Daidzin (DZ) and genistin (GEN) are two major soy isoflavone glycosides isolated from soybeans. Soy products containing isoflavones have recently been widely accepted for commercial use. However, the Japanese Government has suggested that soy isoflavone intake should be limited because of their estrogenic effects due to their interactions with estrogen receptors. In this study, we established a one-step indirect competitive immunochromatographic assay (ICA) for rapid and sensitive detection of total isoflavone glycosides (DZ and GEN) using gold nanoparticles conjugated with a monoclonal antibody against DZ. This assay was able to be completed in 15min following the immersion of a test strip in an analyte solution. Furthermore, the limit of detection for the total amount of isoflavone glycosides was ∼125ngmL(-1). Considering that the major soy isoflavone glycosides found in soy products are DZ and GEN, this study demonstrates the potential use of ICA for the assessments of over consumption of isoflavones in soy supplements and foods, which would increase the safe dietary intake of soy products. PMID:26471543

  11. Detection of irradiated onion by means of the comet assay

    International Nuclear Information System (INIS)

    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 60Co 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)

  12. 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)

  13. Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia.

    Directory of Open Access Journals (Sweden)

    Syed M Jamal

    Full Text Available Rapid and accurate diagnosis of foot-and-mouth disease (FMD and virus serotyping are of paramount importance for control of this disease in endemic areas where vaccination is practiced. Ideally this virus characterization should be achieved without the need for virus amplification in cell culture. Due to the heterogeneity of FMD viruses (FMDVs in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysable probe-based real time reverse transcription quantitative polymerase chain reaction (RT-qPCR assays were developed for specific detection and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnostic assays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of O-PanAsia, A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08. In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 sublineage were also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV and detected the RNA from the target viruses with cycle threshold (CT values comparable with those obtained with the serotype-independent pan-FMDV diagnostic assays. No cross-reactivity was observed in these assays between the heterotypic viruses circulating in the region. The assays reported here have higher diagnostic sensitivity (100% each for serotypes O and Asia-1, and 92% [95% CI = 81.4-100%] for serotype A positive samples and specificity (100% each for serotypes O, A and Asia-1 positive samples for the viruses currently circulating in West Eurasia compared to the serotyping assays reported earlier. Comparisons of the sequences of the primers and probes used in these assays and the corresponding regions of the circulating viruses provided explanations for

  14. Mitochondrial base excision repair assays

    DEFF Research Database (Denmark)

    Maynard, Scott; de Souza-Pinto, Nadja C; Scheibye-Knudsen, Morten;

    2010-01-01

    The main source of mitochondrial DNA (mtDNA) damage is reactive oxygen species (ROS) generated during normal cellular metabolism. The main mtDNA lesions generated by ROS are base modifications, such as the ubiquitous 8-oxoguanine (8-oxoG) lesion; however, base loss and strand breaks may also occur....... Many human diseases are associated with mtDNA mutations and thus maintaining mtDNA integrity is critical. All of these lesions are repaired primarily by the base excision repair (BER) pathway. It is now known that mammalian mitochondria have BER, which, similarly to nuclear BER, is catalyzed by DNA...

  15. A rapid and highly sensitive protocol for the detection of Escherichia coli O157:H7 based on immunochromatography assay combined with the enrichment technique of immunomagnetic nanoparticles

    Directory of Open Access Journals (Sweden)

    Qi H

    2011-11-01

    Full Text Available Hui Qi1, Zhen Zhong1, Han-Xin Zhou1, Chun-Yan Deng1, Hai Zhu2, Jin-Feng Li2, Xi-Li Wang2, Fu-Rong Li1,31Clinical Medical Research Center, The Second Clinical Medical College (Shenzhen People's Hospital, Jinan University, 2Shenzhen Bioeasy Biotechnologies Co, Ltd, 3Shenzhen Institute of Gerontology, Shenzhen, People's Republic of ChinaBackground: Escherichia coli O157:H7 (E. coli O157:H7 is an important pathogenic bacterium that threatens human health. A rapid, simple, highly sensitive, and specific method for the detection of E. coli O157:H7 is necessary.Methods: In the present study, immunomagnetic nanoparticles (IMPs were prepared with nanopure iron as the core, coated with E. coli O157:H7 polyclonal antibodies. These IMPs were used in combination with immunochromatographic assay (ICA and used to establish highly sensitive and rapid kits (IMPs+ICA to detect E. coli O157:H7. The kits were then used to detect E. coli O157:H7 in 150 food samples and were compared with conventional ICA to evaluate their efficacy.Results: The average diameter of IMPs was 56 nm and the amount of adsorbed antibodies was 106.0 µg/mg. The sensitivity of ICA and IMPs+ICA was 105 colony-forming units/mL and 103 CFUs/mL, respectively, for purified E. coli O157:H7 solution. The sensitivity of IMPs+ICA was increased by two orders, and its specificity was similar to ICA.Conclusion: The kits have the potential to offer important social and economic benefits in the screening, monitoring, and control of food safety.Keywords: colloidal gold, immunomagnetic nanoparticles, Escherichia coli O157:H7, immunochromatographic assay

  16. Advances in Assays and Analytical Approaches for Botulinum Toxin Detection

    Energy Technology Data Exchange (ETDEWEB)

    Grate, Jay W.; Ozanich, Richard M.; Warner, Marvin G.; Bruckner-Lea, Cindy J.; Marks, James D.

    2010-08-04

    Methods to detect botulinum toxin, the most poisonous substance known, are reviewed. Current assays are being developed with two main objectives in mind: 1) to obtain sufficiently low detection limits to replace the mouse bioassay with an in vitro assay, and 2) to develop rapid assays for screening purposes that are as sensitive as possible while requiring an hour or less to process the sample an obtain the result. This review emphasizes the diverse analytical approaches and devices that have been developed over the last decade, while also briefly reviewing representative older immunoassays to provide background and context.

  17. Reporter Gene Assay for Detection of Shellfish Toxins

    Institute of Scientific and Technical Information of China (English)

    WEI-DONG YANG; MIN-YI WU; JIE-SHENG LIU; XI-CHUN PENG; HONG-YE LI

    2009-01-01

    Objective To explore the potential reporter gene assay for the detection of sodium channel-specific toxins in shellfish as an alternative for screening harmful algal bloom (HAB) toxins, considering the fact that the existing methods including HPLC and bioassay are inappropriate for identifying HAB toxins which poses a serious problem on human health and shellfish industry. Methods A reporter plasmid pEGFP-c-fos containing c-fos promoter and EGFP was constructed and transfected into T24 cells using LipofectAMINE 2000. Positive transfectants were screened by G418 to produce a pEGFP-c-fos-T24 cell line. After addition of increasing neurotoxic shellfish poison (NSP) or GTX2,3, primary components of paralytic shellfish poison (PSP), changes in expression of EGFP in the cell line were observed under a laser scanning confocal microscope and quantified with Image-pro Plus software. Results Dose-dependent changes in the intensity of green fluorescence were observed for NSP in a range from 0 to 10 ng/mL and for GTX 2,3 from 0 to 16 ng/mL. Conclusion pEGFP-c-fos-T24 can be applied in detecting HAB toxins, and cell-based assay can be used as an alternative for screening sodium channel-specific HAB toxins.

  18. ASSAYS FOR DETECTION OF TELOMERASE ACTIVITY

    OpenAIRE

    Skvortsov, D.; Zvereva, M.; Shpanchenko, O.; Dontsova, O.

    2011-01-01

    Progressive loss of the telomeric ends of chromosomes caused by the semi-conservative mechanism of DNA replication is an important timing mechanism which controls the number of cells doubling. Telomerase is an enzyme which elongates one chain of the telomeric DNA and compensates for its shortening during replication. Therefore, telomerase activity serves as a proliferation marker. Telomerase activity is not detected in most somatic cells, with the exception of embryonic tissues, stem cells, a...

  19. Development and evaluation of a one-step SYBR-Green I-based real-time RT-PCR assay for the detection and quantification of Chikungunya virus in human, monkey and mosquito samples.

    Science.gov (United States)

    Ummul Haninah, A; Vasan, S S; Ravindran, T; Chandru, A; Lee, H L; Shamala Devi, S

    2010-12-01

    This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/μl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999. PMID:21399603

  20. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish

    Science.gov (United States)

    Hardison, D. Ransom; Holland, William C.; McCall, Jennifer R.; Bourdelais, Andrea J.; Baden, Daniel G.; Darius, H. Taiana; Chinain, Mireille; Tester, Patricia A.; Shea, Damian; Flores Quintana, Harold A.; Morris, James A.; Litaker, R. Wayne

    2016-01-01

    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

  1. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

    Science.gov (United States)

    Hardison, D Ransom; Holland, William C; McCall, Jennifer R; Bourdelais, Andrea J; Baden, Daniel G; Darius, H Taiana; Chinain, Mireille; Tester, Patricia A; Shea, Damian; Flores Quintana, Harold A; Morris, James A; Litaker, R Wayne

    2016-01-01

    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

  2. Development of a nanogold-based immunochromatographic assay for detection of morphine in urine using the Amor-HK16 monoclonal antibody.

    Science.gov (United States)

    Dehghannezhad, Ardeshir; Paknejad, Maliheh; Rasaee, Mohammad Javad; Omidfar, Kobra; Seyyed Ebrahimi, Shadi Sadat; Ghahremani, Hossein

    2012-12-01

    A simple, rapid competitive immunochromatography (ICG) strip test was developed to detect morphine in urine samples using a monoclonal antibody produced in-house and conjugated to gold nanoparticles. Hybridoma cells were cultured and the Amor-HK16 monoclonal antibody against morphine was obtained from the supernatant after purification by salting out and passing through a Protein G-Agarose affinity column. Morphine was obtained from morphine sulfate and a C6-hemisuccinate derivative of morphine was prepared, conjugated to bovine serum albumin, and immobilized to a nitrocellulose membrane as the test line. Goat anti-mouse antibody was used as a binder in the control line in the detection zone of the strip. Colloidal gold particles of diameter approximately 20 nm were prepared and conjugated to the monoclonal antibody. The detection limit of the test strip was found to be 2000 ng/mL of morphine in urine samples. Reliability was determined by performing the ICG test on 103 urine samples and comparing the results with those obtained by thin-layer chromatography. The sensitivity of the test was 100%, and the analysis time for the assay was approximately 5 min. The new ICG method was adequately sensitive and accurate for the rapid screening of morphine in urine. PMID:23244319

  3. Towards a high throughput droplet-based agglutination assay

    KAUST Repository

    Kodzius, Rimantas

    2013-10-22

    This work demonstrates the detection method for a high throughput droplet based agglutination assay system. Using simple hydrodynamic forces to mix and aggregate functionalized microbeads we avoid the need to use magnetic assistance or mixing structures. The concentration of our target molecules was estimated by agglutination strength, obtained through optical image analysis. Agglutination in droplets was performed with flow rates of 150 µl/min and occurred in under a minute, with potential to perform high-throughput measurements. The lowest target concentration detected in droplet microfluidics was 0.17 nM, which is three orders of magnitude more sensitive than a conventional card based agglutination assay.

  4. Emerging Technologies and Generic Assays for the Detection of Anti-Drug Antibodies

    Science.gov (United States)

    Elango, Chinnasamy

    2016-01-01

    Anti-drug antibodies induced by biologic therapeutics often impact drug pharmacokinetics, pharmacodynamics response, clinical efficacy, and patient safety. It is critical to assess the immunogenicity risk of potential biotherapeutics in producing neutralizing and nonneutralizing anti-drug antibodies, especially in clinical phases of drug development. Different assay methodologies have been used to detect all anti-drug antibodies, including ELISA, radioimmunoassay, surface plasmon resonance, and electrochemiluminescence-based technologies. The most commonly used method is a bridging assay, performed in an ELISA or on the Meso Scale Discovery platform. In this report, we aim to review the emerging new assay technologies that can complement or address challenges associated with the bridging assay format in screening and confirmation of ADAs. We also summarize generic anti-drug antibody assays that do not require drug-specific reagents for nonclinical studies. These generic assays significantly reduce assay development efforts and, therefore, shorten the assay readiness timeline. PMID:27556048

  5. Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia

    DEFF Research Database (Denmark)

    Jamal, Syed M.; Belsham, Graham

    2015-01-01

    . Due to the heterogeneity of FMD viruses (FMDVs) in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysableprobe-based real time reverse transcription quantitative polymerase chain reaction (RTqPCR) assays were developed for specific detection...... and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnosticassays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of OPan......Asia,A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08)). In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 subline-agewere also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV...

  6. Evaluation of a feline-specific multiplex, bead-based assay for detection of cytokines, chemokines, growth factors, and other immunologically active proteins in serum and plasma samples from cats.

    Science.gov (United States)

    Halpin, Rachel E; Saunders, Rebecca S; Thompson, Beverly J; Rohde Newgent, Allison S; Amorim, Juliana; Melillo, Gabrielle N; DeClue, Amy E

    2016-05-01

    OBJECTIVE To evaluate a feline-specific multiplex, bead-based assay system for detection of recombinant and native proteins in serum samples and in EDTA-treated and heparinized plasma samples. SAMPLE Serum samples and EDTA-treated and heparinized plasma samples from 30 sick cats and 9 healthy client-owned cats and heparinized whole blood samples from 5 healthy purpose-bred cats. PROCEDURES Ability of the assay system to detect 19 recombinant and native immunologically active proteins in plasma and serum samples from healthy and purpose-bred cats was evaluated via spike-and-recovery tests, assessments of inter- and intra-assay variation, linearity results, and leukocyte stimulation. Effects of various concentrations of heparin and serum matrix solution on percentages of analytes recovered were also evaluated. Analyte concentrations in samples from healthy and sick cats were measured and compared between groups. RESULTS Percentages of analytes recovered were unsatisfactory for most assays. Serum and heparinized plasma samples yielded better recovery results than did EDTA-treated plasma samples. Use of serum matrix solution did not improve results. Use of heparin concentrations greater than the recommended range affected the results. Linearity of results was difficult to assess because of the poor recovery. For the analytes that were recovered sufficiently for assessment, linearity appeared to be reasonable despite the limited detection. CONCLUSIONS AND CLINICAL RELEVANCE Poor percentages of analytes recovered and adverse effects of sample protein matrix limited the usefulness of the multiplex, bead-based assay system for measurement of immunologically active proteins in solutions with high protein content; however, recovery results were fairly linear, potentially allowing evaluation of feline plasma or serum samples with high analyte concentrations. PMID:27111017

  7. Gel Mobility Shift Assays to Detect Protein–RNA Interactions

    OpenAIRE

    Yakhnin, Alexander V.; Yakhnin, Helen; Babitzke, Paul

    2012-01-01

    The gel mobility shift assay is a powerful technique for detecting and quantifying protein–RNA interactions. While other techniques such as filter binding and isothermal titration calorimetry (ITC) are available for quantifying protein–RNA interactions, gel shift analysis provides the added advantage that you can visualize the protein–RNA complexes. In the gel shift assay, protein–RNA complexes are typically separated from the unbound RNA using native polyacrylamide gels in Tris/borate/EDTA b...

  8. A Comparison of In-House Real-Time LAMP Assays with a Commercial Assay for the Detection of Pathogenic Bacteria

    OpenAIRE

    Deguo Wang; Yongzhen Wang; Fugang Xiao; Weiyun Guo; Yongqing Zhang; Aiping Wang; Yanhong Liu

    2015-01-01

    Molecular detection of bacterial pathogens based on LAMP methods is a faster and simpler approach than conventional culture methods. Although different LAMP-based methods for pathogenic bacterial detection are available, a systematic comparison of these different LAMP assays has not been performed. In this paper, we compared 12 in-house real-time LAMP assays with a commercialized kit (Isothermal Master Mix) for the detection of Listeria monocytogenes, Salmonella spp, Staphylococcus aureus, E...

  9. Comparison of Two Rapid Diagnostic Assays for Detection of Immunoglobulin M Antibodies to Dengue Virus

    OpenAIRE

    Wu, Shuenn-Jue L.; Paxton, Helene; Hanson, Barbara; Kung, Cheryl G.; Chen, Timothy B.; Rossi, Cindy; David W Vaughn; Murphy, Gerald S.; Hayes, Curtis G.

    2000-01-01

    Two easy-to-use commercial diagnostic assays, a dipstick enzyme-linked immunosorbent assay (ELISA) (Integrated Diagnostics, Baltimore, Md.) and an immunochromatographic card assay (PanBio, Brisbane, Australia) were evaluated for detection of immunoglobulin M (IgM) antibody to dengue virus with an in-house IgM antibody capture microplate ELISA as a reference assay. The dipstick ELISA was based on the indirect-ELISA format using dengue 2 virus as the only antigen and enzyme-labeled goat anti-hu...

  10. A novel fluorescence-based cellular permeability assay.

    Science.gov (United States)

    Chandra, Ankur; Barillas, Samuel; Suliman, Ahmed; Angle, Niren

    2007-04-10

    Vascular permeability is a pathologic process in many disease states ranging from metastatic progression of malignancies to ischemia-reperfusion injury. In order to more precisely study tissue, and more specifically cell layer permeability, our goal was to create a fluorescence-based assay which could quantify permeability without radioactivity or electrical impedance measurements. Human aortic endothelial cells were grown in monolayer culture on Costar-Transwell clear polyester membrane 6-well cell culture inserts. After monolayer integrity was confirmed, vascular endothelial growth factor (VEGF(165)) at varying concentrations with a fixed concentration of yellow-green fluorescent 0.04 microm carboxylate-modified FluoSpheres microspheres were placed in the luminal chamber and incubated for 24 h. When stimulated with VEGF(165) at 20, 40, 80, and 100 ng/ml, this assay system was able to detect increases in trans-layer flux of 8.2+/-2.4%, 16.0+/-3.7%, 41.5+/-4.9%, and 58.6+/-10.1% for each concentration, respectively. This represents the first fluorescence-based permeability assay with the sensitivity to detect changes in the permeability of a cell layer to fluid flux independent of protein flux; as well as being simpler and safer than previous radioactive-and impedance-based permeability assays. With the application of this in vitro assay to a variety of pathologic conditions, both the dynamics and physiology relating to cellular permeability can be more fully investigated. PMID:16962665

  11. An immunofluorescence assay to detect soybean rust urediniospores

    Science.gov (United States)

    An indirect immunofluorescence assay (IF) was developed to detect urediniospores of Phakopsora pachyrhizi and P. meibomiae, utilizing rabbit polyclonal antisera produced in response to intact non-germinated (SBR1A) or germinated (SBR2) urediniospores of P. pachyrhizi. Both antisera were specific to ...

  12. Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Functional Assay for Rapid Detection of Resistance against β-Lactam Antibiotics

    OpenAIRE

    Sparbier, Katrin; Schubert, Sören; Weller, Ulrich; Boogen, Christiane; Kostrzewa, Markus

    2012-01-01

    Resistance against β-lactam antibiotics is a growing challenge for managing severe bacterial infections. The rapid and cost-efficient determination of β-lactam resistance is an important prerequisite for the choice of an adequate antibiotic therapy. β-Lactam resistance is based mainly on the expression/overexpression of β-lactamases, which destroy the central β-lactam ring of these drugs by hydrolysis. Hydrolysis corresponds to a mass shift of +18 Da, which can be easily detected by matrix-as...

  13. A reverse transcription loop-mediated isothermal amplification assay optimized to detect multiple HIV subtypes.

    Directory of Open Access Journals (Sweden)

    Karen E Ocwieja

    Full Text Available Diagnostic methods for detecting and quantifying HIV RNA have been improving, but efficient methods for point-of-care analysis are still needed, particularly for applications in resource-limited settings. Detection based on reverse-transcription loop-mediated isothermal amplification (RT-LAMP is particularly useful for this, because when combined with fluorescence-based DNA detection, RT-LAMP can be implemented with minimal equipment and expense. Assays have been developed to detect HIV RNA with RT-LAMP, but existing methods detect only a limited subset of HIV subtypes. Here we report a bioinformatic study to develop optimized primers, followed by empirical testing of 44 new primer designs. One primer set (ACeIN-26, targeting the HIV integrase coding region, consistently detected subtypes A, B, C, D, and G. The assay was sensitive to at least 5000 copies per reaction for subtypes A, B, C, D, and G, with Z-factors of above 0.69 (detection of the minor subtype F was found to be unreliable. There are already rapid and efficient assays available for detecting HIV infection in a binary yes/no format, but the rapid RT-LAMP assay described here has additional uses, including 1 tracking response to medication by comparing longitudinal values for a subject, 2 detecting of infection in neonates unimpeded by the presence of maternal antibody, and 3 detecting infection prior to seroconversion.

  14. Development of a VHH-Based Erythropoietin Quantification Assay

    DEFF Research Database (Denmark)

    Kol, Stefan; Beuchert Kallehauge, Thomas; Adema, Simon;

    2015-01-01

    Erythropoietin (EPO) quantification during cell line selection and bioreactor cultivation has traditionally been performed with ELISA or HPLC. As these techniques suffer from several drawbacks, we developed a novel EPO quantification assay. A camelid single-domain antibody fragment directed against...... human EPO was evaluated as a capturing antibody in a label-free biolayer interferometry-based quantification assay. Human recombinant EPO can be specifically detected in Chinese hamster ovary cell supernatants in a sensitive and pH-dependent manner. This method enables rapid and robust quantification of...

  15. Mismatch oxidation assay: detection of DNA mutations using a standard UV/Vis microplate reader.

    Science.gov (United States)

    Tabone, Tania; Sallmann, Georgina; Cotton, Richard G H

    2009-01-01

    Simple, low-cost mutation detection assays that are suitable for low-throughput analysis are essential for diagnostic applications where the causative mutation may be different in every family. The mismatch oxidation assay is a simple optical absorbance assay to detect nucleotide substitutions, insertions, and deletions in heteroduplex DNA. The method relies on detecting the oxidative modification products of mismatched thymine and cytosine bases by potassium permanganate as it is reduced to manganese dioxide. This approach, unlike other methods commonly used to detect sequence variants, does not require costly labeled probes or primers, toxic chemicals, or a time-consuming electrophoretic separation step. The oxidation rate, and hence the presence of a sequence variant, is detected by measuring the formation of the potassium permanganate reduction product (hypomanganate diester), which absorbs at the 420-nm visible wavelength, using a standard UV/vis microplate reader. PMID:19768598

  16. Fluidic Force Discrimination Assays: A New Technology for Tetrodotoxin Detection

    Directory of Open Access Journals (Sweden)

    Cy R. Tamanaha

    2010-03-01

    Full Text Available Tetrodotoxin (TTX is a low molecular weight (~319 Da neurotoxin found in a number of animal species, including pufferfish. Protection from toxin tainted food stuffs requires rapid, sensitive, and specific diagnostic tests. An emerging technique for the detection of both proteins and nucleic acids is Fluidic Force Discrimination (FFD assays. This simple and rapid method typically uses a sandwich immunoassay format labeled with micrometer-diameter beads and has the novel capability of removing nonspecifically attached beads under controlled, fluidic conditions. This technique allows for near real-time, multiplexed analysis at levels of detection that exceed many of the conventional transduction methods (e.g., ELISAs. In addition, the large linear dynamic range afforded by FFD should decrease the need to perform multiple sample dilutions, a common challenge for food testing. By applying FFD assays to an inhibition immunoassay platform specific for TTX and transduction via low magnification microscopy, levels of detection of ~15 ng/mL and linear dynamic ranges of 4 to 5 orders of magnitude were achieved. The results from these studies on the first small molecule FFD assay, along with the impact to detection of seafood toxins, will be discussed in this manuscript.

  17. Detection of North American eastern and western equine encephalitis viruses by nucleic acid amplification assays.

    Science.gov (United States)

    Lambert, Amy J; Martin, Denise A; Lanciotti, Robert S

    2003-01-01

    We have developed nucleic acid sequence-based amplification (NASBA), standard reverse transcription PCR (RT-PCR), and TaqMan nucleic acid amplification assays for the rapid detection of North American eastern equine encephalitis (EEE) and western equine encephalitis (WEE) viral RNAs from samples collected in the field and clinical samples. The sensitivities of these assays have been compared to that of virus isolation. While all three types of nucleic acid amplification assays provide rapid detection of viral RNAs comparable to the isolation of viruses in Vero cells, the TaqMan assays for North American EEE and WEE viral RNAs are the most sensitive. We have shown these assays to be specific for North American EEE and WEE viral RNAs by testing geographically and temporally distinct strains of EEE and WEE viruses along with a battery of related and unrelated arthropodborne viruses. In addition, all three types of nucleic acid amplification assays have been used to detect North American EEE and WEE viral RNAs from mosquito and vertebrate tissue samples. The sensitivity, specificity, and rapidity of nucleic acid amplification demonstrate the usefulness of NASBA, standard RT-PCR, and TaqMan assays, in both research and diagnostic settings, to detect North American EEE and WEE viral RNAs. PMID:12517876

  18. Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

    Energy Technology Data Exchange (ETDEWEB)

    Li Li [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Li Xincang [School of Life Sciences, Shandong University, Jinan 250100 (China); Li Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2011-01-24

    An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10{sup -14} mol L{sup -1}. Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

  19. Detection of Fusarium graminearum DNA using a loop-mediated isothermal amplification (LAMP) assay.

    Science.gov (United States)

    Niessen, Ludwig; Vogel, Rudi F

    2010-06-15

    Loop-mediated isothermal amplification (LAMP) of DNA is a simple, cost effective, and rapid method for the specific detection of genomic DNA using a set of six oligonucleotide primers with eight binding sites hybridizing specifically to different regions of a target gene, and a thermophilic DNA polymerase from Geobacillus stearothermophilus for DNA amplification. The method has been applied in various assays for the diagnosis of bacterial and viral infections of humans and animals, sexing of bovine and swine embryos, and in the detection of bacteria from environmental samples. Only recently, first applications for fungal organisms were published. During the current study a LAMP assay was developed for the specific detection of Fusarium graminearum, the major causative agent of Fusarium head blight of small cereals and producer of the mycotoxins deoxynivalenol, nivalenol, and zearalenone. The assay was based on the gaoA gene (galactose oxidase) of the fungus. Amplification of DNA during the reaction was indirectly detected in situ by using calcein fluorescence as a marker without the necessity of time-consuming electrophoretic analysis. The assay was optimized for rapidness, specificity, and sensitivity and was shown to detect the presence of less than 2pg of purified target DNA per reaction within 30 min. Within 132 fungal species tested, exclusively DNA isolated from cultures of F. graminearum (lineages 1-9) resulted in a fluorescent signal after amplification with the LAMP assay. The method was demonstrated to be useful in the analysis of fungal cultures by direct analysis of surface scrapings from agar plate cultures, direct testing of single infected barley grains, and detection of F. graminearum in total genomic DNA isolated from bulk samples of ground wheat grains. Results obtained indicate that LAMP offers an interesting new assay format for the rapid and specific DNA-based detection and identification of agriculturally important toxigenic fungi in pure

  20. A highly scalable peptide-based assay system for proteomics.

    Directory of Open Access Journals (Sweden)

    Igor A Kozlov

    Full Text Available We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays.

  1. Diagnostic sensitivity of two radio receptor assays (TRAK Assay and TRAK Dyno human) for the detection of TSH receptor antibodies

    International Nuclear Information System (INIS)

    Radio receptor assays for the detection of TSH receptor antibodies in serum are typically based on binding the competition of TSH-R antibodies and 125I-labelled-TSH for membrane preparation of thyrocytes (TBII tests). The sensitivity of the available tests utilizing porcine cell membranes was found to be around 80%. A new test (TRAK Dyno human, BRAHMS) utilizes human recombinant TSH receptor and human standard material that is supposed to improve the performance of the test. We have compared the results of these two assays. The sensitivity of the TRAK Assay tested in 356 patients with untreated Grave's disease was found to be 85%, and 97.5% for TRAK Dyno human in 111 newly diagnosed patients. Both tests were performed from the same serum specimen for 60 of the investigated patients. The TRAK Assay was positive in 50 patients (83.2%) and TRAK Dyno human in 59 patients (98.3%). The specificity of the new radio receptor assay was also improved. (author)

  2. Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay

    Science.gov (United States)

    Yao, Xiefeng; Li, Pingfang; Xu, Jinghua; Zhang, Man; Ren, Runsheng; Liu, Guang; Yang, Xingping

    2016-01-01

    Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB) in Cucurbitaceae crops (e.g., cantaloupe, muskmelon, cucumber, and watermelon). GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated amplification (LAMP) assay for D. bryoniae detection based on sequence-characterized amplified regions (GenBank accession nos GQ872461 and GQ872462) common to the two random amplification of polymorphic DNA group genotypes (RGI and RGII) of D. bryoniae; ideal conditions for detection were optimized for completion in 45 min at 63°C. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with those of a conventional polymerase chain reaction (PCR). The sensitivity of the LAMP assay was 1000-fold higher than that of conventional PCR with a detection limit of 0.1 fg μL-1 of targeted DNA. The LAMP assay could be accomplished in about 45 min, with the results visible to the naked eye. The assay showed high specificity in discriminating all D. bryoniae isolates from seven other fungal pathogens that occur in Cucurbitaceae crops. The LAMP assay also detected D. bryoniae infection in young muskmelon leaves with suspected early symptoms of GSB disease. Hence, the technique has great potential for developing rapid and sensitive visual detection methods for the D. bryoniae pathogen in crops and seeds. This method has potential application in early prediction of disease and reducing the risk of epidemics.

  3. Simple and sensitive progesterone detection in human serum using a CdSe/ZnS quantum dot-based direct binding assay.

    Science.gov (United States)

    Oh, Sung-Duk; Duong, Hong Dinh; Rhee, Jong Il

    2015-08-15

    In this study, we developed a CdSe/ZnS quantum dot (QD)-based immunoassay for use in determining the presence of progesterone (P4) in human serum. Hydrophilic QDs were conjugated to anti-progesterone antibody (P4Ab) via ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling reagents. After purification, the P4Ab-QD conjugates were immobilized onto the wells of a 96-well microtiter plate, and a direct-binding immunoassay based on the binding of P4 to immobilized P4Ab-QD conjugates had a detection limit of 0.21 ng/ml and a sensitivity of 1.37 ng/ml, with a linear range of 0.385 to 4.55 ng/ml. The proposed immunoassay was successfully used to determine the P4 concentration in real human serum, and the results showed a good correlation with the accredited radioimmunoassay (RIA). PMID:25963894

  4. An electrochemical aptasensor based on the amplification of two kinds of gold nanocrystals for the assay of L-histidine with picomolar detection limit

    Science.gov (United States)

    Chen, Zhengbo; Guo, Junxia; Li, Jing; Guo, Lin

    2013-07-01

    Au nanocrystals (NCs) enclosed by higher-index facets have high surface chemical activity. They attract much attention because of their excellent biocompatibility. In this work, well-defined elongated tetrahexahedral (ETHH) Au NCs and end-truncated ETHH with high-index facets are successfully prepared by using a single surfactant system of didodecyldimethylammonium bromide (DDAB) and a binary system of cetyltrimethylammoniumbromide and DDAB in two-step seed-mediated growth. The characteristics of high-index facet Au NC modified electronic aptamer-based sensors are presented and they are applicable to a wide range of aptamers. Herein, we only take L-histidine as the representative sensing target. With modification of the Au NCs, a very low detection limit (sub-picomolar) is obtained. In particular, a detailed sensitivity comparison between the modification of end-truncated ETHH and ETHH Au NCs is presented to demonstrate the slight difference in the chemical activities of Au NCs with different high-index facets. Our work sheds light on the large scale fabrication of Au/metallic NCs with high-index facets and also provides a new eye-opening example of engineering ultra-sensitive DNA sensors based on Au NCs.

  5. An electrochemical aptasensor based on the amplification of two kinds of gold nanocrystals for the assay of L-histidine with picomolar detection limit

    International Nuclear Information System (INIS)

    Au nanocrystals (NCs) enclosed by higher-index facets have high surface chemical activity. They attract much attention because of their excellent biocompatibility. In this work, well-defined elongated tetrahexahedral (ETHH) Au NCs and end-truncated ETHH with high-index facets are successfully prepared by using a single surfactant system of didodecyldimethylammonium bromide (DDAB) and a binary system of cetyltrimethylammoniumbromide and DDAB in two-step seed-mediated growth. The characteristics of high-index facet Au NC modified electronic aptamer-based sensors are presented and they are applicable to a wide range of aptamers. Herein, we only take L-histidine as the representative sensing target. With modification of the Au NCs, a very low detection limit (sub-picomolar) is obtained. In particular, a detailed sensitivity comparison between the modification of end-truncated ETHH and ETHH Au NCs is presented to demonstrate the slight difference in the chemical activities of Au NCs with different high-index facets. Our work sheds light on the large scale fabrication of Au/metallic NCs with high-index facets and also provides a new eye-opening example of engineering ultra-sensitive DNA sensors based on Au NCs. (paper)

  6. Improvement of a real-time RT-PCR assay for the detection of enterovirus RNA

    OpenAIRE

    Bruynseels Peggy; Verstrepen Walter A; Piqueur Marian AC; Mertens An H

    2009-01-01

    Abstract We describe an improvement of an earlier reported real-time RT-PCR assay for the detection of enterovirus RNA, based on the 5' exonuclease digestion of a dual-labeled fluorogenic probe by Taq DNA polymerase. A different extraction method, real-time RT-PCR instrument and primer set were evaluated. Our data show that the optimized assay yields a higher sensitivity and reproducibility and resulted in a significant reduced hands-on time per sample.

  7. Improvement of a real-time RT-PCR assay for the detection of enterovirus RNA

    Directory of Open Access Journals (Sweden)

    Bruynseels Peggy

    2009-07-01

    Full Text Available Abstract We describe an improvement of an earlier reported real-time RT-PCR assay for the detection of enterovirus RNA, based on the 5' exonuclease digestion of a dual-labeled fluorogenic probe by Taq DNA polymerase. A different extraction method, real-time RT-PCR instrument and primer set were evaluated. Our data show that the optimized assay yields a higher sensitivity and reproducibility and resulted in a significant reduced hands-on time per sample.

  8. Detection of specific DNA sequences by fluorescence amplification: a color complementation assay.

    OpenAIRE

    Chehab, F. F.; Kan, Y W

    1989-01-01

    We have developed a color complementation assay that allows rapid screening of specific genomic DNA sequences. It is based on the simultaneous amplification of two or more DNA segments with fluorescent oligonucleotide primers such that the generation of a color, or combination of colors, can be visualized and used for diagnosis. Color complementation assay obviates the need for gel electrophoresis and has been applied to the detection of a large and small gene deletion, a chromosomal transloc...

  9. Clinical utility of the Xpert MRSA assay for early detection of methicillin-resistant Staphylococcus aureus

    OpenAIRE

    OH, AE-CHIN; Lee, Jin Kyung; LEE, HA NA; HONG, YOUNG JUN; Chang, Yoon Hwan; Hong, Seok-Il; Kim, Dong Ho

    2012-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for many nosocomial and community-acquired infections, resulting in significant morbidity and mortality. A practical way to limit the spread of MRSA is early detection and proper treatment. However, screening culture for MRSA typically requires 2–3 days. The Xpert MRSA assay (Cepheid, Sunnyvale, CA, USA) is a real-time polymerase chain reaction-based assay developed for screening an MRSA-specific DNA sequence within the staphyl...

  10. Evaluation of 5 ' nuclease assay for detection of Actinobacillus pleuropneumoniae

    DEFF Research Database (Denmark)

    Angen, Øystein; Jensen, J.; Lavritsen, D. T.

    2001-01-01

    Sequence detection by the 5' nuclease TaqMan assay uses online detection of internal fluorogenic probes in closed PCR tubes. Primers and probe were chosen from a part of the omlA gene common to all serotypes of Actinobacillus pleuropneumoniae, which gave an amplicon of 92 bp, The test was evaluated...... with 73 lung isolates and 120 tonsil isolates of A. pleuropneumoniae as well as with a collection of reference strains. By using a C-t value (cycle number in which the fluorescence exceeds the threshold defined by the software) of 30 as the cutoff limit, the 5' nuclease assay represents a test with 100......% sensitivity and 100% specificity, A high degree of reproducibility of the test was demonstrated. If samples with C-t values of less than or equal to 30 are considered positive, the detection limit of the assay was 1 CPU/reaction tube, corresponding to a 10-fold higher number of DNA templates. After cycle 30...

  11. Development of a Calibration Strip for Immunochromatographic Assay Detection Systems.

    Science.gov (United States)

    Gao, Yue-Ming; Wei, Jian-Chong; Mak, Peng-Un; Vai, Mang-I; Du, Min; Pun, Sio-Hang

    2016-01-01

    With many benefits and applications, immunochromatographic (ICG) assay detection systems have been reported on a great deal. However, the existing research mainly focuses on increasing the dynamic detection range or application fields. Calibration of the detection system, which has a great influence on the detection accuracy, has not been addressed properly. In this context, this work develops a calibration strip for ICG assay photoelectric detection systems. An image of the test strip is captured by an image acquisition device, followed by performing a fuzzy c-means (FCM) clustering algorithm and maximin-distance algorithm for image segmentation. Additionally, experiments are conducted to find the best characteristic quantity. By analyzing the linear coefficient, an average value of hue (H) at 14 min is chosen as the characteristic quantity and the empirical formula between H and optical density (OD) value is established. Therefore, H, saturation (S), and value (V) are calculated by a number of selected OD values. Then, H, S, and V values are transferred to the RGB color space and a high-resolution printer is used to print the strip images on cellulose nitrate membranes. Finally, verification of the printed calibration strips is conducted by analyzing the linear correlation between OD and the spectral reflectance, which shows a good linear correlation (R² = 98.78%). PMID:27367694

  12. Development and evaluation of a new PCR assay for detection of Pseudomonas aeruginosa D genotype.

    Science.gov (United States)

    Lødeng, A G G; Ahlén, C; Lysvand, H; Mandal, L H; Iversen, O J

    2006-08-01

    This report describes a new PCR-based assay for the detection of Pseudomonas aeruginosa genotype D in occupational saturation diving systems in the North Sea. This genotype has persisted in these systems for 11 years (1993-2003) and represents 18% of isolates from infections analysed during this period. The new PCR assay was based on sequences obtained after randomly amplified polymorphic DNA (RAPD)-PCR analysis of a group of isolates related to diving that had been identified previously by pulsed-field gel electrophoresis (PFGE). The primer set for the D genotype targets a gene that codes for a hypothetical class 4 protein in the P. aeruginosa PAO1 genome. A primer set able to detect P. aeruginosa at the species level was also designed, based on the 23S-5S rDNA spacer region. The two assays produced 382-bp and 192-bp amplicons, respectively. The PCR assay was evaluated by analysing 100 P. aeruginosa isolates related to diving, representing 28 PFGE genotypes, and 38 clinical and community P. aeruginosa isolates and strains from other species. The assay identified all of the genotype D isolates tested. Two additional diving-relevant genotypes (TP2 and TP27) were also identified, as well as three isolates of non-diving origin. It was concluded that the new PCR assay is a useful tool for early detection and prevention of infections with the D genotype. PMID:16842571

  13. 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.

  14. Development of an EvaGreen-based multiplex real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of six viral pathogens of porcine reproductive and respiratory disorder.

    Science.gov (United States)

    Rao, Pinbin; Wu, Haigang; Jiang, Yonghou; Opriessnig, Tanja; Zheng, Xiaowen; Mo, Yecheng; Yang, Zongqi

    2014-11-01

    Concurrent infection of pigs with two or more pathogens is common in pigs under intensive rearing conditions. Porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Japanese encephalitis virus (JEV) and pseudorabies virus (PRV) are all associated with reproductive or respiratory disorders or both and can cause significant economic losses in pig production worldwide. An EvaGreen-based multiplex real-time PCR (EG-mPCR) with melting curve analysis was developed in this study for simultaneous detection and differentiation of these six viruses in pigs. This method is able to detect and distinguish PCV2, PPV, PRRSV, CSFV, JEV and PRV with the limits of detection ranging from 100 to 500 copies/μL, high reproducibility, and intra-assay and inter-assay variation ranging from 0.11 to 3.20%. After validation, a total of 118 field samples were tested by the newly developed EG-mPCR. PCV2 was identified in 23%, PPV in 15%, PRRSV in 17% and PRV in 5% of the samples. Concurrent PCV2 and PRRSV infection was detected in 6.7%, PCV2 and PPV in 5% and PPV2 and PRRSV infection was detected in 5% of the cases. The agreement of the EG-mPCR and conventional PCR tests was 99.2%. This EG-mPCR will be a useful, rapid, reliable and cost-effective alternative for routine surveillance testing of viral infections in pigs. PMID:25102430

  15. Syphilis detection: evaluation of serological screening and pilot reverse confirmatory assay algorithm in blood donors.

    Science.gov (United States)

    Sommese, Linda; Paolillo, Rossella; Sabia, Chiara; Costa, Dario; De Pascale, Maria Rosaria; Iannone, Carmela; Esposito, Antonella; Schiano, Concetta; Napoli, Claudio

    2016-07-01

    Serological assays are still considered the most useful tests in the diagnosis of syphilis. Since no single serological assay is able to provide a satisfactory result, in our laboratory we have evaluated the usefulness of a commercially-available immunoblot to diagnose syphilis infection among blood donors. From October 2012 to June 2013, 4572 blood donors were screened for syphilis with an automated chemiluminescent microparticle immunoassay (CMIA). To confirm the presence of treponemal antibodies, CMIA-reactive sera were tested by standard Treponema pallidum haemagglutination assay (TPHA). In addition, an alternative confirmatory test - the immunoblot INNO-LIA assay was introduced in our laboratory. Since two additional positives among CMIA-reactive-TPHA-negative samples were found, we concluded that the INNO-LIA immunoblot allowed a better detection of syphilis compared to TPHA. A confirmatory strategy based on the use of two treponemal assays could meet the screening requirements for blood donors as well as in our centre. PMID:26068964

  16. Detection of glucocorticoid bioactivity in bovine urine samples using a reporter gene assay.

    Science.gov (United States)

    Connolly, Lisa; Cai, Kai; Van der Heiden, Edwige; Scippo, Marie-Louise; Muller, Marc; Tarbin, Jonathan; Elliott, Chris

    2009-04-01

    The illegal use of anabolic substances in the meat producing industry is an ongoing problem due to the continual production of new synthetic compounds and/or the practice of low-level cocktail administration to avoid detection by the surveillance schemes of EU member states National Plan surveillance systems. We present a highly sensitive reporter gene assay and sample extraction procedure based on a two step solid phase extraction and high performance liquid chromatography, developed for the detection of glucocorticoid abuse in bovine urine. The assay is capable of detecting compounds with glucocorticoid bioactivity and is extremely sensitive with an EC(50) of 0.79 ngmL(-1) for dexamethasone. New or unknown compounds with glucocorticoid bioactivity and low-level cocktail mixtures are detectable by this assay. Cross-reactivity data for a range of 11beta-hydroxyglucocorticoids has been provided. This assay shows low interference from the 11-keto prohormones and other steroidal hormones. The assay may be suitable for application in other matrices such as hair. In conclusion this screening assay offers advantages over existing analytical techniques. PMID:19286046

  17. DNA & Protein detection based on microbead agglutination

    KAUST Repository

    Kodzius, Rimantas

    2012-06-06

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microparticles in the presence of a specific analyte thus enabling the macroscopic observation. Agglutination-based tests are most often used to explore the antibody-antigen reactions. Agglutination has been used for mode protein assays using a biotin/streptavidin two-component system, as well as a hybridization based two-component assay; however, as our work shows, two-component systems are prone to self-termination of the linking analyte and thus have a lower sensitivity. Three component systems have also been used with DNA hybridization, as in our work; however, their assay requires 48 hours for incubation, while our assay is performed in 5 minutes making it a real candidate for POC testing. We demonstrate three assays: a two-component biotin/streptavidin assay, a three-component hybridization assay using single stranded DNA (ssDNA) molecules and a stepped three-component hybridization assay. The comparison of these three assays shows our simple stepped three-component agglutination assay to be rapid at room temperature and more sensitive than the two-component version by an order of magnitude. An agglutination assay was also performed in a PDMS microfluidic chip where agglutinated beads were trapped by filter columns for easy observation. We developed a rapid (5 minute) room temperature assay, which is based on microbead agglutination. Our three-component assay solves the linker self-termination issue allowing an order of magnitude increase in sensitivity over two–component assays. Our stepped version of the three-component assay solves the issue with probe site saturation thus enabling a wider range of detection. Detection of the agglutinated beads with the naked eye by trapping in microfluidic channels has been shown.

  18. Validation of an Immunodiagnostic Assay for Detection of 13 Streptococcus pneumoniae Serotype-Specific Polysaccharides in Human Urine

    OpenAIRE

    Pride, Michael W.; Huijts, Susanne M.; Wu, Kangjian; Souza, Victor; Passador, Sherry; Tinder, Chunyan; Song, Esther; Elfassy, Arik; McNeil, Lisa; Menton, Ronald; French, Roger; Callahan, Janice; Webber, Chris; Gruber, William C.; Bonten, Marc J. M.

    2012-01-01

    To improve the clinical diagnosis of pneumococcal infection in bacteremic and nonbacteremic community-acquired pneumonia (CAP), a Luminex technology-based multiplex urinary antigen detection (UAD) diagnostic assay was developed and validated. The UAD assay can simultaneously detect 13 different serotypes of Streptococcus pneumoniae by capturing serotype-specific S. pneumoniae polysaccharides (PnPSs) secreted in human urine. Assay specificity is achieved by capturing the polysaccharides with s...

  19. Recombinant Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Antibodies to Crimean-Congo Hemorrhagic Fever Virus

    OpenAIRE

    Saijo, Masayuki; Qing, Tang; Niikura, Masahiro; Maeda, Akihiko; Ikegami, Tetsuro; Prehaud, Christophe; Kurane, Ichiro; Morikawa, Shigeru

    2002-01-01

    The full-length nucleoprotein of Crimean-Congo hemorrhagic fever virus (CCHFV; 482 amino acid residues) was expressed as a His-tagged recombinant protein (His-CCHFV rNP) in the baculovirus system. The His-CCHFV rNP was efficiently expressed in insect cells and purified by Ni2+ column chromatography. Using this substrate, an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was developed. We evaluated the sensitivity and specificity of the IgG ELISA, using serum samples previous...

  20. Fluorescent turn-on detection and assay of water based on 4-(2-dimethylaminoethyloxy)-N-octadecyl-1,8-naphthalimide with aggregation-induced emission enhancement.

    Science.gov (United States)

    Sun, Yang; Liang, Xuhua; Wei, Song; Fan, Jun; Yang, Xiaohui

    2012-11-01

    The photophysical properties of 4-(2-dimethylaminoethyloxy)-N-octadecyl-1,8-naphthalimide (DON) consisting of donor and acceptor units were investigated in different solutions. Changing from a non-polar to a polar solvent increased the solvent interaction and both the excitation and emission spectra were shifted to longer wavelength and intensity decreased through taking advantage of twisted intramolecular charge transfer (TICT). Density functional theory (DFT) calculations and spectral analyses revealed that such fluorophores were capable of sensing protons by intramolecular charge transfer (ICT). Empirical and quantum mechanical calculations showed that the electron donating effect of the dimethylamino group decreased the change in dipole moment on excitation which resulted in a fluorescence quantum yield remarkably enhanced as the solvent polarity increased. In alkaline media the fluorescence of DON was quenched owing to photoinduced electron transfer being disabled in acidic media. The pK(a) of the 1,8-naphthailimide dye was 6.70, which defines the dye as a highly efficient "off-on" switch. DON exhibited a typical aggregation-induced emission enhancement (AIEE) behavior that it is virtually nonemissive in organic solvent but highly luminescent in water, as a result of the restriction of free intramolecular rotation of a C-N bond and the non-planar configuration in the aggregate state. The hydrophobicity of octadecyl group provided DON with a fluorescent response to water based on AIEE and the water-dependent spectral characteristics of DON, and the AIEE of DON caused by the effect of water and formation of J-aggregation states. In the range of 0-79.8% (v/v), the fluorescence intensity of DON in acetone solution increased as a linear function of the water content. The optimum detection limits were of 0.011%, 0.0021%, and 0.0033% of water in acetone, ethanol, and acetonitrile, respectively. Satisfactory reproducibility, reversibility and a short response time

  1. Evaluation of five hepatitis delta virus marker assays for detection of antigen and antibody.

    OpenAIRE

    Bezeaud, A; Rosenswajg, M; Guillin, M C

    1989-01-01

    Five commercially available assays for hepatitis delta (HD) virus markers were compared for sensitivity, specificity, and reproducibility: three assays for antibody (anti-HD), provided by Diagnostics Pasteur, Organon Teknika, and Abbott Laboratories, and two assays for antigen (HD Ag), from Pasteur and Organon Teknika. The assay from Organon Teknika is the less sensitive assay for anti-HD detection. Although the sensitivities of the Pasteur and Abbott assays for anti-HD detection are similar,...

  2. Ultraminiaturized assay for rapid, low cost detection and quantification of clinical and biochemical samples.

    Science.gov (United States)

    Parween, Shahila; Nahar, Pradip

    2016-04-01

    Herein, we report a simple, sensitive, rapid and low-cost ultraminiaturized assay technique for quantitative detection of 1 μl of clinical or biochemical sample on a novel ultraminiaturized assay plate (UAP). UAP is prepared by making tiny cavities on a polypropylene sheet. As UAP cannot immobilize a biomolecule through absorption, we have activated the tiny cavities of UAP by 1-fluoro-2-nitro-4-azidobenzene in a photochemical reaction. Activated UAP (AUAP) can covalently immobilize any biomolecule having an active nucleophilic group such as amino group. Efficacy of AUAP is demonstrated by detecting human IgE, antibody of hepatitis C virus core antigen and oligonucleotides. Quantification is performed by capturing the image of the colored assay solution and digitally quantifying the image by color saturation without using costly NanoDrop spectrophotometer. Image - based detection of human IgE and an oligonucleotide shows an excellent correlation with absorbance - based assay (recorded in a NanoDrop spectrophotometer); it is validated by Pearson's product-moment correlation with correlation coefficient of r = 0.9545088 and r = 0.9947444 respectively. AUAP is further checked by detecting hepatitis C virus Ab where strong correlation of color saturation with absorbance with respect to concentration is observed. Ultraminiaturized assay successfully detects target oligonucleotides by perfectly hybridizing with their respective complementary oligonucleotide probes but not with a random oligonucleotide. Ultraminiaturized assay technique has substantially reduced the requirement of reagents by 100 times and assay timing by 50 times making it a potential alternative to conventional method. PMID:26973054

  3. Multiwalled carbon nanotube modified screen-printed electrodes for the detection of p-aminophenol: Optimisation and application in alkaline phosphatase-based assays

    International Nuclear Information System (INIS)

    Carboxylated multiwalled carbon nanotubes (MWCNT-COOH) were used to modify the working electrode surface of different screen-printed electrodes. The effect of this modification on the electrodic characteristics (double layer capacitance, electroactive area and heterogeneous rate constants for the electron transfer) was evaluated and optimized for the cyclic voltammetric determination of p-aminophenol. The enzymatic hydrolysis of p-aminophenylphosphate was employed for the quantification of alkaline phosphatase, one of the most important label enzymes in immunoassays. Finally, ELISA assays were carried out to quantify pneumolysin using this enzymatic system. Results obtained indicated that low superficial densities of MWCNT-COOH (0.03-0.06 μg mm-2) yielded the same electrodic improvements but with better analytical properties

  4. Multiwalled carbon nanotube modified screen-printed electrodes for the detection of p-aminophenol: Optimisation and application in alkaline phosphatase-based assays

    Energy Technology Data Exchange (ETDEWEB)

    Lamas-Ardisana, Pedro Jose [Departamento de Quimica Fisica y Analitica, Universidad de Oviedo, 33006 Oviedo, Asturias (Spain); Queipo, Paula [Departamento de Fisica, Universidad de Oviedo, 33007 Oviedo, Asturias (Spain); Fanjul-Bolado, Pablo [Departamento de Quimica Fisica y Analitica, Universidad de Oviedo, 33006 Oviedo, Asturias (Spain); Costa-Garcia, Agustin [Departamento de Quimica Fisica y Analitica, Universidad de Oviedo, 33006 Oviedo, Asturias (Spain)], E-mail: costa@fq.uniovi.es

    2008-05-12

    Carboxylated multiwalled carbon nanotubes (MWCNT-COOH) were used to modify the working electrode surface of different screen-printed electrodes. The effect of this modification on the electrodic characteristics (double layer capacitance, electroactive area and heterogeneous rate constants for the electron transfer) was evaluated and optimized for the cyclic voltammetric determination of p-aminophenol. The enzymatic hydrolysis of p-aminophenylphosphate was employed for the quantification of alkaline phosphatase, one of the most important label enzymes in immunoassays. Finally, ELISA assays were carried out to quantify pneumolysin using this enzymatic system. Results obtained indicated that low superficial densities of MWCNT-COOH (0.03-0.06 {mu}g mm{sup -2}) yielded the same electrodic improvements but with better analytical properties.

  5. A new nanoPCR molecular assay for detection of porcine bocavirus.

    Science.gov (United States)

    Wang, Xiaoling; Bai, Aiquan; Zhang, Jing; Kong, Miaomiao; Cui, Yuchao; Ma, Xingjie; Ai, Xia; Tang, Qinghai; Cui, Shangjin

    2014-06-01

    Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid amplification of DNA and has been used for the detection of virus. For detection of porcine bocavirus (PBoV), a sensitive and specific nanoPCR assay was developed with a pair of primers that were designed based on NS1 gene sequences available in GenBank. Under the optimized conditions of the PBoV nanoPCR assay, the nanoPCR assay was 100-fold more sensitive than a conventional PCR assay. The lower detection limit of the nanoPCR assay was about 6.70×10(1) copies. The nanoPCR assay amplified the specific 482-bp fragment of the PBoV NS1 recombinant plasmid but did not produce any product with genomic DNA or cDNA of porcine parvovirus, porcine circovirus type II, porcine reproductive and respiratory syndrome virus, pseudorabies virus, classic swine fever virus, Encephalomyocarditis virus, Porcine Teschovirus or African swine fever virus plasmid. Of 65 clinical samples collected from diseased pigs, 73.8% and 86.2% were determined to be PBoV positive by PBoV conventional PCR and PBoV nanoPCR assay, respectively. Of 36 clinical samples from healthy pigs, 27.8% and 44.4% were PBoV positive by PBoV conventional PCR and PBoV nanoPCR assay, respectively. The nanoPCR assay will be useful for diagnosing PBoV and for studying its epidemiology and pathology. PMID:24642242

  6. Evaluation of a Novel Heminested PCR Assay Based on the Phosphoglucosamine Mutase Gene for Detection of Helicobacter pylori in Saliva and Dental Plaque

    OpenAIRE

    Goosen, C.; Theron, J.; Ntsala, M.; Maree, F F; Olckers, A; Botha, S. J.; Lastovica, A. J.; van der Merwe, S. W.

    2002-01-01

    A novel heminested PCR protocol was developed for the specific detection of Helicobacter pylori at low copy numbers. A set of primers specific for the phosphoglucosamine mutase gene (glmM) of H. pylori produced a 765-bp fragment that was used as template for the heminested primer pair delineating a 496-bp fragment. By using agarose gel electrophoresis for detection of the heminested PCR-amplified products, amplification of H. pylori genomic DNA was achieved at concentrations as low as 0.1 pg,...

  7. Potential Impact of a Microarray-Based Nucleic Acid Assay for Rapid Detection of Gram-Negative Bacteria and Resistance Markers in Positive Blood Cultures

    OpenAIRE

    Mancini, Nicasio; Infurnari, Laura; Ghidoli, Nadia; Valzano, Grazia; Clementi, Nicola; Burioni, Roberto; Clementi, Massimo

    2014-01-01

    We evaluated the Verigene Gram-negative blood culture (BC-GN) test, a microarray that detects Gram-negative bacteria and several resistance genes. A total of 102 positive blood cultures were tested, and the BC-GN test correctly identified 97.9% of the isolates within its panel. Resistance genes (CTX-M, KPC, VIM, and OXA genes) were detected in 29.8% of the isolates, with positive predictive values of 95.8% (95% confidence interval [CI], 87.7% to 98.9%) in Enterobacteriaceae and 100% (95% CI, ...

  8. An enzymatic assay for the detection of natural cytotoxicity

    International Nuclear Information System (INIS)

    The pronase-enhanced release of 14C-labeled DNA was used to assay cytotoxicity of murine natural killer cells. YAC-1 lymphoma cells were labeled with [14C]thymidine and incubated with increasing numbers of spleen cells from nude mice. At harvest, nuclease-free pronase was used to digest damaged target cells. Increases in cytotoxicity of 5-39% were obtained using optimal conditions. The reaction could be successfully inhibited with unlabeled YAC-1 cells. The degree of cytotoxicity was similar to that detected by 51Cr release, but a longer period of incubation was required. (Auth.)

  9. Extended-spectrum β-lactamase (ESBL) detection directly from urine samples with the rapid isothermal amplification-based eazyplex® SuperBug CRE assay: Proof of concept.

    Science.gov (United States)

    Hinić, V; Ziegler, J; Straub, C; Goldenberger, D; Frei, R

    2015-12-01

    A commercially available assay (eazyplex® SuperBug CRE) detecting the most common carbapenemase and ESBL types was evaluated directly on 50 urine samples. Eazyplex® correctly detected ESBL-encoding genes in all 30 urine samples with confirmed ESBL production (sensitivity 100%). Two specimens showed invalid and one specimen false-positive results (specificity 97.9%). PMID:26506282

  10. An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk

    Science.gov (United States)

    A rapid lateral flow fluorescent microspheres immunochromatography test strip (FMs-ICTS) has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MA...

  11. 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

  12. The synchronous active neutron detection system for spent fuel assay

    International Nuclear Information System (INIS)

    The authors have begun to develop a novel technique for active neutron assay of fissile material in spent nuclear fuel. This approach will exploit the unique operating features of a 14-MeV neutron generator developed by Schlumberger. This generator and a novel detection system will be applied to the direct measurement of the fissile material content in spent fuel in place of the indirect measures used at present. The technique they are investigating is termed synchronous active neutron detection (SAND). It closely follows a method that has been used routinely in other branches of physics to detect very small signals in the presence of large backgrounds. Synchronous detection instruments are widely available commercially and are termed open-quotes lock-inclose quotes amplifiers. The authors have implemented a digital lock-in amplifier in conjunction with the Schlumberger neutron generator to explore the possibility of synchronous detection with active neutrons. This approach is possible because the Schlumberger system can operate at up to a 50% duty factor, in effect, a square wave of neutron yield. The results to date are preliminary but quite promising. The system is capable of resolving the fissile material contained in a small fraction of the fuel rods in a cold fuel assembly. It also appears to be quite resilient to background neutron interference. The interrogating neutrons appear to be nonthermal and penetrating. Although a significant amount of work remains to fully explore the relevant physics and optimize the instrument design, the underlying concept appears sound

  13. Reverse transcription loop-mediated isothermal amplification assay for rapid detection of Papaya ringspot virus.

    Science.gov (United States)

    Shen, Wentao; Tuo, Decai; Yan, Pu; Yang, Yong; Li, Xiaoying; Zhou, Peng

    2014-08-01

    Papaya ringspot virus (PRSV) and Papaya leaf distortion mosaic virus (PLDMV), which causes disease symptoms similar to PRSV, threaten commercial production of both non-transgenic-papaya and PRSV-resistant transgenic papaya in China. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect PLDMV was developed previously. In this study, the development of another RT-LAMP assay to distinguish among transgenic, PRSV-infected and PLDMV-infected papaya by detection of PRSV is reported. A set of four RT-LAMP primers was designed based on the highly conserved region of the P3 gene of PRSV. The RT-LAMP method was specific and sensitive in detecting PRSV, with a detection limit of 1.15×10(-6)μg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR. Field application of the RT-LAMP assay demonstrated that samples positive for PRSV were detected only in non-transgenic papaya, whereas samples positive for PLDMV were detected only in commercialized PRSV-resistant transgenic papaya. This suggests that PRSV remains the major limiting factor for non-transgenic-papaya production, and the emergence of PLDMV threatens the commercial transgenic cultivar in China. However, this study, combined with the earlier development of an RT-LAMP assay for PLDMV, will provide a rapid, sensitive and cost-effective diagnostic power to distinguish virus infections in papaya. PMID:24769198

  14. Evaluation of 5 ' nuclease assay for detection of Actinobacillus pleuropneumoniae

    DEFF Research Database (Denmark)

    Angen, Øystein; Jensen, J.; Lavritsen, D. T.

    2001-01-01

    , nonspecific reactions appeared when testing dilutions of DNA templates or pure cultures of A. pleuropneumoniae, as well as when testing tonsil scrapings from specific-pathogen-free herds. The diagnostic sensitivity, as evaluated with 586 tonsil scrapings from animals infected with A. pleuropneumoniae, is the...... equal to 30 must be chosen in order to obtain reliable results. The investigation emphasizes that a thorough evaluation of the criteria used to define a positive test result is necessary.......Sequence detection by the 5' nuclease TaqMan assay uses online detection of internal fluorogenic probes in closed PCR tubes. Primers and probe were chosen from a part of the omlA gene common to all serotypes of Actinobacillus pleuropneumoniae, which gave an amplicon of 92 bp, The test was evaluated...

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

    Science.gov (United States)

    Guo, Limin; Hao, Lihua; Zhao, Qiang

    2016-07-01

    We describe a sensitive aptamer-based sandwich assay for protein detection on microplate by using rolling circle amplification (RCA) coupled with thrombin catalysis. This assay takes advantage of RCA generating long DNA oligonucleotides with repeat thrombin-binding aptamer sequence, specific aptamer affinity binding to achieve multiple thrombin labeling, and enzyme activity of thrombin for signal generation. Protein target is specifically captured by antibody-coated microplate. Then, an oligonucleotide containing an aptamer for protein and a primer sequence is added to form a typical sandwich structure. Following a template encoded with complementary sequence of aptamer for thrombin, RCA reaction extends the primer sequence into a long oligonucleotide. Many thrombin molecules bind with the RCA product. Thrombin catalyzes the conversion of its chromogenic or fluorogenic peptide substrates into detectable products for final quantification of protein targets. We applied this strategy to the detection of a model protein target, platelet-derived growth factor-BB (PDGF-BB). Due to double signal amplifications from RCA and thrombin catalysis, this assay enabled the detection of PDGF-BB as low as 3.1 pM when a fluorogenic peptide substrate was used. This assay provides a new way for signal generation in RCA-involved assay through direct thrombin labeling, circumventing time-consuming preparation of enzyme-conjugate and affinity probes. This method has promise for a variety of analytical applications. PMID:27108282

  16. ELISA assays for the detection of Bothrops lanceolatus venom in envenomed patient plasmas.

    Science.gov (United States)

    Rodriguez-Acosta, A; Uzcategui, W; Azuaje, R; Giron, M E; Aguilar, I

    1998-01-01

    A double antibody sandwich enzyme linked immunosorbant assay (ELISA) was carried out to detect Bothrops Ianceolatus venom in plasma from envenomed patients at various time intervals (0, 6, 12, 18 and 24 hrs). The test could detect Bothrops lanceolatus levels up to 12 ng/mL of envenomed patient plasmas. Elaboration of an easy, fast and species-diagnostic based on this ELISA technique useful to physicians is discussed. PMID:11845439

  17. Detection of radiation treatment of beans using DNA comet assay

    International Nuclear Information System (INIS)

    A simple technique of microgel electrophoresis of single cells (DNA Comet Assay) enabled a quick detection of radiation treatment of several kinds of leguminous beans (azuki, black, black eye, mung, pinto, red kidney and white beans). Each variety was exposed to radiation doses of 0.5, 1 and 5 kGy covering the permissible limits for insect disinfestation. The cells or nuclei from beans were extracted in cold PBS, embedded in agarose on microscope slides, lysed between 15 and 60 min in 2.5% SDS and electrophoresis was carried out at a voltage of 2 V/cm for 2-2.5 min. After silver staining, the slides were evaluated through an ordinary transmission microscope. In irradiated samples, fragmented DNA stretched towards the anode and the damaged cells appeared as a comet. The density of DNA in the tails increased with increasing radiation dose. However, in non-irradiated samples, the large molecules of DNA remained relatively intact and there was only minor or no migration of DNA; the cells were round or had very short tails only. Hence, the DNA comet assay provides an inexpensive, rapid and relatively simple screening method for the detection of irradiated beans

  18. Detection of radiation treatment of beans using DNA comet assay

    Science.gov (United States)

    Khan, Ashfaq A.; Khan, Hasan M.; Delincée, Henry

    2002-03-01

    A simple technique of microgel electrophoresis of single cells (DNA Comet Assay) enabled a quick detection of radiation treatment of several kinds of leguminous beans (azuki, black, black eye, mung, pinto, red kidney and white beans). Each variety was exposed to radiation doses of 0.5, 1 and 5kGy covering the permissible limits for insect disinfestation. The cells or nuclei from beans were extracted in cold PBS, embedded in agarose on microscope slides, lysed between 15 and 60min in 2.5% SDS and electrophoresis was carried out at a voltage of 2V/cm for 2-2.5min. After silver staining, the slides were evaluated through an ordinary transmission microscope. In irradiated samples, fragmented DNA stretched towards the anode and the damaged cells appeared as a comet. The density of DNA in the tails increased with increasing radiation dose. However, in non-irradiated samples, the large molecules of DNA remained relatively intact and there was only minor or no migration of DNA; the cells were round or had very short tails only. Hence, the DNA comet assay provides an inexpensive, rapid and relatively simple screening method for the detection of irradiated beans.

  19. 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.

  20. Two novel nonradioactive polymerase chain reaction-based assays of dried blood spots, genomic DNA, or whole cells for fast, reliable detection of Z and S mutations in the alpha 1-antitrypsin gene

    DEFF Research Database (Denmark)

    Andresen, B S; Knudsen, I; Jensen, P K;

    1992-01-01

    Two new nonradioactive polymerase chain reaction (PCR)-based assays for the Z and S mutations in the alpha 1-antitrypsin gene are presented. The assays take advantage of PCR-mediated mutagenesis, creating new diagnostic restriction enzyme sites for unambiguous discrimination between test samples...... from individuals who are normal, heterozygous, or homozygous for the mutations. We show that the two assays can be performed with purified genomic DNA as well as with boiled blood spots. The new assays were validated by parallel testing with a technique in which PCR is combined with allele......-specific oligonucleotide (ASO) probes. In all cases tested the results obtained by the different techniques were in accordance. The new assays can be used for prenatal diagnostics and can be performed directly with boiled tissue samples. Because the new assays are easy to perform and reliable, we conclude that they are...

  1. How to evaluate PCR assays for the detection of low-level DNA

    DEFF Research Database (Denmark)

    Banch-Clausen, Frederik; Urhammer, Emil; Rieneck, Klaus;

    2015-01-01

    High sensitivity of PCR-based detection of very low copy number DNA targets is crucial. Much focus has been on design of PCR primers and optimization of the amplification conditions. Very important are also the criteria used for determining the outcome of a PCR assay, e.g. how many replicates are...... significantly different from experimental data generated by testing of cell-free foetal DNA. Also, the simplified formula was applicable for fast and accurate assay evaluation. In conclusion, the model can be applied for evaluation of sensitivity of real-time PCR-based detection of low-level DNA, and may also...... distribution describing parameters for singleplex real-time PCR-based detection of low-level DNA. The model was tested against experimental data of diluted cell-free foetal DNA. Also, the model was compared with a simplified formula to enable easy predictions. The model predicted outcomes that were not...

  2. Detection of plant viruses by enzyme-linked immunosorbent assay

    International Nuclear Information System (INIS)

    Investigations were carried out to check the sensitivity, specificity and efficiency of the enzyme-linked immunosorbent assay (ELISA) test for the detection of potato leafroll virus, potato virus Y, potato virus S, potato virus M and beet necrotic yellow vein virus (BNYVV). For the identification of potato viruses, a comparative test of the conventional eye-shoot and ELISA techniques was performed on leaf and tuber material. A good correlation (88-100%) was achieved between the results obtained with these methods and the large differences between the extinction values of infected and healthy plant material (positive results were 13-49 times higher than negative ones). For detection of BNYVV in sugar-beets, the ELISA method and immune electron microscopy (the Derrick method) were compared. Correlation between the results obtained with the two assays was 78 and 87%, respectively. Owing to the low titre of immunized gammaglobulin and the long reaction time, after pipetting, of the p-nitrophenylphosphate, overlapping extinction values of the positive and negative samples occurred in classes 0.100-0.200. Investigations of the host range of BNYVV were carried out by ELISA and inoculation of test plants. Chenopodium quinoa was used as the indicator plant. Six days after mechanical inoculation, plants showed yellow-coloured local lesions with necrotic centres. If the concentration of BNYVV was low in the original plant material, inoculated indicator plants showed no symptoms of virus infection. After reinoculation of a second generation, specific lesions occurred. If high-quality test kits are available, ELISA is a very sensitive and rapid method for the detection of plant viruses. The ELISA technique is as reliable, or even more reliable, than the best conventional methods. (author)

  3. Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens.

    Science.gov (United States)

    Altinok, Ilhan; Capkin, Erol; Kayis, Sevki

    2008-10-15

    A multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of the five major fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida subsp. salmonicida, Flavobacterium columnare, Renibacterium salmoninarum, and Yersinia ruckeri. Each of the five pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The detection limits of the multiplex PCR was in the range of 2, 1, 1, 3, and 1CFU for A. hydrophila, A. salmonicida, F. columnare, R. salmoninarum, and Y. ruckeri, respectively. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria. The multiplex PCR assay was useful for the detection of the bacteria in naturally infected fish. This assay is a sensitive and specific and reproducible diagnostic tool for the simultaneous detection of five pathogenic bacteria that cause disease in fish. Therefore, it could be a useful alternative to the conventional culture based method. PMID:18499358

  4. GeXP analyzer-based multiplex reverse-transcription PCR assay for the simultaneous detection and differentiation of eleven duck viruses

    OpenAIRE

    Zhang, Yan-fang; Xie, Zhi-xun; Xie, Li-ji; Deng, Xian-wen; Xie, Zhi-qin; Luo, Si-si; Huang, Li; Huang, Jiao-ling; Zeng, Ting-Ting

    2015-01-01

    Background Duck viral pathogens primarily include the avian influenza virus (AIV) subtypes H5, H7, and H9; duck hepatitis virus (DHV); duck tembusu virus (DTMUV); egg drop syndrome virus (EDSV); duck enteritis virus (DEV); Newcastle disease virus (NDV); duck circovirus (DuCV); muscovy duck reovirus (MDRV); and muscovy duck parvovirus (MDPV). These pathogens cause great economic losses to China’s duck breeding industry. Result A rapid, specific, sensitive and high-throughput GeXP-based multipl...

  5. Rapid and sensitive detection of major uropathogens in a single-pot multiplex PCR assay.

    Science.gov (United States)

    Padmavathy, B; Vinoth Kumar, R; Patel, Amee; Deepika Swarnam, S; Vaidehi, T; Jaffar Ali, B M

    2012-07-01

    Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant healthcare burden. Escherichia coli is the most common cause of UTI accounting for up to 70 % and a variable contribution from Proteus mirabilis, Pseudomonas aeruginosa and Klebsiella pneumoniae. To establish a complete diagnostic system, we have developed a single-tube multiplex PCR assay (mPCR) for the detection of the above-mentioned four major uropathogens. The sensitivity of the assay was found to be as low as 10(2) cfu/ml of cells. The mPCR evaluated on 280 clinical isolates detected 100 % of E. coli, P. aeruginosa, P. mirabilis and 95 % of K. pneumonia. The assay was performed on 50 urine samples and found to be specific and sensitive for clinical diagnosis. In addition, the mPCR was also validated on spiked urine samples using 40 clinical isolates to demonstrate its application under different strain used in this assay. In total, mPCR reported here is a rapid and simple screening tool that can compete with conventional biochemical-based screening assays that may require 2-3 days for detection. PMID:22526571

  6. Impedimetric detection and lumped element modelling of a hemagglutination assay in microdroplets.

    Science.gov (United States)

    Marcali, Merve; Elbuken, Caglar

    2016-07-01

    Droplet-based microfluidic systems offer tremendous benefits for high throughput biochemical assays. Despite the wide use of electrical detection for microfluidic systems, application of impedimetric sensing for droplet systems is very limited. This is mainly due to the insulating oil-based continuous phase used for most aqueous samples of interest. We present modelling and experimental verification of impedimetric detection of hemagglutination in microdroplets. We have detected agglutinated red blood cells in microdroplets and screened whole blood samples for multiple antibody sera using conventional microelectrodes. We were able to form antibody and whole blood microdroplets in PDMS microchannels without any tedious chemical surface treatment. Following the injection of a blood sample into antibody droplets, we have detected the agglutination-positive and negative droplets in an automated manner. In order to understand the characteristics of impedimetric detection inside microdroplets, we have developed the lumped electrical circuit equivalent of an impedimetric droplet content detection system. The empirical lumped element values are in accordance with similar models developed for single phase electrical impedance spectroscopy systems. The presented approach is of interest for label-free, quantitative analysis of droplets. In addition, the standard electronic equipment used for detection allows miniaturized detection circuitries that can be integrated with a fluidic system for a quantitative microdroplet-based hemagglutination assay that is conventionally performed in well plates. PMID:27270895

  7. Automated detection of irradiated food with the comet assay

    International Nuclear Information System (INIS)

    Food irradiation is the process of exposing food to ionising radiation in order to disinfect, sanitise, sterilise and preserve food or to provide insect disinfestation. Irradiated food should be adequately labelled according to international and national guidelines. In many countries, there are furthermore restrictions to the product-specific maximal dose that can be administered. Therefore, there is a need for methods that allow detection of irradiated food, as well as for methods that provide a reliable dose estimate. In recent years, the comet assay was proposed as a simple, rapid and inexpensive method to fulfil these goals, but further research is required to explore the full potential of this method. In this paper we describe the use of an automated image analysing system to measure DNA comets which allow the discrimination between irradiated and non-irradiated food as well as the set-up of standard dose-response curves, and hence a sufficiently accurate dose estimation. (authors)

  8. 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)

  9. Detection of garlic gamma-irradiated by assay comet

    International Nuclear Information System (INIS)

    The garlic samples were irradiated in a facility with 60Co 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)

  10. Comprehensive TP53-denaturing gradient gel electrophoresis mutation detection assay also applicable to archival paraffin-embedded tissue

    NARCIS (Netherlands)

    Hayes, V M; Bleeker, W; Verlind, E; Timmer, T; Karrenbeld, A; Plukker, J T; Marx, M P; Hofstra, R M; Buys, C H

    1999-01-01

    A comprehensive mutation detection assay is described for the entire coding region and all splice site junctions of TP53. The assay is based on denaturing gradient gel electrophoresis, which follows either multiplex polymerase chain reaction (PCR) applied to DNA extracted from fresh or frozen tissue

  11. Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

    International Nuclear Information System (INIS)

    Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development

  12. Visual and fluorescent assays for selective detection of beta-amyloid oligomers based on the inner filter effect of gold nanoparticles on the fluorescence of CdTe quantum dots.

    Science.gov (United States)

    Xia, Ning; Zhou, Binbin; Huang, Nanbing; Jiang, Mengsha; Zhang, Jiebing; Liu, Lin

    2016-11-15

    Beta-amyloid (Aβ) peptides are the major constituents of senile plaques in the brains of Alzheimer's disease (AD) patients. Aβ monomers (AβMs) can coalesce to form small, soluble oligomers (AβOs), followed by reorganization and assembly into long, thread-like fibrils (AβFs). Recently, soluble AβOs have been regarded as reliable molecular biomarkers for the diagnosis of AD because of their high toxicity for neuronal synapse and high concentration levels in the brains of AD patients. In this work, we reported a label-free, sensitive and selective method for visual and fluorescent detection of AβOs based on the inner filter effect (IFE) of gold nanoparticles (AuNPs) on the fluorescence of CdTe quantum dots (QDs). Specifically, the fluorescence of CdTe QDs was quenched significantly by AuNPs through the IFE. PrP(95-110), an AβOs-specific binding peptide from cellular prion protein, triggered the aggregation and color change of AuNPs suspension; thus, the IFE of AuNPs on the fluorescence of CdTe QDs was weakened and the fluorescence intensity was recovered. However, in the presence of AβOs, the specific interaction of AβOs and PrP(95-110) prevented the absorption of PrP(95-110) onto the surface of AuNPs. As a result, the aggregation of AuNPs was inhibited and the fluorescence intensity of CdTe QDs was quenched again. This label-free method is specific for detection of AβOs but not for AβMs and AβFs. The detection limits were found to be 0.5nM for the visual assay and 0.2nM for the fluorescent detection. We believe that this work would be valuable for many investigations related to AD diagnosis and drug discovery. PMID:27240009

  13. 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.

  14. First direct fluorescence polarization assay for the detection and quantification of spirolides in mussel samples

    International Nuclear Information System (INIS)

    Highlights: → A direct assay based in the binding of nAChR to spirolide toxins by FP is described. → A direct relationship between FP and 13-desMeC in the range of 10-500 nM is obtained. → FP is dependent on the 13, 19-didesMeC in a higher concentration range than 13-desMeC. → 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 μg kg-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.

  15. 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.

  16. High Frequency of Chlamydia trachomatis Mixed Infections Detected by Microarray Assay in South American Samples

    Science.gov (United States)

    Gallo Vaulet, Lucía; Entrocassi, Carolina; Portu, Ana I.; Castro, Erica; Di Bartolomeo, Susana; Ruettger, Anke; Sachse, Konrad; Rodriguez Fermepin, Marcelo

    2016-01-01

    Chlamydia trachomatis is one of the most common sexually transmitted infections worldwide. Based on sequence variation in the ompA gene encoding the major outer membrane protein, the genotyping scheme distinguishes 17 recognized genotypes, i.e. A, B, Ba, C, D, Da, E, F, G, H, I, Ia, J, K, L1, L2, and L3. Genotyping is an important tool for epidemiological tracking of C. trachomatis infections, including the revelation of transmission pathways and association with tissue tropism and pathogenicity. Moreover, genotyping can be useful for clinicians to establish the correct treatment when LGV strains are detected. Recently a microarray assay was described that offers several advantages, such as rapidity, ease of standardization and detection of mixed infections. The aim of this study was to evaluate the performance of the DNA microarray-based assay for C. trachomatis genotyping of clinical samples already typed by PCR-RFLP from South America. The agreement between both typing techniques was 90.05% and the overall genotype distribution obtained with both techniques was similar. Detection of mixed-genotype infections was significantly higher using the microarray assay (8.4% of cases) compared to PCR-RFLP (0.5%). Among 178 samples, the microarray assay identified 10 ompA genotypes, i.e. D, Da, E, F, G, H, I, J, K and L2. The most predominant type was genotype E, followed by D and F. PMID:27082962

  17. Ultrasensitive immunochromatographic assay for the simultaneous detection of five chemicals in drinking water.

    Science.gov (United States)

    Xing, Changrui; Liu, Liqiang; Song, Shanshan; Feng, Min; Kuang, Hua; Xu, Chuanlai

    2015-04-15

    In this paper, we describe the development of a multicomponent lateral-flow assay based on an antibody-antigen reaction for the rapid and simultaneous detection of trace contaminants in water, including a heavy metal, algal toxin, antibiotic, hormone, and pesticide. The representative analytes chosen for the study were lead (Pb(II), microcystin-leucine-arginine (MC-LR), chloramphenicol (CAP), testosterone (T), and chlorothalonil (CTN). Five different antigens were immobilized separately in five test lines on a nitrocellulose membrane. The monoclonal antibodies specifically recognized the corresponding antigens, and there was no cross-reactivity between the antibodies in the detection assay. Samples or standards containing the five analytes were preincubated with the freeze-dried colloidal-gold-labeled monoclonal antibody conjugates to improve the sensitivity of the assay. The results were obtained within 20min with a paper-based sensor. The cut-off values for the strip test were 4ng/mL for Pb(II), 1ng/mL for MC-LR, 0.1ng/mL for CAP, 5ng/mL for T, and 5ng/mL for CTN. The assay was evaluated using spiked water samples, and the accuracy and reproducibility of the results were good. In summary, this lateral-flow device provides an effective and rapid method for the onsite detection of multiple contaminants in water samples, with no treatment or devices required. PMID:25499659

  18. Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

    Science.gov (United States)

    Bead based multiplex assays (BBMA) also referred to as Luminex, MultiAnalyte Profiling or cytometric bead array (CBA) assays, are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several, up to 50-500 analytes within a single, small sample volume). Curren...

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

    Directory of Open Access Journals (Sweden)

    Kawahara Ryuji

    2008-09-01

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

  20. Evaluation of a commercial cytotoxicity assay for detection of Clostridium difficile toxin.

    OpenAIRE

    1986-01-01

    A comparative study was performed to determine the accuracy of Clostridium difficile toxin detection. A commercial cytotoxicity assay (Bartels Immunodiagnostic Supply, Bellevue, Wash.) was compared with conventional microcytotoxicity assays, using Vero and MRC-5 cells. The Bartels system was found to be essentially equivalent to conventional cytotoxicity assays currently being performed for routine C. difficile toxin detection.

  1. Development of a rapid recombinase polymerase amplification assay for detection of Brucella in blood samples.

    Science.gov (United States)

    Ren, Hang; Yang, Mingjuan; Zhang, Guoxia; Liu, Shiwei; Wang, Xinhui; Ke, Yuehua; Du, Xinying; Wang, Zhoujia; Huang, Liuyu; Liu, Chao; Chen, Zeliang

    2016-04-01

    A rapid and sensitive recombinase polymerase amplification (RPA) assay, Bruce-RPA, was developed for detection of Brucella. The assay could detect as few as 3 copies of Brucella per reaction within 20 min. Bruce-RPA represents a candidate point-of-care diagnosis assay for human brucellosis. PMID:26911890

  2. Detection of irradiation treatment of foods using DNA 'comet assay'

    International Nuclear Information System (INIS)

    Microgel electrophoresis of single cells (DNA comet assay) has been investigated to detect irradiation treatment of some food samples. These samples of fresh and frozen rainbow trout, red lentil, gram and sliced almonds were irradiated to 1 or 2 kGy using 10 MeV electron beam from a linear accelerator. Rainbow trout samples yielded good results with samples irradiated to 1 or 2 kGy showing fragmentation of DNA and, therefore, longer comets with no intact cells. Unirradiated samples showed shorter comets with a significant number of intact cells. For rainbow trout stored in a freezer for 11 days the irradiated samples can still be discerned by electrophoresis from unirradiated samples, however, the unirradiated trouts also showed some longer comets besides some intact cells. Radiation treatment of red lentils can also be detected by this method, i.e. no intact cells in 1 or 2 kGy irradiated samples and shorter comets and some intact cells in unirradiated samples. However, the results for gram and sliced almond samples were not satisfactory since some intact DNA cells were observed in irradiated samples as well. Probably, incomplete lysis has led to these deviating results

  3. Detection of Irradiation Treatment of Foods Using DNA `Comet Assay'

    Science.gov (United States)

    Khan, Hasan M.; Delincée, Henry

    1998-06-01

    Microgel electrophoresis of single cells (DNA comet assay) has been investigated to detect irradiation treatment of some food samples. These samples of fresh and frozen rainbow trout, red lentil, gram and sliced almonds were irradiated to 1 or 2 kGy using 10 MeV electron beam from a linear accelerator. Rainbow trout samples yielded good results with samples irradiated to 1 or 2 kGy showing fragmentation of DNA and, therefore, longer comets with no intact cells. Unirradiated samples showed shorter comets with a significant number of intact cells. For rainbow trout stored in a freezer for 11 days the irradiated samples can still be discerned by electrophoresis from unirradiated samples, however, the unirradiated trouts also showed some longer comets besides some intact cells. Radiation treatment of red lentils can also be detected by this method, i.e. no intact cells in 1 or 2 kGy irradiated samples and shorter comets and some intact cells in unirradiated samples. However, the results for gram and sliced almond samples were not satisfactory since some intact DNA cells were observed in irradiated samples as well. Probably, incomplete lysis has led to these deviating results.

  4. Comet assay in the detection of irradiated garlic

    International Nuclear Information System (INIS)

    The increased claim for fresh produce has forced a consensus between nations to pay more attention to the phytosanitary regulations. Inhibition of sprouting of bulbs and tubers by applying ionising radiation is authorised by the National Food Codes in Brazil. The availability of methods for detection of irradiated food will contribute to increase consumers' confidence. A quick and simple screening test to indicate whether a food product has been irradiated or not was utilised in this study. The DNA comet assay was applied to verify whether garlic imported from China had been irradiated or not. This test has already been adopted as a European Standard (EN 13784), for detection of irradiated food. Non-irradiated control samples of garlic and garlic treated with maleic hydrazide were compared with garlic samples irradiated in our department. The unirradiated samples exhibited only limited DNA migration. If samples were irradiated, an increased DNA fragmentation was observed which permitted the discrimination between non-irradiated and irradiated samples. Since the garlic samples from China showed only very limited DNA fragmentation, they were deemed non-irradiated. Thus, this simple screening test was shown to be successful for identification of an irradiation treatment. (author)

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

    Science.gov (United States)

    Nagamine, Kenjiro; Hung, Guo-Chiuan; Li, Bingjie; Lo, Shyh-Ching

    2015-01-01

    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. PMID:26279626

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

    OpenAIRE

    Aysen Durmaz; Nurten Dikmen; Cumhur Gunduz

    2010-01-01

    “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 radiati...

  7. 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. PMID:26687075

  8. Towards the standardisation of the neuroblastoma (neuro-2a) cell-based assay for ciguatoxin-like toxicity detection in fish: application to fish caught in the Canary Islands.

    Science.gov (United States)

    Caillaud, A; Eixarch, H; de la Iglesia, P; Rodriguez, M; Dominguez, L; Andree, K B; Diogène, J

    2012-01-01

    The ouabain/veratridine-dependent neuroblastoma (neuro-2a) cell-based assay (CBA) was applied for the determination of the presence of ciguatoxin (CTX)-like compounds in ciguatera-suspected fish samples caught in the Canary Islands. In order to avoid matrix interferences the maximal concentration of wet weight fish tissue exposed to the neuro-2a cells was set at 20 mg tissue equivalent (TE) ml(-1) according to the sample preparation procedure applied. In the present study, the limit of quantification (LOQ) of CTX1B equivalents in fish extract was set at the limit of detection (LOD), being defined as the concentration of CTX1B equivalents inhibiting 20% cell viability (IC(20)). The LOQ was estimated as 0.0096 ng CTX1B eq.g TE(-1) with 23-31% variability between experiments. These values were deemed sufficient even though quantification given at the IC(50) (the concentration of CTX1B equivalents inhibiting 50% cell viability) is more accurate with a variability of 17-19% between experiments. Among the 13 fish samples tested, four fish samples were toxic to the neuro-2a cells with estimations of the content in CTX1B g(-1) of TE ranging from 0.058 (± 0.012) to 6.23 (± 0.713) ng CTX1B eq.g TE(-1). The high sensitivity and specificity of the assay for CTX1B confirmed its suitability as a screening tool of CTX-like compounds in fish extracts at levels that may cause ciguatera fish poisoning. Species identification of fish samples by DNA sequence analysis was conducted in order to confirm tentatively the identity of ciguatera risk species and it revealed some evidence of inadvertent misidentification. Results presented in this study are a contribution to the standardisation of the neuro-2a CBA and to the risk analysis for ciguatera in the Canary Islands. PMID:22394180

  9. Validation of an immunodiagnostic assay for detection of 13 Streptococcus pneumoniae serotype-specific polysaccharides in human urine.

    Science.gov (United States)

    Pride, Michael W; Huijts, Susanne M; Wu, Kangjian; Souza, Victor; Passador, Sherry; Tinder, Chunyan; Song, Esther; Elfassy, Arik; McNeil, Lisa; Menton, Ronald; French, Roger; Callahan, Janice; Webber, Chris; Gruber, William C; Bonten, Marc J M; Jansen, Kathrin U

    2012-08-01

    To improve the clinical diagnosis of pneumococcal infection in bacteremic and nonbacteremic community-acquired pneumonia (CAP), a Luminex technology-based multiplex urinary antigen detection (UAD) diagnostic assay was developed and validated. The UAD assay can simultaneously detect 13 different serotypes of Streptococcus pneumoniae by capturing serotype-specific S. pneumoniae polysaccharides (PnPSs) secreted in human urine. Assay specificity is achieved by capturing the polysaccharides with serotype-specific monoclonal antibodies (MAbs) on spectrally unique microspheres. Positivity for each serotype was based on positivity cutoff values calculated from a standard curve run on each assay plate together with positive- and negative-control urine samples. The assay is highly specific, since significant signals are detected only when each PnPS was paired with its homologous MAb-coated microspheres. Validation experiments demonstrated excellent accuracy and precision. The UAD assay and corresponding positivity cutoff values were clinically validated by assessing 776 urine specimens obtained from patients with X-ray-confirmed CAP. The UAD assay demonstrated 97% sensitivity and 100% specificity using samples obtained from patients with bacteremic, blood culture-positive CAP. Importantly, the UAD assay identified Streptococcus pneumoniae (13 serotypes) in a proportion of individuals with nonbacteremic CAP, a patient population for which the pneumococcal etiology of CAP was previously difficult to assess. Therefore, the UAD assay provides a specific, noninvasive, sensitive, and reproducible tool to support vaccine efficacy as well as epidemiological evaluation of pneumococcal disease, including CAP, in adults. PMID:22675155

  10. Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

    OpenAIRE

    José Carlos Pelielo De Mattos; Ellen Serri da Motta; Márcia Betania Nunes de Oliveira; Flávio José da Silva Dantas; Adriano Caldeira de Araujo

    2008-01-01

    Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl2) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried ...

  11. Detection of fecal bacteria and source tracking identifiers in environmental waters using rRNA-based RT-qPCR and rDNA-based qPCR assays

    Science.gov (United States)

    The identification of fecal pollution sources is commonly performed using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as “naked DNA” in the environment. To this end, we compared the detection frequency of host specific marke...

  12. Immunological assays for chemokine detection in in-vitro culture of CNS cells

    Directory of Open Access Journals (Sweden)

    Mahajan Supriya D.

    2003-01-01

    Full Text Available Herein we review the various methods currently in use for determining the expression of chemokines by CNS cells in vitro. Chemokine detection assays are used in conjuction with one another to provide a comprehensive, biologically relevant assessment of the chemokines which is necessary for correct data interpretation of a specific observed biological effect. The methods described include bioassays for soluble chemokine receptors, RNA extraction, RT-PCR, Real - time quantitative PCR, gene array analysis, northern blot analysis, Ribonuclease Protection assay, Flow cytometry, ELISPOT, western blot analysis, and ELISA. No single method of analysis meets the criteria for a comprehensive, biologically relevant assessment of the chemokines, therefore more than one assay might be necessary for correct data interpretation, a choice that is based on development of a scientific rationale for the method with emphasis on the reliability and relevance of the method.

  13. Platelet hexosaminidase a enzyme assay effectively detects carriers missed by targeted DNA mutation analysis.

    Science.gov (United States)

    Nakagawa, Sachiko; Zhan, Jie; Sun, Wei; Ferreira, Jose Carlos; Keiles, Steven; Hambuch, Tina; Kammesheidt, Anja; Mark, Brian L; Schneider, Adele; Gross, Susan; Schreiber-Agus, Nicole

    2012-01-01

    Biochemical testing of hexosaminidase A (HexA) enzyme activity has been available for decades and has the ability to detect almost all Tay-Sachs disease (TSD) carriers, irrespective of ethnic background. This is increasingly important, as the gene pool of those who identify as Ashkenazi Jewish is diversifying. Here we describe the analysis of a cohort of 4,325 individuals arising from large carrier screening programs and tested by the serum and/or platelet HexA enzyme assays and by targeted DNA mutation analysis. Our results continue to support the platelet assay as a highly effective method for TSD carrier screening, with a low inconclusive rate and the ability to detect possible disease-causing mutation carriers that would have been missed by targeted DNA mutation analysis. Sequence analysis performed on one such platelet assay carrier, who had one non-Ashkenazi Jewish parent, identified the amino acid change Thr259Ala (A775G). Based on crystallographic modeling, this change is predicted to be deleterious, as threonine 259 is positioned proximal to the HexA alpha subunit active site and helps to stabilize key residues therein. Accordingly, if individuals are screened for TSD in broad-based programs by targeted molecular testing alone, they must be made aware that there is a more sensitive and inexpensive test available that can identify additional carriers. Alternatively, the enzyme assays can be offered as a first tier test, especially when screening individuals of mixed or non-Jewish ancestry. PMID:23430931

  14. Sandwich Enzyme-Linked Immunosorbent Assay for Detecting Sesame Seed in Foods

    Directory of Open Access Journals (Sweden)

    Stef J. Koppelman

    2015-01-01

    Full Text Available Small amounts of sesame can trigger allergic reactions in sesame-allergic patients. Because sesame is a widely used food ingredient, analytical methods are needed to support quality control and food safety programs in the food industry. In this study, polyclonal antibodies against sesame seed proteins were raised, and an enzyme-linked immunosorbent assay (ELISA was developed for the detection and quantification of sesame seed residue in food. A comparison was made between this ELISA and other assays, particularly focusing on recovery of sesame seed residue from different food matrices. The developed ELISA is sensitive with a lower limit of quantification of 0.5 ppm and shows essentially no cross-reactivity with other foods or food ingredients (92 tested. The ELISA has a good recovery for analyzing sesame-based tahini in peanut butter, outperforming one other test. In a baked bread matrix, the ELISA has a low recovery, while two other assays perform better. We conclude that a sensitive and specific ELISA can be constructed based on polyclonal antibodies, which is suitable for detection of small amounts of sesame seed relevant for highly allergic patients. Furthermore, we conclude that different food products may require different assays to ensure adequate quantification of sesame.

  15. Serum Bactericidal Assay: New Role in Salmonella Detection.

    Science.gov (United States)

    Chen, Yu; Wu, Da; Sun, Min; Deng, Mingjun; Cui, Shuhua; Liang, Chengzhu; Geng, Juan; Sun, Tao; Long, Ling; Xiao, Xizhi

    2016-01-01

    While inspecting animal feed for Salmonella contamination, we routinely observed bacterial colonies on selective agars that were similar in appearance to those formed by Salmonella. These were identified as Citrobacter freundii, Proteus mirabilis, and Serratia fonticola using biochemical and serological techniques. Because the presence of these bacterial species confounds identification of Salmonella, we refer to them as "interference bacteria." Polyvalent antisera against these interference bacteria were prepared by immunizing rabbits with a mixture of all three organisms. To minimize or eliminate interference by these bacteria, the polyvalent antisera were introduced between the steps of selective enrichment and Salmonella-selective plating. The antisera raised against the interference bacteria, when combined with neonatal rabbit complement, exhibited specific bactericidal activity against C. freundii, P. mirabilis, and S. fonticola. The respective serum bactericidal assay titers were 2(9), 2(8), and 2(10). In selective broth, polyvalent antisera could also kill the target bacterial cells effectively. We tested 526 samples (186 white fishmeal, 97 red fishmeal, and 243 cattle bone powder) using the polyvalent antisera and found that the rates of contamination of each species of the three respective foods decreased by 58.8, 100, and 83%. Our data indicates that polyvalent sera against C. freundii, P. mirabilis, and S. fonticola can be used as inhibitors to increase the accuracy of Salmonella detection. PMID:26822285

  16. A sensitive in vitro assay for the detection of residual viable rabies virus in inactivated rabies vaccines.

    Science.gov (United States)

    Takayama-Ito, Mutsuyo; Nakamichi, Kazuo; Kinoshita, Hitomi; Kakiuchi, Satsuki; Kurane, Ichiro; Saijo, Masayuki; Lim, Chang-Kweng

    2014-01-01

    Rabies is a viral disease transmitted through bites from rabid animals and can be prevented by vaccines. Clinically used rabies vaccines are prepared from inactivated rabies viruses grown in cell cultures or embryonated eggs. In Japan and across the world, tests that confirm complete inactivation, such as the in vivo suckling mouse assay, in which suckling mice are intracerebrally inoculated with vaccine products, are required for quality control. In this study, we developed a novel cell-based immunofluorescence assay that does not require mice for testing rabies vaccine inactivation for human use. The sensitivity of this cell-based in vitro assay was 5.7 times that of the in vivo suckling mouse assay, with a detection limit of one focus forming units per ml of test sample. This newly developed in vitro assay may replace the established in vivo suckling mouse assay for confirming viral vaccine inactivation. PMID:24321529

  17. Rapid detection of Candida albicans by polymerase spiral reaction assay in clinical blood samples

    Directory of Open Access Journals (Sweden)

    Xiaoqun eJiang

    2016-06-01

    Full Text Available Candida albicans is the most common human yeast pathogen which causes mucosal infections and invasive fungal diseases. Early detection of this pathogen is needed to guide preventative and therapeutic treatment. The aim of this study was to establish a polymerase spiral reaction (PSR assay that rapidly and accurately detects C. albicans and to assess the clinical applicability of PSR-based diagnostic testing. Internal transcribed spacer 2 (ITS2, a region between 5.8S and 28S fungal ribosomal DNA, was used as the target sequence. Four primers were designed for amplification of ITS2 with the PSR method, which was evaluated using real time turbidity monitoring and visual detection using a pH indicator. Fourteen non- C. albicans yeast strains were negative for detection, which indicated the specificity of PSR assay was 100%. A 10-fold serial dilution of C. albicans genomic DNA was subjected to PSR and conventional PCR to compare their sensitivities. The detection limit of PSR was 6.9 pg/µl within 1 h, 10-fold higher than that of PCR (69.0 pg/µl. Blood samples (n=122 were collected from intensive care unit and hematological patients with proven or suspected C. albicans infection at two hospitals in Beijing, China. Both PSR assay and the culture method were used to analyze the samples. Of the 122 clinical samples, 34 were identified as positive by PSR. The result was consistent with those obtained by the culture method. In conclusion, a novel and effective C. albicans detection assay was developed that has a great potential for clinical screening and point-of-care testing.

  18. Application of colloidal gold immunochromatographic assay in rapid detection of mycotoxins

    OpenAIRE

    YING Mei-Rong; Sheng, Hui-Ping; SANG Li-Ya

    2013-01-01

    Colloidal gold immunochromatographic assay(GICA), which is a fast, simple, specific and sensitive detection method and which can make up for the deficiency of trational methods of mycotoxins, is suitable for the rapid detection of mycotoxins. This paper mainly introduced the principle of the colloidal gold immunochromatographic assay, its application and prospect in detection of mycotoxin.

  19. Magnetic Barcode Assay for Genetic Detection of Pathogens

    OpenAIRE

    Liong, Monty; Hoang, Anh N.; Chung, Jaehoon; Gural, Nil; Ford, Christopher B; Min, Changwook; Shah, Rupal R.; Ahmad, Rushdy; Fernandez-Suarez, Marta; Fortune, Sarah M.; Toner, Mehmet; Lee, Hakho; Weissleder, Ralph

    2013-01-01

    The task of rapidly identifying patients infected with Mycobacterium tuberculosis (MTB) in resource-constrained environments remains a challenge. A sensitive and robust platform that does not require bacterial isolation or culture is critical in making informed diagnostic and therapeutic decisions. Here we introduce a platform for the detection of nucleic acids based on a magnetic barcoding strategy. PCR-amplified mycobacterial genes are sequence-specifically captured on microspheres, labeled...

  20. Development and Evaluation of a Novel Taqman Real-Time PCR Assay for Rapid Detection of Mycoplasma bovis: Comparison of Assay Performance with a Conventional PCR Assay and Another Taqman Real-Time PCR Assay

    OpenAIRE

    Hemant Naikare; Daniela Bruno; Debabrata Mahapatra; Alesia Reinisch; Russell Raleigh; Robert Sprowls

    2015-01-01

    The objective of this study was to develop and validate a Taqman real-time PCR assay for the detection of Mycoplasma bovis (M. bovis). Unique primers targeting the highly conserved house-keeping gene (uvrC) were designed and the probe sequence was derived from a previously published microarray study. There was 100% agreement in the outcome between our assay and the other two published assays for M. bovis detection. The analytical limit of detection of our assay is 83 copies of the uvrC gene. ...

  1. Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of Serotypes 40 and 41

    OpenAIRE

    Jothikumar, Narayanan; Cromeans, Theresa L.; Hill, Vincent R.; Lu, Xiaoyan; Sobsey, Mark D.; Erdman, Dean D.

    2005-01-01

    A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV4...

  2. Compact upconverting phosphor detection system for wick assays

    Science.gov (United States)

    Riris, Haris; Van der Laan, Jan E.; Cooper, David E.; Nashold, Karen M.; Carlisle, Clinton B.; Schneider, Luke V.

    1997-05-01

    We describe an integrated detection system based on upconverting phosphor particles bound to capture sites on the inside surfaces of rectangular wick capillaries. This deice can be used with either antibody or nucleic acid to detect specific micro-organisms. The system uses a high- power, 980 nm, semiconductor diode laser to illuminate 200 X 300 X 20 micrometers capture surfaces. The rectangular capillary wicks are held in a tray that is inserted into the detection system, positioning the capture surface at the object plane of the optical system. Phosphorescent light emitted from the capture surface is collected by a high numerical aperture microscope objective and directed through a series of filters onto either a CCD camera or a photomultiplier. A combination of band-reject filters attenuates the 980 nm laser excitation light and its harmonic at 490 nm, and a tunable liquid crystal filter provides for rapid scanning from 400 to 750 nm. The data acquisition and control is controlled by a laptop PC with a custom GUI interface developed using LabWindows/CVI. The system can detect a single phosphor particle bound to a capture surface.

  3. A homogeneous nucleic acid hybridization assay based on strand displacement.

    OpenAIRE

    Vary, C P

    1987-01-01

    A homogeneous nucleic acid hybridization assay which is conducted in solution and requires no separation steps is described. The assay is based on the concept of strand displacement. In the strand displacement assay, an RNA "signal strand" is hybridized within a larger DNA strand termed the "probe strand", which is, in turn, complementary to the target nucleic acid of interest. Hybridization of the target nucleic acid with the probe strand ultimately results in displacement of the RNA signal ...

  4. A reverse transcriptase-PCR assay for detecting filarial infective larvae in mosquitoes.

    Directory of Open Access Journals (Sweden)

    Sandra J Laney

    Full Text Available BACKGROUND: Existing molecular assays for filarial parasite DNA in mosquitoes cannot distinguish between infected mosquitoes that contain any stage of the parasite and infective mosquitoes that harbor third stage larvae (L3 capable of establishing new infections in humans. We now report development of a molecular L3-detection assay for Brugia malayi in vectors based on RT-PCR detection of an L3-activated gene transcript. METHODOLOGY/PRINCIPAL FINDINGS: Candidate genes identified by bioinformatics analysis of EST datasets across the B. malayi life cycle were initially screened by PCR using cDNA libraries as templates. Stage-specificity was confirmed using RNA isolated from infected mosquitoes. Mosquitoes were collected daily for 14 days after feeding on microfilaremic cat blood. RT-PCR was performed with primer sets that were specific for individual candidate genes. Many promising candidates with strong expression in the L3 stage were excluded because of low-level transcription in less mature larvae. One transcript (TC8100, which encodes a particular form of collagen was only detected in mosquitoes that contained L3 larvae. This assay detects a single L3 in a pool of 25 mosquitoes. CONCLUSIONS/SIGNIFICANCE: This L3-activated gene transcript, combined with a control transcript (tph-1, accession # U80971 that is constitutively expressed by all vector-stage filarial larvae, can be used to detect filarial infectivity in pools of mosquito vectors. This general approach (detection of stage-specific gene transcripts from eukaryotic pathogens may also be useful for detecting infective stages of other vector-borne parasites.

  5. Development of a multiplex PCR assay detecting 52 autosomal SNPs

    DEFF Research Database (Denmark)

    Sanchez Sanchez, Juan Jose; Phillips, C.; Børsting, Claus;

    2006-01-01

    An efficient method that can be used to simultaneously amplify a set of genetic loci across the genome with high reliability can provide a valuable tool for single nucleotide polymorphism (SNP) forensic genotyping. A crucial element is the number of individual biochemical reactions that must be...... performed. The SNPforID consortium (www.snpforid.org) was established in 2003 with the principal goal of developing a SNP-based system of DNA analysis that would have comparable discrimination power and ease of use to those of existing short tandem repeat (STR) based techniques. Here, we describe a strategy...... for amplifying 52 genomic DNA fragments, each containing one SNP, in a single tube, and accurately genotyping the PCR product mixture using two single base extension reactions. This multiplex approach reduces the cost of SNP genotyping and requires as little as 0.5 ng of genomic DNA to detect 52 SNPs...

  6. Detection of hypoxic cells in murine tumors using the comet assay. Comparison with a conventional radiobiological assay

    International Nuclear Information System (INIS)

    The comet (single-cell electrophoresis) assay has been developed as a method for measuring DNA damage in single cells after irradiation. We have developed our own methods and image analysis system for the comet assay to identify hypoxic fractions. In vitro, we tested our system using a cultured tumor cell line (SCCVII). In vivo, we compared the hypoxic fractions detected by this assay with those determined by the in vivo-in vitro clonogenic assay using two rodent tumors (SCCVII/C3H, EMT6/KU/balb/c), which exhibit different types of hypoxia: acute and chronic. In vitro, our method could differentiate hypoxic cells from oxic cells, using the parameter of tail moment. In vivo, there were good correlations between the hypoxic fractions determined by the comet assay and by the clonogenic assay, in SCCVII/C3H (r=0.85) and in EMT6/KU/balb/c (r=0.75) tumors. By comparison of the two methods in chronically hypoxic and acutely hypoxic tumors, we further confirmed that the comet assay is clinically useful for estimating hypoxic fractions of solid tumors. (author)

  7. Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of Serotypes 40 and 41

    Science.gov (United States)

    Jothikumar, Narayanan; Cromeans, Theresa L.; Hill, Vincent R.; Lu, Xiaoyan; Sobsey, Mark D.; Erdman, Dean D.

    2005-01-01

    A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples. PMID:15933012

  8. Hybridization assay based on evanescent fluorescence excitation and collection

    Science.gov (United States)

    Sumner, James J.; Mmerole, Robert U.; Stratis-Cullum, Dimitra N.; Yi, Hyunmin; Bentley, William E.; Gillespie, James B.

    2003-08-01

    There is a great need for high throughput and sensitive sensors for genetic analysis. These sensors can be used for varied purposes from monitoring gene expression in organims to speciation of possible pathogens. Consequently, an instrument capable of these tasks would be a great benefit for food and water safety, medical diagnostics and defense of military and civilian populations from biological threats. This work examines the development of a hybridization-based biosensor using a novel tapered fiber optic rpobe. The immobilization of single-stranded, synthetic ologinucleotides utilizing aminoproplytriethoxysilane and glutaraldehyde was implemented on the fiber optic sensor. Hybridization takes place with a complementary analyte sequence followed by a fluorescent, labeled signaling probe to form a sandwich assay. Following hybridization, the fiber is interrogated with a diode laser source and the resulting fluorescence signal is detected using a miniature spectrometer.

  9. Gold Nanoparticle-based Dynamic Light Scattering Assay for Detection of Mercury Ion%基于金纳米粒子的动态光散射法检测溶液中的汞离子

    Institute of Scientific and Technical Information of China (English)

    马立娜; 刘殿骏; 王振新

    2014-01-01

    A gold nanoparticle ( GNP)-based dynamic light scattering ( DLS) assay has been developed for detecting mercury ion ( Hg2+) in aqueous solution. The GNPs are able to maintain monodisperse state in relative high ionic strength ( 0. 1 mol/L NaCl ) aqueous solution because Hg2+ aptamer 5’-TTTCTTCTTTCTTCCCCCCTTGTTTGTTGTTT-3’( Probe DNA ) can adsorb on the GNP surface by electrostatic interaction. In the presence of Hg2+, the Probe DNA molecules easily desorb from GNP surface because of T-Hg2+-T formation. The unprotected GNPs form aggregates in 100 mmol/L NaCl solution, resulting in increased significantly the average hydrodynamic diameter of GNPs. Under the conditions of pH 7. 43, 110 nmol/L aptamer, 100 mmol/L NaCl and 30 minutes incubation time of Hg2+with aptamer, the difference of average hydrodynamic diameter (△D ) of GNPs was increased linearly with logarithm of Hg2+concentration over the range from 0. 1 nmol/L to 5 μmol/L, with a detection limit of 0. 1 nmol/L. Moreover, satisfactory results were obtained when the proposed method was applied to detect Hg2+ in real samples.%发展了一种基于汞离子( Hg2+)适配体( Aptamer)免标记金纳米粒子的动态光散射( DLS)法,用于灵敏、选择性的检测溶液中的Hg2+。 Aptamer 5'-TTTCTTCTTTCTTCCCCCCTTGTTTGTTGTTT-3'与Hg2+的特异性结合使金纳米粒子失去保护,在含有100 mmol/L NaCl的缓冲溶液中发生聚集,金纳米粒子的平均水合粒径变大。在pH=7.43,110 nmol/L Aptamer ,100 mmol/L NaCl,Hg2+与Probe DNA孵育时间为30 min的实验条件下,金纳米粒子水合粒径的变化值(DD)与Hg2+的浓度成正比。检出Hg2+的线性范围为0.1 nmol/L~5μmol/L,检出限达0.1 nmol/L。湖水及矿泉水两种水样加标实验表明本方法能够用于实际水样中Hg2+的检测。

  10. Reliability of a bioluminescence ATP assay for detection of bacteria.

    OpenAIRE

    Selan, L.; Berlutti, F; Passariello, C.; Thaller, M C; Renzini, G

    1992-01-01

    The reliability of bioluminescence assays which employ the luciferin-luciferase ATP-dependent reaction to evaluate bacterial counts was studied, both in vitro and on urine specimens. Bioluminescence and cultural results for the most common urinary tract pathogens were analyzed. Furthermore, the influence of the culture medium, of the assaying method, and of the phase of growth on bioluminescence readings was studied. Results show that Proteus, Providencia, and Morganella strains are not corre...

  11. An optimized one-tube, semi-nested PCR assay for Paracoccidioides brasiliensis detection

    Directory of Open Access Journals (Sweden)

    Amanda de Faveri Pitz

    2013-12-01

    Full Text Available Introduction Herein, we report a one-tube, semi-nested-polymerase chain reaction (OTsn-PCR assay for the detection of Paracoccidioides brasiliensis. Methods We developed the OTsn-PCR assay for the detection of P. brasiliensis in clinical specimens and compared it with other PCR methods. Results The OTsn-PCR assay was positive for all clinical samples, and the detection limit was better or equivalent to the other nested or semi-nested PCR methods for P. brasiliensis detection. Conclusions The OTsn-PCR assay described in this paper has a detection limit similar to other reactions for the molecular detection of P. brasiliensis, but this approach is faster and less prone to contamination than other conventional nested or semi-nested PCR assays.

  12. Enzymatic assay for calmodulins based on plant NAD kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.

    1984-01-01

    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  13. Design of a multiplex PCR assay for the simultaneous detection and confirmation of Neisseria gonorrhoeae.

    LENUS (Irish Health Repository)

    O'Callaghan, Isabelle

    2010-05-01

    To improve the detection of Neisseria gonorrhoeae by designing a multiplex PCR assay using two N gonorrhoeae-specific genes as targets, thereby providing detection and confirmation of a positive result simultaneously.

  14. A novel method for detection of dioxins. Exonuclease protection mediated PCR assay

    Energy Technology Data Exchange (ETDEWEB)

    Xu, S.Q.; Sun, X.; Li, F.; Li, B.S. [Huazhong Univ. of Science and Technology, Wuhan, HB (China). Tongji Medical College

    2004-09-15

    The aromatic hydrocarbon receptor (AhR) is a ligand-actived transcription factor that mediates many of the biologic and toxicologic effects of dioxin-like chemicals (DLCs), such as 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). Numerous AhR-based bioassays for identification and detection of DLCs have been developed in vitro. Such as the chemical-activated luciferase gene expression (CALUX), ethoxyresolufin-O-deethylase (EROD) activity are sometimes represented as the next best system when compared with whole body or in vivo systems. However, cell systems can be affected by the toxic chemical itself during the assay, thus confusing problems couldn't be avoided in the assay. Incorporation of metabolism in cell systems with uncertain consequences prolongs assay complexity and time. Thus these drawbacks limit the utility of cell systems for screening purposes. Most cell-free bioassays require radioactivity, such as the gel retardation of AhR binding (GRAB) assay, or antibody of AhR or ligand, which are unfeasible for some laboratories. Here a cell-free bioanalysis method, Exonuclease Protection Mediated PCR (EPM-PCR) bioassay, was established for detection of AhR ligands based on the binding of the dioxin:AhR complex to the specific DNA. EPM-PCR can provide indirect detection of ligands by quantification of the specific AhR-binding DNA, no necessary of any DNA labeling and sophisticated equipments. This new bioassay not only has the higher sensitivity and specificity, but it is rapid and easy to perform.

  15. Development and validation of a real-time PCR assay for specific and sensitive detection of canid herpesvirus 1.

    Science.gov (United States)

    Decaro, Nicola; Amorisco, Francesca; Desario, Costantina; Lorusso, Eleonora; Camero, Michele; Bellacicco, Anna Lucia; Sciarretta, Rossana; Lucente, Maria Stella; Martella, Vito; Buonavoglia, Canio

    2010-10-01

    A TaqMan-based real-time PCR assay targeting the glycoprotein B-encoding gene was developed for diagnosis of canid herpesvirus 1 (CHV-1) infection. The established assay was highly specific, since no cross-reactions were observed with other canine DNA viruses, including canine parvovirus type 2, canine minute virus, or canine adenovirus types 1 and 2. The detection limit was 10(1) and 1.20 x 10(1) DNA copies per 10 microl(-1) of template for standard DNA and a CHV-1-positive kidney sample, respectively: about 1-log higher than a gel-based PCR assay targeting the thymidine kinase gene. The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. CHV-1 isolates of different geographical origins were recognised by the TaqMan assay. Tissues and clinical samples collected from three pups which died of CHV-1 neonatal infection were also tested, displaying a wide distribution of CHV-l DNA in their organs. Unlike other CHV-1-specific diagnostic methods, this quantitative assay permits simultaneous detection and quantitation of CHV-1 DNA in a wide range of canine tissues and body fluids, thus providing a useful tool for confirmation of a clinical diagnosis, for the study of viral pathogenesis and for evaluation of the efficacy of vaccines and antiviral drugs. PMID:20674611

  16. A smartphone-readable barcode assay for the detection and quantitation of pesticide residues.

    Science.gov (United States)

    Guo, Juan; Wong, Jessica X H; Cui, Caie; Li, Xiaochun; Yu, Hua-Zhong

    2015-08-21

    In this paper, we present a smartphone-readable barcode assay for the qualitative detection of methyl parathion residues, a toxic organophosphorus pesticide that is popularly used in agriculture worldwide. The detection principle is based on the irreversible inhibition of the enzymatic activity of acetylcholinesterase (AchE) by methyl parathion; AchE catalytically hydrolyzes acetylthiocholine iodine to thiocholine that in turn dissociates dithiobis-nitrobenzoate to produce a yellow product (deprotonated thio-nitrobenzoate). The yellow intensity of the product was confirmed to be inversely dependent on the concentration of the pesticide. We have designed a barcode-formatted assay chip by using a PDMS (polydimethylsiloxane) channel plate (as the reaction reservoir), situated under a printed partial barcode, to complete the whole barcode such that it can be directly read by a barcode scanning app installed on a smartphone. The app is able to qualitatively present the result of the pesticide test; the absence or a low concentration of methyl parathion results in the barcode reading as "-", identifying the test as negative for pesticides. Upon obtaining a positive result (the app reads a "+" character), the captured image can be further analyzed to quantitate the methyl parathion concentration in the sample. Besides the portability and simplicity, this mobile-app based colorimetric barcode assay compares favorably with the standard spectrophotometric method. PMID:26087169

  17. Enzyme-linked immunosorbent assay for Salmonella typhimurium in food: feasibility of 1-day Salmonella detection.

    OpenAIRE

    Lee, H. A.; Wyatt, G M; Bramham, S; Morgan, M.R.

    1990-01-01

    A microtitration plate, antibody-capture, enzyme-linked immunosorbent assay was developed for detection of Salmonella typhimurium. The assay utilizes a monoclonal detector antibody which shows no cross-reactions with non-Salmonella species and only a slight cross-reaction with one other Salmonella serotype. By using only one cultural stage (in a nonselective, chemically defined medium) prior to the enzyme-linked immunosorbent assay, low numbers of cells in food (10 cells 25 g-1) were detected...

  18. An improved microbial screening assay for the detection of quinolone residues in egg and poultry muscle

    OpenAIRE

    Pikkemaat, Mariel G; Mulder, Patrick P. J.; Elferink, J.W. Alexander; Nielen, Michel; De Cocq, Angela; Van Egmond, Harry J

    2007-01-01

    Abstract An improved microbiological screening assay for the detection of quinolone residues in poultry muscle and egg samples is presented. The method was validated using fortified tissue samples and is the first microbial assay effectively detecting enrofloxacin, difloxacin, danofloxacin, as well as flumequine and oxolinic acid at or below their EU Maximum Residue Limits (MRL). The accuracy of the assay is shown by analyzing incurred tissue samples containing residue levels arou...

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

    Science.gov (United States)

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

    2016-07-01

    Human rhinoviruses (HRVs) have long been recognized as the cause of more than one-half of acute viral upper respiratory illnesses, and they are associated with more-serious diseases in children, such as asthma, acute otitis media and pneumonia. A rapid and universal test for of HRV infection is in high demand. In this study, a reverse transcription genome exponential amplification reaction (RT-GEAR) assay targeting the HRV 5' untranslated region (UTR) was developed for pan-HRV detection. The reaction was performed in a single tube in one step at 65 °C for 60 min using a real-time fluorometer (Genie(®)II; Optigene). The RT-GEAR assay showed no cross-reactivity with common human enteroviruses, including HEV71, CVA16, CVA6, CVA10, CVA24, CVB5, Echo30, and PV1-3 or with other common respiratory viruses including FluA H3, FluB, PIV1-4, ADV3, RSVA, RSVB and HMPV. With in vitro-transcribed RNA containing the amplified regions of HRV-A60, HRV-B06 and HRV-C07 as templates, the sensitivity of the RT-GEAR assay was 5, 50 and 5 copies/reaction, respectively. Experiments to evaluate the clinical performance of the RT-GEAR assay were also carried out with a panel of 143 previously verified samples, and the results were compared with those obtained using a published semi-nested PCR assay followed by sequencing. The tested panel comprised 91 HRV-negative samples and 52 HRV-positive samples (18 HRV-A-positive samples, 3 HRV-B-positive samples and 31 HRV-C-positive samples). The sensitivity and specificity of the pan-HRVs RT-GEAR assay was 98.08 % and 100 %, respectively. The kappa correlation between the two methods was 0.985. The RT-GEAR assay based on a portable Genie(®)II fluorometer is a sensitive, specific and rapid assay for the universal detection of HRV infection. PMID:27132014

  20. Biotoxin Detection Using Cell-Based Sensors

    OpenAIRE

    Pratik Banerjee; Spyridon Kintzios; Balabhaskar Prabhakarpandian

    2013-01-01

    Cell-based biosensors (CBBs) utilize the principles of cell-based assays (CBAs) by employing living cells for detection of different analytes from environment, food, clinical, or other sources. For toxin detection, CBBs are emerging as unique alternatives to other analytical methods. The main advantage of using CBBs for probing biotoxins and toxic agents is that CBBs respond to the toxic exposures in the manner related to actual physiologic responses of the vulnerable subjects. The results ob...

  1. Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Common Strains of Escherichia coli▿

    OpenAIRE

    Hill, Joshua; Beriwal, Shilpa; Chandra, Ishwad; Paul, Vinod K.; Kapil, Aarti; Singh, Tripti; Wadowsky, Robert M.; Singh, Vinita; Goyal, Ankur; Jahnukainen, Timo; Johnson, James R.; Tarr, Phillip I.; Vats, Abhay

    2008-01-01

    We developed a highly sensitive and specific LAMP assay for Escherichia coli. It does not require DNA extraction and can detect as few as 10 copies. It detected all 36 of 36 E. coli isolates and all 22 urine samples (out of 89 samples tested) that had E. coli. This assay is rapid, low in cost, and simple to perform.

  2. Improved microbial screning assay for the detection of quinolone residues in poultry and eggs

    NARCIS (Netherlands)

    Pikkemaat, M.G.; Mulder, P.P.J.; Elferink, J.W.A.; Cocq, A.; Nielen, M.W.F.; Egmond, van H.J.

    2007-01-01

    An improved microbiological screening assay is reported for the detection of quinolone residues in poultry muscle and eggs. The method was validated using fortified tissue samples and is the first microbial assay to effectively detect enrofloxacin, difloxacin, danofloxacin, as well as flumequine and

  3. Performance of three microimmunofluorescence assays for detection of Chlamydia pneumoniae immunoglobulin M, G, and A antibodies

    DEFF Research Database (Denmark)

    Bennedsen, Mette; Berthelsen, Lene; Lind, Inga

    2002-01-01

    , 100 sera from 100 persons enrolled in the Copenhagen City Heart Study. The accordance among the results of the WRF assay and the two commercial assays was excellent for the immunoglobulin M (IgM) antibody detection rate (98%). The accordance in detection rates for IgG and IgA antibodies in sera from...

  4. Survey of culture, goldengate assay, universal biosensor assay, and 16S rRNA Gene sequencing as alternative methods of bacterial pathogen detection.

    Science.gov (United States)

    Lindsay, Brianna; Pop, Mihai; Antonio, Martin; Walker, Alan W; Mai, Volker; Ahmed, Dilruba; Oundo, Joseph; Tamboura, Boubou; Panchalingam, Sandra; Levine, Myron M; Kotloff, Karen; Li, Shan; Magder, Laurence S; Paulson, Joseph N; Liu, Bo; Ikumapayi, Usman; Ebruke, Chinelo; Dione, Michel; Adeyemi, Mitchell; Rance, Richard; Stares, Mark D; Ukhanova, Maria; Barnes, Bret; Lewis, Ian; Ahmed, Firoz; Alam, Meer Taifur; Amin, Ruhul; Siddiqui, Sabbir; Ochieng, John B; Ouma, Emmanuel; Juma, Jane; Mailu, Eunice; Omore, Richard; O'Reilly, Ciara E; Hannis, James; Manalili, Sheri; Deleon, Jonna; Yasuda, Irene; Blyn, Lawrence; Ranken, Raymond; Li, Feng; Housley, Roberta; Ecker, David J; Hossain, M Anowar; Breiman, Robert F; Morris, J Glenn; McDaniel, Timothy K; Parkhill, Julian; Saha, Debasish; Sampath, Rangarajan; Stine, O Colin; Nataro, James P

    2013-10-01

    Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens. PMID:23884998

  5. Computer-determined assay time based on preset precision

    International Nuclear Information System (INIS)

    Most current assay systems for special nuclear materials (SNM) operate on the principle of a fixed assay time which provides acceptable measurement precision without sacrificing the required throughput of the instrument. Waste items to be assayed for SNM content can contain a wide range of nuclear material. Counting all items for the same preset assay time results in a wide range of measurement precision and wastes time at the upper end of the calibration range. A short time sample taken at the beginning of the assay could optimize the analysis time on the basis of the required measurement precision. To illustrate the technique of automatically determining the assay time, measurements were made with a segmented gamma scanner at the Plutonium Facility of Los Alamos National Laboratory with the assay time for each segment determined by counting statistics in that segment. Segments with very little SNM were quickly determined to be below the lower limit of the measurement range and the measurement was stopped. Segments with significant SNM were optimally assays to the preset precision. With this method the total assay time for each item is determined by the desired preset precision. This report describes the precision-based algorithm and presents the results of measurements made to test its validity

  6. Identification of new quinic acid derivatives as histone deacetylase inhibitors by fluorescence-based cellular assay.

    Science.gov (United States)

    Son, Dohyun; Kim, Chung Sub; Lee, Kang Ro; Park, Hyun-Ju

    2016-05-01

    A fluorescence-based cellular assay system was established to identify potential epigenetic modulator ligands. This assay method is to detect the de-repression of an EGFP reporter in cancer cells by the treatment of HDAC (histone deacetylase) or DNMT (DNA methyltransferase) inhibitor. Using this system, we conducted a preliminary screening of in-house natural product library containing extracts and pure compounds, and identified several active compounds. Among them, novel quinic acid derivatives were recognized as excellent HDAC inhibitors by both enzymatic and cell-based HDAC assays. PMID:26996372

  7. An immunohistochemical assay to detect trophoblasts in frozen feline placenta.

    Science.gov (United States)

    Scott, Veronica L; Wallace, Kedra; Mays, Stephany; Ryan, Peter; Coats, Karen S

    2011-03-01

    The placenta, a fetal endocrine organ, is composed of subpopulations of trophoblasts, including cytotrophoblasts, and syncytiotrophoblasts. Trophoblastic populations can be distinguished based upon their expression of cytokeratin. The purpose of the current study was to develop an immunohistochemistry (IHC) method to identify trophoblasts selectively in frozen feline placental tissue using antibodies specific for cytokeratin. The mouse monoclonal antibody anti-human pan-cytokeratin AE1/AE3 and a commercial detection system were used. Nonspecific immunoreactivity was encountered that could not be eliminated with altered blocking methods. The nonspecific reactivity was attributed to the goat anti-mouse/rabbit immunoglobulin G (IgG) peroxidase polymer included in the commercial kit. Alternatively, a polyclonal rabbit anti-cow cytokeratin wide spectrum screening antibody with goat anti-rabbit IgG polyclonal secondary antibody was used to detect cytokeratin in feline placental tissue. The IHC procedure eliminated nonspecific immunoreactivity while specifically labeling cytokeratin. This new approach provides an IHC method to identify trophoblasts specifically in feline placenta. PMID:21398447

  8. The cyclosporin A washout assay to detect HIV-1 uncoating in infected cells.

    Science.gov (United States)

    Hulme, Amy E; Hope, Thomas J

    2014-01-01

    Uncoating is an early step of HIV-1 replication in which the viral capsid disassembles by p24 capsid (p24(CA)) protein dissociating from the viral complex. Although uncoating is required for HIV-1 replication, many questions remain about the mechanism of this process as well as its impact on other steps in viral replication. Here we describe a recently developed assay to study the process of uncoating in HIV-1-infected cells. The CsA washout assay is a cell-based assay that utilizes the HIV-1 restriction factor TRIM-CypA to detect and inhibit infection of coated viral complexes. Owl monkey kidney (OMK) cells are infected with a GFP reporter virus and TRIM-CypA restriction is switched on at various times postinfection allowing the kinetics of uncoating to be monitored in infected cells. This assay also can be used to examine the effect of different viral or cellular factors on the process of uncoating. PMID:24158812

  9. PCR and real-time PCR assays to detect fungi of Alternaria alternata species.

    Science.gov (United States)

    Kordalewska, Milena; Brillowska-Dąbrowska, Anna; Jagielski, Tomasz; Dworecka-Kaszak, Bożena

    2015-01-01

    Fungi of the Alternaria genus are mostly associated with allergic diseases. However, with a growing number of immunocompromised patients, these fungi, with A. alternata being the most prevalent one, are increasingly recognized as etiological agents of infections (phaeohyphomycoses) in humans. Nowadays, identification of Alternaria spp. requires their pure culture and is solely based on morphological criteria. Clinically, Alternaria infections may be indistinguishable from other fungal diseases. Therefore, a diagnostic result is often delayed or even not achieved at all. In this paper we present easy to perform and interpret PCR and real-time PCR assays enabling detection of A. alternata species. On the basis of alignment of β-tubulin gene sequences, A. alternata-specific primers were designed. DNA from fungal isolates, extracted in a two-step procedure, were used in PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The assays specificity was confirmed, since positive results were obtained for all A. alternata isolates, and no positive results were obtained neither for other molds, dermatophytes, yeast-like fungi, nor human DNA. The assays developed here enable fast and unambiguous identification of A. alternata pathogens. PMID:26610309

  10. Immunoradiometric assay for the detection of circulating antibodies to murine monoclonal antibodies in humans (HAMA)

    International Nuclear Information System (INIS)

    The increasing clinical use of monoclonal antibodies (MAb) has focussed attention on the importance of the generation of human anti-mouse antibodies (HAMA). HAMA can not only be life threatening but can also be associated with reduced image quality and accelerated MAb clearance in vivo as well as interfere with in vitro MAb based assays. The development of a two step immunoradiometric assay (IRMA) for detecting circulating HAMA is reported. Preliminary results have been generated with specific MAb coated polystyrene wells. The appropriately diluted serum sample is first incubated with the MAb coated wells followed by a wash step and a second incubation with 125I labeled goat anti-human IgG. After a final wash, the wells are assayed for 125I and the results expressed as percent of the input bound. The prototype assay is compared with an existing commercially available ELISA kit using patient sera obtained at various time periods up to 7 months after IV MAb injection for radioimmunoscintigraphy. 18 refs., 2 tabs., 1 fig

  11. Immunochromatographic assay for ultrasensitive detection of aflatoxin B₁ in maize by highly luminescent quantum dot beads.

    Science.gov (United States)

    Ren, Meiling; Xu, Hengyi; Huang, Xiaolin; Kuang, Min; Xiong, Yonghua; Xu, Hong; Xu, Yang; Chen, Hongyu; Wang, Andrew

    2014-08-27

    Highly luminescent quantum dot beads (QBs) were synthesized by encapsulating CdSe/ZnS and used for the first time as immunochromatographic assay (ICA) signal amplification probe for ultrasensitive detection of aflatoxin B1 (AFB1) in maize. The challenges to using high brightness QBs as probes for ICA are smooth flow of QBs and nonspecific binding on nitrocellulose (NC) membrane, which are overcome by unique polymer encapsulation of quantum dots (QDs) and surface blocking method. Under optimal conditions, the QB-based ICA (QB-ICA) sensor exhibited dynamic linear detection of AFB1 in maize extract from 5 to 60 pg mL(-1), with a median inhibitory concentration (IC50) of 13.87 ± 0.16 pg mL(-1), that is significantly (39-fold) lower than those of the QD as a signal probe (IC50 = 0.54 ± 0.06 ng mL(-1)). The limit of detection (LOD) for AFB1 using QB-ICA sensor was 0.42 pg mL(-1) in maize extract, which is approximately 2 orders of magnitude better than those of previously reported gold nanoparticle based immunochromatographic assay (AuNP-ICA) and is even comparable with or better than the conventional enzyme-linked immunosorbent assay (ELISA) method. The performance and practicability of our QB-ICA sensor were validated with a commercial ELISA kit and further confirmed with liquid chromatography tandem mass spectrometry (LC-MS/MS). Given its efficient signal amplification performance, the proposed QB-ICA offers great potential for rapid, sensitive, and cost-effective quantitative detection of analytes in food safety monitoring. PMID:25109633

  12. Identification of IgM as a contaminant in lectin-FLISA assays for HCC detection.

    Science.gov (United States)

    Wang, Mengjun; Comunale, Mary Ann; Herrera, Harmin; Betesh, Lucy; Kono, Yuko; Mehta, Anand

    2016-07-29

    Liver disease, in the form of hepatocellular carcinoma (HCC) accounts for > 700,000 deaths worldwide. A major reason for this is late diagnosis of HCC. The currently used biomarker, serum alpha-fetoprotein (AFP) is elevated in 40-60% of those with HCC and other markers that can either compliment or replace AFP are desired. Our previous work has identified a number of proteins that contain altered glycans in HCC. Specifically, these altered glycans were increased levels of core and outer arm fucosylation. To determine the clinical usefulness of those identified glycoproteins, a plate based assay was developed that allowed for the detection of fucosylated glycoforms. While this method was applicable to a number of independent patient sets, it was unable to specifically detect fucosylated glycoforms in many patient samples. That is, some material was present in serum that led to non-specific signal in the lectin- fluorescence -linked immunosorbent assay (lectin-FLISA). To address this issue, a systematic process was undertaken to identify the material. This material was found to be increased levels of lectin reactive IgM. Removal of both IgG and IgM using a multi-step protein A/G incubation and filtration step removed the contaminating signal and allowed for the analysis of specific protein glycoforms. This assay was subsequently used on two sample sets, one that was shown previously to be unable to be tested via a lectin FLISA and in a larger independent sample set. The clinical usefulness of this assay in the early detection of HCC is discussed. PMID:27181357

  13. Validation of real-time PCR assays for bioforensic detection of model plant pathogens.

    Science.gov (United States)

    James, Mindy; Blagden, Trenna; Moncrief, Ian; Burans, James P; Schneider, Katherine; Fletcher, Jacqueline

    2014-03-01

    The U.S. agricultural sector is vulnerable to intentionally introduced microbial threats because of its wide and open distribution and economic importance. To investigate such events, forensically valid assays for plant pathogen detection are needed. In this work, real-time PCR assays were developed for three model plant pathogens: Pseudomonas syringae pathovar tomato, Xylella fastidiosa, and Wheat streak mosaic virus. Validation included determination of the linearity and range, limit of detection, sensitivity, specificity, and exclusivity of each assay. Additionally, positive control plasmids, distinguishable from native signature by restriction enzyme digestion, were developed to support forensic application of the assays. Each assay displayed linear amplification of target nucleic acid, detected 100 fg or less of target nucleic acid, and was specific to its target pathogen. Results obtained with these model pathogens provide the framework for development and validation of similar assays for other plant pathogens of high consequence. PMID:24261870

  14. Dye Labelled Monoclonal Antibody Assay for Detection of Toxic Shock Syndrome Toxin -1 from Staphylococcus Aureus

    Directory of Open Access Journals (Sweden)

    V Javid Khojasteh

    2011-12-01

    Full Text Available Objective: The aim of study was to develop a rapid assay, dye labelled monoclonal antibody assay (DLMAA, using non-radioactive organic synthetic dyes for identification of Toxic Shock Syndrome Toxin-1 (TSST-1 producing strains of Staphylococcus aureus.Materials and Methods: The assay protocol required only two simple steps; addition of TSST-1 antigen to a nitrocellulose membrane and then adding a colloidal dye labelled antibody (D/A suspension detection reagent.Results: The sensitivity and specificity of the assay was determined relative to positive and negative strains compared to an ELISA assay. Overall 100% agreement was found between both assays. The sensitivity for detection of TSST-1 was 30 ng.Conclusion: The DLMAA did not require handling and disposal of radioactive materials. It is a rapid qualitative technique for detection of TSST-1 toxin at room temperature within a short time.

  15. 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

  16. Transcribed single nucleotide polymorphism: Ideal markers for detecting gene imprinting by 5' nuclease assay

    Institute of Scientific and Technical Information of China (English)

    ZHU Guan-shan; WAN Mo-bin; ZHU Zhong-zheng; ZHENG Rui-ying

    2002-01-01

    Objective:To establish a novel approach for quick and highly efficient verification of human gene imprinting. Methods: A pair of dye-labelled probes, 5' nuclease assay was combined with RT-PCR to determine the genotype of a transcribed single nucleotide polymorphism (SNP) rs705 (C>T) of a known imprinted gene, small nuclear ribonucleotide protein N (SNRPN), on both genomic DNA and cDNA of human lymphoblast cell lines. Results: Allele discrimination showed a clear monoallelic expression pattern of SNRPN,which was confirmed by RT-PCR based restriction fragment length polymorphism (RFLPs). Pedigree analysis verified the paternal origin of expressed allele, which was in consistency with previous report. Conclusion: Transcribed SNP is an ideal marker for detecting gene imprinting by 5' nuclease assay. This approach also may be used to discover differential allele expression of non-imprinted genes, finding out gene cis-acting functional polymorphism.

  17. Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

    International Nuclear Information System (INIS)

    Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl2) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl2 in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl2 was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms. (author)

  18. An integrated lateral flow assay for effective DNA amplification and detection at the point of care.

    Science.gov (United States)

    Choi, Jane Ru; Hu, Jie; Gong, Yan; Feng, Shangsheng; Wan Abas, Wan Abu Bakar; Pingguan-Murphy, Belinda; Xu, Feng

    2016-05-10

    Lateral flow assays (LFAs) have been extensively explored in nucleic acid testing (NAT) for medical diagnostics, food safety analysis and environmental monitoring. However, the amount of target nucleic acid in a raw sample is usually too low to be directly detected by LFAs, necessitating the process of amplification. Even though cost-effective paper-based amplification techniques have been introduced, they have always been separately performed from LFAs, hence increasing the risk of reagent loss and cross-contaminations. To date, integrating paper-based nucleic acid amplification into colorimetric LFA in a simple, portable and cost-effective manner has not been introduced. Herein, we developed an integrated LFA with the aid of a specially designed handheld battery-powered system for effective amplification and detection of targets in resource-poor settings. Interestingly, using the integrated paper-based loop-mediated isothermal amplification (LAMP)-LFA, we successfully performed highly sensitive and specific target detection, achieving a detection limit of as low as 3 × 10(3) copies of target DNA, which is comparable to the conventional tube-based LAMP-LFA in an unintegrated format. The device may serve in conjunction with a simple paper-based sample preparation to create a fully integrated paper-based sample-to-answer diagnostic device for point-of-care testing (POCT) in the near future. PMID:27010033

  19. 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. PMID:26141730

  20. Detection of telomerase activity by combination of telomeric repeat amplification protocol and electrochemiluminescence assay

    Institute of Scientific and Technical Information of China (English)

    Xiao Ming Zhou; Li Jia

    2008-01-01

    A highly sensitive telomerase detection method that combines telomeric repeat amplification protocol (TRAP) and magnetic beads based electrochemiluminescence (ECL) assay has been developed. Briefly, telomerase recognizes biotinylated telomerase synthesis primer (B-TS) and synthesizes extension products, which then serve as the templates for PCR amplification using B-TS as the forward primer and Iris-(2'2'-bipyridyl) ruthenium (TBR) labeled ACX (TBR-ACX) as the reversed primer. The amplified product is captured on streptavidin-coated paramagnetic beads and detected by ECL. Telomerase positive HeLa cells were used to validate the feasibility of the method. The experimental results showed down to 10 cancer cells can be detected easily. The method is a useful tool for telomerase activity analysis due to its sensitivity, rapidity, safety, high throughput, and low cost. It can be used for screening a large amount of clinical samples.

  1. 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.

  2. 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. PMID:27014303

  3. An agar gel enzyme assay (AGEA) for simple detection of Salmonella enteritidis antibodies in chicken sera.

    Science.gov (United States)

    Kim, C J; Nagaraja, K V

    1991-01-01

    An agar gel enzyme assay (AGEA) was developed for the detection of antibodies to Salmonella enteritidis (SE). The assay was based on the ability of antibodies to diffuse through an agar gel and react with antigen coated on a polystyrene surface. The antigen-antibody reaction was then made visible by applying an enzyme-conjugated anti-immunoglobulin and the addition, subsequently, of a substrate-containing gel. The color change in circular zones was taken as the indication for the presence of antibodies. The present investigation reports identification of an antigen specific for SE and its use in the development of a relatively simple AGEA procedure. The results of AGEA were compared with those of conventional microagglutination (MA) test and serum plate (SP) test. The percentage agreement between MA and AGEA in positive serum sample was found to be 94.4%, and in negative serum samples it was found to be 88.8%. The present results suggest that the AGEA could be a very useful screening test for the detection of SE antibodies because the assay is inexpensive, specific and simple to perform without much equipment, and give results within a 3-hr period. PMID:1832368

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

    International Nuclear Information System (INIS)

    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-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.

  5. Glycoprotein-Based Enzyme-Linked Immunosorbent Assays for Serodiagnosis of Infectious Laryngotracheitis

    OpenAIRE

    Kanabagatte Basavarajappa, Mallikarjuna; Song, Haichen; Lamichhane, Chinta; Samal, Siba K.

    2015-01-01

    For detection of infectious laryngotracheitis virus (ILTV) antibody, glycoprotein B-, C-, and D-based enzyme-linked immunosorbent assays (B-, C-, and D-ELISAs, respectively) were developed. The B- and D-ELISAs showed enhanced detection of anti-ILTV antibodies in infected chickens compared to that of the commercial ELISA. Furthermore, the D-ELISA was efficient in detecting seroconversion with vectored vaccine, using recombinant Newcastle disease virus (rNDV) expressing glycoprotein D (gD) as t...

  6. SNaPshot Assay for the Detection of the Most Common CFTR Mutations in Infertile Men

    OpenAIRE

    Predrag Noveski; Svetlana Madjunkova; Marija Mircevska; Toso Plaseski; Vanja Filipovski; Dijana Plaseska-Karanfilska

    2014-01-01

    Congenital bilateral absence of vas deferens (CBAVD) is the most common CFTR-related disorder (CFTR-RD) that explains about 1-2% of the male infertility cases. Controversial data have been published regarding the involvement of CFTR mutations in infertile men with non-obstructive azoospermia and oligozoospermia. Here, we describe single base extension (SNaPshot) assay for detection of 11 common CFTR mutations: F508del, G542X, N1303K, 621+1G->T, G551D, R553X, R1162X, W1282X, R117H, 2184insA an...

  7. A Pentaplex PCR Assay for the Detection and Differentiation of Shigella Species

    OpenAIRE

    Suvash Chandra Ojha; Chan Yean Yean; Asma Ismail; Kirnpal-Kaur Banga Singh

    2013-01-01

    The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene...

  8. Sensitive detection of colorectal cancer in peripheral blood by septin 9 DNA methylation assay.

    Directory of Open Access Journals (Sweden)

    Robert Grützmann

    Full Text Available BACKGROUND: Colorectal cancer (CRC is the second leading cause of cancer deaths despite the fact that detection of this cancer in early stages results in over 90% survival rate. Currently less than 45% of at-risk individuals in the US are screened regularly, exposing a need for better screening tests. We performed two case-control studies to validate a blood-based test that identifies methylated DNA in plasma from all stages of CRC. METHODOLOGY/PRINCIPAL FINDINGS: Using a PCR assay for analysis of Septin 9 (SEPT9 hypermethylation in DNA extracted from plasma, clinical performance was optimized on 354 samples (252 CRC, 102 controls and validated in a blinded, independent study of 309 samples (126 CRC, 183 controls. 168 polyps and 411 additional disease controls were also evaluated. Based on the training study SEPT9-based classification detected 120/252 CRCs (48% and 7/102 controls (7%. In the test study 73/126 CRCs (58% and 18/183 control samples (10% were positive for SEPT9 validating the training set results. Inclusion of an additional measurement replicate increased the sensitivity of the assay in the testing set to 72% (90/125 CRCs detected while maintaining 90% specificity (19/183 for controls. Positive rates for plasmas from the other cancers (11/96 and non-cancerous conditions (41/315 were low. The rate of polyp detection (>1 cm was approximately 20%. CONCLUSIONS/SIGNIFICANCE: Analysis of SEPT9 DNA methylation in plasma represents a straightforward, minimally invasive method to detect all stages of CRC with potential to satisfy unmet needs for increased compliance in the screening population. Further clinical testing is warranted.

  9. Real-Time Cytotoxicity Assay for Rapid and Sensitive Detection of Ricin from Complex Matrices

    OpenAIRE

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

    2012-01-01

    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 commun...

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

    OpenAIRE

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

    2012-01-01

    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 ...

  11. The development and application of the two real-time RT-PCR assays to detect the pathogen of HFMD.

    Directory of Open Access Journals (Sweden)

    Aili Cui

    Full Text Available Large-scale Hand, Foot, and Mouth Disease (HFMD outbreaks have frequently occurred in China since 2008, affecting more than one million children and causing several hundred children deaths every year. The pathogens of HFMD are mainly human enteroviruses (HEVs. Among them, human enterovirus 71 (HEV71 and coxsackievirus A16 (CVA16 are the most common pathogens of HFMD. However, other HEVs could also cause HFMD. To rapidly detect HEV71 and CVA16, and ensure detection of all HEVs causing HFMD, two real-time hybridization probe-based RT-PCR assays were developed in this study. One is a multiplex real-time RT-PCR assay, which was developed to detect and differentiate HEV71 specifically from CVA16 directly from clinical specimens within 1-2 h, and the other is a broad-spectrum real-time RT-PCR assay, which targeted almost all HEVs. The experiments confirmed that the two assays have high sensitivity and specificity, and the sensitivity was up to 0.1 TCID50/ml for detection of HEVs, HEV71, and CVA16, respectively. A total of 213 clinical specimens were simultaneously detected by three kinds of assays, including the two real-time RT-PCR assays, direct conventional RT-PCR assay, and virus isolation assay on human rhabdomyosarcoma cells (RD cells. The total positive rate of both HEV71 and CVA16 was 69.48% with real-time RT-PCR assay, 47.42% with RT-PCR assay, and 34.58% with virus isolation assay. One HFMD clinical specimen was positive for HEV, but negative for HEV71 or CVA16, which was identified as Echovirus 11 (Echo11 by virus isolation, RT-PCR, and sequencing for the VP1 gene. The two real-time RT-PCR assays had been applied in 31 provincial HFMD labs to detect the pathogens of HFMD, which has contributed to the rapid identification of the pathogens in the early stages of HFMD outbreaks, and helped to clarify the etiologic agents of HFMD in China.

  12. Evaluation of the VIDAS glutamate dehydrogenase assay for the detection of Clostridium difficile.

    Science.gov (United States)

    Shin, Bo-Moon; Lee, Eun Joo; Moon, Jung Wha; Lee, Seon Yeong

    2016-08-01

    We evaluated the performance of the VIDAS GDH assay for the detection of Clostridium difficile. In total, 350 fecal specimens collected from patients clinically suspected of having CDI were analyzed by C. difficile culture and enzyme-linked fluorescent immunoassay (VIDAS GDH); the results were compared with those of toxigenic C. difficile culture (TC), PCR (Xpert C. difficile assay), and toxin AB EIA (VIDAS CDAB). The numbers of culture-positive and culture-negative samples were 108 and 242, respectively. The concordance between the GDH assay and C. difficile culture was 90.3%. With PCR, 12 more samples were found to be positive in GDH-positive/C. difficile culture-negative specimens. Thus, the concordance between GDH assay and C. difficile culture/PCR was 93.7%. The sensitivity, specificity, positive predictive value, and negative predictive value of the VIDAS GDH assay were 97.2%, 87.2%, 77.2%, and 98.6%, respectively, based on the C. difficile culture, and 97.5%, 91.7%, 86.0%, and 98.6%, respectively, based on C. difficile culture/PCR. Positivity rates of the GDH assay were partially associated with those of semi-quantitative C. difficile cultures, which were maximized in grade 3 (>100 colony-forming unit [CFU]) compared with grade 1 (<10 CFU). We evaluated the two-step or three-step algorithm using GDH assay as a first step. No toxin EIA-positive case was found among GDH-negative samples, and 60.8% (48/79) were TC- and/or PCR-positive among the GDH-positive/toxin EIA-negative samples. Thus, approximately 25% of the 350 samples required a confirmatory test (TC or PCR) in the GDH-toxin EIA algorithm, whereas only 2.3% of the total samples in GDH-PCR algorithm was discrepant and required another confirmatory test like TC. PMID:27282799

  13. Uranium internal exposure evaluation based on urine assay data

    International Nuclear Information System (INIS)

    The difficulties in assessing internal exposures to uranium from urine assay data are described. A simplified application of the ICRP-30 and ICRP Lung Model concepts to the estimation of uranium intake is presented. A discussion follows on the development of a computer code utilizing the ICRP-30-based uranium elimination model with the existing urine assay information. The calculated uranium exposures from 1949 through 1983 are discussed. 13 references, 1 table

  14. Five-Antigen Fluorescent Bead-Based Assay for Diagnosis of Lyme Disease.

    Science.gov (United States)

    Embers, Monica E; Hasenkampf, Nicole R; Barnes, Mary B; Didier, Elizabeth S; Philipp, Mario T; Tardo, Amanda C

    2016-04-01

    The systematically difficult task of diagnosing Lyme disease can be simplified by sensitive and specific laboratory tests. The currently recommended two-tier test for serology is highly specific but falls short in sensitivity, especially in the early acute phase. We previously examined serially collected serum samples fromBorrelia burgdorferi-infected rhesus macaques and defined a combination of antigens that could be utilized for detection of infection at all phases of disease in humans. The fiveB. burgdorferiantigens, consisting of OspC, OspA, DbpA, OppA2, and the C6 peptide, were combined into a fluorescent cytometric bead-based assay for the detection ofB. burgdorferiantigen-specific IgG antibodies. Samples from Lyme disease patients and controls were used to determine the diagnostic value of this assay. Using this sample set, we found that our five-antigen multiplex IgG assay exhibited higher sensitivity (79.5%) than the enzyme immunoassay (EIA) (76.1%), the two-tier test (61.4%), and the C6 peptide enzyme-linked immunosorbent assay (ELISA) (77.2%) while maintaining specificity over 90%. When detection of IgM was added to the bead-based assay, the sensitivity improved to 91%, but at a cost of reduced specificity (78%). These results indicate that the rational combination of antigens in our multiplex assay may offer an improved serodiagnostic test for Lyme disease. PMID:26843487

  15. Automated 5 ' nuclease assay for detection of virulence factors in porcine Escherichia coli

    DEFF Research Database (Denmark)

    Frydendahl, K.; Imberechts, H.; Lehmann, S.

    2001-01-01

    This paper describes the development of a 5' nuclease assay for detection of virulence factor genes responsible for colonization factors and toxins in Escherichia coil isolated from pigs. Colonization factors were F4, F5, F6, F18, F41 and the outer membrane protein intimin. Toxins were heat stable...... reference strains which have previously been examined with phenotypical assays or DNA hybridization. Furthermore, the assay was evaluated by testing porcine E. coil field strains, previously characterized. The 5' nuclease assay correctly detected the presence of virulence genes in all reference strains....... When testing field strains there was generally excellent agreement with results obtained by laboratories in Belgium and Germany. In conclusion, the 5' nuclease assay developed is a fast and specific tool for detection of E. coli virulence genes in the veterinary diagnostic laboratory....

  16. A protein-protein binding assay using coated microtitre plates: increased throughput, reproducibility and speed compared to bead-based assays.

    Science.gov (United States)

    Craig, Tim J; Ciufo, Leonora F; Morgan, Alan

    2004-07-30

    Protein-protein interactions, and the factors affecting them, are of fundamental importance to all biological systems. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITR) are powerful methods for assaying such interactions, but are expensive to implement. In contrast, bead-based pull-down assays using affinity tags such as glutathione-S-transferase (GST), require no specialist equipment. As a result, such assays are the most popular method for analysing protein-protein interactions, despite being time-consuming and prone to variability. In respect of these problems, we have modified this form of binding assay, using glutathione-coated 96-well plates rather than glutathione-Sepharose beads to bind the primary bait protein. Quantitation of bound protein utilises ELISA for purified proteins and scintillation counting for in vitro translated proteins, rather than the SDS-PAGE-based detection methods used in traditional bead-based assays. These modifications result in an approximately 10-fold increase in the number of samples that can be assayed daily, and allow results to be obtained within hours as opposed to days. We validate the modified assay by analysing the equilibrium binding of Munc18 and syntaxin, and also demonstrate that association and dissociation kinetics may be measured using this approach. The method we describe is generally applicable to any protein-protein interaction assay based on affinity tags and is amenable to automation, and so should benefit a wide range of biochemical research. PMID:15236910

  17. Trans-sialidase inhibition assay detects Trypanosoma cruzi infection in different wild mammal species.

    Science.gov (United States)

    Sartor, Paula A; Ceballos, Leonardo A; Orozco, Marcela M; Cardinal, Marta V; Gürtler, Ricardo E; Leguizamón, María S

    2013-08-01

    The detection of Trypanosoma cruzi infection in mammals is crucial for understanding the eco-epidemiological role of the different species involved in parasite transmission cycles. Xenodiagnosis (XD) and hemoculture (HC) are routinely used to detect T. cruzi in wild mammals. Serological methods are much more limited because they require the use of specific antibodies to immunoglobulins of each mammalian species susceptible to T. cruzi. In this study we detected T. cruzi infection by trans-sialidase (TS) inhibition assay (TIA). TIA is based on the antibody neutralization of a recombinant TS that avoids the use of anti-immunoglobulins. TS activity is not detected in the co-endemic protozoan parasites Leishmania spp and T. rangeli. In the current study, serum samples from 158 individuals of nine wild mammalian species, previously tested by XD, were evaluated by TIA. They were collected from two endemic areas in northern Argentina. The overall TIA versus XD co-reactivity was 98.7% (156/158). All 18 samples from XD-positive mammals were TIA-positive (co-positivity, 100%) and co-negativity was 98.5% (138/140). Two XD-negative samples from a marsupial (Didelphis albiventris) and an edentate (Dasypus novemcinctus) were detected by TIA. TIA could be used as a novel tool for serological detection of Trypanosoma cruzi in a wide variety of sylvatic reservoir hosts. PMID:23930975

  18. DEVELOPMENT OF INTERGENIC SPACERS BASED PCR ASSAY FOR DETECTION OF GIARDIALAMBLIA%犬猫贾第虫IGS序列特异性检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    李雅文; 刘远佳; 郑国超; 谭立娉; 胡伟; 罗琴; 林丽琴; 路鹏云; 李国清

    2013-01-01

    The intergenic spacers (IGS) were amplified in PCR from nuclear ribosomal DNAs of two Giardialamblia strains (assemble D from dog and assemble F from cat) isolated from feces of naturally infected pets in Guangzhou. The PCR products were purified, cloned and sequenced. The resulting nucleotide sequences were compared with related sequences in the GenBank databases. The IGS sequences were 1355 bp for the canine Giardia strain and 1388 bp for the feline Giardia strain. The interspecific difference in the IGS sequence might serve as a genetic marker for the identification and differentiation of Giardia lamblia. Furthermore, a PCR detection method was developed based on IGS sequence. The established PCR assay specifically amplified IGS sequence of G. lamblia but not all control parasites such as Toxocaracanis etc. The minimum detection limit of G.lamblia was 82 fg. Total 84 clinical samples were tested using this method. The positive rate of clinical samples was 3.57%, higher than 2.38%by traditional method.%应用PCR技术对广州市某宠物寄养所采集的一株D型犬源贾第虫和一株F型猫源贾第虫的核糖体IGS序列进行了扩增、克隆、测序,将测序结果与GenBank已上传的贾第虫相应序列进行比对分析,基于贾第虫IGS序列建立了具有良好特异性和敏感性的PCR检测方法,并对84份临床粪样进行了检测。结果显示,D型犬源贾第虫和F型猫源贾第虫的IGS序列长分别为1355 bp和1388 bp,贾第虫IGS序列存在多态性现象,种间差异明显,可以作为区分不同基因型的分子标记;建立的PCR方法能特异性扩增犬源贾第虫核糖体IGS序列,而犬蛔虫等对照虫体DNA均不能扩增,该方法对贾第虫DNA的最小检测量为82 fg,对84份临床粪样的检出率为3.57%,比传统镜检法高出2.38%,具有一定的临床应用价值。

  19. Application of a 5 ' nuclease assay for detection of Lawsonia intracellularis in fecal samples from pigs

    DEFF Research Database (Denmark)

    Lindecrona, R. H.; Jensen, Tim Kåre; Andersen, P. H.; Møller, Kristian

    2002-01-01

    fecal samples derived from a herd known to be free from infection with L. intracellularis all tested negative, with a Ct value of 40. By using a Ct value of 36 as the cutoff limit, the detection limit of the assay was 1 L. intracellularis cell per PCR tube. In conclusion, the 5' nuclease assay that has...

  20. Inhibition enzyme-linked immunosorbent assay for detection of Pseudomonas fluorescens on meat surfaces.

    OpenAIRE

    Eriksson, P V; di Paola, G N; Pasetti, M F; Manghi, M A

    1995-01-01

    An inhibition enzyme-linked immunosorbent assay was developed for Pseudomonas fluorescens enumeration of meat surfaces. The assay detected contamination levels as low as 3 x 10(5) bacteria per ml and could be completed within 4 h. It could be used as a framework for a test system for quantifying P. fluorescens spoilage in meat products.

  1. A Simple and Reliable Assay for Detecting Specific Nucleotide Sequences in Plants Using Optical Thin-film Biosensor Chips

    Institute of Scientific and Technical Information of China (English)

    S. Bai; X. Zhong; L. Ma; W. Zheng; L. Fan; N. Wei; X.W. Deng

    2007-01-01

    @@ Here we report the adaptation and optimization of an efficient, accurate and inexpensive assay that employs custom-designed silicon-based optical thin-film biosensor chips to detect unique transgenes in genetically modified (GM) crops and SNP markers in model plant genomes.

  2. Specific PCR Assay for Rapid and Direct Detection of Neisseria meningitidis in Cerebrospinal Fluid Specimens

    Directory of Open Access Journals (Sweden)

    M Qurbanalizadegan

    2010-12-01

    Full Text Available Background: Neisseria meninigitidis is one of the most frequently encountered microorganisms associated with central nervous system infections. The aim of this study was to evaluate a PCR-based assay for specific and rapid detection of N. meninigitidis in CSF specimens.  Methods: Since April 2002 to July 2006, 130 CSF specimens were collected from patients suspected of having bacterial meningitis. Bacterial isolation and identification was carried out according to the standard bacteriological methods.  The PCR was used to amplify a 101bp fragment of capsular transport gene A (ctrA of N. meningitidis. Results: PCR yielded an amplified product with the expected size of 101 base pair fragment. Sensitivity test proved 500 ng of N. meningitidis DNA as the final detection limit and specificity test revealed no cross-reaction for a wide range of res­pira­tory pathogenic organisms. Conclusion: The PCR assay was more sensitive than the bacterial culturing. It might be possible to apply this procedure for rapid diagnosis of meningococci in clinical samples.

  3. An antigen-specific, four-color, B-cell FluoroSpot assay utilizing tagged antigens for detection.

    Science.gov (United States)

    Jahnmatz, Peter; Bengtsson, Theresa; Zuber, Bartek; Färnert, Anna; Ahlborg, Niklas

    2016-06-01

    The FluoroSpot assay, a variant of ELISpot utilizing fluorescent detection, has so far been used primarily for assessment of T cells, where simultaneous detection of several cytokines has allowed a more qualitative analysis of functionally distinct T cells. The potential to measure multiple analytes also presents several advantages when analyzing B cells. Our aim was to develop a B-cell FluoroSpot assay adaptable to studies of a variety of antigens. The assay utilizes anti-IgG antibodies immobilized in 96-well filter membrane plates. During cell culture, IgG antibodies secreted by antibody-secreting cells (ASCs) are captured in the vicinity of each of these cells and the specificity of single ASCs is defined using antigens for detection. The antigens were labeled with biotin or peptide tags enabling secondary detection with fluorophore-conjugated streptavidin or tag-specific antibodies. The assay, utilizing up to four different tag systems and fluorophores simultaneously, was evaluated using hybridomas and immunized splenocytes as ASCs. Assay variants were developed that could: i) identify multiple ASCs with different antigen specificities; ii) detect ASCs showing cross-reactivity with different but related antigens; and iii) define the antigen-specificity and, by including anti-IgG subclass detection reagents, simultaneously determine the IgG subclass of antibodies secreted by ASCs. As demonstrated here, the B-cell FluoroSpot assay using tag-based detection systems provides a versatile and powerful tool to investigate antibody responses by individual cells that can be readily adapted to studies of a variety of antigen-specific ASCs. PMID:26930550

  4. EUROarray human papillomavirus (HPV) assay is highly concordant with other commercial assays for detection of high-risk HPV genotypes in women with high grade cervical abnormalities.

    Science.gov (United States)

    Cornall, A M; Poljak, M; Garland, S M; Phillips, S; Machalek, D A; Tan, J H; Quinn, M A; Tabrizi, S N

    2016-06-01

    The purpose of this study was to evaluate the performance of the EUROIMMUN EUROArray HPV genotyping assay against the Roche Cobas 4800, Roche HPV Amplicor, Roche Linear Array and Qiagen Hybrid Capture 2 assays in the detection of high-risk HPV (HR-HPV) from liquid based cervical cytology samples collected from women undergoing follow-up for abnormal cervical cytology results. Cervical specimens from 404 women undergoing management of high-grade cytological abnormality were evaluated by EUROarray HPV for detection of HR-HPV genotypes and prediction of histologically-confirmed cervical intraepithelial neoplasia grade 2 or higher (≥CIN2). The results were compared to Hybrid Capture 2, Cobas 4800 HPV, Amplicor and Linear Array HPV. Positivity for 14 HR-HPV types was 80.0 % for EUROarray (95 % CI; 75.7-83.8 %). Agreement (κ, 95 % CI) between the EUROarray and other HPV tests for detection of HR-HPV was good to very good [Hybrid Capture κ = 0.62 (0.54-0.71); Cobas κ = 0.81 (0.74-0.88); Amplicor κ = 0.68 (0.60-0.77); Linear Array κ = 0.77 (0.70-0.85)]. For detection of HR-HPV, agreement with EUROarray was 87.90 % (Hybrid Capture), 93.58 % (Cobas), 92.84 % (Amplicor) and 92.59 % (Linear Array). Detection of HR-HPV was not significantly different between EUROarray and any other test (p < 0.001). EUROarray was concordant with other assays evaluated for detection of high-risk HPV and showed sensitivity and specificity for detection of ≥ CIN2 of 86 % and 71 %, respectively. PMID:27048314

  5. Thyroid Histopathology Assessments for the Amphibian Metamorphosis Assay to Detect Thyroid-active Substances

    Science.gov (United States)

    In support of an Organization for Economic Cooperation and Development (OECD) Amphibian Metamorphosis Assay (AMA) Test Guideline for the detection of substances that interact with the hypothalamic-pituitary-thyroid axis, a document was developed that provides a standardized appro...

  6. A Rapid, Self-confirming Assay for HIV: Simultaneous Detection of Anti-HIV Antibodies and Viral RNA

    Science.gov (United States)

    Chen, Zongyuan; Zhu, Hui; Malamud, Daniel; Barber, Cheryl; Ongagna, Yhombi Yvon Serge; Yasmin, Rubina; Modak, Sayli; Janal, Malvin N.; Abrams, William R.; Montagna, Richard A.

    2016-01-01

    Objective We developed a microfluidic system to simultaneously detect host anti-HIV antibodies and viral RNA in the same specimen in order to satisfy two important diagnostic criteria, especially within resource-limited settings. First, the system can detect acute HIV infection and allow immediate confirmation of a seropositive screening result by detection of HIV RNA. It also addresses the well-known "seroconversion window" during early HIV infection when antibodies are not yet detectable and viral loads are at their highest. Methods We first developed and optimized two separate manual assays for the detection of host anti-HIV antibodies and viral RNA and then converted them to the microfluidic system. We optimized a commercially available serologic assay to run within the microfluidic device while we incorporated the isothermal LAMP assay to detect the presence of viral RNA. The microfluidic device and instrumentation were developed to simultaneously perform both assays without any user intervention. Results The finalized system consists of a disposable injection molded and film-laminated microfluidic CARD disposable device and a portable, software controlled instrument, which together can automatically perform all steps of both assays without any user intervention after the initial loading of samples and reagents. The microfluidic CARD cartridge has multiple microchannels, valves, pumps and reservoirs, which perform the immunoassay, isolates viral RNA for detection by magnetic bead based purification, and Reverse Transcriptase loop-mediated isothermal amplification (RT-LAMP). The microfluidic system was able to detect host anti-HIV antibodies and viral RNA in either a blood or saliva sample. Conclusion The ability to detect antibodies and simultaneously confirm a seropositive HIV-RNA result provides healthcare workers with a complete and accurate appraisal of a patient's infection status in the earliest stages of the disease and represents an important tool for

  7. Rapid, specific and quantitative assays for the detection of the endophytic bacterium Methylobacterium mesophilicum in plants.

    Science.gov (United States)

    Lacava, P T; Li, W B; Araújo, W L; Azevedo, J L; Hartung, J S

    2006-06-01

    Xylella fastidiosa is a xylem-limited bacterium that causes citrus variegated chlorosis disease in sweet orange. There is evidence that X. fastidiosa interacts with endophytic bacteria present in the xylem of sweet orange, and that these interactions, particularly with Methylobacterium mesophilicum, may affect disease progress. However, these interactions cannot be evaluated in detail until efficient methods for detection and enumeration of these bacteria in planta are developed. We have previously developed standard and quantitative PCR-based assays specific for X. fastidiosa using the LightCycler system [Li, W.B., Pria Jr., L.P.M.W.D., X. Qin, and J.S. Hartung, 2003. Presence of Xylella fastidiosa in sweet orange fruit and seeds and its transmission to seedlings. Phytopathology 93:953-958.], and now report the development of both standard and quantitative PCR assays for M. mesophilicum. The assays are specific for M. mesophilicum and do not amplify DNA from other species of Methylobacterium or other bacteria commonly associated with citrus or plant tissue. Other bacteria tested included Curtobacterium flaccumfaciens, Pantoea agglomerans, Enterobacter cloacae, Bacillus sp., X. fastidiosa, Xanthomonas axonopodis pv. citri, and Candidatus Liberibacter asiaticus. We have demonstrated that with these methods we can quantitatively monitor the colonization of xylem by M. mesophilicum during the course of disease development in plants artificially inoculated with both bacteria. PMID:16266765

  8. SNaPshot assay for the detection of the most common CFTR mutations in infertile men.

    Directory of Open Access Journals (Sweden)

    Predrag Noveski

    Full Text Available Congenital bilateral absence of vas deferens (CBAVD is the most common CFTR-related disorder (CFTR-RD that explains about 1-2% of the male infertility cases. Controversial data have been published regarding the involvement of CFTR mutations in infertile men with non-obstructive azoospermia and oligozoospermia. Here, we describe single base extension (SNaPshot assay for detection of 11 common CFTR mutations: F508del, G542X, N1303K, 621+1G->T, G551D, R553X, R1162X, W1282X, R117H, 2184insA and 1717-1G->A and IVS8polyT variants. The assay was validated on 50 previously genotyped samples and was used to screen a total of 369 infertile men with different impairment of spermatogenesis and 136 fertile controls. Our results show that double heterozygosity of cystic fibrosis (CF and CFTR-related disorder (CFTR-RD mutations are found in a high percentage (22.7% of infertile men with obstructive azoospermia, but not in other studied groups of infertile men. The SNaPshot assay described here is an inexpensive, fast and robust method for primary screening of the most common CFTR mutations both in patients with classical CF and CFTR-RD. It can contribute to better understanding of the role of CFTR mutations in impaired spermatogenesis, ultimately leading to improved management of infertile men.

  9. Development of an ELISA microarray assay for the sensitive and simultaneous detection of ten biodefense toxins.

    Energy Technology Data Exchange (ETDEWEB)

    Jenko, Kathryn; Zhang, Yanfeng; Kostenko, Yulia; Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Marks, James D.; Varnum, Susan M.

    2014-10-21

    Plant and microbial toxins are considered bioterrorism threat agents because of their extreme toxicity and/or ease of availability. Additionally, some of these toxins are increasingly responsible for accidental food poisonings. The current study utilized an ELISA-based protein antibody microarray for the multiplexed detection of ten biothreat toxins, botulinum neurotoxins (BoNT) A, B, C, D, E, F, ricin, shiga toxins 1 and 2 (Stx), and staphylococcus enterotoxin B (SEB), in buffer and complex biological matrices. The multiplexed assay displayed a sensitivity of 1.3 pg/mL (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg/mL (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg/mL (BoNT/D, ricin). All assays demonstrated high accuracy (75-120 percent recovery) and reproducibility (most coefficients of variation < 20%). Quantification curves for the ten toxins were also evaluated in clinical samples (serum, plasma, nasal fluid, saliva, stool, and urine) and environmental samples (apple juice, milk and baby food) with overall minimal matrix effects. The multiplex assays were highly specific, with little crossreactivity observed between the selected toxin antibodies. The results demonstrate a multiplex microarray that improves current immunoassay sensitivity for biological warfare agents in buffer, clinical, and environmental samples.

  10. Development of a real-time multiplex PCR assay for the detection of multiple Salmonella serotypes in chicken samples

    Directory of Open Access Journals (Sweden)

    Whyte Paul

    2008-09-01

    Full Text Available Abstract Background A real-time multiplex PCR assay was developed for the detection of multiple Salmonella serotypes in chicken samples. Poultry-associated serotypes detected in the assay include Enteritidis, Gallinarum, Typhimurium, Kentucky and Dublin. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in parallel. The real-time PCR based method comprised a pre-enrichment step in Buffered Peptone Water (BPW overnight, followed by a shortened selective enrichment in Rappaport Vasilliadis Soya Broth (RVS for 6 hours and subsequent DNA extraction. Results The real-time multiplex PCR assay and traditional cultural method showed 100% inclusivity and 100% exclusivity on all strains tested. The real-time multiplex PCR assay was as sensitive as the traditional cultural method in detecting Salmonella in artificially contaminated chicken samples and correctly identified the serotype. Artificially contaminated chicken samples resulted in a detection limit of between 1 and 10 CFU per 25 g sample for both methods. A total of sixty-three naturally contaminated chicken samples were investigated by both methods and relative accuracy, relative sensitivity and relative specificity of the real-time PCR method were determined to be 89, 94 and 87%, respectively. Thirty cultures blind tested were correctly identified by the real-time multiplex PCR method. Conclusion Real-time PCR methodology can contribute to meet the need for rapid identification and detection methods in food testing laboratories.

  11. Evaluation of Elecsys Syphilis Assay for Routine and Blood Screening and Detection of Early Infection.

    Science.gov (United States)

    Kremastinou, J; Polymerou, V; Lavranos, D; Aranda Arrufat, A; Harwood, J; Martínez Lorenzo, M J; Ng, K P; Queiros, L; Vereb, I; Cusini, M

    2016-09-01

    Treponema pallidum infections can have severe complications if not diagnosed and treated at an early stage. Screening and diagnosis of syphilis require assays with high specificity and sensitivity. The Elecsys Syphilis assay is an automated treponemal immunoassay for the detection of antibodies against T. pallidum The performance of this assay was investigated previously in a multicenter study. The current study expands on that evaluation in a variety of diagnostic settings and patient populations, at seven independent laboratories. The samples included routine diagnostic samples, blood donation samples, samples from patients with confirmed HIV infections, samples from living organ or bone marrow donors, and banked samples, including samples previously confirmed as syphilis positive. This study also investigated the seroconversion sensitivity of the assay. With a total of 1,965 syphilis-negative routine diagnostic samples and 5,792 syphilis-negative samples collected from blood donations, the Elecsys Syphilis assay had specificity values of 99.85% and 99.86%, respectively. With 333 samples previously identified as syphilis positive, the sensitivity was 100% regardless of disease stage. The assay also showed 100% sensitivity and specificity with samples from 69 patients coinfected with HIV. The Elecsys Syphilis assay detected infection in the same bleed or earlier, compared with comparator assays, in a set of sequential samples from a patient with primary syphilis. In archived serial blood samples collected from 14 patients with direct diagnoses of primary syphilis, the Elecsys Syphilis assay detected T. pallidum antibodies for 3 patients for whom antibodies were not detected with the Architect Syphilis TP assay, indicating a trend for earlier detection of infection, which may have the potential to shorten the time between infection and reactive screening test results. PMID:27358468

  12. A Rapid, Sensitive Assay to Detect EGFR Mutation in Small Biopsy Specimens from Lung Cancer

    OpenAIRE

    Yatabe, Yasushi; Hida, Toyoaki; Horio, Yoshitsugu; Kosaka, Takayuki; Takahashi, Takashi; Mitsudomi, Tetsuya

    2006-01-01

    It has been demonstrated that lung cancers, specifically a subset of pulmonary adenocarcinomas, with epidermal growth factor receptor (EGFR) mutation are highly sensitive to EGFR-targeted drugs. Therefore, a rapid, sensitive assay for mutation detection using routine pathological specimens is demanded in clinical practice to predict the response. We therefore developed a new assay for detecting EGFR mutation using only a paraffin section of a small biopsy specimen. The method was very sensiti...

  13. Acoustic detection of DNA conformation in genetic assays combined with PCR

    OpenAIRE

    Papadakis, G.; Tsortos, A.; Kordas, A.; Tiniakou, I.; E. Morou; Vontas, J.; Kardassis, D; Gizeli, E.

    2013-01-01

    Application of PCR to multiplexing assays is not trivial; it requires multiple fluorescent labels for amplicon detection and sophisticated software for data interpretation. Alternative PCR-free methods exploiting new concepts in nanotechnology exhibit high sensitivities but require multiple labeling and/or amplification steps. Here, we propose to simplify the problem of simultaneous analysis of multiple targets in genetic assays by detecting directly the conformation, rather than mass, of tar...

  14. Gold nanoparticles-based colorimetric and visual creatinine assay

    International Nuclear Information System (INIS)

    We demonstrate a selective and sensitive method for determination of creatinine using citrate-stabilized gold nanoparticles (AuNPs) as a colorimetric probe. It is based on a direct cross-linking reaction that occurs between creatinine and AuNPs that causes aggregation of AuNPs and results in a color change from wine red to blue. The absorption peak is shifted from 520 to 670 nm. Under the optimized conditions, the shift in the absorption peak is related the logarithm of the creatinine concentration in the 0.1 to 20 mM range, and the instrumental detection limit (LOD) is 80 μM. This LOD is about one order of magnitude better than that that of the Jaffé method (720 μM). The assay displays good selectivity over interfering substances including various inorganic ions, organic small compounds, proteins, and biothiols. It was successfully employed to the determination of creatinine in spiked human urine. (author)

  15. A 252Cf based nondestructive assay system for fissile material

    International Nuclear Information System (INIS)

    A modulated 252Cf source assay system 'Shuffler' based on fast-or-thermal-neutron interrogation combined with delayed-neutron counting has been developed for the assay of fissile material. The 252Cf neutron source is repetitively transferred from the interrogation position to a shielded position while the delayed neutrons are counted in a high efficiency 3He neutron well-counter. For samples containing plutonium, this well-counter is also used in the passive coincidence mode to assay the effective 240Pu content. The design of an optimized neutron tailoring assembly for fast-neutron interrogation using a Monte Carlo Neutron Computer Code is described. The Shuffler system has been applied to the assay of fuel pellets, inventory samples, irradiated fuel and plutonium mixed-oxide fuel. The system can assay samples with fissile contents from a few milligrams up to several kilograms using thermal-neutron interrogation for the low mass samples and fast-neutron interrogation for the high mass samples. Samples containing 235U-238U, or 233U-Th, or UO2-PuO2 fuel mixtures have been assayed with the Shuffler system. (Auth.)

  16. 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 ...

  17. Colorimetric peroxidase mimetic assay for uranyl detection in sea water

    KAUST Repository

    Zhang, Dingyuan

    2015-03-04

    Uranyl (UO2 2+) is a form of uranium in aqueous solution that represents the greatest risk to human health because of its bioavailability. Different sensing techniques have been used with very sensitive detection limits especially the recently reported uranyl-specific DNAzymes systems. However, to the best of our knowledge, few efficient detection methods have been reported for uranyl sensing in seawater. Herein, gold nanoclusters (AuNCs) are employed in an efficient spectroscopic method to detect uranyl ion (UO2 2+) with a detection limit of 1.86 ÎM. In the absence of UO2 2+, the BSA-stabilized AuNCs (BSA-AuNCs) showed an intrinsic peroxidase-like activity. In the presence of UO2 2+, this activity can be efficiently restrained. The preliminary quenching mechanism and selectivity of UO2 2+ was also investigated and compared with other ions. This design strategy could be useful in understanding the binding affinity of protein-stabilized AuNCs to UO2 2+ and consequently prompt the recycling of UO2 2+ from seawater.

  18. Development of internally controlled duplex real-time NASBA diagnostics assays for the detection of microorganisms associated with bacterial meningitis.

    Science.gov (United States)

    Clancy, Eoin; Coughlan, Helena; Higgins, Owen; Boo, Teck Wee; Cormican, Martin; Barrett, Louise; Smith, Terry J; Reddington, Kate; Barry, Thomas

    2016-08-01

    Three duplex molecular beacon based real-time Nucleic Acid Sequence Based Amplification (NASBA) assays have been designed and experimentally validated targeting RNA transcripts for the detection and identification of Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae respectively. Each real-time NASBA diagnostics assay includes an endogenous non-competitive Internal Amplification Control (IAC) to amplify the splice variant 1 mRNA of the Homo sapiens TBP gene from human total RNA. All three duplex real-time NASBA diagnostics assays were determined to be 100% specific for the target species tested for. Also the Limits of Detection (LODs) for the H. influenzae, N. meningitidis and S. pneumoniae duplex real-time NASBA assays were 55.36, 0.99, and 57.24 Cell Equivalents (CE) respectively. These robust duplex real-time NASBA diagnostics assays have the potential to be used in a clinical setting for the rapid (<60min) specific detection and identification of the most prominent microorganisms associated with bacterial meningitis in humans. PMID:27319375

  19. The PapilloCheck Assay for Detection of High-Grade Cervical Intraepithelial Neoplasia.

    Science.gov (United States)

    Crosbie, Emma J; Bailey, Andrew; Sargent, Alex; Gilham, Clare; Peto, Julian; Kitchener, Henry C

    2015-11-01

    Human papillomavirus (HPV) testing is used in primary cervical screening, as an adjunct to cervical cytology for the management of low grade abnormal cytology, and in a test of cure. PapilloCheck (Greiner Bio-One) is a PCR-based DNA microarray system that can individually identify 24 HPV types, including the 13 high-risk (HR) types identified by Hybrid Capture 2 (HC2). Here, we compare PapilloCheck with HC2 for the detection of high-grade cervical intraepithelial neoplasia (CIN2+) in a total of 8,610 cervical cytology samples from the ARTISTIC population-based cervical screening study. We performed a retrospective analysis of 3,518 cytology samples from round 1 ARTISTIC enriched for underlying CIN2+ (n = 723) and a prospective analysis of 5,092 samples from round 3 ARTISTIC. Discrepant results were tested using the Roche reverse line blot (RLB) or Linear Array (LA) assay. The relative sensitivity and specificity of HR PapilloCheck compared with that of HC2 for the detection of CIN2+ in women aged over 30 years were 0.94 (95% confidence interval [CI], 0.91, 0.97) and 1.05 (95% CI, 1.04, 1.05), respectively. HC2 missed 44/672 (7%) CIN2+ lesions, while HR PapilloCheck missed 74/672 (11%) CIN2+ lesions. Thirty-six percent of HC2-positive normal cytology samples were HR HPV negative by both PapilloCheck and RLB/LA, indicating that the use of HR PapilloCheck rather than HC2 in population-based primary screening would reduce the number of additional tests required (e.g., reflex cytology) in women where underlying CIN2+ is extremely unlikely. HR PapilloCheck could be a suitable HPV detection assay for use in the cervical screening setting. PMID:26338859

  20. Evaluation of Commercially Available Chikungunya Virus Immunoglobulin M Detection Assays.

    Science.gov (United States)

    Johnson, Barbara W; Goodman, Christin H; Holloway, Kimberly; de Salazar, P Martinez; Valadere, Anne M; Drebot, Michael A

    2016-07-01

    Commercial chikungunya virus (CHIKV)-specific IgM detection kits were evaluated at the Centers for Disease Control and Prevention (CDC), the Public Health Agency of Canada National Microbiology Laboratory, and the Caribbean Public Health Agency (CARPHA). The Euroimmun Anti-CHIKV IgM ELISA kit had ≥ 95% concordance with all three reference laboratory results. The limit of detection for low CHIK IgM+ samples, as measured by serial dilution of seven sera up to 1:12,800 ranged from 1:800 to 1:3,200. The Euroimmun IIFT kit evaluated at CDC and CARPHA performed well, but required more retesting of equivocal results. The InBios CHIKjj Detect MAC-ELISA had 100% and 98% concordance with CDC and CARPHA results, respectively, and had equal sensitivity to the CDC MAC-ELISA to 1:12,800 dilution in serially diluted samples. The Abcam Anti-CHIKV IgM ELISA had high performance at CARPHA, but at CDC, performance was inconsistent between lots. After replacement of the biotinylated IgM antibody controls with serum containing CHIKV-specific IgM and additional quality assurance/control measures, the Abcam kit was rereleased and reevaluated at CDC. The reformatted Abcam kit had 97% concordance with CDC results and limit of detection of 1:800 to 1:3,200. Two rapid tests and three other CHIKV MAC-ELISAs evaluated at CDC had low sensitivity, as the CDC CHIKV IgM in-house positive controls were below the level of detection. In conclusion, laboratories have options for CHIKV serological diagnosis using validated commercial kits. PMID:26976887

  1. Development and validation of an oligonucleotide ligation assay to detect lamivudine resistance in hepatitis B virus.

    Science.gov (United States)

    Beck, Ingrid A; Payant, Rachel; Ngo-Giang-Huong, Nicole; Khamduang, Woottichai; Laomanit, Laddawan; Jourdain, Gonzague; Frenkel, Lisa M

    2016-07-01

    Treatment of chronic hepatitis B virus (HBV) infection with lamivudine-monotherapy rapidly selects mutant variants in a high proportion of individuals. Monitoring lamivudine resistance by consensus sequencing is costly and insensitive for detection of minority variants. An oligonucleotide ligation assay (OLA) for HBV lamivudine-resistance was developed and compared to consensus sequencing. Both assays detected drug resistance mutations in 35/64 (54.7%) specimens evaluated, and OLA detected minority mutants in an additional six (9.4%). OLA may offer a sensitive and inexpensive alternative to consensus sequencing for detection of HBV drug resistance in resource-limited settings. PMID:27025356

  2. Automated 5 ' nuclease assay for detection of virulence factors in porcine Escherichia coli

    DEFF Research Database (Denmark)

    Frydendahl, K.; Imberechts, H.; Lehmann, S.

    2001-01-01

    (STa, STb, EAST1) and heat labile LT) enterotoxins and the verocytotoxin variant 2e (VT2e). To correctly identify false negative results, an endogenous internal control targeting the E. coil 16S rRNA gene was incorporated in each test tube. The assay was evaluated using a collection of E. coil...... reference strains which have previously been examined with phenotypical assays or DNA hybridization. Furthermore, the assay was evaluated by testing porcine E. coil field strains, previously characterized. The 5' nuclease assay correctly detected the presence of virulence genes in all reference strains....... When testing field strains there was generally excellent agreement with results obtained by laboratories in Belgium and Germany. In conclusion, the 5' nuclease assay developed is a fast and specific tool for detection of E. coli virulence genes in the veterinary diagnostic laboratory....

  3. Multicenter evaluation of the nitrate reductase assay for drug resistance detection of Mycobacterium tuberculosis.

    Science.gov (United States)

    Martin, Anandi; Montoro, Ernesto; Lemus, Dihadenys; Simboli, Norberto; Morcillo, Nora; Velasco, Maritza; Chauca, José; Barrera, Lucía; Ritacco, Viviana; Portaels, Françoise; Palomino, Juan Carlos

    2005-11-01

    The performance of the nitrate reductase assay was evaluated in a multicenter laboratory study to detect resistance of Mycobacterium tuberculosis to the first-line anti-tuberculosis drugs rifampicin, isoniazid, ethambutol and streptomycin using a set of coded isolates. Compared with the gold standard proportion method on Löwenstein-Jensen medium, the assay was highly accurate in detecting resistance to rifampicin, isoniazid and ethambutol with an accuracy of 98%, 96.6% and 97.9%, respectively. For streptomycin, discrepant results were obtained with an overall accuracy of 85.3%. The assay proved easy to be implemented in countries with limited laboratory facilities. PMID:15893391

  4. Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples

    Institute of Scientific and Technical Information of China (English)

    LI Jin-ping; LI Yan; SHI Yan-xia; XIE Xue-wen; Chai A-li; LI Bao-ju

    2013-01-01

    A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA (rDNA) and internal transcribed spacer (ITS). A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P. brassicae. The positive plasmid pB12 was obtained and used as the template to create standard curve. The specificity, sensitivity, and reproducibility of real-time PCR were evaluated respectively. Naturally and artificially infested soil samples containing different concentrations of P. brassicae were detected. The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration. The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%. The detection limit of P. brassicae genomic DNA was approximately 40 copies per 25μL. The sensitivity of the assay was at least 100-fold higher than conventional PCR. Only DNA from P. brassicae could be amplified and detected using this assay, suggesting the highly specific of this assay. The coefficient of variation was less than 3%, indicating the PCR method revealed high reproducibility. The detection limit in soil samples corresponded to 1 000 resting spores g-1 soil. Bait plants were used to validate the real-time PCR assay. This developed real-time PCR assay allows for fast and sensitive detection of P. brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P. brassicae.

  5. Biosensors and Bio-Bar Code Assays Based on Biofunctionalized Magnetic Microbeads

    Directory of Open Access Journals (Sweden)

    Jean-Pierre Cloarec

    2007-04-01

    Full Text Available This review paper reports the applications of magnetic microbeads in biosensors and bio-bar code assays. Affinity biosensors are presented through different types of transducing systems: electrochemical, piezo electric or magnetic ones, applied to immunodetection and genodetection. Enzymatic biosensors are based on biofunctionalization through magnetic microbeads of a transducer, more often amperometric, potentiometric or conductimetric. The bio-bar code assays relie on a sandwich structure based on specific biological interaction of a magnetic microbead and a nanoparticle with a defined biological molecule. The magnetic particle allows the separation of the reacted target molecules from unreacted ones. The nanoparticles aim at the amplification and the detection of the target molecule. The bio-bar code assays allow the detection at very low concentration of biological molecules, similar to PCR sensitivity.

  6. International collaborative study to compare reverse transcriptase PCR assays for detection and genotyping of noroviruses.

    Science.gov (United States)

    Vinjé, Jan; Vennema, Harry; Maunula, Leena; von Bonsdorff, Carl-Henrik; Hoehne, Marina; Schreier, Eckart; Richards, Alison; Green, Jon; Brown, David; Beard, Suzanne S; Monroe, Stephan S; de Bruin, Erwin; Svensson, Lennart; Koopmans, Marion P G

    2003-04-01

    To allow more rapid and internationally standardized assessment of the spread of noroviruses (previously called Norwalk-like viruses [NLVs]) as important food-borne pathogens, harmonization of methods for their detection is needed. Diagnosis of NLVs in clinical diagnostic laboratories is usually performed by reverse transciptase PCR (RT-PCR) assays. In the present study, the performance of five different RT-PCR assays for the detection of NLVs was evaluated in an international collaborative study by five laboratories in five countries with a coded panel of 91 fecal specimens. The assays were tested for their sensitivity, detection limit, and ease of standardization. In total, NLVs could be detected by at least one RT-PCR assay in 69 (84%) of the samples that originally tested positive. Sensitivity ranged from 52 to 73% overall and from 54 to 100% and 58 to 85% for genogroup I and II viruses, respectively. In all, 64% of the false-negative results were obtained with a set of diluted stools (n = 20) that may have lost quality upon storage. Sensitivity was improved when these samples were excluded from analysis. No one single assay stood out as the best, although the p1 assay demonstrated the most satisfactory overall performance. To promote comparability of data, this assay will be recommended for newly starting groups in future collaborative studies. PMID:12682125

  7. A cell culture assay for the detection of cardiotoxicity

    International Nuclear Information System (INIS)

    An important step in minimizing the number of animal experiments in medical research is the study of in vitro model systems. The authors propose the use of shock protein formation, which is a cellular response to cell-damaging stress as an assay to monitor cardiotoxicity. Isolated and cultured cardiac myocytes were prepared by a trypsin digestion method from 18-day-old fetal mice. These cells respond to typical substances inducing shock protein formation in other cellular systems as well as to known cardiotoxins with the de novo synthesis of shock proteins. Pharmaceuticals relevant in transplant medicine were tested for possible cardiotoxic effects: Cyclosporine A evokes shock protein formation at subtherapeutic concentrations. Azathioprine and methyl-prednisolone exert the same effect but at concentration ranges highly above the therapeutic level. The ability to induce shock protein synthesis obviously seems to be restricted to toxic drugs. The data presented demonstrate that the proposed in vitro model system for cardiotoxicity is animal saving and sensitive

  8. Low dose effects detected by micronucleus assay in lymphocytes

    International Nuclear Information System (INIS)

    The effects of low doses of X-rays between 0.01 and 1 Gy were studied on whole blood samples of various individuals using the cytokinesis-blocked lymphocyte micronucleus assay as an endpoint. The adaptive response could be induced in G0 cells by 0.01 Gy followed by 1 Gy challenging dose within a time period of 8 hours, in vitro. The probability distribution of micronucleus increments in those samples which had received very low doses in the range 0.01-0.05 Gy proved to be of asymmetrical type (i.e. lognormal) -very likely to the same shape which has been verified for unirradiated (control) population - while the variable turned to be normally distributed at or above 1 Gy. Profound changes have been experienced in the main characteristics of the linear dose - response relationship and in regression parameters, as well, when successively lessened dose ranges were studied toward 0.01 Gy. In the range below ∼ 0.2 Gy the response were found to be unrelated to the absorbed dose. These findings suggest that in (very) low dose range a higher attention should be needed to biological parameters like repair, protective mechanisms and antioxidant capacities, rather than to the absorbed radiation energy only. (author)

  9. A cell culture assay for the detection of cardiotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Loew-Friedrich, Iv.; von Bredow, F.; Schoeppe, W. (Department of Nephrology, Hospital of the Johann Wolfgang Goethe-University, Frankfurt am Main (Germany))

    1991-04-01

    An important step in minimizing the number of animal experiments in medical research is the study of in vitro model systems. The authors propose the use of shock protein formation, which is a cellular response to cell-damaging stress as an assay to monitor cardiotoxicity. Isolated and cultured cardiac myocytes were prepared by a trypsin digestion method from 18-day-old fetal mice. These cells respond to typical substances inducing shock protein formation in other cellular systems as well as to known cardiotoxins with the de novo synthesis of shock proteins. Pharmaceuticals relevant in transplant medicine were tested for possible cardiotoxic effects: Cyclosporine A evokes shock protein formation at subtherapeutic concentrations. Azathioprine and methyl-prednisolone exert the same effect but at concentration ranges highly above the therapeutic level. The ability to induce shock protein synthesis obviously seems to be restricted to toxic drugs. The data presented demonstrate that the proposed in vitro model system for cardiotoxicity is animal saving and sensitive.

  10. Quantification of Analyte Concentration from a Paper-Based Lateral Flow Assay Device Using Reflective Sensors

    OpenAIRE

    Chiu, Megan Z; Linnes, Jacqueline

    2015-01-01

    Paper-based point-of-care (POC) diagnostics is a growing field in global health due to the extreme portability, accuracy, affordability, and ease of use of these tests. Advancements in recent years have led to more accurate detection and improved functionality using multistep molecular diagnostics. Many such assays utilize lateral flow detection strips for visualization of diagnostic results by eye, which limits the results to qualitative Yes/No readouts. This project focused on combining rec...

  11. Development and Application of Nested PCR Assay for Detection of Dairy Cattle-Derived Cyclospora sp.

    Institute of Scientific and Technical Information of China (English)

    XIAO Shu-min; LI Guo-qing; LI Wei-hua; ZHOU Rong-qiong; YANG Jian-wei

    2007-01-01

    To develop a nested PCR assay for the detection of cattle-derived Cyclospora sp.,two pairs of primers were designed on the basis of the cattle-derived Cyclospora sp.sequences.These primers selectively amplified a 168-bp DNA fragment of the 18S rRNA gene of cattle-derived Cyclospora sp.With these primers,a nested PCR assay for the detection of cattlederived Cyclospora sp.was developed.The nested PCR assay was specific and there is no cross-reaction with other parasites,such as Eimeria spp.,Cryptosporidium spp.,Giardia sp.,Toxoplasma sp.,Trichuris sp.and cattle ciliate.The assay was able to detect as low as 2.85 x 10-2 fg of the control positive DNA.The results of the detection of clinical samples indicated that the assay coincided with microscopic examination.The results show that the nested PCR assay will be an effective tool for the detection of Cyclospora sp.in cattle feces.

  12. Bioanalytical method transfer considerations of chromatographic-based assays.

    Science.gov (United States)

    Williard, Clark V

    2016-07-01

    Bioanalysis is an important part of the modern drug development process. The business practice of outsourcing and transferring bioanalytical methods from laboratory to laboratory has increasingly become a crucial strategy for successful and efficient delivery of therapies to the market. This chapter discusses important considerations when transferring various types of chromatographic-based assays in today's pharmaceutical research and development environment. PMID:27277876

  13. Polymerase Chain Reaction (Pcr) Assay to Detect Hepatitis C Virus

    International Nuclear Information System (INIS)

    Research on the detection of hepatitis C virus in blood serum using PCR technique has been carried out. Amount of 50 blood serum from laboratory of Indonesia Red Cross (Palang Merah Indonesia = PMI) and RSCM hospital as samples, were used in this research. Lysis of virus cell and extraction of RNA virus as a preliminary treatment of the sample, was done with BOOM method using guanidine thiocyanate and diatomaceous earth, respectively. Synthesis of cDNA from RNA as an extraction product mentioned above, was carried out by means of reverse-transcriptase and RNA-se inhibitor. Amplification of cDNA was done with nested PCR technique that was performed with two times PCR processes using two pairs of oligonucleotide primers for each process. The amplified DNA was detected by agarose gel electrophoresis and ethidium bromide staining. Subsequently, the DNA was visualized with UV transilluminator. Result shows that of 50 blood serum samples, 13 serum were positive for RNA HCV that were performed with the present of specific DNA band on agarose gel. (author)

  14. Development of a reverse transcription loop-mediated isothermal amplification assay for the detection of vesicular stomatitis New Jersey virus: Use of rapid molecular assays to differentiate between vesicular disease viruses.

    Science.gov (United States)

    Fowler, Veronica L; Howson, Emma L A; Madi, Mikidache; Mioulet, Valérie; Caiusi, Chiara; Pauszek, Steven J; Rodriguez, Luis L; King, Donald P

    2016-08-01

    Vesicular stomatitis (VS) is endemic in Central America and northern regions of South America, where sporadic outbreaks in cattle and pigs can cause clinical signs that are similar to foot-and-mouth disease (FMD). There is therefore a pressing need for rapid, sensitive and specific differential diagnostic assays that are suitable for decision making in the field. RT-LAMP assays have been developed for vesicular diseases such as FMD and swine vesicular disease (SVD) but there is currently no RT-LAMP assay that can detect VS virus (VSV), nor are there any multiplex RT-LAMP assays which permit rapid discrimination between these 'look-a-like' diseases in situ. This study describes the development of a novel RT-LAMP assay for the detection of VSV focusing on the New Jersey (VSNJ) serotype, which has caused most of the recent VS cases in the Americas. This RT-LAMP assay was combined in a multiplex format combining molecular lateral-flow devices for the discrimination between FMD and VS. This assay was able to detect representative VSNJV's and the limit of detection of the singleplex and multiplex VSNJV RT-LAMP assays were equivalent to laboratory based real-time RT-PCR assays. A similar multiplex RT-LAMP assay was developed to discriminate between FMDV and SVDV, showing that FMDV, SVDV and VSNJV could be reliably detected within epithelial suspensions without the need for prior RNA extraction, providing an approach that could be used as the basis for a rapid and low cost assay for differentiation of FMD from other vesicular diseases in the field. PMID:27118518

  15. Visual Detection of Brucella spp. in Spiked Bovine Semen Using Loop-Mediated Isothermal Amplification (LAMP) Assay.

    Science.gov (United States)

    Prusty, Bikash R; Chaudhuri, Pallab; Chaturvedi, V K; Saini, Mohini; Mishra, B P; Gupta, Praveen K

    2016-06-01

    Several pathogens including Brucella spp. are shed in semen of infected bulls and can be transmitted to cows through contaminated semen during artificial insemination. The present study reports omp2a and bcsp31 gene based loop-mediated isothermal amplification (LAMP) assays for detection of Brucella genomic DNA in semen from infected bulls. The positive results could be interpreted visually by change in colour of reaction mixture containing hydroxyl naphthol blue (HNB) dye from violet to sky blue. LAMP assays based on omp2a and bcsp31 could detect as little as 10 and 100 fg of B. abortus S19 genomic DNA, respectively. Sensitivity of omp2a and bcsp31 LAMP assays for direct detection of organisms in bovine semen was 2.28 × 10(1) CFU and 2.28 × 10(2) CFU of B. abortus S19 in spiked bovine semen, respectively. The omp2a LAMP assay was found equally sensitive to TaqMan probe based real-time PCR and 100 times more sensitive than conventional PCR in identifying Brucella in spiked semen. The diagnostic applicability of the omp2a LAMP assay was evaluated with seventy-nine bovine semen samples and results were re-evaluated through TaqMan probe based real-time PCR and conventional PCR. Taken together, the omp2a LAMP assay is easy to perform, rapid and sensitive in diagnosis of Brucella spp. in bovine semen. PMID:27570305

  16. Optimized Pan-species and speciation duplex real-time PCR assays for Plasmodium parasites detection in malaria vectors.

    Directory of Open Access Journals (Sweden)

    Maurice Marcel Sandeu

    Full Text Available BACKGROUND: An accurate method for detecting malaria parasites in the mosquito's vector remains an essential component in the vector control. The Enzyme linked immunosorbent assay specific for circumsporozoite protein (ELISA-CSP is the gold standard method for the detection of malaria parasites in the vector even if it presents some limitations. Here, we optimized multiplex real-time PCR assays to accurately detect minor populations in mixed infection with multiple Plasmodium species in the African malaria vectors Anopheles gambiae and Anopheles funestus. METHODS: Complementary TaqMan-based real-time PCR assays that detect Plasmodium species using specific primers and probes were first evaluated on artificial mixtures of different targets inserted in plasmid constructs. The assays were further validated in comparison with the ELISA-CSP on 200 field caught Anopheles gambiae and Anopheles funestus mosquitoes collected in two localities in southern Benin. RESULTS: The validation of the duplex real-time PCR assays on the plasmid mixtures demonstrated robust specificity and sensitivity for detecting distinct targets. Using a panel of mosquito specimen, the real-time PCR showed a relatively high sensitivity (88.6% and specificity (98%, compared to ELISA-CSP as the referent standard. The agreement between both methods was "excellent" (κ=0.8, P<0.05. The relative quantification of Plasmodium DNA between the two Anopheles species analyzed showed no significant difference (P=0, 2. All infected mosquito samples contained Plasmodium falciparum DNA and mixed infections with P. malariae and/or P. ovale were observed in 18.6% and 13.6% of An. gambiae and An. funestus respectively. Plasmodium vivax was found in none of the mosquito samples analyzed. CONCLUSION: This study presents an optimized method for detecting the four Plasmodium species in the African malaria vectors. The study highlights substantial discordance with traditional ELISA-CSP pointing out the

  17. Avian haemosporidian parasites (Haemosporida): A comparative analysis of different polymerase chain reaction assays in detection of mixed infections.

    Science.gov (United States)

    Bernotienė, Rasa; Palinauskas, Vaidas; Iezhova, Tatjana; Murauskaitė, Dovilė; Valkiūnas, Gediminas

    2016-04-01

    Mixed infections of different species and genetic lineages of haemosporidian parasites (Haemosporida) predominate in wildlife, and such infections are particularly virulent. However, currently used polymerase chain reaction (PCR)-based detection methods often do not read mixed infections. Sensitivity of different PCR assays in detection of mixed infections has been insufficiently tested, but this knowledge is essential in studies addressing parasite diversity in wildlife. Here, we applied five different PCR assays, which are broadly used in wildlife avian haemosporidian research, and compared their sensitivity in detection of experimentally designed mixed infections of Haemoproteus and Plasmodium parasites. Three of these PCR assays use primer sets that amplify fragments of cytochrome b gene (cyt b), one of cytochrome oxidase subunit I (COI) gene, and one target apicoplast genome. We collected blood from wild-caught birds and, using microscopic and PCR-based methods applied in parallel, identified single infections of ten haemosporidian species with similar parasitemia. Then, we prepared 15 experimental mixes of different haemosporidian parasites, which often are present simultaneously in wild birds. Similar concentration of total DNA was used in each parasite lineage during preparation of mixes. Positive amplifications were sequenced, and the presence of mixed infections was reported by visualising double-base calling in sequence electropherograms. This study shows that the use of each single PCR assay markedly underestimates biodiversity of haemosporidian parasites. The application of at least 3 PCR assays in parallel detected the majority, but still not all lineages present in mixed infections. We determined preferences of different primers in detection of parasites belonging to different genera of haemosporidians during mixed infections. PMID:26821298

  18. Development of a PCR assay to detect mycoplasma contamination in cord blood hematopoietic stem cells.

    Directory of Open Access Journals (Sweden)

    Reza Tabatabaei-Qomi

    2014-08-01

    Full Text Available Contamination of cell lines and biological products is one of the major problems of cell culture techniques. Rapid detection of mycoplasma contamination in cell culture is an important part of quality control standards in related laboratories. The aim of this study was to evaluate the efficacy of PCR in detection of myroplasma as contaminants in cell cultures and other biological products.PCR assays were optimized for 16 S rRNA target gene. Also the utilized PCR method was evaluated in terms of sensitivity and specificity. Finally, a simple DNA extraction and PCR analysis of 164 cell culture of adipose tissue derived mesenchymal stem cells were performed.A 715 bp product was amplified and subsequently was confirmed by sequencing. The technique could detect 10 copies of the target DNA. No cross-reactivity with genomic DNA of other microorganisms was observed.The PCR technique in this study was based on 16S rRNA gene. It was highly sensitive and specific since it was able to detected Mycoplasma contamination in cell cultures.

  19. Serological assays based on recombinant viral proteins for the diagnosis of arenavirus hemorrhagic fevers.

    Science.gov (United States)

    Fukushi, Shuetsu; Tani, Hideki; Yoshikawa, Tomoki; Saijo, Masayuki; Morikawa, Shigeru

    2012-10-01

    The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW) and New World (NW) complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF) in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs) derived from these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs) to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture) ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV)-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses. PMID:23202455

  20. Serological Assays Based on Recombinant Viral Proteins for the Diagnosis of Arenavirus Hemorrhagic Fevers

    Directory of Open Access Journals (Sweden)

    Masayuki Saijo

    2012-10-01

    Full Text Available The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW and New World (NW complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs derived from these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.

  1. Early detection of influenza virus by using a fluorometric assay of infected tissue culture.

    OpenAIRE

    Pachucki, C T; Creticos, C

    1988-01-01

    A fluorometric substrate, 4-methylumbelliferyl-alpha-ketoside of N-acetylneuramide, was used directly on clinical specimens and infected tissue culture 24 h after inoculation for the detection of influenza viral neuraminidase. Viral neuraminidase was detected in infected tissue culture but not in clinical specimens. The sensitivity of the assay on tissue culture was 92%, and the specificity was 96%.

  2. Indirect solid-phase immunosorbent assay for detection of arenavirus antigens and antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, A.P.; Rezapkin, G.V.; Dzagurova, T.K.; Tkachenko, E.A. (Institute of Poliomyelitis anU Viral Encephalities of the U.S.S.R. Academy of Medical Sciences, Moscow)

    1984-05-01

    Indirect enzyme-linked immunosorbent assay (ELISA) and solid phase radioimmunoassay (SPRIA) using either enti-human or anti-mouse IgG labelled with horseradish peroxidase and /sup 125/I, respectively, were developed for the detection of Junin, Machupo, Tacaribe, Amapari, Tamiami, Lassa and LCM arenaviruses. Both methods allow high sensitivity detection of arenavirus antigens and antibodies.

  3. Detection of Trichomonas vaginalis in vaginal specimens by direct immunofluorescence assay.

    OpenAIRE

    Smith, R F

    1986-01-01

    Vaginal discharge specimens from 105 women were examined by wet mount, culture, and a new direct immunofluorescence assay to detect Trichomonas vaginalis. The organism was detected by culture in 31 patients, by direct immunofluorescence in 26 patients, and by wet mount in 21 patients.

  4. Review on porcine endogenous retrovirus detection assays--impact on quality and safety of xenotransplants.

    Science.gov (United States)

    Godehardt, Antonia W; Rodrigues Costa, Michael; Tönjes, Ralf R

    2015-01-01

    Xenotransplantation of porcine organs, tissues, and cells inherits a risk for xenozoonotic infections. Viable tissues and cells intended for transplantation have to be considered as potentially contaminated non-sterile products. The demands on microbial testing, based on the regulatory requirements, are often challenging due to a restricted shelf life or the complexity of the product itself. In Europe, the regulatory framework for xenogeneic cell therapy is based on the advanced therapy medicinal products (ATMP) regulation (2007), the EMA CHMP Guideline on xenogeneic cell-based medicinal products (2009), as well as the WHO and Council of Europe recommendations. In the USA, FDA guidance for industry (2003) regulates the use of xenotransplants. To comply with the regulations, validated test methods need to be established that reveal the microbial status of a transplant within its given shelf life, complemented by strictly defined action alert limits and supported by breeding in specific pathogen-free (SPF) facilities. In this review, we focus on assays for the detection of the porcine endogenous retroviruses PERV-A/-B/-C, which exhibit highly polymorphic proviral loci in pig genomes. PERVs are transmitted vertically and cannot be completely eliminated by breeding or gene knock out technology. PERVs entail a public health concern that will persist even if no evidence of PERV infection of xenotransplant recipients in vivo has been revealed yet. Nevertheless, infectious risks must be minimized by full assessment of pigs as donors by combining different molecular screening assays for sensitive and specific detection as well as a functional analysis of the infectivity of PERV including an adequate monitoring of recipients. PMID:25641488

  5. Characterization of a nested polymerase chain reaction assay for detection of parvovirus B19.

    OpenAIRE

    Patou, G.; Pillay, D.; Myint, S; Pattison, J.

    1993-01-01

    The characterization and application of a nested polymerase chain reaction (PCR) assay for the detection of human parvovirus B19 DNA is described. The assay was evaluated with 149 diagnostic serum samples (collected up to 150 days after the onset of symptoms) previously tested by dot blot hybridization for B19 DNA and by class-specific capture radioimmunoassays for the detection of B19 immunoglobulin M (IgM) and IgG. B19 DNA was detectable by the PCR in 70% of the sera. There was a statistica...

  6. Short report : Development of a molecular assay to detect predation on Anopheles gambiae complex larval stages

    OpenAIRE

    Schielke, E.; Costantini, Carlo; Carchini, G.; Sagnon, N.; J. Powell; Caccone, A

    2007-01-01

    We developed a molecular assay to detect predation on Anopheles gambiae sensu lato (s.l.) mosquitoes. This intergenic spacer ribosomal DNA polymerase chain reaction assay and restriction enzyme analysis uses An. gambiae-specific primers to detect mosquito DNA in the DNA extracts from whole invertebrate predators, which enables identification of species (An. gambiae s.s. versus An. arabiensis) and molecular forms (M versus S in An. gambiae s.s.). We show that An. gambiae s.l. DNA can be detect...

  7. Genotype MTBDR plus assay for molecular detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Soniya Sharma

    2014-01-01

    Full Text Available Aim: This study was performed for the rapid identification of Mycobacterium tuberculosis complex and its resistance to rifampicin and isoniazid, directly from the sputum samples of pulmonary tuberculosis patients. Materials and Methods: A commercially available genotype MTBDR plus assay was used for the identification and detection of mutations in Mycobacterial isolates. A total of 100 sputum samples of pulmonary tuberculosis patients were analyzed by using the genotype MTBDR plus assay. The MTBDR plus assay is designed to detect the mutations in the hotspot region of rpoB gene, katG and regulatory region of inhA gene. Results: The genotype MTBDR plus assay detected 22% multidrug resistant (MDR, 2% rifampicin (RMP monoresistant and 1% isoniazid (INH monoresistant isolates. In 22 MDR isolates, the codons most frequently involved in RMP-associated mutations were codon 531 (54.55%, 516 (31.82% and 526 (13.63%, and 90.90% of MDR isolates showed KatG S315T mutations and 9.1% showed inhA C-15T mutations associated with INH resistance. Conclusion: The new genotype MTBDR plus assay represents a rapid, reliable tool for the detection of MDR-TB, wherein results are obtained in 5 h allowing early and appropriate treatment, which is essential to cut the transmission path and reduce the spread of MDR-TB. The genotype MTBDR plus assay can readily be included in a routine laboratory work for the early diagnosis and control of MDR-TB.

  8. 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. PMID:24792836

  9. Zebrafish-based reporter gene assays reveal different estrogenic activities in river waters compared to a conventional human-derived assay.

    Science.gov (United States)

    Sonavane, Manoj; Creusot, Nicolas; Maillot-Maréchal, Emmanuelle; Péry, Alexandre; Brion, François; Aїt-Aïssa, Selim

    2016-04-15

    Endocrine disrupting chemicals (EDCs) act on the endocrine system through multiple mechanisms of action, among them interaction with estrogen receptors (ERs) is a well-identified key event in the initiation of adverse outcomes. As the most commonly used estrogen screening assays are either yeast- or human-cell based systems, the question of their (eco)toxicological relevance when assessing risks for aquatic species can be raised. The present study addresses the use of zebrafish (zf) derived reporter gene assays, both in vitro (i.e. zf liver cell lines stably expressing zfERα, zfERβ1 and zfERβ2 subtypes) and in vivo (i.e. transgenic cyp19a1b-GFP zf embryos), to assess estrogenic contaminants in river waters. By investigating 20 French river sites using passive sampling, high frequencies of in vitro zfER-mediated activities in water extracts were measured. Among the different in vitro assays, zfERβ2 assay was the most sensitive and responsive one, enabling the detection of active compounds at all investigated sites. In addition, comparison with a conventional human-based in vitro assay highlighted sites that were able to active zfERs but not human ER, suggesting the occurrence of zf-specific ER ligands. Furthermore, a significant in vivo estrogenic activity was detected at the most active sites in vitro, with a good accordance between estradiol equivalent (E2-EQ) concentrations derived from both in vitro and in vivo assays. Overall, this study shows the relevance and usefulness of such novel zebrafish-based assays as screening tools to monitor estrogenic activities in complex mixtures such as water extracts. It also supports their preferred use compared to human-based assays to assess the potential risks caused by endocrine disruptive chemicals for aquatic species such as fish. PMID:26851879

  10. A time-resolved luminescent competitive assay to detect L-selectin using aptamers as recognition elements

    Energy Technology Data Exchange (ETDEWEB)

    Cywiński, Piotr J., E-mail: piotr.cywinski@iap.fraunhofer.de [Functional Materials and Devices, Fraunhofer Institute for Applied Polymer Research, Geiselberstr.69, 14476 Potsdam-Golm (Germany); Department of Physical Chemistry, Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam-Golm (Germany); Olejko, Lydia; Löhmannsröben, Hans-Gerd [Department of Physical Chemistry, Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam-Golm (Germany)

    2015-08-05

    L-selectin is a protein with potential importance for numerous diseases and clinical disorders. In this paper, we present a new aptamer-based luminescent assay developed to detect L-selectin. The sensing system working principle is based on Förster Resonance Energy Transfer (FRET) from a donor terbium complex (TbC) to an acceptor cyanine dye (Cy5). In the present approach, the biotinylated aptamer is combined with Cy5-labelled streptavidin (Cy5-Strep) to yield an aptamer-based acceptor construct (Apta-Cy5-Strep), while L-selectin is conjugated using luminescent TbC. Upon aptamer binding to the TbC-labelled L-selectin (L-selectin-TbC), permanent donor-acceptor proximity is established which allows for radiationless energy transfer to occur. However, when unlabelled L-selectin is added, it competes with the L-selectin-TbC and the FRET signal decreases as the L-selectin concentration increases. FRET from the TbC to Cy5 was observed with time-gated time-resolved luminescence spectroscopy. A significant change in the corrected luminescence signal was observed in the dynamic range of 10–500 ng/mL L-selectin, the concentration range relevant for accelerated cognitive decline of Alzheimer's disease, with a limit of detection (LOD) equal to 10 ng/mL. The aptasensor-based assay is homogeneous and can be realized within one hour. Therefore, this method has the potential to become an alternative to tedious heterogeneous analytical methods, e.g. based on enzyme-linked immunosorbent assay (ELISA). - Highlights: • Tb-based FRET assay with aptamers toward a protein is presented for the first time. • L-selectin can be detected in concentrations relevant for the Alzheimer's Disease. • The assay can be realized in one hour with the LOD equal to 10 ng/ml.

  11. A time-resolved luminescent competitive assay to detect L-selectin using aptamers as recognition elements

    International Nuclear Information System (INIS)

    L-selectin is a protein with potential importance for numerous diseases and clinical disorders. In this paper, we present a new aptamer-based luminescent assay developed to detect L-selectin. The sensing system working principle is based on Förster Resonance Energy Transfer (FRET) from a donor terbium complex (TbC) to an acceptor cyanine dye (Cy5). In the present approach, the biotinylated aptamer is combined with Cy5-labelled streptavidin (Cy5-Strep) to yield an aptamer-based acceptor construct (Apta-Cy5-Strep), while L-selectin is conjugated using luminescent TbC. Upon aptamer binding to the TbC-labelled L-selectin (L-selectin-TbC), permanent donor-acceptor proximity is established which allows for radiationless energy transfer to occur. However, when unlabelled L-selectin is added, it competes with the L-selectin-TbC and the FRET signal decreases as the L-selectin concentration increases. FRET from the TbC to Cy5 was observed with time-gated time-resolved luminescence spectroscopy. A significant change in the corrected luminescence signal was observed in the dynamic range of 10–500 ng/mL L-selectin, the concentration range relevant for accelerated cognitive decline of Alzheimer's disease, with a limit of detection (LOD) equal to 10 ng/mL. The aptasensor-based assay is homogeneous and can be realized within one hour. Therefore, this method has the potential to become an alternative to tedious heterogeneous analytical methods, e.g. based on enzyme-linked immunosorbent assay (ELISA). - Highlights: • Tb-based FRET assay with aptamers toward a protein is presented for the first time. • L-selectin can be detected in concentrations relevant for the Alzheimer's Disease. • The assay can be realized in one hour with the LOD equal to 10 ng/ml

  12. Activity-based assay for ricin-like toxins

    Science.gov (United States)

    Keener, William K.; Ward, Thomas E.

    2007-02-06

    A method of detecting N-glycosylase activity in a sample involves incubating an oligodeoxyribonucleotide substrate containing a deoxyadenosine or deoxyuridine residue with the sample to be tested such that the N-glycosylase, if present, hydrolyzes the deoxyadenosine or deoxyuridine residue to result in an N-glycosylase product having an abasic site. A primer is annealed to the N-glycosylase product, and the primer is extended with a DNA polymerase, such as Taq DNA polymerase, that pauses at abasic sites. The resulting extension products are melted from the N-glycosylase product, allowed to form hairpins due to self-complementarity, and further extended in the presence of labeled precursors to result in labeled products. Extension products synthesized from undigested substrate as template do not result in labeled products. Thus, detection of labeled products results in detection of N-glycosylase activity. Oligodeoxyribonucleotide substrates, primer, and positive controls and a kit for N-glycosylase assay are also disclosed.

  13. Comparison of real-time PCR detection chemistries and cycling modes using Mon810 event-specific assays as model.

    Science.gov (United States)

    La Paz, José Luis; Esteve, Teresa; Pla, Maria

    2007-05-30

    The most widely accepted methods for accurate quantitative detection of genetically modified organisms rely on real-time PCR. Various detection chemistries are available for real-time PCR. They include sequence-unspecific DNA labeling dyes such SYBR-Green I and the use of both universal (e.g., AmpliFluor) and sequence-specific double-labeled probes, the latter comprising hybridization (e.g., Molecular Beacon) and hydrolysis (e.g., TaqMan or MGB) probes. Also, new real-time PCR devices and reagents allowing fast cycling reactions exist. Five Mon810 real-time PCR assays were developed in which the event specificity was based on the detection of transgene and plant rearranged sequences found to 3' flank the insertion site. Every assay was specifically designed for one particular detection chemistry, that is, AmpliFluor, Molecular Beacon, MGB, TaqMan, and SYBR-Green I. When possible, the assays were adapted to fast cycling mode. All assays displayed satisfactory performance parameters, although Molecular Beacon, MGB, and TaqMan chemistries were the most suitable for quantification purposes in both conventional and fast cycling modes. PMID:17488028

  14. Detection of TGEV Antibody by Enzyme-Linked Immunosorbent Assay Using Recombinant Nucleocapsid Proteins

    Institute of Scientific and Technical Information of China (English)

    YU Li-yun; HOU Xi-lin

    2005-01-01

    An enzyme linked immunosorbent assays (ELISA) based on recombinant nucleocapsid (N) protein generated in Escherichia coli was evaluated for its sensitivity and specificity for diagnosis of transmissible gastroenteritis virus (TGEV) infection.The N gene encoding the N protein was cloned and expressed as a fusion protein with His tag protein in E. coli. The recombinant N protein migrated at 42 kDa and reacted with His6 tag specific monoclonal antibody by immunoblotting.Recombinant N protein ELISA (rnELISA) demonstrated 97.5% specificity among 80 TGEV-free individuals, and 97.3%sensitivity ranging among 110 clinical samples with TGEV. Taken together, these results indicated that nucleocapsid may be a useful antigen for the sera-diagnosis of TGEV and it was also suggested that the ELISA is a highly sensitive and specific test for detecting antibodies against TGEV.

  15. Nondestructive assay holdup measurements with the Ortec detective

    Energy Technology Data Exchange (ETDEWEB)

    Vo, Duc [Los Alamos National Laboratory; Wenz, Tracy [Los Alamos National Laboratory; Bracken, David [Los Alamos National Laboratory

    2009-01-01

    facility, we used a new technique that we tentatively named 'Room Holdup Measurement' to do holdup measurements of the site. This technique uses the portable, electric-cooled high-purity germanium detectors from Ortec (the Detectives) to measure the activities of the isotopes.

  16. Comparison of three commercially available assays for detection of varicella-zoster virus antibody.

    OpenAIRE

    Martins, T B; Jaskowski, T D; Schroder, C; Streeter, B; Hill, H R

    1994-01-01

    Three commercially available diagnostic assays for the detection of antibodies to varicella-zoster virus were evaluated to determine which would be the most suitable for our clinical laboratory. Three different methods were examined: an enzyme-linked immunosorbent assay (ELISA), latex agglutination (LA), and an indirect fluorescent-antibody technique. For the 141 serum specimens tested, the ELISA had an agreement of 90.1% and LA had an agreement of 92.2% with the indirect immunofluorescent-an...

  17. Improvement in the specificity of assays for detection of antibody to hepatitis B core antigen.

    OpenAIRE

    Weare, J A; Robertson, E F; Madsen, G; Hu, R; Decker, R H

    1991-01-01

    Reducing agents dramatically alter the specificity of competitive assays for antibody to hepatitis B core antigen (anti-HBc). A specificity improvement was demonstrated with a new assay which utilizes microparticle membrane capture and chemiluminescence detection as well as a current radioimmunoassay procedure (Corab: Abbott Laboratories, Abbott Park, Ill.). The effect was most noticeable with elevated negative and weakly reactive samples. In both systems, reductants increased separation of a...

  18. Microsphere Suspension Array Assays for Detection and Differentiation of Hendra and Nipah Viruses

    OpenAIRE

    Foord, Adam J.; John R. White; Axel Colling; Heine, Hans G.

    2013-01-01

    Microsphere suspension array systems enable the simultaneous fluorescent identification of multiple separate nucleotide targets in a single reaction. We have utilized commercially available oligo-tagged microspheres (Luminex MagPlex-TAG) to construct and evaluate multiplexed assays for the detection and differentiation of Hendra virus (HeV) and Nipah virus (NiV). Both these agents are bat-borne zoonotic paramyxoviruses of increasing concern for veterinary and human health. Assays were develop...

  19. Development of a consensus reverse transcription PCR assay for the specific detection of tortoise picornaviruses.

    Science.gov (United States)

    Marschang, Rachel E; Ihász, Katalin; Kugler, Renáta; Lengyel, György; Fehér, Enikő; Marton, Szilvia; Bányai, Krisztián; Aqrawi, Tara; Farkas, Szilvia L

    2016-05-01

    Picornaviruses (PVs) of different terrestrial tortoise species, previously designated as Virus "X," have been frequently detected from various tissues by virus isolation in Terrapene heart cell culture as the preferred laboratory method for diagnosis. Here, we describe the development of 2 diagnostic reverse transcription (RT)-PCR-based assays for the identification and characterization of tortoise PVs belonging to the tentative genus Topivirus To test the novel diagnostic systems, PVs were isolated from swab and tissue samples collected in Germany, Italy, and Hungary between 2000 and 2013. All 25 tested isolates gave positive results with both novel consensus primer sets. Sequencing of the amplified products confirmed that all studied viruses were members of the new proposed genus Topivirus Phylogenetic analyses clearly distinguished 2 lineages within the genus. Based on sequence analysis, no association was observed between the geographic distribution and genetic relatedness. Furthermore, no strict host specificity was indicated. The PCR-based diagnosis may provide a time-saving and sensitive method to detect tortoise PVs, and evaluation of PV presence in these animals may help control virus spread. PMID:27034342

  20. A simple colorimetric assay for specific detection of glutathione-S transferase activity associated with DDT resistance in mosquitoes.

    Directory of Open Access Journals (Sweden)

    Evangelia Morou

    Full Text Available BACKGROUND: Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations. METHODOLOGY/PRINCIPAL FINDINGS: We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically. CONCLUSIONS/SIGNIFICANCE: The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control.

  1. Development of fluorescence-based high-throughput screening assays: choice of appropriate instrumentation

    Science.gov (United States)

    Burns, David J.; Alder, Elisabeth; Fan, Yi-Hong; McKeegan, Evelyn; Warrior, Usha; Beutel, Bruce

    1998-04-01

    Fluorescence-based assays have become increasingly popular in high throughput screening for a variety of reasons (e.g. sensitivity). However, new screening technologies are pushing the limits of conventional fluorescence plate readers. For example, instruments that have optical sensitivities beyond most of the commercially available plate readers are required to reproducibly measure the fluorescence generated by the green fluorescent protein (GFP)--a novel reporter gene. Also, miniaturization of screening formats (with densities higher than the conventional 96-well plate) requires high resolution instrumentation to measure fluorescence. Several assays based on optical fluorescence measurements have been developed and screened in our Biological Screening group. These assays include various fluorescence-based protease assays (standard end-point and kinetic modes) and a functional cell-based screen using the green fluorescent protein as a reporter gene. The choice of instrumentation was the critical factor in the performance and success of each of these arrays. Data will be presented for the cell- based reporter assay including the type of instrumentation (fluorescence plate readers; fluorescence imaging systems) used for detection of GFP fluorescence.

  2. 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. PMID:25822163

  3. Detection of hypoxia in human brain tumor xenografts using a modified comet assay

    OpenAIRE

    Jingli Wang; Jack Klem; Wyrick, Jan B; Tomoko Ozawa; Erin Cunningham; Jay Golinveaux; Allen, Max J; Lamborn, Kathleen R.; Dennis F. Deen

    2003-01-01

    We used the standard comet assay successfully to generate in vitro dose-response curves under oxic and hypoxic conditions. We then made mixtures of cells that had been irradiated with 3 and 9 Gy of X-rays to simulate two subpopulations in a tumor, but efforts to accurately detect and quantify the subpopulations using the standard comet assay were unsuccessful. Therefore, we investigated a modified comet assay to determine whether it could be used for measuring hypoxia in our model systems. U2...

  4. Ultrasensitive isolation, identification and quantification of DNA-protein adducts by ELISA-based RADAR assay.

    Science.gov (United States)

    Kiianitsa, Kostantin; Maizels, Nancy

    2014-07-01

    Enzymes that form transient DNA-protein covalent complexes are targets for several potent classes of drugs used to treat infectious disease and cancer, making it important to establish robust and rapid procedures for analysis of these complexes. We report a method for isolation of DNA-protein adducts and their identification and quantification, using techniques compatible with high-throughput screening. This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA-protein covalent complexes in living cells. Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA-protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA-protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA-protein adducts by direct ELISA assay. The ELISA-based RADAR assay can detect Top1-DNA and Top2a-DNA adducts in human cells, and gyrase-DNA adducts in Escherichia coli. This approach will be useful for discovery and characterization of new drugs to treat infectious disease and cancer, and for development of companion diagnostics assays for individualized medicine. PMID:24914050

  5. Application of the DNA ''comet assay'' to detect irradiation treatment of foods

    International Nuclear Information System (INIS)

    Since treatment with ionising radiation causes DNA fragmentation, microgel electrophoresis of single cells (''comet assay'') offers a simple and rapid tool for identification of irradiated foods. The principle is based on migration of DNA in an agarose gel exposed to an electric field. Single cells or nuclei are embedded in the agarose and after lysis, intact DNA will virtually not move out of the cell upon electrophoresis, whereas if DNA has been fragmented, the fragments are able to migrate and ''comets'' following the cells become visible after staining. The advantages of this test, however, for foods not exposed to heat are its speed and simplicity, as the electrophoretic separation only requires a few minutes. DNA is visualised by silver staining, avoiding the need for a fluorescence microscope. Thus the method requires only relatively cheap equipment - in contrast to other methods for identification of irradiated food such as electron spin resonance (ESR) spectroscopy or gas chromatography/mass spectrometry (GC/MS). The DNA ''comet assay'' , therefore, seems suitable as a pre-screening test to detect whether food has been radiation processed. Suspected samples may subsequently be analysed by established, but more expensive techniques. (author)

  6. Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay.

    Science.gov (United States)

    Wolter, Justin M; Kotagama, Kasuen; Babb, Cody S; Mangone, Marco

    2015-01-01

    Luminescent Identification of Functional Elements in 3'UTRs (3'LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3'UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3'LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3'LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3'UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3'LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets. PMID:26066857

  7. Detecting ricin: sensitive luminescent assay for ricin A-chain ribosome depurination kinetics.

    Science.gov (United States)

    Sturm, Matthew B; Schramm, Vern L

    2009-04-15

    Ricin is a family member of the lethal ribosome-inactivating proteins (RIP) found in plants. Ricin toxin A-chain (RTA) from castor beans catalyzes the hydrolytic depurination of a single base from a GAGA tetraloop of eukaryotic rRNA to release a single adenine from the sarcin-ricin loop (SRL). Protein synthesis is inhibited by loss of the elongation factor binding site resulting in cell death. We report a sensitive coupled assay for the measurement of adenine released from ribosomes or small stem-loop RNAs by RTA catalysis. Adenine phosphoribosyl transferase (APRTase) and pyruvate orthophosphate dikinase (PPDK) convert adenine to ATP for quantitation by firefly luciferase. The resulting AMP is cycled to ATP to give sustained luminescence proportional to adenine concentration. Subpicomole adenine quantitation permits the action of RTA on eukaryotic ribosomes to be followed in continuous, high-throughput assays. Facile analysis of RIP catalytic activity will have applications in plant toxin detection, inhibitor screens, mechanistic analysis of depurinating agents on oligonucleotides and intact ribosomes, and in cancer immunochemotherapy. Kinetic analysis of the catalytic action of RTA on rabbit reticulocyte 80S ribosomes establishes a catalytic efficiency of 2.6 x 10(8) M(-1) s(-1), a diffusion limited reaction indicating catalytic perfection even with large reactants. PMID:19364139

  8. Gold nanoparticles mediated colorimetric assay for HIV-Tat protein detection

    Science.gov (United States)

    Hashwan, Saeed S. Ba; Ruslinda, A. Rahim; Fatin, M. F.; Gopinath, Subash C. B.; Thivina, V.; Tony, V. C. S.; Arshad, M. K. Md.; Hashim, U.

    2016-07-01

    Gold-nanoparticle (AuNP) based colorimetric assays have been formulated for different biomolecular interactions. With this assay the probe such as antibody immobilized on the Au surface and in the presence of appropriate binding partner (antigen), will interact with each other on the Au surface. By following this strategy, herein we formulated a detection system with two anti-HIV-Tat antibodies, Mono (McAb) - and polyclonal (PcAb) by immobilizing them independently with different AuNPs. Under this condition, these two antibodies are under dispersed condition, and in the presence of HIV-Tat antigen, these molecules will be connected and forms the aggregation of AuNPs. This strategy yield rapid results, can be monitored by the spectral changes in UV-Vis spectrophotometry. Experiments were performed with two different methods using two anti-HIV-Tats monoclonal and one Polyclonal antibody against the antigen HIV-Tat. Between these methods conjugation of HIV-Tat and McAb on the AuNP followed by addition of PcAb yielded better results.

  9. Evaluation of Diagnos Malaria Stix test (antigen detection assay) for diagnosis of malaria.

    Science.gov (United States)

    Khan, Haris M; Shujatullah, Fatima; Shahid, M; Raza, Adil; Malik, Ritu

    2010-06-01

    Malaria is one of the most common parasitic infection in India. The diagnosis largely depends on peripheral blood smear examination. Newer diagnostic methods like various antigen detection assays are now in use for prompt diagnosis and treatment. This study was done to determine the effectiveness of Diagnos Malaria Stix (antigen detection) assay in diagnosis of malaria. This involves detection of PfHRP-2 antigen and P.V. specific pLDH antigen. 162 patients with signs and symptoms of malaria included in the study. Leishman stained blood smear examination was done for all patients. Commercially available Diagnos Malaria Stix assay was used. Diagnos Malaria Stix showed sensitivity, specificity positive and negative predictive values of 100% each while Sensitivity, specificity, positive and negative predictive values of Leishman stained blood smear examination were 45.45%, 100%, 100% and 92% respectively. PMID:22471175

  10. Acoustic detection of DNA conformation in genetic assays combined with PCR.

    Science.gov (United States)

    Papadakis, G; Tsortos, A; Kordas, A; Tiniakou, I; Morou, E; Vontas, J; Kardassis, D; Gizeli, E

    2013-01-01

    Application of PCR to multiplexing assays is not trivial; it requires multiple fluorescent labels for amplicon detection and sophisticated software for data interpretation. Alternative PCR-free methods exploiting new concepts in nanotechnology exhibit high sensitivities but require multiple labeling and/or amplification steps. Here, we propose to simplify the problem of simultaneous analysis of multiple targets in genetic assays by detecting directly the conformation, rather than mass, of target amplicons produced in the same PCR reaction. The new methodology exploits acoustic wave devices which are shown to be able to characterize in a fully quantitative manner multiple double stranded DNAs of various lengths. The generic nature of the combined acoustic/PCR platform is shown using real samples and, specifically, during the detection of SNP genotyping in Anopheles gambiae and gene expression quantification in treated mice. The method possesses significant advantages to TaqMan assay and real-time PCR regarding multiplexing capability, speed, simplicity and cost. PMID:23778520

  11. Sequence diversity within the HA-1 gene as detected by melting temperature assay without oligonucleotide probes

    Directory of Open Access Journals (Sweden)

    Mattiuz Pier

    2005-10-01

    Full Text Available Abstract Background The minor histocompatibility antigens (mHags are self-peptides derived from common cellular proteins and presented by MHC class I and II molecules. Disparities in mHags are a potential risk for the development of graft-versus-host disease (GvHD in the recipients of bone marrow from HLA-identical donors. Two alleles have been identified in the mHag HA-1. The correlation between mismatches of the mHag HA-1 and GvHD has been suggested and methods to facilitate large-scale testing were afterwards developed. Methods We used sequence specific primer (SSP PCR and direct sequencing to detect HA-1 gene polymorphisms in a sample of 131 unrelated Italian subjects. We then set up a novel melting temperature (Tm assay that may help identification of HA-1 alleles without oligonucleotide probes. Results We report the frequencies of HA-1 alleles in the Italian population and the presence of an intronic 5 base-pair deletion associated with the immunogeneic allele HA-1H. We also detected novel variable sites with respect to the consensus sequence of HA-1 locus. Even though recombination/gene conversion events are documented, there is considerable linkage disequilibrium in the data. The gametic associations between HA-1R/H alleles and the intronic 5-bp ins/del polymorphism prompted us to try the Tm analysis with SYBR® Green I. We show that the addition of dimethylsulfoxide (DMSO during the assay yields distinct patterns when amplicons from HA-1H homozygotes, HA-1R homozygotes, and heterozygotes are analysed. Conclusion The possibility to use SYBR® Green I to detect Tm differences between allelic variants is attractive but requires great caution. We succeeded in allele discrimination of the HA-1 locus using a relatively short (101 bp amplicon, only in the presence of DMSO. We believe that, at least in certain assets, Tm assays may benefit by the addition of DMSO or other agents affecting DNA strand conformation and stability.

  12. Integration of agglutination assay for protein detection in microfluidic disc using Blu-ray optical pickup unit and optical fluid scanning

    DEFF Research Database (Denmark)

    Uddin, Rokon; Burger, Robert; Donolato, Marco;

    2015-01-01

    We present a novel strategy for thrombin detection by combining a magnetic bead based agglutination assay and low-cost microfluidic disc. The detection method is based on an optomagnetic readout system implemented using a Blu-ray optical pickup unit (OPU) as main optoelectronic component. The ass...

  13. A novel SERRS sandwich-hybridization assay to detect specific DNA target.

    Directory of Open Access Journals (Sweden)

    Cécile Feuillie

    Full Text Available In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR. In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.

  14. Rapid and Sensitive Detection of PRRSV by a Reverse Transcription-Loop-mediated Isothermal Amplification Assay

    Institute of Scientific and Technical Information of China (English)

    Lei Zhang; Ye-bing Liu; Lei Chen; Jian-huan Wang; Yi-bao Ning

    2011-01-01

    A real-time monitoring reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of prototypic,prevalent North American porcine reproductive and respiratory syndrome virus (PRRSV)strains.As a higher sensitivity and specificity method than reverse transcription polymerase chain reaction (RT-PCR),the RT-LAMP method only used a turbidimeter,exhibited a detection limit corresponding to a 10-4 dilution of template RNA extracted from 250 μL of 105 of the 50% tissue culture infective dose (TCID50) of PRRSV containing cells,and no cross-reactivity was observed with other related viruses including porcine circovirus type 2,swine influenza virus,porcine rotavirus and classical swine fever virus.From forty-two field samples,33 samples in the RT-LAMP assay was detected positive,whereas three of which were not detected by RT-PCR.Furthermore,in 33 strains of PRRSV,an identical detection rate was observed with the RT-LAMP assay to what were isolated using porcine alveolar macrophages.These findings demonstrated that the RT-LAMP assay has potential clinical applications for the detection of highly pathogenic PRRSV isolates,especially in developing countries.

  15. Detection of Brugia malayi in laboratory and wild-caught Mansonioides mosquitoes (Diptera: Culicidae) using Hha I PCR assay.

    Science.gov (United States)

    Hoti, S L; Vasuki, V; Lizotte, M W; Patra, K P; Ravi, G; Vanamail, P; Manonmani, A; Sabesan, S; Krishnamoorthy, K; Williams, S A

    2001-04-01

    An Hha 1 based polymerase chain reaction (PCR) assay developed for the detection of Brugia malayi, the causative agent of Brugian lymphatic filariasis, was evaluated for its sensitivity in the laboratory and for its usefulness in measuring changes in transmission of the disease in the field. Laboratory studies showed that the new assay was highly sensitive in comparison with the standard dissection and microscopy technique. The assay can detect as little as 4 pg of parasite DNA or a single microfilaria in pools of up to 100 mosquitoes. The optimum pool size for convenience was found to be 50 mosquitoes per pool. The efficacy of PCR assay was evaluated in filariasis control programmes in operation in endemic areas of Kerala State, South India. The infection rates obtained by the Hha I PCR assay and the conventional dissection and microscopy technique were 1.2% and 1.7% respectively in operational areas and 8.3% and 4.4% respectively, in check areas, which were not significantly different (P used as a new epidemiological tool for assessing parasite infection in field-collected mosquitoes. PMID:11260722

  16. Novel Phosphorylation Assay Based on Multi-Functionalized Soluble Nanopolymer

    OpenAIRE

    Iliuk, Anton; Martinez, Juan S.; Hall, Mark C.; Tao, W. Andy

    2011-01-01

    Quantitative phosphorylation analysis is essential to understanding cellular signal transductions. Here we present a novel technology for the highly efficient assay of protein phosphorylation in high throughput format without the use of phospho-specific antibodies. The technique is based on a water-soluble, nanosize polymer, termed pIMAGO that is multi-functionalized with titanium (IV)ions for specific binding to phosphoproteins, and with biotin groups that allow for enzyme-linked spectrometr...

  17. Development of a versatile organophosphorous-hydrolase-based assay for organophosphate pesticides

    Science.gov (United States)

    Rogers, Kim R.; Wang, Yi; Mulchandani, Ashok; Mulchandani, P.; Chen, Wilfred

    1999-02-01

    We report a rapid and versatile organophosphorus hydrolase (OPH)-based method for measurement of organophosphate pesticides. This assay is based on a substrate-dependant change in pH near the active site of the enzyme. The pH change is monitored using fluorescein isothiocyanate (FITC) which is covalently immobilized to the enzyme. This method employs FITC-labeled enzyme adsorbed to polymethylmethacrylate beads. Analytes were measured using a microbead fluorescence analyzer. The dynamic concentration range for the assay extends from 25 (mu) M to 400 (mu) M for paraoxon with a detection limit of 8 (mu) M. This assay compared favorably to an HPLC method for monitoring the concentration of coumaphos in bioremediation filtrate samples.

  18. Glycoprotein-based enzyme-linked immunosorbent assays for serodiagnosis of infectious laryngotracheitis.

    Science.gov (United States)

    Kanabagatte Basavarajappa, Mallikarjuna; Song, Haichen; Lamichhane, Chinta; Samal, Siba K

    2015-05-01

    For detection of infectious laryngotracheitis virus (ILTV) antibody, glycoprotein B-, C-, and D-based enzyme-linked immunosorbent assays (B-, C-, and D-ELISAs, respectively) were developed. The B- and D-ELISAs showed enhanced detection of anti-ILTV antibodies in infected chickens compared to that of the commercial ELISA. Furthermore, the D-ELISA was efficient in detecting seroconversion with vectored vaccine, using recombinant Newcastle disease virus (rNDV) expressing glycoprotein D (gD) as the vaccine vector. PMID:25694519

  19. Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay

    International Nuclear Information System (INIS)

    Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg-1 almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg-1. We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg-1 almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg-1. Further, between 100 and 100,000 mg kg-1 spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n = 5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and potentially

  20. Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay

    Energy Technology Data Exchange (ETDEWEB)

    Roeder, Martin; Vieths, Stefan [Division of Allergology, Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, 63225 Langen (Germany); Holzhauser, Thomas, E-mail: holth@pei.de [Division of Allergology, Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, 63225 Langen (Germany)

    2011-01-24

    Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg{sup -1} almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg{sup -1}. We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg{sup -1} almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg{sup -1}. Further, between 100 and 100,000 mg kg{sup -1} spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n = 5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a

  1. 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. PMID:25553930

  2. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.

    Science.gov (United States)

    Wu, Yuling; Li, Jia J; Kim, Hyun Jun; Liu, Xu; Liu, Weiyi; Akhgar, Ahmad; Bowen, Michael A; Spitz, Susan; Jiang, Xu-Rong; Roskos, Lorin K; White, Wendy I

    2015-11-01

    Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function. PMID:26205082

  3. Performance of Simplexa Dengue Molecular Assay Compared to Conventional and SYBR Green RT-PCR for Detection of Dengue Infection in Indonesia

    OpenAIRE

    R Tedjo Sasmono; Aryati Aryati; Puspa Wardhani; Benediktus Yohan; Hidayat Trimarsanto; Sukmal Fahri; Setianingsih, Tri Y.; Febrina Meutiawati

    2014-01-01

    Diagnostic tests based on detection of dengue virus (DENV) genome are available with varying sensitivities and specificities. The Simplexa Dengue assay (Focus Diagnostics) is a newly developed real-time RT-PCR method designed to detect and serotype DENV simultaneously. To assess the performance of the Simplexa Dengue assay, we performed comparison with conventional RT-PCR and SYBR Green real-time RT-PCR on patients sera isolated from eight cities across Indonesia, a dengue endemic country. A ...

  4. A real-time PCR assay for the detection of atypical strains of Chlamydiaceae from pigeons.

    Directory of Open Access Journals (Sweden)

    Aleksandar Zocevic

    Full Text Available Recent evidence of the occurrence of atypical Chlamydiaceae strains in pigeons, different from the established Chlamydiaceae, requires the development of a specific and rapid detection tool to investigate their prevalence and significance. Here is described a new real-time PCR assay that allows specific detection of atypical Chlamydiaceae from pigeons. The assay has been used to assess the dissemination of these strains in field samples collected from Parisian pigeon populations in 2009. The results suggest a limited dissemination compared to the usually higher prevalence of Chlamydia psittaci that is the main species associated with avian chlamydiosis.

  5. Detection of antibodies and antigens of human parvovirus B19 by enzyme-linked immunosorbent assay.

    OpenAIRE

    Anderson, L J; Tsou, C; Parker, R. A.; Chorba, T L; Wulff, H; Tattersall, P; Mortimer, P P

    1986-01-01

    Acute-phase serum from a patient with aplastic crisis provided sufficient human parvovirus B19 to make a monoclonal antibody against B19 and to develop antigen and immunoglobulin M (IgM) and IgG antibody detection enzyme-linked immunosorbent assays (ELISAs). The indirect capture antibody method was used for all three assays. Antigen was detected in 8 of 29 sera drawn within 2 days of onset of illness from patients with aplastic crisis. These sera had high titers of virus by electron microscop...

  6. First field trial of an immunoradiometric assay for the detection of malaria sporozoites in mosquitoes

    International Nuclear Information System (INIS)

    An immunoradiometric assay (IRMA) using a monoclonal antibody to the major surface protein of Plasmodium falciparum sporozoites was used to assess the P. falciparum sporozoite rate in a West African population of Anopheles gambiae (s.1.). Unlike current dissection techniques, the IRMA could detect sporozoite antigen in dried as well as fresh mosquitoes. In a controlled comparison, the sensitivity of the IRMA was comparable that of the dissection technique. Additionally, the IRMA was species specific and quantitative. Sensitivity of the assay was sufficient to detect sporozoite infections resulting from the development of a single oocyst

  7. Panfungal PCR Assay for Detection of Fungal Infection in Human Blood Specimens

    OpenAIRE

    Van Burik, Jo-Anne; Myerson, David; Schreckhise, Randall W.; Bowden, Raleigh A.

    1998-01-01

    A novel panfungal PCR assay which detects the small-subunit rRNA gene sequence of the two major fungal organism groups was used to test whole-blood specimens obtained from a series of blood or bone marrow transplant recipients. The 580-bp PCR product was identified after amplification by panfungal primers and hybridization to a 245-bp digoxigenin-labeled probe. The lower limit of detection of the assay was approximately four organisms per milliliter of blood. Multiple whole-blood specimens fr...

  8. Capture-S, a nontreponemal solid-phase erythrocyte adherence assay for serological detection of syphilis.

    OpenAIRE

    Stone, D L; Moheng, M C; Rolih, S; Sinor, L T

    1997-01-01

    A solid-phase erythrocyte adherence assay has been developed for the serological detection of reagin antibodies in syphilis. Capture-S (Immucor, Inc., Norcross, Ga.) is a nontreponemal, qualitative screening test for the detection of immunoglobulin G (IgG) and IgM antilipid antibodies in serum or plasma samples from blood donors. The Capture-S assay utilizes a modified Venereal Disease Research Laboratory antigen bound to microtitration wells and anti-IgG- plus anti-IgM-coated indicator eryth...

  9. Sensitive microplate assay for the detection of proteolytic enzymes using radiolabeled gelatin

    International Nuclear Information System (INIS)

    A sensitive, microplate assay is described for the detection of a wide range of proteolytic enzymes, using radio-iodine-labeled gelatin as substrate. The technique uses the Bolton-Hunter reagent to label the substrate, which is then coated onto the wells of polyvinyl chloride microtiter plates. By measuring the radioactivity released the assay is able to detect elastase, trypsin, and collagenase in concentrations of 1 ng/ml or less, while the microtiter format permits multiple sample handling and minimizes sample volumes required for analysis

  10. Comparison of three multiplex PCR assays for the detection of respiratory viral infections: evaluation of xTAG respiratory virus panel fast assay, RespiFinder 19 assay and RespiFinder SMART 22 assay

    Directory of Open Access Journals (Sweden)

    Dabisch-Ruthe Mareike

    2012-07-01

    Full Text Available Abstract Background A broad spectrum of pathogens is causative for respiratory tract infections, but symptoms are mostly similar. Therefore, the identification of the causative viruses and bacteria is only feasible using multiplex PCR or several monoplex PCR tests in parallel. Methods The analytical sensitivity of three multiplex PCR assays, RespiFinder-19, RespiFinder-SMART-22 and xTAG-Respiratory-Virus-Panel-Fast-Assay (RVP, were compared to monoplex real-time PCR with quantified standardized control material. All assays include the most common respiratory pathogens. Results To compare the analytical sensitivity of the multiplex assays, samples were inoculated with 13 different quantified viruses in the range of 101 to 105 copies/ml. Concordant results were received for rhinovirus, whereas the RVP detected influenzavirus, RSV and hMPV more frequently in low concentrations. The RespiFinder-19 and the RespiFinder-SMART-22 showed a higher analytical sensitivity for adenoviruses and coronaviruses, whereas the RVP was incapable to detect adenovirus and coronavirus in concentrations of 104 copies/ml. The RespiFinder-19 and RespiFinder-SMART-22A did not detect influenzaviruses (104 copies/ml and RSV (103 copies/ml. The detection of all 13 viruses in one sample was only achieved using monoplex PCR. To analyze possible competitive amplification reactions between the different viruses, samples were further inoculated with only 4 different viruses in one sample. Compared to the detection of 13 viruses in parallel, only a few differences were found. The incidence of respiratory viruses was compared in tracheal secretion (TS samples (n = 100 of mechanically ventilated patients in winter (n = 50 and summer (n = 50. In winter, respiratory viruses were detected in 32 TS samples (64% by RespiFinder-19, whereas the detection rate with RVP was only 22%. The most frequent viruses were adenovirus (32% and PIV-2 (20%. Multiple infections were detected

  11. Comparison of the Vitek Immunodiagnostic Assay System with Three Immunoassay Systems for Detection of Cytomegalovirus-Specific Immunoglobulin G

    OpenAIRE

    Hopson, D K; Niles, A C; Murray, P R

    1993-01-01

    The Vitek Immunodiagnostic Assay System (VIDAS; bioMerieux Vitek Inc., Hazelwood, Mo.) was evaluated for its ability to detect anticytomegalovirus immunoglobulin G (IgG) and was compared with the following assay systems: Abbott IMx Cytomegalovirus IgG antibody assay (Abbott Laboratories, Abbott Park, Ill.), Whittaker Cytomegelisa II IgG assay (Whittaker Bioproducts, Walkersville, Md.), and Whittaker FIAX Cytomegalovirus IgG assay (Whittaker Bioproducts). Samples were considered positive if at...

  12. Variability in assays used for detection of lentiviral infection in bobcats (Lynx rufus), pumas (Puma concolor), and ocelots (Leopardus pardalis)

    Science.gov (United States)

    Franklin, S.P.; Troyer, J.L.; TerWee, J.A.; Lyren, L.M.; Kays, R.W.; Riley, S.P.D.; Boyce, W.M.; Crooks, K.R.; VandeWoude, S.

    2007-01-01

    Although lentiviruses similar to feline immunodeficiency virus (FIV) are known to infect numerous felid species, the relative utility of assays used for detecting lentiviral infection has not been compared for many of these hosts. We tested bobcats (Lynx rufus), pumas (Felis concolor), and ocelots (Leopardus pardalis) for exposure to lentivirus using five different assays: puma lentivirus (PLV), African lion lentivirus (LLV), and domestic cat FIV-based immunoblots, a commercially available enzyme-linked immunosorbent assay (ELISA) kit, and nested polymerase chain reaction (PCR). Puma lentivirus immunoblots identified more seropositive individuals than the other antibody-detection assays. The commercial ELISA provided a fair ability to recognize seropositive samples when compared with PLV immunoblot for screening bobcats and ocelots, but not pumas. Polymerase chain reaction identified fewer positive samples than PLV immunoblot for all three species. Immunoblot results were equivalent whether the sample tested was serum, plasma, or whole blood. The results from this study and previous investigations suggest that the PLV immunoblot has the greatest ability to detect reactive samples when screening wild felids of North America and is unlikely to produce false positive results. However, the commercial ELISA kit may provide ap adequate alternative for screening of some species and is more easily adapted to field conditions. ?? Wildlife Disease Association 2007.

  13. Variability in assays used for detection of lentiviral infection in bobcats (Lynx rufus), pumas (Puma concolor), and ocelots (Leopardus pardalis).

    Science.gov (United States)

    Franklin, Samuel P; Troyer, Jennifer L; Terwee, Julie A; Lyren, Lisa M; Kays, Roland W; Riley, Seth P D; Boyce, Walter M; Crooks, Kevin R; Vandewoude, Sue

    2007-10-01

    Although lentiviruses similar to feline immunodeficiency virus (FIV) are known to infect numerous felid species, the relative utility of assays used for detecting lentiviral infection has not been compared for many of these hosts. We tested bobcats (Lynx rufus), pumas (Felis concolor), and ocelots (Leopardus pardalis) for exposure to lentivirus using five different assays: puma lentivirus (PLV), African lion lentivirus (LLV), and domestic cat FIV-based immunoblots, a commercially available enzyme-linked immunosorbent assay (ELISA) kit, and nested polymerase chain reaction (PCR). Puma lentivirus immunoblots identified more seropositive individuals than the other antibody-detection assays. The commercial ELISA provided a fair ability to recognize seropositive samples when compared with PLV immunoblot for screening bobcats and ocelots, but not pumas. Polymerase chain reaction identified fewer positive samples than PLV immunoblot for all three species. Immunoblot results were equivalent whether the sample tested was serum, plasma, or whole blood. The results from this study and previous investigations suggest that the PLV immunoblot has the greatest ability to detect reactive samples when screening wild felids of North America and is unlikely to produce false positive results. However, the commercial ELISA kit may provide an adequate alternative for screening of some species and is more easily adapted to field conditions. PMID:17984266

  14. Comparison of Onclarity Human Papillomavirus (HPV) Assay with Hybrid Capture II HPV DNA Assay for Detection of Cervical Intraepithelial Neoplasia Grade 2 and 3 Lesions

    DEFF Research Database (Denmark)

    Bottari, F; Sideri, M; Gulmini, C; Igidbashian, S; Tricca, A; Casadio, C; Carinelli, S; Boveri, S; Ejegod, D; Bonde, J; Sandri, M T

    2015-01-01

    Analytical and clinical performance validation is essential before introduction of a new human papillomavirus (HPV) assay into clinical practice. This study compares the new BD Onclarity HPV assay, which detects E6/E7 DNA from 14 high-risk HPV types, to the Hybrid Capture II (HC2) HPV DNA test, t...

  15. Comparison of heparin and EDTA transport tubes for detection of cytomegalovirus in leukocytes by shell vial assay, pp65 antigenemia assay, and PCR.

    OpenAIRE

    Storch, G A; Gaudreault-Keener, M; Welby, P C

    1994-01-01

    The anticoagulants heparin and EDTA were compared for inhibitory effects on the detection of cytomegalovirus from washed leukocytes in specimen transport tubes. Evaluation was made by the centrifugation/shell vial culture technique, the pp65 antigenemia assay, and PCR. For each assay, the results with heparin and EDTA were equivalent.

  16. A Two-Tube Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Sixteen Human Respiratory Virus Types/Subtypes

    OpenAIRE

    2013-01-01

    There is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens. With an intended application in provincial Centers for Diseases Control and Prevention, in this study, we present a two-tube multiplex RT-PCR assay (two-tube assay) using automatic electrophoresis to simultaneously detect sixteen common respiratory viruses. The specificity and the sensitivity of the assay were tested. The assay could detect 20–200 copies per reaction when each viral type was...

  17. Bluetongue virus: comparative evaluation of enzyme-linked immunosorbent assay, immunodiffusion, and serum neutralization for detection of viral antibodies.

    OpenAIRE

    Poli, G.; Stott, J.; Liu, Y. S.; Manning, J S

    1982-01-01

    Comparative studies on the detection of bovine serum immunoglobulin G antibodies to bluetongue virus with an enzyme-linked immunosorbent assay, an immunodiffusion method, and a serum neutralization assay demonstrated complete concordance between the enzyme-linked immunosorbent assay and the serum neutralization assay results. However, the immunodiffusion method failed to detect bluetongue virus antibody in a substantial number of sera found to possess bluetongue virus immunoglobulin G with th...

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

    Directory of Open Access Journals (Sweden)

    King Ting Lim

    2013-01-01

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

  19. An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi

    OpenAIRE

    Kellermann, Gottfried H.; Lehmann, Paul V; Diana R. Roen; Chenggang Jin

    2013-01-01

    Lyme Borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi that is transmitted through the bite of infected ticks. Both B cell-mediated humoral immunity and T cell immunity develop during natural Borrelia infection. However, compared with humoral immunity, the T cell response to Borrelia infection has not been well elucidated. In this study, a novel T cell-based assay was developed and validated for the sensitive detection of antigen-specific T cell response to B....

  20. 12C6+ ion beam induced DNA damage in human hepatocyte L02 cells detected by comet assay

    International Nuclear Information System (INIS)

    Human hepatocyte L02 cells were irradiated by the carbon ion beam with LET of 30 keV/μm and DNA strand breaks were detected immediately after the irradiation using comet assay. Based on the comet images, all the indexes of comet assay including head DNA%, tail DNA%, comet length, tail length, tail moment and olive tail moment were analyzed with CASP and SPSS 11.5 code. Statistically significant dose-effect relationships could be observed in all the indexes of comet assay and TM increased with increasing the radiation dose. These experimental results suggest that carbon ion beam with intermediate LET value would cause remarkable DNA strand breaks immediately and the damage increases in a dose-dependent manner. This work provides basic data and evidence for the risk assessment of heavy ion radiation to healthy tissue. (authors)

  1. High resolution melting curve assay for rapid detection of drug-resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Nagai, Yuhki; Iwade, Yoshito; Hayakawa, Eri; Nakano, Manabu; Sakai, Takashi; Mitarai, Satoshi; Katayama, Masahiko; Nosaka, Tetsuya; Yamaguchi, Tetsuo

    2013-12-01

    We developed and evaluated a high resolution melting (HRM) curve assay by using real-time PCR for the detection of the most frequent mutations of Mycobacterium tuberculosis, which are responsible for the resistance of four anti-TB drugs: rifampicin, isoniazid, ethambutol, and streptomycin. The HRM assay was successfully used for the detection of dominant mutations: A516V, H526A, H526T, S531L, L533P, and A516G/S531L in rpoB; S315T, and S315A in katG; -15C/T, and -8T/C in mab-inhA; M306I in embB; K88Q and K43R in rpsL; and 513A/C in rrs. We were able to discriminate the mutant from the wild type by analyzing the melting-curve shape in 40 clinical M. tuberculosis isolates, and the results of the HRM assay were completely consistent with those of DNA sequencing. This HRM assay is a simple, rapid, and cost-effective method that can be performed in a closed tube. Therefore, our assay is a potentially useful tool for the rapid detection of drug-resistant M. tuberculosis. PMID:23793795

  2. Enzyme assays using sensor arrays based on ion-selective carbon nanotube field-effect transistors.

    Science.gov (United States)

    Melzer, K; Bhatt, V Deep; Jaworska, E; Mittermeier, R; Maksymiuk, K; Michalska, A; Lugli, P

    2016-10-15

    In the fields of clinical diagnostics and point-of-care diagnosis as well as food and environmental monitoring there is a high demand for reliable high-throughput, rapid and highly sensitive assays for a simultaneous detection of several analytes in complex and low-volume samples. Sensor platforms based on solution-processable electrolyte-gated carbon nanotube field-effect transistors (CNT-FETs) are a simple and cost-effective alternative for conventional assays. In this work we demonstrate a selective as well as direct detection of the products of an enzyme-substrate interaction, here the for metabolic processes important urea-urease system, with sensors based on spray-coated CNT-FETs. The selective and direct detection is achieved by immobilizing the enzyme urease via certain surface functionalization techniques on the sensor surface and further modifying the active interfaces with polymeric ion-selective membranes as well as pH-sensitive layers. Thereby, we can avoid the generally applied approach for a field-effect based detection of enzyme reactions via detecting changes in the pH value due to an on-going enzymatic reaction and directly detect selectively the products of the enzymatic conversion. Thus, we can realize a buffering-capacity independent monitoring of changes in the substrate concentration. PMID:27140308

  3. 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

  4. Fluorescence-quenching-based homogeneous caspase-3 activity assay using photon upconversion

    Energy Technology Data Exchange (ETDEWEB)

    Vuojola, Johanna, E-mail: johanna.vuojola@utu.fi [Department of Biotechnology, University of Turku, Tykistoekatu 6A, FI-20520 Turku (Finland); Riuttamaeki, Terhi; Kulta, Essi; Arppe, Riikka; Soukka, Tero [Department of Biotechnology, University of Turku, Tykistoekatu 6A, FI-20520 Turku (Finland)

    2012-05-06

    Highlights: Black-Right-Pointing-Pointer We demonstrate the use of photon upconversion in a caspase-3 activity assay. Black-Right-Pointing-Pointer The separation-free assay uses an internally quenched substrate peptide. Black-Right-Pointing-Pointer UCPs enable simple instrumentation and total elimination of autofluorescence. Black-Right-Pointing-Pointer A sensitive assay with high signal-to-background ratios was achieved. Black-Right-Pointing-Pointer Suitable for high-throughput screening through miniaturization and white plates. - Abstract: Caspase proteases are key mediators in apoptosis and thus of great interest in pharmaceutical industry. Enzyme-activity assays are commonly employed in the screening of protease inhibitors that are potential drug candidates. Conventional homogeneous fluorescence-based assays are susceptible to autofluorescence originating from biological material. This background autofluorescence can be eliminated by using upconverting phosphors (UCPs) that emit visible light upon excitation at near-infrared. In the assay energy was transferred from a UCP-donor to a conventional fluorophore acceptor that resided at one end of a caspase-3-specific substrate peptide. Attached to the other end was a quencher molecule that was used to attenuate the acceptor emission through intramolecular energy transfer in an intact peptide. In non-inhibitory conditions the enzyme reaction separated the fluorophore from the quencher and the emission of the fluorophore was recovered. The method was applied for the detection and characterization of a known caspase-3 inhibitor Z-DEVD-FMK, and the assay gave IC{sub 50} values of approximately 13 nM for this inhibitor. We have demonstrated the applicability of UCPs on a fluorescence-quenching-based homogeneous enzyme-activity assay for the detection of caspase-3 inhibitors. The use of near-infrared excitable UCPs enables inexpensive instrumentation and total elimination of autofluorescence, while the use of an

  5. Development of a molecular assay to detect predation on Anopheles gambiae complex larval stages.

    Science.gov (United States)

    Schielke, Erika; Costantini, Carlo; Carchini, Gianmaria; Sagnon, N'falé; Powell, Jeffrey; Caccone, Adalgisa

    2007-09-01

    We developed a molecular assay to detect predation on Anopheles gambiae sensu lato (s.l.) mosquitoes. This intergenic spacer ribosomal DNA polymerase chain reaction assay and restriction enzyme analysis uses An. gambiae-specific primers to detect mosquito DNA in the DNA extracts from whole invertebrate predators, which enables identification of species (An. gambiae s.s. versus An. arabiensis) and molecular forms (M versus S in An. gambiae s.s.). We show that An. gambiae s.l. DNA can be detected after ingestion by members of the families Lestidae (order Odonata) after four hours, Libellulidae (order Odonata) after six hours, and Notonectidae (order Hemiptera) after 24 hours. This method is an improvement over previously published methods because of ease of execution and increased time of detection after ingestion. PMID:17827361

  6. Detection and identification of entamoeba species in stool samples by a reverse line hybridization assay.

    Science.gov (United States)

    Verweij, Jaco J; Laeijendecker, Daphne; Brienen, Eric A T; van Lieshout, Lisette; Polderman, Anton M

    2003-11-01

    Classically, detection of Entamoeba histolytica is performed by microscopic examination for characteristic cysts and/or trophozoites in fecal preparations. Differentiation of E. histolytica cysts and those of nonpathogenic amoebic species is made on the basis of the appearance and the size of the cysts. However, by classical means objective tools for confirmation and quality control do not exist. Therefore, a reverse line blot hybridization assay was developed to detect a variety of Entamoeba species and genetic variants known to infect humans. The assay was performed after amplification with general Entamoeba-specific primers. The assay could identify four genetic variants of Entamoeba polecki-like cysts as well as E. histolytica, Entamoeba dispar, Entamoeba hartmanni, Entamoeba moshkovskii and Entamoeba coli and even mixed infections in a range of controls and fecal samples. This technique can be used as an additional standard for diagnosis, epidemiology, and quality control for amoebic infections. PMID:14605136

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

    Science.gov (United States)

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

    2016-01-01

    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. PMID:27609471

  8. The application of reporter gene assays for the detection of endocrine disruptors in sport supplements

    International Nuclear Information System (INIS)

    The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC50 of 0.01 ng mL-1 and 0.16 ng mL-1 respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC-MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC-MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally active compounds the

  9. The application of reporter gene assays for the detection of endocrine disruptors in sport supplements

    Energy Technology Data Exchange (ETDEWEB)

    Plotan, Monika; Elliott, Christopher T. [Institute of Agri-Food and Land Use, School of Biological Sciences, Queen' s University Belfast, Belfast BT95AG, Northern Ireland (United Kingdom); Scippo, Marie Louise [Department of Food Sciences, University of Liege, 4000 Liege (Belgium); Muller, Marc [Molecular Biology and Genetic Engineering GIGA-R, University of Liege, 4000 Liege (Belgium); Antignac, Jean-Philippe [LABERCA, ENVN, USC INRA 2013, BP 50707, 44 307, Nantes (France); Malone, Edward [The State Laboratory, Young' s Cross, Celbridge, Co. Kildare (Ireland); Bovee, Toine F.H. [RIKILT Institute of Food Safety, P.O. Box 230, AE Wageningen 6700 (Netherlands); Mitchell, Samuel [Agri-Food and Biosciences Institute, Belfast BT9 5PX (United Kingdom); Connolly, Lisa, E-mail: l.connolly@qub.ac.uk [Institute of Agri-Food and Land Use, School of Biological Sciences, Queen' s University Belfast, Belfast BT95AG, Northern Ireland (United Kingdom)

    2011-08-26

    The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC{sub 50} of 0.01 ng mL{sup -1} and 0.16 ng mL{sup -1} respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC-MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC-MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally

  10. A species-specific polymerase chain reaction assay for rapid and sensitive detection of Colletotrichum capsici.

    Science.gov (United States)

    Torres-Calzada, C; Tapia-Tussell, R; Quijano-Ramayo, A; Martin-Mex, R; Rojas-Herrera, R; Higuera-Ciapara, I; Perez-Brito, D

    2011-09-01

    Colletotrichum capsici is an important fungal species that causes anthracnose in many genera of plants causing severe economic losses worldwide. A primer set was designed based on the sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a conventional PCR assay. The primer set (CcapF/CcapR) amplified a single product of 394 bp with DNA extracted from 20 Mexican isolates of C. capsici. The specificity of primers was confirmed by the absence of amplified product with DNA of four other Colletotrichum species and eleven different fungal genera. This primer set is capable of amplifying only C. capsici from different contaminated tissues or fungal structures, thereby facilitating rapid diagnoses as there is no need to isolate and cultivate the fungus in order to identify it. The sensitivity of detection with this PCR method was 10 pg of genomic DNA from the pathogen. This is the first report of a C. capsici-specific primer set. It allows rapid pathogen detection and provides growers with a powerful tool for a rational selection of fungicides to control anthracnose in different crops and in the post-harvest stage. PMID:21253896

  11. Detection and Identification of Entamoeba Species in Stool Samples by a Reverse Line Hybridization Assay

    OpenAIRE

    Verweij, Jaco J.; Laeijendecker, Daphne; Brienen, Eric A. T.; van Lieshout, Lisette; Polderman, Anton M.

    2003-01-01

    Classically, detection of Entamoeba histolytica is performed by microscopic examination for characteristic cysts and/or trophozoites in fecal preparations. Differentiation of E. histolytica cysts and those of nonpathogenic amoebic species is made on the basis of the appearance and the size of the cysts. However, by classical means objective tools for confirmation and quality control do not exist. Therefore, a reverse line blot hybridization assay was developed to detect a variety of Entamoeba...

  12. Specific Dot-Immunobinding Assay for Detection and Enumeration of Thiobacillus ferrooxidans

    OpenAIRE

    Arredondo, Renato; Jerez, Carlos A.

    1989-01-01

    A specific and very sensitive dot-immunobinding assay for the detection and enumeration of the bioleaching microorganism Thiobacillus ferrooxidans was developed. Nitrocellulose spotted with samples was incubated with polyclonal antisera against whole T. ferrooxidans cells and then in 125I-labeled protein A or 125I-labeled goat antirabbit immunoglobulin G; incubation was followed by autoradiography. Since a minimum of 103 cells per dot could be detected, the method offers the possibility of si...

  13. A Novel SERRS Sandwich-Hybridization Assay to Detect Specific DNA Target

    OpenAIRE

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

    2011-01-01

    International audience In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to ...

  14. A Novel SERRS Sandwich-Hybridization Assay to Detect Specific DNA Target

    OpenAIRE

    Feuillie, Cecile; Merheb, Mohamad; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Haenni, Catherine

    2011-01-01

    In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors...

  15. PCR Assay To Detect Bacillus anthracis Spores in Heat-Treated Specimens

    OpenAIRE

    Fasanella, A.; Losito, S.; Adone, R.; Ciuchini, F.; Trotta, T.; Altamura, S. A.; D. Chiocco; Ippolito, G

    2003-01-01

    Recent interest in anthrax is due to its potential use in bioterrorism and as a biowarfare agent against civilian populations. The development of rapid and sensitive techniques to detect anthrax spores in suspicious specimens is the most important aim for public health. With a view to preventing exposure of laboratory workers to viable Bacillus anthracis spores, this study evaluated the suitability of PCR assays for detecting anthrax spores previously inactivated at 121°C for 45 min. The resu...

  16. Evaluation of the Hologic Panther Transcription-Mediated Amplification Assay for Detection of Mycoplasma genitalium.

    Science.gov (United States)

    Tabrizi, S N; Costa, A M; Su, J; Lowe, P; Bradshaw, C S; Fairley, C K; Garland, S M

    2016-08-01

    The detection of Mycoplasma genitalium was evaluated on 1,080 urine samples by the use of a Panther instrument. Overall sensitivity, specificity, positive predictive values, and negative predictive values were 100%, 99.4%, 93.6%, and 100%, respectively. Detection of M. genitalium by the use of the Panther transcription-mediated amplification assay offers a simple, accurate, and sensitive platform for diagnostic laboratories. PMID:27307453

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

    Directory of Open Access Journals (Sweden)

    Maíra Bidart Macedo

    2012-09-01

    Full Text Available 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.

  18. Rapid microfluidic assay for the detection of botulinum neurotoxin in animal sera

    Science.gov (United States)

    The potent botulinum neurotoxins (BoNTs) represent a threat to public health and safety. Botulism is a disease caused by BoNT intoxication that results in muscle paralysis that can be fatal. Sensitive assays capable of detecting BoNTs from different substrates and settings are essential to limit f...

  19. An Immunological Assay for Detection and Enumeration of Thermophilic Biomining Microorganisms

    OpenAIRE

    Amaro, Ana M.; Hallberg, Kevin B.; Lindström, E. Börje; Jerez, Carlos A.

    1994-01-01

    A specific, fast, and sensitive nonradioactive immunobinding assay for the detection and enumeration of the moderate thermophile Thiobacillus caldus and the thermophilic archaeon Sulfolobus acidocaldarius was developed. It employs enhanced chemiluminescence or peroxidase-conjugated immunoglobulins in a dot or slot blotting system and is very convenient for monitoring thermophilic bioleaching microorganisms in effluents from industrial bioleaching processes.

  20. 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...