Immunoliposome-based immunomagnetic concentration and separation assay for rapid detection of Cronobacter sakazakii.

PubMed

Shukla, Shruti; Lee, Gibaek; Song, Xinjie; Park, Sunhyun; Kim, Myunghee

2016-03-15

This study aimed to develop an immunoliposome-based immunomagnetic concentration and separation assay for the rapid detection of Cronobacter sakazakii (C. sakazakii), an acute opportunistic foodborne pathogenic bacterium, in both pure culture and infant formula. To develop the assay, magnetic nanoparticles (diameter 30 nm) were coated with immunoglobulin G (IgG), specifically anti-C. sakazakii IgG, and applied for the sensitive and efficient detection of C. sakazakii using immunoliposomes. The binding efficiency of anti-C. sakazakii IgG to the magnetic nanoparticles was 86.23 ± 0.59%. The assay developed in this study detected as few as 3.3 × 10(3) CFUmL(-1) of C. sakazakii in pure culture within 2h 30 min; in comparison, an indirect non-competitive enzyme-linked immunosorbent assay was able to detect 6.2 × 10(5) CFUmL(-1) of C. sakazakii in pure culture after 17 h. The developed assay did not show any cross-reactivity with other Cronobacter spp. or pathogens belonging to other genera. In addition, the method was able to detect 10(3) CFUmL(-1) of C. sakazakii in infant formula without any pre-incubation. These results confirm that the immunoliposome-based immunomagnetic concentration and separation assay may facilitate highly sensitive, efficient, and rapid detection of C. sakazakii. Copyright © 2015 Elsevier B.V. All rights reserved.

Duplex detection of the Mycobacterium tuberculosis complex and medically important non-tuberculosis mycobacteria by real-time PCR based on the rnpB gene.

PubMed

Abdeldaim, Guma; Svensson, Erik; Blomberg, Jonas; Herrmann, Björn

2016-11-01

A duplex real-time PCR based on the rnpB gene was developed for Mycobacterium spp. The assay was specific for the Mycobacterium tuberculosis complex (MTB) and also detected all 19 tested species of non-tuberculous mycobacteria (NTM). The assay was evaluated on 404 clinical samples: 290 respiratory samples and 114 from tissue and other non-respiratory body sites. M. tuberculosis was detected by culture in 40 samples and in 30 samples by the assay. The MTB assay showed a sensitivity similar to Roche Cobas Amplicor MTB-PCR (Roche Molecular Systems, Pleasanton, CA, USA). There were only nine samples with non-tuberculous mycobacteria detected by culture. Six of them were detected by the PCR assay. © 2016 APMIS. Published by John Wiley & Sons Ltd.

Rapid detection of highly pathogenic porcine reproductive and respiratory syndrome virus by a fluorescent probe-based isothermal recombinase polymerase amplification assay.

PubMed

Yang, Yang; Qin, Xiaodong; Sun, Yingjun; Chen, Ting; Zhang, Zhidong

2016-12-01

A novel fluorescent probe-based real-time reverse transcription recombinase polymerase amplification (real-time RT-RPA) assay was developed for rapid detection of highly pathogenic type 2 porcine reproductive and respiratory syndrome virus (HP-PRRSV). The sensitivity analysis showed that the detection limit of RPA was 70 copies of HP-PRRSV RNA/reaction. The real-time RT-RPA highly specific amplified HP-PRRSV with no cross-reaction with classic PRRSV, classic swine fever virus, pseudorabies virus, and foot-and-mouth disease virus. Assessment with 125 clinical samples showed that the developed real-time RT-RPA assay was well correlated with real-time RT-qPCR assays for detection of HP-PRRSV. These results suggest that the developed real-time RT-RPA assay is suitable for rapid detection of HP-PRRSV.

Evaluation of a real-time PCR assay based on the single-copy SAG1 gene for the detection of Toxoplasma gondii.

PubMed

Yu, Haijie; Huang, Bin; Zhuo, Xunhui; Chen, Xueqiu; Du, Aifang

2013-11-08

Real-time PCR-based detection of Toxoplasma gondii is very sensitive and convenient for diagnosing toxoplasmosis. However, the performance of the PCR assays could be influenced by the target gene chosen. Here we evaluate a real-time PCR assay using double-stranded DNA dyes (SYBR(®) Green I assay) with a new set of primers targeting the SAG1 gene for the fast and specific detection of T. gondii. The assay showed higher sensitivity than conventional PCR protocols using T. gondii DNA as template. The detection limit of the developed real-time PCR assay was in the order of 1 tachyzoite. The assay was also assessed by experimentally infected mice and showed positive results for blood (25%), spleen (50%) and lung (50%) as early as 1 dpi. The specificity of the assay was confirmed by using DNA from Neospora caninum, Escherichia coli, Babesia bovis, Trypanosoma brucei, Cryptosporidium parvum, and Toxocara canis. Assay applicability was successfully tested in blood samples collected from slaughtered pigs. These results indicate that, based on SYBR(®) green I, the quantitative SAG1 assay may also be useful in the study of the pathogenicity, immunoprophylaxis, and treatment of T. gondii. Copyright © 2013 Elsevier B.V. All rights reserved.

A high-throughput colorimetric assay for glucose detection based on glucose oxidase-catalyzed enlargement of gold nanoparticles

NASA Astrophysics Data System (ADS)

Xiong, Yanmei; Zhang, Yuyan; Rong, Pengfei; Yang, Jie; Wang, Wei; Liu, Dingbin

2015-09-01

We developed a simple high-throughput colorimetric assay to detect glucose based on the glucose oxidase (GOx)-catalysed enlargement of gold nanoparticles (AuNPs). Compared with the currently available glucose kit method, the AuNP-based assay provides higher clinical sensitivity at lower cost, indicating its great potential to be a powerful tool for clinical screening of glucose.We developed a simple high-throughput colorimetric assay to detect glucose based on the glucose oxidase (GOx)-catalysed enlargement of gold nanoparticles (AuNPs). Compared with the currently available glucose kit method, the AuNP-based assay provides higher clinical sensitivity at lower cost, indicating its great potential to be a powerful tool for clinical screening of glucose. Electronic supplementary information (ESI) available: Experimental section and additional figures. See DOI: 10.1039/c5nr03758a

Highly sensitive detection of target molecules using a new fluorescence-based bead assay

NASA Astrophysics Data System (ADS)

Scheffler, Silvia; Strauß, Denis; Sauer, Markus

2007-07-01

Development of immunoassays with improved sensitivity, specificity and reliability are of major interest in modern bioanalytical research. We describe the development of a new immunomagnetic fluorescence detection (IM-FD) assay based on specific antigen/antibody interactions and on accumulation of the fluorescence signal on superparamagnetic PE beads in combination with the use of extrinsic fluorescent labels. IM-FD can be easily modified by varying the order of coatings and assay conditions. Depending on the target molecule, antibodies (ABs), entire proteins, or small protein epitopes can be used as capture molecules. The presence of target molecules is detected by fluorescence microscopy using fluorescently labeled secondary or detection antibodies. Here, we demonstrate the potential of the new assay detecting the two tumor markers IGF-I and p53 antibodies in the clinically relevant concentration range. Our data show that the fluorescence-based bead assay exhibits a large dynamic range and a high sensitivity down to the subpicomolar level.

Detection of HLA Antibodies in Organ Transplant Recipients – Triumphs and Challenges of the Solid Phase Bead Assay

PubMed Central

Tait, Brian D.

2016-01-01

This review outlines the development of human leukocyte antigen (HLA) antibody detection assays and their use in organ transplantation in both antibody screening and crossmatching. The development of sensitive solid phase assays such as the enzyme-linked immunosorbent assay technique, and in particular the bead-based technology has revolutionized this field over the last 10–15 years. This revolution however has created a new paradigm in clinical decision making with respect to the detection of low level pretransplant HLA sensitization and its clinical relevance. The relative sensitivities of the assays used are discussed and the relevance of conflicting inter-assay results. Each assay has its advantages and disadvantages and these are discussed. Over the last decade, the bead-based assay utilizing the Luminex® fluorocytometer instrument has become established as the “gold standard” for HLA antibody testing. However, there are still unresolved issues surrounding this technique, such as the presence of denatured HLA molecules on the beads which reveal cryptic epitopes and the issue of appropriate fluorescence cut off values for positivity. The assay has been modified to detect complement binding (CB) in addition to non-complement binding (NCB) HLA antibodies although the clinical relevance of the CB and NCB IgG isotypes is not fully resolved. The increase sensitivity of the Luminex® bead assay over the complement-dependent cytotoxicity crossmatch has permitted the concept of the “virtual crossmatch” whereby the crossmatch is predicted to a high degree of accuracy based on the HLA antibody specificities detected by the solid phase assay. Dialog between clinicians and laboratory staff on an individual patient basis is essential for correct clinical decision making based on HLA antibody results obtained by the various techniques. PMID:28018342

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

PubMed

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

2014-06-01

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

Advantages and application of label-free detection assays in drug screening.

PubMed

Cunningham, Brian T; Laing, Lance G

2008-08-01

Adoption is accelerating for a new family of label-free optical biosensors incorporated into standard format microplates owing to their ability to enable highly sensitive detection of small molecules, proteins and cells for high-throughput drug discovery applications. Label-free approaches are displacing other detection technologies owing to their ability to provide simple assay procedures for hit finding/validation, accessing difficult target classes, screening the interaction of cells with drugs and analyzing the affinity of small molecule inhibitors to target proteins. This review describes several new drug discovery applications that are under development for microplate-based photonic crystal optical biosensors and the key issues that will drive adoption of the technology. Microplate-based optical biosensors are enabling a variety of cell-based assays, inhibition assays, protein-protein binding assays and protein-small molecule binding assays to be performed with high-throughput and high sensitivity.

Application of a molecular beacon based real-time isothermal amplification (MBRTIA) technology for simultaneous detection of Bacillus cereus and Staphylococcus aureus.

PubMed

Mandappa, I M; Joglekar, Prasanna; Manonmani, H K

2015-07-01

A multiplex real-time isothermal amplification assay was developed using molecular beacons for the detection of Bacillus cereus and Staphylococcus aureus by targeting four important virulence genes. A correlation between targeting highly accessible DNA sequences and isothermal amplification based molecular beacon efficiency and sensitivity was demonstrated using phi(Φ)29 DNA polymerase at a constant isothermal temperature of 30 °C. It was very selective and consistently detected down to 10(1) copies of DNA. The specificity and sensitivity of this assay, when tested with pure culture were high, surpassing those of currently used PCR assays for the detection of these organisms. The molecular beacon based real-time isothermal amplification (MBRTIA) assay could be carried out entirely in 96 well plates or well strips, enabling a rapid and high-throughput detection of food borne pathogens.

Novel drug and soluble target tolerant antidrug antibody assay for therapeutic antibodies bearing the P329G mutation.

PubMed

Wessels, Uwe; Schick, Eginhard; Ritter, Mirko; Kowalewsky, Frank; Heinrich, Julia; Stubenrauch, Kay

2017-06-01

Bridging immunoassays for detection of antidrug antibodies (ADAs) are typically susceptible to high concentrations of residual drug. Sensitive drug-tolerant assays are, therefore, needed. An immune complex assay to detect ADAs against therapeutic antibodies bearing Pro329Gly mutation was established. The assay uses antibodies specific for the Pro329Gly mutation for capture and human soluble Fcγ receptor for detection. When compared with a bridging assay, the new assay showed similar precision, high sensitivity to IgG1 ADA and dramatically improved drug tolerance. However, it was not able to detect early (IgM-based) immune responses. Applied in combination with a bridging assay, the novel assay serves as orthogonal assay for immunogenicity assessment and allows further characterization of ADA responses.

A high-throughput multiplex method adapted for GMO detection.

PubMed

Chaouachi, Maher; Chupeau, Gaëlle; Berard, Aurélie; McKhann, Heather; Romaniuk, Marcel; Giancola, Sandra; Laval, Valérie; Bertheau, Yves; Brunel, Dominique

2008-12-24

A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences (approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

Development of binding assays in microfabricated picoliter vials: an assay for biotin.

PubMed

Grosvenor, A L; Feltus, A; Conover, R C; Daunert, S; Anderson, K W

2000-06-01

A homogeneous binding assay for the detection of biotin in picoliter vials was developed using the photoprotein aequorin as the label. The binding assay was based on the competition of free biotin with biotinylated aequorin (AEQ-biotin) for avidin. A sequential protocol was used, and modification of the assay to reduce the number of steps was examined. Results showed that detection limits on the order of 10(-14) mol of biotin were possible. Reducing the number of steps provided similar detection limits but only if the amount of avidin used was decreased. These binding assays based on picoliter volumes have potential applications in a variety of fields, including microanalysis and single-cell analysis, where the amount of sample is limited. In addition, these assays are suitable for the high-throughput screening of biopharmaceuticals.

Preliminary Validation of Direct Detection of Foot-And-Mouth Disease Virus within Clinical Samples Using Reverse Transcription Loop-Mediated Isothermal Amplification Coupled with a Simple Lateral Flow Device for Detection

PubMed Central

Waters, Ryan A.; Fowler, Veronica L.; Armson, Bryony; Nelson, Noel; Gloster, John; Paton, David J.; King, Donald P.

2014-01-01

Rapid, field-based diagnostic assays are desirable tools for the control of foot-and-mouth disease (FMD). Current approaches involve either; 1) Detection of FMD virus (FMDV) with immuochromatographic antigen lateral flow devices (LFD), which have relatively low analytical sensitivity, or 2) portable RT-qPCR that has high analytical sensitivity but is expensive. Loop-mediated isothermal amplification (LAMP) may provide a platform upon which to develop field based assays without these drawbacks. The objective of this study was to modify an FMDV-specific reverse transcription–LAMP (RT-LAMP) assay to enable detection of dual-labelled LAMP products with an LFD, and to evaluate simple sample processing protocols without nucleic acid extraction. The limit of detection of this assay was demonstrated to be equivalent to that of a laboratory based real-time RT-qPCR assay and to have a 10,000 fold higher analytical sensitivity than the FMDV-specific antigen LFD currently used in the field. Importantly, this study demonstrated that FMDV RNA could be detected from epithelial suspensions without the need for prior RNA extraction, utilising a rudimentary heat source for amplification. Once optimised, this RT-LAMP-LFD protocol was able to detect multiple serotypes from field epithelial samples, in addition to detecting FMDV in the air surrounding infected cattle, pigs and sheep, including pre-clinical detection. This study describes the development and evaluation of an assay format, which may be used as a future basis for rapid and low cost detection of FMDV. In addition it provides providing “proof of concept” for the future use of LAMP assays to tackle other challenging diagnostic scenarios encompassing veterinary and human health. PMID:25165973

Development of an Assay for the Detection of PrPres in Blood and Urine Based on PMCA Assay an ELISA Methods

DTIC Science & Technology

2005-09-01

constitute the basis for a TSE diagnostic human plasma assay platform. We have established a collaboration with Biotraces to develop an assay based on...their MPD instrument. One of the problems with the Biotraces ’ test was its inability to detect low levels of recombinant PrP. Biotraces has now succeeded...data and decide based on these new results whether to continue the collaboration with Biotraces . As soon we have reached a decision, we will inform

Development of a taqman-based real-time PCR assay for the rapid and specific detection of novel duck- origin goose parvovirus.

PubMed

Wang, Jianchang; Wang, Jinfeng; Cui, Yuan; Nan, Huizhu; Yuan, Wanzhe

2017-08-01

A real-time PCR assay was developed for specific detection of novel duck-origin goose parvovirus (N-GPV), the etiological agent of duck beak atrophy and dwarfism syndrome (BADS). The detection limit of the assay was 10 2 copies. The assay was useful in the prevention and control of BADS. Copyright © 2017 Elsevier Ltd. All rights reserved.

A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples

PubMed Central

Kumanan, Vijayarani; Nugen, Sam R.; Baeumner, Antje J.

2009-01-01

A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (101 to 106) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortuitum, M. scrofulaceum, M. intracellulare, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR. PMID:19255522

Fluorescence-based bioassays for the detection and evaluation of food materials.

PubMed

Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

2015-10-13

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials.

Fluorescence-Based Bioassays for the Detection and Evaluation of Food Materials

PubMed Central

Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

2015-01-01

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials. PMID:26473869

Development of a fluorescent probe-based recombinase polymerase amplification assay for rapid detection of Orf virus.

PubMed

Yang, Yang; Qin, Xiaodong; Wang, Guangxiang; Zhang, Yuen; Shang, Youjun; Zhang, Zhidong

2015-12-02

Orf virus (ORFV) is the causative agent of Orf (also known as contagious ecthyma or contagious papular dermatitis), a severe infectious skin disease in goats, sheep and other ruminants. The rapid detection of ORFV is of great importance in disease control and highly needed. A isothermal molecular diagnostic approach, termed recombinase polymerase amplification (RPA), is considered as an novel and rapid alternative techonology to PCR assay. In the present study, a novel fluorescent probe based on RPA assay (ORFV exo RPA assay) was developed. The developed ORFV exo RPA assay was capable of as low as 100 copies of ORFV DNA /reaction and was highly specific, with no cross-reaction with closely related viruses (capripox virus, foot-and-mouth disease virus or peste des petits ruminants virus). Further assessment with clinical samples showed that the developed ORFV exo RPA assay has good correlation with qPCR assays for detection of ORFV. These results suggest that the developed ORFV exo RPA assay is suitable for rapid detection of ORFV.

Validation of a Rapid Bacteria Endospore Enumeration System for Planetary Protection Application

NASA Astrophysics Data System (ADS)

Chen, Fei; Kern, Roger; Kazarians, Gayane; Venkateswaran, Kasthuri

NASA monitors spacecraft surfaces to assure that the presence of bacterial endospores meets strict criteria at launch, to minimize the risk of inadvertent contamination of the surface of Mars. Currently, the only approved method for enumerating the spores is a culture based assay that requires three days to produce results. In order to meet the demanding schedules of spacecraft assembly, a more rapid spore detection assay is being considered as an alternate method to the NASA standard culture-based assay. The Millipore Rapid Microbiology Detection System (RMDS) has been used successfully for rapid bioburden enumeration in the pharmaceutical and food industries. The RMDS is rapid and simple, shows high sensitivity (to 1 colony forming unit [CFU]/sample), and correlates well with traditional culture-based methods. It combines membrane filtration, adenosine triphosphate (ATP) bioluminescence chemistry, and image analysis based on photon detection with a Charge Coupled Device (CCD) camera. In this study, we have optimized the assay conditions and evaluated the use of the RMDS as a rapid spore detection tool for NASA applications. In order to select for spores, the samples were subjected to a heat shock step before proceeding with the RMDS incubation protocol. Seven species of Bacillus (nine strains) that have been repeatedly isolated from clean room environments were assayed. All strains were detected by the RMDS in 5 hours and these assay times were repeatedly demonstrated along with low image background noise. Validation experiments to compare the Rapid Sore Assay (RSA) and NASA standard assay (NSA) were also performed. The evaluation criteria were modeled after the FDA Guideline of Process Validation, and Analytical Test Methods. This body of research demonstrates that the Rapid Spore Assay (RSA) is quick, and of equivalent sensitivity to the NASA standard assay, potentially reducing the assay time for bacterial endospores from over 72 hours to less than 8 hours. Accordingly, JPL has produced a report recommending that NASA adopt the RSA method as a suitable alternative to the NASA standard assay.

Rapid Diagnostic Assay for Intact Influenza Virus Using a High Affinity Hemagglutinin Binding Protein.

PubMed

Anderson, Caitlin E; Holstein, Carly A; Strauch, Eva-Maria; Bennett, Steven; Chevalier, Aaron; Nelson, Jorgen; Fu, Elain; Baker, David; Yager, Paul

2017-06-20

Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 10 7 and 1.34 × 10 7 CEID 50 /mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.

Quantitative PCR detection of Batrachochytrium dendrobatidis DNA from sediments and water

USGS Publications Warehouse

Kirshtein, Julie D.; Anderson, Chauncey W.; Wood, J.S.; Longcore, Joyce E.; Voytek, Mary A.

2007-01-01

The fungal pathogen Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, a disease implicated in amphibian declines on 5 continents. Polymerase chain reaction (PCR) primer sets exist with which amphibians can be tested for this disease, and advances in sampling techniques allow non-invasive testing of animals. We developed filtering and PCR based quantitative methods by modifying existing PCR assays to detect Bd DNA in water and sediments, without the need for testing amphibians; we tested the methods at 4 field sites. The SYBR based assay using Boyle primers (SYBR/Boyle assay) and the Taqman based assay using Wood primers performed similarly with samples generated in the laboratory (Bd spiked filters), but the SYBR/Boyle assay detected Bd DNA in more field samples. We detected Bd DNA in water from 3 of 4 sites tested, including one pond historically negative for chytridiomycosis. Zoospore equivalents in sampled water ranged from 19 to 454 l-1 (nominal detection limit is 10 DNA copies, or about 0.06 zoospore). We did not detect DNA of Bd from sediments collected at any sites. Our filtering and amplification methods provide a new tool to investigate critical aspects of Bd in the environment. ?? Inter-Research 2007.

Molecular method for detection of total coliforms in drinking water samples.

PubMed

Maheux, Andrée F; Boudreau, Dominique K; Bisson, Marc-Antoine; Dion-Dupont, Vanessa; Bouchard, Sébastien; Nkuranga, Martine; Bergeron, Michel G; Rodriguez, Manuel J

2014-07-01

This work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting the lacZ, wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species of Enterobacteriaceae encountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas the lacZ, wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culture-based methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Molecular Method for Detection of Total Coliforms in Drinking Water Samples

PubMed Central

Boudreau, Dominique K.; Bisson, Marc-Antoine; Dion-Dupont, Vanessa; Bouchard, Sébastien; Nkuranga, Martine; Bergeron, Michel G.; Rodriguez, Manuel J.

2014-01-01

This work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting the lacZ, wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species of Enterobacteriaceae encountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas the lacZ, wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culture-based methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality. PMID:24771030

Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis

PubMed Central

Bercovier, Herve; Fishman, Yolanta; Nahary, Ronen; Sinai, Sharon; Zlotkin, Amir; Eyngor, Marina; Gilad, Oren; Eldar, Avi; Hedrick, Ronald P

2005-01-01

Background Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary. Results A robust and sensitive PCR assay based on a defined gene sequence of KHV was developed to improve the diagnosis of KHV infection. From a KHV genomic library, a hypothetical thymidine kinase gene (TK) was identified, subcloned and expressed as a recombinant protein. Preliminary characterization of the recombinant TK showed that it has a kinase activity using dTTP but not dCTP as a substrate. A PCR assay based on primers selected from the defined DNA sequence of the TK gene was developed and resulted in a 409 bp amplified fragment. The TK based PCR assay did not amplify the DNAs of other fish herpesviruses such as Herpesvirus cyprini (CHV) and the channel catfish virus (CCV). The TK based PCR assay was specific for the detection of KHV and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. The TK based PCR was compared to previously described PCR assays and to viral culture in diseased fish and was shown to be the most sensitive method of diagnosis of KHV infection. Conclusion The TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was more sensitive for the detection of KHV than previously described PCR assays; it was as sensitive as virus isolation which is the golden standard method for KHV diagnosis and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. PMID:15774009

Highly Stable Lyophilized Homogeneous Bead-Based Immunoassays for On-Site Detection of Bio Warfare Agents from Complex Matrices.

PubMed

Mechaly, Adva; Marx, Sharon; Levy, Orly; Yitzhaki, Shmuel; Fisher, Morly

2016-06-21

This study shows the development of dry, highly stable immunoassays for the detection of bio warfare agents in complex matrices. Thermal stability was achieved by the lyophilization of the complete, homogeneous, bead-based immunoassay in a special stabilizing buffer, resulting in a ready-to-use, simple assay, which exhibited long shelf and high-temperature endurance (up to 1 week at 100 °C). The developed methodology was successfully implemented for the preservation of time-resolved fluorescence, Alexa-fluorophores, and horse radish peroxidase-based bead assays, enabling multiplexed detection. The multiplexed assay was successfully implemented for the detection of Bacillus anthracis, botulinum B, and tularemia in complex matrices.

High-throughput receptor-based assay for the detection of spirolides by chemiluminescence.

PubMed

Rodríguez, Laura P; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Botana, Luis M

2013-12-01

The spirolides are marine toxins that belong to a new class of macrocyclic imines produced by dinoflagellates. In this study a previously described solid-phase receptor-based assay for the detection of spirolides was optimized for high-throughput screening and prevalidated. This method is based on the competition between 13-desmethyl spirolide C and biotin-α-bungarotoxin immobilized on a streptavidin-coated surface, for binding to nicotinic acetylcholine receptors. In this inhibition assay the amount of nAChR bound to the well surface is quantified using a specific antibody, followed by a second anti-mouse IgG antibody labeled with horseradish peroxidase (HRP). The assay protocol was optimized for 384-well microplates, which allowed a reduction of the amount of reagents per sample and an increase of the number of samples per plate versus previously published receptor-based assays. The sensitivity of the assay for 13-desmethyl spirolide C ranged from 5 to 150 ng mL(-1). The performance of the assay in scallop extracts was adequate, with an estimated detection limit for 13-desmethyl spirolide C of 50 μg kg(-1) of shellfish meat. The recovery rate of 13-desmethyl spirolide C for spiked samples with this assay was 80% and the inter-assay coefficient of variation was 8%. This 384-well microplate, chemiluminescence method can be used as a high-throughput screening assay to detect 13-desmethyl spirolide C in shellfish meat in order to reduce the number of samples to be processed through bioassays or analytical methods. Copyright © 2013 Elsevier Ltd. All rights reserved.

Development of DNAzyme-based PCR signal cascade amplification for visual detection of Listeria monocytogenes in food.

PubMed

Liu, Zhanmin; Yao, Chenhui; Yang, Cuiyun; Wang, Yanming; Wan, Sibao; Huang, Junyi

2018-05-16

Listeria monocytogenes is an important foodborne pathogen, and it can cause severe diseases. Rapid detection of L. monocytogenes is crucial to control this pathogen. A simple and robust strategy based on the cascade of PCR and G-quadruplex DNAzyme catalyzed reaction was used to detect L. monocytogenes. In the presence of hemin and the aptamer formed during PCR, the catalytic horseradish peroxidase-mimicking G-quadruplex DNAzymes allow the colorimetric responses of target DNA from L. monocytogenes. This assay can detect genomic DNA of L. monocytogenes specifically with as low as 50 pg/reaction with the naked eye. Through 20 pork samples assay, visual detection assay had the same results as conventional detection methods, and had a good performance. This is a powerful demonstration of the ability of G-quadruplex DNAzyme to be used for PCR-based assay with significant advantages of high sensitivity, low cost and simple manipulation over existing approaches and offers the opportunity for application in pathogen detection. Copyright © 2018 Elsevier Inc. All rights reserved.

Development of a highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus 71 in hand, foot, and mouth disease.

PubMed

Niu, Peihua; Qi, Shunxiang; Yu, Benzhang; Zhang, Chen; Wang, Ji; Li, Qi; Ma, Xuejun

2016-11-01

Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand, foot, and mouth disease (HFMD). A commercial TaqMan probe-based real-time PCR assay has been widely used for the differential detection of EV71 despite its relatively high cost and failure to detect samples with a low viral load (Ct value > 35). In this study, a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of EV71 in HFMD was developed. The sensitivity and specificity of this assay were evaluated using a reference EV71 stock and a panel of controls consisting of coxsackievirus A16 (CVA16) and common respiratory viruses, respectively. The clinical performance of this assay was evaluated and compared with those of a commercial TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional two-step nested RT-PCR assay. The limit of detection for the RTN RT-PCR assay was 0.01 TCID50/ml, with a Ct value of 38.3, which was the same as that of the traditional two-step nested RT-PCR assay and approximately tenfold lower than that of the qRT-PCR assay. When testing the reference strain EV71, this assay showed favorable detection reproducibility and no obvious cross-reactivity. The testing results of 100 clinical throat swabs from HFMD-suspected patients revealed that 41 samples were positive for EV71 by both RTN RT-PCR and traditional two-step nested RT-PCR assays, whereas only 29 were EV71 positive by qRT-PCR assay.

Evaluation of SYBR green I based visual loop-mediated isothermal amplification (LAMP) assay for genus and species-specific diagnosis of malaria in P. vivax and P. falciparum endemic regions.

PubMed

Singh, Ruchi; Singh, Dhirendra Pratap; Savargaonkar, Deepali; Singh, Om P; Bhatt, Rajendra M; Valecha, Neena

2017-01-01

Loop-mediated isothermal amplification (LAMP) is an emerging nucleic acid based diag- nostic approach that is easily adaptable to the field settings with limited technical resources. This study was aimed to evaluate the LAMP assay for the detection and identification of Plasmodium falciparum and P. vivax infection in malaria suspected cases using genus and species-specific assay. The 18S rRNA-based LAMP assay was evaluated for diagnosis of genus Plasmodium, and species- specific diagnosis of P. falciparum and P. vivax, infection employing 317 malaria suspected cases, and the results were compared with those obtained by 18S nested PCR (n-PCR). All the samples were confirmed by microscopy for the presence of Plasmodium parasite. The n-PCR was positive in all Plasmodium-infected cases (n=257; P. falciparum=133; P. vivax=124) and negative in microscopy negative cases (n=58) except for two cases which were positive for P. vivax, giving a sen- sitivity of 100% (95% CI: 97.04-100%) and a specificity of 100% (95% CI: 88.45-99.5%). Genus-specific LAMP assay missed 11 (3.2%) microscopy and n-PCR confirmed vivax malaria cases. Considering PCR results as a refer- ence, LAMP was 100% sensitive and specific for P. falciparum, whereas it exhibited 95.16% sensitivity and 96.7% specificity for P. vivax. The n-PCR assay detected 10 mixed infection cases while species-specific LAMP detected five mixed infection cases of P. vivax and P. falciparum, which were not detected by microscopy. Genus-specific LAMP assay displayed low sensitivity. Falciparum specific LAMP assay displayed high sensitivity whereas vivax specific LAMP assay displayed low sensitivity. Failed detection of vivax cases otherwise confirmed by the n-PCR assay indicates exploitation of new targets and improved detection methods to attain 100% results for P. vivax detection.

Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures

PubMed Central

Wiklander, Oscar P. B.; Bostancioglu, R. Beklem; Welsh, Joshua A.; Zickler, Antje M.; Murke, Florian; Corso, Giulia; Felldin, Ulrika; Hagey, Daniel W.; Evertsson, Björn; Liang, Xiu-Ming; Gustafsson, Manuela O.; Mohammad, Dara K.; Wiek, Constanze; Hanenberg, Helmut; Bremer, Michel; Gupta, Dhanu; Björnstedt, Mikael; Giebel, Bernd; Nordin, Joel Z.; Jones, Jennifer C.; EL Andaloussi, Samir; Görgens, André

2018-01-01

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell’s activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.

Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures.

PubMed

Wiklander, Oscar P B; Bostancioglu, R Beklem; Welsh, Joshua A; Zickler, Antje M; Murke, Florian; Corso, Giulia; Felldin, Ulrika; Hagey, Daniel W; Evertsson, Björn; Liang, Xiu-Ming; Gustafsson, Manuela O; Mohammad, Dara K; Wiek, Constanze; Hanenberg, Helmut; Bremer, Michel; Gupta, Dhanu; Björnstedt, Mikael; Giebel, Bernd; Nordin, Joel Z; Jones, Jennifer C; El Andaloussi, Samir; Görgens, André

2018-01-01

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell's activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.

Luminescence resonance energy transfer-based nucleic acid hybridization assay on cellulose paper with upconverting phosphor as donors.

PubMed

Zhou, Feng; Noor, M Omair; Krull, Ulrich J

2014-03-04

A bioassay based on DNA hybridization on cellulose paper is a promising format for gene fragment detection that may be suited for in-field and rapid diagnostic applications. We demonstrate for the first time that luminescence resonance energy transfer (LRET) associated with upconverting phosphors (UCPs) can be used to develop a paper-based DNA hybridization assay with high sensitivity, selectivity and fast response. UCPs with strong green emission were synthesized and subsequently functionalized with streptavidin (UCP-strep). UCP-strep particles were immobilized on cellulose paper, and then biotinylated single-stranded oligonucleotide probes were conjugated onto the UCPs via streptavidin-biotin linkage. The UCPs served as donors that were LRET-paired with Cy3-labeled target DNA. Selective DNA hybridization enabled the proximity required for LRET-sensitized emission from Cy3, which was used as the detection signal. Hybridization was complete within 2 min, and the limit of detection of the method was 34 fmol, which is a significant improvement in comparison to an analogous fluorescence resonance energy transfer (FRET) assay based on quantum dots. The assay exhibited excellent resistance to nonspecific adsorption of noncomplementary short/long DNA and protein. The selectivity of the assay was further evaluated by one base pair mismatched (1BPM) DNA detection, where a maximum signal ratio of 3.1:1 was achieved between fully complementary and 1BPM samples. This work represents a preliminary but significant step for the development of paper-based UCP-LRET nucleic acid hybridization assays, which offer potential for lowering the limit of detection of luminescent hybridization assays due to the negligible background signal associated with optical excitation by near-infrared (NIR) light.

Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage.

PubMed

Talarico, Sarah; Safaeian, Mahboobeh; Gonzalez, Paula; Hildesheim, Allan; Herrero, Rolando; Porras, Carolina; Cortes, Bernal; Larson, Ann; Fang, Ferric C; Salama, Nina R

2016-08-01

Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool-based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning. Stool-based ddPCR assays for H. pylori 16S gene detection and cagA virulence gene typing were tested using a collection of 50 matched stool and serum samples from Costa Rican volunteers and 29 H. pylori stool antigen-tested stool samples collected at a US hospital. The stool-based H. pylori 16S ddPCR assay had a sensitivity of 84% and 100% and a specificity of 100% and 71% compared to serology and stool antigen tests, respectively. The stool-based cagA genotyping assay detected cagA in 22 (88%) of 25 stools from CagA antibody-positive individuals and four (16%) of 25 stools from CagA antibody-negative individuals from Costa Rica. All 26 of these samples had a Western-type cagA allele. Presence of serum CagA antibodies was correlated with a significantly higher load of H. pylori in the stool. The stool-based ddPCR assays are a sensitive, noninvasive method for detection, quantification, and partial genotyping of H. pylori. The quantitative nature of ddPCR-based H. pylori detection revealed significant variation in bacterial load among individuals that correlates with presence of the cagA virulence gene. These stool-based ddPCR assays will facilitate future population-based epidemiologic studies of this important human pathogen. © 2015 John Wiley & Sons Ltd.

Sensitive assays enable detection of serum IgG antibodies against Clostridium difficile toxin A and toxin B in healthy subjects and patients with Clostridium difficile infection.

PubMed

Zhao, Xuemei; Bender, Florent; Shukla, Rajiv; Kang, John J; Caro-Aguilar, Ivette; Laterza, Omar F

2016-04-01

Pathogenic Clostridium difficile produces two proinflammatory exotoxins, toxin A and toxin B. Low level of serum antitoxin IgG antibodies is a risk factor for the development of primary and recurrent C. difficile infection (CDI). We developed and validated two sensitive, titer-based electrochemiluminescence assays for the detection of serum antibody levels against C. difficile toxins A and B. These assays demonstrated excellent precision. The sensitivity of the assays allowed the detection of antitoxin A and antitoxin B IgG antibodies in all tested serum samples during assay validation. The validated titer-based assays enable assessment of antitoxin A and antitoxin B IgG antibodies as potential biomarkers to identify patients with CDI at increased risk for CDI recurrence.

Comparative Evaluation of Veriflow®Listeria Species to USDA Culture-Based Method for the Detection of Listeria spp. in Food and Environmental Samples.

PubMed

Joelsson, Adam C; Terkhorn, Shawn P; Brown, Ashley S; Puri, Amrita; Pascal, Benjamin J; Gaudioso, Zara E; Siciliano, Nicholas A

2017-09-01

Veriflow® Listeria species (Veriflow LS) is a molecular-based assay for the presumptive detection of Listeria spp. from environmental surfaces (stainless steel, sealed concrete, plastic, and ceramic tile) and ready-to-eat (RTE) food matrixes (hot dogs and deli meat). The assay utilizes a PCR detection method coupled with a rapid, visual, flow-based assay that develops in 3 min post-PCR amplification and requires only a 24 h enrichment for maximum sensitivity. The Veriflow LS system eliminates the need for sample purification, gel electrophoresis, or fluorophore-based detection of target amplification and does not require complex data analysis. This Performance Tested MethodSM validation study demonstrated the ability of the Veriflow LS assay to detect low levels of artificially inoculated Listeria spp. in six distinct environmental and food matrixes. In each unpaired reference comparison study, probability of detection analysis indicated that there was no significant difference between the Veriflow LS method and the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guide Chapter 8.08 reference method. Fifty-one strains of various Listeria spp. were detected in the inclusivity study, and 35 nonspecific organisms went undetected in the exclusivity study. The study results show that the Veriflow LS is a sensitive, selective, and robust assay for the presumptive detection of Listeria spp. sampled from environmental surfaces (stainless steel, sealed concrete, plastic, and ceramic tile) and RTE food matrixes (hot dogs and deli meat).

A visual assay and spectrophotometric determination of LLM-105 explosive using detection of gold nanoparticle aggregation at two pH values.

PubMed

He, Yi; Cheng, Yang

2016-08-01

We report a simple, rapid, and sensitive assay for visual and spectrophotometric detection of the 2,6-diamino-3,5-dinitropyrazine-1-oxide (LLM-105) explosive. The assay is based on different interactions between LLM-105 and gold nanoparticle (AuNP) dispersions at two pH values, leading to the formation of dispersed or aggregated AuNPs. Two AuNP dispersions at two pH values were applied to recognize and detect LLM-105 instead of traditional AuNP dispersion under an aptotic pH to improve the anti-interference ability. The developed assay showed excellent sensitivity with a detection limit of 3 ng/mL, and the presence of as low as 0.2 μg/mL LLM-105 can be directly detected with the bare eye. This sensitivity is about six orders of magnitude higher than that of the reported traditional assays. Additionally, the assay exhibited good selectivity toward LLM-105 over other explosives, sulfur-containing compounds, and amines. Graphical abstract A simple, sensitive, and selective assay for LLM-105 was developed based on the pH-dependent interaction between the LLM-105 explosive and gold nanoparticle dispersion.

Bio-barcode gel assay for microRNA

NASA Astrophysics Data System (ADS)

Lee, Hyojin; Park, Jeong-Eun; Nam, Jwa-Min

2014-02-01

MicroRNA has been identified as a potential biomarker because expression level of microRNA is correlated with various cancers. Its detection at low concentrations would be highly beneficial for cancer diagnosis. Here, we develop a new type of a DNA-modified gold nanoparticle-based bio-barcode assay that uses a conventional gel electrophoresis platform and potassium cyanide chemistry and show this assay can detect microRNA at aM levels without enzymatic amplification. It is also shown that single-base-mismatched microRNA can be differentiated from perfectly matched microRNA and the multiplexed detection of various combinations of microRNA sequences is possible with this approach. Finally, differently expressed microRNA levels are selectively detected from cancer cells using the bio-barcode gel assay, and the results are compared with conventional polymerase chain reaction-based results. The method and results shown herein pave the way for practical use of a conventional gel electrophoresis for detecting biomolecules of interest even at aM level without polymerase chain reaction amplification.

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides.

PubMed

Ito, Takao; Suzaki, Koichi

2017-01-01

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides

PubMed Central

Suzaki, Koichi

2017-01-01

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays. PMID:28957362

Analytical platform evaluation for quantification of ERG in prostate cancer using protein and mRNA detection methods

DOE PAGES

He, Jintang; Schepmoes, Athena A.; Shi, Tujin; ...

2015-01-01

Background: The established methods for detecting prostate cancer (CaP) are based on tests using PSA (blood), PCA3 (urine), and AMACR (tissue) as biomarkers in patient samples. The demonstration of ERG oncoprotein overexpression due to gene fusion in CaP has thus provided ERG as an additional biomarker. Based on this, we hypothesized that ERG protein quantification methods can be of use in the diagnosis of prostate cancer. Methods: Therefore, an antibody-free assay for ERG3 protein detection was developed based on PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry. We utilized TMPRSS2-ERG positive VCaP and TMPRSS2-ERGmore » negative LNCaP cells to simulate three different sample types (cells, tissue, and post-DRE urine sediment). Results: Recombinant ERG3 protein spiked into LNCaP cell lysates could be detected at levels as low as 20 pg by PRISM-SRM analysis. The sensitivity of the PRISM-SRM assay was around approximately 10,000 VCaP cells in a mixed cell population model of VCaP and LNCaP cells. Interestingly, ERG protein could be detected in as few as 600 VCaP cells spiked into female urine. The sensitivity of the in-house enzyme-linked immunosorbent assay (ELISA) was similar to the PRISM-SRM assay, with detection of 30 pg of purified recombinant ERG3 protein and 10,000 VCaP cells. On the other hand, qRT-PCR exhibited a higher sensitivity, as TMPRSS2-ERG transcripts were detected in as few as 100 VCaP cells, in comparison to NanoString methodologies which detected ERG from 10,000 cells. Conclusions: Based on this data, we propose that the detection of both ERG transcriptional products with RNA-based assays, as well as protein products of ERG using PRISM-SRM assays, may be of clinical value in developing diagnostics and prognostics assays for prostate cancer given their sensitivity, specificity, and reproducibility.« less

Rapid detection of Enterovirus and Coxsackievirus A10 by a TaqMan based duplex one-step real time RT-PCR assay.

PubMed

Chen, Jingfang; Zhang, Rusheng; Ou, Xinhua; Yao, Dong; Huang, Zheng; Li, Linzhi; Sun, Biancheng

2017-06-01

A TaqMan based duplex one-step real time RT-PCR (rRT-PCR) assay was developed for the rapid detection of Coxsackievirus A10 (CV-A10) and other enterovirus (EVs) in clinical samples. The assay was fully evaluated and found to be specific and sensitive. When applied in 115 clinical samples, a 100% diagnostic sensitivity in CV-A10 detection and 97.4% diagnostic sensitivity in other EVs were found. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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.

Novel Time-Resolved Fluorescence Europium Nanoparticle Immunoassay for Detection of Human Immunodeficiency Virus-1 Group O Viruses Using Microplate and Microchip Platforms.

PubMed

Haleyur Giri Setty, Mohan Kumar; Liu, Jikun; Mahtani, Prerna; Zhang, Panhe; Du, Bingchen; Ragupathy, Viswanath; Devadas, Krishnakumar; Hewlett, Indira K

2016-06-01

Accurate detection and quantification of HIV-1 group O viruses have been challenging for currently available HIV assays. We have developed a novel time-resolved fluorescence (TRF) europium nanoparticle immunoassay for HIV-1 group O detection using a conventional microplate enzyme-linked immunosorbent assay (ELISA) and a microchip platform. We screened several antibodies for optimal reactivity with several HIV-1 group O strains and identified antibodies that can detect all the strains of HIV-1 group O that were available for testing. The antibodies were used to develop a conventional ELISA format assay and an in-house developed europium nanoparticle-based assay for sensitivity. The method was evaluated on both microwell plate and microchip platforms. We identified two specific and sensitive antibodies among the six we screened. The antibodies, C65691 and ANT-152, were able to quantify 15 and detect all 17 group O viruses, respectively, as they were broadly cross-reactive with all HIV-1 group O strains and yielded better signals compared with other antibodies. We have developed a sensitive assay that reflects the actual viral load in group O samples by using an appropriate combination of p24 antibodies that enhance group O detection and a highly sensitive TRF-based europium nanoparticle for detection. The combination of ANT-152 and C65690M in the ratio 3:1 was able to give significantly higher signals in our europium-based assay compared with using any single antibody.

Gold Nanoparticle Labeling Based ICP-MS Detection/Measurement of Bacteria, and Their Quantitative Photothermal Destruction

PubMed Central

Lin, Yunfeng

2015-01-01

Bacteria such as Salmonella and E. coli present a great challenge in public health care in today’s society. Protection of public safety against bacterial contamination and rapid diagnosis of infection require simple and fast assays for the detection and elimination of bacterial pathogens. After utilizing Salmonella DT104 as an example bacterial strain for our investigation, we report a rapid and sensitive assay for the qualitative and quantitative detection of bacteria by using antibody affinity binding, popcorn shaped gold nanoparticle (GNPOPs) labeling, surfance enchanced Raman spectroscopy (SERS), and inductively coupled plasma mass spectrometry (ICP-MS) detection. For qualitative analysis, our assay can detect Salmonella within 10 min by Raman spectroscopy; for quantitative analysis, our assay has the ability to measure as few as 100 Salmonella DT104 in a 1 mL sample (100 CFU/mL) within 40 min. Based on the quantitative detection, we investigated the quantitative destruction of Salmonella DT104, and the assay’s photothermal efficiency in order to reduce the amount of GNPOPs in the assay to ultimately to eliminate any potential side effects/toxicity to the surrounding cells in vivo. Results suggest that our assay may serve as a promising candidate for qualitative and quantitative detection and elimination of a variety of bacterial pathogens. PMID:26417447

Solid-phase proximity ligation assays for individual or parallel protein analyses with readout via real-time PCR or sequencing.

PubMed

Nong, Rachel Yuan; Wu, Di; Yan, Junhong; Hammond, Maria; Gu, Gucci Jijuan; Kamali-Moghaddam, Masood; Landegren, Ulf; Darmanis, Spyros

2013-06-01

Solid-phase proximity ligation assays share properties with the classical sandwich immunoassays for protein detection. The proteins captured via antibodies on solid supports are, however, detected not by single antibodies with detectable functions, but by pairs of antibodies with attached DNA strands. Upon recognition by these sets of three antibodies, pairs of DNA strands brought in proximity are joined by ligation. The ligated reporter DNA strands are then detected via methods such as real-time PCR or next-generation sequencing (NGS). We describe how to construct assays that can offer improved detection specificity by virtue of recognition by three antibodies, as well as enhanced sensitivity owing to reduced background and amplified detection. Finally, we also illustrate how the assays can be applied for parallel detection of proteins, taking advantage of the oligonucleotide ligation step to avoid background problems that might arise with multiplexing. The protocol for the singleplex solid-phase proximity ligation assay takes ~5 h. The multiplex version of the assay takes 7-8 h depending on whether quantitative PCR (qPCR) or sequencing is used as the readout. The time for the sequencing-based protocol includes the library preparation but not the actual sequencing, as times may vary based on the choice of sequencing platform.

Toxin detection using a fiber-optic-based biosensor

NASA Astrophysics Data System (ADS)

Ogert, Robert A.; Shriver-Lake, Lisa C.; Ligler, Frances S.

1993-05-01

Using an evanescent wave fiber optic-based biosensor developed at Naval Research Laboratory, ricin toxin can be detected in the low ng/ml range. Sensitivity was established at 1 - 5 ng/ml using a two-step assay. The two-step assay showed enhanced signal levels in comparison to a one-step assay. A two-step assay utilizes a 10 minute incubation of an immobilized affinity purified anti-ricin antibody fiber optic probe in the ricin sample before placement in a solution of fluorophore-labeled goat anti-ricin antibodies. The specific fluorescent signal is obtained by the binding of the fluorophore-labeled antibodies to ricin which is bound by the immobilized antibodies on the fiber optic probe. The toxin can be detected directly from urine and river water using this fiber optic assay.

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

NASA Astrophysics Data System (ADS)

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.

Rapid, highly sensitive detection of herpes simplex virus-1 using multiple antigenic peptide-coated superparamagnetic beads.

PubMed

Ran, Ying-Fen; Fields, Conor; Muzard, Julien; Liauchuk, Viktoryia; Carr, Michael; Hall, William; Lee, Gil U

2014-12-07

A sensitive, rapid, and label free magnetic bead aggregation (MBA) assay has been developed that employs superparamagnetic (SPM) beads to capture, purify, and detect model proteins and the herpes simplex virus (HSV). The MBA assay is based on monitoring the aggregation state of a population of SPM beads using light scattering of individual aggregates. A biotin-streptavidin MBA assay had a femtomolar (fM) level sensitivity for analysis times less than 10 minutes, but the response of the assay becomes nonlinear at high analyte concentrations. A MBA assay for the detection of HSV-1 based on a novel peptide probe resulted in the selective detection of the virus at concentrations as low as 200 viral particles (vp) per mL in less than 30 min. We define the parameters that determine the sensitivity and response of the MBA assay, and the mechanism of enhanced sensitivity of the assay for HSV. The speed, relatively low cost, and ease of application of the MBA assay promise to make it useful for the identification of viral load in resource-limited and point-of-care settings where molecular diagnostics cannot be easily implemented.

Enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence.

PubMed Central

Crouch, C F

1995-01-01

AIMS--To evaluate the clinical performance of enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence. METHODS--Classification of routine clinical samples from the originating laboratories was compared with that obtained using the chemiluminescence based assays. Resolution of discordant results was achieved by testing in alternative enzyme immunoassays (IgM) or by an independent laboratory using the dye test (IgG). RESULTS--Compared with resolved data, the IgM assay was found to be highly specific (100%) with a cut off selected to give optimal performance with respect to both the early detection of specific IgM and the detection of persistent levels of specific IgM (sensitivity 98%). Compared with resolved data, the IgG assay was shown to have a sensitivity and a specificity of 99.4%. CONCLUSIONS--The Amerlite Toxo IgM assay possesses high levels of sensitivity and specificity. Assay interference due to rheumatoid factor like substances is not a problem. The Amerlite Toxo IgG assay possesses good sensitivity and specificity, but is less sensitive for the detection of seroconversion than methods detecting both IgG and IgM. PMID:7560174

An improved Abbott ARCHITECT assay for the detection of hepatitis B virus surface antigen (HBsAg).

PubMed

Lou, Sheng C; Pearce, Sandra K; Lukaszewska, Teresa X; Taylor, Russell E; Williams, Gregg T; Leary, Thomas P

2011-05-01

The sensitive and accurate detection of hepatitis B virus surface antigen (HBsAg) is critical to the identification of infection and the prevention of transfusion transmitted disease. Improvement in HBsAg assay sensitivity is essential to reduce the window to detect an acute HBV infection. Additionally, the sensitive detection of HBsAg mutants that continue to evolve due to vaccine escape, immune selection and an error prone reverse transcriptase is a necessity. A fully automated HBsAg prototype assay on the Abbott ARCHITECT instrument was developed to improve sensitivity and mutant detection. This magnetic microparticle-based assay utilizes anti-HBsAg monoclonal antibodies to capture antigen present in serum or plasma. Captured antigen is then detected using anti-HBsAg antibody conjugated with the chemiluminescent compound, acridinium. The sensitivity of the ARCHITECT HBsAg prototype assay was improved as compared to the current ARCHITECT, PRISM, and competitor HBsAg assays. The enhancement in assay sensitivity was demonstrated by the use of commercially available HBV seroconversion panels. The prototype assay detected more panel members (185 of 383) vs. the current ARCHITECT (171), PRISM (181), or competitor HBsAg assays (73/140 vs. 62/140, respectively). The ARCHITECT prototype assay also efficiently detected all mutants evaluated. Finally, the sensitivity improvement did not compromise the specificity of the assay (99.94%). An improved Abbott ARCHITECT HBsAg prototype assay with enhanced detection of HBsAg and HBsAg mutants, as well as equivalent specificity was developed for the detection, diagnosis, and management of HBV infection. Copyright © 2011 Elsevier B.V. All rights reserved.

Recombinase polymerase amplification-based assay to diagnose Giardia in stool samples.

PubMed

Crannell, Zachary Austin; Cabada, Miguel Mauricio; Castellanos-Gonzalez, Alejandro; Irani, Ayesha; White, Arthur Clinton; Richards-Kortum, Rebecca

2015-03-01

Giardia duodenalis is one of the most commonly identified parasites in stool samples. Although relatively easy to treat, giardiasis can be difficult to detect as it presents similar to other diarrheal diseases. Here, we present a recombinase polymerase amplification-based Giardia (RPAG) assay to detect the presence of Giardia in stool samples. The RPAG assay was characterized on the bench top using stool samples spiked with Giardia cysts where it showed a limit-of-detection nearly as low as the gold standard polymerase chain reaction assay. The RPAG assay was then tested in the highlands of Peru on 104 stool samples collected from the surrounding communities where it showed 73% sensitivity and 95% specificity against a polymerase chain reaction and microscopy composite gold standard. Further improvements in clinical sensitivity will be needed for the RPAG assay to have clinical relevance. © The American Society of Tropical Medicine and Hygiene.

Recombinase Polymerase Amplification-Based Assay to Diagnose Giardia in Stool Samples

PubMed Central

Crannell, Zachary Austin; Cabada, Miguel Mauricio; Castellanos-Gonzalez, Alejandro; Irani, Ayesha; White, Arthur Clinton; Richards-Kortum, Rebecca

2015-01-01

Giardia duodenalis is one of the most commonly identified parasites in stool samples. Although relatively easy to treat, giardiasis can be difficult to detect as it presents similar to other diarrheal diseases. Here, we present a recombinase polymerase amplification-based Giardia (RPAG) assay to detect the presence of Giardia in stool samples. The RPAG assay was characterized on the bench top using stool samples spiked with Giardia cysts where it showed a limit-of-detection nearly as low as the gold standard polymerase chain reaction assay. The RPAG assay was then tested in the highlands of Peru on 104 stool samples collected from the surrounding communities where it showed 73% sensitivity and 95% specificity against a polymerase chain reaction and microscopy composite gold standard. Further improvements in clinical sensitivity will be needed for the RPAG assay to have clinical relevance. PMID:25510713

Functional Assays for Ricin Detection

NASA Astrophysics Data System (ADS)

Ezan, Eric; Duriez, Elodie; Fenaille, François; Becher, François

In this review, we provide background information on ricin structure, present available functional assays for other toxins that are potential biothreat agents, and finish by describing the functional assay of ricin itself. Using appropriate sample preparation and optimized detection based on N-glycosidase activity, we demonstrate that specific detection of whole ricin at a level of around 0.1 ng/mL is possible and applicable to environmental samples.

Low-Cost Charged-Coupled Device (CCD) Based Detectors for Shiga Toxins Activity Analysis.

PubMed

Rasooly, Reuven; Prickril, Ben; Bruck, Hugh A; Rasooly, Avraham

2017-01-01

To improve food safety there is a need to develop simple, low-cost sensitive devices for detection of food-borne pathogens and their toxins. We describe a simple, low-cost webcam-based detector which can be used for various optical detection modalities, including fluorescence, chemiluminescence, densitometry, and colorimetric assays. The portable battery-operated CCD-based detection system consists of four modules: (1) a webcam to measure and record light emission, (2) a sample plate to perform assays, (3) a light emitting diode (LED) for illumination, and (4) a portable computer to acquire and analyze images. To demonstrate the technology, we used a cell based assay for fluorescence detection of the activity of the food borne Shiga toxin type 2 (Stx2), differentiating between biologically active toxin and inactive toxin which is not a risk. The assay is based on Shiga toxin inhibition of cell protein synthesis measured through inhibition of the green fluorescent protein (GFP). In this assay, GFP emits light at 509 nm when excited with a blue LED equipped with a filter at 486 nm. The emitted light is then detected with a green filter at 535 nm. Toxin activity is measured through a reduction in the 509 nm emission. In this system the level of detection (LOD) for Stx2 was 0.1 pg/ml, similar to the LOD of commercial fluorometers. These results demonstrate the utility and potential of low cost detectors for toxin activity. This approach could be readily adapted to the detection of other food-borne toxins.

Evaluation of two commercial real-time PCR assays for detecting Campylobacter in broiler carcass rinses.

USDA-ARS?s Scientific Manuscript database

Traditional plating methods are reliable means for Campylobacter identification from poultry samples but automated gene-based detection systems now available can reduce assay time, data collection and analysis. Bio-Rad and DuPont Qualicon recently introduced Campylobacter assays for their real-time ...

Phone camera detection of glucose blood level based on magnetic particles entrapped inside bubble wrap.

PubMed

Martinkova, Pavla; Pohanka, Miroslav

2016-12-18

Glucose is an important diagnostic biochemical marker of diabetes but also for organophosphates, carbamates, acetaminophens or salicylates poisoning. Hence, innovation of accurate and fast detection assay is still one of priorities in biomedical research. Glucose sensor based on magnetic particles (MPs) with immobilized enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) was developed and the GOx catalyzed reaction was visualized by a smart-phone-integrated camera. Exponential decay concentration curve with correlation coefficient 0.997 and with limit of detection 0.4 mmol/l was achieved. Interfering and matrix substances were measured due to possibility of assay influencing and no effect of the tested substances was observed. Spiked plasma samples were also measured and no influence of plasma matrix on the assay was proved. The presented assay showed complying results with reference method (standard spectrophotometry based on enzymes glucose oxidase and peroxidase inside plastic cuvettes) with linear dependence and correlation coefficient 0.999 in concentration range between 0 and 4 mmol/l. On the grounds of measured results, method was considered as highly specific, accurate and fast assay for detection of glucose.

Detection of proteins using a colorimetric bio-barcode assay.

PubMed

Nam, Jwa-Min; Jang, Kyung-Jin; Groves, Jay T

2007-01-01

The colorimetric bio-barcode assay is a red-to-blue color change-based protein detection method with ultrahigh sensitivity. This assay is based on both the bio-barcode amplification method that allows for detecting miniscule amount of targets with attomolar sensitivity and gold nanoparticle-based colorimetric DNA detection method that allows for a simple and straightforward detection of biomolecules of interest (here we detect interleukin-2, an important biomarker (cytokine) for many immunodeficiency-related diseases and cancers). The protocol is composed of the following steps: (i) conjugation of target capture molecules and barcode DNA strands onto silica microparticles, (ii) target capture with probes, (iii) separation and release of barcode DNA strands from the separated probes, (iv) detection of released barcode DNA using DNA-modified gold nanoparticle probes and (v) red-to-blue color change analysis with a graphic software. Actual target detection and quantification steps with premade probes take approximately 3 h (whole protocol including probe preparations takes approximately 3 days).

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

PubMed

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

2016-08-03

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

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

PubMed

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. Copyright © 2016 Elsevier Inc. All rights reserved.

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

PubMed

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

2017-09-01

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

A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates

PubMed Central

Wilson, Peter M.; LaBonte, Melissa J.; Russell, Jared; Louie, Stan; Ghobrial, Andrew A.; Ladner, Robert D.

2011-01-01

Current methods for measuring deoxyribonucleoside triphosphates (dNTPs) employ reagent and labor-intensive assays utilizing radioisotopes in DNA polymerase-based assays and/or chromatography-based approaches. We have developed a rapid and sensitive 96-well fluorescence-based assay to quantify cellular dNTPs utilizing a standard real-time PCR thermocycler. This assay relies on the principle that incorporation of a limiting dNTP is required for primer-extension and Taq polymerase-mediated 5–3′ exonuclease hydrolysis of a dual-quenched fluorophore-labeled probe resulting in fluorescence. The concentration of limiting dNTP is directly proportional to the fluorescence generated. The assay demonstrated excellent linearity (R2 > 0.99) and can be modified to detect between ∼0.5 and 100 pmol of dNTP. The limits of detection (LOD) and quantification (LOQ) for all dNTPs were defined as <0.77 and <1.3 pmol, respectively. The intra-assay and inter-assay variation coefficients were determined to be <4.6% and <10%, respectively with an accuracy of 100 ± 15% for all dNTPs. The assay quantified intracellular dNTPs with similar results obtained from a validated LC–MS/MS approach and successfully measured quantitative differences in dNTP pools in human cancer cells treated with inhibitors of thymidylate metabolism. This assay has important application in research that investigates the influence of pathological conditions or pharmacological agents on dNTP biosynthesis and regulation. PMID:21576234

Negative interferences by calcium dobesilate in the detection of five serum analytes involving Trinder reaction-based assays

PubMed Central

Wu, Jie; Zhao, Fang; Xia, Liangyu; Cheng, Xinqi; Liu, Qian; Liu, Li; Xu, Ermu; Qiu, Ling

2018-01-01

Previously, we reported the strong negative interference of calcium dobesilate, a vasoprotective agent, in creatinine assays involving the Trinder reaction. It is hypothesized that a similar effect occurs in the detection of uric acid (UA), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). The interferences of calcium dobesilate during the detection of the five serum analytes were investigated on automated systems/analysers, and the effects were compared among eight different assay systems for each analyte. A calcium dobesilate standard was added into two sets of the blank serum pools of each analyte at final concentrations of 0, 2, 4, 8, 16, 32, and 64 μg/mL. The percentage deviation of each analyte value was calculated between each drug concentration and the drug-free samples. The clinically acceptable error levels for UA, TC, TG, HDL-C, and LDL-C were defined as ±4.87%, ±4.1%, ±9.57%, ±5.61%, and ±5.46%, respectively. The observed interference was concentration dependent for each analyte. In the presence of 16 μg/mL calcium dobesilate, which was within the therapeutic range, all seven Trinder reaction-based UA assay systems, two TG assay systems, two HDL-C assay systems and one TC assay system exhibited negative drug interferences. Calcium dobesilate negatively interferes with the detection of UA, TG, TC, and HDL-C in assay systems based on the Trinder reaction. The effect was most significant in UA and TG detection. PMID:29432460

Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling

PubMed Central

Nakatsuma, Akira; Kaneda, Mugiho; Kodama, Hiromi; Morikawa, Mika; Watabe, Satoshi; Nakaishi, Kazunari; Yamashita, Masakane; Yoshimura, Teruki; Miura, Toshiaki; Ninomiya, Masaki; Ito, Etsuro

2015-01-01

To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10-18 moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10-18 moles of the p24/assay corresponds to ca. 103 copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (102 copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA. PMID:26098695

Electrochemical detection of microRNAs via gap hybridization assay.

PubMed

Pöhlmann, Christopher; Sprinzl, Mathias

2010-06-01

MicroRNAs have recently been associated with cancer development by acting as tumor suppressors or oncogenes and could therefore be applied as molecular markers for early diagnosis of cancer. In this work, we established a rapid, selective, and sensitive gap hybridization assay for detection of mature microRNAs based on four components DNA/RNA hybridization and electrochemical detection using esterase 2-oligodeoxynucleotide conjugates. Complementary binding of microRNA to a gap built of capture and detector oligodeoxynucleotide, the reporter enzyme is brought to the vicinity of the electrode and produces enzymatically an electrochemical signal. In the absence of microRNA, the gap between capture and detector oligodeoxynucleotide is not filled, and missing base stacking energy destabilizes the hybridization complex. The gap hybridization assay demonstrates selective detection of miR-16 within a mixture of other miRNAs, including the feasibility of single mismatch discrimination. Applying the biosensor assay, a detection limit of 2 pM or 2 amol of miR-16 was obtained. Using isolated total RNA from human breast adenocarcinoma MCF-7 cells, the assay detected specifically miR-21 and miR-16 in parallel, and higher expression of oncogene miR-21 compared to miR-16 was demonstrated. Including RNA isolation, the gap hybridization assay was developed with a total assay time of 60 min and without the need for reverse transcription PCR amplification of the sample. The characteristics of the assay developed in this work could satisfy the need for rapid and easy methods for early cancer marker detection in clinical diagnostics.

The applicability of TaqMan-based quantitative real-time PCR assays for detecting and enumeratIng Cryptosporidium spp. oocysts in the environment

EPA Science Inventory

Molecular detection methods such as PCR have been extensively used to type Cryptosporidium oocysts detected in the environment. More recently, studies have developed quantitative real-time PCR assays for detection and quantification of microbial contaminants in water as well as ...

A reporter system for replication-competent gammaretroviruses: the inGluc-MLV-DERSE assay

PubMed Central

Aloia, Amanda L.; Duffy, Lisa; Pak, Vladimir; Lee, KyeongEun; Sanchez-Martinez, Silvia; Derse, David; Heidecker, Gisela; Cornetta, Kenneth; Rein, Alan

2012-01-01

While novel retroviral vectors for use in gene-therapy products are reducing the potential for formation of replication-competent retrovirus (RCR), it remains crucial to screen products for RCR for both research and clinical purposes. For clinical grade gammaretrovirus-based vectors, RCR screening is achieved by an extended S+L− or marker rescue assay, while standard methods for replication-competent lentivirus detection are still in development. In this report, we describe a rapid and sensitive method for replication-competent gammaretrovirus detection. We used this assay to detect three members of the gammaretrovirus family and compared the sensitivity of our assay with well-established methods for retrovirus detection, including the extended S+L− assay. Results presented here demonstrate that this assay should be useful for gene-therapy product testing. PMID:22402321

Development and application of two independent real-time PCR assays to detect clinically relevant Mucorales species.

PubMed

Springer, Jan; Goldenberger, Daniel; Schmidt, Friderike; Weisser, Maja; Wehrle-Wieland, Elisabeth; Einsele, Hermann; Frei, Reno; Löffler, Jürgen

2016-03-01

PCR-based detection of Mucorales species could improve diagnosis of suspected invasive fungal infection, leading to a better patient outcome. This study describes two independent probe-based real-time PCR tests for detection of clinically relevant Mucorales, targeting specific fragments of the 18S and the 28S rRNA genes. Both assays have a short turnaround time, allow fast, specific and very sensitive detection of clinically relevant Mucorales and have the potential to be used as quantitative tests. They were validated on various clinical samples (fresh and formalin-fixed paraffin-embedded specimens, mainly biopsies, n = 17). The assays should be used as add-on tools to complement standard techniques; a combined approach of both real-time PCR assays has 100 % sensitivity. Genus identification by subsequent sequencing is possible for amplicons of the 18S PCR assay. In conclusion, combination of the two independent Mucorales assays described in this study, 18S and 28S, detected all clinical samples associated with proven Mucorales infection (n = 10). Reliable and specific identification of Mucorales is a prerequisite for successful antifungal therapy as these fungi show intrinsic resistance to voriconazole and caspofungin.

Development and performance assessment of a luminex xMAP® direct hybridization assay for the detection and identification of indoor air fungal contamination.

PubMed

Libert, Xavier; Packeu, Ann; Bureau, Fabrice; Roosens, Nancy H; De Keersmaecker, Sigrid C J

2017-01-01

Considered as a public health problem, indoor fungal contamination is generally monitored using classical protocols based on culturing. However, this culture dependency could influence the representativeness of the fungal population detected in an analyzed sample as this includes the dead and uncultivable fraction. Moreover, culture-based protocols are often time-consuming. In this context, molecular tools are a powerful alternative, especially those allowing multiplexing. In this study a Luminex xMAP® assay was developed for the simultaneous detection of 10 fungal species which are most frequently in indoor air and that may cause health problems. This xMAP® assay was found to be sensitive, i.e. its limit of detection is ranging between 0.05 and 0.01 ng of gDNA. The assay was subsequently tested with environmental air samples which were also analyzed with a classical protocol. All the species identified with the classical method were also detected with the xMAP® assay, however in a shorter time frame. These results demonstrate that the Luminex xMAP® fungal assay developed in this study could contribute to the improvement of public health and specifically to the indoor fungal contamination treatment.

Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species

PubMed Central

McCuiston, Jamie L.; Hudson, Laura C.; Subbotin, Sergei A.; Davis, Eric L.; Warfield, Colleen Y.

2007-01-01

A PCR-based diagnostic assay was developed for early detection and identification of Aphelenchoides fragariae directly in host plant tissues using the species-specific primers AFragFl and AFragRl that amplify a 169-bp fragment in the internal transcribed spacer (ITS1) region of ribosomal DNA. These species-specific primers did not amplify DNA from Aphelenchoides besseyi or Aphelenchoides ritzemabosi. The PCR assay was sensitive, detecting a single nematode in a background of plant tissue extract. The assay accurately detected A. fragariae in more than 100 naturally infected, ornamental plant samples collected in North Carolina nurseries, garden centers and landscapes, including 50 plant species not previously reported as hosts of Aphelenchoides spp. The detection sensitivity of the PCR-based assay was higher for infected yet asymptomatic plants when compared to the traditional, water extraction method for Aphelenchoides spp. detection. The utility of using NaOH extraction for rapid preparation of total DNA from plant samples infected with A. fragariae was demonstrated. PMID:19259510

Comparative Evaluation of Veriflow® Listeria monocytogenes to USDA and AOAC Culture Based Methods for the Detection of Listeria monocytogenes in Food.

PubMed

Joelsson, Adam C; Brown, Ashley S; Puri, Amrita; Keough, Martin P; Gaudioso, Zara E; Siciliano, Nicholas A; Snook, Adam E

2015-01-01

Veriflow® Listeria monocytogenes (LM) is a molecular based assay for the presumptive detection of Listeria monocytogenes from environmental surfaces, dairy, and ready-to-eat (RTE) food matrixes (hot dogs and deli meat). The assay utilizes a PCR detection method coupled with a rapid, visual, flow-based assay that develops in 3 min post PCR amplification and requires only 24 h of enrichment for maximum sensitivity. The Veriflow LM system eliminates the need for sample purification, gel electrophoresis, or fluorophore-based detection of target amplification, and does not require complex data analysis. This Performance Tested Method(SM) validation study demonstrated the ability of the Veriflow LM method to detect low levels of artificially inoculated L. monocytogenes in seven distinct environmental and food matrixes. In each unpaired reference comparison study, probability of detection analysis indicated no significant difference between the Veriflow LM method and the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook 8.08 or AOAC 993.12 reference method. Fifty strains of L. monocytogenes were detected in the inclusivity study, while 39 nonspecific organisms were undetected in the exclusivity study. The study results show that Veriflow LM is a sensitive, selective, and robust assay for the presumptive detection of L. monocytogenes sampled from environmental, dairy, or RTE (hot dogs and deli meat) food matrixes.

Challenging loop-mediated isothermal amplification (LAMP) technique for molecular detection of Toxoplasma gondii.

PubMed

Fallahi, Shirzad; Mazar, Zahra Arab; Ghasemian, Mehrdad; Haghighi, Ali

2015-05-01

To compare analytical sensitivity and specificity of a newly described DNA amplification technique, LAMP and nested PCR assay targeting the RE and B1 genes for the detection of Toxoplasma gondii (T. gondii) DNA. The analytical sensitivity of LAMP and nested-PCR was obtained against10-fold serial dilutions of T. gondii DNA ranging from 1 ng to 0.01 fg. DNA samples of other parasites and human chromosomal DNA were used to determine the specificity of molecular assays. After testing LAMP and nested-PCR in duplicate, the detection limit of RE-LAMP, B1-LAMP, RE-nested PCR and B1-nested PCR assays was one fg, 100 fg, 1 pg and 10 pg of T. gondii DNA respectively. All the LAMP assays and nested PCRs were 100% specific. The RE-LAMP assay revealed the most sensitivity for the detection of T. gondii DNA. The obtained results demonstrate that the LAMP technique has a greater sensitivity for detection of T. gondii. Furthermore, these findings indicate that primers based on the RE are more suitable than those based on the B1 gene. However, the B1-LAMP assay has potential as a diagnostic tool for detection of T. gondii. Copyright © 2015 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

Rapid molecular detection of invasive species in ballast and harbor water by integrating environmental DNA and light transmission spectroscopy.

PubMed

Egan, Scott P; Grey, Erin; Olds, Brett; Feder, Jeffery L; Ruggiero, Steven T; Tanner, Carol E; Lodge, David M

2015-04-07

Invasive species introduced via the ballast water of commercial ships cause enormous environmental and economic damage worldwide. Accurate monitoring for these often microscopic and morphologically indistinguishable species is challenging but critical for mitigating damages. We apply eDNA sampling, which involves the filtering and subsequent DNA extraction of microscopic bits of tissue suspended in water, to ballast and harbor water sampled during a commercial ship's 1400 km voyage through the North American Great Lakes. Using a lab-based gel electrophoresis assay and a rapid, field-ready light transmission spectroscopy (LTS) assay, we test for the presence of two invasive species: quagga (Dreissena bugensis) and zebra (D. polymorpha) mussels. Furthermore, we spiked a set of uninfested ballast and harbor samples with zebra mussel tissue to further test each assay's detection capabilities. In unmanipulated samples, zebra mussel was not detected, while quagga mussel was detected in all samples at a rate of 85% for the gel assay and 100% for the LTS assay. In the spiked experimental samples, both assays detected zebra mussel in 94% of spiked samples and 0% of negative controls. Overall, these results demonstrate that eDNA sampling is effective for monitoring ballast-mediated invasions and that LTS has the potential for rapid, field-based detection.

Non-Enzymatic Detection of Bacterial Genomic DNA Using the Bio-Barcode Assay

PubMed Central

Hill, Haley D.; Vega, Rafael A.; Mirkin, Chad A.

2011-01-01

The detection of bacterial genomic DNA through a non-enzymatic nanomaterials based amplification method, the bio-barcode assay, is reported. The assay utilizes oligonucleotide functionalized magnetic microparticles to capture the target of interest from the sample. A critical step in the new assay involves the use of blocking oligonucleotides during heat denaturation of the double stranded DNA. These blockers bind to specific regions of the target DNA upon cooling, and prevent the duplex DNA from re-hybridizing, which allows the particle probes to bind. Following target isolation using the magnetic particles, oligonucleotide functionalized gold nanoparticles act as target recognition agents. The oligonucleotides on the nanoparticle (barcodes) act as amplification surrogates. The barcodes are then detected using the Scanometric method. The limit of detection for this assay was determined to be 2.5 femtomolar, and this is the first demonstration of a barcode type assay for the detection of double stranded, genomic DNA. PMID:17927207

Urine sample used for detection of toxoplasma gondii infection by loop-mediated isothermal amplification (LAMP).

PubMed

Hu, Xin; Pan, Chang-Wang; Li, Ya-Fei; Wang, Han; Tan, Feng

2012-02-01

In this study, a loop-mediated isothermal amplification (LAMP) assay was established to detect Toxoplasma gondii DNA in mice infected with T. gondii PRU strain. This LAMP assay was based on the sequence of highly repetitive B1 gene. The detection limit of T. gondii LAMP assay was 1 pg of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. The LAMP assay was also highly specific for T. gondii and able to detect T. gondii DNA in urine of mice treated with dexamethasone at 90 day post infection (p.i.), although this assay could not detect the DNA in mice urine 2-6 days p.i. These results demonstrated that LAMP is effective for evaluation of therapy effectiveness for T. gondii infection. The established LAMP assay may represent a useful and practical tool for the routine diagnosis and therapeutic evaluation of human toxoplasmosis.

Novel Time-Resolved Fluorescence Europium Nanoparticle Immunoassay for Detection of Human Immunodeficiency Virus-1 Group O Viruses Using Microplate and Microchip Platforms

PubMed Central

Liu, Jikun; Mahtani, Prerna; Zhang, Panhe; Du, Bingchen; Ragupathy, Viswanath; Devadas, Krishnakumar

2016-01-01

Abstract Accurate detection and quantification of HIV-1 group O viruses have been challenging for currently available HIV assays. We have developed a novel time-resolved fluorescence (TRF) europium nanoparticle immunoassay for HIV-1 group O detection using a conventional microplate enzyme-linked immunosorbent assay (ELISA) and a microchip platform. We screened several antibodies for optimal reactivity with several HIV-1 group O strains and identified antibodies that can detect all the strains of HIV-1 group O that were available for testing. The antibodies were used to develop a conventional ELISA format assay and an in-house developed europium nanoparticle-based assay for sensitivity. The method was evaluated on both microwell plate and microchip platforms. We identified two specific and sensitive antibodies among the six we screened. The antibodies, C65691 and ANT-152, were able to quantify 15 and detect all 17 group O viruses, respectively, as they were broadly cross-reactive with all HIV-1 group O strains and yielded better signals compared with other antibodies. We have developed a sensitive assay that reflects the actual viral load in group O samples by using an appropriate combination of p24 antibodies that enhance group O detection and a highly sensitive TRF-based europium nanoparticle for detection. The combination of ANT-152 and C65690M in the ratio 3:1 was able to give significantly higher signals in our europium-based assay compared with using any single antibody. PMID:26978478

Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas▿ †

PubMed Central

Hodgetts, Jennifer; Boonham, Neil; Mumford, Rick; Dickinson, Matthew

2009-01-01

Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays. PMID:19270148

Development of a Microsphere-based Immunoassay for Serological Detection of African Horse Sickness Virus and Comparison with Other Diagnostic Techniques.

PubMed

Sánchez-Matamoros, A; Beck, C; Kukielka, D; Lecollinet, S; Blaise-Boisseau, S; Garnier, A; Rueda, P; Zientara, S; Sánchez-Vizcaíno, J M

2016-12-01

African horse sickness (AHS) is a viral disease that causes high morbidity and mortality rates in susceptible Equidae and therefore significant economic losses. More rapid, sensitive and specific assays are required by diagnostic laboratories to support effective surveillance programmes. A novel microsphere-based immunoassay (Luminex assay) in which beads are coated with recombinant AHS virus (AHSV) structural protein 7 (VP7) has been developed for serological detection of antibodies against VP7 of any AHSV serotype. The performance of this assay was compared with that of a commercial enzyme-linked immunosorbent assay (ELISA) and commercial lateral flow assay (LFA) on a large panel of serum samples from uninfected horses (n = 92), from a reference library of all AHSV serotypes (n = 9), on samples from horses experimentally infected with AHSV (n = 114), and on samples from West African horses suspected of having AHS (n = 85). The Luminex assay gave the same negative results as ELISA when used to test the samples from uninfected horses. Both assays detected antibodies to all nine AHSV serotypes. In contrast, the Luminex assay detected a higher rate of anti-VP7 positivity in the West African field samples than did ELISA or LFA. The Luminex assay detected anti-VP7 positivity in experimentally infected horses at 7 days post-infection, compared to 13 days for ELISA. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple ASHV antigens can be detected simultaneously. This would be useful for serotyping or for differentiating infected from vaccinated animals. © 2015 Blackwell Verlag GmbH.

Development of internally controlled duplex real-time NASBA diagnostics assays for the detection of microorganisms associated with bacterial meningitis.

PubMed

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Real-Time Reverse Transcription–Polymerase Chain Reaction Assay for SARS-associated Coronavirus

PubMed Central

Emery, Shannon L.; Bowen, Michael D.; Newton, Bruce R.; Winchell, Jonas M.; Meyer, Richard F.; Tong, Suxiang; Cook, Byron T.; Holloway, Brian P.; McCaustland, Karen A.; Rota, Paul A.; Bankamp, Bettina; Lowe, Luis E.; Ksiazek, Tom G.; Bellini, William J.; Anderson, Larry J.

2004-01-01

A real-time reverse transcription–polymerase chain reaction (RT-PCR) assay was developed to rapidly detect the severe acute respiratory syndrome–associated coronavirus (SARS-CoV). The assay, based on multiple primer and probe sets located in different regions of the SARS-CoV genome, could discriminate SARS-CoV from other human and animal coronaviruses with a potential detection limit of <10 genomic copies per reaction. The real-time RT-PCR assay was more sensitive than a conventional RT-PCR assay or culture isolation and proved suitable to detect SARS-CoV in clinical specimens. Application of this assay will aid in diagnosing SARS-CoV infection. PMID:15030703

Evaluation of a novel ultra-sensitive nanoparticle probe-based assay for ricin detection.

PubMed

Yin, Hui-qiong; Jia, Min-xian; Shi, Li-jun; Liu, Jun; Wang, Rui; Lv, Mao-min; Ma, Yu-yuan; Zhao, Xiong; Zhang, Jin-gang

2014-01-01

A gold nanoparticle (GNP) probe-based assay (GNPA) modified from the bio-barcode assay (BCA) was developed for ultrasensitive detection of ricin, a potential biothreat agent. In the GNPA, a chain of ricin was captured by a GNP probe coated with polyclonal antibodies and single-stranded signal DNA. A magnetic microparticle (MMP) probe coated with ricin A chain monoclonal antibody was then added to form an immuno-complex. After being magnetically separated, the immuno-complex containing the single-stranded signal DNA was characterized by PCR and real-time PCR. A detection limit of 10(-2) fg/ml was determined for the ricin A chain; this is eight orders of magnitude more sensitive than that achieved with an ELISA and two orders more sensitive than that obtained with the BCA. The coefficients of variation (CV) of the intra- and inter-assay values ranged from 3.82-6.46%. The results here show that this novel assay is an ultrasensitive method for detection of ricin proteins and may be suitable for the ultrasensitive detection of other proteins.

A highly sensitive and selective diagnostic assay based on virus nanoparticles

NASA Astrophysics Data System (ADS)

Park, Jin-Seung; Cho, Moon Kyu; Lee, Eun Jung; Ahn, Keum-Young; Lee, Kyung Eun; Jung, Jae Hun; Cho, Yunjung; Han, Sung-Sik; Kim, Young Keun; Lee, Jeewon

2009-04-01

Early detection of the protein marker troponin I in patients with a higher risk of acute myocardial infarction can reduce the risk of death from heart attacks. Most troponin assays are currently based on the conventional enzyme linked immunosorbent assay and have detection limits in the nano- and picomolar range. Here, we show that by combining viral nanoparticles, which are engineered to have dual affinity for troponin antibodies and nickel, with three-dimensional nanostructures including nickel nanohairs, we can detect troponin levels in human serum samples that are six to seven orders of magnitude lower than those detectable using conventional enzyme linked immunosorbent assays. The viral nanoparticle helps to orient the antibodies for maximum capture of the troponin markers. High densities of antibodies on the surfaces of the nanoparticles and nanohairs lead to greater binding of the troponin markers, which significantly enhances detection sensitivities. The nickel nanohairs are re-useable and can reproducibly differentiate healthy serum from unhealthy ones. We expect other viral nanoparticles to form similar highly sensitive diagnostic assays for a variety of other protein markers.

The Androgen Receptor and Its Use in Biological Assays: Looking Toward Effect-Based Testing and Its Applications

PubMed Central

Cadwallader, Amy B.; Lim, Carol S.; Rollins, Douglas E.; Botrè, Francesco

2015-01-01

Steroid abuse is a growing problem among amateur and professional athletes. Because of an inundation of newly and illegally synthesized steroids with minor structural modifications and other designer steroid receptor modulators, there is a need to develop new methods of detection which do not require prior knowledge of the abused steroid structure. The number of designer steroids currently being abused is unknown because detection methods in general are only identifying substances with a known structure. The detection of doping is moving away from merely checking for exposure to prohibited substance toward detecting an effect of prohibited substances, as biological assays can do. Cell-based biological assays are the next generation of assays which should be utilized by antidoping laboratories; they can detect androgenic anabolic steroid and other human androgen receptor (hAR) ligand presence without knowledge of their structure and assess the relative biological activity of these compounds. This review summarizes the hAR and its action and discusses its relevance to sports doping and its use in biological assays. PMID:22080898

Quantitation of secreted proteins using mCherry fusion constructs and a fluorescent microplate reader.

PubMed

Duellman, Tyler; Burnett, John; Yang, Jay

2015-03-15

Traditional assays for secreted proteins include methods such as Western blot and enzyme-linked immunosorbent assay (ELISA) detection of the protein in the cell culture medium. We describe a method for the detection of a secreted protein based on fluorescent measurement of an mCherry fusion reporter. This microplate reader-based mCherry fluorescence detection method has a wide dynamic range of 4.5 orders of magnitude and a sensitivity that allows detection of 1 to 2fmol fusion protein. Comparison with the Western blot detection method indicated greater linearity, wider dynamic range, and a similar lower detection threshold for the microplate-based fluorescent detection assay of secreted fusion proteins. An mCherry fusion protein of matrix metalloproteinase-9 (MMP-9), a secreted glycoprotein, was created and expressed by transfection of human embryonic kidney (HEK) 293 cells. The cell culture medium was assayed for the presence of the fluorescent signal up to 32 h after transfection. The secreted MMP-9-mCherry fusion protein was detected 6h after transfection with a linear increase in signal intensity over time. Treatment with chloroquine, a drug known to inhibit the secretion of many proteins, abolished the MMP-9-mCherry secretion, demonstrating the utility of this method in a biological experiment. Copyright © 2014 Elsevier Inc. All rights reserved.

An Enzyme-Free Signal Amplification Technique for Ultrasensitive Colorimetric Assay of Disease Biomarkers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ye, Haihang; Yang, Kuikun; Tao, Jing

Enzyme-based colorimetric assays have been widely used in research labs and clinical diagnosis for decades. Nevertheless, as constrained by the performance of enzymes, their detection sensitivity has not been substantially improved in recent years, which inhibits many critical applications such as early detection of cancers. In this work, we demonstrate an enzyme-free signal amplification technique, based on gold vesicles encapsulated with Pd-Ir nanoparticles as peroxidase mimics, for colorimetric assay of disease biomarkers with significantly enhanced sensitivity. This technique overcomes the intrinsic limitations of enzymes, thanks to the superior catalytic efficiency of peroxidase mimics and the efficient loading and release ofmore » these mimics. Using human prostate surface antigen as a model biomarker, we demonstrated that the enzyme-free assay could reach a limit of detection at the femtogram/mL level, which is over 10 3-fold lower than that of conventional enzyme-based assay when the same antibodies and similar procedure were used.« less

An Enzyme-Free Signal Amplification Technique for Ultrasensitive Colorimetric Assay of Disease Biomarkers

DOE PAGES

Ye, Haihang; Yang, Kuikun; Tao, Jing; ...

2017-01-30

Enzyme-based colorimetric assays have been widely used in research labs and clinical diagnosis for decades. Nevertheless, as constrained by the performance of enzymes, their detection sensitivity has not been substantially improved in recent years, which inhibits many critical applications such as early detection of cancers. In this work, we demonstrate an enzyme-free signal amplification technique, based on gold vesicles encapsulated with Pd-Ir nanoparticles as peroxidase mimics, for colorimetric assay of disease biomarkers with significantly enhanced sensitivity. This technique overcomes the intrinsic limitations of enzymes, thanks to the superior catalytic efficiency of peroxidase mimics and the efficient loading and release ofmore » these mimics. Using human prostate surface antigen as a model biomarker, we demonstrated that the enzyme-free assay could reach a limit of detection at the femtogram/mL level, which is over 10 3-fold lower than that of conventional enzyme-based assay when the same antibodies and similar procedure were used.« less

Development of a polymerase chain reaction-based assay for the detection of Alternaria fungal contamination in food products.

PubMed

Zur, G; Hallerman, E M; Sharf, R; Kashi, Y

1999-10-01

Alternaria sp. are important fungal contaminants of vegetable, fruit, and grain products, including Alternaria alternata, a contaminant of tomato products. To date, the Howard method, based on microscopic observation of fungal filaments, has been the standard examination for inspection of tomato products. We report development of a polymerase chain reaction (PCR)-based method for detection of Alternaria DNA. PCR primers were designed to anneal to the internal transcribed regions ITS1 and ITS2 of the 5.8S rRNA gene of Alternaria but not to other microbial or tomato DNA. We demonstrate use of the PCR assay to detect Alternaria DNA in experimentally infested and commercially obtained tomato sauce and tomato powder. Use of the PCR method offers a rapid and sensitive assay for the presence of Alternaria DNA in tomato products. The apparent breakdown of DNA in tomato sauce may limit the utility of the assay to freshly prepared products. The assay for tomato powder is not affected by storage time.

Human papillomavirus detection and typing using a nested-PCR-RFLP assay.

PubMed

Coser, Janaina; Boeira, Thaís da Rocha; Fonseca, André Salvador Kazantzi; Ikuta, Nilo; Lunge, Vagner Ricardo

2011-01-01

It is clinically important to detect and type human papillomavirus (HPV) in a sensitive and specific manner. Development of a nested-polymerase chain reaction-restriction fragment length polymorphism (nested-PCR-RFLP) assay to detect and type HPV based on the analysis of L1 gene. Analysis of published DNA sequence of mucosal HPV types to select sequences of new primers. Design of an original nested-PCR assay using the new primers pair selected and classical MY09/11 primers. HPV detection and typing in cervical samples using the nested-PCR-RFLP assay. The nested-PCR-RFLP assay detected and typed HPV in cervical samples. Of the total of 128 clinical samples submitted to simple PCR and nested-PCR for detection of HPV, 37 (28.9%) were positive for the virus by both methods and 25 samples were positive only by nested-PCR (67.5% increase in detection rate compared with single PCR). All HPV positive samples were effectively typed by RFLP assay. The method of nested-PCR proved to be an effective diagnostic tool for HPV detection and typing.

An isothermal based recombinase polymerase amplification assay for rapid, sensitive and robust indexing of citrus yellow mosaic virus.

PubMed

Kumar, P V; Sharma, S K; Rishi, N; Ghosh, D K; Baranwal, V K

Management of viral diseases relies on definite and sensitive detection methods. Citrus yellow mosaic virus (CYMV), a double stranded DNA virus of the genus Badnavirus, causes yellow mosaic disease in citrus plants. CYMV is transmitted through budwood and requires a robust and simplified indexing protocol for budwood certification programme. The present study reports development and standardization of an isothermal based recombinase polymerase amplification (RPA) assay for a sensitive, rapid, easy, and cost-effective method for detection and diagnosis of CYMV. Two different oligonucleotide primer sets were designed from ORF III (coding for polyprotein) and ORF II (coding for virion associated protein) regions of CYMV to perform amplification assays. Comparative evaluation of RPA, PCR and immuno-capture recombinase polymerase amplification (IC-RPA) based assays were done using purified DNA and plant crude sap. CYMV infection was efficiently detected from the crude sap in RPA and IC-RPA assays. The primer set used in RPA was specific and did not show any cross-amplification with banana streak MY virus (BSMYV), another Badnavirus species. The results from the present study indicated that RPA assay can be used easily in routine indexing of citrus planting material. To the best of our knowledge, this is the first report on development of a rapid and simplified isothermal detection assay for CYMV and can be utilized as an effective technique in quarantine and budwood certification process.

Rapid and specific detection of porcine parvovirus by isothermal recombinase polymerase amplification assays.

PubMed

Yang, Yang; Qin, Xiaodong; Zhang, Wei; Li, Yanmin; Zhang, Zhidong

2016-10-01

Porcine parvovirus (PPV) is a major cause of swine reproductive failure and reported in many countries worldwide. Recombinase polymerase amplification (RPA) assays using a real-time fluorescent detection (PPV real-time RPA assay) and a lateral flow dipstick (PPV RPA LFD assay) were developed targeting PPV NS1 gene. The detection limit of PPV real-time RPA assay was 300 copies per reaction within 9 min at 38 °C, while the RPA LFD assay has a detection limit of 400 copies per reaction in less than 20 min at 38 °C. In both assays, there were no cross-reactions with porcine circovirus type 2, pseudorabies virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus, and foot-and-mouth disease virus. Based on a total of 128 clinical samples examined, the sensitivity and the specificity of the developed RPA assays for identification of PPV was 94.4% and 100%, respectively, when compared to real-time (qPCR) assay. Therefore, the RPA assay provides a rapid, sensitive and specific alternative for PPV detection. Copyright © 2016 Elsevier Ltd. All rights reserved.

A nucleic acid strand displacement system for the multiplexed detection of tuberculosis-specific mRNA using quantum dots

NASA Astrophysics Data System (ADS)

Gliddon, H. D.; Howes, P. D.; Kaforou, M.; Levin, M.; Stevens, M. M.

2016-05-01

The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis.The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis. Electronic supplementary information (ESI) available: Base pair mismatch tuning of CProbes. Binding capacity of the QDs. Theoretical limit of detection (LOD) for the monoplex systems. Kinetics of strand displacement. Kinetics of QProbe-CProbe binding. LOD and saturation point calculations. See DOI: 10.1039/c6nr00484a

Biosensors: Viruses for ultrasensitive assays

NASA Astrophysics Data System (ADS)

Donath, Edwin

2009-04-01

A three-dimensional assay based on genetically engineered viral nanoparticles and nickel nanohairs can detect much lower levels of protein markers associated with heart attacks than conventional assays.

Development and validation of a real-time PCR assay for the detection of anguillid herpesvirus 1.

PubMed

van Beurden, S J; Voorbergen-Laarman, M A; Roozenburg, I; van Tellingen, J; Haenen, O L M; Engelsma, M Y

2016-01-01

Anguillid herpesvirus 1 (AngHV1) causes a haemorrhagic disease with increased mortality in wild and farmed European eel, Anguilla anguilla (L.) and Japanese eel Anguilla japonica, Temminck & Schlegel). Detection of AngHV1 is currently based on virus isolation in cell culture, antibody-based typing assays or conventional PCR. We developed, optimized and concisely validated a diagnostic TaqMan probe based real-time PCR assay for the detection of AngHV1. The primers and probe target AngHV1 open reading frame 57, encoding the capsid protease and scaffold protein. Compared to conventional PCR, the developed real-time PCR is faster, less labour-intensive and has a reduced risk of cross-contamination. The real-time PCR assay was shown to be analytically sensitive and specific and has a high repeatability, efficiency and r(2) -value. The diagnostic performance of the assay was determined by testing 10% w/v organ suspensions and virus cultures from wild and farmed European eels from the Netherlands by conventional and real-time PCR. The developed real-time PCR assay is a useful tool for the rapid and sensitive detection of AngHV1 in 10% w/v organ suspensions from wild and farmed European eels. © 2015 John Wiley & Sons Ltd.

Live Cell Visualization of Multiple Protein-Protein Interactions with BiFC Rainbow.

PubMed

Wang, Sheng; Ding, Miao; Xue, Boxin; Hou, Yingping; Sun, Yujie

2018-05-18

As one of the most powerful tools to visualize PPIs in living cells, bimolecular fluorescence complementation (BiFC) has gained great advancement during recent years, including deep tissue imaging with far-red or near-infrared fluorescent proteins or super-resolution imaging with photochromic fluorescent proteins. However, little progress has been made toward simultaneous detection and visualization of multiple PPIs in the same cell, mainly due to the spectral crosstalk. In this report, we developed novel BiFC assays based on large-Stokes-shift fluorescent proteins (LSS-FPs) to detect and visualize multiple PPIs in living cells. With the large excitation/emission spectral separation, LSS-FPs can be imaged together with normal Stokes shift fluorescent proteins to realize multicolor BiFC imaging using a simple illumination scheme. We also further demonstrated BiFC rainbow combining newly developed BiFC assays with previously established mCerulean/mVenus-based BiFC assays to achieve detection and visualization of four PPI pairs in the same cell. Additionally, we prove that with the complete spectral separation of mT-Sapphire and CyOFP1, LSS-FP-based BiFC assays can be readily combined with intensity-based FRET measurement to detect ternary protein complex formation with minimal spectral crosstalk. Thus, our newly developed LSS-FP-based BiFC assays not only expand the fluorescent protein toolbox available for BiFC but also facilitate the detection and visualization of multiple protein complex interactions in living cells.

A Multiplex Microsphere-Based Immunoassay Increases the Sensitivity of SIV-Specific Antibody Detection in Serum Samples and Mucosal Specimens Collected from Rhesus Macaques Infected with SIVmac239.

PubMed

Powell, Rebecca L R; Ouellette, Ian; Lindsay, Ross W; Parks, Christopher L; King, C Richter; McDermott, Adrian B; Morrow, Gavin

2013-06-01

Results from recent HIV-1 vaccine studies have indicated that high serum antibody (Ab) titers may not be necessary for Ab-mediated protection, and that Abs localized to mucosal sites might be critical for preventing infection. Enzyme-linked immunosorbent assay (ELISA) has been used for decades as the gold standard for Ab measurement, though recently, highly sensitive microsphere-based assays have become available, with potential utility for improved detection of Abs. In this study, we assessed the Bio-Plex(®) Suspension Array System for the detection of simian immunodeficiency virus (SIV)-specific Abs in rhesus macaques (RMs) chronically infected with SIV, whose serum or mucosal SIV-specific Ab titers were negative by ELISA. We developed a SIVmac239-specific 4-plex bead array for the simultaneous detection of Abs binding to Env, Gag, Pol, and Nef. The 4-plex assay was used to quantify SIV-specific serum IgG and rectal swab IgA titers from control (SIV-naive) and SIVmac239-infected RMs. The Bio-Plex assay specifically detected anti-SIV Abs in specimens from SIV-infected animals for all four analytes when compared to SIV-naive control samples (p≤0.04). Furthermore, in 70% of Env and 79% of Gag ELISA-negative serum samples, specific Ab was detected using the Bio-Plex assay. Similarly, 71% of Env and 48% of Gag ELISA-negative rectal swab samples were identified as positive using the Bio-Plex assay. Importantly, assay specificity (i.e., probability of true positives) was comparable to ELISA (94%-100%). The results reported here indicate that microsphere-based methods provide a substantial improvement over ELISA for the detection of Ab responses, aid in detecting specific Abs when analyzing samples containing low levels of Abs, such as during the early stages of a vaccine trial, and may be valuable in attempts to link protective efficacy of vaccines with induced Ab responses.

Rapid polymerase chain reaction-based screening assay for bacterial biothreat agents.

PubMed

Yang, Samuel; Rothman, Richard E; Hardick, Justin; Kuroki, Marcos; Hardick, Andrew; Doshi, Vishal; Ramachandran, Padmini; Gaydos, Charlotte A

2008-04-01

To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens. The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria. The UniProbe detected the presence of all tested Eubacteria (31/31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus. A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents.

Sensitive high-throughput screening for the detection of reducing sugars.

PubMed

Mellitzer, Andrea; Glieder, Anton; Weis, Roland; Reisinger, Christoph; Flicker, Karlheinz

2012-01-01

The exploitation of renewable resources for the production of biofuels relies on efficient processes for the enzymatic hydrolysis of lignocellulosic materials. The development of enzymes and strains for these processes requires reliable and fast activity-based screening assays. Additionally, these assays are also required to operate on the microscale and on the high-throughput level. Herein, we report the development of a highly sensitive reducing-sugar assay in a 96-well microplate screening format. The assay is based on the formation of osazones from reducing sugars and para-hydroxybenzoic acid hydrazide. By using this sensitive assay, the enzyme loads and conversion times during lignocellulose hydrolysis can be reduced, thus allowing higher throughput. The assay is about five times more sensitive than the widely applied dinitrosalicylic acid based assay and can reliably detect reducing sugars down to 10 μM. The assay-specific variation over one microplate was determined for three different lignocellulolytic enzymes and ranges from 2 to 8%. Furthermore, the assay was combined with a microscale cultivation procedure for the activity-based screening of Pichia pastoris strains expressing functional Thermomyces lanuginosus xylanase A, Trichoderma reesei β-mannanase, or T. reesei cellobiohydrolase 2. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Paraffin section immunocytochemistry and cytosol-based ligand-binding assays for ER and PR detection in breast cancer: the time has come for more objectivity.

PubMed

Goussard, J

1998-10-23

The importance of the receptor level in breast cancer as an indicator of hormone response has been extensively studied for more than 20 years. Besides cytosol-based ligand-binding assays (dextran-coated charcoal assay, DCC), new methods using monoclonal antibodies raised against estrogen and progesterone receptors allow for the detection of receptors both in cytosol extracts (enzyme immunoassay, EIA) and in tissue sections (immunocytochemical assay, ICA). The biochemical assays (DCC and EIA) as well as the immunochemical detection (ICA) have specific qualities and produce original information which is useful for the therapeutic decision. While DCC gives a measure of the receptor level, whatever the real source of synthesis (normal and/or neoplastic tissue), ICA locates the positive cells and their relative proportion in the tumor. Both methods present their own advantages and disadvantages which are summarized in this study.

A simple and rapid method for detection of Trypanosoma evansi in the dromedary camel using a nested polymerase chain reaction

PubMed Central

Aradaib, Imadeldin E; Majid, Ali A

2006-01-01

A nested polymerase chain reaction (nPCR)-based assay, was developed and evaluated for rapid detection of Trypanosoma evansi in experimentally infected mice and naturally infected camels (Camelus dromedarius). Four oligonucleotide primers (TE1, TE2, TE3 and TE4), selected from nuclear repetitive gene of T. evansi, were designed and used for PCR amplifications. The first amplification, using a pair of outer primers TE1 and TE2, produced a 821-bp primary PCR product from T. evansi DNA. The second amplification, using nested (internal) pair of primers TE3 and TE4, produced a 270-bp PCR product. T. evansi DNAs extracted from blood samples of experimentally infected mice and naturally infected Sudanese breed of dromedary camels were detected by this nested PCR-based assay. The nested primers TE3 and TE4 increased the sensitivity of the PCR assay and as little as 10 fg of T. evansi DNA (equivalent to a single copy of the putative gene of the parasite) was amplified and visualized onto ethidium bromide-stained agarose gels. Amplification products were not detected when the PCR-based assay was applied to DNA from other blood parasites including Thieleria annulata, Babesia bigemina or nucleic acid free samples. Application of this nPCR-based assay to clinical samples resulted in direct detection of T. evansi from a variety of tissue samples collected from experimentally infected mice and blood from naturally infected camels. The described nPCR-based assay provides a valuable tool to study the epidemiology of T. evansi infection in camels and other susceptible animal populations. PMID:16712737

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

USDA-ARS?s Scientific Manuscript database

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...

Assay Portal | Office of Cancer Clinical Proteomics Research

Cancer.gov

The CPTAC Assay Portal serves as a centralized public repository of "fit-for-purpose," multiplexed quantitative mass spectrometry-based proteomic targeted assays. Targeted proteomic assays eliminate issues that are commonly observed using conventional protein detection systems.

PCR-free quantitative detection of genetically modified organism from raw materials – A novel electrochemiluminescence-based bio-barcode method

PubMed Central

Zhu, Debin; Tang, Yabing; Xing, Da; Chen, Wei R.

2018-01-01

Bio-barcode assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio-barcode assay requires lengthy experimental procedures including the preparation and release of barcode DNA probes from the target-nanoparticle complex, and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio-barcode assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2’2’-bipyridyl) ruthenium (TBR)-labele barcode DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products. PMID:18386909

PCR-free quantitative detection of genetically modified organism from raw materials. An electrochemiluminescence-based bio bar code method.

PubMed

Zhu, Debin; Tang, Yabing; Xing, Da; Chen, Wei R

2008-05-15

A bio bar code assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio bar code assay requires lengthy experimental procedures including the preparation and release of bar code DNA probes from the target-nanoparticle complex and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio bar code assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2,2'-bipyridyl) ruthenium (TBR)-labeled bar code DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products.

A universal array-based multiplexed test for cystic fibrosis carrier screening.

PubMed

Amos, Jean A; Bridge-Cook, Philippa; Ponek, Victor; Jarvis, Michael R

2006-01-01

Cystic fibrosis is a multisystem autosomal recessive disorder with high carrier frequencies in caucasians and significant, but lower, carrier frequencies in other ethnicities. Based on technology that allows high detection of mutations in caucasians and significant detection in other ethnic groups, the American College of Medical Genetics (ACMG) and American College of Obstetricians and Gynecologists (ACOG) have recommended pan-ethnic cystic fibrosis carrier screening for all reproductive couples. This paper discusses carrier screening using the Tag-It multiplex mutation platform and the Cystic Fibrosis Mutation Detection Kit. The Tag-It cystic fibrosis assay is a multiplexed genotyping assay that detects a panel of 40 cystic fibrosis transmembrane conductance regulator mutations including the 23 mutations recommended by the ACMG and ACOG for population screening. A total of 16 additional mutations detected by the Tag-It cystic fibrosis assay may also be common. The assay method is described in detail, and its performance in a genetics reference laboratory performing high-volume cystic fibrosis carrier screening is assessed.

A novel double recognition enzyme-linked immunosorbent assay based on the nucleocapsid protein for early detection of European porcine reproductive and respiratory syndrome virus infection.

PubMed

Venteo, A; Rebollo, B; Sarraseca, J; Rodriguez, M J; Sanz, A

2012-04-01

Precise and rapid detection of porcine reproductive respiratory syndrome virus (PRRSV) infection in swine farms is critical. Improvement of control procedures, such as testing incoming gilt and surveillance of seronegative herds requires more rapid and sensitive methods. However, standard serological techniques detect mainly IgG antibodies. A double recognition enzyme-linked immunosorbent assay (DR-ELISA) was developed for detection of antibodies specific to European PRRSV. This new assay can recognize both IgM and IgG antibodies to PRSSV which might be useful for detecting in routine surveillance assays pigs that are in the very early stages of infection and missed by conventional assays detecting only IgG antibodies. DR-ELISA is based on the double recognition of antigen by antibody. In this study, the recombinant nucleocapsid protein (N) of PRRSV was used both as the coating and the enzyme-conjugated antigen. To evaluate the sensitivity of the assay at early stages of the infection, sera from 69 pigs infected with PRRSV were collected during successive days post infection (pi) and tested. While standard methods showed low sensitivity rates before day 14 pi, DR-ELISA detected 88.4% seropositive samples at day 7 showing greater sensitivity at early stages of the infection. Further studies were carried out to assess the efficiency of the new assay, and the results showed DR-ELISA to be a sensitive and accurate method for early diagnosis of EU-PRRSV infection. Copyright © 2012 Elsevier B.V. All rights reserved.

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.

PubMed

Rininsland, Frauke; Stankewicz, Casey; Weatherford, Wendy; McBranch, Duncan

2005-05-31

High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that are used in the high-throughput community to determine assay robustness (Z'-value) demonstrate the suitability of this format for high-throughput screening applications for detection of inhibitors of enzyme activity. The QTL Lightspeed protein detection system provides a simple mix and measure "turn on" assay for the detection of kinase activity using natural protein substrates. The platform is robust and allows for identification of inhibitors of kinase activity.

Development of a reverse transcription quantitative polymerase chain reaction-based assay for broad coverage detection of African and Asian Zika virus lineages.

PubMed

Yang, Yang; Wong, Gary; Ye, Baoguo; Li, Shihua; Li, Shanqin; Zheng, Haixia; Wang, Qiang; Liang, Mifang; Gao, George F; Liu, Lei; Liu, Yingxia; Bi, Yuhai

2017-06-01

The Zika virus (ZIKV) is an arbovirus that has spread rapidly worldwide within recent times. There is accumulating evidence that associates ZIKV infections with Guillain-Barré Syndrome (GBS) and microcephaly in humans. The ZIKV is genetically diverse and can be separated into Asian and African lineages. A rapid, sensitive, and specific assay is needed for the detection of ZIKV across various pandemic regions. So far, the available primers and probes do not cover the genetic diversity and geographic distribution of all ZIKV strains. To this end, we have developed a one-step quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay based on conserved sequences in the ZIKV envelope (E) gene. The detection limit of the assay was determined to be five RNA transcript copies and 2.94 × 10 -3 50% tissue culture infectious doses (TCID 50 ) of live ZIKV per reaction. The assay was highly specific and able to detect five different ZIKV strains covering the Asian and African lineages without nonspecific amplification, when tested against other flaviviruses. The assay was also successful in testing for ZIKV in clinical samples. Our assay represents an improvement over the current methods available for the detection ZIKV and would be valuable as a diagnostic tool in various pandemic regions.

Effective surveillance for early classical swine fever virus detection will utilize both virus and antibody detection capabilities.

PubMed

Panyasing, Yaowalak; Kedkovid, Roongtham; Thanawongnuwech, Roongroje; Kittawornrat, Apisit; Ji, Ju; Giménez-Lirola, Luis; Zimmerman, Jeffrey

2018-03-01

Early recognition and rapid elimination of infected animals is key to controlling incursions of classical swine fever virus (CSFV). In this study, the diagnostic characteristics of 10 CSFV assays were evaluated using individual serum (n = 601) and/or oral fluid (n = 1417) samples collected from -14 to 28 days post inoculation (DPI). Serum samples were assayed by virus isolation (VI), 2 commercial antigen-capture enzyme-linked immunosorbent assays (ELISA), virus neutralization (VN), and 3 antibody ELISAs. Both serum and oral fluid samples were tested with 3 commercial real-time reverse transcription-polymerase chain reaction (rRT-PCR) assays. One or more serum samples was positive by VI from DPIs 3 to 21 and by antigen-capture ELISAs from DPIs 6 to 17. VN-positive serum samples were observed at DPIs ≥ 7 and by antibody ELISAs at DPIs ≥ 10. CSFV RNA was detected in serum samples from DPIs 2 to 28 and in oral fluid samples from DPIs 4 to 28. Significant differences in assay performance were detected, but most importantly, no single combination of sample and assay was able to dependably identify CSFV-inoculated pigs throughout the 4-week course of the study. The results show that effective surveillance for CSFV, especially low virulence strains, will require the use of PCR-based assays for the detection of early infections (<14 days) and antibody-based assays, thereafter. Copyright © 2018 Elsevier B.V. All rights reserved.

A high resolution melting (HRM) technology-based assay for cost-efficient clinical detection and genotyping of herpes simplex virus (HSV)-1 and HSV-2.

PubMed

Lieveld, M; Carregosa, A; Benoy, I; Redzic, N; Berth, M; Vanden Broeck, D

2017-10-01

Genital herpes can be caused by two very similar viruses, herpes simplex virus (HSV)-1 or HSV-2. These two HSV types cannot be distinguished clinically, but genotyping is recommended in the first-episodes of genital herpes to guide counselling and management. Quantitative polymerase chain reaction (qPCR) is the preferred diagnostic method for HSV typing. However, commercial qPCR methods use expensive fluorescent labeled probes for detection. Furthermore, most low-cost methods are not able to differentiate between HSV-1 and -2. The aim of this study was to develop a high resolution melting (HRM) technology-based assay for sensitive HSV-1 and HSV-2 detection and genotyping. Using a panel of 46 clinical specimens, the performance of the HRM assay was compared to two commercial HSV tests: the HRM assay detected HSV in all 23 positive samples, with no false positive results (100% concordance with HSV I/II Real-TM assay). Additionally, the HRM assay correctly genotyped both HSV types in a subset of these clinical samples, as determined by the Realstar HSV PCR Kit. The HSV HRM assay provides a cost-effective alternative method to conventional more expensive assays and can be used in routine clinical specimens, in cases where it is particularly necessary to detect and distinguish HSV-1 from -2. Copyright © 2017 Elsevier B.V. All rights reserved.

Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koopman, R P; Langlois, R G; Nasarabadi, S

2002-04-17

This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flowmore » cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.« less

Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents.

PubMed

Aitichou, Mohamed; Saleh, Sharron; Kyusung, Park; Huggins, John; O'Guinn, Monica; Jahrling, Peter; Ibrahim, Sofi

2008-11-01

A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6-carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible.

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

PubMed Central

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.

2012-01-01

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 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

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

PubMed

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.

Comparison of the BD Viper System with XTR Technology to the Gen-Probe APTIMA COMBO 2 Assay using the TIGRIS DTS system for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine specimens.

PubMed

Mushanski, Linda M; Brandt, Ken; Coffin, Nicolette; Levett, Paul N; Horsman, Gregory B; Rank, Elliot L

2012-07-01

Performances of the BD ProbeTec Chlamydia trachomatis (CT)/Neisseria Gonorrhoeae (GC) Q(x) Amplified DNA Assay reagents on a BD Viper System with XTR Technology and APTIMA COMBO 2 Assay reagents on a TIGRIS DTS platform, for detection of both CT and GC were compared. A total of 1018 first-void urine specimens were tested for the presence of CT and GC DNA using the 2 assays. CT was detected in 143 specimens (14%). Eight specimens exhibited discordant results, and they were divided equally between the 2 assays. Based on the original results, the overall agreement for CT was 99.2%, with 97.1% and 99.5% in agreement with positive and negative specimens, respectively. Cohen's Kappa was 0.967. GC was detected in 27 specimens (2.6%). Two specimens exhibited discordant results, and they were divided equally between the 2 assays. Based on the original results, the overall agreement was 99.8%, with 96.2% and 99.9% in agreement for positive and negative specimens, respectively. Cohen's Kappa was 0.961. There was a high level of agreement between the systems for both CT and GC detection.

Enhanced Reverse Transcription-PCR Assay for Detection of Norovirus Genogroup I

PubMed Central

Dreier, Jens; Störmer, Melanie; Mäde, Dietrich; Burkhardt, Sabine; Kleesiek, Knut

2006-01-01

We have developed a one-tube reverse transcription (RT)-PCR method using the real-time TaqMan PCR system for the detection of norovirus genogroup I (NV GGI). By introduction of a novel probe based on locked nucleic acid technology, we enhanced the sensitivity of the assay compared to those of conventional TaqMan probes. The sensitivity of the NV GGI RT-PCR was determined by probit analysis with defined RNA standards and quantified norovirus isolates to 711 copies/ml (95% detection limit). In order to detect PCR inhibition, we included a heterologous internal control (IC) system based on phage MS2. This internally controlled RT-PCR was tested on different real-time PCR platforms, LightCycler, Rotorgene, Mastercycler EP realplex, and ABI Prism. Compared to the assay without an IC, the duplex RT-PCR exhibited no reduction in sensitivity in clinical samples. In combination with an established NV GGII real-time RT-PCR, we used the novel assay in a routine assay for diagnosis of clinical and food-borne norovirus infection. We applied this novel assay to analyze outbreaks of nonbacterial acute gastroenteritis. Norovirus of GGI was detected in these outbreaks. Sequence and similarity plot analysis of open reading frame 1 (ORF1) and ORF2 showed two genotypes, GGI/2 and GGI/4, in semiclosed communities. PMID:16891482

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

USDA-ARS?s Scientific Manuscript database

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...

groEL is a suitable genetic marker for detecting Vibrio parahaemolyticus by loop-mediated isothermal amplification assay.

PubMed

Siddique, M P; Jang, W J; Lee, J M; Ahn, S H; Suraiya, S; Kim, C H; Kong, I S

2017-08-01

A groEL gene-based loop-mediated isothermal amplification (LAMP) assay was developed to detect Vibrio parahaemolyticus in contaminated seafood and water. The assay was optimized and conducted at 63°C for 40 min using Bacillus stearothermophilus (Bst) DNA polymerase, large fragment. Amplification was analysed via multiple detection methods, including opacity, formation of white precipitate, DNA intercalating dyes (ethidium bromide and SYBR Green I), metal ion-binding indicator dye, calcein, and 2% agarose gel electrophoresis. A characteristic ladder-like band pattern on agarose gel and the desired colour changes when using different dyes were observed in positive cases, and these were species-specific for V. parahaemolyticus when compared with other closely related Vibrio spp. The limit of detection (LoD) of this assay was 100 fg per reaction, 100-fold higher than that for conventional polymerase chain reaction (PCR). When tested on artificially contaminated seafood and seawater, the LoDs of the LAMP assay were 120 and 150 fg per reaction respectively, and those of conventional PCR were 120 and 150 pg per reaction respectively. Based on our results, the groEL gene-based LAMP assay is rapid, specific, sensitive, and reliable for detecting V. parahaemolyticus, and it could be used in field diagnosis. The loop-mediated isothermal amplification (LAMP) assay using groEL gene (an abundant, highly conserved gene and member of the groESL chaperone gene family) provided rapid, species-specific and highly sensitive method for detecting Vibrio parahaemolyticus, the leading causal agent of seafood-borne diseases worldwide. Moreover, groEL LAMP revealed high efficiency than conventional PCR assay for V. parahaemolyticus using template both from pure culture and artificially contaminated seafood and water, which indicated the applicability in the field and environmental screening purpose for the organism. © 2017 The Society for Applied Microbiology.

Aptamer-based detection of plasma proteins by an electrochemical assay coupled to magnetic beads.

PubMed

Centi, Sonia; Tombelli, Sara; Minunni, Maria; Mascini, Marco

2007-02-15

The DNA thrombin aptamer has been extensively investigated, and the coupling of this aptamer to different transduction principles has demonstrated the wide applicability of aptamers as bioreceptors in bioanalytical assays. The goal of this work was to design an aptamer-based sandwich assay with electrochemical detection for thrombin analysis in complex matrixes, using a simple target capturing step by aptamer-functionalized magnetic beads. The conditions for the aptamer immobilization and for the protein binding have been first optimized by surface plasmon resonance, and then transferred to the electrochemical-based assay performed onto screen-printed electrodes. The assay was then applied to the analysis of thrombin in buffer, spiked serum, and plasma and high sensitivity and specificity were found. Moreover, thrombin was generated in situ in plasma by the conversion of its precursor prothrombin, and the formation of thrombin was followed at different times. The concentrations detected by the electrochemical assay were in agreement with a simulation software that mimics the formation of thrombin over time (thrombogram). The proposed work demonstrates that the high specificity of aptamers together with the use of magnetic beads are the key features for aptamer-based analysis in complex matrixes, opening the possibility of a real application to diagnostics or medical investigation.

Exploring target-specific primer extension in combination with a bead-based suspension array for multiplexed detection and typing using Streptococcus suis as a model pathogen

PubMed Central

van der Wal, Fimme J.; Achterberg, René P.; van Solt-Smits, Conny; Bergervoet, Jan H. W.; de Weerdt, Marjanne; Wisselink, Henk J.

2017-01-01

We investigated the feasibility of an assay based on target-specific primer extension, combined with a suspension array, for the multiplexed detection and typing of a veterinary pathogen in animal samples, using Streptococcus suis as a model pathogen. A procedure was established for simultaneous detection of 6 S. suis targets in pig tonsil samples (i.e., 4 genes associated with serotype 1, 2, 7, or 9, the generic S. suis glutamate dehydrogenase gene [gdh], and the gene encoding the extracellular protein factor [epf]). The procedure was set up as a combination of protocols: DNA isolation from porcine tonsils, a multiplex PCR, a multiplex target-specific primer extension, and finally a suspension array as the readout. The resulting assay was compared with a panel of conventional PCR assays. The proposed multiplex assay can correctly identify the serotype of isolates and is capable of simultaneous detection of multiple targets in porcine tonsillar samples. The assay is not as sensitive as the current conventional PCR assays, but with the correct sampling strategy, the assay can be useful for screening pig herds to establish which S. suis serotypes are circulating in a pig population. PMID:28980519

High Throughput Multiplex PCR and Probe-based Detection with Luminex Beads for Seven Intestinal Parasites

PubMed Central

Taniuchi, Mami; Verweij, Jaco J.; Noor, Zannatun; Sobuz, Shihab U.; van Lieshout, Lisette; Petri, William A.; Haque, Rashidul; Houpt, Eric R.

2011-01-01

Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and copro-antigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites—Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis—were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites. PMID:21292910

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

PubMed Central

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

2012-01-01

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 current study, we have developed an enzyme-linked immunosorbent assay (ELISA)-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotypes A, B, C, D, E, and F. With engineered high-affinity antibodies, the BoNT assays have sensitivities in buffer ranging from 1.3 fM (0.2 pg/ml) to 14.7 fM (2.2 pg/ml). Using clinical and food matrices (serum and milk), the microarray is capable of detecting BoNT serotypes A to F to similar 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, food, and environmental samples. PMID:22935296

Performance of the BioPlex 2200 HIV Ag-Ab assay for identifying acute HIV infection.

PubMed

Eshleman, Susan H; Piwowar-Manning, Estelle; Sivay, Mariya V; Debevec, Barbara; Veater, Stephanie; McKinstry, Laura; Bekker, Linda-Gail; Mannheimer, Sharon; Grant, Robert M; Chesney, Margaret A; Coates, Thomas J; Koblin, Beryl A; Fogel, Jessica M

Assays that detect HIV antigen (Ag) and antibody (Ab) can be used to screen for HIV infection. To compare the performance of the BioPlex 2200 HIV Ag-Ab assay and two other Ag/Ab combination assays for detection of acute HIV infection. Samples were obtained from 24 individuals (18 from the US, 6 from South Africa); these individuals were classified as having acute infection based on the following criteria: positive qualitative RNA assay; two negative rapid tests; negative discriminatory test. The samples were tested with the BioPlex assay, the ARCHITECT HIV Ag/Ab Combo test, the Bio-Rad GS HIV Combo Ag-Ab EIA test, and a viral load assay. Twelve (50.0%) of 24 samples had RNA detected only ( > 40 to 13,476 copies/mL). Ten (43.5%) samples had reactive results with all three Ag/Ab assays, one sample was reactive with the ARCHITECT and Bio-Rad assays, and one sample was reactive with the Bio-Rad and BioPlex assays. The 11 samples that were reactive with the BioPlex assay had viral loads from 83,010 to >750,000 copies/mL; 9/11 samples were classified as Ag positive/Ab negative by the BioPlex assay. Detection of acute HIV infection was similar for the BioPlex assay and two other Ag/Ab assays. All three tests were less sensitive than a qualitative RNA assay and only detected HIV Ag when the viral load was high. The BioPlex assay detected acute infection in about half of the cases, and identified most of those infections as Ag positive/Ab negative. Copyright © 2018 Elsevier B.V. All rights reserved.

Highly sensitive immunoassay of anti-cyclic citrullinated peptide marker using surface-enhanced Raman scattering detection

NASA Astrophysics Data System (ADS)

Chon, H.; Lee, S.; Wang, R.; Bang, S.-Y.; Lee, H.-S.; Bae, S.-C.; Hong, S. H.; Yoon, Y. H.; Lim, D.; Choo, J.

2015-07-01

We report a highly sensitive anti-cyclic citrullinated peptide (anti-CCP) detection method for early diagnosis of rheumatoid arthritis (RA) using surface-enhanced Raman scattering (SERS)-based immunoassay. Herein, cyclic citrullinated peptide (CCP)-conjugated magnetic beads and anti-human IgG-conjugated hollow gold nanospheres (HGNs) were used as substrates and SERS nano-tags, respectively. First, its detection sensitivity was evaluated using anti-CCP standard solutions. Then quantitative anti-CCP levels, determined by the SERS-based assay, were compared with those obtained from three commercially available anti-CCP assay kits (Immunoscan CCPlus, ImmunnLisa™ CCP and BioPlex™ 2200) to assess its potential utility as a clinical tool. Finally, clinical samples from 20 RA patients were investigated using them. In the SERS-based assay, the anti-CCP level in human serum was successfully determined by monitoring the characteristic Raman peak intensity of SERS nano-tags. The diagnostic performance of our SERS-based immunoassay for clinical samples shows a good agreement with those measured by three commercial anti-CCP kits. In addition, our SERS-based assay results are more consistent in the low concentration range (0-25 U/mL) than those achieved by the commercial kits. Accordingly, it is estimated that the SERS-based assay is a potentially useful diagnostic tool for early diagnosis of RA.

The use of upconverting phosphors in point-of-care (POC) testing

NASA Astrophysics Data System (ADS)

Tanke, Hans J.; Zuiderwijk, Michel; Wiesmeijer, Karien C.; Breedveld, Robert N.; Abrams, William R.; de Dood, Claudia J.; Tjon Kon Fat, Elisa M.; Corstjens, Paul L. A. M.

2014-03-01

Point-of-care (POC) testing is increasingly applied as a cost effective alternative to many diagnostic tests. Key in POC testing is to create sufficient assay sensitivity with relatively low cost reagents and equipment. For this purpose we have employed a unique reporter, upconverting phosphor (UCP) particles, in combination with lateral flow (LF) assays. UCPs, submicron ceramic particles doped with rare earth ions (lanthanides), convert infrared to visible light and do not suffer from autofluorescence which limits conventional fluorescence based assays. Low cost handheld readers and microfluidics were evaluated in various applications. Designed assays are well suited for applications outside diagnostic laboratories, in resource poor settings, and can even be used by patients at home. Using two distinctly different UCP-LF assay formats, we focussed on assays for infectious diseases based on the detection of pathogen-specific antibodies and/or antigens including nucleic acids to demonstrate active infection with HIV. Only minor adaptation of the standard UCP-LF assay format is needed to render the format suitable for applications involving low affinity capture antibodies (e.g. in the detection of neurotoxin, botulism), capture of small molecules (e.g. detection of melatonin, a key hormone in chronopharmacology) or the use of dry UCP reagents (e.g. detection of protein based fruit-ripening markers, of economic interest in agriculture). Finally, we anticipate on developments in healthcare (personalized medicine) by discussing the potential of one of the UCP-LF assay formats to measure serum trough levels of immunodrugs (e.g. infliximab or adalimumab) in patients treated for inflammatory bowel disease and rheumatoid arthritis.

Fluorescence-based recombination assay for sensitive and specific detection of genotoxic carcinogens in human cells.

PubMed

Ireno, Ivanildce C; Baumann, Cindy; Stöber, Regina; Hengstler, Jan G; Wiesmüller, Lisa

2014-05-01

In vitro genotoxicity tests are known to suffer from several shortcomings, mammalian cell-based assays, in particular, from low specificities. Following a novel concept of genotoxicity detection, we developed a fluorescence-based method in living human cells. The assay quantifies DNA recombination events triggered by DNA double-strand breaks and damage-induced replication fork stalling predicted to detect a broad spectrum of genotoxic modes of action. To maximize sensitivities, we engineered a DNA substrate encompassing a chemoresponsive element from the human genome. Using this substrate, we screened various human tumor and non-transformed cell types differing in the DNA damage response, which revealed that detection of genotoxic carcinogens was independent of the p53 status but abrogated by apoptosis. Cell types enabling robust and sensitive genotoxicity detection were selected for the generation of reporter clones with chromosomally integrated DNA recombination substrate. Reporter cell lines were scrutinized with 21 compounds, stratified into five sets according to the established categories for identification of carcinogenic compounds: genotoxic carcinogens ("true positives"), non-genotoxic carcinogens, compounds without genotoxic or carcinogenic effect ("true negatives") and non-carcinogenic compounds, which have been reported to induce chromosomal aberrations or mutations in mammalian cell-based assays ("false positives"). Our results document detection of genotoxic carcinogens in independent cell clones and at levels of cellular toxicities <60 % with a sensitivity of >85 %, specificity of ≥90 % and detection of false-positive compounds <17 %. Importantly, through testing cyclophosphamide in combination with primary hepatocyte cultures, we additionally provide proof-of-concept for the identification of carcinogens requiring metabolic activation using this novel assay system.

[Detection of Plasmodium falciparum by using magnetic nanoparticles separation-based quantitative real-time PCR assay].

PubMed

Wang, Fei; Tian, Yin; Yang, Jing; Sun, Fu-Jun; Sun, Ning; Liu, Bi-Yong; Tian, Rui; Ge, Guang-Lu; Zou, Ming-qiang; Deng, Cong-liang; Liu, Yi

2014-10-01

To establish a magnetic nanoparticles separation-based quantitative real-time PCR (RT-PCR) assay for fast and accurate detection of Plasmodium falciparum and providing a technical support for improving the control and prevention of imported malaria. According to the conserved sequences of the P. falciparum genome 18SrRNA, the species-specific primers and probe were designed and synthetized. The RT-PCR was established by constructing the plasmid standard, fitting the standard curve and using magnetic nanoparticles separation. The sensitivity and specificity of the assay were evaluated. The relationship between the threshold cycle (Ct) and logarithm of initial templates copies was linear over a range of 2.5 x 10(1) to 2.5 x 10(8) copies/μl (R2 = 0.999). Among 13 subjects of entry frontier, a P. falciparum carrier with low load was detected by using the assay and none was detected with the conventional examinations (microscopic examinations and rapid tests). This assay shows a high sensitivity in detection of P. falciparum, with rapid and accurate characteristics, and is especially useful in diagnosis of P. falciparum infectors with low parasitaemia at entry-exit frontier ports.

Utilization of microparticles in next-generation assays for microflow cytometers.

PubMed

Kim, Jason S; Ligler, Frances S

2010-11-01

Micron-sized particles have primarily been used in microfabricated flow cytometers for calibration purposes and proof-of-concept experiments. With increasing frequency, microparticles are serving as a platform for assays measured in these small analytical devices. Light scattering has been used to measure the agglomeration of antibody-coated particles in the presence of an antigen. Impedance detection is another technology being integrated into microflow cytometers for microparticle-based assays. Fluorescence is the most popular detection method in flow cytometry, enabling highly sensitive multiplexed assays. Finally, magnetic particles have also been used to measure antigen levels using a magnetophoretic micro-device. We review the progress of microparticle-based assays in microflow cytometry in terms of the advantages and limitations of each approach.

Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification.

PubMed

Hammond, Rosemarie W; Zhang, Shulu

2016-10-01

A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39°C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in infected leaf and seed tissues. The performance of the AmplifyRP(®) Acceler8™ RT-RPA diagnostic assay, utilizing a lateral flow strip contained within an amplicon detection chamber, was evaluated and the results were compared with a standard RT-PCR assay. The AmplifyRP(®) Acceler8™ assay was specific for TCDVd in leaf and seed tissues, its sensitivity was comparable to conventional RT-PCR in leaf tissues, and it does not require extensive sample purification, specialized equipment, or technical expertise. This is the first report utilizing an RT-RPA assay to detect viroids and the assay can be used both in the laboratory and in the field for TCDVd detection. Published by Elsevier B.V.

A Fluorescence Quenching Assay Based on Molecular Beacon Formation through a Ligase Detection Reaction for Facile and Rapid Detection of Point Mutations.

PubMed

Sawamura, Kensuke; Hashimoto, Masahiko

2017-01-01

A fluorescence quenching assay based on a ligase detection reaction was developed for facile and rapid detection of point mutations present in a mixed population of non-variant DNA. If the test DNA carried a targeted mutation, then the two allele-specific primers were ligated to form a molecular beacon resulting in the expected fluorescence quenching signatures. Using this method, we successfully detected as low as 5% mutant DNA in a mixture of wild-type DNA (t test at 99% confidence level).

A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY STYRENE OXIDE

EPA Science Inventory

A rapid and simple assay to detect DNA damage to calf thymus DNA caused by styrene oxide (SO) is reported. This assay is based on changes observed in the melting and annealing behavior of the damaged DNA. The melting annealing process was monitored using a fluorescence indicat...

A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY RADIATION, CHEMICAL MUTAGENS AND ENZYMES

EPA Science Inventory

A simple and rapid assay to detect DNA damage is reported. This novel assay is based on changes in melting/annealing behavior and facilitated using certain dyes that increase their fluorescence upon association with double stranded (ds)DNA. Damage caused by ultraviolet (UV) ra...

Inferring biomarkers for Mycobacterium avium subsp. paratuberculosis infection and disease progression in cattle using experimental data

NASA Astrophysics Data System (ADS)

Magombedze, Gesham; Shiri, Tinevimbo; Eda, Shigetoshi; Stabel, Judy R.

2017-03-01

Available diagnostic assays for Mycobacterium avium subsp. paratuberculosis (MAP) have poor sensitivities and cannot detect early stages of infection, therefore, there is need to find new diagnostic markers for early infection detection and disease stages. We analyzed longitudinal IFN-γ, ELISA-antibody and fecal shedding experimental sensitivity scores for MAP infection detection and disease progression. We used both statistical methods and dynamic mathematical models to (i) evaluate the empirical assays (ii) infer and explain biological mechanisms that affect the time evolution of the biomarkers, and (iii) predict disease stages of 57 animals that were naturally infected with MAP. This analysis confirms that the fecal test is the best marker for disease progression and illustrates that Th1/Th2 (IFN-γ/ELISA antibodies) assays are important for infection detection, but cannot reliably predict persistent infections. Our results show that the theoretical simulated macrophage-based assay is a potential good diagnostic marker for MAP persistent infections and predictor of disease specific stages. We therefore recommend specifically designed experiments to test the use of a based assay in the diagnosis of MAP infections.

Recommendations for the design, optimization, and qualification of cell-based assays used for the detection of neutralizing antibody responses elicited to biological therapeutics.

PubMed

Gupta, Shalini; Indelicato, Stephen R; Jethwa, Vijay; Kawabata, Thomas; Kelley, Marian; Mire-Sluis, Anthony R; Richards, Susan M; Rup, Bonita; Shores, Elizabeth; Swanson, Steven J; Wakshull, Eric

2007-04-10

The administration of biological therapeutics can evoke some level of immune response to the drug product in the receiving subjects. An immune response comprised of neutralizing antibodies can lead to loss of efficacy or potentially more serious clinical sequelae. Therefore, it is important to monitor the immunogenicity of biological therapeutics throughout the drug product development cycle. Immunoassays are typically used to screen for the presence and development of anti-drug product antibodies. However, in-vitro cell-based assays prove extremely useful for the characterization of immunoassay-positive samples to determine if the detected antibodies have neutralizing properties. This document provides scientific recommendations based on the experience of the authors for the development of cell-based assays for the detection of neutralizing antibodies in non-clinical and clinical studies.

Using detergent to enhance detection sensitivity of African trypanosomes in human CSF and blood by loop-mediated isothermal amplification (LAMP).

PubMed

Grab, Dennis J; Nikolskaia, Olga V; Inoue, Noboru; Thekisoe, Oriel M M; Morrison, Liam J; Gibson, Wendy; Dumler, J Stephen

2011-08-01

The loop-mediated isothermal amplification (LAMP) assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. While many LAMP-based assays are sufficiently sensitive to detect DNA well below the amount present in a single parasite, the detection limit of the assay is restricted by the number of parasites present in the volume of sample assayed; i.e. 1 per µL or 10(3) per mL. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by simply adding detergent to the samples prior to LAMP assay. For proof of principle we used two different LAMP assays capable of detecting 0.1 fg genomic DNA (0.001 parasite). The assay was tested on dilution series of intact bloodstream form Trypanosoma brucei rhodesiense in human cerebrospinal fluid (CSF) or blood with or without the addition of the detergent Triton X-100 and 60 min incubation at ambient temperature. With human CSF and in the absence of detergent, the LAMP detection limit for live intact parasites using 1 µL of CSF as the source of template was at best 10(3) parasites/mL. Remarkably, detergent enhanced LAMP assay reaches sensitivity about 100 to 1000-fold lower; i.e. 10 to 1 parasite/mL. Similar detergent-mediated increases in LAMP assay analytical sensitivity were also found using DNA extracted from filter paper cards containing blood pretreated with detergent before card spotting or blood samples spotted on detergent pretreated cards. This simple procedure for the enhanced detection of live African trypanosomes in biological fluids by LAMP paves the way for the adaptation of LAMP for the economical and sensitive diagnosis of other protozoan parasites and microorganisms that cause diseases that plague the developing world.

SYBR green-based one step quantitative real-time polymerase chain reaction assay for the detection of Zika virus in field-caught mosquitoes.

PubMed

Tien, Wei-Ping; Lim, Gareth; Yeo, Gladys; Chiang, Suzanna Nicole; Chong, Chee-Seng; Ng, Lee-Ching; Hapuarachchi, Hapuarachchige Chanditha

2017-09-19

The monitoring of vectors is one of the key surveillance measures to assess the risk of arbovirus transmission and the success of control strategies in endemic regions. The recent re-emergence of Zika virus (ZIKV) in the tropics, including Singapore, emphasizes the need to develop cost-effective, rapid and accurate assays to monitor the virus spread by mosquitoes. As ZIKV infections largely remain asymptomatic, early detection of ZIKV in the field-caught mosquitoes enables timely implementation of appropriate mosquito control measures. We developed a rapid, sensitive and specific real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for the detection of ZIKV in field-caught mosquitoes. The primers and PCR cycling conditions were optimized to minimize non-specific amplification due to cross-reactivity with the genomic material of Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex tritaeniorhynchus, Culex sitiens and Anopheles sinensis, as well as accompanying microbiota. The performance of the assay was further evaluated with a panel of flaviviruses and alphaviruses as well as in field-caught Ae. aegypti mosquitoes confirmed to be positive for ZIKV. As compared to a probe-based assay, the newly developed assay demonstrated 100% specificity and comparable detection sensitivity for ZIKV in mosquitoes. Being a SYBR Green-based method, the newly-developed assay is cost-effective and easy to adapt, thus is applicable to large-scale vector surveillance activities in endemic countries, including those with limited resources and expertise. The amplicon size (119 bp) also allows sequencing to confirm the virus type. The primers flank relatively conserved regions of ZIKV genome, so that, the assay is able to detect genetically diverse ZIKV strains. Our findings, therefore, testify the potential use of the newly-developed assay in vector surveillance programmes for ZIKV in endemic regions.

Molecular Detection of Antimicrobial Resistance

PubMed Central

Fluit, Ad C.; Visser, Maarten R.; Schmitz, Franz-Josef

2001-01-01

The determination of antimicrobial susceptibility of a clinical isolate, especially with increasing resistance, is often crucial for the optimal antimicrobial therapy of infected patients. Nucleic acid-based assays for the detection of resistance may offer advantages over phenotypic assays. Examples are the detection of the methicillin resistance-encoding mecA gene in staphylococci, rifampin resistance in Mycobacterium tuberculosis, and the spread of resistance determinants across the globe. However, molecular assays for the detection of resistance have a number of limitations. New resistance mechanisms may be missed, and in some cases the number of different genes makes generating an assay too costly to compete with phenotypic assays. In addition, proper quality control for molecular assays poses a problem for many laboratories, and this results in questionable results at best. The development of new molecular techniques, e.g., PCR using molecular beacons and DNA chips, expands the possibilities for monitoring resistance. Although molecular techniques for the detection of antimicrobial resistance clearly are winning a place in routine diagnostics, phenotypic assays are still the method of choice for most resistance determinations. In this review, we describe the applications of molecular techniques for the detection of antimicrobial resistance and the current state of the art. PMID:11585788

A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis.

PubMed

Ashraf, Aqeela; Imran, Muhammad; Yaqub, Tahir; Tayyab, Muhammad; Shehzad, Wasim; Thomson, Peter C

2017-06-01

For rapid and simultaneous detection of nine bovine mastitic pathogens, a sensitive and specific multiplex PCR assay was developed. The assay was standardized using reference strains and validated on mastitic milk cultures which were identified to species level based on 16S rRNA sequencing. Multiplex PCR assay also efficiently detected the target bacterial strains directly from milk. The detection limit of the assay was up to 50 pg for DNA isolated from pure cultures and 10 4  CFU/ml for spiked milk samples. As estimated by latent class analysis, the assay was sensitive up to 88% and specific up to 98% for targeted mastitic pathogens, compared with the bacterial culture method and the 16S rRNA sequence analysis. This novel molecular assay could be useful for monitoring and maintaining the bovine udder health, ensuring the bacteriological safety of milk, and conducting epidemiological studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development of field-based real-time reverse transcription-polymerase chain reaction assays for detection of Chikungunya and O'nyong-nyong viruses in mosquitoes.

PubMed

Smith, Darci R; Lee, John S; Jahrling, Jordan; Kulesh, David A; Turell, Michael J; Groebner, Jennifer L; O'Guinn, Monica L

2009-10-01

Chikungunya (CHIK) and O'nyong-nyong (ONN) are important emerging arthropod-borne diseases. Molecular diagnosis of these two viruses in mosquitoes has not been evaluated, and the effects of extraneous mosquito tissue on assay performance have not been tested. Additionally, no real-time reverse transcription-polymerase chain reaction (RT-PCR) assay exists for detecting ONN virus (ONNV) RNA. We describe the development of sensitive and specific real-time RT-PCR assays for detecting CHIK and ONN viral RNA in mosquitoes, which have application for field use. In addition, we compared three methods for primer/probe design for assay development by evaluating their sensitivity and specificity. This comparison resulted in development of virus-specific assays that could detect less than one plaque-forming unit equivalent of each of the viruses in mosquitoes. The use of these assays will aid in arthropod-borne disease surveillance and in the control of the associated diseases.

Evaluation of a Real-Time PCR Test for the Detection and Discrimination of Theileria Species in the African Buffalo (Syncerus caffer)

PubMed Central

Chaisi, Mamohale E.; Janssens, Michiel E.; Vermeiren, Lieve; Oosthuizen, Marinda C.; Collins, Nicola E.; Geysen, Dirk

2013-01-01

A quantitative real-time PCR (qPCR) assay based on the cox III gene was evaluated for the simultaneous detection and discrimination of Theileria species in buffalo and cattle blood samples from South Africa and Mozambique using melting curve analysis. The results obtained were compared to those of the reverse line blot (RLB) hybridization assay for the simultaneous detection and differentiation of Theileria spp. in mixed infections, and to the 18S rRNA qPCR assay results for the specific detection of Theileria parva. Theileria parva, Theileria sp. (buffalo), Theileria taurotragi, Theileria buffeli and Theileria mutans were detected by the cox III assay. Theileria velifera was not detected from any of the samples analysed. Seventeen percent of the samples had non-species specific melting peaks and 4.5% of the samples were negative or below the detection limit of the assay. The cox III assay identified more T. parva and Theileria sp. (buffalo) positive samples than the RLB assay, and also detected more T. parva infections than the 18S assay. However, only a small number of samples were positive for the benign Theileria spp. To our knowledge T. taurotragi has never been identified from the African buffalo, its identification in some samples by the qPCR assay was unexpected. Because of these discrepancies in the results, cox III qPCR products were cloned and sequenced. Sequence analysis indicated extensive inter- and intra-species variations in the probe target regions of the cox III gene sequences of the benign Theileria spp. and therefore explains their low detection. The cox III assay is specific for the detection of T. parva infections in cattle and buffalo. Sequence data generated from this study can be used for the development of a more inclusive assay for detection and differentiation of all variants of the mildly pathogenic and benign Theileria spp. of buffalo and cattle. PMID:24146782

Evaluation of a real-time PCR test for the detection and discrimination of theileria species in the African buffalo (Syncerus caffer).

PubMed

Chaisi, Mamohale E; Janssens, Michiel E; Vermeiren, Lieve; Oosthuizen, Marinda C; Collins, Nicola E; Geysen, Dirk

2013-01-01

A quantitative real-time PCR (qPCR) assay based on the cox III gene was evaluated for the simultaneous detection and discrimination of Theileria species in buffalo and cattle blood samples from South Africa and Mozambique using melting curve analysis. The results obtained were compared to those of the reverse line blot (RLB) hybridization assay for the simultaneous detection and differentiation of Theileria spp. in mixed infections, and to the 18S rRNA qPCR assay results for the specific detection of Theileria parva. Theileria parva, Theileria sp. (buffalo), Theileria taurotragi, Theileria buffeli and Theileria mutans were detected by the cox III assay. Theileria velifera was not detected from any of the samples analysed. Seventeen percent of the samples had non-species specific melting peaks and 4.5% of the samples were negative or below the detection limit of the assay. The cox III assay identified more T. parva and Theileria sp. (buffalo) positive samples than the RLB assay, and also detected more T. parva infections than the 18S assay. However, only a small number of samples were positive for the benign Theileria spp. To our knowledge T. taurotragi has never been identified from the African buffalo, its identification in some samples by the qPCR assay was unexpected. Because of these discrepancies in the results, cox III qPCR products were cloned and sequenced. Sequence analysis indicated extensive inter- and intra-species variations in the probe target regions of the cox III gene sequences of the benign Theileria spp. and therefore explains their low detection. The cox III assay is specific for the detection of T. parva infections in cattle and buffalo. Sequence data generated from this study can be used for the development of a more inclusive assay for detection and differentiation of all variants of the mildly pathogenic and benign Theileria spp. of buffalo and cattle.

Novel PCR Assays Complement Laser Biosensor-Based Method and Facilitate Listeria Species Detection from Food.

PubMed

Kim, Kwang-Pyo; Singh, Atul K; Bai, Xingjian; Leprun, Lena; Bhunia, Arun K

2015-09-08

The goal of this study was to develop the Listeria species-specific PCR assays based on a house-keeping gene (lmo1634) encoding alcohol acetaldehyde dehydrogenase (Aad), previously designated as Listeria adhesion protein (LAP), and compare results with a label-free light scattering sensor, BARDOT (bacterial rapid detection using optical scattering technology). PCR primer sets targeting the lap genes from the species of Listeria sensu stricto were designed and tested with 47 Listeria and 8 non-Listeria strains. The resulting PCR primer sets detected either all species of Listeria sensu stricto or individual L. innocua, L. ivanovii and L. seeligeri, L. welshimeri, and L. marthii without producing any amplified products from other bacteria tested. The PCR assays with Listeria sensu stricto-specific primers also successfully detected all species of Listeria sensu stricto and/or Listeria innocua from mixed culture-inoculated food samples, and each bacterium in food was verified by using the light scattering sensor that generated unique scatter signature for each species of Listeria tested. The PCR assays based on the house-keeping gene aad (lap) can be used for detection of either all species of Listeria sensu stricto or certain individual Listeria species in a mixture from food with a detection limit of about 10⁴ CFU/mL.

Novel PCR Assays Complement Laser Biosensor-Based Method and Facilitate Listeria Species Detection from Food

PubMed Central

Kim, Kwang-Pyo; Singh, Atul K.; Bai, Xingjian; Leprun, Lena; Bhunia, Arun K.

2015-01-01

The goal of this study was to develop the Listeria species-specific PCR assays based on a house-keeping gene (lmo1634) encoding alcohol acetaldehyde dehydrogenase (Aad), previously designated as Listeria adhesion protein (LAP), and compare results with a label-free light scattering sensor, BARDOT (bacterial rapid detection using optical scattering technology). PCR primer sets targeting the lap genes from the species of Listeria sensu stricto were designed and tested with 47 Listeria and 8 non-Listeria strains. The resulting PCR primer sets detected either all species of Listeria sensu stricto or individual L. innocua, L. ivanovii and L. seeligeri, L. welshimeri, and L. marthii without producing any amplified products from other bacteria tested. The PCR assays with Listeria sensu stricto-specific primers also successfully detected all species of Listeria sensu stricto and/or Listeria innocua from mixed culture-inoculated food samples, and each bacterium in food was verified by using the light scattering sensor that generated unique scatter signature for each species of Listeria tested. The PCR assays based on the house-keeping gene aad (lap) can be used for detection of either all species of Listeria sensu stricto or certain individual Listeria species in a mixture from food with a detection limit of about 104 CFU/mL. PMID:26371000

Visual colorimetric detection of tin(II) and nitrite using a molybdenum oxide nanomaterial-based three-input logic gate.

PubMed

Du, Jiayan; Zhao, Mengxin; Huang, Wei; Deng, Yuequan; He, Yi

2018-05-09

We report a molybdenum oxide (MoO 3 ) nanomaterial-based three-input logic gate that uses Sn 2+ , NO 2 - , and H + ions as inputs. Under acidic conditions, Sn 2+ is able to reduce MoO 3 nanosheets, generating oxygen-vacancy-rich MoO 3-x nanomaterials along with strong localized surface plasmon resonance (LSPR) and an intense blue solution as the output signal. When NO 2 - is introduced, the redox reaction between the MoO 3 nanosheets and Sn 2+ is strongly inhibited because the NO 2 - consumes both H + and Sn 2+ . The three-input logic gate was employed for the visual colorimetric detection of Sn 2+ and NO 2 - under different input states. The colorimetric assay's limit of detection for Sn 2+ and the lowest concentration of NO 2 - detectable by the assay were found to be 27.5 nM and 0.1 μM, respectively. The assay permits the visual detection of Sn 2+ and NO 2 - down to concentrations as low as 2 μM and 25 μM, respectively. The applicability of the logic-gate-based colorimetric assay was demonstrated by using it to detect Sn 2+ and NO 2 - in several water sources.

Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

PubMed Central

Worbs, Sylvia; Fiebig, Uwe; Zeleny, Reinhard; Schimmel, Heinz; Rummel, Andreas; Luginbühl, Werner; Dorner, Brigitte G.

2015-01-01

In the framework of the EU project EQuATox, a first international proficiency test (PT) on the detection and quantification of botulinum neurotoxins (BoNT) was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo/in vitro approaches (mouse bioassay, hemidiaphragm assay and Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult, resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently considered as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need for certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphragm assay delivered quantitative results superior to the mouse bioassay. PMID:26703724

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

PubMed

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

2017-12-01

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

A loop-mediated isothermal amplification assay for rapid and sensitive detection of bovine papular stomatitis virus.

PubMed

Kurosaki, Yohei; Okada, Sayaka; Nakamae, Sayuri; Yasuda, Jiro

2016-12-01

Bovine papular stomatitis virus (BPSV) causes pustular cutaneous disease in cattle worldwide. This paper describes the development of a specific loop-mediated isothermal amplification (LAMP) assay to detect BPSV which did not cross-react with other parapoxviruses. To assess analytical sensitivity of this LAMP assay, DNA was extracted from serially diluted BPSV from which the infectious titer was determined by a novel assay based on calf kidney epithelial cells. The LAMP assay had equivalent analytical sensitivity to quantitative PCR, and could detect as few as 86 copies of viral DNA per reaction. These results suggest that the assay is a specific and sensitive technique to rapidly diagnose bovine papular stomatitis in domestic animals. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparisons of VLP-Based ELISA, Neutralization Assays with Native HPV, and Neutralization Assays with PsV in Detecting HPV Antibody Responses in HIV-Infected Women

PubMed Central

Du, Ping; Brendle, Sarah; Milici, Janice; Camacho, Fabian; Zurlo, John; Christensen, Neil; Meyers, Craig

2015-01-01

Objective Human papillomavirus (HPV)-associated cancers are important public health problems in HIV-infected people. Assays based on HPV virus-like particles (VLP) and pseudoviruses (PsV) are commonly used to examine HPV antibody responses in HIV-infected people, but neutralization assays with native HPV have not been utilized and a comparison of these three assays is lacking. We evaluated the agreement of assays using VLP, native HPV and PsV in detecting HPV16 and 18 antibodies in HIV-infected women. Methods The VLP-based ELISA (VLP-ELISA) was used to detect antibody responses to HPV16 and 18 and cottontail rabbit papillomavirus (CRPV) VLP antigens. Neutralization assays with native HPV (NA-HPV) and with PsV (NA-PsV) were conducted to examine HPV16 or 18 neutralizing antibodies. Intra class correlation coefficients (ICC) and kappa coefficients were used to assess the agreements of seropositivity between the assays. Results The seroprevalence detected by the VLP-ELISA, NA-HPV and NA-PsV in 94 HIV-infected women was 35%, 51% and 27% for HPV16 and 14%, 44% and 21% for HPV18. Cross-reactivity between HPV16 and HPV18 was 0.35, 0.04 and 0.33 (kappa coefficients) for the VLP-ELISA, NA-HPV and NA-PsV. The agreements of seropositivity between the three assays were low. Six women who were HPV16 DNA positive were seropositive by the NA-HPV but only two were HPV16 seropositive by the VLP-ELISA or NA-PsV. One HPV18 DNA positive woman was seropositive by all three assays. Repeated tests indicated excellent reproducibility of the NA-HPV. Conclusion HPV serology results vary across different assays. The NA-HPV appears to be a sensitive and reliable approach in detecting natural HPV antibodies in HIV-infected women. The NA-HPV can be applied in both HPV natural history studies and vaccine studies in HIV-infected people. PMID:26085957

Development and in-use evaluation of a novel Luminex MicroPlex microsphere-based (TRIOL) assay for simultaneous identification of Mycobacterium tuberculosis and detection of first-line and second-line anti-tuberculous drug resistance in China.

PubMed

Yin, Feifei; Chan, Jasper Fuk-Woo; Zhu, Qixuan; Fu, Ruijia; Chen, Jonathan Hon-Kwan; Choi, Garnet Kwan-Yue; Tee, Kah-Meng; Li, Lihua; Qian, Shiuyun; Yam, Wing-Cheong; Lu, Gang; Yuen, Kwok-Yung

2017-04-01

Rapid and accurate diagnostic assays with simultaneous microbial identification and drug resistance detection are essential for optimising treatment and control of tuberculosis. We developed a novel multiplex (TRIOL, Tuberculosis-Rifampicin-Isoniazid-Ofloxacin-Luminex) assay using the Luminex xMAP system that simultaneously identifies Mycobacterium tuberculosis and detects resistance to first-line and second-line anti-tuberculous drugs, and compared its performance with that by PCR sequencing, using phenotypic drug susceptibility testing as the gold standard. Identification of M. tuberculosis by the TRIOL assay was highly sensitive (100%) and specific (100%). The overall drug-specific specificities were excellent (100%). The overall sensitivity of the TRIOL assay was lower than that of the PCR-sequencing assays (72.4% vs 82.8%) because of a lower sensitivity of detecting rifampicin resistance (71.4% vs 92.9%). The sensitivity of detecting isoniazid and ofloxacin resistance was as good as the PCR-sequencing assays. Importantly, the TRIOL assay did not miss any mutations that were included in the assay. All of the resistant isolates that were missed had uncommon mutations or unknown resistance mechanisms that were not included in the assay. The TRIOL assay has higher throughput, lower cost and is less labour intensive than the PCR-sequencing assays. The TRIOL assay is advantageous in having the capability to detect resistance to multiple drugs and an open-architecture system that allows addition of more specific primers to detect uncommon mutations. Inclusion of additional primers for the identification of non-tuberculous mycobacteria, spoligotyping and improvement of rifampicin resistance detection would enhance the use of the TRIOL assay in future clinical and epidemiological studies. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

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

PubMed

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

2016-05-13

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

A Portable Smart-Phone Readout Device for the Detection of Mercury Contamination Based on an Aptamer-Assay Nanosensor.

PubMed

Xiao, Wei; Xiao, Meng; Fu, Qiangqiang; Yu, Shiting; Shen, Haicong; Bian, Hongfen; Tang, Yong

2016-11-08

The detection of environmental mercury (Hg) contamination requires complex and expensive instruments and professional technicians. We present a simple, sensitive, and portable Hg 2+ detection system based on a smartphone and colorimetric aptamer nanosensor. A smartphone equipped with a light meter app was used to detect, record, and process signals from a smartphone-based microwell reader (MR S-phone), which is composed of a simple light source and a miniaturized assay platform. The colorimetric readout of the aptamer nanosensor is based on a specific interaction between the selected aptamer and Hg 2+ , which leads to a color change in the reaction solution due to an aggregation of gold nanoparticles (AuNPs). The MR S-phone-based AuNPs-aptamer colorimetric sensor system could reliably detect Hg 2+ in both tap water and Pearl River water samples and produced a linear colorimetric readout of Hg 2+ concentration in the range of 1 ng/mL-32 ng/mL with a correlation of 0.991, and a limit of detection (LOD) of 0.28 ng/mL for Hg 2+ . The detection could be quickly completed in only 20 min. Our novel mercury detection assay is simple, rapid, and sensitive, and it provides new strategies for the on-site detection of mercury contamination in any environment.

A Portable Smart-Phone Readout Device for the Detection of Mercury Contamination Based on an Aptamer-Assay Nanosensor

PubMed Central

Xiao, Wei; Xiao, Meng; Fu, Qiangqiang; Yu, Shiting; Shen, Haicong; Bian, Hongfen; Tang, Yong

2016-01-01

The detection of environmental mercury (Hg) contamination requires complex and expensive instruments and professional technicians. We present a simple, sensitive, and portable Hg2+ detection system based on a smartphone and colorimetric aptamer nanosensor. A smartphone equipped with a light meter app was used to detect, record, and process signals from a smartphone-based microwell reader (MR S-phone), which is composed of a simple light source and a miniaturized assay platform. The colorimetric readout of the aptamer nanosensor is based on a specific interaction between the selected aptamer and Hg2+, which leads to a color change in the reaction solution due to an aggregation of gold nanoparticles (AuNPs). The MR S-phone-based AuNPs-aptamer colorimetric sensor system could reliably detect Hg2+ in both tap water and Pearl River water samples and produced a linear colorimetric readout of Hg2+ concentration in the range of 1 ng/mL–32 ng/mL with a correlation of 0.991, and a limit of detection (LOD) of 0.28 ng/mL for Hg2+. The detection could be quickly completed in only 20 min. Our novel mercury detection assay is simple, rapid, and sensitive, and it provides new strategies for the on-site detection of mercury contamination in any environment. PMID:27834794

Rapid specific and visible detection of porcine circovirus type 3 using loop-mediated isothermal amplification (LAMP).

PubMed

Zheng, S; Wu, X; Shi, J; Peng, Z; Gao, M; Xin, C; Liu, Y; Wang, S; Xu, S; Han, H; Yu, J; Sun, W; Cong, X; Li, J; Wang, J

2018-06-01

In this study, a rapid and specific assay for the detection of porcine circovirus type 3 (PCV3) was established using loop-mediated isothermal amplification (LAMP). Four primers were specifically designed to amplify PCV3. The LAMP assay was effectively optimized to amplify PCV3 by water bath at 60°C for 60 min. The detection limit was approximately 1 × 10 1 copy in this LAMP assay. Compared to porcine circovirus type 2 (PCV2), both gE and gD genes of pseudorabies virus (PRV) and porcine parvovirus (PPV), the LAMP assay showed a high specific detection of PCV3. A visible detection method was developed using SYBR Green I to recognize the results rapidly. Based on the detection of 20 clinical tissue samples, the LAMP assay was more practical and convenient than classical PCR due to its simplicity, high sensitivity, rapidity, specificity, visibility and cost efficiency. © 2018 Blackwell Verlag GmbH.

Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR).

PubMed

Turner, Andrew; Sasse, Jurgen; Varadi, Aniko

2016-10-19

Inherited disorders of haemoglobin are the world's most common genetic diseases, resulting in significant morbidity and mortality. The large number of mutations associated with the haemoglobin beta gene (HBB) makes gene scanning by High Resolution Melting (HRM) PCR an attractive diagnostic approach. However, existing HRM-PCR assays are not able to detect all common point mutations and have only a very limited ability to detect larger gene rearrangements. The aim of the current study was to develop a HBB assay, which can be used as a screening test in highly heterogeneous populations, for detection of both point mutations and larger gene rearrangements. The assay is based on a combination of conventional HRM-PCR and a novel Gene Ratio Analysis Copy Enumeration (GRACE) PCR method. HRM-PCR was extensively optimised, which included the use of an unlabelled probe and incorporation of universal bases into primers to prevent interference from common non-pathological polymorphisms. GRACE-PCR was employed to determine HBB gene copy numbers relative to a reference gene using melt curve analysis to detect rearrangements in the HBB gene. The performance of the assay was evaluated by analysing 410 samples. A total of 44 distinct pathological genotypes were detected. In comparison with reference methods, the assay has a sensitivity of 100 % and a specificity of 98 %. We have developed an assay that detects both point mutations and larger rearrangements of the HBB gene. This assay is quick, sensitive, specific and cost effective making it suitable as an initial screening test that can be used for highly heterogeneous cohorts.

Development of a multiplex assay for genus and species-specific detection of Phytophthora based on differences in mitochondrial gene order

USDA-ARS?s Scientific Manuscript database

The availability of a molecular diagnostic assay for Phytophthora that is specific, sensitive, has both genus and species specific detection capabilities multiplexed and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory ef...

Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories

PubMed Central

2012-01-01

Background Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium Dichelobacter nodosus. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet is fundamental to diagnosis of footrot, but D. nodosus should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop a TaqMan-based real-time PCR assay for detection of D. nodosus and to compare its performance with culturing and conventional PCR. Methods A D. nodosus-specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126 samples, and to a conventional PCR method by analysing 224 samples. A selection of PCR-products was cloned and sequenced in order to verify that they had been identified correctly. Results The developed assay had a detection limit of 3.9 fg of D. nodosus genomic DNA. This result was obtained at all three laboratories and corresponds to approximately three copies of the D. nodosus genome per reaction. The assay showed 100% inclusivity and 100% exclusivity for the strains tested. The real-time PCR assay found 54.8% more positive samples than by culturing and 8% more than conventional PCR. Conclusions The developed real-time PCR assay has good specificity and sensitivity for detection of D. nodosus, and the results are easy to interpret. The method is less time-consuming than either culturing or conventional PCR. PMID:22293440

The development of a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay using TaqMan technology for the pan detection of bluetongue virus (BTV).

PubMed

Mulholland, Catherine; McMenamy, Michael J; Hoffmann, Bernd; Earley, Bernadette; Markey, Bryan; Cassidy, Joseph; Allan, Gordon; Welsh, Michael D; McKillen, John

2017-07-01

Bluetongue virus (BTV) is an infectious, non-contagious viral disease of domestic and wild ruminants that is transmitted by adult females of certain Culicoides species. Since 2006, several serotypes including BTV-1, 2, 4, 6, 8, 9 and 16, have spread from the Mediterranean basin into Northern Europe for the first time. BTV-8 in particular, caused a major epidemic in northern Europe. As a result, it is evident that most European countries are at risk of BTV infection. The objective of this study was to develop and validate a real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay based on TaqMan technology for the detection of representative strains of all BTV serotypes. Primers and probes were based on genome segment 10 of the virus, the NS3 gene. The assay was tested for sensitivity, and specificity. The analytical sensitivity of the rRT-PCR assay was 200 copies of RNA per reaction. The assay did not amplify the closely related orbivirus epizootic hemorrhagic disease virus (EHDV) but successfully detected all BTV reference strains including clinical samples from animals experimentally infected with BTV-8. This real time RT-PCR assay offers a sensitive, specific and rapid alternative assay for the pan detection of BTV that could be used as part of a panel of diagnostic assays for the detection of all serotypes of BTV. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Development of a single-tube loop-mediated isothermal amplification assay for detection of four pathogens of bacterial meningitis.

PubMed

Huy, Nguyen Tien; Hang, Le Thi Thuy; Boamah, Daniel; Lan, Nguyen Thi Phuong; Van Thanh, Phan; Watanabe, Kiwao; Huong, Vu Thi Thu; Kikuchi, Mihoko; Ariyoshi, Koya; Morita, Kouichi; Hirayama, Kenji

2012-12-01

Several loop-mediated isothermal amplification (LAMP) assays have been developed to detect common causative pathogens of bacterial meningitis (BM). However, no LAMP assay is reported to detect Streptococcus agalactiae and Streptococcus suis, which are also among common pathogens of BM. Moreover, it is laborious and expensive by performing multiple reactions for each sample to detect bacterial pathogen. Thus, we aimed to design and develop a single-tube LAMP assay capable of detecting multiple bacterial species, based on the nucleotide sequences of the 16S rRNA genes of the bacteria. The nucleotide sequences of the 16S rRNA genes of main pathogens involved in BM were aligned to identify conserved regions, which were further used to design broad range specific LAMP assay primers. We successfully designed a set of broad range specific LAMP assay primers for simultaneous detection of four species including Staphylococcus aureus, Streptococcus pneumoniae, S. suis and S. agalactiae. The broad range LAMP assay was highly specific without cross-reactivity with other bacteria including Haemophilus influenzae, Neisseria meningitidis and Escherichia coli. The sensitivity of our LAMP assay was 100-1000 times higher compared with the conventional PCR assay. The bacterial species could be identified after digestion of the LAMP products with restriction endonuclease DdeI and HaeIII. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

A homogeneous biochemiluminescent assay for detection of influenza

NASA Astrophysics Data System (ADS)

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

2015-05-01

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

Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

PubMed

Pu, Xinzhu; Wang, Zemin; Klaunig, James E

2015-08-06

Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. Copyright © 2015 John Wiley & Sons, Inc.

Serological assays based on recombinant viral proteins for the diagnosis of arenavirus hemorrhagic fevers.

PubMed

Fukushi, Shuetsu; Tani, Hideki; Yoshikawa, Tomoki; Saijo, Masayuki; Morikawa, Shigeru

2012-10-12

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.

An aptamer-based bio-barcode assay with isothermal recombinase polymerase amplification for cytochrome-c detection and anti-cancer drug screening.

PubMed

Loo, Jacky F C; Lau, P M; Ho, H P; Kong, S K

2013-10-15

Based on a recently reported ultra-sensitive bio-barcode (BBC) assay, we have developed an aptamer-based bio-barcode (ABC) alternative to detect a cell death marker cytochrome-c (Cyto-c) and its subsequent application to screen anti-cancer drugs. Aptamer is a short single-stranded DNA selected from a synthetic DNA library by virtue of its high binding affinity and specificity to its target based on its unique 3D structure from the nucleotide sequence after folding. In the BBC assay, an antigen (Ag) in analytes is captured by a micro-magnetic particle (MMP) coated with capturing antibodies (Abs). Gold nanoparticles (NPs) with another recognition Ab against the same target and hundreds of identical DNA molecules of known sequence are subsequently added to allow the formation of sandwich structures ([MMP-Ab1]-Ag-[Ab2-NP-DNA]). After isolating the sandwiches by a magnetic field, the DNAs hybridized to their complementary DNAs covalently bound on the NPs are released from the sandwiches after heating. Acting as an Ag identification tag, these bio-barcode DNAs with known DNA sequence are then amplified by polymerase chain reaction (PCR) and detected by fluorescence. In our ABC assay, we employed a Cyto-c-specific aptamer to substitute both the recognition Ab and barcode DNAs on the NPs in the BBC assay; and a novel isothermal recombinase polymerase amplification for the time-consuming PCR. The detection limit of our ABC assay for the Cyto-c was found to be 10 ng/mL and this new assay can be completed within 3h. Several potential anti-cancer drugs have been tested in vitro for their efficacy to kill liver cancer with or without multi-drug resistance. © 2013 Elsevier B.V. All rights reserved.

Recommended Mass Spectrometry-Based Strategies to Identify Ricin-Containing Samples.

PubMed

Kalb, Suzanne R; Schieltz, David M; Becher, François; Astot, Crister; Fredriksson, Sten-Åke; Barr, John R

2015-11-25

Ricin is a protein toxin produced by the castor bean plant (Ricinus communis) together with a related protein known as R. communis agglutinin (RCA120). Mass spectrometric (MS) assays have the capacity to unambiguously identify ricin and to detect ricin's activity in samples with complex matrices. These qualitative and quantitative assays enable detection and differentiation of ricin from the less toxic RCA120 through determination of the amino acid sequence of the protein in question, and active ricin can be monitored by MS as the release of adenine from the depurination of a nucleic acid substrate. In this work, we describe the application of MS-based methods to detect, differentiate and quantify ricin and RCA120 in nine blinded samples supplied as part of the EQuATox proficiency test. Overall, MS-based assays successfully identified all samples containing ricin or RCA120 with the exception of the sample spiked with the lowest concentration (0.414 ng/mL). In fact, mass spectrometry was the most successful method for differentiation of ricin and RCA120 based on amino acid determination. Mass spectrometric methods were also successful at ranking the functional activities of the samples, successfully yielding semi-quantitative results. These results indicate that MS-based assays are excellent techniques to detect, differentiate, and quantify ricin and RCA120 in complex matrices.

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

Treesearch

William R. Kenealy; Thomas W. Jeffries

2003-01-01

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

Glycoprotein-Based Enzyme-Linked Immunosorbent Assays for Serodiagnosis of Infectious Laryngotracheitis

PubMed Central

Kanabagatte Basavarajappa, Mallikarjuna; Song, Haichen; Lamichhane, Chinta

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 the vaccine vector. PMID:25694519

Rectal swab sampling followed by an enrichment culture-based real-time PCR assay to detect Salmonella enterocolitis in children.

PubMed

Lin, L-H; Tsai, C-Y; Hung, M-H; Fang, Y-T; Ling, Q-D

2011-09-01

Although routine bacterial culture is the traditional reference standard method for the detection of Salmonella infection in children with diarrhoea, it is a time-consuming procedure that usually only gives results after 3-4 days. Some molecular detection methods can improve the turn-around time to within 24 h, but these methods are not applied directly from stool or rectal swab specimens as routine diagnostic methods for the detection of gastrointestinal pathogens. In this study, we tested the feasibility of a bacterial enrichment culture-based real-time PCR assay method for detecting and screening for diarrhoea in children caused by Salmonella. Our results showed that the minimum real-time PCR assay time required to detect enriched bacterial culture from a swab was 3 h. In all children with suspected Salmonella diarrhoea, the enrichment culture-based real-time PCR achieved 85.4% sensitivity and 98.1% specificity, as compared with the 53.7% sensitivity and 100% specificity of detection with the routine bacterial culture method. We suggest that rectal swab sampling followed by enrichment culture-based real-time PCR is suitable as a rapid method for detecting and screening for Salmonella in paediatric patients. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

Development of a β-Lactoglobulin Sensor Based on SPR for Milk Allergens Detection.

PubMed

Ashley, Jon; D'Aurelio, Roberta; Piekarska, Monika; Temblay, Jeff; Pleasants, Mike; Trinh, Linda; Rodgers, Thomas L; Tothill, Ibtisam E

2018-03-27

A sensitive and label-free surface plasmon resonance (SPR) based sensor was developed in this work for the detection of milk allergens. β-lactoglobulin (BLG) protein was used as the biomarker for cow milk detection. This is to be used directly in final rinse samples of cleaning in-place (CIP) systems of food manufacturers. The affinity assay was optimised and characterised before a standard curve was performed in pure buffer conditions, giving a detection limit of 0.164 µg mL -1 as a direct binding assay. The detection limit can be further enhanced through the use of a sandwich assay and amplification with nanomaterials. However, this was not required here, as the detection limit achieved exceeded the required allergen detection levels of 2 µg mL -1 for β-lactoglobulin. The binding affinities of the polyclonal antibody for BLG, expressed by the dissociation constant (K D ), were equal to 2.59 × 10 -9 M. The developed SPR-based sensor offers several advantages in terms of label-free detection, real-time measurements, potential on-line system and superior sensitivity when compared to ELISA-based techniques. The method is novel for this application and could be applied to wider food allergen risk management decision(s) in food manufacturing.

A recombinase polymerase amplification-based assay for rapid detection of African swine fever virus.

PubMed

Wang, Jianchang; Wang, Jinfeng; Geng, Yunyun; Yuan, Wanzhe

2017-10-01

A recombinase polymerase amplification (RPA)-based method was developed for rapid and specific detection of African swine fever virus (ASFV), the etiologic agent of African swine fever, a devastating disease of swine. Primers and the exo probe targeting the conserved region of the P72 gene of ASFV were designed and the reaction was run on the Genie III scanner device. Using recombinant plasmid DNA containing the P72 gene as template, we showed that the amplified product could be detected in less than 10 min and that the detection limit was 10 2 copies DNA/reaction [same detection limit as real-time polymerase chain reaction (PCR)]. The RPA assay did not cross-detect CSFV, PCV-2, PRV, PRRSV, or FMDV, common viruses seen in pigs. Tests of recombinant plasmid-spiked serum samples revealed that RPA and real-time PCR had the same diagnostic rate. The RPA assay, which is simple, cost-effective, and fast, is a promising alternative to real-time PCR for ASFV detection.

A recombinase polymerase amplification-based assay for rapid detection of African swine fever virus

PubMed Central

Wang, Jianchang; Wang, Jinfeng; Geng, Yunyun; Yuan, Wanzhe

2017-01-01

A recombinase polymerase amplification (RPA)-based method was developed for rapid and specific detection of African swine fever virus (ASFV), the etiologic agent of African swine fever, a devastating disease of swine. Primers and the exo probe targeting the conserved region of the P72 gene of ASFV were designed and the reaction was run on the Genie III scanner device. Using recombinant plasmid DNA containing the P72 gene as template, we showed that the amplified product could be detected in less than 10 min and that the detection limit was 102 copies DNA/reaction [same detection limit as real-time polymerase chain reaction (PCR)]. The RPA assay did not cross-detect CSFV, PCV-2, PRV, PRRSV, or FMDV, common viruses seen in pigs. Tests of recombinant plasmid-spiked serum samples revealed that RPA and real-time PCR had the same diagnostic rate. The RPA assay, which is simple, cost-effective, and fast, is a promising alternative to real-time PCR for ASFV detection. PMID:29081590

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

PubMed

Dong, Huahuang; Liu, Jianli; Zhu, Hong; Ou, Chin-Yih; Xing, Wenge; Qiu, Maofeng; Zhang, Guiyun; Xiao, Yao; Yao, Jun; Pan, Pinliang; Jiang, Yan

2012-08-31

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. 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. 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. 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 with HIV.

Simultaneous detection of multiple biomarkers by means of SERS on polymer nanopillar gold arrays

NASA Astrophysics Data System (ADS)

Morasso, Carlo; Picciolini, Silvia; Mehn, Dora; Pellacani, Paola; Frangolho, Ana; Marchesini, Gerardo; Vanna, Renzo; Gualerzi, Alice; Bedoni, Marzia; Marabelli, Franco; Gramatica, Furio

2016-03-01

The detection of biomarkers by means of Surface Enhanced Raman Spectroscopy (SERS) is foreseen to became a very important tool in the clinical practice because of its excellent sensitivity and potential for the simultaneous detection of multiple biomarkers. In the present paper we describe how it was possible to build a sensor for the detection of genetic biomarkers involved in acute myeloid leukemia. The assay is based on the use of a specifically designed SERS substrate made of a 2D crystal structure of polymeric pillars embedded in a gold layer. This substrate is characterized by good enhancing properties coupled with an excellent homogeneity. The SERS substrate was conjugated with DNA probes complementary to a target sequence and used in a sandwich assay with gold nanoparticles labeled with a second DNA probe and a Raman reporter. The so developed assay allowed the detection of a leukemia biomarker (WT1 gene) and an housekeeping gene with low picomolar sensitivity. At last, we optimized the assay in order to tackle one of the main limitations of SERS based assay: the loss of signal that is observed when the Raman spectra are collected in liquid. Combining a preferential functionalization on the polymeric pillars with a different height of the polymer pillars from the gold layer the assay demonstrated its effectiveness even when measured in buffer.

Identification of biological agents using surface enhanced Raman scattering

NASA Astrophysics Data System (ADS)

Paxon, Tracy L.; Duthie, R. Scott; Renko, Casey; Burns, Andrew A.; Lesaicherre, Marie L.; Mondello, Frank J.

2011-05-01

GE Global Research Center, in collaboration with Morpho Detection, Inc. has developed an assay scheme for the identification of biological agents using Surface Enhanced Raman Scattering (SERS). Specifically, unique spectroscopic signatures are generated using SERS tags consisting of individual glass-encapsulated gold nanoparticles and surfacebound reporter molecules. These SERS tags are modified with a capture moiety specific to the antigen of interest, and serve as a spectroscopic label in a bead-based sandwich assay. Assays are being developed for a variety of pathogens and this paper will focus on aspects of assay development, optimization, stabilization and validation. Results on the development of an assay to detect Ricin toxin will be presented, and preliminary feasibility studies for the detection of additional pathogens will be discussed.

Rapid determination of trace copper in animal feed based on micro-plate colorimetric reaction and statistical partitioning correction.

PubMed

Niu, Yiming; Wang, Jiayi; Zhang, Chi; Chen, Yiqiang

2017-04-15

The objective of this study was to develop a micro-plate based colorimetric assay for rapid and high-throughput detection of copper in animal feed. Copper ion in animal feed was extracted by trichloroacetic acid solution and reduced to cuprous ion by hydroxylamine. The cuprous ion can chelate with 2,2'-bicinchoninic acid to form a Cu-BCA complex which was detected with high sensitivity by micro-plate reader at 354nm. The whole assay procedure can be completed within 20min. To eliminate matrix interference, a statistical partitioning correction approach was proposed, which makes the detection of copper in complex samples possible. The limit of detection was 0.035μg/mL and the detection range was 0.1-10μg/mL of copper in buffer solution. Actual sample analysis indicated that this colorimetric assay produced results consistent with atomic absorption spectrometry analysis. These results demonstrated that the developed assay can be used for rapid determination of copper in animal feed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Biosensors and Bio-Bar Code Assays Based on Biofunctionalized Magnetic Microbeads

PubMed Central

Jaffrezic-Renault, Nicole; Martelet, Claude; Chevolot, Yann; Cloarec, Jean-Pierre

2007-01-01

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.

Four human Plasmodium species quantification using droplet digital PCR.

PubMed

Srisutham, Suttipat; Saralamba, Naowarat; Malleret, Benoit; Rénia, Laurent; Dondorp, Arjen M; Imwong, Mallika

2017-01-01

Droplet digital polymerase chain reaction (ddPCR) is a partial PCR based on water-oil emulsion droplet technology. It is a highly sensitive method for detecting and delineating minor alleles from complex backgrounds and provides absolute quantification of DNA targets. The ddPCR technology has been applied for detection of many pathogens. Here the sensitive assay utilizing ddPCR for detection and quantification of Plasmodium species was investigated. The assay was developed for two levels of detection, genus specific for all Plasmodium species and for specific Plasmodium species detection. The ddPCR assay was developed based on primers and probes specific to the Plasmodium genus 18S rRNA gene. Using ddPCR for ultra-sensitive P. falciparum assessment, the lower level of detection from concentrated DNA obtained from a high volume (1 mL) blood sample was 11 parasites/mL. For species identification, in particular for samples with mixed infections, a duplex reaction was developed for detection and quantification P. falciparum/ P. vivax and P. malariae/ P. ovale. Amplification of each Plasmodium species in the duplex reaction showed equal sensitivity to singleplex single species detection. The duplex ddPCR assay had higher sensitivity to identify minor species in 32 subpatent parasitaemia samples from Cambodia, and performed better than real-time PCR. The ddPCR assay shows high sensitivity to assess very low parasitaemia of all human Plasmodium species. This provides a useful research tool for studying the role of the asymptomatic parasite reservoir for transmission in regions aiming for malaria elimination.

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

PubMed

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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

USDA-ARS?s Scientific Manuscript database

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...

Paper-based solid-phase nucleic acid hybridization assay using immobilized quantum dots as donors in fluorescence resonance energy transfer.

PubMed

Noor, M Omair; Shahmuradyan, Anna; Krull, Ulrich J

2013-02-05

A paper-based solid-phase assay is presented for transduction of nucleic acid hybridization using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) were FRET-paired with Cy3 acceptor. Hybridization of Cy3-labeled oligonucleotide targets provided the proximity required for FRET-sensitized emission from Cy3, which served as an analytical signal. The assay exhibited rapid transduction of nucleic acid hybridization within minutes. Without any amplification steps, the limit of detection of the assay was found to be 300 fmol with the upper limit of the dynamic range at 5 pmol. The implementation of glutathione-coated QDs for the development of nucleic acid hybridization assay integrated on a paper-based platform exhibited excellent resistance to nonspecific adsorption of oligonucleotides and showed no reduction in the performance of the assay in the presence of large quantities of noncomplementary DNA. The selectivity of nucleic acid hybridization was demonstrated by single-nucleotide polymorphism (SNP) detection at a contrast ratio of 19 to 1. The reuse of paper over multiple cycles of hybridization and dehybridization was possible, with less than 20% reduction in the performance of the assay in five cycles. This work provides an important framework for the development of paper-based solid-phase QD-FRET nucleic acid hybridization assays that make use of a ratiometric approach for detection and analysis.

Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay

NASA Astrophysics Data System (ADS)

Zhou, Huijuan; Wu, Baoyan

2008-12-01

The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

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

PubMed Central

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

2017-01-01

Late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating diseases affecting potato and tomato worldwide. Early diagnosis of the P. infestans pathogen causing late blight should be the top priority for addressing disease epidemics and management. In this study, we performed a loop-mediated isothermal amplification (LAMP) assay, conventional polymerase chain reaction (PCR), nested PCR, and real-time PCR to verify and compare the sensitivity and specificity of the reaction based on the Ypt1 (Ras-related protein) gene of P. infestans. In comparison with the PCR-based assays, the LAMP technique led to higher specificity and sensitivity, using uncomplicated equipment with an equivalent time frame. All 43 P. infestans isolates, yielded positive detection results using LAMP assay showing no cross reaction with other Phytophthora spp., oomycetes or fungal pathogens. The LAMP assay yielded the lowest detectable DNA concentration (1.28 × 10-4 ng μL-1), being 10 times more sensitive than nested PCR (1.28 × 10-3 ng μL-1), 100 times more sensitive than real-time PCR (1.28 × 10-2 ng μL-1) and 103 times more sensitive than the conventional PCR assay (1.28 × 10-1 ng μL-1). In the field experiment, the LAMP assay outperformed the other tests by amplifying only diseased tissues (leaf and stem), and showing no positive reaction in healthy tissues. Overall, the LAMP assay developed in this study provides a specific, sensitive, simple, and effective visual method for detection of the P. infestans pathogen, and is therefore suitable for application in early prediction of the disease to reduce the risk of epidemics. PMID:29051751

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

PubMed

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

2017-01-01

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

Micromotor-based on-off fluorescence detection of sarin and soman simulants.

PubMed

Singh, Virendra V; Kaufmann, Kevin; Orozco, Jahir; Li, Jinxing; Galarnyk, Michael; Arya, Gaurav; Wang, Joseph

2015-06-30

Self-propelled micromotor-based fluorescent "On-Off" detection of nerve agents is described. The motion-based assay utilizes Si/Pt Janus micromotors coated with fluoresceinamine toward real-time "on-the-fly" field detection of sarin and soman simulants.

Detection and characterization of recombinant DNA expressing vip3A-type insecticidal gene in GMOs--standard single, multiplex and construct-specific PCR assays.

PubMed

Singh, Chandra K; Ojha, Abhishek; Bhatanagar, Raj K; Kachru, Devendra N

2008-01-01

Vegetative insecticidal protein (Vip), a unique class of insecticidal protein, is now part of transgenic plants for conferring resistance against lepidopteron pests. In order to address the imminent regulatory need for detection and labeling of vip3A carrying genetically modified (GM) products, we have developed a standard single PCR and a multiplex PCR assay. As far as we are aware, this is the first report on PCR-based detection of a vip3A-type gene (vip-s) in transgenic cotton and tobacco. Our assay involves amplification of a 284-bp region of the vip-s gene. This assay can possibly detect as many as 20 natural wild-type isolates bearing a vip3A-like gene and two synthetic genes of vip3A in transgenic plants. The limit of detection as established by our assay for GM trait (vip-s) is 0.1%. Spiking with nontarget DNA originating from diverse plant sources had no inhibitory effect on vip-s detection. Since autoclaving of vip-s bearing GM leaf samples showed no deterioration/interference in detection efficacy, the assay seems to be suitable for processed food products as well. The vip-s amplicon identity was reconfirmed by restriction endonuclease assay. The primer set for vip-s was equally effective in a multiplex PCR assay format (duplex, triplex and quadruplex), used in conjunction with the primer sets for the npt-II selectable marker gene, Cauliflower mosaic virus 35S promoter and nopaline synthetase terminator, enabling concurrent detection of the transgene, regulatory sequences and marker gene. Further, the entire transgene construct was amplified using the forward primer of the promoter and the reverse primer of the terminator. The resultant amplicon served as a template for nested PCR to confirm the construct integrity. The method is suitable for screening any vip3A-carrying GM plant and food. The availability of a reliable PCR assay method prior to commercial release of vip3A-based transgenic crops and food would facilitate rapid and efficient regulatory compliance.

Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis

PubMed Central

Sun, Chongyun; Li, Chao; Wang, Xiaochen; Liu, Haican; Zhang, Pingping; Zhao, Xiuqin; Wang, Xinrui; Jiang, Yi; Yang, Ruifu; Wan, Kanglin; Zhou, Lei

2015-01-01

Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a “probe dropout” manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories. PMID:26599667

Development of a bead-based Luminex assay using lipopolysaccharide specific monoclonal antibodies to detect biological threats from Brucella species.

PubMed

Silbereisen, Angelika; Tamborrini, Marco; Wittwer, Matthias; Schürch, Nadia; Pluschke, Gerd

2015-10-05

Brucella, a Gram-negative bacterium, is classified as a potential bioterrorism agent mainly due to the low dose needed to cause infection and the ability to transmit the bacteria via aerosols. Goats/sheep, cattle, pigs, dogs, sheep and rodents are infected by B. melitensis, B. abortus, B. suis, B. canis, B. ovis and B. neotomae, respectively, the six classical Brucella species. Most human cases are caused by B. melitensis and B. abortus. Our aim was to specifically detect Brucellae with 'smooth' lipopolysaccharide (LPS) using a highly sensitive monoclonal antibody (mAb) based immunological assay. To complement molecular detection systems for potential bioterror agents, as required by international biodefense regulations, sets of mAbs were generated by B cell hybridoma technology and used to develop immunological assays. The combination of mAbs most suitable for an antigen capture assay format was identified and an immunoassay using the Luminex xMAP technology was developed. MAbs specific for the LPS O-antigen of Brucella spp. were generated by immunising mice with inactivated B. melitensis or B. abortus cells. Most mAbs recognised both B. melitensis and B. abortus and antigen binding was not impeded by inactivation of the bacterial cells by γ irradiation, formalin or heat treatment, a step required to analyse the samples immunologically under biosafety level two conditions. The Luminex assay recognised all tested Brucella species with 'smooth' LPS with detection limits of 2×10(2) to 8×10(4) cells per mL, depending on the species tested. Milk samples spiked with Brucella spp. cells were identified successfully using the Luminex assay. In addition, the bead-based immunoassay was integrated into a multiplex format, allowing for simultaneous, rapid and specific detection of Brucella spp., Bacillus anthracis, Francisella tularensis and Yersinia pestis within a single sample. Overall, the robust Luminex assay should allow detection of Brucella spp. in both natural outbreak and bio-threat situations.

Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis

PubMed Central

Hanson, Erin K.; Ballantyne, Jack

2014-01-01

Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen).  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive, screening of biological evidence. PMID:24715968

Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis.

PubMed

Hanson, Erin K; Ballantyne, Jack

2013-01-01

Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen).  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive, screening of biological evidence.

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia

PubMed Central

Martin, Jaclyn; Carrillo, Yisel; Talley, Justin L.; Ochoa-Corona, Francisco M.

2018-01-01

Background The importance of tick and flea-borne rickettsia infections is increasingly recognized worldwide. While increased focus has shifted in recent years to the development of point-of-care diagnostics for various vector-borne diseases in humans and animals, little research effort has been devoted to their integration into vector surveillance and control programs, particularly in resource-challenged countries. One technology which may be helpful for large scale vector surveillance initiatives is loop-mediated isothermal amplification (LAMP). The aim of this study was to develop a LAMP assay to detect spotted fever group (SFG) rickettsia DNA from field-collected ticks and fleas and compare with published end-point PCR results. Methodology/Principal findings A Spotted Fever Group rickettsia-specific loop-mediated isothermal amplification (SFGR-LAMP) assay was developed using primers based on a region of the R. rickettsii 17kDa protein gene. The sensitivity, specificity, and reproducibility of the assay were evaluated. The assay was then compared with the results of end-point PCR assays for pooled tick and flea samples obtained from field-based surveillance studies. The sensitivity of the SFGR-LAMP assay was 0.00001 ng/μl (25μl volume) which was 10 times more sensitive than the 17kDa protein gene end-point PCR used as the reference method. The assay only recognized gDNA from SFG and transitional group (TRG) rickettsia species tested but did not detect gDNA from typhus group (TG) rickettsia species or closely or distantly related bacterial species. The SFGR-LAMP assay detected the same positives from a set of pooled tick and flea samples detected by end-point PCR in addition to two pooled flea samples not detected by end-point PCR. Conclusions/significance To our knowledge, this is the first study to develop a functional LAMP assay to initially screen for SFG and TRG rickettsia pathogens in field-collected ticks and fleas. With a high sensitivity and specificity, the results indicate the potential use as a field-based surveillance tool for tick and flea-borne rickettsial pathogens in resource-challenged countries. PMID:29390021

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia.

PubMed

Noden, Bruce H; Martin, Jaclyn; Carrillo, Yisel; Talley, Justin L; Ochoa-Corona, Francisco M

2018-01-01

The importance of tick and flea-borne rickettsia infections is increasingly recognized worldwide. While increased focus has shifted in recent years to the development of point-of-care diagnostics for various vector-borne diseases in humans and animals, little research effort has been devoted to their integration into vector surveillance and control programs, particularly in resource-challenged countries. One technology which may be helpful for large scale vector surveillance initiatives is loop-mediated isothermal amplification (LAMP). The aim of this study was to develop a LAMP assay to detect spotted fever group (SFG) rickettsia DNA from field-collected ticks and fleas and compare with published end-point PCR results. A Spotted Fever Group rickettsia-specific loop-mediated isothermal amplification (SFGR-LAMP) assay was developed using primers based on a region of the R. rickettsii 17kDa protein gene. The sensitivity, specificity, and reproducibility of the assay were evaluated. The assay was then compared with the results of end-point PCR assays for pooled tick and flea samples obtained from field-based surveillance studies. The sensitivity of the SFGR-LAMP assay was 0.00001 ng/μl (25μl volume) which was 10 times more sensitive than the 17kDa protein gene end-point PCR used as the reference method. The assay only recognized gDNA from SFG and transitional group (TRG) rickettsia species tested but did not detect gDNA from typhus group (TG) rickettsia species or closely or distantly related bacterial species. The SFGR-LAMP assay detected the same positives from a set of pooled tick and flea samples detected by end-point PCR in addition to two pooled flea samples not detected by end-point PCR. To our knowledge, this is the first study to develop a functional LAMP assay to initially screen for SFG and TRG rickettsia pathogens in field-collected ticks and fleas. With a high sensitivity and specificity, the results indicate the potential use as a field-based surveillance tool for tick and flea-borne rickettsial pathogens in resource-challenged countries.

Simultaneous detection of eight avian influenza A virus subtypes by multiplex reverse transcription-PCR using a GeXP analyser.

PubMed

Li, Meng; Xie, Zhixun; Xie, Zhiqin; Liu, Jiabo; Xie, Liji; Deng, Xianwen; Luo, Sisi; Fan, Qing; Huang, Li; Huang, Jiaoling; Zhang, Yanfang; Zeng, Tingting; Wang, Sheng

2018-04-18

Recent studies have demonstrated that at least eight subtypes of avian influenza virus (AIV) can infect humans, including H1, H2, H3, H5, H6, H7, H9 and H10. A GeXP analyser-based multiplex reverse transcription (RT)-PCR (GeXP-multiplex RT-PCR) assay was developed in our recent studies to simultaneously detect these eight AIV subtypes using the haemagglutinin (HA) gene. The assay consists of chimeric primer-based PCR amplification with fluorescent labelling and capillary electrophoresis separation. RNA was extracted from chick embryo allantoic fluid or liquid cultures of viral isolates. In addition, RNA synthesised via in vitro transcription was used to determine the specificity and sensitivity of the assay. After selecting the primer pairs, their concentrations and GeXP-multiplex RT-PCR conditions were optimised. The established GeXP-multiplex RT-PCR assay can detect as few as 100 copies of premixed RNA templates. In the present study, 120 clinical specimens collected from domestic poultry at live bird markets and from wild birds were used to evaluate the performance of the assay. The GeXP-multiplex RT-PCR assay specificity was the same as that of conventional RT-PCR. Thus, the GeXP-multiplex RT-PCR assay is a rapid and relatively high-throughput method for detecting and identifying eight AIV subtypes that may infect humans.

Clinical evaluation of a helicase-dependant amplification (HDA)-based commercial assay for the simultaneous detection of HSV-1 and HSV-2.

PubMed

Teo, Jeanette W P; Chiang, Donald; Jureen, Roland; Lin, Raymond T P

2015-11-01

In this study, we evaluate the performance of a commercial assay, the AmpliVue HSV 1+2 Assay (Quidel), which employs HDA for the detection of both HSV-1 and HSV-2. The assay was tested on 307 clinical specimens (genital, oral, and dermal). When compared to shell vial virus culture and immunofluorescence typing of HSV, the positive percent agreement and negative percent agreement values were 98.2% and 90.9%, respectively. Excellent assay performance was demonstrated. Copyright © 2015 Elsevier Inc. All rights reserved.

Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma, Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases.

PubMed

Ueno, Tomohiro; Niimi, Hideki; Yoneda, Noriko; Yoneda, Satoshi; Mori, Masashi; Tabata, Homare; Minami, Hiroshi; Saito, Shigeru; Kitajima, Isao

2015-01-01

Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate. We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples. Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR. We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and the prevention of preterm delivery.

Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma, Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases

PubMed Central

Yoneda, Noriko; Yoneda, Satoshi; Mori, Masashi; Tabata, Homare; Minami, Hiroshi; Saito, Shigeru; Kitajima, Isao

2015-01-01

Background Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate. Methods We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples. Results Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR. Conclusions We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and the prevention of preterm delivery. PMID:26042418

Development of a multiplex assay for genus- and species-specific detection of Phytophthora based on differences in mitochondrial gene order

Treesearch

G. J. Bilodeau; F. N. Martin; M. D. Coffey; C. L. Blomquist

2014-01-01

A molecular diagnostic assay for Phytophthora spp. that is specific, sensitive, has both genus- and species-specific detection capabilities multiplexed, and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory efforts. To address this need, a marker system was developed...

Development of Assays for Detecting Significant Prostate Cancer Based on Molecular Alterations Associated with Cancer in Non-Neoplastic Prostate Tissue

DTIC Science & Technology

2016-12-01

Award Number: W81XWH-11-1-0744 TITLE: Development of Assays for Detecting Significant Prostate Cancer Based on Molecular Alterations Associated...Significant Prostate Cancer Based on Molecular Alterations Associated with Cancer in Non- Neoplastic Prostate Tissue 5b. GRANT NUMBER 10623678 5c...Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The goal of this project was to develop molecular models to

An accurate and inexpensive color-based assay for detecting severe anemia in a limited-resource setting

PubMed Central

McGann, Patrick T.; Tyburski, Erika A.; de Oliveira, Vysolela; Santos, Brigida; Ware, Russell E.; Lam, Wilbur A.

2016-01-01

Severe anemia is an important cause of morbidity and mortality among children in resource-poor settings, but laboratory diagnostics are often limited in these locations. To address this need, we developed a simple, inexpensive, and color-based point-of-care (POC) assay to detect severe anemia. The purpose of this study was to evaluate the accuracy of this novel POC assay to detect moderate and severe anemia in a limited-resource setting. The study was a cross-sectional study conducted on children with sickle cell anemia in Luanda, Angola. The hemoglobin concentrations obtained by the POC assay were compared to reference values measured by a calibrated automated hematology analyzer. A total of 86 samples were analyzed (mean hemoglobin concentration 6.6 g/dL). There was a strong correlation between the hemoglobin concentrations obtained by the POC assay and reference values obtained from an automated hematology analyzer (r=0.88, P<0.0001). The POC assay demonstrated excellent reproducibility (r=0.93, P<0.0001) and the reagents appeared to be durable in a tropical setting (r=0.93, P<0.0001). For the detection of severe anemia that may require blood transfusion (hemoglobin <5 g/dL), the POC assay had sensitivity of 88.9% and specificity of 98.7%. These data demonstrate that an inexpensive (<$0.25 USD) POC assay accurately estimates low hemoglobin concentrations and has the potential to become a transformational diagnostic tool for severe anemia in limited-resource settings. PMID:26317494

An exo probe-based recombinase polymerase amplification assay for the rapid detection of porcine parvovirus.

PubMed

Wang, Jian-Chang; Liu, Li-Bing; Han, Qing-An; Wang, Jin-Feng; Yuan, Wan-Zhe

2017-10-01

Recombinase polymerase amplification (RPA), an isothermal amplification technology, has been developed as an alternative to PCR in pathogen detection. A real-time RPA assay (rt-RPA) was developed to detect the porcine parvovirus (PPV) using primers and exo probe specific for the VP2 gene. The amplification was performed at 39°C for 20min. There was no cross-reaction with other pathogens tested. Using the recombinant plasmid pPPV-VP2 as template, the analytical sensitivity was 103 copies. The assay performance was evaluated by testing 115 field samples by rt-RPA and a real-time PCR assay. The diagnostic agreement between assays was 100%, and PPV DNA was detected in 94 samples. The R 2 value of rt-RPA and real-time PCR was 0.909 by linear regression analysis. The developed rt-RPA assay provides a useful alternative tool for rapid, simple and reliable detection of PPV in diagnostic laboratories and at point-of-care, especially in remote and rural areas. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparison of Laboratory-Developed and Commercial Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assays for Almond (Prunus dulcis) Detection and Quantification.

PubMed

Liu, Changqi; Chhabra, Guneet S; Zhao, Jing; Zaffran, Valerie D; Gupta, Sahil; Roux, Kenneth H; Gradziel, Thomas M; Sathe, Shridhar K

2017-10-01

A commercially available monoclonal antibody (mAb)-based direct sandwich enzyme-linked immunosorbent assay (ELISA) kit (BioFront Technologies, Tallahassee, Fla., U.S.A.) was compared with an in-house developed mAb 4C10-based ELISA for almond detection. The assays were comparable in sensitivity (limit of detection < 1 ppm full fat almond, limit of quantification < 5 ppm full fat almond), specificity (no cross-reactivity with 156 tested foods at a concentration of 100000 ppm whole sample), and reproducibility (intra- and interassay variability < 15% CV). The target antigens were stable and detectable in whole almond seeds subjected to autoclaving, blanching, frying, microwaving, and dry roasting. The almond recovery ranges for spiked food matrices were 84.3% to 124.6% for 4C10 ELISA and 81.2% to 127.4% for MonoTrace ELISA. The almond recovery ranges for commercial and laboratory prepared foods with declared/known almond amount were 30.9% to 161.2% for 4C10 ELISA and 38.1% to 207.6% for MonoTrace ELISA. Neither assay registered any false-positive or negative results among the tested commercial and laboratory prepared samples. Ability to detect and quantify trace amounts of almonds is important for improving safety of almond sensitive consumers. Two monoclonal antibody-based ELISAs were compared for almond detection. The information is useful to food industry, regulatory agencies, scientific community, and almond consumers. © 2017 Institute of Food Technologists®.

Detection of Alternaria fungal contamination in cereal grains by a polymerase chain reaction-based assay.

PubMed

Zur, Gideon; Shimoni, Eyal; Hallerman, Eric; Kashi, Yechezkel

2002-09-01

Alternaria sp. are important fungal contaminants of grain products; they secrete four structural classes of compounds that are toxic or carcinogenic to plants and animals and cause considerable economic losses to growers and the food-processing industry. Alternaria toxins have been detected by high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay, and other techniques. Here, we report the development of a polymerase chain reaction (PCR)-based method for the detection of Alternaria DNA. PCR primers were designed to anneal to the ITS1 and ITS2 regions of the 5.8S rDNA gene of Alternaria alternata or Alternaria solani but not to other microbial or plant DNA. We compared the sensitivity of PCR in detecting Alternaria DNA, that of the HPLC method in detecting Alternaria alternariol and alternariol methyl ether toxins, and that of the morphological examination of mycelia and conidia in experimentally infested corn samples. The sensitivity of toxin detection for HPLC was above the level of contamination in a set of commercially obtained grain samples, resulting in negative scores for all samples, while the PCR-based method and mold growth plating followed by morphological identification of Alternaria gave parallel, positive results for 8 of 10 samples. The PCR assay required just 8 h, enabling the rapid and simultaneous testing of many samples at a low cost. PCR-based evidence for the presence of Alternaria DNA followed by positive assay results for Alternaria toxins would support the rejection of a shipment of grain.

Development of Multiple Cell-Based Assays for the Detection of Histone H3 Lys27 Trimethylation (H3K27me3)

PubMed Central

Lu, Lihui; Wu, Jianghong

2013-01-01

Abstract Posttranslational modification of histone proteins in eukaryotes plays an important role in gene transcription and chromatin structure. Dysregulation of the enzymes involved in histone modification has been linked to many cancer forms, making this target class a potential new area for therapeutics. A reliable assay to monitor small-molecule inhibition of various epigenetic enzymes should play a critical role in drug discovery to fight cancer. However, it has been challenging to develop cell-based assays for high-throughput screening (HTS) and compound profiling. Recently, two homogeneous cell-based assay kits using the AlphaLISA® and LanthaScreen® technologies to detect trimethyl histone H3 Lysine 27 have become commercially available, and a heterogeneous cell assay with modified dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA®) format has been reported. To compare their pros and cons, we evaluated, optimized, and validated these three assay formats in three different cell lines and compared their activities with traditional Western blot detection of histone methylation inhibition by using commercial and in-house small-molecule inhibitors. Our data indicate that, although all four formats produced acceptable results, the homogeneous AlphaLISA assay was best suited for HTS and compound profiling due to its wider window and ease of automation. The DELFIA and Western blot assays were useful as validation tools to confirm the cell activities and eliminate potential false-positive compounds. PMID:23992119

Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform

PubMed Central

Renner, Lars D.; Zan, Jindong; Hu, Linda I.; Martinez, Manuel; Resto, Pedro J.; Siegel, Adam C.; Torres, Clint; Hall, Sara B.; Slezak, Tom R.

2016-01-01

ABSTRACT An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting. IMPORTANCE This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying excellent sensitivity and specificity, the assay systems that we demonstrate may enable future diagnoses of bacterial infection to guide the development of effective chemotherapies and may have a role in areas beyond health where rapid detection is valuable, including in industrial processing and manufacturing, food security, agriculture, and water quality testing. PMID:27986722

Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform.

PubMed

Renner, Lars D; Zan, Jindong; Hu, Linda I; Martinez, Manuel; Resto, Pedro J; Siegel, Adam C; Torres, Clint; Hall, Sara B; Slezak, Tom R; Nguyen, Tuan H; Weibel, Douglas B

2017-02-15

An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting. This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying excellent sensitivity and specificity, the assay systems that we demonstrate may enable future diagnoses of bacterial infection to guide the development of effective chemotherapies and may have a role in areas beyond health where rapid detection is valuable, including in industrial processing and manufacturing, food security, agriculture, and water quality testing. Copyright © 2017 Renner et al.

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

USGS Publications Warehouse

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.

A novel strategy for screening-out raw milk contaminated with Mycobacterium bovis on dairy farms by double-tagging PCR and electrochemical genosensing.

PubMed

Lermo, Anabel; Liébana, Susana; Campoy, Susana; Fabiano, Silvia; García, M Inés; Soutullo, Adriana; Zumárraga, Martín J; Alegret, Salvador; Pividori, M Isabel

2010-06-01

A highly sensitive assay for rapidly screening-out Mycobacterium bovis in contaminated samples was developed based on electrochemical genosensing. The assay consists of specific amplification and double-tagging of the IS6110 fragment, highly related to M. bovis, followed by electrochemical detection of the amplified product. PCR amplification was carried out using a labeled set of primers and resulted in a amplicon tagged at each terminus with both biotin and digoxigenin. Two different electrochemical platforms for the detection of the double-tagged amplicon were evaluated: (i) an avidin biocomposite (Av-GEB) and (ii) a magneto sensor (m-GEC) combined with streptavidin magnetic beads. In both cases, the double- tagged amplicon was immobilized through its biotinylated end and electrochemically detected, using an antiDig-HRP conjugate, through its digoxigenin end. The assay was determined to be highly sensitive, based on the detection of 620 and 10 fmol of PCR amplicon using the Av-GEB and m-GEC strategies, respectively. Moreover, the m-GEC assay showed promising features for the detection of M. bovis on dairy farms by screening for the presence of the bacterium's DNA in milk samples. The obtained results are discussed and compared with respect to those of inter-laboratory PCR assays and tuberculin skin testing.

Setting up a probe based, closed tube real-time PCR assay for focused detection of variable sequence alterations.

PubMed

Becságh, Péter; Szakács, Orsolya

2014-10-01

During diagnostic workflow when detecting sequence alterations, sometimes it is important to design an algorithm that includes screening and direct tests in combination. Normally the use of direct test, which is mainly sequencing, is limited. There is an increased need for effective screening tests, with "closed tube" during the whole process and therefore decreasing the risk of PCR product contamination. The aim of this study was to design such a closed tube, detection probe based screening assay to detect different kind of sequence alterations in the exon 11 of the human c-kit gene region. Inside this region there are variable possible deletions and single nucleotide changes. During assay setup, more probe chemistry formats were screened and tested. After some optimization steps the taqman probe format was selected.

Comparison of human immunodeficiency virus type 1 tropism profiles in clinical samples by the Trofile and MT-2 assays.

PubMed

Coakley, Eoin; Reeves, Jacqueline D; Huang, Wei; Mangas-Ruiz, Marga; Maurer, Irma; Harskamp, Agnes M; Gupta, Soumi; Lie, Yolanda; Petropoulos, Christos J; Schuitemaker, Hanneke; van 't Wout, Angélique B

2009-11-01

The recent availability of CCR5 antagonists as anti-human immunodeficiency virus (anti-HIV) therapeutics has highlighted the need to accurately identify CXCR4-using variants in patient samples when use of this new drug class is considered. The Trofile assay (Monogram Biosciences) has become the method that is the most widely used to define tropism in the clinic prior to the use of a CCR5 antagonist. By comparison, the MT-2 assay has been used since early in the HIV epidemic to define tropism in clinical specimens. Given that there are few data from direct comparisons of these two assays, we evaluated the performance of the plasma-based Trofile assay and the peripheral blood mononuclear cell (PBMC)-based MT-2 assay for the detection of CXCR4 use in defining the tropism of HIV isolates derived from clinical samples. The various samples used for this comparison were derived from participants of the Amsterdam Cohort Studies on HIV infection and AIDS who underwent consecutive MT-2 assay testing of their PBMCs at approximately 3-month intervals. This unique sample set was specifically selected because consecutive MT-2 assays had demonstrated a shift from negative to positive in PBMCs, reflecting the first emergence of CXCR4-using virus in PBMCs above the level of detection of the assay in these individuals. Trofile testing was performed with clonal HIV type 1 (HIV-1) variants (n = 21), MT-2 cell culture-derived cells (n = 20) and supernatants (n = 42), and plasma samples (n = 76). Among the clonal HIV-1 variants and MT-2 cell culture-derived samples, the results of the Trofile and MT-2 assays demonstrated a high degree of concordance (95% to 98%). Among consecutive plasma samples, detection of CXCR4-using virus was at or before the time of first detection by the MT-2 assay in 5/10 patients by the original Trofile assay and in 9/10 patients by the enhanced-sensitivity Trofile assay. Differences in the time to the first detection of CXCR4 use between the MT-2 assay (PBMCs) and the original Trofile assay (plasma) were greatly reduced by the enhanced-sensitivity Trofile assay, suggesting that sensitivity for the detection of minor CXCR4-using variants may be a more important determinant of discordant findings than compartmentalization. The similarities in performance of the enhanced-sensitivity Trofile and MT-2 assays suggest that either may be an appropriate methodology to define tropism in patient specimens.

Development of a multiplex allele-specific primer PCR assay for simultaneous detection of QoI and CAA fungicide resistance alleles in Plasmopara viticola populations.

PubMed

Aoki, Yoshinao; Hada, Yosuke; Suzuki, Shunji

2013-02-01

DNA-based diagnosis has become a common tool for the evaluation of fungicide resistance in obligate phytopathogenic fungus Plasmopara viticola. A multiplex allele-specific primer PCR assay has been developed for the rapid detection of fungicide resistance in P. viticola populations. With this assay, a glycine-to-alanine substitution at codon 143 of the P. viticola cytochrome b gene, which conferred QoI fungicide resistance, and a glycine-to-serine substitution at codon 1105 of the P. viticola cellulose synthase gene PvCesA3, which conferred CAA fungicide resistance, were detected simultaneously. It is suggested that the present assay is a reliable tool for the rapid and simultaneous detection of QoI and CAA fungicide resistance alleles in P. viticola populations. The assay required only 2 h from the sampling of symptoms to the detection of resistance alleles to both fungicides. Copyright © 2012 Society of Chemical Industry.

Evaluation of a dual-probe real time PCR system for detection of mandarin in commercial orange juice.

PubMed

Pardo, Miguel Angel

2015-04-01

A dual-probe real time PCR assay, based on the simultaneous detection of two TaqMan® probes, was evaluated for the detection of mandarin in orange juice. A single conserved polymorphism, located at the 314 position of intron belongs to chloroplast trnL gene, was confirmed by sequencing in 30 mandarin, 28 orange cultivars and 13 hybrids. The assay was also successfully evaluated in a blind trial against analysing 60 samples from different industrial processes in different countries around the world. The detection limit of the assay was established in 1% presence of mandarin detectable in processed orange juice and with a 100% precision. The quantitative application of the assay on citrus mixtures was also investigated, pointing out that the number of chloroplast DNA copies is too variable for its possible use as quantitative analysis. This assay can be employed as a routine methodology to control the accidental mixing during industrial processes and to deter intentional fraud. Copyright © 2014 Elsevier Ltd. All rights reserved.

Rapid and sensitive approach to simultaneous detection of genomes of hepatitis A, B, C, D and E viruses.

PubMed

Kodani, Maja; Mixson-Hayden, Tonya; Drobeniuc, Jan; Kamili, Saleem

2014-10-01

Five viruses have been etiologically associated with viral hepatitis. Nucleic acid testing (NAT) remains the gold standard for diagnosis of viremic stages of infection. NAT methodologies have been developed for all hepatitis viruses; however, a NAT-based assay that can simultaneously detect all five viruses is not available. We designed TaqMan card-based assays for detection of HAV RNA, HBV DNA, HCV RNA, HDV RNA and HEV RNA. The performances of individual assays were evaluated on TaqMan Array Cards (TAC) for detecting five viral genomes simultaneously. Sensitivity and specificity were determined by testing 329 NAT-tested clinical specimens. All NAT-positive samples for HCV (n = 32), HDV (n = 28) and HEV (n = 14) were also found positive in TAC (sensitivity, 100%). Forty-three of 46 HAV-NAT positive samples were also positive in TAC (sensitivity, 94%), while 36 of 39 HBV-NAT positive samples were positive (sensitivity, 92%). No false-positives were detected for HBV (n = 32), HCV (n = 36), HDV (n = 30), and HEV (n = 31) NAT-negative samples (specificity 100%), while 38 of 41 HAV-NAT negative samples were negative by TAC (specificity 93%). TAC assay was concordant with corresponding individual NATs for hepatitis A-E viral genomes and can be used for their detection simultaneously. The TAC assay has potential for use in hepatitis surveillance, for screening of donor specimens and in outbreak situations. Wider availability of TAC-ready assays may allow for customized assays, for improving acute jaundice surveillance and for other purposes for which there is need to identify multiple pathogens rapidly. Published by Elsevier B.V.

Detection of Histoplasma capsulatum from clinical specimens by cycling probe-based real-time PCR and nested real-time PCR.

PubMed

Muraosa, Yasunori; Toyotome, Takahito; Yahiro, Maki; Watanabe, Akira; Shikanai-Yasuda, Maria Aparecida; Kamei, Katsuhiko

2016-05-01

We developed new cycling probe-based real-time PCR and nested real-time PCR assays for the detection of Histoplasma capsulatum that were designed to detect the gene encoding N-acetylated α-linked acidic dipeptidase (NAALADase), which we previously identified as an H. capsulatum antigen reacting with sera from patients with histoplasmosis. Both assays specifically detected the DNAs of all H. capsulatum strains but not those of other fungi or human DNA. The limited of detection (LOD) of the real-time PCR assay was 10 DNA copies when using 10-fold serial dilutions of the standard plasmid DNA and 50 DNA copies when using human serum spiked with standard plasmid DNA. The nested real-time PCR improved the LOD to 5 DNA copies when using human serum spiked with standard plasmid DNA, which represents a 10-fold higher than that observed with the real-time PCR assay. To assess the ability of the two assays to diagnose histoplasmosis, we analyzed a small number of clinical specimens collected from five patients with histoplasmosis, such as sera (n = 4), formalin-fixed paraffin-embedded (FFPE) tissue (n = 4), and bronchoalveolar lavage fluid (BALF) (n = 1). Although clinical sensitivity of the real-time PCR assay was insufficiently sensitive (33%), the nested real-time PCR assay increased the clinical sensitivity (77%), suggesting it has a potential to be a useful method for detecting H. capsulatum DNA in clinical specimens. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Multiplexed detection of DNA sequences using a competitive displacement assay in a microfluidic SERRS-based device.

PubMed

Yazdi, Soroush H; Giles, Kristen L; White, Ian M

2013-11-05

We demonstrate sensitive and multiplexed detection of DNA sequences through a surface enhanced resonance Raman spectroscopy (SERRS)-based competitive displacement assay in an integrated microsystem. The use of the competitive displacement scheme, in which the target DNA sequence displaces a Raman-labeled reporter sequence that has lower affinity for the immobilized probe, enables detection of unlabeled target DNA sequences with a simple single-step procedure. In our implementation, the displacement reaction occurs in a microporous packed column of silica beads prefunctionalized with probe-reporter pairs. The use of a functionalized packed-bead column in a microfluidic channel provides two major advantages: (i) immobilization surface chemistry can be performed as a batch process instead of on a chip-by-chip basis, and (ii) the microporous network eliminates the diffusion limitations of a typical biological assay, which increases the sensitivity. Packed silica beads are also leveraged to improve the SERRS detection of the Raman-labeled reporter. Following displacement, the reporter adsorbs onto aggregated silver nanoparticles in a microfluidic mixer; the nanoparticle-reporter conjugates are then trapped and concentrated in the silica bead matrix, which leads to a significant increase in plasmonic nanoparticles and adsorbed Raman reporters within the detection volume as compared to an open microfluidic channel. The experimental results reported here demonstrate detection down to 100 pM of the target DNA sequence, and the experiments are shown to be specific, repeatable, and quantitative. Furthermore, we illustrate the advantage of using SERRS by demonstrating multiplexed detection. The sensitivity of the assay, combined with the advantages of multiplexed detection and single-step operation with unlabeled target sequences makes this method attractive for practical applications. Importantly, while we illustrate DNA sequence detection, the SERRS-based competitive displacement assay is applicable to detection of a variety of biological macromolecules, including proteins and proteolytic enzymes.

Validation of a rapid bacteria endospore enumeration system for use with spacecraft assembly

NASA Astrophysics Data System (ADS)

Chen, F.; Kuhlman, G.; Kirschner, L.; Kazarians, G.; Matsuyama, A.; Pickett, M.; Venkateswaran, K.; Kastner, J.; Kern, R.

NASA planetary protection policy sets forth strict limits on the number of bacterial endospores that can be present on a spacecraft at launch Currently the only approved method for counting the spores is a culture based assay that requires three days to produce results a timeframe that can be at odds with the rapid pace and rigorous deadlines of spacecraft assembly A possible alternative to the traditional culture based approach is the Millipore Rapid Microbiology Detection System RMDS which has previously been used for process and contamination control in the pharmaceutical and food industries The RMDS is rapid and simple shows high sensitivity 1 colony forming unit CFU sample and correlates well with traditional culture-based methods It combines membrane filtration adenosine triphosphate ATP bioluminescence chemistry and image analysis based on photon detection with a Charge Coupled Device CCD camera In this study we have optimized the assay condition and evaluated the use of the RMDS as a rapid spore detection tool for NASA applications Seven species of Bacillus nine strains that have been repeatedly isolated from clean room environments were assayed In order to select for spores the samples were subjected to a heat shock step before proceeding with the RMDS incubation protocol All strains were detected by the RMDS in sim 5 hours and these assay times were repeatedly demonstrated along with low image background noise The RMDS-based spore detection method is undergoing the final stages of validation and is

Incorporating Aptamers in the Multiple Analyte Profiling Assays (xMAP): Detection of C-Reactive Protein.

PubMed

Bernard, Elyse D; Nguyen, Kathy C; DeRosa, Maria C; Tayabali, Azam F; Aranda-Rodriguez, Rocio

2017-01-01

Aptamers are short oligonucleotide sequences used in detection systems because of their high affinity binding to a variety of macromolecules. With the introduction of aptamers over 25 years ago came the exploration of their use in many different applications as a substitute for antibodies. Aptamers have several advantages; they are easy to synthesize, can bind to analytes for which it is difficult to obtain antibodies, and in some cases bind better than antibodies. As such, aptamer applications have significantly expanded as an adjunct to a variety of different immunoassay designs. The Multiple-Analyte Profiling (xMAP) technology developed by Luminex Corporation commonly uses antibodies for the detection of analytes in small sample volumes through the use of fluorescently coded microbeads. This technology permits the simultaneous detection of multiple analytes in each sample tested and hence could be applied in many research fields. Although little work has been performed adapting this technology for use with apatmers, optimizing aptamer-based xMAP assays would dramatically increase the versatility of analyte detection. We report herein on the development of an xMAP bead-based aptamer/antibody sandwich assay for a biomarker of inflammation (C-reactive protein or CRP). Protocols for the coupling of aptamers to xMAP beads, validation of coupling, and for an aptamer/antibody sandwich-type assay for CRP are detailed. The optimized conditions, protocols and findings described in this research could serve as a starting point for the development of new aptamer-based xMAP assays.

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

PubMed

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

2015-06-02

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

Magnetic Bead Based Immunoassay for Autonomous Detection of Toxins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Y; Hara, C A; Knize, M G

2008-05-01

As a step towards toward the development of a rapid, reliable analyzer for bioagents in the environment, we are developing an automated system for the simultaneous detection of a group of select agents and toxins. To detect toxins, we modified and automated an antibody-based approach previously developed for manual medical diagnostics that uses fluorescent eTag{trademark} reporter molecules and is suitable for highly multiplexed assays. Detection is based on two antibodies binding simultaneously to a single antigen, one of which is labeled with biotin while the other is conjugated to a fluorescent eTag{trademark} through a cleavable linkage. Aqueous samples are incubatedmore » with the mixture of antibodies along with streptavidin-coated magnetic beads coupled to a photo-activatable porphyrin complex. In the presence of antigen, a molecular complex is formed where the cleavable linkage is held in proximity to the photoactivable group. Upon excitation at 680 nm, free radicals are generated, which diffuse and cleave the linkage, releasing the eTags{trademark}. Released eTags{trademark} are analyzed using capillary gel electrophoresis with laser-induced fluorescence detection. Limits of detection for ovalbumin and botulinum toxoid individually were 4 ng/mL (or 80 pg) and 16 ng/mL (or 320 pg), respectively, using the manual assay. In addition, we demonstrated the use of pairs of antibodies from different sources in a single assay to decrease the rate of false positives. Automation of the assay was demonstrated on a flow-through format with higher LODs of 125 ng/mL (or 2.5 ng) each of a mixture of ovalbumin and botulinum toxoid. This versatile assay can be easily modified with the appropriate antibodies to detect a wide range of toxins and other proteins.« less

Rapid and specific detection of Salmonella in water samples using real-time PCR and High Resolution Melt (HRM) curve analysis.

PubMed

van Blerk, G N; Leibach, L; Mabunda, A; Chapman, A; Louw, D

2011-01-01

A real-time PCR assay combined with a pre-enrichment step for the specific and rapid detection of Salmonella in water samples is described. Following amplification of the invA gene target, High Resolution Melt (HRM) curve analysis was used to discriminate between products formed and to positively identify invA amplification. The real-time PCR assay was evaluated for specificity and sensitivity. The assay displayed 100% specificity for Salmonella and combined with a 16-18 h non-selective pre-enrichment step, the assay proved to be highly sensitive with a detection limit of 1.0 CFU/ml for surface water samples. The detection assay also demonstrated a high intra-run and inter-run repeatability with very little variation in invA amplicon melting temperature. When applied to water samples received routinely by the laboratory, the assay showed the presence of Salmonella in particularly surface water and treated effluent samples. Using the HRM based assay, the time required for Salmonella detection was drastically shortened to less than 24 h compared to several days when using standard culturing methods. This assay provides a useful tool for routine water quality monitoring as well as for quick screening during disease outbreaks.

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

PubMed

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

2015-06-02

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.

Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains.

PubMed

He, Peiyan; Chen, Zhongwen; Luo, Jianyong; Wang, Henghui; Yan, Yong; Chen, Lixia; Gao, Wenjie

2014-01-01

Foodborne disease caused by pathogenic Vibrio parahaemolyticus has become a serious public health problem in many countries. Rapid diagnosis and the identification of pathogenic V. parahaemolyticus are very important in the context of public health. In this study, an EvaGreen-based multiplex real-time PCR assay was established for the detection of pathogenic V. parahaemolyticus. This assay targeted three genetic markers of V. parahaemolyticus (species-specific gene toxR and virulence genes tdh and trh). The assay could unambiguously identify pathogenic V. parahaemolyticus with a minimum detection limit of 1.4 pg genomic DNA per reaction (concentration giving a positive multiplex real-time PCR result in 95% of samples). The specificity of the assay was evaluated using 72 strains of V. parahaemolyticus and other bacteria. A validation of the assay with clinical samples confirmed its sensitivity and specificity. Our data suggest the newly established multiplex real-time PCR assay is practical, cost-effective, specific, sensitive and capable of high-throughput detection of pathogenic V. parahaemolyticus. Copyright © 2014. Published by Elsevier Ltd.

Solid-phase receptor-based assay for the detection of cyclic imines by chemiluminescence, fluorescence, or colorimetry.

PubMed

Rodríguez, Laura P; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Antelo, Alvaro; Vieytes, Mercedes R; Botana, Luis M

2011-08-01

The spirolides and gymnodimines are marine phycotoxins included in the group of cyclic imines. The toxicity of these compounds to humans is still unknown, although their toxicity by intraperitoneal injection in rodents is very high. A receptor-based method was developed using the competition of the 13-desmethyl spirolide C with biotin-labeled α-bungarotoxin for binding to nicotinic acetylcholine receptors and the immobilization of the α-bungarotoxin-receptor complex on streptavidin-coated surfaces. The quantification of the immobilized receptor can be achieved using a specific antibody. Finally, after the addition of a secondary antibody labeled with horseradish peroxidase, three alternative substrates of this enzyme generate a chemiluminescent, fluorescent, or colorimetric signal. The assay performs well in shellfish extracts and the detection range is 5-150 nM of 13-desmethyl spirolide C in shellfish extracts, which is at least 5 times more sensitive than the existing fluorescence polarization assay. This assay can also detect gymnodimine, although with 10 times lower sensitivity than the spirolide. The detection of cyclic imines with microplate assays would be useful for screening purposes in order to reduce the number of samples to be processed by bioassays or analytical methods.

Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes.

PubMed

Cai, Xian-Quan; Yu, Hai-Qiong; Bai, Jian-Shan; Tang, Jian-Dong; Hu, Xu-Chu; Chen, Ding-Hu; Zhang, Ren-Li; Chen, Mu-Xin; Ai, Lin; Zhu, Xing-Quan

2012-03-01

Clonorchiasis caused by the oriental liver fluke Clonorchis sinensis is a fish-borne zoonosis endemic in a number of countries. This article describes the development of a TaqMan based real-time PCR assay for detection of C. sinensis DNA in human feces and in fishes. Primers targeting the first internal transcribed spacer (ITS-1) sequence of the fluke were highly specific for C. sinensis, as evidenced by the negative amplification of closely related trematodes in the test with the exception of Opisthorchis viverrini. The detection limit of the assay was 1pg of purified genomic DNA, 5EPG (eggs per gram feces) or one metacercaria per gram fish filet. The assay was evaluated by testing 22 human fecal samples and 37 fish tissues microscopically determined beforehand, and the PCR results were highly in agreement with the microscopic results. This real-time PCR assay provides a useful tool for the sensitive detection of C. sinensis DNA in human stool and aquatic samples in China and other endemic countries where O. viverrini and Opisthorchis felineus are absent. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

A Paper-Based Sandwich Format Hybridization Assay for Unlabeled Nucleic Acid Detection Using Upconversion Nanoparticles as Energy Donors in Luminescence Resonance Energy Transfer.

PubMed

Zhou, Feng; Noor, M Omair; Krull, Ulrich J

2015-09-24

Bioassays based on cellulose paper substrates are gaining increasing popularity for the development of field portable and low-cost diagnostic applications. Herein, we report a paper-based nucleic acid hybridization assay using immobilized upconversion nanoparticles (UCNPs) as donors in luminescence resonance energy transfer (LRET). UCNPs with intense green emission served as donors with Cy3 dye as the acceptor. The avidin functionalized UCNPs were immobilized on cellulose paper and subsequently bioconjugated to biotinylated oligonucleotide probes. Introduction of unlabeled oligonucleotide targets resulted in a formation of probe-target duplexes. A subsequent hybridization of Cy3 labeled reporter with the remaining single stranded portion of target brought the Cy3 dye in close proximity to the UCNPs to trigger a LRET-sensitized emission from the acceptor dye. The hybridization assays provided a limit of detection (LOD) of 146.0 fmol and exhibited selectivity for one base pair mismatch discrimination. The assay was functional even in undiluted serum samples. This work embodies important progress in developing DNA hybridization assays on paper. Detection of unlabeled targets is achieved using UCNPs as LRET donors, with minimization of background signal from paper substrates owing to the implementation of low energy near-infrared (NIR) excitation.

A Simple Colorimetric Assay for Specific Detection of Glutathione-S Transferase Activity Associated with DDT Resistance in Mosquitoes

PubMed Central

Rajatileka, Shavanti; Steven, Andrew; Hemingway, Janet; Ranson, Hilary; Paine, Mark; Vontas, John

2010-01-01

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. PMID:20824165

Comparison of three PCR-based assays for SNP genotyping in sugar beet

USDA-ARS?s Scientific Manuscript database

Background: PCR allelic discrimination technologies have broad applications in the detection of single nucleotide polymorphisms (SNPs) in genetics and genomics. The use of fluorescence-tagged probes is the leading method for targeted SNP detection, but assay costs and error rates could be improved t...

Pseudoperonospora cubensis and P. humuli detection using species-specific probes and high definition melt curve analysis

USDA-ARS?s Scientific Manuscript database

Three assays were developed for molecular differentiation of Pseudoperonospora cubensis and P. humuli, causal agents of cucurbit and hop downy mildew, respectively, for detection of airborne sporangia and diagnosis of symptomatic leaf tissue. The assays were based on previously identified single nuc...

Medically relevant assays with a simple smartphone and tablet based fluorescence detection system.

PubMed

Wargocki, Piotr; Deng, Wei; Anwer, Ayad G; Goldys, Ewa M

2015-05-20

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.

Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells

PubMed Central

Cirelli, Kimberly M.; Dan, Jennifer M.; Morou, Antigoni; Daigneault, Audrey; Brassard, Nathalie; Silvestri, Guido; Routy, Jean-Pierre; Havenar-Daughton, Colin; Crotty, Shane

2017-01-01

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells. PMID:29065175

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

PubMed

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

2017-06-01

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

Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells.

PubMed

Reiss, Samantha; Baxter, Amy E; Cirelli, Kimberly M; Dan, Jennifer M; Morou, Antigoni; Daigneault, Audrey; Brassard, Nathalie; Silvestri, Guido; Routy, Jean-Pierre; Havenar-Daughton, Colin; Crotty, Shane; Kaufmann, Daniel E

2017-01-01

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells.

Competitive immunochromatographic assay for the detection of thiodiglycol sulfoxide, a degradation product of sulfur mustard.

PubMed

Sathe, Manisha; Srivastava, Shruti; Merwyn, S; Agarwal, G S; Kaushik, M P

2014-10-21

An immunochromatographic assay (ICA) based on the competitive antigen-coated format using colloidal gold as the label was developed for the detection of thiodiglycol sulfoxide (TDGO), an important metabolite and degradation compound of sulphur mustard (SM). The ICA test strip consisted of a membrane with a detection zone, a sample pad and an absorbent pad. The membrane was separately coated with hapten-OVA conjugate (test line) and anti-rabbit mouse IgG (control line). The visual detection limit for TDGO by ICA detection was found to be 10 μg mL(-1). For validation, the ICA results obtained for spiked water samples were in good agreement with those obtained by indirect competitive inhibition enzyme-linked immunosorbent assay (ELISA) for TDGO. The assay time for detection was less than 10 min. The developed ICA has the potential to be a useful on-site screening tool for the retrospective detection of SM in environmental samples.

Rapid detection of porcine circovirus type 2 using a TaqMan-based real-time PCR.

PubMed

Zhao, Kai; Han, Fangting; Zou, Yong; Zhu, Lianlong; Li, Chunhua; Xu, Yan; Zhang, Chunling; Tan, Furong; Wang, Jinbin; Tao, Shiru; He, Xizhong; Zhou, Zongqing; Tang, Xueming

2010-12-31

Porcine circovirus type 2 (PCV2) and the associated disease postweaning multisystemic wasting syndrome (PMWS) have caused heavy losses in global agriculture in recent decades. Rapid detection of PCV2 is very important for the effective prophylaxis and treatment of PMWS. To establish a sensitive, specific assay for the detection and quantitation of PCV2, we designed and synthesized specific primers and a probe in the open reading frame 2. The assay had a wide dynamic range with excellent linearity and reliable reproducibility, and detected between 102 and 1010 copies of the genomic DNA per reaction. The coefficient of variation for Ct values varied from 0.59% to 1.05% in the same assay and from 1.9% to 4.2% in 10 different assays. The assay did not cross-react with porcine circovirus type 1, porcine reproductive and respiratory, porcine epidemic diarrhea, transmissible gastroenteritis of pigs and rotavirus. The limits of detection and quantitation were 10 and 100 copies, respectively. Using the established real-time PCR system, 39 of the 40 samples we tested were detected as positive.

Comparative Evaluation of Veriflow® Salmonella Species to USDA and FDA Culture-Based Methods for the Detection of Salmonella spp. in Food and Environmental Samples.

PubMed

Puri, Amrita; Joelsson, Adam C; Terkhorn, Shawn P; Brown, Ashley S; Gaudioso, Zara E; Siciliano, Nicholas A

2017-09-01

Veriflow® Salmonella species (Veriflow SS) is a molecular-based assay for the presumptive detection of Salmonella spp. from environmental surfaces (stainless steel, sealed concrete, plastic, and ceramic tile), dairy (2% milk), raw meat (20% fat ground beef), chicken carcasses, and ready-to-eat (RTE) food (hot dogs). The assay utilizes a PCR detection method coupled with a rapid, visual, flow-based assay that develops in 3 min post-PCR amplification and requires only an 18 h enrichment for maximum sensitivity. The Veriflow SS system eliminates the need for sample purification, gel electrophoresis, or fluorophore-based detection of target amplification and does not require complex data analysis. This Performance Tested MethodSM validation study demonstrated the ability of the Veriflow SS method to detect low levels of artificially inoculated or naturally occurring Salmonella spp. in eight distinct environmental and food matrixes. In each reference comparison study, probability of detection analysis indicated that there was no significant difference between the Veriflow SS method and the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook Chapter 4.06 and the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 5 reference methods. A total of 104 Salmonella strains were detected in the inclusivity study, and 35 nonspecific organisms went undetected in the exclusivity study. The study results show that the Veriflow SS method is a sensitive, selective, and robust assay for the presumptive detection of Salmonella spp. sampled from environmental surfaces (stainless steel, sealed concrete, plastic, and ceramic tile), dairy (2% milk), raw meat (20% fat ground beef), chicken carcasses, and RTE food (hot dogs).

Optimization and validation of a high throughput method for detecting neutralizing antibodies against human papillomavirus (HPV) based on pseudovirons.

PubMed

Nie, Jianhui; Huang, Weijin; Wu, Xueling; Wang, Youchun

2014-09-01

The pseudoviron-based neutralization assay is accepted as the gold standard to evaluate the functional humoral immune response against HPV. The goal of this study was to develop and optimize a human papillomavirus (HPV) neutralization assay using HPV pseudovirons with Gaussia luciferase (Gluc) as the reporter gene. For this purpose, high-titers Gluc pseudovirons were generated by cotransfecting 293TT cells with HPV structural genes and Gluc expressing plasmids. Six types of neutralizing monoclonal antibodies, vaccines immunized serum samples and WHO international antibody standard were used to validate the new developed assay. The ideal circumstances of the assay were identified for cell counts (30,000/well for 96-well plate), pseudoviron inoculating size (100 times RLU above background) and incubation time (72 hr). The sensitivity of the Gluc assay was comparable to secreted alkaline phosphatase (SEAP) assay and higher than the green florescent protein (GFP) assay. The non-specific background for different types of sample was significantly different (rabbit sera > human sera > mouse sera, P < 0.01). The non-specific neutralization effects were not attributed to IgG antibody. The cutoff value for this assay was determined as 50% inhibition at a dilution of 1:40. Without requirements of sample dilution and different incubation times at different temperature before processing, the detection time was shortened from more than 90 min to less than 5 min for a 96-well plate compared with the SEAP-based assay. With the advantages of short detection time and easy-to-use procedure, the newly developed assay is more suitable for large sero-epidemiological studies or clinical trials and more amenable to automation. © 2014 Wiley Periodicals, Inc.

Comparison of Human Immunodeficiency Virus Type 1 Tropism Profiles in Clinical Samples by the Trofile and MT-2 Assays▿

PubMed Central

Coakley, Eoin; Reeves, Jacqueline D.; Huang, Wei; Mangas-Ruiz, Marga; Maurer, Irma; Harskamp, Agnes M.; Gupta, Soumi; Lie, Yolanda; Petropoulos, Christos J.; Schuitemaker, Hanneke; van 't Wout, Angélique B.

2009-01-01

The recent availability of CCR5 antagonists as anti-human immunodeficiency virus (anti-HIV) therapeutics has highlighted the need to accurately identify CXCR4-using variants in patient samples when use of this new drug class is considered. The Trofile assay (Monogram Biosciences) has become the method that is the most widely used to define tropism in the clinic prior to the use of a CCR5 antagonist. By comparison, the MT-2 assay has been used since early in the HIV epidemic to define tropism in clinical specimens. Given that there are few data from direct comparisons of these two assays, we evaluated the performance of the plasma-based Trofile assay and the peripheral blood mononuclear cell (PBMC)-based MT-2 assay for the detection of CXCR4 use in defining the tropism of HIV isolates derived from clinical samples. The various samples used for this comparison were derived from participants of the Amsterdam Cohort Studies on HIV infection and AIDS who underwent consecutive MT-2 assay testing of their PBMCs at approximately 3-month intervals. This unique sample set was specifically selected because consecutive MT-2 assays had demonstrated a shift from negative to positive in PBMCs, reflecting the first emergence of CXCR4-using virus in PBMCs above the level of detection of the assay in these individuals. Trofile testing was performed with clonal HIV type 1 (HIV-1) variants (n = 21), MT-2 cell culture-derived cells (n = 20) and supernatants (n = 42), and plasma samples (n = 76). Among the clonal HIV-1 variants and MT-2 cell culture-derived samples, the results of the Trofile and MT-2 assays demonstrated a high degree of concordance (95% to 98%). Among consecutive plasma samples, detection of CXCR4-using virus was at or before the time of first detection by the MT-2 assay in 5/10 patients by the original Trofile assay and in 9/10 patients by the enhanced-sensitivity Trofile assay. Differences in the time to the first detection of CXCR4 use between the MT-2 assay (PBMCs) and the original Trofile assay (plasma) were greatly reduced by the enhanced-sensitivity Trofile assay, suggesting that sensitivity for the detection of minor CXCR4-using variants may be a more important determinant of discordant findings than compartmentalization. The similarities in performance of the enhanced-sensitivity Trofile and MT-2 assays suggest that either may be an appropriate methodology to define tropism in patient specimens. PMID:19687240

Development of On-Line High Performance Liquid Chromatography (HPLC)-Biochemical Detection Methods as Tools in the Identification of Bioactives

PubMed Central

Malherbe, Christiaan J.; de Beer, Dalene; Joubert, Elizabeth

2012-01-01

Biochemical detection (BCD) methods are commonly used to screen plant extracts for specific biological activities in batch assays. Traditionally, bioactives in the most active extracts were identified through time-consuming bio-assay guided fractionation until single active compounds could be isolated. Not only are isolation procedures often tedious, but they could also lead to artifact formation. On-line coupling of BCD assays to high performance liquid chromatography (HPLC) is gaining ground as a high resolution screening technique to overcome problems associated with pre-isolation by measuring the effects of compounds post-column directly after separation. To date, several on-line HPLC-BCD assays, applied to whole plant extracts and mixtures, have been published. In this review the focus will fall on enzyme-based, receptor-based and antioxidant assays. PMID:22489144

Glycoprotein-based enzyme-linked immunosorbent assays for serodiagnosis of infectious laryngotracheitis.

PubMed

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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fowlkes, Jason Davidson; Owens, Elizabeth T; Standaert, Robert F

2009-01-01

Identifying and characterizing protein interactions are fundamental steps towards understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the co-localization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecularmore » tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin (Imp ) and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified co-localization assay is sensitive enough to detect protein interactions with Kd values that span over four orders of magnitude (1nM to 15 M). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris« less

Hyperthermostable binding molecules on phage: Assay components for point-of-care diagnostics for active tuberculosis infection.

PubMed

Zhao, Ning; Spencer, John; Schmitt, Margaret A; Fisk, John D

2017-03-15

Tuberculosis is the leading cause of death from infectious disease worldwide. The low sensitivity, extended processing time, and high expense of current diagnostics are major challenges to the detection and treatment of tuberculosis. Mycobacterium tuberculosis ornithine transcarbamylase (Mtb OTC, Rv1656) has been identified in the urine of patients with active TB infection and is a promising target for point-of-care diagnostics. Specific binding proteins with low nanomolar affinities for Mtb OTC were selected from a phage display library built upon a hyperthermostable Sso7d scaffold. Phage particles displaying Sso7d variants were utilized to generate a sandwich ELISA-based assay for Mtb OTC. The assay response is linear between 2 ng/mL and 125 ng/mL recombinant Mtb OTC and has a limit of detection of 400 pg/mL recombinant Mtb OTC. The assay employing a phage-based detection reagent is comparable to commercially-available antibody-based biosensors. Importantly, the assay maintains functionality at both neutral and basic pH in presence of salt and urea over the range of concentrations typical for human urine. Phage-based diagnostic systems may feature improved physical stability and cost of production relative to traditional antibody-based reagents, without sacrificing specificity and sensitivity. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of four novel isothermal amplification assays towards simple and rapid genotyping of chloroquine resistant Plasmodium falciparum.

PubMed

Chahar, Madhvi; Anvikar, Anup; Dixit, Rajnikant; Valecha, Neena

2018-07-01

Loop mediated isothermal amplification (LAMP) assay is sensitive, prompt, high throughput and field deployable technique for nucleic acid amplification under isothermal conditions. In this study, we have developed and optimized four different visualization methods of loop-mediated isothermal amplification (LAMP) assay to detect Pfcrt K76T mutants of P. falciparum and compared their important features for one-pot in-field applications. Even though all the four tested LAMP methods could successfully detect K76T mutants of P. falciparum, however considering the time, safety, sensitivity, cost and simplicity, the malachite green and HNB based methods were found more efficient. Among four different visual dyes uses to detect LAMP products accurately, hydroxynaphthol blue and malachite green could produce long stable color change and brightness in a close tube-based approach to prevent cross-contamination risk. Our results indicated that the LAMP offers an interesting novel and convenient best method for the rapid, sensitive, cost-effective, and fairly user friendly tool for detection of K76T mutants of P. falciparum and therefore presents an alternative to PCR-based assays. Based on our comparative analysis, better field based LAMP visualization method can be chosen easily for the monitoring of other important drug targets (Kelch13 propeller region). Copyright © 2018 Elsevier Inc. All rights reserved.

Recommended Mass Spectrometry-Based Strategies to Identify Ricin-Containing Samples

PubMed Central

Kalb, Suzanne R.; Schieltz, David M.; Becher, François; Astot, Crister; Fredriksson, Sten-Åke; Barr, John R.

2015-01-01

Ricin is a protein toxin produced by the castor bean plant (Ricinus communis) together with a related protein known as R. communis agglutinin (RCA120). Mass spectrometric (MS) assays have the capacity to unambiguously identify ricin and to detect ricin’s activity in samples with complex matrices. These qualitative and quantitative assays enable detection and differentiation of ricin from the less toxic RCA120 through determination of the amino acid sequence of the protein in question, and active ricin can be monitored by MS as the release of adenine from the depurination of a nucleic acid substrate. In this work, we describe the application of MS-based methods to detect, differentiate and quantify ricin and RCA120 in nine blinded samples supplied as part of the EQuATox proficiency test. Overall, MS-based assays successfully identified all samples containing ricin or RCA120 with the exception of the sample spiked with the lowest concentration (0.414 ng/mL). In fact, mass spectrometry was the most successful method for differentiation of ricin and RCA120 based on amino acid determination. Mass spectrometric methods were also successful at ranking the functional activities of the samples, successfully yielding semi-quantitative results. These results indicate that MS-based assays are excellent techniques to detect, differentiate, and quantify ricin and RCA120 in complex matrices. PMID:26610568

Phosphodiesterase (PDE5) inhibition assay for rapid detection of erectile dysfunction drugs and analogs in sexual enhancement products.

PubMed

Santillo, Michael F; Mapa, Mapa S T

2018-02-28

Products marketed as dietary supplements for sexual enhancement are frequently adulterated with phosphodiesterase-5 (PDE5) inhibitors, which are erectile dysfunction drugs or their analogs that can cause adverse health effects. Due to widespread adulteration, a rapid screening assay was developed to detect PDE5 inhibitors in adulterated products. The assay employs fluorescence detection and is based on measuring inhibition of PDE5 activity, the pharmacological mechanism shared among the adulterants. Initially, the assay reaction scheme was established and characterized, followed by analysis of 9 representative PDE5 inhibitors (IC 50 , 0.4-4.0 ng mL -1 ), demonstrating sensitive detection in matrix-free solutions. Next, dietary supplements serving as matrix blanks (n = 25) were analyzed to determine matrix interference and establish a threshold value; there were no false positives. Finally, matrix blanks were spiked with 9 individual PDE5 inhibitors, along with several mixtures. All 9 adulterants were successfully detected (≤ 5 % false negative rate; n = 20) at a concentration of 1.00 mg g -1 , which is over 5 times lower than concentrations commonly encountered in adulterated products. A major distinction of the PDE5 inhibition assay is the ability to detect adulterants without prior knowledge of their chemical structures, demonstrating a broad-based detection capability that can address a continuously evolving threat of new adulterants. The PDE5 inhibition assay can analyze over 40 samples simultaneously within 15 minutes and involves a single incubation step and simple data analysis, all of which are advantageous for combating the widespread adulteration of sex-enhancement products. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Rapid and accurate detection of KRAS mutations in colorectal cancers using the isothermal-based optical sensor for companion diagnostics

PubMed Central

Koo, Bonhan; Lee, Tae Yoon; Lee, Jeong Hoon; Shin, Yong; Lim, Seok-Byung

2017-01-01

Although KRAS mutational status testing is becoming a companion diagnostic tool for managing patients with colorectal cancer (CRC), there are still several difficulties when analyzing KRAS mutations using the existing assays, particularly with regard to low sensitivity, its time-consuming, and the need for large instruments. We developed a rapid, sensitive, and specific mutation detection assay based on the bio-photonic sensor termed ISAD (isothermal solid-phase amplification/detection), and used it to analyze KRAS gene mutations in human clinical samples. To validate the ISAD-KRAS assay for use in clinical diagnostics, we examined for hotspot KRAS mutations (codon 12 and codon 13) in 70 CRC specimens using PCR and direct sequencing methods. In a serial dilution study, ISAD-KRAS could detect mutations in a sample containing only 1% of the mutant allele in a mixture of wild-type DNA, whereas both PCR and direct sequencing methods could detect mutations in a sample containing approximately 30% of mutant cells. The results of the ISAD-KRAS assay from 70 clinical samples matched those from PCR and direct sequencing, except in 5 cases, wherein ISAD-KRAS could detect mutations that were not detected by PCR and direct sequencing. We also found that the sensitivity and specificity of ISAD-KRAS were 100% within 30 min. The ISAD-KRAS assay provides a rapid, highly sensitive, and label-free method for KRAS mutation testing, and can serve as a robust and near patient testing approach for the rapid detection of patients most likely to respond to anti-EGFR drugs. PMID:29137388

Detecting the Presence of Bacterial DNA and RNA by Polymerase Chain Reaction to Diagnose Suspected Periprosthetic Joint Infection after Antibiotic Therapy.

PubMed

Fang, Xin-Yu; Li, Wen-Bo; Zhang, Chao-Fan; Huang, Zi-da; Zeng, Hui-Yi; Dong, Zheng; Zhang, Wen-Ming

2018-02-01

To explore the diagnostic efficiency of DNA-based and RNA-based quantitative polymerase chain reaction (qPCR) analyses for periprosthetic joint infection (PJI). To determine the detection limit of DNA-based and RNA-based qPCR in vitro, Staphylococcus aureus and Escherichia coli strains were added to sterile synovial fluid obtained from a patient with knee osteoarthritis. Serial dilutions of samples were analyzed by DNA-based and RNA-based qPCR. Clinically, patients who were suspected of having PJI and eventually underwent revision arthroplasty in our hospital from July 2014 to December 2016 were screened. Preoperative puncture or intraoperative collection was performed on patients who met the inclusion and exclusion criteria to obtain synovial fluid. DNA-based and RNA-based PCR analyses and culture were performed on each synovial fluid sample. The patients' demographic characteristics, medical history, and laboratory test results were recorded. The diagnostic efficiency of both PCR assays was compared with culture methods. The in vitro analysis demonstrated that DNA-based qPCR assay was highly sensitive, with the detection limit being 1200 colony forming units (CFU)/mL of S. aureus and 3200 CFU/mL of E. coli. Meanwhile, The RNA-based qPCR assay could detect 2300 CFU/mL of S. aureus and 11 000 CFU/mL of E. coli. Clinically, the sensitivity, specificity, and accuracy were 65.7%, 100%, and 81.6%, respectively, for the culture method; 81.5%, 84.8%, and 83.1%, respectively, for DNA-based qPCR; and 73.6%, 100%, and 85.9%, respectively, for RNA-based qPCR. DNA-based qPCR could detect suspected PJI with high sensitivity after antibiotic therapy. RNA-based qPCR could reduce the false positive rates of DNA-based assays. qPCR-based methods could improve the efficiency of PJI diagnosis. © 2018 Chinese Orthopaedic Association and John Wiley & Sons Australia, Ltd.

Development of immunoglobulin class-specific enzyme-linked immunosorbent assays for measuring antibodies against avian rotavirus.

PubMed

Myers, T J; Schat, K A; Mockett, A P

1989-01-01

Immunoglobulin class-specific enzyme-linked immunosorbent assays were developed for detecting antibodies against avian rotavirus in serum, intestinal contents, and bile from experimentally infected specific-pathogen-free (SPF) chickens. Both indirect and antibody-capture (AbC) assays were developed based on monoclonal antibodies specific for chicken IgG, IgM, and IgA. Treatment of purified rotavirus with sodium thiocyanate before coating the plate improved the rotavirus-specific reading in the indirect assay. Use of Immunolon 2 plates facilitated attachment of monoclonal antibodies to the plate in the AbC assay. Addition of 5% powdered skim milk to the diluent buffer reduced nonspecific background readings. The indirect assay was superior for detecting rotavirus-specific IgG, whereas the AbC assay was better for detecting rotavirus-specific IgM and IgA. The presence of intestinal contents in the assay wells did not reduce the measurable titers of IgG, IgM, or IgA. These assays showed that SPF chickens produced systemic and mucosal antibodies against avian rotavirus.

Development of a biomimetic enzyme-linked immunosorbent assay based on molecularly imprinted polymers on paper for the detection of carbaryl.

PubMed

Zhang, Can; Cui, Hanyu; Han, Yufeng; Yu, Fangfang; Shi, Xiaoman

2018-02-01

A biomimetic enzyme-linked immunosorbent assay (BELISA) which was based on molecularly imprinted polymers on paper (MIPs-paper) with specific recognition was developed. As a detector, the surface of paper was modified with γ-MAPS by hydrolytic action and anchored the MIP layer on γ-MAPS modified-paper by copolymerization to construct the artificial antibody Through a series of experimentation and verification, we successful got the MIPs-paper and established BELISA for the detection of carbaryl. The development of MIPs-paper based on BELISA was applied to detect carbaryl in real samples and validated by an enzyme-linked immunosorbent assay (ELISA) based on anti-carbaryl biological antibody. The results of these two methods (BELISA and ELISA) were well correlated (R 2 =0.944). The established method of MIPs-paper BELISA exhibits the advantages of low cost, higher stability and being re-generable, which can be applied as a convenient tool for the fast and efficient detection of carbaryl. Copyright © 2017. Published by Elsevier Ltd.

LC-MS-based characterization of the peptide reactivity of chemicals to improve the in vitro prediction of the skin sensitization potential.

PubMed

Natsch, Andreas; Gfeller, Hans

2008-12-01

A key step in the skin sensitization process is the formation of a covalent adduct between skin sensitizers and endogenous proteins and/or peptides in the skin. Based on this mechanistic understanding, there is a renewed interest in in vitro assays to determine the reactivity of chemicals toward peptides in order to predict their sensitization potential. A standardized peptide reactivity assay yielded a promising predictivity. This published assay is based on high-performance liquid chromatography with ultraviolet detection to quantify peptide depletion after incubation with test chemicals. We had observed that peptide depletion may be due to either adduct formation or peptide oxidation. Here we report a modified assay based on both liquid chromatography-mass spectrometry (LC-MS) analysis and detection of free thiol groups. This approach allows simultaneous determination of (1) peptide depletion, (2) peptide oxidation (dimerization), (3) adduct formation, and (4) thiol reactivity and thus generates a more detailed characterization of the reactivity of a molecule. Highly reactive molecules are further discriminated with a kinetic measure. The assay was validated on 80 chemicals. Peptide depletion could accurately be quantified both with LC-MS detection and depletion of thiol groups. The majority of the moderate/strong/extreme sensitizers formed detectable peptide adducts, but many sensitizers were also able to catalyze peptide oxidation. Whereas adduct formation was only observed for sensitizers, this oxidation reaction was also observed for two nonsensitizing fragrance aldehydes, indicating that peptide depletion might not always be regarded as sufficient evidence for rating a chemical as a sensitizer. Thus, this modified assay gives a more informed view of the peptide reactivity of chemicals to better predict their sensitization potential.

Development of a pan-rickettsial molecular diagnostic test based on recombinase polymerase amplification assay.

PubMed

Kissenkötter, Jonas; Hansen, Sören; Böhlken-Fascher, Susanne; Ademowo, Olusegun George; Oyinloye, Oladapo Elijah; Bakarey, Adeleye Solomon; Dobler, Gerhard; Tappe, Dennis; Patel, Pranav; Czerny, Claus-Peter; Abd El Wahed, Ahmed

2018-03-01

Rickettsioses are zoonotic vector-transmitted bacterial infections leading to flu-like symptoms and can progress to severe illness in humans. The gold standard for diagnosis of rickettsial infections is the indirect immunofluorescence assay, a serological method which is not suitable for pathogen identification during the acute phase of the disease. Therefore, several real-time PCR assays were developed. These assays are very sensitive, but require high-equipped laboratories and well-trained personnel. Hence, in this study, a rapid point-of-need detection method was developed to detect all Rickettsia species. The 23S and 16S rRNA genes were targeted to develop a recombinase polymerase amplification (RPA) assay. Both 23S and 16S_RPA assays required between seven to ten minutes to amplify and detect one or ten DNA molecules/reaction, respectively. The 16S_RPA assay detected all tested species, whereas the 23S_RPA assay identified only species of the spotted fever and transitional rickettsial groups. All results were compared with real-time PCR assays directed against the same rickettsial genes. The RPA assays are easy to handle and produced quicker results in comparison to real-time PCRs. Both RPA assays were implemented in a mobile suitcase laboratory to ease the use in rural areas. This method can help to provide rapid management of rickettsial infections. Copyright © 2017 Elsevier Inc. All rights reserved.

Autoantibody Approach for Serum-Based Detection of Head and Neck Cancer — EDRN Public Portal

Cancer.gov

Our long term goal is to improve survival of patients with head and neck squamous cell carcinoma (HNSCC) through early detection using simple noninvasive serum assays in an ELISA-like platform. The objective of this proposal is to improve and confirm the validity of a diagnostic serum assay based on a panel of cancer-specific biomarkers for early cancer detection in patients with HNSCC. Our central hypothesis is that the detection of antibody responses to HNSCC-specific antigens, using a panel of biomarkers, can provide sufficient sensitivity and specificity suitable for clinical testing in the primary setting to screen and diagnose HNSCC in high risk populations to improve early detection.

Field evaluation of a recombinase polymerase amplification assay for the diagnosis of Schistosoma japonicum infection in Hunan province of China.

PubMed

Xing, Weiwei; Yu, Xinling; Feng, Jingtao; Sun, Kui; Fu, Wenliang; Wang, Yuanyuan; Zou, Minji; Xia, Wenrong; Luo, Zhihong; He, Hongbin; Li, Yuesheng; Xu, Donggang

2017-02-21

Current diagnostic methods for Schistosoma japonicum infection are insensitive for low-density infections. Therefore, a new diagnostic assay based on recombinase polymerase amplification (RPA) technology was established and assessed for field applification. The S.japonicum RPA assay was developed to target highly repetitive retrotransposon SjR2 gene of S japonicum, and its sensitivity and specificity were assessed by serial dilution of S. japonicum genomic DNA and other related worm genomic DNA respectively. The RPA diagnostic validity was first evaluated in 60 fecal samples from healthy people and patients, and then compared with other diagnostic tests in 200 high-risk individuals living in endemic areas. The real time RPA assay could detect 0.9 fg S. japonicum DNA within 15 min and distinguish S. japonicum from other worms. The validity analysis of RPA for the detection of S. japonicum in stool samples from 30 S. japonicum-infected patients and 30 healthy persons indicated 100% sensitivity and specificity. When testing 200 fecal or serum samples from a high-risk population, the percentage sensitivity of RPA was 100%, whereas that of indirect hemagglutination assay (IHA) and enzyme-linked immunosorbent assay (ELISA) were 80.3% and 85.2% respectively. In addition, the RPA presented better consistency with the stool-based tests than IHA and ELISA. Overall, the RPA was superior to other detection methods with respect to detection time, sensitivity, and convenience. This is the first time we applied the RPA technology to the field evaluation of S. japonicum infection. And the results suggest that RPA-based assays can be used as a promising point-of-care test for the diagnosis of schistosomiasis.

Detection of liver kidney microsomal type 1 antibody using molecularly based immunoassays

PubMed Central

Kerkar, N; Ma, Y; Davies, E T; Cheeseman, P; Mieli-Vergani, G; Vergani, D

2002-01-01

Aims: To assess the diagnostic value of two commercial molecularly based immunoassays detecting liver kidney microsomal type 1 antibody (LKM1). Methods: The performance of Varelisa and LKM1 enzyme linked immunosorbent assay (ELISA) was compared with immunofluorescence, and two validated research techniques—an in house ELISA and a radioligand assay measuring antibodies to P4502D6. Thirty serum samples from three patients with autoimmune hepatitis type 2 covering immunofluorescence titres of 1/10 to 1/10 240 and 55 LKM1 negative controls were tested. Results: All 30 sera that were LKM1 positive by immunofluorescence were positive by the in house ELISA, the radioligand assay, and LKM1-ELISA, and 29 were also positive by Varelisa. None of the 55 sera negative for LKM1 by immunofluorescence was positive by the in house ELISA and radioligand assay, but one was positive by Varelisa and 14 were positive using the LKM1-ELISA. Agreement between immunofluorescence, the in house ELISA, the radioligand assay, and Varelisa was high (κ > 0.8), and agreement between immunofluorescence and LKM1-ELISA was moderate (κ = 0.63). Conclusion: The assay kit marketed as Varelisa allows accurate detection of LKM1. PMID:12461054

Detection of liver kidney microsomal type 1 antibody using molecularly based immunoassays.

PubMed

Kerkar, N; Ma, Y; Davies, E T; Cheeseman, P; Mieli-Vergani, G; Vergani, D

2002-12-01

To assess the diagnostic value of two commercial molecularly based immunoassays detecting liver kidney microsomal type 1 antibody (LKM1). The performance of Varelisa and LKM1 enzyme linked immunosorbent assay (ELISA) was compared with immunofluorescence, and two validated research techniques-an in house ELISA and a radioligand assay measuring antibodies to P4502D6. Thirty serum samples from three patients with autoimmune hepatitis type 2 covering immunofluorescence titres of 1/10 to 1/10 240 and 55 LKM1 negative controls were tested. All 30 sera that were LKM1 positive by immunofluorescence were positive by the in house ELISA, the radioligand assay, and LKM1-ELISA, and 29 were also positive by Varelisa. None of the 55 sera negative for LKM1 by immunofluorescence was positive by the in house ELISA and radioligand assay, but one was positive by Varelisa and 14 were positive using the LKM1-ELISA. Agreement between immunofluorescence, the in house ELISA, the radioligand assay, and Varelisa was high (kappa > 0.8), and agreement between immunofluorescence and LKM1-ELISA was moderate (kappa = 0.63). The assay kit marketed as Varelisa allows accurate detection of LKM1.

A Melting Curve-Based Multiplex RT-qPCR Assay for Simultaneous Detection of Four Human Coronaviruses

PubMed Central

Wan, Zhenzhou; Zhang, Ya’nan; He, Zhixiang; Liu, Jia; Lan, Ke; Hu, Yihong; Zhang, Chiyu

2016-01-01

Human coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1 are common respiratory viruses associated with acute respiratory infection. They have a global distribution. Rapid and accurate diagnosis of HCoV infection is important for the management and treatment of hospitalized patients with HCoV infection. Here, we developed a melting curve-based multiplex RT-qPCR assay for simultaneous detection of the four HCoVs. In the assay, SYTO 9 was used to replace SYBR Green I as the fluorescent dye, and GC-modified primers were designed to improve the melting temperature (Tm) of the specific amplicon. The four HCoVs were clearly distinguished by characteristic melting peaks in melting curve analysis. The detection sensitivity of the assay was 3 × 102 copies for HCoV-OC43, and 3 × 101 copies for HCoV-NL63, HCoV-229E and HCoV-HKU1 per 30 μL reaction. Clinical evaluation and sequencing confirmation demonstrated that the assay was specific and reliable. The assay represents a sensitive and reliable method for diagnosis of HCoV infection in clinical samples. PMID:27886052

A Melting Curve-Based Multiplex RT-qPCR Assay for Simultaneous Detection of Four Human Coronaviruses.

PubMed

Wan, Zhenzhou; Zhang, Ya'nan; He, Zhixiang; Liu, Jia; Lan, Ke; Hu, Yihong; Zhang, Chiyu

2016-11-23

Human coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1 are common respiratory viruses associated with acute respiratory infection. They have a global distribution. Rapid and accurate diagnosis of HCoV infection is important for the management and treatment of hospitalized patients with HCoV infection. Here, we developed a melting curve-based multiplex RT-qPCR assay for simultaneous detection of the four HCoVs. In the assay, SYTO 9 was used to replace SYBR Green I as the fluorescent dye, and GC-modified primers were designed to improve the melting temperature (Tm) of the specific amplicon. The four HCoVs were clearly distinguished by characteristic melting peaks in melting curve analysis. The detection sensitivity of the assay was 3 × 10² copies for HCoV-OC43, and 3 × 10¹ copies for HCoV-NL63, HCoV-229E and HCoV-HKU1 per 30 μL reaction. Clinical evaluation and sequencing confirmation demonstrated that the assay was specific and reliable. The assay represents a sensitive and reliable method for diagnosis of HCoV infection in clinical samples.

Comparison of isothermal helicase-dependent amplification and PCR for the detection of Mycobacterium tuberculosis by an electrochemical genomagnetic assay.

PubMed

Barreda-García, Susana; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Lobo-Castañón, M Jesús

2016-12-01

Methods for the early and sensitive detection of pathogenic bacteria suited to low-resource settings could impact diagnosis and management of diseases. Helicase-dependent isothermal amplification (HDA) is an ideal tool for this purpose, especially when combined with a sequence-specific detection method able to improve the selectivity of the assay. The implementation of this approach requires that its analytical performance is shown to be comparable with the gold standard method, polymerase chain reaction (PCR). In this study, we optimize and compare the asymmetric amplification of an 84-base-long DNA sequence specific for Mycobacterium tuberculosis by PCR and HDA, using an electrochemical genomagnetic assay for hybridization-based detection of the obtained single-stranded amplicons. The results indicate the generalizability of the magnetic platform with electrochemical detection for quantifying amplification products without previous purification. Moreover, we demonstrate that under optimal conditions the same gene can be amplified by either PCR or HDA, allowing the detection of as low as 30 copies of the target gene sequence with acceptable reproducibility. Both assays have been applied to the detection of M. tuberculosis in sputum, urine, and pleural fluid samples with comparable results. Simplicity and isothermal nature of HDA offer great potential for the development of point-of-care devices. Graphical Abstract Comparative evaluation of isothermal helicase-dependent amplification and PCR for electrochemical detection of Mycobacterium tuberculosis.

Electrochemical detection of leukemia oncogenes using enzyme-loaded carbon nanotube labels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Ai Cheng; Du, Dan; Chen, Baowei

2014-09-07

Here we describe an ultrasensitive electrochemical nucleic acids assay amplified by carbon nanotubes (CNTs)-based labels for the detection of human acute lymphocytic leukemia (ALL) related p185 BCR-ABL fusion transcript. The carboxylated CNTs were functionalized with horseradish peroxidase (HRP) molecules and target-specific detection probes (DP) via diimide-activated amidation, and used to label and amplify target hybridization signal. The activity of captured HRP was monitored by square-wave voltammetry measuring the electroactive enzymatic product in the presence of 2-aminophenol and hydrogen peroxide substrate solution. The effect of DP and HRP loading of the CNT-based labels on its signal-to-noise ratio of electrochemical detection wasmore » studied systematically for the first time. Under optimized conditions, the signal-amplified assay achieved a detection limit of 83 fM targets oligonuecleotides and a 4-order wide dynamic range of target concentration. The resulting assay allowed a robust discrimination between the perfect match and a three-base mismatch sequence. When subjected to full-length (491 bp) DNA oncogene, the approach demonstrated a detection limit of approximately 33 pg of the target gene. The high sensitivity and specificity of assay enabled PCR-free detection of target transcripts in as little as 65 ng of mRNA extracted from positive ALL cell lines SUP-B15, in comparison to those obtained from negative cell lines HL-60. The approach holds promise for simple, low cost and ultrasensitive electrochemical nucleic acids detection in portable devices, point-of-care and early disease diagnostic applications.« less

New immuno-PCR assay for detection of low concentrations of shiga toxin 2 and its variants.

PubMed

Zhang, Wenlan; Bielaszewska, Martina; Pulz, Matthias; Becker, Karsten; Friedrich, Alexander W; Karch, Helge; Kuczius, Thorsten

2008-04-01

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains secrete toxins that are major virulence factors and diagnostic targets, but some STEC strains secrete Stx in amounts that cannot be detected using conventional cell cytotoxicity or immunological assays. Therefore, there is an urgent need for more-sensitive Stx detection methods. We describe the development of an assay that can detect low concentrations of Stx2 and its variants. An immuno-PCR Stx2 assay was developed based on an enzyme immunoassay (EIA) combining antibody capture and DNA amplification to increase the signal. The immuno-PCR assay detected 10 pg/ml of purified Stx2, compared to 1 ng/ml Stx2 detected by commercial EIA. Consequently, immuno-PCR detected Stx2 and its variants in STEC strains that produce the toxins at levels that are nondetectable by using the EIA, as well as the Stx2 in EIA-negative enriched stool cultures from patients. Our data demonstrate that the immuno-PCR developed here is a highly sensitive and specific method for the detection of trace amounts of Stx2 and Stx2 variants. It is therefore suitable for use by clinical microbiological laboratories to improve the toxin detection in clinical samples.

New Immuno-PCR Assay for Detection of Low Concentrations of Shiga Toxin 2 and Its Variants▿

PubMed Central

Zhang, Wenlan; Bielaszewska, Martina; Pulz, Matthias; Becker, Karsten; Friedrich, Alexander W.; Karch, Helge; Kuczius, Thorsten

2008-01-01

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains secrete toxins that are major virulence factors and diagnostic targets, but some STEC strains secrete Stx in amounts that cannot be detected using conventional cell cytotoxicity or immunological assays. Therefore, there is an urgent need for more-sensitive Stx detection methods. We describe the development of an assay that can detect low concentrations of Stx2 and its variants. An immuno-PCR Stx2 assay was developed based on an enzyme immunoassay (EIA) combining antibody capture and DNA amplification to increase the signal. The immuno-PCR assay detected 10 pg/ml of purified Stx2, compared to 1 ng/ml Stx2 detected by commercial EIA. Consequently, immuno-PCR detected Stx2 and its variants in STEC strains that produce the toxins at levels that are nondetectable by using the EIA, as well as the Stx2 in EIA-negative enriched stool cultures from patients. Our data demonstrate that the immuno-PCR developed here is a highly sensitive and specific method for the detection of trace amounts of Stx2 and Stx2 variants. It is therefore suitable for use by clinical microbiological laboratories to improve the toxin detection in clinical samples. PMID:18272709

LAMP-based group specific detection of aflatoxin producers within Aspergillus section Flavi in food raw materials, spices, and dried fruit using neutral red for visible-light signal detection.

PubMed

Niessen, Ludwig; Bechtner, Julia; Fodil, Sihem; Taniwaki, Marta H; Vogel, Rudi F

2018-02-02

Aflatoxins can be produced by 21 species within sections Flavi (16 species), Ochraceorosei (2), and Nidulantes (3) of the fungal genus Aspergillus. They pose risks to human and animal health due to high toxicity and carcinogenicity. Detecting aflatoxin producers can help to assess toxicological risks associated with contaminated commodities. Species specific molecular assays (PCR and LAMP) are available for detection of major producers, but fail to detect species of minor importance. To enable rapid and sensitive detection of several aflatoxin producing species in a single analysis, a nor1 gene-specific LAMP assay was developed. Specificity testing showed that among 128 fungal species from 28 genera, 15 aflatoxigenic species in section Flavi were detected, including synonyms of A. flavus and A. parasiticus. No cross reactions were found with other tested species. The detection limit of the assay was 9.03pg of A. parasiticus genomic DNA per reaction. Visual detection of positive LAMP reactions under daylight conditions was facilitated using neutral red to allow unambiguous distinction between positive and negative assay results. Application of the assay to the detection of A. parasiticus conidia revealed a detection limit of 211 conidia per reaction after minimal sample preparation. The usefulness of the assay was demonstrated in the analysis of aflatoxinogenic species in samples of rice, nuts, raisins, dried figs, as well as powdered spices. Comparison of LAMP results with presence/absence of aflatoxins and aflatoxin producing fungi in 50 rice samples showed good correlation between these parameters. Our study suggests that the developed LAMP assay is a rapid, sensitive and user-friendly tool for surveillance and quality control in our food industry. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates.

PubMed

Smiljanic, M; Kaase, M; Ahmad-Nejad, P; Ghebremedhin, B

2017-07-10

Carbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study aimed to establish and evaluate a multiplex real-time PCR assay for the simultaneous detection of carbapenemase gene variants in gram-negative rods and to compare the performance with a commercial RT-PCR assay (Check-Direct CPE). 116 carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii isolates were genotyped for carbapenemase genes by PCR and sequencing. The defined isolates were used for the validation of the in-house RT-PCR by use of designed primer pairs and probes. Among the carbapenem-resistant isolates the genes bla KPC , bla VIM , bla NDM or bla OXA were detected. Both RT-PCR assays detected all bla KPC , bla VIM and bla NDM in the isolates. The in-house RT-PCR detected 53 of 67 (79.0%) whereas the commercial assay detected only 29 (43.3%) of the OXA genes. The in-house sufficiently distinguished the most prevalent OXA types (23-like and 48-like) in the melting curve analysis and direct detection of the genes from positive blood culture vials. The Check-Direct CPE and the in-house RT-PCR assay detected the carbapenem resistance from solid culture isolates. Moreover, the in-house assay enabled the identification of carbapenemase genes directly from positive blood-culture vials. However, we observed insufficient detection of various OXA genes in both assays. Nevertheless, the in-house RT-PCR detected the majority of the OXA type genes in Enterobacteriaceae and A. baumannii.

Bacteriophage-Based Pathogen Detection

NASA Astrophysics Data System (ADS)

Ripp, Steven

Considered the most abundant organism on Earth, at a population approaching 1031, bacteriophage, or phage for short, mediate interactions with myriad bacterial hosts that has for decades been exploited in phage typing schemes for signature identification of clinical, food-borne, and water-borne pathogens. With over 5,000 phage being morphologically characterized and grouped as to susceptible host, there exists an enormous cache of bacterial-specific sensors that has more recently been incorporated into novel bio-recognition assays with heightened sensitivity, specificity, and speed. These assays take many forms, ranging from straightforward visualization of labeled phage as they attach to their specific bacterial hosts to reporter phage that genetically deposit trackable signals within their bacterial hosts to the detection of progeny phage or other uniquely identifiable elements released from infected host cells. A comprehensive review of these and other phage-based detection assays, as directed towards the detection and monitoring of bacterial pathogens, will be provided in this chapter.

Nano-immunoassay with improved performance for detection of cancer biomarkers

DOE PAGES

Krasnoslobodtsev, Alexey V.; Torres, Maria P.; Kaur, Sukhwinder; ...

2015-01-01

Nano-immunoassay utilizing surface-enhanced Raman scattering (SERS) effect is a promising analytical technique for the early detection of cancer. In its current standing the assay is capable of discriminating samples of healthy individuals from samples of pancreatic cancer patients. Further improvements in sensitivity and reproducibility will extend practical applications of the SERS-based detection platforms to wider range of problems. In this report, we discuss several strategies designed to improve performance of the SERS-based detection system. We demonstrate that reproducibility of the platform is enhanced by using atomically smooth mica surface as a template for preparation of capture surface in SERS sandwichmore » immunoassay. Furthermore, the assay's stability and sensitivity can be further improved by using either polymer or graphene monolayer as a thin protective layer applied on top of the assay addresses. The protective layer renders the signal to be more stable against photo-induced damage and carbonaceous contamination.« less

Current technologies for detection of ricin in different matrices

USDA-ARS?s Scientific Manuscript database

Ricin is a convenient, potent, and available toxin for terrorist acts. The importance of detecting it in various matrices is obvious. This chapter reviews methods for ricin detection based on the mechanisms used for assay development. Five detection approaches are reviewed: 1. Antibody-based metho...

Serum creatinine detection by a conducting-polymer-based electrochemical sensor to identify allograft dysfunction.

PubMed

Wei, Fang; Cheng, Scott; Korin, Yael; Reed, Elaine F; Gjertson, David; Ho, Chih-ming; Gritsch, H Albin; Veale, Jeffrey

2012-09-18

Kidney transplant recipients who have abnormally high creatinine levels in their blood often have allograft dysfunction secondary to rejection. Creatinine has become the preferred marker for renal dysfunction and is readily available in hospital clinical settings. We developed a rapid and accurate polymer-based electrochemical point-of-care (POC) assay for creatinine detection from whole blood to identify allograft dysfunction. The creatinine concentrations of 19 blood samples from transplant recipients were measured directly from clinical serum samples by the conducting polymer-based electrochemical (EC) sensor arrays. These measurements were compared to the traditional clinical laboratory assay. The time required for detection was <5 min from sample loading. Sensitivity of the detection was found to be 0.46 mg/dL of creatinine with only 40 μL sample in the creatinine concentration range of 0 mg/dL to 11.33 mg/dL. Signal levels that were detected electrochemically correlated closely with the creatinine blood concentration detected by the UCLA Ronald Reagan Medical Center traditional clinical laboratory assay (correlation coefficient = 0.94). This work is encouraging for the development of a rapid and accurate POC device for measuring creatinine levels in whole blood.

Novel Cell-Based Assays for Detecting Low Levels of Active Ricin Following Decontamination

DTIC Science & Technology

2011-12-01

fluorescent protein, are powerful tools, which have been used for detection assays for ricin protein ( DeWet , 1987). Zhao et al. (2005) have reported a...toxic Type 2 Ribosome-Inactivating Proteins. FEBS Lett. 2004, 563, pp 219–222. DeWet , J.R. et al. Firefly Luciferase Gene: Structure and

75 FR 32184 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-07

... ELISA for Detection of Serum Levels of Soluble IL-15 Receptor Alpha Description of Invention: The invention is an ELISA based assay that can be used in the clinical setting to detect the presence of soluble...-15R expression. Advantages: The assay is in the industry accepted ELISA format. This non-radioactive...

Development of Two FhSAP2 Recombinant–Based Assays for Immunodiagnosis of Human Chronic Fascioliasis

PubMed Central

Shin, Sun Hee; Hsu, Angel; Chastain, Holly M.; Cruz, Lorna A.; Elder, Eric S.; Sapp, Sarah G. H.; McAuliffe, Isabel; Espino, Ana M.; Handali, Sukwan

2016-01-01

In the United States, infection with Fasciola hepatica has been identified as an emerging disease, primarily in immigrants, refugees, and travelers. The laboratory test of choice for diagnosis of fascioliasis is detection of disease specific antibodies, most commonly uses excretory-secretory antigens for detection of IgG antibodies. Recently, recombinant proteins such as F. hepatica antigen (FhSAP2) have been used to detect IgG antibodies. The glutathione S-transferase (GST)–FhSAP2 recombinant antigen was used to develop Western blot (WB) and fluorescent bead-based (Luminex) assays to detect F. hepatica total IgG and IgG4 antibodies. The sensitivity and specificity of GST-FhSAP2 total IgG and IgG4 WB were similar at 94% and 98%, respectively. For the IgG Luminex assay, the sensitivity and specificity were 94% and 97%, and for the IgG4, the values were 100% and 99%, respectively. In conclusion, the GST-FhSAP2 antigen performs well in several assay formats and can be used for clinical diagnosis. PMID:27549636

Development of Two FhSAP2 Recombinant-Based Assays for Immunodiagnosis of Human Chronic Fascioliasis.

PubMed

Shin, Sun Hee; Hsu, Angel; Chastain, Holly M; Cruz, Lorna A; Elder, Eric S; Sapp, Sarah G H; McAuliffe, Isabel; Espino, Ana M; Handali, Sukwan

2016-10-05

In the United States, infection with Fasciola hepatica has been identified as an emerging disease, primarily in immigrants, refugees, and travelers. The laboratory test of choice for diagnosis of fascioliasis is detection of disease specific antibodies, most commonly uses excretory-secretory antigens for detection of IgG antibodies. Recently, recombinant proteins such as F. hepatica antigen (FhSAP2) have been used to detect IgG antibodies. The glutathione S-transferase (GST)-FhSAP2 recombinant antigen was used to develop Western blot (WB) and fluorescent bead-based (Luminex) assays to detect F. hepatica total IgG and IgG 4 antibodies. The sensitivity and specificity of GST-FhSAP2 total IgG and IgG 4 WB were similar at 94% and 98%, respectively. For the IgG Luminex assay, the sensitivity and specificity were 94% and 97%, and for the IgG 4 , the values were 100% and 99%, respectively. In conclusion, the GST-FhSAP2 antigen performs well in several assay formats and can be used for clinical diagnosis. © The American Society of Tropical Medicine and Hygiene.

Visual detection of Brucella in bovine biological samples using DNA-activated gold nanoparticles

PubMed Central

Kumar, Satish; Kaur, Gurpreet; Ali, Syed Atif; Shrivastava, Sameer; Gupta, Praveen K.; Cooper, Jonathan M.; Chaudhuri, Pallab

2017-01-01

Brucellosis is a bacterial disease, which, although affecting cattle primarily, has been associated with human infections, making its detection an important challenge. The existing gold standard diagnosis relies on the culture of bacteria which is a lengthy and costly process, taking up to 45 days. New technologies based on molecular diagnosis have been proposed, either through dip-stick, immunological assays, which have limited specificity, or using nucleic acid tests, which enable to identify the pathogen, but are impractical for use in the field, where most of the reservoir cases are located. Here we demonstrate a new test based on hybridization assays with metal nanoparticles, which, upon detection of a specific pathogen-derived DNA sequence, yield a visual colour change. We characterise the components used in the assay with a range of analytical techniques and show sensitivities down to 1000 cfu/ml for the detection of Brucella. Finally, we demonstrate that the assay works in a range of bovine samples including semen, milk and urine, opening up the potential for its use in the field, in low-resource settings. PMID:28719613

C. trachomatis in female reproductive tract infections and RFLP-based genotyping: a 16-year study from a tertiary care hospital.

PubMed

Gita, Satpathy; Suneeta, Mittal; Anjana, Sharma; Niranjan, Nayak; Sujata, Mohanty; Pandey, R M

2011-01-01

Presence of Chlamydia trachomatis in endocervix was determined in 2466 women attending a tertiary care hospital in New Delhi, India over a period of 16 years, using a monoclonal-based direct immunofluorescence assay, tissue culture isolation, and a conventional PCR assay. Chlamydia antigen could be detected in 391 out of 2466 (15.85%) of patients studied; in 27.27% women with PID, 16.74% women with cervicitis, 16.03% women with infertility, and 12.06% women with adverse pregnancy outcomes, respectively. There was a statistically significant decreasing trend in Chlamydia antigen positivity between the years 1994-1999 and 2000-2004; the apparent decline in antigen positivity between the years 2000-2004 and 2005-2010 was not statistically significant. Antigen detection assay detected equal number of positives as the PCR assay; tissue culture isolation demonstrated lower positivity. In a few representative specimens from cervicitis patients, genotyping was done using RFLP pattern analysis of C. trachomatis MOMP gene amplified by PCR assay, all of these belonged to Chlamydia trachomatis serovar E.

C. trachomatis in Female Reproductive Tract Infections and RFLP-Based Genotyping: A 16-Year Study from a Tertiary Care Hospital

PubMed Central

Gita, Satpathy; Suneeta, Mittal; Anjana, Sharma; Niranjan, Nayak; Sujata, Mohanty; Pandey, R. M.

2011-01-01

Presence of Chlamydia trachomatis in endocervix was determined in 2466 women attending a tertiary care hospital in New Delhi, India over a period of 16 years, using a monoclonal-based direct immunofluorescence assay, tissue culture isolation, and a conventional PCR assay. Chlamydia antigen could be detected in 391 out of 2466 (15.85%) of patients studied; in 27.27% women with PID, 16.74% women with cervicitis, 16.03% women with infertility, and 12.06% women with adverse pregnancy outcomes, respectively. There was a statistically significant decreasing trend in Chlamydia antigen positivity between the years 1994–1999 and 2000–2004; the apparent decline in antigen positivity between the years 2000–2004 and 2005–2010 was not statistically significant. Antigen detection assay detected equal number of positives as the PCR assay; tissue culture isolation demonstrated lower positivity. In a few representative specimens from cervicitis patients, genotyping was done using RFLP pattern analysis of C. trachomatis MOMP gene amplified by PCR assay, all of these belonged to Chlamydia trachomatis serovar E. PMID:21747643

Options in human papillomavirus (HPV) detection for cervical cancer screening: comparison between full genotyping and a rapid qualitative HPV-DNA assay in Ghana.

PubMed

Obiri-Yeboah, Dorcas; Adu-Sarkodie, Yaw; Djigma, Florencia; Akakpo, Kafui; Aniakwa-Bonsu, Ebenezer; Amoako-Sakyi, Daniel; Jacques, Simpore; Mayaud, Philippe

2017-01-01

Modern cervical cancer screening increasingly relies on the use of molecular techniques detecting high-risk oncogenic human papillomavirus (hr-HPV). A major challenge for developing countries like Ghana has been the unavailability and costs of HPV DNA-based testing. This study compares the performance of care HPV, a semi-rapid and affordable qualitative detection assay for 14 hr-HPV genotypes, with HPV genotyping, for the detection of cytological cervical squamous intraepithelial lesions (SIL). A study comparing between frequency matched HIV-1 seropositive and HIV-seronegative women was conducted in the Cape Coast Teaching Hospital, Ghana. A systematic sampling method was used to select women attending clinics in the hospital. Cervical samples were tested for HPV by care HPV and Anyplex-II HPV28 genotyping assay, and by conventional cytology. A total of 175 paired results (94 from HIV-1 seropositive and 81 from HIV-seronegative women) were analyzed based on the ability of both tests to detect the 14 hr-HPV types included in the care HPV assay. The inter-assay concordance was 94.3% (95%CI: 89.7-97.2%, kappa = 0.88), similar by HIV serostatus. The care HPV assay was equally sensitive among HIV-1 seropositive and seronegative women (97.3% vs. 95.7%, p  = 0.50) and slightly more specific among HIV-seronegative women (85.0% vs. 93.1%, p  = 0.10). care HPV had good sensitivity (87.5%) but low specificity (52.1%) for the detection of low SIL or greater lesions, but its performance was superior to genotyping (87.5 and 38.8%, respectively). Reproducibility of care HPV, tested on 97 samples by the same individual was 82.5% (95%CI: 73.4-89.4%). The performance characteristics of care HPV compared to genotyping suggest that this simpler and cheaper HPV detection assay could offer a suitable alternative for HPV screening in Ghana.

Comparative study of β-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.).

PubMed

Vera-Jimenez, N I; Pietretti, D; Wiegertjes, G F; Nielsen, M E

2013-05-01

The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by β-glucans. Copyright © 2013 Elsevier Ltd. All rights reserved.

Hepatitis E Virus (HEV) Detection and Quantification by a Real-Time Reverse Transcription-PCR Assay Calibrated to the World Health Organization Standard for HEV RNA

PubMed Central

Germer, Jeffrey J.; Ankoudinova, Irina; Belousov, Yevgeniy S.; Mahoney, Walt; Dong, Chen; Meng, Jihong; Mandrekar, Jayawant N.

2017-01-01

ABSTRACT Hepatitis E virus (HEV) has emerged as a cause of chronic hepatitis among immunocompromised patients. Molecular assays have become important tools for the diagnosis and management of these chronically infected patients. A real-time reverse transcription-quantitative PCR (RT-qPCR) assay utilizing Pleiades probe chemistry and an RNA internal control for the simultaneous detection and quantification of HEV RNA in human serum was developed based on an adaptation of a previously described and broadly reactive primer set targeting the overlapping open reading frame 2/3 (ORF2/3) nucleotide sequence of HEV. A chimeric bovine viral diarrhea virus construct containing an HEV RNA insert (SynTura HEV) was developed, value assigned with the first World Health Organization (WHO) international standard for HEV RNA (code 6329/10), and used to prepare working assay calibrators and controls, which supported an assay quantification range of 100 to 5,000,000 IU/ml. The analytical sensitivity (95% detection rate) of this assay was 25.2 IU/ml (95% confidence interval [CI], 19.2 to 44.1 IU/ml). The assay successfully amplified 16 different HEV sequences with significant nucleotide mismatching in primer/probe binding regions, while evaluation of a WHO international reference panel for HEV genotypes (code 8578/13) showed viral load results falling within the result ranges generated by WHO collaborative study participants for all panel members (genotypes 1 to 4). Broadly reactive RT-qPCR primers targeting HEV ORF2/3 were successfully adapted for use in an assay based on Pleiades probe chemistry. The availability of secondary standards calibrated to the WHO HEV international standard can improve the standardization and performance of assays for the detection and quantification of HEV RNA. PMID:28228493

A review of Theileria diagnostics and epidemiology

PubMed Central

Mans, Ben J.; Pienaar, Ronel; Latif, Abdalla A.

2015-01-01

An extensive range of serological and molecular diagnostic assays exist for most of the economically important Theileira species such as T. annulata, T. equi, T. lestoquardi, T. parva, T. uilenbergi and other more benign species. Diagnostics of Theileria is considered with regard to sensitivity and specificity of current molecular and serological assays and their use in epidemiology. In the case of serological assays, cross-reactivity of genetically closely related species reduces the use of the gold standard indirect fluorescent antibody test (IFAT). Development of antigen-specific assays does not necessarily address this problem, since closely related species will potentially have similar antigens. Even so, serological assays remain an important line of enquiry in epidemiological surveys. Molecular based assays have exploded in the last decade with significant improvements in sensitivity and specificity. In this review, the current interpretation of what constitute a species in Theileria and its impact on accurate molecular diagnostics is considered. Most molecular assays based on conventional or real-time PCR technology have proven to be on standard with regard to analytical sensitivity. However, consideration of the limits of detection in regard to total blood volume of an animal indicates that most assays may only detect >400,000 parasites/L blood. Even so, natural parasitaemia distribution in carrier-state animals seems to be above this limit of detection, suggesting that most molecular assays should be able to detect the majority of infected individuals under endemic conditions. The potential for false-negative results can, however, only be assessed within the biological context of the parasite within its vertebrate host, i.e. parasitaemia range in the carrier-state that will support infection of the vector and subsequent transmission. PMID:25830110

A Multi-Receptor and Multi-Species Assay for Potential Endocrine Disruptor Targets (SLAS meeting)

EPA Science Inventory

Screening methods for detecting potential endocrine disrupting chemicals rely chiefly on transactivation assays targeting nuclear receptors such as the estrogen (ER) and androgen receptors (AR). These assays are predominately human-based; yet environmental exposure can affect div...

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

PubMed Central

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

2017-01-01

Vibrio cholerae serogroups O1 and O139 are etiological agents of cholera, a serious and acute diarrheal disease, and rapid detection of V. cholerae is a key method for preventing and controlling cholera epidemics. Here, a point of care testing (POCT) method called Vch-UPT-LF, which is an up-converting phosphor technology-based lateral flow (UPT-LF) assay with a dual-target detection mode, was developed to detect V. cholerae O1 and O139 simultaneously from one sample loading. Although applying an independent reaction pair made both detection results for the two Vch-UPT-LF detection channels more stable, the sensitivity slightly declined from 104 to 105 colony-forming units (CFU) mL−1 compared with that of the single-target assay, while the quantification ranges covering four orders of magnitude were maintained. The strip showed excellent specificity for seven Vibrio species that are highly related genetically, and nine food-borne species whose transmission routes are similar to those of V. cholerae. The legitimate arrangement of the two adjacent test lines lessened the mutual impact of the quantitation results between the two targets, and the quantification values did not differ by more than one order of magnitude when the samples contained high concentrations of both V. cholerae O1 and O139. Under pre-incubation conditions, 1×101 CFU mL−1 of V. cholerae O1 or O139 could be detected in fewer than 7 h, while the Vch-UPT-LF assay exhibited sensitivity as high as a real-time fluorescent polymerase chain reaction with fewer false-positive results. Therefore, successful development of Vch-UPT-LF as a dual-target assay for quantitative detection makes this assay a good candidate POCT method for the detection and surveillance of epidemic cholera. PMID:28662147

Real-Time Cytotoxicity Assay for Rapid and Sensitive Detection of Ricin from Complex Matrices

PubMed Central

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

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 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 sample matrices. PMID:22532852

Prevalence of antibodies to type A influenza virus in wild avian species using two serologic assays

USGS Publications Warehouse

Brown, Justin D.; Luttrell, M. Page; Berghaus, Roy D.; Kistler, Whitney; Keeler, Shamus P.; Howey, Andrea; Wilcox, Benjamin; Hall, Jeffrey S.; Niles, Larry; Dey, Amanda; Knutsen, Gregory; Fritz, Kristen; Stallknecht, David E.

2010-01-01

Serologic testing to detect antibodies to avian influenza (AI) virus has been an underused tool for the study of these viruses in wild bird populations, which traditionally has relied on virus isolation and reverse transcriptase-polymerase chain reaction (RT-PCR). In a preliminary study, a recently developed commercial blocking enzyme-linked immunosorbent assay (bELISA) had sensitivity and specificity estimates of 82% and 100%, respectively, for detection of antibodies to AI virus in multiple wild bird species after experimental infection. To further evaluate the efficacy of this commercial bELISA and the agar gel immunodiffusion (AGID) test for AI virus antibody detection in wild birds, we tested 2,249 serum samples collected from 62 wild bird species, representing 10 taxonomic orders. Overall, the bELISA detected 25.4% positive samples, whereas the AGID test detected 14.8%. At the species level, the bELISA detected as many or more positive serum samples than the AGID in all 62 avian species. The majority of positive samples, detected by both assays, were from species that use aquatic habitats, with the highest prevalence from species in the orders Anseriformes and Charadriiformes. Conversely, antibodies to AI virus were rarely detected in the terrestrial species. The serologic data yielded by both assays are consistent with the known epidemiology of AI virus in wild birds and published reports of host range based on virus isolation and RT-PCR. The results of this research are also consistent with the aforementioned study, which evaluated the performance of the bELISA and AGID test on experimental samples. Collectively, the data from these two studies indicate that the bELISA is a more sensitive serologic assay than the AGID test for detecting prior exposure to AI virus in wild birds. Based on these results, the bELISA is a reliable species-independent assay with potentially valuable applications for wild bird AI surveillance.

Template reporter bacteriophage platform and multiple bacterial detection assays based thereon

NASA Technical Reports Server (NTRS)

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.

Gold nanoparticle-based simple colorimetric and ultrasensitive dynamic light scattering assay for the selective detection of Pb(II) from paints, plastics, and water samples.

PubMed

Beqa, Lule; Singh, Anant Kumar; Khan, Sadia Afrin; Senapati, Dulal; Arumugam, Sri Ranjini; Ray, Paresh Chandra

2011-03-01

Pb (II) is a common water pollutant with high toxicity. According to the CDC, about 310,000 U.S. children of ages 1-5 have high levels of lead in their blood that it is due to the exposure to lead from plastic toys and other products. As a result, the development of ultrasensitive assays for the real-time detection of Pb(II) from plastic toys and paints is very important for water controlling, clinical toxicology and industrial processes. Driven by the need to detect trace amounts of Pb(II) from water samples, we report a label-free, highly selective and ultra sensitive glutathione modified gold nanoparticle based dynamic light scattering (DLS) probe for Pb(II) recognition in 100 ppt level from aqueous solution with excellent discrimination against other heavy metals. The sensitivity of our assay to detect Pb(II) level in water is almost 2 orders of magnitude higher than the EPA standard limit. We have also demonstrated that our DLS assay is capable of measuring the amount of Pb(II) in paint, plastic toys, and water from MS river. A possible mechanism and operating principles of our DLS assay have been discussed. Ultimately, this nanotechnology driven assay could have enormous potential applications in rapid, on-site monitoring of Pb(II) from day-to-day sample.

Bacteriophage-based assays for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a meta-analysis.

PubMed

Pai, Madhukar; Kalantri, Shriprakash; Pascopella, Lisa; Riley, Lee W; Reingold, Arthur L

2005-10-01

To summarize, using meta-analysis, the accuracy of bacteriophage-based assays for the detection of rifampicin resistance in Mycobacterium tuberculosis. By searching multiple databases and sources we identified a total of 21 studies eligible for meta-analysis. Of these, 14 studies used phage amplification assays (including eight studies on the commercial FASTPlaque-TB kits), and seven used luciferase reporter phage (LRP) assays. Sensitivity, specificity, and agreement between phage assay and reference standard (e.g. agar proportion method or BACTEC 460) results were the main outcomes of interest. When performed on culture isolates (N=19 studies), phage assays appear to have relatively high sensitivity and specificity. Eleven of 19 (58%) studies reported sensitivity and specificity estimates > or =95%, and 13 of 19 (68%) studies reported > or =95% agreement with reference standard results. Specificity estimates were slightly lower and more variable than sensitivity; 5 of 19 (26%) studies reported specificity <90%. Only two studies performed phage assays directly on sputum specimens; although one study reported sensitivity and specificity of 100 and 99%, respectively, another reported sensitivity of 86% and specificity of 73%. Current evidence is largely restricted to the use of phage assays for the detection of rifampicin resistance in culture isolates. When used on culture isolates, these assays appear to have high sensitivity, but variable and slightly lower specificity. In contrast, evidence is lacking on the accuracy of these assays when they are directly applied to sputum specimens. If phage-based assays can be directly used on clinical specimens and if they are shown to have high accuracy, they have the potential to improve the diagnosis of MDR-TB. However, before phage assays can be successfully used in routine practice, several concerns have to be addressed, including unexplained false positives in some studies, potential for contamination and indeterminate results.

Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays.

PubMed

Glais, Laurent; Jacquot, Emmanuel

2015-01-01

Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay.

Corynebacterium Prosthetic Joint Infection

PubMed Central

Cazanave, Charles; Greenwood-Quaintance, Kerryl E.; Hanssen, Arlen D.

2012-01-01

Identification of Corynebacterium species may be challenging. Corynebacterium species are occasional causes of prosthetic joint infection (PJI), but few data are available on the subject. Based on the literature, C. amycolatum, C. aurimucosum, C. jeikeium, and C. striatum are the most common Corynebacterium species that cause PJI. We designed a rapid PCR assay to detect the most common human Corynebacterium species, with a specific focus on PJI. A polyphosphate kinase gene identified using whole-genome sequence was targeted. The assay differentiates the antibiotic-resistant species C. jeikeium and C. urealyticum from other species in a single assay. The assay was applied to a collection of human Corynebacterium isolates from multiple clinical sources, and clinically relevant species were detected. The assay was then tested on Corynebacterium isolates specifically associated with PJI; all were detected. We also describe the first case of C. simulans PJI. PMID:22337986

Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation.

PubMed

Kusakawa, Shinji; Yasuda, Satoshi; Kuroda, Takuya; Kawamata, Shin; Sato, Yoji

2015-12-08

Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human cell-processed therapeutic products (hCTPs). The soft agar colony formation (SACF) assay, which is a well-known in vitro assay for the detection of malignant transformed cells, is applicable for the quality assessment of hCTPs. Here we established an image-based screening system for the SACF assay using a high-content cell analyzer termed the digital SACF assay. Dual fluorescence staining of formed colonies and the dissolution of soft agar led to accurate detection of transformed cells with the imaging cytometer. Partitioning a cell sample into multiple wells of culture plates enabled digital readout of the presence of colonies and elevated the sensitivity for their detection. In practice, the digital SACF assay detected impurity levels as low as 0.00001% of the hCTPs, i.e. only one HeLa cell contained in 10,000,000 human mesenchymal stem cells, within 30 days. The digital SACF assay saves time, is more sensitive than in vivo tumorigenicity tests, and would be useful for the quality control of hCTPs in the manufacturing process.

Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation

PubMed Central

Kusakawa, Shinji; Yasuda, Satoshi; Kuroda, Takuya; Kawamata, Shin; Sato, Yoji

2015-01-01

Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human cell-processed therapeutic products (hCTPs). The soft agar colony formation (SACF) assay, which is a well-known in vitro assay for the detection of malignant transformed cells, is applicable for the quality assessment of hCTPs. Here we established an image-based screening system for the SACF assay using a high-content cell analyzer termed the digital SACF assay. Dual fluorescence staining of formed colonies and the dissolution of soft agar led to accurate detection of transformed cells with the imaging cytometer. Partitioning a cell sample into multiple wells of culture plates enabled digital readout of the presence of colonies and elevated the sensitivity for their detection. In practice, the digital SACF assay detected impurity levels as low as 0.00001% of the hCTPs, i.e. only one HeLa cell contained in 10,000,000 human mesenchymal stem cells, within 30 days. The digital SACF assay saves time, is more sensitive than in vivo tumorigenicity tests, and would be useful for the quality control of hCTPs in the manufacturing process. PMID:26644244

Development and testing of real-time PCR assays for determining fecal loading and source identification (cattle, human, etc.) in surface water and groundwater

NASA Astrophysics Data System (ADS)

McKay, L. D.; Layton, A.; Gentry, R.

2004-12-01

A multi-disciplinary group of researchers at the University of Tennessee is developing and testing a series of microbial assay methods based on real-time PCR to detect fecal bacterial concentrations and host sources in water samples. Real-time PCR is an enumeration technique based on the unique and conserved nucleic acid sequences present in all organisms. The first research task was development of an assay (AllBac) to detect total amount of Bacteroides, which represents up to 30 percent of fecal mass. Subsequent assays were developed to detect Bacteroides from cattle (BoBac) and humans (HuBac) using 16sRNA genes based on DNA sequences in the national GenBank, as well as sequences from local fecal samples. The assays potentially have significant advantages over conventional bacterial source tracking methods because: 1. unlike traditional enumeration methods, they do not require bacterial cultivation; 2. there are no known non-fecal sources of Bacteroides; 3. the assays are quantitative with results for total concentration and for each species expressed in mg/l; and 4. they show little regional variation within host species, meaning that they do not require development of extensive local gene libraries. The AllBac and BoBac assays have been used in a study of fecal contamination in a small rural watershed (Stock Creek) near Knoxville, TN, and have proven useful in identification of areas where cattle represent a significant fecal input and in development of BMPs. It is expected that these types of assays (and future assays for birds, hogs, etc.) could have broad applications in monitoring fecal impacts from Animal Feeding Operations, as well as from wildlife and human sources.

A sensitive and accurate quantification method for the detection of hepatitis B virus covalently closed circular DNA by the application of a droplet digital polymerase chain reaction amplification system.

PubMed

Mu, Di; Yan, Liang; Tang, Hui; Liao, Yong

2015-10-01

To develop a sensitive and accurate assay system for the quantification of covalently closed circular HBV DNA (cccDNA) for future clinical monitoring of cccDNA fluctuation during antiviral therapy in the liver of infected patients. A droplet digital PCR (ddPCR)-based assay system detected template DNA input at the single copy level (or ~10(-5) pg of plasmid HBV DNA) by using serially diluted plasmid HBV DNA samples. Compared with the conventional quantitative PCR assay in the detection of cccDNA, which required at least 50 ng of template DNA input, a parallel experiment applying a ddPCR system demonstrates that the lowest detection limit of cccDNA from HepG2.215 cellular DNA samples is around 1 ng, which is equivalent to 0.54 ± 0.94 copies of cccDNA. In addition, we demonstrated that the addition of cccDNA-safe exonuclease and utilization of cccDNA-specific primers in the ddPCR assay system significantly improved the detection accuracy of HBV cccDNA from HepG2.215 cellular DNA samples. The ddPCR-based cccDNA detection system is a sensitive and accurate assay for the quantification of cccDNA in HBV-transfected HepG2.215 cellular DNA samples and may represent an important method for future application in monitoring cccDNA fluctuation during antiviral therapy.

UroMark-a urinary biomarker assay for the detection of bladder cancer.

PubMed

Feber, Andrew; Dhami, Pawan; Dong, Liqin; de Winter, Patricia; Tan, Wei Shen; Martínez-Fernández, Mónica; Paul, Dirk S; Hynes-Allen, Antony; Rezaee, Sheida; Gurung, Pratik; Rodney, Simon; Mehmood, Ahmed; Villacampa, Felipe; de la Rosa, Federico; Jameson, Charles; Cheng, Kar Keung; Zeegers, Maurice P; Bryan, Richard T; James, Nicholas D; Paramio, Jesus M; Freeman, Alex; Beck, Stephan; Kelly, John D

2017-01-01

Bladder cancer (BC) is one of the most common cancers in the western world and ranks as the most expensive to manage, due to the need for cystoscopic examination. BC shows frequent changes in DNA methylation, and several studies have shown the potential utility of urinary biomarkers by detecting epigenetic alterations in voided urine. The aim of this study is to develop a targeted bisulfite next-generation sequencing assay to diagnose BC from urine with high sensitivity and specificity. We defined a 150 CpG loci biomarker panel from a cohort of 86 muscle-invasive bladder cancers and 30 normal urothelium. Based on this panel, we developed the UroMark assay, a next-generation bisulphite sequencing assay and analysis pipeline for the detection of bladder cancer from urinary sediment DNA. The 150 loci UroMark assay was validated in an independent cohort ( n  = 274, non-cancer ( n  = 167) and bladder cancer ( n  = 107)) voided urine samples with an AUC of 97%. The UroMark classifier sensitivity of 98%, specificity of 97% and NPV of 97% for the detection of primary BC was compared to non-BC urine. Epigenetic urinary biomarkers for detection of BC have the potential to revolutionise the management of this disease. In this proof of concept study, we show the development and utility of a novel high-throughput, next-generation sequencing-based biomarker for the detection of BC-specific epigenetic alterations in urine.

Microwell hybridization assay for detection of PCR products from Mycobacterium tuberculosis complex and the recombinant Mycobacterium smegmatis strain 1008 used as an internal control.

PubMed Central

Kox, L F; Noordhoek, G T; Kunakorn, M; Mulder, S; Sterrenburg, M; Kolk, A H

1996-01-01

A microwell hybridization assay was developed for the detection of the PCR products from both Mycobacterium tuberculosis complex bacteria and the recombinant Mycobacterium smegmatis strain 1008 that is used as an internal control to monitor inhibition in the PCR based on the M. tuberculosis complex-specific insertion sequence IS6110. The test is based on specific detection with digoxigenin-labeled oligonucleotide probes of biotinylated PCR products which are captured in a microtiter plate coated with streptavidin. The captured PCR products are hybridized separately with two probes, one specific for the PCR product from IS6110 from M. tuberculosis complex and the other specific for the PCR fragment from the modified IS6110 fragment from the recombinant M. smegmatis 1008. The microwell hybridization assay discriminates perfectly between the two types of amplicon. The amount of PCR product that can be detected by this assay is 10 times less than that which can be detected by agarose gel electrophoresis. The test can be performed in 2 h. It is much faster and less laborious than Southern blot hybridization. Furthermore, the interpretation of results is objective. The assay was used with 172 clinical samples in a routine microbiology laboratory, and the results were in complete agreement with those of agarose gel electrophoresis and Southern blot hybridization. PMID:8862568

Clinical evaluation of a Mucorales-specific real-time PCR assay in tissue and serum samples.

PubMed

Springer, Jan; Lackner, Michaela; Ensinger, Christian; Risslegger, Brigitte; Morton, Charles Oliver; Nachbaur, David; Lass-Flörl, Cornelia; Einsele, Hermann; Heinz, Werner J; Loeffler, Juergen

2016-12-01

Molecular diagnostic assays can accelerate the diagnosis of fungal infections and subsequently improve patient outcomes. In particular, the detection of infections due to Mucorales is still challenging for laboratories and physicians. The aim of this study was to evaluate a probe-based Mucorales-specific real-time PCR assay (Muc18S) using tissue and serum samples from patients suffering from invasive mucormycosis (IMM). This assay can detect a broad range of clinically relevant Mucorales species and can be used to complement existing diagnostic tests or to screen high-risk patients. An advantage of the Muc18S assay is that it exclusively detects Mucorales species allowing the diagnosis of Mucorales DNA without sequencing within a few hours. In paraffin-embedded tissue samples this PCR-based method allowed rapid identification of Mucorales in comparison with standard methods and showed 91 % sensitivity in the IMM tissue samples. We also evaluated serum samples, an easily accessible material, from patients at risk from IMM. Mucorales DNA was detected in all patients with probable/proven IMM (100 %) and in 29 % of the possible cases. Detection of IMM in serum could enable an earlier diagnosis (up to 21 days) than current methods including tissue samples, which were gained mainly post-mortem. A screening strategy for high-risk patients, which would enable targeted treatment to improve patient outcomes, is therefore possible.

A fluorometric lateral flow assay for visual detection of nucleic acids using a digital camera readout.

PubMed

Magiati, Maria; Sevastou, Areti; Kalogianni, Despina P

2018-06-04

A fluorometric lateral flow assay has been developed for the detection of nucleic acids. The fluorophores phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were used as labels, while a common digital camera and a colored vinyl-sheet, acting as a cut-off optical filter, are used for fluorescence imaging. After DNA amplification by polymerase chain reaction (PCR), the biotinylated PCR product is hybridized to its complementary probe that carries a poly(dA) tail at 3΄ edge and then applied to the lateral flow strip. The hybrids are captured to the test zone of the strip by immobilized poly(dT) sequences and detected by streptavidin-fluorescein and streptavidin-phycoerythrin conjugates, through streptavidin-biotin interaction. The assay is widely applicable, simple, cost-effective, and offers a large multiplexing potential. Its performance is comparable to assays based on the use of streptavidin-gold nanoparticles conjugates. As low as 7.8 fmol of a ssDNA and 12.5 fmol of an amplified dsDNA target were detectable. Graphical abstract Schematic presentation of a fluorometric lateral flow assay based on fluorescein and phycoerythrin fluorescent labels for the detection of single-stranded (ssDNA) and double-stranded DNA (dsDNA) sequences and using a digital camera readout. SA: streptavidin, BSA: Bovine Serum Albumin, B: biotin, FITC: fluorescein isothiocyanate, PE: phycoerythrin, TZ: test zone, CZ: control zone.

Rapid visual detection of cyprinid herpesvirus 2 by recombinase polymerase amplification combined with a lateral flow dipstick.

PubMed

Wang, H; Sun, M; Xu, D; Podok, P; Xie, J; Jiang, Ys; Lu, Lq

2018-05-28

Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus 2 (CyHV-2), causes significant losses in crucian carp (Carassius carassius) aquaculture. Rapid and convenient DNA assay detection of CyHV-2 is useful for field diagnosis. Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that can amplify DNA within 30 min at ~37°C by simulating in vivo DNA recombination. Herein, a rapid and convenient detection assay based on RPA with a lateral flow dipstick (LFD) was developed for detecting CyHV-2. The highly conserved ORF72 of CyHV-2 was targeted by specific and sensitive primers and probes. The optimized assay takes only 15 min at 38°C using a water bath, with analysis of products by 2% agarose gel electrophoresis within 30 min. A simple lateral flow strip based on the unique probe in reaction buffer was developed for visualization. The entire RPA-LFD assay takes 50 min less than the routine PCR method, is 100 times more sensitive and displays no cross-reaction with other aquatic viruses. The combined isothermal RPA and lateral flow assay (RPA-LFD) provides a simple, rapid, reliable method that could improve field diagnosis of CyHV-2 when resources are limited. © 2018 John Wiley & Sons Ltd.

Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling.

PubMed

Plagnol, Vincent; Woodhouse, Samuel; Howarth, Karen; Lensing, Stefanie; Smith, Matt; Epstein, Michael; Madi, Mikidache; Smalley, Sarah; Leroy, Catherine; Hinton, Jonathan; de Kievit, Frank; Musgrave-Brown, Esther; Herd, Colin; Baker-Neblett, Katherine; Brennan, Will; Dimitrov, Peter; Campbell, Nathan; Morris, Clive; Rosenfeld, Nitzan; Clark, James; Gale, Davina; Platt, Jamie; Calaway, John; Jones, Greg; Forshew, Tim

2018-01-01

Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications.

Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling

PubMed Central

Howarth, Karen; Lensing, Stefanie; Smith, Matt; Epstein, Michael; Madi, Mikidache; Smalley, Sarah; Leroy, Catherine; Hinton, Jonathan; de Kievit, Frank; Musgrave-Brown, Esther; Herd, Colin; Baker-Neblett, Katherine; Brennan, Will; Dimitrov, Peter; Campbell, Nathan; Morris, Clive; Rosenfeld, Nitzan; Clark, James; Gale, Davina; Platt, Jamie; Calaway, John; Jones, Greg

2018-01-01

Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications. PMID:29543828

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

PubMed

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

2017-10-01

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

Application of a Homogenous Assay for the Detection of 2,4,6-Trinitrotoluene to Environmental Water Samples

PubMed Central

Goldman, Ellen R.; Egge, Adrienne L.; Medintz, Igor L.; Lassman, Michael E.; Anderson, George P.

2005-01-01

A homogeneous assay was used to detect 2,4,6-trinitrotoluene (TNT) spiked into environmental water samples. This assay is based on changes in fluorescence emission intensity when TNT competitively displaces a fluorescently labeled, TNT analog bound to an anti-TNT antibody. The effectiveness of the assay was highly dependent on the source of the sample being tested. As no correlation between pH and assay performance was observed, ionic strength was assumed to be the reason for variation in assay results. Addition of 10x phosphate-buffered saline to samples to increase their ionic strength to that of our standard laboratory buffer (about 0.17 M) significantly improved the range over which the assay functioned in several river water samples. PMID:15915298

Comparison of reverse transcriptase PCR, reverse transcriptase loop-mediated isothermal amplification, and culture-based assays for Salmonella detection from pork processing environments.

PubMed

Techathuvanan, Chayapa; Draughon, Frances Ann; D'Souza, Doris Helen

2011-02-01

Novel rapid Salmonella detection assays without the need for sophisticated equipment or labor remain in high demand. Real-time reverse transcriptase PCR (RT-PCR) assays, though rapid and sensitive, require expensive thermocyclers, while a novel RT loop-mediated isothermal amplification (RT-LAMP) method requires only a simple water bath. Our objective was to compare the detection sensitivity of Salmonella Typhimurium from the pork processing environment by RT-LAMP, RT-PCR, and culture-based assays. Carcass and surface swabs and carcass rinses were obtained from a local processing plant. Autoclaved carcass rinses (500 ml) were spiked with Salmonella Typhimurium and filtered. Filters were placed in stomacher bags containing tetrathionate broth (TTB) and analyzed with or without 10-h enrichment at 37 °C. Natural swabs were stomached with buffered peptone water, and natural carcass rinses were filtered, preenriched, and further enriched in TTB. Serially-diluted enriched samples were enumerated by spread plating on xylose lysine Tergitol 4 agar. RNA was extracted from 5 ml of enriched TTB with TRIzol. RT-LAMP assay using previously described invA primers was conducted at 62 °C for 90 min in a water bath with visual detection and by gel electrophoresis. SYBR Green I-based-real-time RT-PCR was carried out with invA primers followed by melt temperature analysis. The results of RT-LAMP detection for spiked carcass rinses were comparable to those of RT-PCR and cultural plating, with detection limits of 1 log CFU/ml, although they were obtained significantly faster, within 24 h including preenrichment and enrichment. RT-LAMP showed 4 of 12 rinse samples positive, while RT-PCR showed 1 of 12 rinse samples positive. For swabs, 6 of 27 samples positive by RT-LAMP and 5 of 27 by RT-PCR were obtained. This 1-day RT-LAMP assay shows promise for routine Salmonella screening by the pork industry. Copyright ©, International Association for Food Protection

Visual Non-Instrumental On-Site Detection of Fumonisin B1, B2, and B3 in Cereal Samples Using a Clean-Up Combined with Gel-Based Immunoaffinity Test Column Assay

PubMed Central

Wu, Hesen; Ji, Weihong; Li, Zhi; Chu, Fangyu; Wang, Shuo

2018-01-01

A visual immunoaffinity test column (IATC) assay was developed to detect fumonisins in cereal samples for spot tests without the need for special instruments. The developed IATC assay had equivalent recognition capability for fumonisin B1 (FB1), fumonisin B2 (FB2), or fumonisin B3 (FB3), and exhibited no cross-reactivity with aflatoxin B1, ochratoxin A, zearalenone, or the T-2 toxin. The sample pretreatment was accomplished more rapidly and with greater ease, the entire assay procedure was completed in approximately 10 min, including sample pretreatment and testing. The limits of detection (LODs) of the IATC assay to detect fumonisins in the maize, barley, oat, and millet samples were 20 μg kg−1. The results of the spiked maize, barley, oat, and millet and real maize samples by the IATC assay agreed well with the results obtained by the commercial fumonisin enzyme-linked immunosorbent assay (ELISA) test kit and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. The developed IATC assay can serve as a useful screening tool for the rapid, qualitative, and semi-quantitative detection of the total content of fumonisins (sum of FB1, FB2, and FB3) in cereal samples on-site. PMID:29671825

A multiplex protein-free lateral flow assay for detection of microRNAs based on unmodified molecular beacons.

PubMed

Javani, Atefeh; Javadi-Zarnaghi, Fatemeh; Rasaee, Mohammad Javad

2017-11-15

Lateral flow assays (LFAs) have promising potentials for point-of-care applications. Recently, many LFAs have been reported that are based on hybridization of oligonucleotide strands. Mostly, biotinylated capture DNAs are immobilized on the surface of a nitrocellulose membrane via streptavidin interactions. During the assay, stable colorful complexes get formed that are visible by naked eyes. Here, we present an inexpensive and unique design of LFA that applies unmodified oligonucleotides at capture lines. The presented LFA do not utilize streptavidin or any other affinity protein. We employ structural switch of molecular beacons (MB) in combination with base stacking hybridization (BSH) phenomenon. The unique design of the reported LFA provided high selectivity for target oligonucleotides. We validated potential applications of the system for detection of DNA mimics of two microRNAs in multiplex assays. Copyright © 2017 Elsevier Inc. All rights reserved.

Rapid colorimetric assay for detection of Listeria monocytogenes in food samples using LAMP formation of DNA concatemers and gold nanoparticle-DNA probe complex

NASA Astrophysics Data System (ADS)

Wachiralurpan, Sirirat; Sriyapai, Thayat; Areekit, Supatra; Sriyapai, Pichapak; Augkarawaritsawong, Suphitcha; Santiwatanakul, Somchai; Chansiri, Kosum

2018-04-01

ABSTRACT Listeria monocytogenes is a major foodborne pathogen of global health concern. Herein, the rapid diagnosis of L. monocytogenes has been achieved using loop-mediated isothermal amplification (LAMP) based on the phosphatidylcholine-phospholipase C gene (plcB). Colorimetric detection was then performed through the formation of DNA concatemers and a gold nanoparticle/DNA probe complex (GNP/DNA probe). The overall detection process was accomplished within approximately 1 h with no need for complicated equipment. The limits of detection for L. monocytogenes in the forms of purified genomic DNA and pure culture were 800 fg and 2.82 CFU mL-1, respectively. No cross reactions were observed from closely related bacteria species. The LAMP-GNP/DNA probe assay was applied to the detection of 200 raw chicken meat samples and compared to routine standard methods. The data revealed that the specificity, sensitivity and accuracy were 100%, 90.20% and 97.50%, respectively. The present assay was 100% in conformity with LAMP-agarose gel electrophoresis assay. Five samples that were negative by both assays appeared to have the pathogen at below the level of detection. The assay can be applied as a rapid direct screening method for L. monocytogenes.

"Dip-and-read" paper-based analytical devices using distance-based detection with color screening.

PubMed

Yamada, Kentaro; Citterio, Daniel; Henry, Charles S

2018-05-15

An improved paper-based analytical device (PAD) using color screening to enhance device performance is described. Current detection methods for PADs relying on the distance-based signalling motif can be slow due to the assay time being limited by capillary flow rates that wick fluid through the detection zone. For traditional distance-based detection motifs, analysis can take up to 45 min for a channel length of 5 cm. By using a color screening method, quantification with a distance-based PAD can be achieved in minutes through a "dip-and-read" approach. A colorimetric indicator line deposited onto a paper substrate using inkjet-printing undergoes a concentration-dependent colorimetric response for a given analyte. This color intensity-based response has been converted to a distance-based signal by overlaying a color filter with a continuous color intensity gradient matching the color of the developed indicator line. As a proof-of-concept, Ni quantification in welding fume was performed as a model assay. The results of multiple independent user testing gave mean absolute percentage error and average relative standard deviations of 10.5% and 11.2% respectively, which were an improvement over analysis based on simple visual color comparison with a read guide (12.2%, 14.9%). In addition to the analytical performance comparison, an interference study and a shelf life investigation were performed to further demonstrate practical utility. The developed system demonstrates an alternative detection approach for distance-based PADs enabling fast (∼10 min), quantitative, and straightforward assays.

Optical reading of contaminants in aqueous media based on gold nanoparticles.

PubMed

Du, Jianjun; Zhu, Bowen; Peng, Xiaojun; Chen, Xiaodong

2014-09-10

With increasing trends of global population growth, urbanization, pollution over-exploitation, and climate change, the safe water supply has become a global issue and is threatening our society in terms of sustainable development. Therefore, there is a growing need for a water-monitoring platform with the capability of rapidness, specificity, low-cost, and robustness. This review summarizes the recent developments in the design and application of gold nanoparticles (AuNPs) based optical assays to detect contaminants in aqueous media with a high performance. First, a brief discussion on the correlation between the optical reading strategy and the optical properties of AuNPs is presented. Then, we summarize the principle behind AuNP-based optical assays to detect different contaminants, such as toxic metal ion, anion, and pesticides, according to different optical reading strategies: colorimetry, scattering, and fluorescence. Finally, the comparison of these assays and the outlook of AuNP-based optical detection are discussed. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Simultaneous Analytical Method for Duplex Identification of Porcine and Horse in the Meat Products by EvaGreen based Real-time PCR.

PubMed

Sakalar, Ergün; Ergün, Seyma Özçirak; Akar, Emine

2015-01-01

A duplex real-time polymerase chain reaction (PCR) based assay for the detection of porcine and horse meat in sausages was designed by using EvaGreen fluorescent dye. Primers were selected from mitochondrial 12S rRNA and 16S rRNA genes which are powerful regions for identification of horse and porcine meat. DNA from reference samples and industrial products was successfully extracted using the GIDAGEN® Multi-Fast DNA Isolation Kit. Genomes were identified based on their specific melting peaks (Mp) which are 82.5℃ and 78℃ for horse and porcine, respectively. The assay used in this study allowed the detection of as little as 0.0001% level of horse meat and 0.001% level of porcine meat in the experimental admixtures. These findings indicate that EvaGreen based duplex real-time PCR is a potentially sensitive, reliable, rapid and accurate assay for the detection of meat species adulterated with porcine and horse meats.

Improving colorimetric assays through protein enzyme-assisted gold nanoparticle amplification.

PubMed

Xie, Xiaoji; Xu, Wei; Liu, Xiaogang

2012-09-18

The discovery of the DNA-mediated assembly of gold nanoparticles was a great moment in the history of science; this understanding and chemical control enabled the rational design of functional nanomaterials as novel probes in biodetection. In contrast with conventional probes such as organic dyes, gold nanoparticles exhibit high photostability and unique size-dependent optical properties. Because of their high extinction coefficients and strong distance dependent optical properties, these nanoparticles have emerged over the past decade as a promising platform for rapid, highly sensitive colorimetric assays that allow for the visual detection of low concentrations of metal ions, small molecules, and biomacromolecules. These discoveries have deepened our knowledge of biological phenomena and facilitated the development of many new diagnostic and therapeutic tools. Despite these many advances and continued research efforts, current nanoparticle-based colorimetric detection systems still suffer from several drawbacks, such as limited sensitivity and selectivity. This Account describes the recent development of colorimetric assays based on protein enzyme-assisted gold nanoparticle amplification. The benefits of such detection systems include significantly improved detection sensitivity and selectivity. First, we discuss the general design of enzyme-modified nanoparticle systems in colorimetric assays. We show that a quantitative understanding of the unique properties of different enzymes is paramount for effective biological assays. We then examine the assays for nucleic acid detection based on different types of enzymes, including endonucleases, ligases, and polymerases. For each of these assays, we identify the underlying principles that contribute to the enhanced detection capability of nanoparticle systems and illustrate them with selected examples. Furthermore, we demonstrate that the combination of gold nanoparticles and specific enzymes can probe enzyme dynamics and function with high specificity, offering substantial advantages in both sensitivity and specificity over conventional detection methods. The screening of nuclease, methyltransferase, protease, and kinase activities can be colorimetrically performed in a straightforward manner. Finally, we discuss examples of colorimetric assays for metal ions and small molecules that constitute important advances toward visual monitoring of enzyme catalytic functions and gene expression. Although these enzyme-assisted assay methods hold great promise for myriad applications in biomedicine and bioimaging, the application of the described techniques in vivo faces formidable challenges. In addition, researchers do not fully understand the interactions of gold nanoparticles with enzyme molecules. This understanding will require the development of new techniques to probe enzyme substrate dynamics at the particle interface with higher spatial resolution and chemical specificity.

A novel MALDI–TOF based methodology for genotyping single nucleotide polymorphisms

PubMed Central

Blondal, Thorarinn; Waage, Benedikt G.; Smarason, Sigurdur V.; Jonsson, Frosti; Fjalldal, Sigridur B.; Stefansson, Kari; Gulcher, Jeffery; Smith, Albert V.

2003-01-01

A new MALDI–TOF based detection assay was developed for analysis of single nucleotide polymorphisms (SNPs). It is a significant modification on the classic three-step minisequencing method, which includes a polymerase chain reaction (PCR), removal of excess nucleotides and primers, followed by primer extension in the presence of dideoxynucleotides using modified thermostable DNA polymerase. The key feature of this novel assay is reliance upon deoxynucleotide mixes, lacking one of the nucleotides at the polymorphic position. During primer extension in the presence of depleted nucleotide mixes, standard thermostable DNA polymerases dissociate from the template at positions requiring a depleted nucleotide; this principal was harnessed to create a genotyping assay. The assay design requires a primer- extension primer having its 3′-end one nucleotide upstream from the interrogated site. The assay further utilizes the same DNA polymerase in both PCR and the primer extension step. This not only simplifies the assay but also greatly reduces the cost per genotype compared to minisequencing methodology. We demonstrate accurate genotyping using this methodology for two SNPs run in both singleplex and duplex reactions. We term this assay nucleotide depletion genotyping (NUDGE). Nucleotide depletion genotyping could be extended to other genotyping assays based on primer extension such as detection by gel or capillary electrophoresis. PMID:14654708

Marine neurotoxins: state of the art, bottlenecks, and perspectives for mode of action based methods of detection in seafood.

PubMed

Nicolas, Jonathan; Hendriksen, Peter J M; Gerssen, Arjen; Bovee, Toine F H; Rietjens, Ivonne M C M

2014-01-01

Marine biotoxins can accumulate in fish and shellfish, representing a possible threat for consumers. Many marine biotoxins affect neuronal function essentially through their interaction with ion channels or receptors, leading to different symptoms including paralysis and even death. The detection of marine biotoxins in seafood products is therefore a priority. Official methods for control are often still using in vivo assays, such as the mouse bioassay. This test is considered unethical and the development of alternative assays is urgently required. Chemical analyses as well as in vitro assays have been developed to detect marine biotoxins in seafood. However, most of the current in vitro alternatives to animal testing present disadvantages: low throughput and lack of sensitivity resulting in a high number of false-negative results. Thus, there is an urgent need for the development of new in vitro tests that would allow the detection of marine biotoxins in seafood products at a low cost, with high throughput combined with high sensitivity, reproducibility, and predictivity. Mode of action based in vitro bioassays may provide tools that fulfil these requirements. This review covers the current state of the art of such mode of action based alternative assays to detect neurotoxic marine biotoxins in seafood. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Development of a recombinase polymerase amplification assay for Vibrio parahaemolyticus detection with an internal amplification control.

PubMed

Yang, Huan-Lan; Wei, Shuang; Gooneratne, Ravi; Mutukumira, Anthony N; Ma, Xue-Jun; Tang, Shu-Ze; Wu, Xi-Yang

2018-04-01

A novel RPA-IAC assay using recombinase polymerase and an internal amplification control (IAC) for Vibrio parahaemolyticus detection was developed. Specific primers were designed based on the coding sequence for the toxR gene in V. parahaemolyticus. The recombinase polymerase amplification (RPA) reaction was conducted at a constant low temperature of 37 °C for 20 min. Assay specificity was validated by using 63 Vibrio strains and 10 non-Vibrio bacterial species. In addition, a competitive IAC was employed to avoid false-negative results, which co-amplified simultaneously with the target sequence. The sensitivity of the assay was determined as 3 × 10 3 CFU/mL, which is decidedly more sensitive than the established PCR method. This method was then used to test seafood samples that were collected from local markets. Seven out of 53 different raw seafoods were detected as V. parahaemolyticus-positive, which were consistent with those obtained using traditional culturing method and biochemical assay. This novel RPA-IAC assay provides a rapid, specific, sensitive, and more convenient detection method for V. parahaemolyticus.

Gold nanoparticles-based protease assay

PubMed Central

Guarise, Cristian; Pasquato, Lucia; De Filippis, Vincenzo; Scrimin, Paolo

2006-01-01

We describe here a simple assay that allows the visual detection of a protease. The method takes advantage of the high molar absorptivity of the plasmon band of gold colloids and is based on the color change of their solution when treated with dithiols. We used C- and N-terminal cysteinyl derivatives of a peptide substrate exploiting its selective recognition and cleavage by a specific protease. Contrary to the native ones, cleaved peptides are unable to induce nanoparticles aggregation; hence, the color of the solution does not change. The detection of two proteases is reported: thrombin (involved in blood coagulation and thrombosis) and lethal factor (an enzyme component of the toxin produced by Bacillus anthracis). The sensitivity of this nanoparticle-based assay is in the low nanomolar range. PMID:16537471

Gold nanoparticles-based protease assay.

PubMed

Guarise, Cristian; Pasquato, Lucia; De Filippis, Vincenzo; Scrimin, Paolo

2006-03-14

We describe here a simple assay that allows the visual detection of a protease. The method takes advantage of the high molar absorptivity of the plasmon band of gold colloids and is based on the color change of their solution when treated with dithiols. We used C- and N-terminal cysteinyl derivatives of a peptide substrate exploiting its selective recognition and cleavage by a specific protease. Contrary to the native ones, cleaved peptides are unable to induce nanoparticles aggregation; hence, the color of the solution does not change. The detection of two proteases is reported: thrombin (involved in blood coagulation and thrombosis) and lethal factor (an enzyme component of the toxin produced by Bacillus anthracis). The sensitivity of this nanoparticle-based assay is in the low nanomolar range.

A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis.

PubMed

Sonkar, Subash C; Sachdev, Divya; Mishra, Prashant K; Kumar, Anita; Mittal, Pratima; Saluja, Daman

2016-12-15

The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of culture and a novel 5' Taq nuclease assay for direct detection of Campylobacter fetus subsp. venerealis in clinical specimens from cattle.

PubMed

McMillen, Lyle; Fordyce, Geoffry; Doogan, Vivienne J; Lew, Ala E

2006-03-01

A Campylobacter fetus subsp. venerealis-specific 5' Taq nuclease PCR assay using a 3' minor groove binder-DNA probe (TaqMan MGB) was developed based on a subspecies-specific fragment of unknown identity (S. Hum, K. Quinn, J. Brunner, and S. L. On, Aust. Vet. J. 75:827-831, 1997). The assay specifically detected four C. fetus subsp. venerealis strains with no observed cross-reaction with C. fetus subsp. fetus-related Campylobacter species or other bovine venereal microflora. The 5' Taq nuclease assay detected approximately one single cell compared to 100 and 10 cells in the conventional PCR assay and 2,500 and 25,000 cells from selective culture from inoculated smegma and mucus, respectively. The respective detection limits following the enrichments from smegma and mucus were 5,000 and 50 cells/inoculum for the conventional PCR compared to 500 and 50 cells/inoculum for the 5' Taq nuclease assay. Field sampling confirmed the sensitivity and the specificity of the 5' Taq nuclease assay by detecting an additional 40 bulls that were not detected by culture. Urine-inoculated samples demonstrated comparable detection of C. fetus subsp. venerealis by both culture and the 5' Taq nuclease assay; however, urine was found to be less effective than smegma for bull sampling. Three infected bulls were tested repetitively to compare sampling tools, and the bull rasper proved to be the most suitable, as evidenced by the improved ease of specimen collection and the consistent detection of higher levels of C. fetus subsp. venerealis. The 5' Taq nuclease assay demonstrates a statistically significant association with culture (chi2 = 29.8; P < 0.001) and significant improvements for the detection of C. fetus subsp. venerealis-infected animals from crude clinical extracts following prolonged transport.

Development of an Assay for the Detection of PrPres in Blood and Urine Based on PMCA Assay and ELISA Methods

DTIC Science & Technology

2008-09-01

the Origen Analyzer (BioVeris), the DELFIA (Wallac/PE) and the MPD ELISA ( BioTraces ). BioTraces had the most sensitive assay in which 125I was used...investigations we decided to abandon the BioTraces assay and focused on a more practical and also sensitive assay provided by the Origen Analyzer

HIGHLY SENSITIVE ASSAY FOR ANTICHOLINESTERASE COMPOUNDS USING 96 WELL PLATE FORMAT

EPA Science Inventory

The rapid and sensitive detection of organophosphate insecticides using a 96 well plate format is reported. Several features of this assay make it attractive for development as a laboratory-based or field screening assay. Acetylcholinesterase (AChE) was stabilized in a gelati...

Direct duplex real-time loop mediated isothermal amplification assay for the simultaneous detection of cow and goat species origin of milk and yogurt products for field use.

PubMed

Kim, Mi-Ju; Kim, Hae-Yeong

2018-04-25

A multiple loop-mediated isothermal amplification (LAMP) method was developed to detect cow and goat milk in the field using a portable fluorescence device. For rapid on-site detection, this duplex LAMP assay was used in combination with direct amplification, without DNA extraction. The cow- and goat-specific LAMP primer sets were designed based on the mitochondrial cytochrome b gene, and showed specificity against 13 other animal species in the reactions. The sensitivity of the duplex LAMP assay for cow and goat was 0.1 and 1 pg, respectively. The detection limit for both target species in milk mixtures was 2%. This assay successfully amplified and identified the two target species in 24 samples of commercial milk and yogurt products, with 30 min sampling-to-result analysis time. Therefore, this direct duplex real-time LAMP assay is useful for on-site simultaneous detection of cow and goat milk in commercial products, a capability needed to confirm accurate labeling. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparative genome analysis identifies novel nucleic acid diagnostic targets for use in the specific detection of Haemophilus influenzae.

PubMed

Coughlan, Helena; Reddington, Kate; Tuite, Nina; Boo, Teck Wee; Cormican, Martin; Barrett, Louise; Smith, Terry J; Clancy, Eoin; Barry, Thomas

2015-10-01

Haemophilus influenzae is recognised as an important human pathogen associated with invasive infections, including bloodstream infection and meningitis. Currently used molecular-based diagnostic assays lack specificity in correctly detecting and identifying H. influenzae. As such, there is a need to develop novel diagnostic assays for the specific identification of H. influenzae. Whole genome comparative analysis was performed to identify putative diagnostic targets, which are unique in nucleotide sequence to H. influenzae. From this analysis, we identified 2H. influenzae putative diagnostic targets, phoB and pstA, for use in real-time PCR diagnostic assays. Real-time PCR diagnostic assays using these targets were designed and optimised to specifically detect and identify all 55H. influenzae strains tested. These novel rapid assays can be applied to the specific detection and identification of H. influenzae for use in epidemiological studies and could also enable improved monitoring of invasive disease caused by these bacteria. Copyright © 2015 Elsevier Inc. All rights reserved.

Detection of dopamine in dopaminergic cell using nanoparticles-based barcode DNA analysis.

PubMed

An, Jeung Hee; Kim, Tae-Hyung; Oh, Byung-Keun; Choi, Jeong Woo

2012-01-01

Nanotechnology-based bio-barcode-amplification analysis may be an innovative approach to dopamine detection. In this study, we evaluated the efficacy of this bio-barcode DNA method in detecting dopamine from dopaminergic cells. Herein, a combination DNA barcode and bead-based immunoassay for neurotransmitter detection with PCR-like sensitivity is described. This method relies on magnetic nanoparticles with antibodies and nanoparticles that are encoded with DNA, and antibodies that can sandwich the target protein captured by the nanoparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated in order to remove the conjugated barcode DNA. The DNA barcodes were then identified via PCR analysis. The dopamine concentration in dopaminergic cells can be readily and rapidly detected via the bio-barcode assay method. The bio-barcode assay method is, therefore, a rapid and high-throughput screening tool for the detection of neurotransmitters such as dopamine.

Development and Application of a Saccharomyces cerevisiae-Expressed Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against Infectious Bronchitis Virus

PubMed Central

Gibertoni, Aliandra M.; Montassier, Maria de Fátima S.; Sena, Janete A. D.; Givisiez, Patrícia E. N.; Furuyama, Cibele R. A. G.; Montassier, Hélio J.

2005-01-01

A Saccharomyces cerevisiae-expressed nucleocapsid (N) polypeptide of the M41 strain of infectious bronchitis virus (IBV) was used as antigen in a recombinant yeast-expressed N protein-based enzyme-linked immunosorbent assay (Y-N-ELISA). The Y-N-ELISA was rapid, sensitive, and specific for detecting chicken serum antibodies to IBV, and it compared favorably with a commercial ELISA. PMID:15815038

A Novel Pan-Flavivirus Detection and Identification Assay Based on RT-qPCR and Microarray

PubMed Central

Sachse, Konrad; Ziegler, Ute; Keller, Markus

2017-01-01

The genus Flavivirus includes arthropod-borne viruses responsible for a large number of infections in humans and economically important animals. While RT-PCR protocols for specific detection of most Flavivirus species are available, there has been also a demand for a broad-range Flavivirus assay covering all members of the genus. It is particularly challenging to balance specificity at genus level with equal sensitivity towards each target species. In the present study, a novel assay combining a SYBR Green-based RT-qPCR with a low-density DNA microarray has been developed. Validation experiments confirmed that the RT-qPCR exhibited roughly equal sensitivity of detection and quantification for all flaviviruses tested. These PCR products are subjected to hybridization on a microarray carrying 84 different oligonucleotide probes that represent all known Flavivirus species. This assay has been used as a screening and confirmation tool for Flavivirus presence in laboratory and field samples, and it performed successfully in international External Quality Assessment of NAT studies. Twenty-six Flavivirus strains were tested with the assay, showing equivalent or superior characteristics compared with the original or even with species-specific RT-PCRs. As an example, test results on West Nile virus detection in a panel of 340 mosquito pool samples from Greece are presented. PMID:28626758

SERS Assay for Copper(II) Ions Based on Dual Hot-Spot Model Coupling with MarR Protein: New Cu2+-Specific Biorecognition Element.

PubMed

Wang, Yulong; Su, Zhenhe; Wang, Limin; Dong, Jinbo; Xue, Juanjuan; Yu, Jiao; Wang, Yuan; Hua, Xiude; Wang, Minghua; Zhang, Cunzheng; Liu, Fengquan

2017-06-20

We have developed a rapid and ultrasensitive surface-enhanced Raman scattering (SERS) assay for Cu 2+ detection using the multiple antibiotic resistance regulator (MarR) as specific bridging molecules in a SERS hot-spot model. In the assay, Cu 2+ induces formation of MarR tetramers, which provide Au nanoparticle (NP)-AuNP bridges, resulting in the formation of SERS hot spots. 4-Mercaptobenzoic acid (4-MBA) was used as a Raman reporter. The addition of Cu 2+ increased the Raman intensity of 4-MBA. Use of a dual hot-spot signal-amplification strategy based on AuNP-AgNP heterodimers combined through antigen-antibody reactions increased the sensitivity of the sensing platform by 50-fold. The proposed method gave a linear response for Cu 2+ detection in the range of 0.5-1000 nM, with a detection limit of 0.18 nM, which is 5 orders of magnitude lower than the U.S. Environmental Protection Agency limit for Cu 2+ in drinking water (20 μM). In addition, all analyses can be completed in less than 15 min. The high sensitivity, high specificity, and rapid detection capacity of the SERS assay therefore provide a combined advantage over current assays.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathryn L.

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)more » 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.« less

SensiScreen® KRAS exon 2-sensitive simplex and multiplex real-time PCR-based assays for detection of KRAS exon 2 mutations

PubMed Central

Guldmann-Christensen, Mariann; Hauge Kyneb, Majbritt; Voogd, Kirsten; Andersen, Christina; Epistolio, Samantha; Merlo, Elisabetta; Yding Wolff, Tine; Hamilton-Dutoit, Stephen; Lorenzen, Jan; Christensen, Ulf Bech

2017-01-01

Activating mutations in codon 12 and codon 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) gene are implicated in the development of several human cancer types and influence their clinical evaluation, treatment and prognosis. Numerous different methods for KRAS genotyping are currently available displaying a wide range of sensitivities, time to answer and requirements for laboratory equipment and user skills. Here we present SensiScreen® KRAS exon 2 simplex and multiplex CE IVD assays, that use a novel real-time PCR-based method for KRAS mutation detection based on PentaBase’s proprietary DNA analogue technology and designed to work on standard real-time PCR instruments. By means of the included BaseBlocker™ technology, we show that SensiScreen® specifically amplifies the mutated alleles of interest with no or highly subdued amplification of the wild type allele. Furthermore, serial dilutions of mutant DNA in a wild type background demonstrate that all SensiScreen® assays display a limit of detection that falls within the range of 0.25–1%. Finally, in three different colorectal cancer patient populations, SensiScreen® assays confirmed the KRAS genotype previously determined by commonly used methods for KRAS mutation testing, and notably, in two of the populations, SensiScreen® identified additional mutant positive cases not detected by common methods. PMID:28636636

Recent advancements in chemical luminescence-based lab-on-chip and microfluidic platforms for bioanalysis.

PubMed

Mirasoli, Mara; Guardigli, Massimo; Michelini, Elisa; Roda, Aldo

2014-01-01

Miniaturization of analytical procedures through microchips, lab-on-a-chip or micro total analysis systems is one of the most recent trends in chemical and biological analysis. These systems are designed to perform all the steps in an analytical procedure, with the advantages of low sample and reagent consumption, fast analysis, reduced costs, possibility of extra-laboratory application. A range of detection technologies have been employed in miniaturized analytical systems, but most applications relied on fluorescence and electrochemical detection. Chemical luminescence (which includes chemiluminescence, bioluminescence, and electrogenerated chemiluminescence) represents an alternative detection principle that offered comparable (or better) analytical performance and easier implementation in miniaturized analytical devices. Nevertheless, chemical luminescence-based ones represents only a small fraction of the microfluidic devices reported in the literature, and until now no review has been focused on these devices. Here we review the most relevant applications (since 2009) of miniaturized analytical devices based on chemical luminescence detection. After a brief overview of the main chemical luminescence systems and of the recent technological advancements regarding their implementation in miniaturized analytical devices, analytical applications are reviewed according to the nature of the device (microfluidic chips, microchip electrophoresis, lateral flow- and paper-based devices) and the type of application (micro-flow injection assays, enzyme assays, immunoassays, gene probe hybridization assays, cell assays, whole-cell biosensors). Copyright © 2013 Elsevier B.V. All rights reserved.

Improving membrane based multiplex immunoassays for semi-quantitative detection of multiple cytokines in a single sample

PubMed Central

2014-01-01

Background Inflammatory mediators can serve as biomarkers for the monitoring of the disease progression or prognosis in many conditions. In the present study we introduce an adaptation of a membrane-based technique in which the level of up to 40 cytokines and chemokines can be determined in both human and rodent blood in a semi-quantitative way. The planar assay was modified using the LI-COR (R) detection system (fluorescence based) rather than chemiluminescence and semi-quantitative outcomes were achieved by normalizing the outcomes using the automated exposure settings of the Odyssey readout device. The results were compared to the gold standard assay, namely ELISA. Results The improved planar assay allowed the detection of a considerably higher number of analytes (n = 30 and n = 5 for fluorescent and chemiluminescent detection, respectively). The improved planar method showed high sensitivity up to 17 pg/ml and a linear correlation of the normalized fluorescence intensity with the results from the ELISA (r = 0.91). Conclusions The results show that the membrane-based technique is a semi-quantitative assay that correlates satisfactorily to the gold standard when enhanced by the use of fluorescence and subsequent semi-quantitative analysis. This promising technique can be used to investigate inflammatory profiles in multiple conditions, particularly in studies with constraints in sample sizes and/or budget. PMID:25022797

Development of an LC-MS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis.

PubMed

Deng, Gejing; Gu, Rong-Fang; Marmor, Stephen; Fisher, Stewart L; Jahic, Haris; Sanyal, Gautam

2004-06-29

An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (> or = 10 fold), lower limit of quantitation (LOQ) (0.02 microM versus 1 microM) and wider linear dynamic range (> or = 4 fold) than the MG assay. Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated. Copyright 2004 Elsevier B.V.

Rapid and field-deployable biological and chemical Raman-based identification

NASA Astrophysics Data System (ADS)

Botonjic-Sehic, Edita; Paxon, Tracy L.; Boudries, Hacene

2011-06-01

Pathogen detection using Raman spectroscopy is achieved through the use of a sandwich immunoassay. Antibody-modified magnetic beads are used to capture and concentrate target analytes in solution and surface-enhanced Raman spectroscopy (SERS) tags are conjugated with antibodies and act as labels to enable specific detection of biological pathogens. The rapid detection of biological pathogens is critical to first responders, thus assays to detect E.Coli and Anthrax have been developed and will be reported. The problems associated with pathogen detection resulting from the spectral complexity and variability of microorganisms are overcome through the use of SERS tags, which provide an intense, easily recognizable, and spectrally consistent Raman signal. The developed E. coli assay has been tested with 5 strains of E. coli and shows a low limit of detection, on the order of 10 and 100 c.f.u. per assay. Additionally, the SERS assay utilizes magnetic beads to collect the labeled pathogens into the focal point of the detection laser beam, making the assay robust to commonly encountered white powder interferants such as flour, baking powder, and corn starch. The reagents were also found to be stable at room temperature over extended periods of time with testing conducted over a one year period. Finally, through a specialized software algorithm, the assays are interfaced to the Raman instrument, StreetLab Mobile, for rapid-field-deployable biological identification.

Biochip-Based Detection of KRAS Mutation in Non-Small Cell Lung Cancer

PubMed Central

Kriegshäuser, Gernot; Fabjani, Gerhild; Ziegler, Barbara; Zöchbauer-Müller, Sabine; End, Adelheid; Zeillinger, Robert

2011-01-01

This study is aimed at evaluating the potential of a biochip assay to sensitively detect KRAS mutation in DNA from non-small cell lung cancer (NSCLC) tissue samples. The assay covers 10 mutations in codons 12 and 13 of the KRAS gene, and is based on mutant-enriched PCR followed by reverse-hybridization of biotinylated amplification products to an array of sequence-specific probes immobilized on the tip of a rectangular plastic stick (biochip). Biochip hybridization identified 17 (21%) samples to carry a KRAS mutation of which 16 (33%) were adenocarcinomas and 1 (3%) was a squamous cell carcinoma. All mutations were confirmed by DNA sequencing. Using 10 ng of starting DNA, the biochip assay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. Our results suggest that the biochip assay is a sensitive alternative to protocols currently in use for KRAS mutation testing on limited quantity samples. PMID:22272089

Simple, rapid and sensitive detection of Orientia tsutsugamushi by loop-isothermal DNA amplification.

PubMed

Paris, Daniel H; Blacksell, Stuart D; Newton, Paul N; Day, Nicholas P J

2008-12-01

We present a loop-mediated isothermal PCR assay (LAMP) targeting the groEL gene, which encodes the 60kDa heat shock protein of Orientia tsutsugamushi. Evaluation included testing of 63 samples of contemporary in vitro isolates, buffy coats and whole blood samples from patients with fever. Detection limits for LAMP were assessed by serial dilutions and quantitation by real-time PCR assay based on the same target gene: three copies/microl for linearized plasmids, 26 copies/microl for VERO cell culture isolates, 14 copies/microl for full blood samples and 41 copies/microl for clinical buffy coats. Based on a limited sample number, the LAMP assay is comparable in sensitivity with conventional nested PCR (56kDa gene), with limits of detection well below the range of known admission bacterial loads of patients with scrub typhus. This inexpensive method requires no sophisticated equipment or sample preparation, and may prove useful as a diagnostic assay in financially poor settings; however, it requires further prospective validation in the field setting.

A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva† †Electronic supplementary information (ESI) available: Optimization of Mg2+ and ATMND concentrations for our CBSA-based ATMND-binding assay; ATMND-reported calibration curve for CBSA-5325 at various cocaine concentrations; ATMND binding affinity for the cocaine-assembled CBSA-5325; K D of 38-GC and different 38-GC mutants for cocaine as characterized by ITC; stem length effects on cocaine-induced CBSA assembly; spectra of CBSA-5335-based fluorescence detection of cocaine in 1× binding buffer; characterization of cocaine binding affinity of CBSA-5335 and PSA using ITC; fluorescence detection of cocaine in saliva with our fluorophore/quencher modified CBSA-5335; calibration curve of our CBSA-5335-based fluorophore/quencher assay in 1× binding buffer and 10% saliva at cocaine concentrations ranging from 0 to 10 μM; bias and precision of the CBSA-5335-based fluorophore/quencher assay; comparison of amplification-free split-aptamer assays for cocaine detection; sequence ID and DNA sequences used in this work. See DOI: 10.1039/c6sc01833e Click here for additional data file.

PubMed Central

Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav; Lou, Xinhui

2017-01-01

Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding. We have generated a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-binding affinity of our CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples. PMID:28451157

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

PubMed Central

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

2012-01-01

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

Polymerase chain reaction-hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation.

PubMed

Xu, Chen; Zhang, Nan; Huo, Qianyu; Chen, Minghui; Wang, Rengfeng; Liu, Zhili; Li, Xue; Liu, Yunde; Bao, Huijing

2016-04-15

In this article, we discuss the polymerase chain reaction (PCR)-hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA-BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase-streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR-hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR-hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular beacon probes-base multiplex NASBA Real-time for detection of HIV-1 and HCV.

PubMed

Mohammadi-Yeganeh, S; Paryan, M; Mirab Samiee, S; Kia, V; Rezvan, H

2012-06-01

Developed in 1991, nucleic acid sequence-based amplification (NASBA) has been introduced as a rapid molecular diagnostic technique, where it has been shown to give quicker results than PCR, and it can also be more sensitive. This paper describes the development of a molecular beacon-based multiplex NASBA assay for simultaneous detection of HIV-1 and HCV in plasma samples. A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used for primers and molecular beacon design. The performance features of HCV/HIV-1 multiplex NASBA assay including analytical sensitivity and specificity, clinical sensitivity and clinical specificity were evaluated. The analysis of scalar concentrations of the samples indicated that the limit of quantification of the assay was <1000 copies/ml for HIV-1 and <500 copies/ml for HCV with 95% confidence interval. Multiplex NASBA assay showed a 98% sensitivity and 100% specificity. The analytical specificity study with BLAST software demonstrated that the primers do not attach to any other sequences except for that of HIV-1 or HCV. The primers and molecular beacon probes detected all HCV genotypes and all major variants of HIV-1. This method may represent a relatively inexpensive isothermal method for detection of HIV-1/HCV co-infection in monitoring of patients.

Highly labeled methylene blue-ds DNA silica nanoparticles for signal enhancement of immunoassays: application to the sensitive detection of bacteria in human platelet concentrates.

PubMed

Bonnet, Romaric; Farre, Carole; Valera, Lionel; Vossier, Ludivine; Léon, Fanny; Dagland, Typhaine; Pouzet, Agnès; Jaffrézic-Renault, Nicole; Fareh, Jeannette; Fournier-Wirth, Chantal; Chaix, Carole

2018-05-15

A nanoparticle-based electrochemical sandwich immunoassay was developed for bacteria detection in platelet concentrates. For the assay, magnetic beads were functionalized with antibodies to allow the specific capture of bacteria from the complex matrix, and innovative methylene blue-DNA/nanoparticle assemblies provided the electrochemical response for amplified detection. This nanoparticular system was designed as a temperature-sensitive nano-tool for electrochemical detection. First, oligonucleotide-functionalized nanoparticles were obtained by direct synthesis of the DNA strands on the nanoparticle surface using an automated oligonucleotide synthesizer. Densely packed DNA coverage was thus obtained. Then, DNA duplexes were constructed on the NP surface with a complementary strand bearing a 3 methylene blue tag. This strategy ultimately produced highly functionalized nanoparticles with electrochemical markers. These assemblies enabled amplification of the electrochemical signal, resulting in a very good sensitivity. A proof-of-concept was carried out for E. coli detection in human platelet concentrates. Bacterial contamination of this complex biological matrix is the highest residual infectious risk in blood transfusion. The development of a rapid assay that could reach 10-102 CFU mL-1 sensitivity is a great challenge. The nanoparticle-based electrochemical sandwich immunoassay carried out on a boron doped diamond electrode proved to be sensitive for E. coli detection in human platelets. Two antibody pairs were used to develop either a generic assay against certain Gram negative strains or a specific assay for E. coli. The methylene blue-DNA/nanoparticles amplify sensitivity ×1000 compared with the assay run without NPs for electrochemical detection. A limit of detection of 10 CFU mL-1 in a biological matrix was achieved for E. coli using the highly specific antibody pair.

Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification

USDA-ARS?s Scientific Manuscript database

A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39 °C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in ...

Chemical & Biological Point Detection Decontamination

DTIC Science & Technology

2002-04-01

high priority in biological defense. Research on multivalent assays is also ongoing. Biased libraries, generated from immunized animals, or unbiased ...2003 TBD decontamination and modeling and simulation I I The Chem-Bio Point Detection Roadmap The summary level updated and expanded Bio Point... Molecular Imprinted Polymer Sensor, Dendrimer-based Antibody Assays, Pyrolysis-GC-ion mobility spectrometry, and surface enhanced Raman spectroscopy. Data

One-step reverse transcription loop mediated isothermal amplification assay for detection of Apple chlorotic leaf spot virus

USDA-ARS?s Scientific Manuscript database

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of Apple chlorotic leaf spot virus (ACLSV) was developed. In this method, a set of four primers was designed based on the conserved regions in the coat protein gene of ACLSV, and was synthesized for the ...

Biochemical Assay Detects Feeding Damage to Loblolly Pine Seeds Caused by the Leaffooted Pine Seed Bug (Hemiptera: Coreidae)

Treesearch

Cameron G. Lait; Daniel R. Miller; Sarah L. Bates; John H. Borden; Allison R. Kermode

2003-01-01

A large number of proteins in salivary gland extracts of the leaffooted pine seed bug, Leptoglossus corculus Say, were strongly recognized by a polyclonal antibody-based assay developed for detecting saliva of the western conifer seed bug, Lepfoglossus occidentalis Heidemann, in lodgepole pine, Pinus contorta var...

Rationalizing and advancing the 3-MPBA SERS sandwich assay for rapid detection of bacteria in environmental and food matrices.

PubMed

Pearson, Brooke; Mills, Alexander; Tucker, Madeline; Gao, Siyue; McLandsborough, Lynne; He, Lili

2018-06-01

Bacterial foodborne illness continues to be a pressing issue in our food supply. Rapid detection methods are needed for perishable foods due to their short shelf lives and significant contribution to foodborne illness. Previously, a sensitive and reliable surface-enhanced Raman spectroscopy (SERS) sandwich assay based on 3-mercaptophenylboronic acid (3-MBPA) as a capturer and indicator molecule was developed for rapid bacteria detection. In this study, we explored the advantages and constraints of this assay over the conventional aerobic plate count (APC) method and further developed methods for detection in real environmental and food matrices. The SERS sandwich assay was able to detect environmental bacteria in pond water and on spinach leaves at higher levels than the APC method. In addition, the SERS assay appeared to have higher sensitivity to quantify bacteria in the stationary phase. On the other hand, the APC method was more sensitive to cell viability. Finally, a method to detect bacteria in a challenging high-sugar juice matrix was developed to enhance bacteria capture. This study advanced the SERS technique for real applications in environment and food matrices. Copyright © 2017 Elsevier Ltd. All rights reserved.

A sandwich-type optical immunosensor based on the alkaline phosphatase enzyme for Salmonella thypimurium detection

NASA Astrophysics Data System (ADS)

Widyastuti, E.; Puspitasari Schonherr, M. F.; Masruroh, A.; Anggraeni, R. A.; Nisak, Y. K.; Mursidah, S.

2018-03-01

Salmonella is pathogenic bacteria that caused foodborne diseases which being called Salmonellosis. Prevalence of Salmonellosis that being caused by Salmonella thypimurium in Indonesia is quite high. However, detection of Salmonella bacteria in food still limited, complicated, and required a lot time. Sensitive optical assay for Salmonella thypimurium paper based detection has been developed by integrating sandwich assay between antibody-antigen complex and alkaline phosphatase enzyme that produce visible bluish-purple colour with presence of NBT-BCIP substrate. The results showed that Limit of Quantitation of detection is 105 CFU mL-1 with detection time 15 minutes. Linearity test between Colour intensity that produced from Salmonella concentration presence on samples showed that detection has good linearity. Selectivity test exhibited excellent sensitivity with good discrimination against Escherichia coli.

Comparison of the Third Wave Invader Human Papillomavirus (HPV) Assay and the Digene HPV Hybrid Capture 2 Assay for Detection of High-Risk HPV DNA▿

PubMed Central

Ginocchio, C. C.; Barth, D.; Zhang, F.

2008-01-01

This study compared the clinical performance of the Digene Hybrid Capture 2 (HC2) assay to that of a prototype Third Wave Invader human papillomavirus (HPV) (IHPV) analyte-specific reagent-based assay for the detection of oncogenic or “high-risk” (HR) HPV DNA using liquid-based cytology specimens. In total, 821 ThinPrep vials were tested using both assays. In accordance with the type-specific probes contained within each test, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the IHPV assay were 95.9%, 97.6%, 97.5%, and 96.1%, respectively, and those for the HC2 assay were 98.1%, 86.2%, 87.1%, and 97.9%. Overall, the sensitivity and NPV were comparable between the assays, but the IHPV assay demonstrated a better specificity and PPV, since the IHPV assay had fewer false-positive HR HPV results. The incorporation of an internal control to evaluate the cellularity of the test material is an important feature of the IHPV assay and should reduce the risk of false-negative results due to insufficient sample collection rather than the lack of HR HPV DNA. An additional benefit of the IHPV assay was the smaller sample volume required (1 ml versus 4 ml for the HC2 assay). PMID:18367578

Multiplex qPCR for reliable detection and differentiation of Burkholderia mallei and Burkholderia pseudomallei.

PubMed

Janse, Ingmar; Hamidjaja, Raditijo A; Hendriks, Amber C A; van Rotterdam, Bart J

2013-02-14

Burkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance. Signature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated. A single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction. The new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification.

Biosensor-based microRNA detection: techniques, design, performance, and challenges.

PubMed

Johnson, Blake N; Mutharasan, Raj

2014-04-07

The current state of biosensor-based techniques for amplification-free microRNA (miRNA) detection is critically reviewed. Comparison with non-sensor and amplification-based molecular techniques (MTs), such as polymerase-based methods, is made in terms of transduction mechanism, associated protocol, and sensitivity. Challenges associated with miRNA hybridization thermodynamics which affect assay selectivity and amplification bias are briefly discussed. Electrochemical, electromechanical, and optical classes of miRNA biosensors are reviewed in terms of transduction mechanism, limit of detection (LOD), time-to-results (TTR), multiplexing potential, and measurement robustness. Current trends suggest that biosensor-based techniques (BTs) for miRNA assay will complement MTs due to the advantages of amplification-free detection, LOD being femtomolar (fM)-attomolar (aM), short TTR, multiplexing capability, and minimal sample preparation requirement. Areas of future importance in miRNA BT development are presented which include focus on achieving high measurement confidence and multiplexing capabilities.

Evaluation of Various Campylobacter-Specific Quantitative PCR (qPCR) Assays for Detection and Enumeration of Campylobacteraceae in Irrigation Water and Wastewater via a Miniaturized Most-Probable-Number–qPCR Assay

PubMed Central

Banting, Graham S.; Braithwaite, Shannon; Scott, Candis; Kim, Jinyong; Jeon, Byeonghwa; Ashbolt, Nicholas; Ruecker, Norma; Tymensen, Lisa; Charest, Jollin; Pintar, Katarina; Checkley, Sylvia

2016-01-01

ABSTRACT Campylobacter spp. are the leading cause of bacterial gastroenteritis worldwide, and water is increasingly seen as a risk factor in transmission. Here we describe a most-probable-number (MPN)–quantitative PCR (qPCR) assay in which water samples are centrifuged and aliquoted into microtiter plates and the bacteria are enumerated by qPCR. We observed that commonly used Campylobacter molecular assays produced vastly different detection rates. In irrigation water samples, detection rates varied depending upon the PCR assay and culture method used, as follows: 0% by the de Boer Lv1-16S qPCR assay, 2.5% by the Van Dyke 16S and Jensen glyA qPCR assays, and 75% by the Linton 16S endpoint PCR when cultured at 37°C. Primer/probe specificity was the major confounder, with Arcobacter spp. routinely yielding false-positive results. The primers and PCR conditions described by Van Dyke et al. (M. I. Van Dyke, V. K. Morton, N. L. McLellan, and P. M. Huck, J Appl Microbiol 109:1053–1066, 2010, http://dx.doi.org/10.1111/j.1365-2672.2010.04730.x) proved to be the most sensitive and specific for Campylobacter detection in water. Campylobacter occurrence in irrigation water was found to be very low (<2 MPN/300 ml) when this Campylobacter-specific qPCR was used, with the most commonly detected species being C. jejuni, C. coli, and C. lari. Campylobacters in raw sewage were present at ∼102/100 ml, with incubation at 42°C required for reducing microbial growth competition from arcobacters. Overall, when Campylobacter prevalence and/or concentration in water is reported using molecular methods, considerable validation is recommended when adapting methods largely developed for clinical applications. Furthermore, combining MPN methods with molecular biology-based detection algorithms allows for the detection and quantification of Campylobacter spp. in environmental samples and is potentially suited to quantitative microbial risk assessment for improved public health disease prevention related to food and water exposures. IMPORTANCE The results of this study demonstrate the importance of assay validation upon data interpretation of environmental monitoring for Campylobacter when using molecular biology-based assays. Previous studies describing Campylobacter prevalence in Canada utilized primers that we have determined to be nonspecific due to their cross-amplification of Arcobacter spp. As such, Campylobacter prevalence may have been vastly overestimated in other studies. Additionally, the development of a quantitative assay described in this study will allow accurate determination of Campylobacter concentrations in environmental water samples, allowing more informed decisions to be made about water usage based on quantitative microbial risk assessment. PMID:27235434

Development of an upconverting chelate assay

NASA Astrophysics Data System (ADS)

Xiao, Xudong; Haushalter, Jeanne P.; Kotz, Kenneth T.; Faris, Gregory W.

2005-04-01

We report progress on performing a cell-based assay for the detection of EGFR on cell surfaces by using upconverting chelates. An upconversion microscope has been developed for performing assays and testing optical response. A431 cells are labeled with europium DOTA and imaged using this upconverting microscope.

Prospective assessment of early fetal loss using an immunoenzymometric screening assay for detection of urinary human chorionic gonadotropin.

PubMed

Taylor, C A; Overstreet, J W; Samuels, S J; Boyers, S P; Canfield, R E; O'Connor, J F; Hanson, F W; Lasley, B L

1992-06-01

To develop an economical, nonradiometric immunoenzymometric assay (IEMA) for the detection of urinary human chorionic gonadotropin (hCG) in studies of early fetal loss. To be effective, the IEMA must have a sensitivity equal to the standard immunoradiometric assay (IRMA) and sufficient specificity to eliminate the need for screening most nonconceptive cycles with the expensive and labor-intensive IRMA. Two different assays were used to measure hCG in daily early morning urine samples from potential conceptive cycles. Women undergoing donor artificial insemination (AI) were evaluated in a prospective study. Ninety-two women volunteers were selected on the basis of apparent normal reproductive health. Artificial insemination with nonfrozen donor semen was performed by cervical cup twice each menstrual cycle at 48-hour intervals, and daily urine samples were self-collected throughout the menstrual cycle. An IEMA was developed to detect urinary hCG using the same antibodies as in the standard IRMA; a study was designed to determine whether this nonradiometric assay could successfully detect the early fetal loss that was detected by the IRMA. Of 224 menstrual cycles analyzed by both assays, a total of six early fetal losses were detected by the IRMA. When the tentative screening rule was set to allow all six of these losses and 95% of future losses to be detected by the IEMA, an additional 34 false-positive results were detected by the IEMA. The specificity of the IEMA with this rule was calculated to be 84%. An IEMA based on the same antibodies used for the standard IRMA can serve as an efficient screening assay for the detection of early fetal loss. When the IEMA is used in this manner, nearly 80% of screened menstrual cycles can be eliminated without further testing by the IRMA.

Molecular beacon-based real-time PCR method for detection of porcine DNA in gelatin and gelatin capsules.

PubMed

Mohamad, Nurhidayatul Asma; Mustafa, Shuhaimi; Khairil Mokhtar, Nur Fadhilah; El Sheikha, Aly Farag

2018-03-05

The pharmaceutical industry has boosted gelatin consumption worldwide. This is supported by the availability of cost-effective gelatin production from porcine by-products. However, cross-contamination of gelatin materials, where porcine gelatin was unintentionally included in the other animal sources of gelatin, has caused significant concerns about halal authenticity. The real-time polymerase chain reaction (PCR) has enabled a highly specific and sensitive animal species detection method in various food products. Hence, such a technique was employed in the present study to detect and quantify porcine DNA in gelatin using a molecular beacon probe, with differences in performance between mitochondrial (cytochrome b gene) and chromosomal DNA-(MPRE42 repetitive element) based porcine-specific PCR assays being compared. A higher sensitivity was observed in chromosomal DNA (MPRE-PCR assay), where this assay allows the detection of gelatin DNA at amounts as as low as 1 pg, whereas mitochondrial DNA (CBH-PCR assay) can only detect at levels down to 10 pg of gelatin DNA. When an analysis with commercial gelatin and gelatin capsule samples was conducted, the same result was observed, with a significantly more sensitive detection being provided by the repetitive element of chromosomal DNA. The present study has established highly sensitive DNA-based porcine detection systems derived from chromosomal DNA that are feasible for highly processed products such as gelatin and gelatin capsules containing a minute amount of DNA. This sensitive detection method can also be implemented to assist the halal authentication process of various food products available on the market. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

T2 Magnetic Resonance Assay-Based Direct Detection of Three Lyme Disease-Related Borrelia Species in Whole-Blood Samples.

PubMed

Snyder, Jessica L; Giese, Heidi; Bandoski-Gralinski, Cheryl; Townsend, Jessica; Jacobson, Beck E; Shivers, Robert; Schotthoefer, Anna M; Fritsche, Thomas R; Green, Clayton; Callister, Steven M; Branda, John A; Lowery, Thomas J

2017-08-01

In early Lyme disease (LD), serologic testing is insensitive and seroreactivity may reflect active or past infection. In this study, we evaluated a novel assay for the direct detection of three species of Borrelia spirochetes in whole blood. The T2 magnetic resonance (T2MR) assay platform was used to amplify Borrelia DNA released from intact spirochetes and to detect amplicon. Analytical sensitivity was determined from blood spiked with known concentrations of spirochetes, and the assay's limit of detection was found to be in the single-cell-per-milliliter range: 5 cells/ml for B. afzelii and 8 cells/ml for Borrelia burgdorferi and Borrelia garinii Clinical samples ( n = 66) from confirmed or suspected early LD patients were also analyzed. B. burgdorferi was detected using T2MR in 2/2 (100%) of blood samples from patients with confirmed early LD, based on the presence of erythema migrans and documentation of seroconversion or a positive real-time blood PCR. T2MR detected B. burgdorferi in blood samples from 17/54 (31%) of patients with probable LD, based on the presence of erythema migrans without documented seroconversion or of documented seroconversion in patients with a compatible clinical syndrome but without erythema migrans. Out of 21 clinical samples tested by real-time PCR, only 1 was positive and 13 were negative with agreement with T2MR. An additional 7 samples that were negative by real-time PCR were positive with T2MR. Therefore, T2MR enables a low limit of detection (LoD) for Borrelia spp. in whole blood samples and is able to detect B. burgdorferi in clinical samples. Copyright © 2017 American Society for Microbiology.

T2 Magnetic Resonance Assay-Based Direct Detection of Three Lyme Disease-Related Borrelia Species in Whole-Blood Samples

PubMed Central

Giese, Heidi; Bandoski-Gralinski, Cheryl; Townsend, Jessica; Jacobson, Beck E.; Shivers, Robert; Schotthoefer, Anna M.; Fritsche, Thomas R.; Green, Clayton; Callister, Steven M.; Branda, John A.

2017-01-01

ABSTRACT In early Lyme disease (LD), serologic testing is insensitive and seroreactivity may reflect active or past infection. In this study, we evaluated a novel assay for the direct detection of three species of Borrelia spirochetes in whole blood. The T2 magnetic resonance (T2MR) assay platform was used to amplify Borrelia DNA released from intact spirochetes and to detect amplicon. Analytical sensitivity was determined from blood spiked with known concentrations of spirochetes, and the assay's limit of detection was found to be in the single-cell-per-milliliter range: 5 cells/ml for B. afzelii and 8 cells/ml for Borrelia burgdorferi and Borrelia garinii. Clinical samples (n = 66) from confirmed or suspected early LD patients were also analyzed. B. burgdorferi was detected using T2MR in 2/2 (100%) of blood samples from patients with confirmed early LD, based on the presence of erythema migrans and documentation of seroconversion or a positive real-time blood PCR. T2MR detected B. burgdorferi in blood samples from 17/54 (31%) of patients with probable LD, based on the presence of erythema migrans without documented seroconversion or of documented seroconversion in patients with a compatible clinical syndrome but without erythema migrans. Out of 21 clinical samples tested by real-time PCR, only 1 was positive and 13 were negative with agreement with T2MR. An additional 7 samples that were negative by real-time PCR were positive with T2MR. Therefore, T2MR enables a low limit of detection (LoD) for Borrelia spp. in whole blood samples and is able to detect B. burgdorferi in clinical samples. PMID:28566314

Highly Sensitive Multiplex Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA

PubMed Central

Giachetti, C.; Linnen, J. M.; Kolk, D. P.; Dockter, J.; Gillotte-Taylor, K.; Park, M.; Ho-Sing-Loy, M.; McCormick, M. K.; Mimms, L. T.; McDonough, S. H.

2002-01-01

Various nucleic acid assays have been developed and implemented for diagnostics and therapeutic monitoring of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections. The high-throughput, semiautomated assays described here were developed to provide a method suitable for screening plasma specimens for the presence of HIV-1 and HCV RNAs. Three assays were developed: a multiplex HIV-1/HCV assay for simultaneous detection of HIV-1 and HCV, and discriminatory assays for specific detection of HIV-1 and HCV. The assay systems utilize three proprietary technologies: (i) target capture-based sample preparation, (ii) transcription-mediated amplification (TMA), and (iii) hybridization protection assay (HPA). An internal control is incorporated into each reaction to control for every step of the assay and identify random false-negative reactions. The assays demonstrated a sensitivity of at least 100 copies/ml for each target, and they detected with similar sensitivity all major variants of HCV and HIV-1, including HIV-1 group O strains. Assay sensitivity for one virus was not affected by the presence of the other. The specificity of these TMA-driven assays was ≥99.5% in both normal donor specimens and plasma containing potentially interfering substances or other blood-borne pathogens. Statistical receiver operating characteristic plots of 1 − specificity versus sensitivity data determined very wide analyte cutoff values for each assay at the point at which the assay specificity and sensitivity were both ≥99.5%. The sensitivity, specificity, and throughput capability predict that these assays will be valuable for large-volume plasma screening, either in a blood bank setting or in other diagnostic applications. PMID:12089255

Rapid detection of Prunus necrotic ringspot virus using magnetic nanoparticle-assisted reverse transcription loop-mediated isothermal amplification.

PubMed

Zong, Xiaojuan; Wang, Wenwen; Wei, Hairong; Wang, Jiawei; Chen, Xin; Xu, Li; Zhu, Dongzi; Tan, Yue; Liu, Qingzhong

2014-11-01

Prunus necrotic ringspot virus (PNRSV) has seriously reduced the yield of Prunus species worldwide. In this study, a highly efficient and specific two-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect PNRSV. Total RNA was extracted from sweet cherry leaf samples using a commercial kit based on a magnetic nanoparticle technique. Transcripts were used as the templates for the assay. The results of this assay can be detected using agarose gel electrophoresis or by assessing in-tube fluorescence after adding SYBR Green I. The assay is highly specific for PNRSV, and it is more sensitive than reverse-transcription polymerase chain reaction (RT-PCR). Restriction enzyme digestion verified further the reliability of this RT-LAMP assay. To our knowledge, this is the first report of the application of RT-LAMP to PNRSV detection in Prunus species. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) by real-time, isothermal recombinase polymerase amplification assay.

PubMed

Xia, Xiaoming; Yu, Yongxin; Hu, Linghao; Weidmann, Manfred; Pan, Yingjie; Yan, Shuling; Wang, Yongjie

2015-04-01

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) causes mortality or runt deformity syndrome in penaeid shrimps and is responsible for significant economic losses in the shrimp aquaculture industry. Here, we describe a novel real-time isothermal recombinase polymerase amplification (RPA) assay developed for IHHNV detection. Using IHHNV plasmid standards and DNA samples from a variety of organisms, we evaluated the ability of the IHHNV-RPA assay to detect IHHNV based on analysis of its sensitivity, specificity, rapidity, and reproducibility. Probit analysis of eight independent experimental replicates indicated satisfactory performance of the RPA assay, which is sufficiently sensitive to detect as few as 4 copies of the IHHNV genome within 7 min at 39 °C with 95 % reliability. Therefore, this rapid RPA method has great potential for applications, either in field use or as a point of care diagnostic technique.

Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?

PubMed

Morton, C Oliver; White, P Lewis; Barnes, Rosemary A; Klingspor, Lena; Cuenca-Estrella, Manuel; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem; Mengoli, Carlo; Caliendo, Angela M; Cogliati, Massimo; Debets-Ossenkopp, Yvette; Gorton, Rebecca; Hagen, Ferry; Halliday, Catriona; Hamal, Petr; Harvey-Wood, Kathleen; Jaton, Katia; Johnson, Gemma; Kidd, Sarah; Lengerova, Martina; Lass-Florl, Cornelia; Linton, Chris; Millon, Laurence; Morrissey, C Orla; Paholcsek, Melinda; Talento, Alida Fe; Ruhnke, Markus; Willinger, Birgit; Donnelly, J Peter; Loeffler, Juergen

2017-06-01

A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Development of an updated PCR assay for detection of African swine fever virus.

PubMed

Luo, Yuzi; Atim, Stella A; Shao, Lina; Ayebazibwe, Chrisostom; Sun, Yuan; Liu, Yan; Ji, Shengwei; Meng, Xing-Yu; Li, Su; Li, Yongfeng; Masembe, Charles; Ståhl, Karl; Widén, Frederik; Liu, Lihong; Qiu, Hua-Ji

2017-01-01

Due to the current unavailability of vaccines or treatments for African swine fever (ASF), which is caused by African swine fever virus (ASFV), rapid and reliable detection of the virus is essential for timely implementation of emergency control measures and differentiation of ASF from other swine diseases with similar clinical presentations. Here, an improved PCR assay was developed and evaluated for sensitive and universal detection of ASFV. Primers specific for ASFV were designed based on the highly conserved region of the vp72 gene sequences of all ASFV strains available in GenBank, and the PCR assay was established and compared with two OIE-validated PCR tests. The analytic detection limit of the PCR assay was 60 DNA copies per reaction. No amplification signal was observed for several other porcine viruses. The novel PCR assay was more sensitive than two OIE-validated PCR assays when testing 14 strains of ASFV representing four genotypes (I, V, VIII and IX) from diverse geographical areas. A total of 62 clinical swine blood samples collected from Uganda were examined by the novel PCR, giving a high agreement (59/62) with a superior sensitive universal probe library-based real-time PCR. Eight out of 62 samples tested positive, and three samples with higher Ct values (39.15, 38.39 and 37.41) in the real-time PCR were negative for ASFV in the novel PCR. In contrast, one (with a Ct value of 29.75 by the real-time PCR) and two (with Ct values of 29.75 and 33.12) ASFV-positive samples were not identified by the two OIE-validated PCR assays, respectively. Taken together, these data show that the novel PCR assay is specific, sensitive, and applicable for molecular diagnosis and surveillance of ASF.

Comparison of the genexpert enterovirus assay (GXEA) with real-time one step RT-PCR for the detection of enteroviral RNA in the cerebrospinal fluid of patients with meningitis.

PubMed

Hong, JiYoung; Kim, Ahyoun; Hwang, Seoyeon; Cheon, Doo-Sung; Kim, Jong-Hyen; Lee, June-Woo; Park, Jae-Hak; Kang, Byunghak

2015-02-13

Enteroviruses (EVs) are the leading cause of aseptic meningitis worldwide. Detection of enteroviral RNA in clinical specimens has been demonstrated to improve the management of patient care, especially that of neonates and young children. To establish a sensitive and reliable assay for routine laboratory diagnosis, we compared the sensitivity and specificity of the GeneXpert Enterovirus Assay (GXEA) with that of the reverse transcription polymerase chain reaction (RT-PCR) based assay referred to as real-time one step RT-PCR (RTo-PCR). The sensitivity/specificity produced by GXEA and RTo-PCR were 100%/100% and 65%/100%, respectively. Both methods evaluated in this article can be used for detection of enterovirus in clinical specimens and these nucleic acid amplification methods are useful assays for the diagnosis of enteroviral infection.

BRAF mutation testing in solid tumors: a methodological comparison.

PubMed

Weyant, Grace W; Wisotzkey, Jeffrey D; Benko, Floyd A; Donaldson, Keri J

2014-09-01

Solid tumor genotyping has become standard of care for the characterization of proto-oncogene mutational status, which has traditionally been accomplished with Sanger sequencing. However, companion diagnostic assays and comparable laboratory-developed tests are becoming increasingly popular, such as the cobas 4800 BRAF V600 Mutation Test and the INFINITI KRAS-BRAF assay, respectively. This study evaluates and validates the analytical performance of the INFINITI KRAS-BRAF assay and compares concordance of BRAF status with two reference assays, the cobas test and Sanger sequencing. DNA extraction from FFPE tissue specimens was performed followed by multiplex PCR amplification and fluorescent label incorporation using allele-specific primer extension. Hybridization to a microarray, signal detection, and analysis were then performed. The limits of detection were determined by testing dilutions of mutant BRAF alleles within wild-type background DNA, and accuracy was calculated based on these results. The INFINITI KRAS-BRAF assay produced 100% concordance with the cobas test and Sanger sequencing and had sensitivity equivalent to the cobas assay. The INFINITI assay is repeatable with at least 95% accuracy in the detection of mutant and wild-type BRAF alleles. These results confirm that the INFINITI KRAS-BRAF assay is comparable to traditional sequencing and the Food and Drug Administration-approved companion diagnostic assay for the detection of BRAF mutations. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

A Review of Conventional PCR Assays for the Detection of Selected Phytopathogens of Wheat.

PubMed

Kuzdraliński, Adam; Kot, Anna; Szczerba, Hubert; Nowak, Michał; Muszyńska, Marta

2017-01-01

Infection of phyllosphere (stems, leaves, husks, and grains) by pathogenic fungi reduces the wheat yield and grain quality. Detection of the main wheat pathogenic fungi provides information about species composition and allows effective and targeted plant treatment. Since conventional procedures for the detection of these organisms are unreliable and time consuming, diagnostic DNA-based methods are required. Nucleic acid amplification technologies are independent of the morphological and biochemical characteristics of fungi. Microorganisms do not need to be cultured. Therefore, a number of PCR-based methodologies have been developed for the identification of key pathogenic fungi, such as Fusarium spp., Puccinia spp., Zymoseptoria tritici, Parastagonospora nodorum, Blumeria graminis f. sp. tritici, and Pyrenophora tritici-repentis. This article reviews frequently used DNA regions for fungus identification and discusses already known PCR assays for detection of the aforementioned wheat pathogens. We demonstrate that PCR-based wheat pathogen identification assays require further research. In particular, the number of diagnostic tests for Fusarium graminearum, Puccinia spp., and P. tritici-repentis are insufficient. © 2017 S. Karger AG, Basel.

Development of a F actin-based live-cell fluorimetric microplate assay for diarrhetic shellfish toxins.

PubMed

Leira, F; Alvarez, C; Cabado, A G; Vieites, J M; Vieytes, M R; Botana, L M

2003-06-15

A new cytotoxicity assay for detection and quantitation of diarrhetic shellfish toxins (DSP) is presented. This assay is based upon fluorimetric determination of F-actin depolymerization induced by okadaic acid (OA)-class compounds in the BE(2)-M17 neuroblastoma cell line. No interferences were observed with other marine toxins such as saxitoxin, domoic acid, or yessotoxin, thus indicating a good specificity of the assay as expected by the direct relationship between protein phosphatase inhibition and cytoskeletal changes. The proposed method is rapid (<2h) and shows a linear response in the range of 50-300 nM OA. The detection limit of the assay for crude methanolic extracts of bivalves lies between 0.2 and 1.0 microg OA per gram of digestive glands, depending on the type of samples (fresh or canned), thus being similar to that of the mouse bioassay. The performance of this assay has been evaluated by comparative analysis of 32 toxic mussel samples by the F-actin assay, mouse bioassay, HPLC and PP2A inhibition assay. Results obtained by the F-actin method showed no differences with HPLC and significant correlation with PP2A inhibition assay (r(2)=0.71). No false negative results were obtained with this new cell assay, which also showed optimum reproducibility.

Evaluation of a new T2 Magnetic Resonance assay for rapid detection of emergent fungal pathogen Candida auris on clinical skin swab samples.

PubMed

Sexton, D Joseph; Bentz, Meghan L; Welsh, Rory M; Litvintseva, Anastasia P

2018-06-25

Candida auris is a multidrug-resistant pathogenic yeast whose recent emergence is of increasing public-health concern. C. auris can colonize multiple body sites, including patients' skin, and survive for weeks in the healthcare environment, facilitating patient-to-patient transmission and fueling healthcare-associated outbreaks. Rapid and accurate detection of C. auris colonization is essential for timely implementation of infection control measures and prevent transmission. Currently, axilla/groin composite swabs, used to assess colonization status, are processed using a culture-based method that is sensitive and specific but requires 14 days. This delay limits the opportunity to respond and highlights the need for a faster alternative. The culture-independent T2 Magnetic Resonance (T2MR) system is a rapid diagnostic platform shown to detect target pathogens of interest from unprocessed blood samples in <5 hours. In this study, a new C. auris-specific T2 assay was evaluated for screening of the skin surveillance samples. Inclusivity and limit of detection of the T2 C. auris assay were assessed with spiked samples in a representative skin flora background. The T2 C. auris assay recognized isolates from each of the 4 known clades of C. auris and consistently detected cells at 5 CFU/mL. Finally, 89 clinical axilla/groin swab samples were processed with the T2 C. auris assay. The culture-based diagnostic assay was used as a gold standard to determine performance statistics including sensitivity (0.89) and specificity (0.98). Overall, the T2 C. auris assay performed well as a rapid diagnostic and could help expedite the detection of C. auris in patient skin swabs. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Polymerase chain reaction-based detection of myc transduction in feline leukemia virus-infected cats.

PubMed

Sumi, Ryosuke; Miyake, Ariko; Endo, Taiji; Ohsato, Yoshiharu; Ngo, Minh Ha; Nishigaki, Kazuo

2018-04-01

Feline lymphomas are associated with the transduction and activation of cellular proto-oncogenes, such as c-myc, by feline leukemia virus (FeLV). We describe a polymerase chain reaction assay for detection of myc transduction usable in clinical diagnosis. The assay targets c-myc exons 2 and 3, which together result in a FeLV-specific fusion gene following c-myc transduction. When this assay was conducted on FeLV-infected feline tissues submitted for clinical diagnosis of tumors, myc transduction was detected in 14% of T-cell lymphoma/leukemias. This newly established system could become a useful diagnostic tool in veterinary medicine.

Advances in the detection of telomerase activity using isothermal amplification

PubMed Central

Zhang, Xiaojin; Lou, Xiaoding; Xia, Fan

2017-01-01

Telomerase plays a significantly important role in keeping the telomere length of a chromosome. Telomerase overexpresses in nearly all tumor cells, suggesting that telomerase could be not only a promising biomarker but also a potential therapeutic target for cancers. Therefore, numerous efforts focusing on the detection of telomerase activity have been reported from polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assays to PCR-free assays such as isothermal amplification in recent decade. In this review, we highlight the strategies for the detection of telomerase activity using isothermal amplification and discuss some of the challenges in designing future telomerase assays as well. PMID:28638472

Accurate Detection of Streptococcus pyogenes at the Point of Care Using the cobas Liat Strep A Nucleic Acid Test.

PubMed

Wang, Fangnian; Tian, Yu; Chen, Lingjun; Luo, Robert; Sickler, Joanna; Liesenfeld, Oliver; Chen, Shuqi

2017-10-01

The performance of a polymerase chain reaction-based point-of-care assay, the cobas Strep A Nucleic Acid Test for use on the cobas Liat System (cobas Liat Strep A assay), for the detection of group A Streptococcus bacteria was evaluated in primary care settings. Throat swab specimens from 427 patients were tested with the cobas Liat Strep A assay and a rapid antigen detection test (RADT) by existing medical staff at 5 primary care clinics, and results were compared with bacterial culture. The cobas Liat Strep A assay demonstrated equivalent sensitivity (97.7%) and specificity (93.3%) to reference culture with a 15-minute turnaround time. In comparison to RADTs, the cobas Liat Strep A assay showed improved sensitivity (97.7% Liat vs 84.5% RADT). The Clinical Laboratory Improvement Amendments-waived cobas Liat Strep A assay demonstrated the ease of use and improved turnaround time of RADTs along with the sensitivity of culture.

A suitable method to detect potential fraud of bringing Malayan box turtle (Cuora amboinensis) meat into the food chain.

PubMed

Ali, Md Eaqub; Asing; Hamid, Sharifah Bee Abd; Razzak, Md Abdur; Rashid, Nur Raifana Abd; Al Amin, Md; Mustafa, Shuhaimi

2015-01-01

Malayan box turtle (Cuora amboinensis) has been a wildlife-protected vulnerable turtle species in Malaysia since 2005. However, because of its purported usage in traditional medicine, tonic foods and feeds, clandestine black market trade is rampant. Several polymerase chain reaction (PCR) assays for the taxonomic detection and classification of turtle species have been proposed. These assays are based on long-length target amplicons which are assumed to break down under compromised states and, hence, might not be suitable for the forensic tracing and tracking of turtle trafficking. For the first time this paper develops a very short-amplicon-length PCR assay (120 bp) for the detection of Malayan box turtle meat in raw, processed and mixed matrices, and experimental evidence is produced that such an assay is not only more stable and reliable but also more sensitive than those previously published. We checked the assay specificity against 20 different species and no cross-species detection was observed. The possibility of any false-negative detection was eliminated by a universal endogenous control for eukaryotes. The assay detection limit was 0.0001 ng of box turtle DNA from pure meat and 0.01% turtle meat in binary and ternary admixtures and commercial meatballs. Superior target stability and sensitivity under extreme treatments of boiling, autoclaving and microwave cooking suggested that this newly developed assay would be suitable for any forensic and/or archaeological identification of Malayan box turtle species, even in severely degraded specimens. Further, in silico studies indicated that the assay has the potential to be used as a universal probe for the detection of nine Cuora species, all of which are critically endangered.

Colorimetric detection with aptamer-gold nanoparticle conjugates coupled to an android-based color analysis application for use in the field.

PubMed

Smith, Joshua E; Griffin, Daniel K; Leny, Juliann K; Hagen, Joshua A; Chávez, Jorge L; Kelley-Loughnane, Nancy

2014-04-01

The feasibility of using aptamer-gold nanoparticle conjugates (Apt-AuNPs) to design colorimetric assays for in the field detection of small molecules was investigated. An assay to detect cocaine was designed using two clones of a known cocaine-binding aptamer. The assay was based on the AuNPs difference in affinity for single-stranded DNA (non-binding) and double stranded DNA (target bound). In the first assay, a commonly used design was followed, in which the aptamer and target were incubated to allow binding followed by exposure to the AuNPs. Interactions between the non-bound analytes and the AuNPs surface resulted in a number of false positives. The assay was redesigned by incubating the AuNPs and the aptamer prior to target addition to passivate the AuNPs surface. The adsorbed aptamer was able to bind the target while preventing non-specific interactions. The assay was validated with a number of masking and cutting agents and other controlled substances showing minimal false positives. Studies to improve the assay performance in the field were performed, showing that assay activity could be preserved for up to 2 months. To facilitate the assay analysis, an android application for automatic colorimetric characterization was developed. The application was validated by challenging the assay with cocaine standards of different concentrations, and comparing the results to a conventional plate reader, showing outstanding agreement. Finally, the rapid identification of cocaine in mixtures mimicking street samples was demonstrated. This work established that Apt-AuNPs can be used to design robust assays to be used in the field. Copyright © 2013 Elsevier B.V. All rights reserved.

Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes.

PubMed

Bassler, H A; Flood, S J; Livak, K J; Marmaro, J; Knorr, R; Batt, C A

1995-10-01

A PCR-based assay for Listeria monocytogenes that uses the hydrolysis of an internal fluorogenic probe to monitor the amplification of the target has been formatted. The fluorogenic 5' nuclease PCR assay takes advantage of the endogenous 5' --> 3' nuclease activity of Taq DNA polymerase to digest a probe which is labelled with two fluorescent dyes and hybridizes to the amplicon during PCR. When the probe is intact, the two fluorophores interact such that the emission of the reporter dye is quenched. During amplification, the probe is hydrolyzed, relieving the quenching of the reporter and resulting in an increase in its fluorescence intensity. This change in reporter dye fluorescence is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. We have applied the fluorogenic 5' nuclease PCR assay to detect L. monocytogenes, using an 858-bp amplicon of hemolysin (hlyA) as the target. Maximum sensitivity was achieved by evaluating various fluorogenic probes and then optimizing the assay components and cycling parameters. With crude cell lysates, the total assay could be completed in 3 h with a detection limit of approximately 50 CFU. Quantification was linear over a range of 5 x 10(1) to 5 x 10(5) CFU.

Detection limits and cost comparisons of human- and gull-associated conventional and quantitative PCR assays in artificial and environmental waters.

PubMed

Riedel, Timothy E; Zimmer-Faust, Amity G; Thulsiraj, Vanessa; Madi, Tania; Hanley, Kaitlyn T; Ebentier, Darcy L; Byappanahalli, Muruleedhara; Layton, Blythe; Raith, Meredith; Boehm, Alexandria B; Griffith, John F; Holden, Patricia A; Shanks, Orin C; Weisberg, Stephen B; Jay, Jennifer A

2014-04-01

Some molecular methods for tracking fecal pollution in environmental waters have both PCR and quantitative PCR (qPCR) assays available for use. To assist managers in deciding whether to implement newer qPCR techniques in routine monitoring programs, we compared detection limits (LODs) and costs of PCR and qPCR assays with identical targets that are relevant to beach water quality assessment. For human-associated assays targeting Bacteroidales HF183 genetic marker, qPCR LODs were 70 times lower and there was no effect of target matrix (artificial freshwater, environmental creek water, and environmental marine water) on PCR or qPCR LODs. The PCR startup and annual costs were the lowest, while the per reaction cost was 62% lower than the Taqman based qPCR and 180% higher than the SYBR based qPCR. For gull-associated assays, there was no significant difference between PCR and qPCR LODs, target matrix did not effect PCR or qPCR LODs, and PCR startup, annual, and per reaction costs were lower. Upgrading to qPCR involves greater startup and annual costs, but this increase may be justified in the case of the human-associated assays with lower detection limits and reduced cost per sample. Copyright © 2014 Elsevier Ltd. All rights reserved.

Detection limits and cost comparisons of human- and gull-associated conventional and quantitative PCR assays in artificial and environmental waters

USGS Publications Warehouse

Riedel, Timothy E.; Zimmer-Faust, Amity G.; Thulsiraj, Vanessa; Madi, Tania; Hanley, Kaitlyn T.; Ebentier, Darcy L.; Byappanahalli, Muruleedhara N.; Layton, Blythe; Raith, Meredith; Boehm, Alexandria B.; Griffith, John F.; Holden, Patricia A.; Shanks, Orin C.; Weisberg, Stephen B.; Jay, Jennifer A.

2014-01-01

Some molecular methods for tracking fecal pollution in environmental waters have both PCR and quantitative PCR (qPCR) assays available for use. To assist managers in deciding whether to implement newer qPCR techniques in routine monitoring programs, we compared detection limits (LODs) and costs of PCR and qPCR assays with identical targets that are relevant to beach water quality assessment. For human-associated assays targeting Bacteroidales HF183 genetic marker, qPCR LODs were 70 times lower and there was no effect of target matrix (artificial freshwater, environmental creek water, and environmental marine water) on PCR or qPCR LODs. The PCR startup and annual costs were the lowest, while the per reaction cost was 62% lower than the Taqman based qPCR and 180% higher than the SYBR based qPCR. For gull-associated assays, there was no significant difference between PCR and qPCR LODs, target matrix did not effect PCR or qPCR LODs, and PCR startup, annual, and per reaction costs were lower. Upgrading to qPCR involves greater startup and annual costs, but this increase may be justified in the case of the human-associated assays with lower detection limits and reduced cost per sample.

Advanced yellow fever virus genome detection in point-of-care facilities and reference laboratories.

PubMed

Domingo, Cristina; Patel, Pranav; Yillah, Jasmin; Weidmann, Manfred; Méndez, Jairo A; Nakouné, Emmanuel Rivalyn; Niedrig, Matthias

2012-12-01

Reported methods for the detection of the yellow fever viral genome are beset by limitations in sensitivity, specificity, strain detection spectra, and suitability to laboratories with simple infrastructure in areas of endemicity. We describe the development of two different approaches affording sensitive and specific detection of the yellow fever genome: a real-time reverse transcription-quantitative PCR (RT-qPCR) and an isothermal protocol employing the same primer-probe set but based on helicase-dependent amplification technology (RT-tHDA). Both assays were evaluated using yellow fever cell culture supernatants as well as spiked and clinical samples. We demonstrate reliable detection by both assays of different strains of yellow fever virus with improved sensitivity and specificity. The RT-qPCR assay is a powerful tool for reference or diagnostic laboratories with real-time PCR capability, while the isothermal RT-tHDA assay represents a useful alternative to earlier amplification techniques for the molecular diagnosis of yellow fever by field or point-of-care laboratories.

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

PubMed

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

2015-02-01

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

Validation of T47D-KBluc cell assay for detection of estrogen receptor agonists and antagonists

EPA Science Inventory

There is growing concern of exposure to fish, wildlife, and humans to environmental estrogens and their potential impact on reproductive health. Cell-based assays are useful tools to determine the estrogenic activity of chemicals. Confidence in in vitro assay results is strengthe...

Validation of T47D-KBluc cell assay for detection of estrogen receptor agonists and antagonists###

EPA Science Inventory

There is growing concern of exposure to fish, wildlife, and humans to environmental estrogens and their potential impact on reproductive health. Cell-based assays are useful tools to determine the estrogenic activity of chemicals. Confidence in in vitro assay results is strengthe...

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT, PCB DETECTION TECHNOLOGY, HYBRIZYME DELFIA TM ASSAY

EPA Science Inventory

The DELFIA PCB Assay is a solid-phase time-resolved fluoroimmunoassay based on the sequential addition of sample extract and europium-labeled PCB tracer to a monoclonal antibody reagent specific for PCBs. In this assay, the antibody reagent and sample extract are added to a strip...

Simple fluorescence-based detection of protein kinase A activity using a molecular beacon probe.

PubMed

Ma, Changbei; Lv, Xiaoyuan; Wang, Kemin; Jin, Shunxin; Liu, Haisheng; Wu, Kefeng; Zeng, Weimin

2017-11-02

Protein kinase A was detected by quantifying the amount of ATP used after a protein kinase reaction. The ATP assay was performed using the T4 DNA ligase and a molecular beacon (MB). In the presence of ATP, DNA ligase catalyzed the ligation of short DNA. The ligation product then hybridized to MB, resulting in a fluorescence enhancement of the MB. This assay was capable of determining protein kinase A in the range of 12.5∼150 nM, with a detection limit of 1.25 nM. Furthermore, this assay could also be used to investigate the effect of genistein on protein kinase A. It was a universal, non-radioisotopic, and homogeneous method for assaying protein kinase A.

Development of an enzyme-linked immunosorbent assay for detection of cellular and in vivo LRRK2 S935 phosphorylation

PubMed Central

Delbroek, Lore; Van Kolen, Kristof; Steegmans, Liesbeth; da Cunha, Raquel; Mandemakers, Wim; Daneels, Guy; De Bock, Pieter-Jan; Zhang, Jinwei; Gevaert, Kris; De Strooper, Bart; Alessi, Dario R.; Verstreken, Patrik; Moechars, Diederik W.

2014-01-01

After the discovery of kinase activating mutations in leucine-rich repeat kinase 2 (LRRK2) as associated with autosomal dominant forms of Parkinson’s disease, inhibition of the kinase is being extensively explored as a disease modifying strategy. As signaling properties and substrate(s) of LRRK2 are poorly documented, autophosphorylation has been an important readout for the enzyme’s activity. Western blotting using anti-phospho-S910 or S935 LRRK2 antibodies showed effectiveness in demonstrating inhibitory effects of compounds. In this communication we describe two types of enzyme-linked immunosorbent assays (ELISA) to determine LRRK2 protein levels and kinase activity. Both assays take advantage of the sensitivity of the earlier described total and pS935 antibodies for detection (Nichols et al., Biochem. J. 2010) [10]. The first assay is based on anti-GFP-based capturing of overexpressed LRRK2 and is highly suitable to show cellular effects of kinase inhibitors in a 96-well format. In the other platform anti-LRRK2-based capturing allows detection of endogenously expressed LRRK2 in rat tissue with no significant signal in tissue from LRRK2 knockout rats. Furthermore, both assays showed a significant reduction in pS935 levels on cellular and transgenic R1441C/G LRRK2. With the anti-LRRK2 ELISA we were able to detect LRRK2 phosphorylation in human peripheral blood mononuclear cells (PBMC). To conclude, we report two sensitive assays to monitor LRRK2 expression and kinase activity in samples coming from cellular and in vivo experimental settings. Both can show their value in drug screening and biomarker development but will also be useful in the elucidation of LRRK2-mediated signaling pathways. PMID:23313773

Spectral surface plasmon resonance biosensor for detection of staphylococcal enterotoxin B in milk.

PubMed

Homola, Jirí; Dostálek, Jakub; Chen, Shengfu; Rasooly, Avraham; Jiang, Shaoyi; Yee, Sinclair S

2002-05-05

This work evaluates a newly developed wavelength modulation-based SPR biosensor for the detection of staphylococcal enterotoxin B (SEB) in milk. Two modes of operation of the SPR biosensor are described: direct detection of SEB and sandwich assay. In the sandwich assay detection mode, secondary antibodies are bound to the already captured toxin to amplify sensor response. Samples including SEB in buffer and SEB in milk were analyzed in this work. The SPR biosensor has been shown to be capable of directly detecting concentrations of SEB in buffer as low as 5 ng/ml. In sandwich detection mode, the lowest detection limit was determined to be 0.5 ng/ml for both buffer and milk samples. The reported wavelength modulation-based SPR sensor provides a generic platform which can be tailored for detection of various foodborne pathogens and agents for food analysis and testing.

One-step multiplex RT-qPCR detects three citrus viroids from different genera in a wide range of hosts.

PubMed

Osman, Fatima; Dang, Tyler; Bodaghi, Sohrab; Vidalakis, Georgios

2017-07-01

A one-step multiplex reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) based on species-specific minor groove binding (MGB) probes, was developed for the simultaneous detection, identification, and quantification of three citrus viroids belonging to different genera. Citrus exocortis viroid (Pospiviroid), Hop stunt viroid (Hostuviroid), and Citrus bark cracking viroid (Cocadviroid) cause a variety of maladies in agriculturally significant crops. Therefore, reliable assays for their detection are essential tools for various government and industry organizations implementing disease management programs. Singleplex qPCR primers and MGB probes were designed individually for the detection of the three targeted viroids, and subsequently combined in a one-step multiplex RT-qPCR reaction. A wide host range of woody plants, including citrus, grapevines, apricots, plums and herbaceous plants such as tomato, cucumber, eggplant and chrysanthemum different world regions were used to validate the assay. Single, double and triple viroid infections were identified in the tested samples. The developed multiplex RT-qPCR assay was compared with a previously reported SYBR Green I RT-qPCR for the universal detection of citrus viroids. Both assays accurately identified all citrus viroid infected samples. The multiplex assay complemented the SYBR Green I universal detection assay by differentiating among citrus viroid species in the positive samples. The developed multiplex RT-qPCR assay has the potential to simultaneously detect each targeted viroid and could be used in high throughput screenings for citrus viroids in field surveys, germplasm banks, nurseries and other viroid disease management programs. Copyright © 2017. Published by Elsevier B.V.

Enzyme-free and isothermal detection of microRNA based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction signal amplification.

PubMed

Oishi, Motoi

2015-05-01

An enzyme-free and isothermal microRNA (miRNA) detection method has been developed based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction (HCR) on magnetic beads (MBs). The click-chemical ligation between an azide-modified probe DNA and a dibenzocyclooctyne-modified probe DNA occurred through the hybridization of target miRNA (miR-141). HCR on MBs was performed by the addition of DNA hairpin monomers (H1 and H2). After magnetic separation and denaturation/rehybridization of HCR products ([H1/H2] n ), the resulting HCR products were analyzed by the fluorescence emitted from an intercalative dye, allowing amplification of the fluorescent signal. The proposed assay had a limit of detection of 0.55 fmol, which was 230-fold more sensitive than that of the HCR on the MBs coupled with a conventional sandwich hybridization assay (without click-chemical ligation) (limit of detection 127 fmol). Additionally, the proposed assay could discriminate between miR-141 and other miR-200 family members. In contrast to quantitative reverse transcription polymerase chain reaction techniques using enzymes and thermal cycling, this is an enzyme-free assay that can be conducted under isothermal conditions and can specifically detect miR-141 in fetal bovine serum.

Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time reverse-transcription polymerase chain reaction and virus isolation

PubMed Central

Goodell, Christa K.; Zhang, Jianqiang; Strait, Erin; Harmon, Karen; Patnayak, Devi; Otterson, Tracy; Culhane, Marie; Christopher-Hennings, Jane; Clement, Travis; Leslie-Steen, Pamela; Hesse, Richard; Anderson, Joe; Skarbek, Kevin; Vincent, Amy; Kitikoon, Pravina; Swenson, Sabrina; Jenkins-Moore, Melinda; McGill, Jodi; Rauh, Rolf; Nelson, William; O’Connell, Catherine; Shah, Rohan; Wang, Chong; Main, Rodger; Zimmerman, Jeffrey J.

2016-01-01

The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 assays based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) and 7 assays based on virus isolation (VI). The OF specimens were inoculated with H1N1 or H3N2 IAV and serially diluted 10-fold (10−1 to 10−8). Eight participating laboratories received 180 randomized OF samples (10 replicates × 8 dilutions × 2 IAV subtypes plus 20 IAV-negative samples) and performed the rRT-PCR and VI procedure(s) of their choice. Analysis of the results with a mixed-effect logistic-regression model identified dilution and assay as variables significant (P < 0.0001) for IAV detection in OF by rRT-PCR or VI. Virus subtype was not significant for IAV detection by either rRT-PCR (P = 0.457) or VI (P = 0.101). For rRT-PCR the cycle threshold (Ct) values increased consistently with dilution but varied widely. Therefore, it was not possible to predict VI success on the basis of Ct values. The success of VI was inversely related to the dilution of the sample; the assay was generally unsuccessful at lower virus concentrations. Successful swine health monitoring and disease surveillance require assays with consistent performance, but significant differences in reproducibility were observed among the assays evaluated. PMID:26733728

A Highly Sensitive Chemiluminometric Assay for Real-Time Detection of Biological Hydrogen Peroxide Formation.

PubMed

Zhu, Hong; Jia, Zhenquan; Trush, Michael A; Li, Y Robert

2016-05-01

Hydrogen peroxide (H 2 O 2 ) is a major reactive oxygen species (ROS) produced by various cellular sources, especially mitochondria. At high levels, H 2 O 2 causes oxidative stress, leading to cell injury, whereas at low concentrations, this ROS acts as an important second messenger to participate in cellular redox signaling. Detection and measurement of the levels or rates of production of cellular H 2 O 2 are instrumental in studying the biological effects of this major ROS. While a number of assays have been developed over the past decades for detecting and/or quantifying biological H 2 O 2 formation, none has been shown to be perfect. Perhaps there is no perfect assay for sensitively and accurately quantifying H 2 O 2 as well as other ROS in cells, wherein numerous potential reactants are present to interfere with the reliable measurement of the specific ROS. In this context, each assay has its own advantages and intrinsic limitations. This article describes a highly sensitive assay for real-time detection of H 2 O 2 formation in cultured cells and isolated mitochondria. This assay is based on the luminol/horseradish peroxidase-dependent chemiluminescence that is inhibitable by catalase. The article discusses the usefulness and shortcomings of this chemiluminometric assay in detecting biological H 2 O 2 formation induced by beta-lapachone redox cycling with both cells and isolated mitochondria.

Evaluation of the BD Max Cdiff assay for the detection of toxigenic Clostridium difficile in human stool specimens.

PubMed

Putsathit, Papanin; Morgan, Justin; Bradford, Damien; Engelhardt, Nelly; Riley, Thomas V

2015-02-01

The Becton Dickinson (BD) PCR-based GeneOhm Cdiff assay has demonstrated a high sensitivity and specificity for detecting Clostridium difficile. Recently, the BD Max platform, using the same principles as BD GeneOhm, has become available in Australia. This study aimed to investigate the sensitivity and specificity of BD Max Cdiff assay for the detection of toxigenic C. difficile in an Australian setting. Between December 2013 and January 2014, 406 stool specimens from 349 patients were analysed with the BD Max Cdiff assay. Direct and enrichment toxigenic culture were performed on bioMérieux ChromID C. difficile agar as a reference method. isolates from specimens with discrepant results were further analysed with an in-house PCR to detect the presence of toxin genes. The overall prevalence of toxigenic C. difficile was 7.2%. Concordance between the BD Max assay and enrichment culture was 98.5%. The sensitivity, specificity, positive predictive value and negative predictive value for the BD Max Cdiff assay were 95.5%, 99.0%, 87.5% and 99.7%, respectively, when compared to direct culture, and 91.7%, 99.0%, 88.0% and 99.4%, respectively, when compared to enrichment culture. The new BD Max Cdiff assay appeared to be an excellent platform for rapid and accurate detection of toxigenic C. difficile.

Development of a panel of seven duplex real-time PCR assays for detecting 13 streptococcal superantigens.

PubMed

Yang, Peng; Peng, Xiaomin; Cui, Shujuan; Shao, Junbin; Zhu, Xuping; Zhang, Daitao; Liang, Huijie; Wang, Quanyi

2013-07-30

Streptococcal superantigens (SAgs) are the major virulence factors of infection in humans for group A Streptococcus (GAS) bacteria. A panel consisting of seven duplex real-time PCR assays was developed to simultaneously detect 13 streptococcal SAgs and one internal control which may be important in the control of GAS-mediated diseases. Primer and probe sequences were selected based on the highly conserved region from an alignment of nucleotide sequences of the 13 streptococcal SAgs. The reaction conditions of the duplex real-time PCR were optimized and the specificity of the duplex assays was evaluated using SAg positive strains. The limit of detection of the duplex assays was determined by using 10-fold serial dilutions of the DNA of 13 streptococcal SAgs and compared to a conventional polymerase chain reaction (PCR) method for evaluating the duplex assays sensitivity. Using the duplex assays, we were able to differentiate between 13 SAgs from Streptococcus strains and other non-Streptococcus bacteria without cross-reaction. On the other hand, the limit of detection of the duplex assays was at least one or two log dilutions lower than that of the conventional PCR. The panel was highly specific (100%) and the limit of detection of these duplex groups was at least ten times lower than that obtained by using a conventional PCR method.

Comparative Assessment of Anti-HLA Antibodies Using Two Commercially Available Luminex-Based Assays.

PubMed

Clerkin, Kevin J; See, Sarah B; Farr, Maryjane A; Restaino, Susan W; Serban, Geo; Latif, Farhana; Li, Lingzhi; Colombo, Paolo C; Vlad, George; Ray, Bryan; Vasilescu, Elena R; Zorn, Emmanuel

2017-11-01

Allospecific anti-HLA antibodies (Abs) are associated with rejection of solid organ grafts. The 2 main kits to detect anti-HLA Ab in patient serum are commercialized by Immucor and One Lambda/ThermoFisher. We sought to compare the performance of both platforms. Background-adjusted mean fluorescence intensity (MFI) values were used from both platforms to compare sera collected from 125 pretransplant and posttransplant heart and lung transplant recipients. Most HLA class I (94.5%) and HLA class II (89%) Abs with moderate to high MFI titer (≥4000) were detected by both assays. A modest correlation was observed between MFI values obtained from the 2 assays for both class I ( r = 0.3, r 2 = 0.09, P < 0.0001) and class II Ab ( r = 0.707, r 2 = 0.5, P < 0.0001). Both assays detected anti-class I and II Ab that the other did not; however, no specific HLA allele was detected preferentially by either of the 2 assays. For a limited number of discrepant sera, dilution resulted in comparable reactivity profiles between the 2 platforms. Immucor and One Lambda/ThermoFisher assays have a similar, albeit nonidentical, ability to detect anti-HLA Ab. Although the correlation between the assays was present, significant variances exist, some of which can be explained by a dilution-sensitive "prozone" effect.

Comparative Assessment of Anti-HLA Antibodies Using Two Commercially Available Luminex-Based Assays

PubMed Central

Clerkin, Kevin J.; See, Sarah B.; Farr, Maryjane A.; Restaino, Susan W.; Serban, Geo; Latif, Farhana; Li, Lingzhi; Colombo, Paolo C.; Vlad, George; Ray, Bryan; Vasilescu, Elena R.; Zorn, Emmanuel

2017-01-01

Background Allospecific anti-HLA antibodies (Abs) are associated with rejection of solid organ grafts. The 2 main kits to detect anti-HLA Ab in patient serum are commercialized by Immucor and One Lambda/ThermoFisher. We sought to compare the performance of both platforms. Methods Background-adjusted mean fluorescence intensity (MFI) values were used from both platforms to compare sera collected from 125 pretransplant and posttransplant heart and lung transplant recipients. Results Most HLA class I (94.5%) and HLA class II (89%) Abs with moderate to high MFI titer (≥4000) were detected by both assays. A modest correlation was observed between MFI values obtained from the 2 assays for both class I (r = 0.3, r2 = 0.09, P < 0.0001) and class II Ab (r = 0.707, r2 = 0.5, P < 0.0001). Both assays detected anti–class I and II Ab that the other did not; however, no specific HLA allele was detected preferentially by either of the 2 assays. For a limited number of discrepant sera, dilution resulted in comparable reactivity profiles between the 2 platforms. Conclusions Immucor and One Lambda/ThermoFisher assays have a similar, albeit nonidentical, ability to detect anti-HLA Ab. Although the correlation between the assays was present, significant variances exist, some of which can be explained by a dilution-sensitive “prozone” effect. PMID:29184907

An eDNA Assay to Monitor a Globally Invasive Fish Species from Flowing Freshwater.

PubMed

Adrian-Kalchhauser, Irene; Burkhardt-Holm, Patricia

2016-01-01

Ponto-Caspian gobies are a flock of five invasive fish species that have colonized freshwaters and brackish waters in Europe and North America. One of them, the round goby Neogobius melanostomus, figures among the 100 worst invaders in Europe. Current methods to detect the presence of Ponto-Caspian gobies involve catching or sighting the fish. These approaches are labor intense and not very sensitive. Consequently, populations are usually detected only when they have reached high densities and when management or containment efforts are futile. To improve monitoring, we developed an assay based on the detection of DNA traces (environmental DNA, or eDNA) of Ponto-Caspian gobies in river water. The assay specifically detects invasive goby DNA and does not react to any native fish species. We apply the assay to environmental samples and demonstrate that parameters such as sampling depth, sampling location, extraction protocol, PCR protocol and PCR inhibition greatly impact detection. We further successfully outline the invasion front of Ponto-Caspian gobies in a large river, the High Rhine in Switzerland, and thus demonstrate the applicability of the assay to lotic environments. The eDNA assay requires less time, equipment, manpower, skills, and financial resources than the conventional monitoring methods such as electrofishing, angling or diving. Samples can be taken by untrained individuals, and the assay can be performed by any molecular biologist on a conventional PCR machine. Therefore, this assay enables environment managers to map invaded areas independently of fishermen's' reports and fish community monitorings.

One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae.

PubMed

Settypalli, Tirumala Bharani Kumar; Lamien, Charles Euloge; Spergser, Joachim; Lelenta, Mamadou; Wade, Abel; Gelaye, Esayas; Loitsch, Angelika; Minoungou, Germaine; Thiaucourt, Francois; Diallo, Adama

2016-01-01

Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%-4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in 17 samples, PPRV in 45, and PM in six samples. In addition, three samples showed a co-infection of PPRV and PM. Overall, the one-step multiplex RT-qPCR assay developed will be a valuable tool for rapid detection of individual and co-infections of the targeted pathogens with high specificity and sensitivity.

Comparison of methods for the detection of coliphages in recreational water at two California, United States beaches.

PubMed

Rodríguez, Roberto A; Love, David C; Stewart, Jill R; Tajuba, Julianne; Knee, Jacqueline; Dickerson, Jerold W; Webster, Laura F; Sobsey, Mark D

2012-04-01

Methods for detection of two fecal indicator viruses, F+ and somatic coliphages, were evaluated for application to recreational marine water. Marine water samples were collected during the summer of 2007 in Southern California, United States from transects along Avalon Beach (n=186 samples) and Doheny Beach (n=101 samples). Coliphage detection methods included EPA method 1601 - two-step enrichment (ENR), EPA method 1602 - single agar layer (SAL), and variations of ENR. Variations included comparison of two incubation times (overnight and 5-h incubation) and two final detection steps (lysis zone assay and a rapid latex agglutination assay). A greater number of samples were positive for somatic and F+ coliphages by ENR than by SAL (p<0.01). The standard ENR with overnight incubation and detection by lysis zone assay was the most sensitive method for the detection of F+ and somatic coliphages from marine water, although the method takes up to three days to obtain results. A rapid 5-h enrichment version of ENR also performed well, with more positive samples than SAL, and could be performed in roughly 24h. Latex agglutination-based detection methods require the least amount of time to perform, although the sensitivity was less than lysis zone-based detection methods. Rapid culture-based enrichment of coliphages in marine water may be possible by further optimizing culture-based methods for saline water conditions to generate higher viral titers than currently available, as well as increasing the sensitivity of latex agglutination detection methods. Copyright © 2012 Elsevier B.V. All rights reserved.

Difference between beta1-adrenoceptor autoantibodies of human and animal origin-Limitations detecting beta1-adrenoceptor autoantibodies using peptide based ELISA technology.

PubMed

Wenzel, Katrin; Schulze-Rothe, Sarah; Müller, Johannes; Wallukat, Gerd; Haberland, Annekathrin

2018-01-01

Cell-based analytics for the detection of the beta1-adrenoceptor autoantibody (beta1-AAB) are functional, yet difficult to handle, and should be replaced by easily applicable, routine lab methods. Endeavors to develop solid-phase-based assays such as ELISA to exploit epitope moieties for trapping autoantibodies are ongoing. These solid-phase-based assays, however, are often unreliable when used with human patient material, in contrast to animal derived autoantibodies. We therefore tested an immunogen peptide-based ELISA for the detection of beta1-AAB, and compared commercially available goat antibodies against the 2nd extracellular loop of human beta1-adrenoceptor (ADRB1-AB) to autoantibodies enriched from patient material. The functionality of these autoantibodies was tested in a cell based assay for comparison and their structural appearance was investigated using 2D gel electrophoresis. The ELISA showed a limit of detection for ADRB1-AB of about 1.5 nmol antibody/L when spiked in human control serum and only about 25 nmol/L when spiked in species identical (goat) matrix material. When applied to samples of human origin, the ELISA failed to identify the specific beta1-AABs. A low concentration of beta1-AAB, together with structural inconsistency of the patient originated samples as seen from the 2D Gel appearance, might contribute to the failure of the peptide based ELISA technology to detect human beta1-AABs.

Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate.

PubMed

Schmitt, Martin L; Ladwein, Kathrin I; Carlino, Luca; Schulz-Fincke, Johannes; Willmann, Dominica; Metzger, Eric; Schilcher, Pierre; Imhof, Axel; Schüle, Roland; Sippl, Wolfgang; Jung, Manfred

2014-07-01

Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry. © 2014 Society for Laboratory Automation and Screening.

Novel Surface-Enhanced Raman Scattering-based Assays for Ultra-sensitive Detection of Human Pluripotent Stem Cells

PubMed Central

Han, Jingjia; Qian, Ximei; Wu, Qingling; Jha, Rajneesh; Duan, Jinshuai; Yang, Zhou; Maher, Kevin O.; Nie, Shuming; Xu, Chunhui

2017-01-01

Human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, but their derivatives need to be rigorously evaluated for residual stem cells to prevent teratoma formation. Here, we report the development of novel surface-enhanced Raman scattering (SERS)-based assays that can detect trace numbers of undifferentiated hPSCs in mixed cell populations in a highly specific, ultra-sensitive, and time-efficient manner. By targeting stem cell surface markers SSEA-5 and TRA-1-60 individually or simultaneously, these SERS assays were able to identify as few as 1 stem cell in 106 cells, a sensitivity (0.0001%) which was ~2,000 to 15,000-fold higher than that of flow cytometry assays. Using the SERS assay, we demonstrate that the aggregation of hPSC-based cardiomyocyte differentiation cultures into 3D spheres significantly reduced SSEA-5+ and TRA-1-60+ cells compared with parallel 2D cultures. Thus, SERS may provide a powerful new technology for quality control of hPSC-derived products for preclinical and clinical applications. PMID:27509304

An Ultrasensitive Gold Nanoparticle-based Lateral Flow Test for the Detection of Active Botulinum Neurotoxin Type A

NASA Astrophysics Data System (ADS)

Liu, Jing; Gao, Shan; Kang, Lin; Ji, Bin; Xin, Wenwen; Kang, Jingjing; Li, Ping; Gao, Jie; Wang, Hanbin; Wang, Jinglin; Yang, Hao

2017-03-01

Botulism is a severe and potentially lethal paralytic disease caused by several botulinum neurotoxin-producing Clostridia spp. In China, the majority of the cases caused by botulism were from less-developed rural areas. Here, we designed specific substrate peptides and reconfigured gold nanoparticle-based lateral flow test strip (LFTS) to develop an endopeptidase-based lateral flow assay for the diagnosis of botulism. We performed this lateral flow assay on botulinum neurotoxin-spiked human serum samples. The as-prepared LFTS had excellent performance in the detection of botulinum neurotoxin using only 1 μL of simulated serum, and its sensitivity and specificity were comparable to that of mouse lethality assay. Moreover, the assay takes only half a day and does not require highly trained laboratory staff, specialized facility, or equipment. Finally, our LFTS can be potentially extended to other serotypes of BoNTs by designing specific substrate peptides against the different types of BoNTs. Overall, we demonstrate a strategy by which LFTS and endopeptidase activity assays can be integrated to achieve facile and economic diagnosis of botulism in resource-limited settings.

Sources of Blood Meals of Sylvatic Triatoma guasayana near Zurima, Bolivia, Assayed with qPCR and 12S Cloning

PubMed Central

Lucero, David E.; Ribera, Wilma; Pizarro, Juan Carlos; Plaza, Carlos; Gordon, Levi W.; Peña, Reynaldo; Morrissey, Leslie A.; Rizzo, Donna M.; Stevens, Lori

2014-01-01

Background In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia. Methodology/Principal Findings We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors). Conclusions/Significance We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors. PMID:25474154

Measuring oxidative damage to DNA and its repair with the comet assay.

PubMed

Collins, Andrew R

2014-02-01

Single cell gel electrophoresis, or the comet assay, was devised as a sensitive method for detecting DNA strand breaks, at the level of individual cells. A simple modification, incorporating a digestion of DNA with a lesion-specific endonuclease, makes it possible to measure oxidised bases. With the inclusion of formamidopyrimidine DNA glycosylase to recognise oxidised purines, or Nth (endonuclease III) to detect oxidised pyrimidines, the comet assay has been used extensively in human biomonitoring to monitor oxidative stress, usually in peripheral blood mononuclear cells. There is evidence to suggest that the enzymic approach is more accurate than chromatographic methods, when applied to low background levels of base oxidation. However, there are potential problems of over-estimation (because the enzymes are not completely specific) or under-estimation (failure to detect lesions that are close together). Attempts have been made to improve the inter-laboratory reproducibility of the comet assay. In addition to measuring DNA damage, the assay can be used to monitor the cellular or in vitro repair of strand breaks or oxidised bases. It also has applications in assessing the antioxidant status of cells. In its various forms, the comet assay is now an invaluable tool in human biomonitoring and genotoxicity testing. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. Copyright © 2013 Elsevier B.V. All rights reserved.

Gold nanoparticle-based enzyme-linked antibody-aptamer sandwich assay for detection of Salmonella Typhimurium.

PubMed

Wu, Wenhe; Li, Jun; Pan, Dun; Li, Jiang; Song, Shiping; Rong, Mingge; Li, Zixi; Gao, Jimin; Lu, Jianxin

2014-10-08

Enzyme-linked immunosorbent assay (ELISA) provides a convenient means for the detection of Salmonella enterica serovar Typhimurium (STM), which is important for rapid diagnosis of foodborne pathogens. However, conventional ELISA is limited by antibody-antigen immunoreactions and suffers from poor sensitivity and tedious sample pretreatment. Therefore, development of novel ELISA remains challenging. Herein, we designed a comprehensive strategy for rapid, sensitive, and quantitative detection of STM with high specificity by gold nanoparticle-based enzyme-linked antibody-aptamer sandwich (nano-ELAAS) method. STM was captured and preconcentrated from samples with aptamer-modified magnetic particles, followed by binding with detector antibodies. Then nanoprobes carrying a large amount of reporter antibodies and horseradish peroxidase molecules were used for colorimetric signal amplification. Under the optimized reaction conditions, the nano-ELAAS assay had a quantitative detection range from 1 × 10(3) to 1 × 10(8) CFU mL(-1), a limit of detection of 1 × 10(3) CFU mL(-1), and a selectivity of >10-fold for STM in samples containing other bacteria at higher concentration with an assay time less than 3 h. In addition, the developed nanoprobes were improved in terms of detection range and/or sensitivity when compared with two commercial enzyme-labeled antibody signal reporters. Finally, the nano-ELAAS method was demonstrated to work well in milk samples, a common source of STM contamination.

Rapid, simple and direct detection of Meloidogyne hapla from infected root galls using loop-mediated isothermal amplification combined with FTA technology

PubMed Central

Peng, Huan; Long, Haibo; Huang, Wenkun; Liu, Jing; Cui, Jiangkuan; Kong, Lingan; Hu, Xianqi; Gu, Jianfeng; Peng, Deliang

2017-01-01

The northern root-knot nematode (Meloidogyne hapla) is a damaging nematode that has caused serious economic losses worldwide. In the present study, a sensitive, simple and rapid method was developed for detection of M. hapla in infested plant roots by combining a Flinders Technology Associates (FTA) card with loop-mediated isothermal amplification (LAMP). The specific primers of LAMP were designed based on the distinction of internal transcribed spacer (ITS) sequences between M. hapla and other Meloidogyne spp. The LAMP assay can detect nematode genomic DNA at concentrations low to 1/200 000, which is 100 times more sensitive than conventional PCR. The LAMP was able to highly specifically distinguish M. hapla from other closely related nematode species. Furthermore, the advantages of the FTA-LAMP assay to detect M. hapla were demonstrated by assaying infected root galls that were artificially inoculated. In addition, M. hapla was successfully detected from six of forty-two field samples using FTA-LAMP technology. This study was the first to provide a simple diagnostic assay for M. hapla using the LAMP assay combined with FTA technology. In conclusion, the new FTA-LAMP assay has the potential for diagnosing infestation in the field and managing the pathogen M. hapla. PMID:28368036

Rapid, simple and direct detection of Meloidogyne hapla from infected root galls using loop-mediated isothermal amplification combined with FTA technology.

PubMed

Peng, Huan; Long, Haibo; Huang, Wenkun; Liu, Jing; Cui, Jiangkuan; Kong, Lingan; Hu, Xianqi; Gu, Jianfeng; Peng, Deliang

2017-04-03

The northern root-knot nematode (Meloidogyne hapla) is a damaging nematode that has caused serious economic losses worldwide. In the present study, a sensitive, simple and rapid method was developed for detection of M. hapla in infested plant roots by combining a Flinders Technology Associates (FTA) card with loop-mediated isothermal amplification (LAMP). The specific primers of LAMP were designed based on the distinction of internal transcribed spacer (ITS) sequences between M. hapla and other Meloidogyne spp. The LAMP assay can detect nematode genomic DNA at concentrations low to 1/200 000, which is 100 times more sensitive than conventional PCR. The LAMP was able to highly specifically distinguish M. hapla from other closely related nematode species. Furthermore, the advantages of the FTA-LAMP assay to detect M. hapla were demonstrated by assaying infected root galls that were artificially inoculated. In addition, M. hapla was successfully detected from six of forty-two field samples using FTA-LAMP technology. This study was the first to provide a simple diagnostic assay for M. hapla using the LAMP assay combined with FTA technology. In conclusion, the new FTA-LAMP assay has the potential for diagnosing infestation in the field and managing the pathogen M. hapla.

A paper-based resonance energy transfer nucleic acid hybridization assay using upconversion nanoparticles as donors and quantum dots as acceptors.

PubMed

Doughan, Samer; Uddayasankar, Uvaraj; Krull, Ulrich J

2015-06-09

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reduction amination to develop a luminescence resonance energy transfer (LRET)-based nucleic acid hybridization assay. This first account of covalent immobilization of UCNPs on paper for a bioassay reports an optically responsive method that is sensitive, reproducible and robust. The immobilized UCNPs were decorated with oligonucleotide probes to capture HPRT1 housekeeping gene fragments, which in turn brought reporter conjugated quantum dots (QDs) in close proximity to the UCNPs for LRET. This sandwich assay could detect unlabeled oligonucleotide target, and had a limit of detection of 13 fmol and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs, which are excellent LRET acceptors, demonstrated improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was attributed to the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The kinetics of hybridization were determined to be diffusion limited and full signal development occurred within 3 min. Copyright © 2015 Elsevier B.V. All rights reserved.

A simple clot based assay for detection of procoagulant cell-derived microparticles.

PubMed

Patil, Rucha; Ghosh, Kanjaksha; Shetty, Shrimati

2016-05-01

Cell-derived microparticles (MPs) are important biomarkers in many facets of medicine. However, the MP detection methods used till date are costly and time consuming. The main aim of this study was to standardize an in-house clot based screening method for MP detection which would not only be specific and sensitive, but also inexpensive. Four different methods of MP assessment were performed and the results correlated. Using the flow cytometry technique as the gold standard, 25 samples with normal phosphatidylserine (PS) expressing MP levels and 25 samples with elevated levels were selected, which was cross checked by the commercial STA Procoag PPL clotting time (CT) assay. A simple recalcification time and an in-house clot assay were the remaining two tests. The in-house test measures the CT after the addition of calcium chloride to MP rich plasma, following incubation with Russell viper venom and phospholipid free plasma. The CT obtained by the in-house assay significantly correlated with the results obtained by flow cytometry (R2=0.87, p<0.01). Though preliminary, the in-house assay seems to be efficient, inexpensive and promising. It could definitely be utilized routinely for procoagulant MP assessment in various clinical settings.

A simple, one-tube assay for the simultaneous detection and diagnosis of ten Australian poultry Eimeria.

PubMed

Godwin, Rosamond M; Morgan, Jess A T

2014-02-01

Coccidiosis is a costly worldwide enteric disease of chickens caused by parasites of the genus Eimeria. At present, there are seven described species that occur globally and a further three undescribed, operational taxonomic units (OTUs X, Y, and Z) that are known to infect chickens from Australia. Species of Eimeria have both overlapping morphology and pathology and frequently occur as mixed-species infections. This makes definitive diagnosis with currently available tests difficult and, to date, there is no test for the detection of the three OTUs. This paper describes the development of a PCR-based assay that is capable of detecting all ten species of Eimeria, including OTUs X, Y, and Z in field samples. The assay is based on a single set of generic primers that amplifies a single diagnostic fragment from the mitochondrial genome of each species. This one-tube assay is simple, low-cost, and has the capacity to be high throughput. It will therefore be of great benefit to the poultry industry for Eimeria detection and control, and the confirmation of identity and purity of vaccine strains. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Standardization of a cytometric p24-capture bead-assay for the detection of main HIV-1 subtypes.

PubMed

Merbah, Mélanie; Onkar, Sayali; Grivel, Jean-Charles; Vanpouille, Christophe; Biancotto, Angélique; Bonar, Lydia; Sanders-Buell, Eric; Kijak, Gustavo; Michael, Nelson; Robb, Merlin; Kim, Jerome H; Tovanabutra, Sodsai; Chenine, Agnès-Laurence

2016-04-01

The prevailing method to assess HIV-1 replication and infectivity is to measure the production of p24 Gag protein by enzyme-linked immunosorbent assay (ELISA). Since fluorescent bead-based technologies offer a broader dynamic range and higher sensitivity, this study describes a p24 capture Luminex assay capable of detecting HIV-1 subtypes A-D, circulating recombinant forms (CRF) CRF01_AE and CRF02_AG, which together are responsible for over 90% of HIV-1 infections worldwide. The success of the assay lies in the identification and selection of a cross-reactive capture antibody (clone 183-H12-5C). Fifty-six isolates that belonged to six HIV-1 subtypes and CRFs were successfully detected with p-values below 0.021; limits of detection ranging from 3.7 to 3 × 104 pg/ml. The intra- and inter-assay variation gave coefficient of variations below 6 and 14%, respectively. The 183-bead Luminex assay also displayed higher sensitivity of 91% and 98% compared to commercial p24 ELISA and a previously described Luminex assay. The p24 concentrations measured by the 183-bead Luminex assay showed a significant correlation (R=0.92, p<0.0001) with the data obtained from quantitative real time PCR. This newly developed p24 assay leverages the advantages of the Luminex platform, which include smaller sample volume and simultaneous detection of up to 500 analytes in a single sample, and delivers a valuable tool for the field. Copyright © 2016 Elsevier B.V. All rights reserved.

Multiplex Droplet Digital PCR Quantification of Recurrent Somatic Mutations in Diffuse Large B-Cell and Follicular Lymphoma.

PubMed

Alcaide, Miguel; Yu, Stephen; Bushell, Kevin; Fornika, Daniel; Nielsen, Julie S; Nelson, Brad H; Mann, Koren K; Assouline, Sarit; Johnson, Nathalie A; Morin, Ryan D

2016-09-01

A plethora of options to detect mutations in tumor-derived DNA currently exist but each suffers limitations in analytical sensitivity, cost, or scalability. Droplet digital PCR (ddPCR) is an appealing technology for detecting the presence of specific mutations based on a priori knowledge and can be applied to tumor biopsies, including formalin-fixed paraffin embedded (FFPE) tissues. More recently, ddPCR has gained popularity in its utility in quantifying circulating tumor DNA. We have developed a suite of novel ddPCR assays for detecting recurrent mutations that are prevalent in common B-cell non-Hodgkin lymphomas (NHLs), including diffuse large B-cell lymphoma, follicular lymphoma, and lymphoplasmacytic lymphoma. These assays allowed the differentiation and counting of mutant and wild-type molecules using one single hydrolysis probe. We also implemented multiplexing that allowed the simultaneous detection of distinct mutations and an "inverted" ddPCR assay design, based on employing probes matching wild-type alleles, capable of detecting the presence of multiple single nucleotide polymorphisms. The assays successfully detected and quantified somatic mutations commonly affecting enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) (Y641) and signal transducer and activator of transcription 6 (STAT6) (D419) hotspots in fresh tumor, FFPE, and liquid biopsies. The "inverted" ddPCR approach effectively reported any single nucleotide variant affecting either of these 2 hotspots as well. Finally, we could effectively multiplex hydrolysis probes targeting 2 additional lymphoma-related hotspots: myeloid differentiation primary response 88 (MYD88; L265P) and cyclin D3 (CCND3; I290R). Our suite of ddPCR assays provides sufficient analytical sensitivity and specificity for either the invasive or noninvasive detection of multiple recurrent somatic mutations in B-cell NHLs. © 2016 American Association for Clinical Chemistry.

Sequence-based methods for detecting and evaluating the human gut mycobiome.

PubMed

Suhr, M J; Banjara, N; Hallen-Adams, H E

2016-03-01

We surveyed the fungal microbiota in 16 faecal samples from healthy humans with a vegetarian diet. Fungi were identified using molecular cloning, 454 pyrosequencing and a Luminex analyte-specific reagent (ASR) assay, all targeting the ITS region of the rRNA genes. Fungi were detected in each faecal sample and at least 46 distinct fungal operational taxonomic units (OTUs) were detected, from two phyla - Ascomycota and Basidiomycota. Fusarium was the most abundant genus, followed by Malassezia, Penicillium, Aspergillus and Candida. Commonly detected fungi such as Aspergillus and Penicillium, as well as known dietary fungi Agaricus bisporus and Ophiocordyceps sinensis, are presumed to be transient, allochthonous members due to their abundance in the environment or dietary associations. No single method identified the full diversity of fungi in all samples; pyrosequencing detected more distinct OTUs than the other methods, but failed to detect OTUs in some samples that were detected by cloning and/or ASR assays. ASRs were limited by the commercially available assays, but the potential to design new, optimized assays, coupled with speed and cost, makes the ASR method worthy of further study. Fungi play a role in human gut ecology and health. The field lags immensely behind bacterial gut microbiota research, and studies continue to identify new fungi in faecal samples from healthy humans. However, many of these 'new' species are incapable of growth in the human GI tract, let alone making a meaningful contribution to the gut microbial community. Fungi actually inhabiting and impacting the gut likely constitute a small set of species, and an optimized, targeted, probe-based assay may prove to be the most sensible way of quantifying their abundances. © 2015 The Society for Applied Microbiology.

Is Endotoxemia in Stable Hemodialysis Patients an Artefact? Limitations of the Limulus Amebocyte Lysate Assay and Role of (1→3)-β-D Glucan

PubMed Central

Zhang, Yonglong; Patidar, Ashish; Vilar, Enric; Finkelman, Malcolm; Farrington, Ken

2016-01-01

Background Elevated blood endotoxin levels are frequently reported in the dialysis population and are strongly linked with inflammation, a major predictor of mortality. Virtually all studies have employed the Limulus Amoebocyte Lysate (LAL) assay to detect endotoxin. However this assay is not endotoxin-specific and can be activated by (1→3)-β-glucan (BG), a component of fungal cell walls leading to false positive signals. Very few studies have taken account of this. We examined the influence of BG-based activation of the LAL assay on the detection of endotoxemia in this setting. Method We measured plasma endotoxin levels in 50 hemodialysis patients with and without the use of BG-blocking buffers. These buffers inhibit BG activation of the LAL assay to ensure that any signal detected is endotoxin-specific. Blood samples were measured for BG, interleukin-6 (IL-6), tumor necrosis factor-alfa (TNF-α) to examine the association between endotoxin signals, BG and inflammation. Results Endotoxin signals were detected in 50% of patients. On repeat measurement with a BG-blocking buffer, all detected endotoxin signals were extinguished. No patient had detectable endotoxemia. Plasma BG levels were significantly elevated in 58% of patients and were higher in those with detectable endotoxin signals using the LAL assay without BG-blocking buffers (78vs.54pg/mL;p<0.001). Endotoxin signal and BG levels did not correlate with levels of TNF-α or IL-6. Conclusion Use of the LAL assay for blood endotoxin detection in dialysis patients has its limitations due to high blood BG. Endotoxemia frequently reported in non-infected hemodialysis patients may be artefactual due to BG interference. PMID:27764208

Periplasmic binding protein-based detection of maltose using liposomes: a new class of biorecognition elements in competitive assays.

PubMed

Edwards, Katie A; Baeumner, Antje J

2013-03-05

A periplasmic binding protein (PBP) was investigated as a novel binding species in a similar manner to an antibody in a competitive enzyme linked immunosorbent assay (ELISA), resulting in a highly sensitive and specific assay utilizing liposome-based signal amplification. PBPs are located at high concentrations (10(-4) M) between the inner and outer membranes of gram negative bacteria and are involved in the uptake of solutes and chemotaxis of bacteria toward nutrient sources. Previous sensors relying on PBPs took advantage of the change in local environment or proximity of site-specific fluorophore labels resulting from the significant conformational shift of these proteins' two globular domains upon target binding. Here, rather than monitoring conformational shifts, we have instead utilized the maltose binding protein (MBP) in lieu of an antibody in an ELISA. To our knowledge, this is the first PBP-based sensor without the requirement for engineering site-specific modifications within the protein. MBP conjugated fluorescent dye-encapsulating liposomes served to provide recognition and signal amplification in a competitive assay for maltose using amylose magnetic beads in a microtiter plate-based format. The development of appropriate binding buffers and competitive surfaces are described, with general observations expected to extend to PBPs for other analytes. The resulting assay was specific for d-(+)-maltose versus other sugar analogs including d-(+)-raffinose, sucrose, d-trehalose, d-(+)-xylose, d-fructose, 1-thio-β-d-glucose sodium salt, d-(+)-galactose, sorbitol, glycerol, and dextrose. Cross-reactivity with d-lactose and d-(+)-glucose occurred only at concentrations >10(4)-fold greater than d-(+)-maltose. The limit of detection was 78 nM with a dynamic range covering over 3 orders of magnitude. Accurate detection of maltose as an active ingredient in a pharmaceutical preparation was demonstrated. This method offers a significant improvement over existing enzymatic detection approaches that cannot discriminate between maltose and glucose and over existing fluorescence resonance energy transfer (FRET)-based detection methods that are sensitivity limited. In addition, it opens up a new strategy for the development of biosensors to difficult analytes refractory to immunological detection.

Bead-based competitive fluorescence immunoassay for sensitive and rapid diagnosis of cyanotoxin risk in drinking water.

PubMed

Yu, Hye-Weon; Jang, Am; Kim, Lan Hee; Kim, Sung-Jo; Kim, In S

2011-09-15

Due to the increased occurrence of cyanobacterial blooms and their toxins in drinking water sources, effective management based on a sensitive and rapid analytical method is in high demand for security of safe water sources and environmental human health. Here, a competitive fluorescence immunoassay of microcystin-LR (MCYST-LR) is developed in an attempt to improve the sensitivity, analysis time, and ease-of-manipulation of analysis. To serve this aim, a bead-based suspension assay was introduced based on two major sensing elements: an antibody-conjugated quantum dot (QD) detection probe and an antigen-immobilized magnetic bead (MB) competitor. The assay was composed of three steps: the competitive immunological reaction of QD detection probes against analytes and MB competitors, magnetic separation and washing, and the optical signal generation of QDs. The fluorescence intensity was found to be inversely proportional to the MCYST-LR concentration. Under optimized conditions, the proposed assay performed well for the identification and quantitative analysis of MCYST-LR (within 30 min in the range of 0.42-25 μg/L, with a limit of detection of 0.03 μg/L). It is thus expected that this enhanced assay can contribute both to the sensitive and rapid diagnosis of cyanotoxin risk in drinking water and effective management procedures.

Use of culture- and ELISA-based toxin assay for detecting Clostridium Difficile, a neglected pathogen: A single-center study from a tertiary care setting.

PubMed

Lall, Sujata; Nataraj, Gita; Mehta, Preeti

2017-01-01

Clostridium difficile is a Gram-positive spore-bearing anaerobic bacillus increasingly associated with both community- and hospital-acquired colitis and diarrhea. It is the most common identifiable bacterial cause of healthcare-associated diarrhea associated with antibiotic use and one of the most common anaerobic infections. The diagnosis of C. difficile infection includes detection of toxin A/B in stool specimens by direct enzyme immunoassay, culture of pathogen from the stool specimens using a selective agar Cycloserine-Cefoxitin fructose agar (CCFA), tissue culture assay, and detection of glutamate dehydrogenase an enzyme produced by C. difficile. With few reports from India on this disease, the present study was planned to throw more light on the prevalence and utility of laboratory diagnostic methods for C. difficile-associated diarrhea (CDAD). After taking approval from the Ethics Committee, 150 patients with antibiotic-associated diarrhea were taken as a study group and fifty patients with exposure to antibiotics but who did not develop diarrhea were taken as controls. Stool specimen was processed for both culture on CCFA and toxin detection by IVD Tox A + B ELISA. Only four specimens were culture positive, whereas 13 were ELISA positive. All culture-positive isolates were toxigenic. C. difficile was neither isolated nor its toxin detected in the control group. Culture- and toxin-based assays may not detect all cases of CDAD. Based on the results of the present study, culture does not provide any additional yield over toxin assay. Better diagnostic modalities would be required to prove CDAD.

MethylMeter(®): bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples.

PubMed

McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

2016-06-01

Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter(®). Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas.

Accurate inclusion mass screening: a bridge from unbiased discovery to targeted assay development for biomarker verification.

PubMed

Jaffe, Jacob D; Keshishian, Hasmik; Chang, Betty; Addona, Theresa A; Gillette, Michael A; Carr, Steven A

2008-10-01

Verification of candidate biomarker proteins in blood is typically done using multiple reaction monitoring (MRM) of peptides by LC-MS/MS on triple quadrupole MS systems. MRM assay development for each protein requires significant time and cost, much of which is likely to be of little value if the candidate biomarker is below the detection limit in blood or a false positive in the original discovery data. Here we present a new technology, accurate inclusion mass screening (AIMS), designed to provide a bridge from unbiased discovery to MS-based targeted assay development. Masses on the software inclusion list are monitored in each scan on the Orbitrap MS system, and MS/MS spectra for sequence confirmation are acquired only when a peptide from the list is detected with both the correct accurate mass and charge state. The AIMS experiment confirms that a given peptide (and thus the protein from which it is derived) is present in the plasma. Throughput of the method is sufficient to qualify up to a hundred proteins/week. The sensitivity of AIMS is similar to MRM on a triple quadrupole MS system using optimized sample preparation methods (low tens of ng/ml in plasma), and MS/MS data from the AIMS experiments on the Orbitrap can be directly used to configure MRM assays. The method was shown to be at least 4-fold more efficient at detecting peptides of interest than undirected LC-MS/MS experiments using the same instrumentation, and relative quantitation information can be obtained by AIMS in case versus control experiments. Detection by AIMS ensures that a quantitative MRM-based assay can be configured for that protein. The method has the potential to qualify large number of biomarker candidates based on their detection in plasma prior to committing to the time- and resource-intensive steps of establishing a quantitative assay.

Sensitive and quantitative detection of botulinum neurotoxin in neurons derived from mouse embryonic stem cells.

PubMed

Pellett, Sabine; Du, Zhong-wei; Pier, Christina L; Tepp, William H; Zhang, Su-chun; Johnson, Eric A

2011-01-07

Botulinum neurotoxins (BoNTs), the most poisonous protein toxins known, represent a serious bioterrorism threat but are also used as a unique and important bio-pharmaceutical to treat an increasing myriad of neurological disorders. The only currently accepted detection method by the United States Food and Drug Administration for biological activity of BoNTs and for potency determination of pharmaceutical preparations is the mouse bioassay (MBA). Recent advances have indicated that cell-based assays using primary neuronal cells can provide an equally sensitive and robust detection platform as the MBA to reliably and quantitatively detect biologically active BoNTs. This study reports for the first time a BoNT detection assay using mouse embryonic stem cells to produce a neuronal cell culture. The data presented indicate that this assay can reliably detect BoNT/A with a similar sensitivity as the MBA. Published by Elsevier Inc.

A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus.

PubMed

Jothikumar, Narayanan; Cromeans, Theresa L; Robertson, Betty H; Meng, X J; Hill, Vincent R

2006-01-01

Hepatitis E virus (HEV) is transmitted by the fecal-oral route and causes sporadic and epidemic forms of acute hepatitis. Large waterborne HEV epidemics have been documented exclusively in developing countries. At least four major genotypes of HEV have been reported worldwide: genotype 1 (found primarily in Asian countries), genotype 2 (isolated from a single outbreak in Mexico), genotype 3 (identified in swine and humans in the United States and many other countries), and genotype 4 (identified in humans, swine and other animals in Asia). To better detect and quantitate different HEV strains that may be present in clinical and environmental samples, we developed a rapid and sensitive real-time RT-PCR assay for the detection of HEV RNA. Primers and probes for the real-time RT-PCR were selected based on the multiple sequence alignments of 27 sequences of the ORF3 region. Thirteen HEV isolates representing genotypes 1-4 were used to standardize the real-time RT-PCR assay. The TaqMan assay detected as few as four genome equivalent (GE) copies of HEV plasmid DNA and detected as low as 0.12 50% pig infectious dose (PID50) of swine HEV. Different concentrations of swine HEV (120-1.2PID50) spiked into a surface water concentrate were detected in the real-time RT-PCR assay. This is the first reporting of a broadly reactive TaqMan RT-PCR assay for the detection of HEV in clinical and environmental samples.

Etiologic Diagnosis of Lower Respiratory Tract Bacterial Infections Using Sputum Samples and Quantitative Loop-Mediated Isothermal Amplification

PubMed Central

Peng, Peichao; Cheng, Xiaoxing; Wang, Guoqing; Qian, Minping; Gao, Huafang; Han, Bei; Chen, Yusheng; Hu, Yinghui; Geng, Rong; Hu, Chengping; Zhang, Wei; Yang, Jingping; Wan, Huanying; Yu, Qin; Wei, Liping; Li, Jiashu; Tian, Guizhen; Wang, Qiuyue; Hu, Ke; Wang, Siqin; Wang, Ruiqin; Du, Juan; He, Bei; Ma, Jianjun; Zhong, Xiaoning; Mu, Lan; Cai, Shaoxi; Zhu, Xiangdong; Xing, Wanli; Yu, Jun; Deng, Minghua; Gao, Zhancheng

2012-01-01

Etiologic diagnoses of lower respiratory tract infections (LRTI) have been relying primarily on bacterial cultures that often fail to return useful results in time. Although DNA-based assays are more sensitive than bacterial cultures in detecting pathogens, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. Here we report a nationwide cohort study on 2986 suspected LRTI patients across P. R. China. We compared the performance of a DNA-based assay qLAMP (quantitative Loop-mediated isothermal AMPlification) with that of standard bacterial cultures in detecting a panel of eight common respiratory bacterial pathogens from sputum samples. Our qLAMP assay detects the panel of pathogens in 1047(69.28%) patients from 1533 qualified patients at the end. We found that the bacterial titer quantified based on qLAMP is a predictor of probability that the bacterium in the sample can be detected in culture assay. The relatedness of the two assays fits a logistic regression curve. We used a piecewise linear function to define breakpoints where latent pathogen abruptly change its competitive relationship with others in the panel. These breakpoints, where pathogens start to propagate abnormally, are used as cutoffs to eliminate the influence of contaminations from normal flora. With help of the cutoffs derived from statistical analysis, we are able to identify causative pathogens in 750 (48.92%) patients from qualified patients. In conclusion, qLAMP is a reliable method in quantifying bacterial titer. Despite the fact that there are always latent bacteria contaminated in sputum samples, we can identify causative pathogens based on cutoffs derived from statistical analysis of competitive relationship. Trial Registration ClinicalTrials.gov NCT00567827 PMID:22719933

Genotypic and Phenotypic Characterization of Escherichia coli Isolates from Feces, Hands, and Soils in Rural Bangladesh via the Colilert Quanti-Tray System

PubMed Central

Islam, M. Aminul; Pickering, Amy J.; Roy, Subarna; Fuhrmeister, Erica R.; Ercumen, Ayse; Harris, Angela; Bishai, Jason; Schwab, Kellogg J.

2014-01-01

The increased awareness of the role of environmental matrices in enteric disease transmission has resulted in the need for rapid, field-based methods for fecal indicator bacteria and pathogen detection. Evidence of the specificity of β-glucuronidase-based assays for detection of Escherichia coli from environmental matrices relevant to enteric pathogen transmission in developing countries, such as hands, soils, and surfaces, is limited. In this study, we quantify the false-positive rate of a β-glucuronidase-based E. coli detection assay (Colilert) for two environmental reservoirs in Bangladeshi households (hands and soils) and three fecal composite sources (cattle, chicken, and humans). We investigate whether or not the isolation source of E. coli influences phenotypic and genotypic characteristics. Phenotypic characteristics include results of biochemical assays provided by the API-20E test; genotypic characteristics include the Clermont phylogroup and the presence of enteric and/or environmental indicator genes sfmH, rfaI, and fucK. Our findings demonstrate no statistically significant difference in the false-positive rate of Colilert for environmental compared to enteric samples. E. coli isolates from all source types are genetically diverse, representing six of the seven phylogroups, and there is no difference in relative frequency of phylogroups between enteric and environmental samples. We conclude that Colilert, and likely other β-glucuronidase-based assays, is appropriate for detection of E. coli on hands and in soils with low false-positive rates. Furthermore, E. coli isolated from hands and soils in Bangladeshi households are diverse and indistinguishable from cattle, chicken, and human fecal isolates, using traditional biochemical assays and phylogrouping. PMID:25548044

Genotypic and phenotypic characterization of Escherichia coli isolates from feces, hands, and soils in rural Bangladesh via the Colilert Quanti-Tray System.

PubMed

Julian, Timothy R; Islam, M Aminul; Pickering, Amy J; Roy, Subarna; Fuhrmeister, Erica R; Ercumen, Ayse; Harris, Angela; Bishai, Jason; Schwab, Kellogg J

2015-03-01

The increased awareness of the role of environmental matrices in enteric disease transmission has resulted in the need for rapid, field-based methods for fecal indicator bacteria and pathogen detection. Evidence of the specificity of β-glucuronidase-based assays for detection of Escherichia coli from environmental matrices relevant to enteric pathogen transmission in developing countries, such as hands, soils, and surfaces, is limited. In this study, we quantify the false-positive rate of a β-glucuronidase-based E. coli detection assay (Colilert) for two environmental reservoirs in Bangladeshi households (hands and soils) and three fecal composite sources (cattle, chicken, and humans). We investigate whether or not the isolation source of E. coli influences phenotypic and genotypic characteristics. Phenotypic characteristics include results of biochemical assays provided by the API-20E test; genotypic characteristics include the Clermont phylogroup and the presence of enteric and/or environmental indicator genes sfmH, rfaI, and fucK. Our findings demonstrate no statistically significant difference in the false-positive rate of Colilert for environmental compared to enteric samples. E. coli isolates from all source types are genetically diverse, representing six of the seven phylogroups, and there is no difference in relative frequency of phylogroups between enteric and environmental samples. We conclude that Colilert, and likely other β-glucuronidase-based assays, is appropriate for detection of E. coli on hands and in soils with low false-positive rates. Furthermore, E. coli isolated from hands and soils in Bangladeshi households are diverse and indistinguishable from cattle, chicken, and human fecal isolates, using traditional biochemical assays and phylogrouping. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion.

PubMed

Aslanoglou, Despoina; George, Emily W; Freyberg, Zachary

2018-05-10

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved Förster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved Förster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

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

PubMed

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

2018-02-01

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

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

PubMed

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.

Detection of the enzymatically-active polyhydroxyalkanoate synthase subunit gene, phaC, in cyanobacteria via colony PCR.

PubMed

Lane, Courtney E; Benton, Michael G

2015-12-01

A colony PCR-based assay was developed to rapidly determine if a cyanobacterium of interest contains the requisite genetic material, the PHA synthase PhaC subunit, to produce polyhydroxyalkanoates (PHAs). The test is both high throughput and robust, owing to an extensive sequence analysis of cyanobacteria PHA synthases. The assay uses a single detection primer set and a single reaction condition across multiple cyanobacteria strains to produce an easily detectable positive result - amplification via PCR as evidenced by a band in electrophoresis. In order to demonstrate the potential of the presence of phaC as an indicator of a cyanobacteria's PHA accumulation capabilities, the ability to produce PHA was assessed for five cyanobacteria with a traditional in vivo PHA granule staining using an oxazine dye. The confirmed in vivo staining results were then compared to the PCR-based assay results and found to be in agreement. The colony PCR assay was capable of successfully detecting the phaC gene in all six of the diverse cyanobacteria tested which possessed the gene, while exhibiting no undesired product formation across the nine total cyanobacteria strains tested. The colony PCR quick prep provides sufficient usable DNA template such that this assay could be readily expanded to assess multiple genes of interest simultaneously. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed

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. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of a high-throughput detection system for HIV-1 using real-time NASBA based on molecular beacons

NASA Astrophysics Data System (ADS)

van Beuningen, Rinie; Marras, Salvatore A.; Kramer, Fred R.; Oosterlaken, Tom; Weusten, Jos; Borst, G.; van de Wiel, Paul

2001-04-01

HIV-1 viral load assays require accuracy and sensitivity at low RNA levels with the capability to detect all subtypes. Furthermore, the assay should be easy to perform and fast to be useful for routine diagnostics. In order to meet these demands we have combined isothermal NASBA amplification with molecular beacon probes for real-time detection and quantitation of HIV-1 RNA. Quantitation is based on co-amplification of the HIV-1 RNA in the clinical sample and a synthetic calibrator RNA which is amplified by the same primer set but detected with a differently labeled molecular beacon. The entire procedure is simple and analysis of 48 samples requires less than 1» hours with minimal hands-on time. A fluorescent plate reader is used for real-time detection and isothermal amplification. The linearity and precision of the assay was determined with the VQC HIV-1 type B standard of the Central Laboratory of the Dutch Red Cross Blood Banks, The Netherlands. Sensitivity was shown to be 50 copies per ml (cps/ml). The average assay precision was 0,19 log10 over a range of 100-300,000 cps/ml tested at nine concentrations. The linearity of dilution series of 15 cultured HIV-1 gag clades A-H was shown. The specificity was 100% on non HIV-1 samples HIV-2, HTLV-1 and HTLV-2. The assay robustness in terms of valid results was 99%. In conclusion, the new real-time NASBA assay meets state-of-the-art HIV-1 viral load performance requirements combined with a high level of user convenience.

Recombinant protein-based assays for detection of antibodies to severe acute respiratory syndrome coronavirus spike and nucleocapsid proteins.

PubMed

Haynes, Lia M; Miao, Congrong; Harcourt, Jennifer L; Montgomery, Joel M; Le, Mai Quynh; Dryga, Sergey A; Kamrud, Kurt I; Rivers, Bryan; Babcock, Gregory J; Oliver, Jennifer Betts; Comer, James A; Reynolds, Mary; Uyeki, Timothy M; Bausch, Daniel; Ksiazek, Thomas; Thomas, William; Alterson, Harold; Smith, Jonathan; Ambrosino, Donna M; Anderson, Larry J

2007-03-01

Recombinant severe acute respiratory syndrome (SARS) nucleocapsid and spike protein-based immunoglobulin G immunoassays were developed and evaluated. Our assays demonstrated high sensitivity and specificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms. These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological and antibody kinetic studies.

Recombinant Protein-Based Assays for Detection of Antibodies to Severe Acute Respiratory Syndrome Coronavirus Spike and Nucleocapsid Proteins▿

PubMed Central

Haynes, Lia M.; Miao, Congrong; Harcourt, Jennifer L.; Montgomery, Joel M.; Le, Mai Quynh; Dryga, Sergey A.; Kamrud, Kurt I.; Rivers, Bryan; Babcock, Gregory J.; Oliver, Jennifer Betts; Comer, James A.; Reynolds, Mary; Uyeki, Timothy M.; Bausch, Daniel; Ksiazek, Thomas; Thomas, William; Alterson, Harold; Smith, Jonathan; Ambrosino, Donna M.; Anderson, Larry J.

2007-01-01

Recombinant severe acute respiratory syndrome (SARS) nucleocapsid and spike protein-based immunoglobulin G immunoassays were developed and evaluated. Our assays demonstrated high sensitivity and specificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms. These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological and antibody kinetic studies. PMID:17229882

Recommendations for the validation of cell-based assays used for the detection of neutralizing antibody immune responses elicited against biological therapeutics.

PubMed

Gupta, Shalini; Devanarayan, Viswanath; Finco, Deborah; Gunn, George R; Kirshner, Susan; Richards, Susan; Rup, Bonita; Song, An; Subramanyam, Meena

2011-07-15

The administration of biological therapeutics may result in the development of anti-drug antibodies (ADAs) in treated subjects. In some cases, ADA responses may result in the loss of therapeutic efficacy due to the formation of neutralizing ADAs (NAbs). An important characteristic of anti-drug NAbs is their direct inhibitory effect on the pharmacological activity of the therapeutic. Neutralizing antibody responses are of particular concern for biologic products with an endogenous homolog whose activity can be potentially dampened or completely inhibited by the NAbs leading to an autoimmune-type deficiency syndrome. Therefore, it is important that ADAs are detected and characterized appropriately using sensitive and reliable methods. The design, development and optimization of cell-based assays used for detection of NAbs have been published previously by Gupta et al. 2007 [1]. This paper provides recommendations on best practices for the validation of cell-based NAb assay and suggested validation parameters based on the experience of the authors. Copyright © 2011 Elsevier B.V. All rights reserved.

Renilla luciferase-labeled Annexin V: a new probe for detection of apoptotic cells.

PubMed

Nazari, Mahboobeh; Emamzadeh, Rahman; Hosseinkhani, Saman; Cevenini, Luca; Michelini, Elisa; Roda, Aldo

2012-11-07

The Ca(2+)-dependent binding of Annexin V to phosphatidylserine on cell surfaces is a reliable marker for apoptosis that is widely used in flow cytometry based apoptosis assays. In this paper, we report a new class of Annexin V-based probes for apoptosis. Luciferase from Renilla reniformis (RLuc) was linked to Annexin V and expressed successfully in a soluble form in Escherichia coli BL21 (DE3). The new probe, Rluc/Annexin V, was purified and functionally assayed for detection of apoptosis in actinomycin D-induced apoptotic Jurkat cells. Moreover, the spontaneous apoptosis in neutrophils was shown using the new probe. The results indicate that Rluc/Annexin V can bind to the apoptotic cells, and the signal of Renilla luciferase can be detected by luminometric measurements. The availability of Rluc/Annexin V may be of potential commercial interest for improving current apoptosis assays.

Development of a paper-based vertical flow SERS assay for citrulline detection using aptamer-conjugated gold nanoparticles

NASA Astrophysics Data System (ADS)

Locke, Andrea; Deutz, Nicolaas; Coté, Gerard

2018-02-01

Research toward development of point-of-care (POC) technologies is emerging as a means for diagnosis and monitoring of patients outside the hospital. These POC devices typically utilize assays capable of detecting low level biomarkers indicative of specific diseases. L-citrulline, an α-amino acid produced in the intestinal mucosa cells, is one such biomarker typically found circulating within the plasma at physiological concentrations of 40 μM. Researchers have found that intestinal enterocyte malfunction causes its level to be significantly lowered, establishing it as a potential diagnostic biomarker for gut function. Our research group has proposed the development of a surface enhanced Raman spectroscopy (SERS) based assay, using vertical flow paper fluidics, for citrulline detection. The assay consists of a fluorescently active, Raman reporter labeled aptamer conjugated on gold nanoparticles. The aptamer changes its confirmation on binding to its target, which in turn changes the distance between the Raman active molecule and the nanoparticle surface. These particles were embedded within a portable chip consisting of cellulose-based paper. After the chips were loaded with different concentrations of free L-citrulline in phosphate buffer, time was given for the assay to interact with the sample. A handheld Raman spectrometer (638 nm; Ocean Optics) was used to measure the SERS intensity. Results showed decrease in intensity with increasing concentration of L-citrulline (0-50μM).

Development of a SYBR green I based RT-PCR assay for yellow fever virus: application in assessment of YFV infection in Aedes aegypti.

PubMed

Dash, Paban Kumar; Boutonnier, Alain; Prina, Eric; Sharma, Shashi; Reiter, Paul

2012-01-22

Yellow Fever virus (YFV) is an important arboviral pathogen in much of sub-Saharan Africa and the tropical Americas. It is the prototype member of the genus Flavivirus and is transmitted primarily by Aedes (Stegomyia) mosquitoes. The incidence of human infections in endemic areas has risen in recent years. Prompt and dependable identification of YFV is a critical component of response to suspect cases. We developed a one-step SYBR Green I-based real-time quantitative RT-PCR (qRT-PCR) assay targeting the 5'NTR and capsid-gene junction--for rapid detection and quantification of YFV. The detection limit was 1 PFU/mL, 10-fold more sensitive than conventional RT-PCR, and there was no cross-reactivity with closely related flaviviruses or with alphaviruses. Viral load in samples was determined by standard curve plotted from cycle threshold (Ct) values and virus concentration. The efficacy of the assay in mosquitoes was assessed with spiked samples. The utility of the assay for screening of pooled mosquitoes was also confirmed. Replication of a Cameroon isolate of YFV in Ae. aegypti revealed a marked variation in susceptibility among different colonies at different days post infection (pi). The SYBR Green-1 based qRT-PCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of YFV than other currently used methods.

A toxin-free enzyme-linked immunosorbent assay for the analysis of aflatoxins based on a VHH surrogate standard.

PubMed

Wang, Yanru; Li, Peiwu; Zhang, Qi; Hu, Xiaofeng; Zhang, Wen

2016-09-01

A toxin-free enzyme-linked immunosorbent assay (ELISA) for aflatoxins was developed using an anti-idiotype nanobody VHH 2-5 as surrogate standard. Anti-idiotype nanobody VHH 2-5 was generated by immunizing an alpaca with anti-aflatoxin monoclonal antibody 1C11. This assay was used to detect aflatoxins in agro-products after a simple extraction with 75 % methanol/H2O. Aflatoxin concentration was calculated by a two-step approach: the concentration of VHH 2-5 was first obtained by a four-parameter logistic regression from the detected absorbance value at 450 nm, and then converted to aflatoxin concentration by a linear equation. The assay exhibits a limit of detection (LOD) of 0.015 ng mL(-1), which is better than or comparable with conventional immunoassays. The performance of our VHH surrogate-based ELISA was further validated with a high-performance liquid chromatography (HPLC) method for total aflatoxins determination in 20 naturally contaminated peanut samples, displaying a good correlation (R (2) = 0.988). In conclusion, the proposed assay represents a first example applying an anti-idiotype VHH antibody as a standard surrogate in ELISA. With the advantages of high stability and ease of production, the VHH antibody-based standard surrogate can be extended in the future to immunoassays for other highly toxic compounds. Graphical Abstract ᅟ.

A Novel Assay for the Identification of NOTCH1 PEST Domain Mutations in Chronic Lymphocytic Leukemia

PubMed Central

Petroni, Roberta Cardoso; Muto, Nair Hideko; Sitnik, Roberta; de Carvalho, Flavia Pereira; Bacal, Nydia Strachman; Velloso, Elvira Deolinda Rodrigues Pereira; Oliveira, Gislaine Borba; Pinho, João Renato Rebello; Torres, Davi Coe; Mansur, Marcela Braga; Hassan, Rocio; Lorand-Metze, Irene Gyongyvér Heidemarie; Chiattone, Carlos Sérgio; Hamerschlak, Nelson; Mangueira, Cristovão Luis Pitangueira

2016-01-01

Aims. To develop a fast and robust DNA-based assay to detect insertions and deletions mutations in exon 34 that encodes the PEST domain of NOTCH1 in order to evaluate patients with chronic lymphocytic leukemia (CLL). Methods. We designed a multiplexed allele-specific polymerase chain reaction (PCR) combined with a fragment analysis assay to detect specifically the mutation c.7544_7545delCT and possibly other insertions and deletions in exon 34 of NOTCH1. Results. We evaluated our assay in peripheral blood samples from two cohorts of patients with CLL. The frequency of NOTCH1 mutations was 8.4% in the first cohort of 71 unselected CLL patients. We then evaluated a second cohort of 26 CLL patients with known cytogenetic abnormalities that were enriched for patients with trisomy 12. NOTCH1 mutations were detected in 43.7% of the patients with trisomy 12. Conclusions. We have developed a fast and robust assay combining allele-specific PCR and fragment analysis able to detect NOTCH1 PEST domain insertions and deletions. PMID:28074183

Development and validation of duplex, triplex, and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed.

PubMed

Huber, Ingrid; Block, Annette; Sebah, Daniela; Debode, Frédéric; Morisset, Dany; Grohmann, Lutz; Berben, Gilbert; Stebih, Dejan; Milavec, Mojca; Zel, Jana; Busch, Ulrich

2013-10-30

Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis.

Diagnostic tools based on minor groove binder probe technology for rapid identification of vaccinal and field strains of canine parvovirus type 2b.

PubMed

Decaro, Nicola; Martella, Vito; Elia, Gabriella; Desario, Costantina; Campolo, Marco; Buonavoglia, Domenico; Bellacicco, Anna Lucia; Tempesta, Maria; Buonavoglia, Canio

2006-12-01

TaqMan-based diagnostic tests have been developed for the identification of canine parvovirus type 2 (CPV-2) strains in the faeces of dogs with diarrhoea, including a minor groove binder (MGB) probe assay for identification of type 2-based vaccines and field strains (types 2a, 2b and 2c). Since type 2b vaccines have been licensed recently in Europe, two novel MGB assays were developed for discrimination between type 2b vaccines and field strains of CPV. Such assays have been found to be highly sensitive, specific and reproducible, allowing for simultaneous detection of type 2b vaccinal and field strains present in the same specimens. These new assays will help resolution of the diagnostic problems related to the detection of a type 2b strain in the faeces of dogs shortly after the administration of a type 2b vaccine.

Simultaneous detection of antibodies to five Actinobacillus pleuropneumoniae serovars using bead-based multiplex analysis.

PubMed

Berger, Sanne Schou; Lauritsen, Klara Tølbøll; Boas, Ulrik; Lind, Peter; Andresen, Lars Ole

2017-11-01

We developed and made a preliminary validation of a bead-based multiplexed immunoassay for simultaneous detection of porcine serum antibodies to Actinobacillus pleuropneumoniae serovars 1, 2, 6, 7, and 12. Magnetic fluorescent beads were coupled with A. pleuropneumoniae antigens and tested with a panel of serum samples from experimentally infected pigs and with serum samples from uninfected and naturally infected pigs. The multiplex assay was compared to in-house ELISAs and complement fixation (CF) tests, which have been used for decades as tools for herd classification in the Danish Specific Pathogen Free system. Assay specificities and sensitivities as well as the corresponding cutoff values were determined using receiver operating characteristic (ROC) curve analysis, and the A. pleuropneumoniae multiplex assay showed good correlation with the in-house ELISAs and CF tests with areas under ROC curves ≥ 0.988. Benefits of multiplexed assays compared to ELISAs and CF tests include reduced serum sample volumes needed for analysis, less labor, and shorter assay time.

Monochromosomal hybrid cell assay for evaluating the genotoxicity of environmental chemicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sandhu, S.S.; Gudi, R.D.; Athwal, R.S.

1988-12-01

The development and utilization of a monochromosomal hybrid cell assay for detecting aneuploidy and chromosomal aberrations are described. The monochromosomal hybrid cell lines were produced by a two-step process involving transfer of a marker bacterial gene to a human chromosome and then by integration of that human chromosome into a mouse complement of chromosomes through microcell fusion. For chemically induced aneuploidy, the segregation of a single human chromosome among mouse chromosomes is used as a cytogenetic marker. The genetic assay for aneuploidy is based on the ability of the cells to grow in a medium that selects for the lossmore » of the human chromosome. The assay for clastogenicity is based on survival of the cells after treatment with the chemicals in medium that selects for retention of the human chromosome but loss of its segment containing diphtheria toxin locus. The assays greatly simplify the detection of chromosomal aberrations induced by environmental factors at low-dose levels.« less

Exo-Dye-based assay for rapid, inexpensive, and sensitive detection of DNA-binding proteins.

PubMed

Chen, Zaozao; Ji, Meiju; Hou, Peng; Lu, Zuhong

2006-07-07

We reported herein a rapid, inexpensive, and sensitive technique for detecting sequence-specific DNA-binding proteins. In this technique, the common exonuclease III (ExoIII) footprinting assay is coupled with simple SYBR Green I staining for monitoring the activities of DNA-binding proteins. We named this technique as ExoIII-Dye-based assay. In this assay, a duplex probe was designed to detect DNA-binding protein. One side of the probe contains one protein-binding site, and another side of it contains five protruding bases at 3' end for protection from ExoIII digestion. If a target protein is present, it will bind to binding sites of probe and produce a physical hindrance to ExoIII, which protects the duplex probe from digestion of ExoIII. SYBR Green I will bind to probe, which results in high fluorescence intensity. On the contrary, in the absence of the target protein, the naked duplex probe will be degraded by ExoIII. SYBR Green I will be released, which results in a low fluorescence intensity. In this study, we employed this technique to successfully detect transcription factor NF-kappaB in crude cell extracts. Moreover, it could also be used to evaluate the binding affinity of NF-kappaB. This technique has therefore wide potential application in research, medical diagnosis, and drug discovery.

Rapid and amplification-free detection of fish pathogens by utilizing a molecular beacon-based microfluidic system.

PubMed

Su, Yi-Chih; Wang, Chih-Hung; Chang, Wen-Hsin; Chen, Tzong-Yueh; Lee, Gwo-Bin

2015-01-15

Nervous necrosis virus (NNV) and iridovirus are highly infectious pathogens that can cause lethal diseases in various species of fish. These infectious diseases have no effective treatments and the mortality rate is over 80%, which could cause dramatic economic losses in the aquaculture industry. Conventional diagnostic methods of NNV or iridovirus infected fishes, such as virus culture, enzyme-linked immunosorbent assays and nucleic acid assays usually require time-consuming and complex procedures performed by specialized technicians with delicate laboratory facilities. Rapid, simple, accurate and on-site detection of NNV and iridovirus infections would enable timely preventive measures such as immediate sacrifice of infected fishes, and is therefore critically needed for the aquaculture industry. In this study, a microfluidic-based assay that employ magnetic beads conjugated with viral deoxyribonucleic acid (DNA) capturing probes and fluorescent DNA molecular beacons were developed to rapidly detect NNV and iridovirus. Importantly, this new assay was realized in an integrated microfluidic system with a custom-made control system. With this approach, direct and automated NNV and iridovirus detection from infected fishes can be achieved in less than 30 min. Therefore, this molecular-beacon based microfluidic system presents a potentially promising tool for rapid diagnosis of fish pathogens in the field in the future. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection of bovine viral diarrhoea virus nucleic acid, but not infectious virus, in bovine serum used for human vaccine manufacture.

PubMed

Laassri, Majid; Mee, Edward T; Connaughton, Sarah M; Manukyan, Hasmik; Gruber, Marion; Rodriguez-Hernandez, Carmen; Minor, Philip D; Schepelmann, Silke; Chumakov, Konstantin; Wood, David J

2018-06-22

Bovine viral diarrhoea virus (BVDV) is a cattle pathogen that has previously been reported to be present in bovine raw materials used in the manufacture of biological products for human use. Seven lots of trivalent measles, mumps and rubella (MMR) vaccine and 1 lot of measles vaccine from the same manufacturer, together with 17 lots of foetal bovine serum (FBS) from different vendors, 4 lots of horse serum, 2 lots of bovine trypsin and 5 lots of porcine trypsin were analysed for BVDV using recently developed techniques, including PCR assays for BVDV detection, a qRT-PCR and immunofluorescence-based virus replication assays, and deep sequencing to identify and genotype BVDV genomes. All FBS lots and one lot of bovine-derived trypsin were PCR-positive for the presence of BVDV genome; in contrast all vaccine lots and the other samples were negative. qRT-PCR based virus replication assay and immunofluorescence-based infection assay detected no infectious BVDV in the PCR-positive samples. Complete BVDV genomes were generated from FBS samples by deep sequencing, and all were BVDV type 1. These data confirmed that BVDV nucleic acid may be present in bovine-derived raw materials, but no infectious virus or genomic RNA was detected in the final vaccine products. Copyright © 2018 International Alliance for Biological Standardization. All rights reserved.

International ring trial to detect anti-Trichinella IgG by ELISA on pig sera.

USDA-ARS?s Scientific Manuscript database

In the present study, the sensitivity and specificity of the ELISA assay determined by the CRLP validation was 100% and 98.29%, respectively. The assay was reproducible, moreover, based on the receiver-operator characteristic (ROC) curve, the sensitivity and specificity of the assay reached 97.5% an...

High-performance liquid chromatography-mass spectrometry-based acetylcholinesterase assay for the screening of inhibitors in natural extracts.

PubMed

de Jong, Camiel F; Derks, Rico J E; Bruyneel, Ben; Niessen, Wilfried; Irth, Hubertus

2006-04-21

The present paper describes a High-performance liquid chromatography-mass spectrometry (LC-MS) methodology for the screening of acetylcholinesterase (AChE) inhibitors in natural extracts. AChE activity of sample components is monitored by a post-column biochemical assay that is based on the separate, sequential mixing of AChE and acetylcholine, respectively, with the HPLC eluate. AChE inhibitors are detected by measuring a decrease of product formation using electrospray MS. Ammonium bicarbonate was used as buffer in order to achieve optimum compatibility between biochemical assay and MS detection conditions. The assay is robust and stable for over 13 h and compares favourably with other AChE assays in terms of stability and sensitivity. IC(50) values of 9-aminoacridine, galanthamine, gallamine, (-)-huperzine A and thioflavin T were determined to be 0.12, 0.38, 6.4, 0.46 and 3.2 microM, respectively. The assay was used to effectively identify an AChE inhibitor present in a crude extract of Narcissus c.v. "Bridal Crown".

Simultaneous detection of ricin and abrin DNA by real-time PCR (qPCR).

PubMed

Felder, Eva; Mossbrugger, Ilona; Lange, Mirko; Wölfel, Roman

2012-09-01

Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5'-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

A dynamic sandwich assay on magnetic beads for selective detection of single-nucleotide mutations at room temperature.

PubMed

Wang, Junxiu; Xiong, Guoliang; Ma, Liang; Wang, Shihui; Zhou, Xu; Wang, Lei; Xiao, Lehui; Su, Xin; Yu, Changyuan

2017-08-15

Single-nucleotide mutation (SNM) has proven to be associated with a variety of human diseases. Development of reliable methods for the detection of SNM is crucial for molecular diagnosis and personalized medicine. The sandwich assays are widely used tools for detecting nucleic acid biomarkers due to their low cost and rapid signaling. However, the poor hybridization specificity of signal probe at room temperature hampers the discrimination of mutant and wild type. Here, we demonstrate a dynamic sandwich assay on magnetic beads for SNM detection based on the transient binding between signal probe and target. By taking the advantage of mismatch sensitive thermodynamics of transient DNA binding, the dynamic sandwich assay exhibits high discrimination factor for mutant with a broad range of salt concentration at room temperature. The beads used in this assay serve as a tool for separation, and might be helpful to enhance SNM selectivity. Flexible design of signal probe and facile magnetic separation allow multiple-mode downstream analysis including colorimetric detection and isothermal amplification. With this method, BRAF mutations in the genomic DNA extracted from cancer cell lines were tested, allowing sensitive detection of SNM at very low abundances (0.1-0.5% mutant/wild type). Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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

2017-02-01

Rose rosette disease caused by Rose rosette virus (RRV; genus Emaravirus) is the most economically relevant disease of Knock Out ® series roses in the U.S. As there are no effective chemical control options for the disease, the most critical disease management strategies include the use of virus free clean plants for propagation and early detection and destruction of infected plants. The current diagnostic techniques for RRV including end-point reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR (RT-qPCR) are highly sensitive, but limited to diagnostic labs with the equipment and expertise; and is time consuming. To address this limitation, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay based on multiple gene targets for specific detection of RRV was developed. The assay is highly specific and did not cross react with other viruses belonging to the inclusive and exclusive genus. Dilution assays using the in vitro transcripts showed that the primer sets designed (RPA-267, RPA-131, and RPA-321) are highly sensitive, consistently detecting RRV with a detection limit of 1fg/μL. Testing of the infected plants using the primer sets indicated that the virus could be detected from leaves, stems and petals of roses. The primer pair RPA-267 produced 100% positive detection of the virus from infected leaf tissues, while primer set RPA-131 produced 100% detection from stems and petals. The primer set RPA-321 produced 83%, 87.5% and 75% positive detection from leaves, petals and stem tissues, respectively. In addition, the assay has been efficiently used in the detection of RRV infecting Knock Out ® roses, collected from different states in the U.S. The assay can be completed in 20min as compared to the end-point RT-PCR assay (3-4h) and RT-qPCR (1.5h). The RT-RPA assay is reliable, rapid, highly sensitive, and can be easily used in diagnostic laboratories for detection of RRV with no need for any special equipment. Copyright © 2016 Elsevier B.V. All rights reserved.

PCR-Mediated Detection and Quantification of the Goss's Wilt Pathogen Clavibacter michiganensis subsp. nebraskensis Via a Novel Gene Target.

PubMed

McNally, R Ryan; Ishimaru, Carol A; Malvick, Dean K

2016-12-01

Goss's leaf blight and wilt of maize (corn) is a significant and reemerging disease caused by the bacterium Clavibacter michiganensis subsp. nebraskensis. Despite its importance, molecular tools for diagnosing and studying this disease remain limited. We report the identification of CMN_01184 as a novel gene target and its use in conventional PCR (cPCR) and SYBR green-based quantitative PCR (qPCR) assays for specific detection and quantification of C. michiganensis subsp. nebraskensis. The cPCR and qPCR assays based on primers targeting CMN_01184 specifically amplified only C. michiganensis subsp. nebraskensis among a diverse collection of 129 bacterial and fungal isolates, including multiple maize bacterial and fungal pathogens, environmental organisms from agricultural fields, and all known subspecies of C. michiganensis. Specificity of the assays for detection of only C. michiganensis subsp. nebraskensis was also validated with field samples of C. michiganensis subsp. nebraskensis-infected and uninfected maize leaves and C. michiganensis subsp. nebraskensis-infested and uninfested soil. Detection limits were determined at 30 and 3 ng of pure C. michiganensis subsp. nebraskensis DNA, and 100 and 10 CFU of C. michiganensis subsp. nebraskensis for the cPCR and qPCR assays, respectively. Infection of maize leaves by C. michiganensis subsp. nebraskensis was quantified from infected field samples and was standardized using an internal maize DNA control. These novel, specific, and sensitive PCR assays based on CMN_01184 are effective for diagnosis of Goss's wilt and for studies of the epidemiology and host-pathogen interactions of C. michiganensis subsp. nebraskensis.

Integrated sample-to-detection chip for nucleic acid test assays.

PubMed

Prakash, R; Pabbaraju, K; Wong, S; Tellier, R; Kaler, K V I S

2016-06-01

Nucleic acid based diagnostic techniques are routinely used for the detection of infectious agents. Most of these assays rely on nucleic acid extraction platforms for the extraction and purification of nucleic acids and a separate real-time PCR platform for quantitative nucleic acid amplification tests (NATs). Several microfluidic lab on chip (LOC) technologies have been developed, where mechanical and chemical methods are used for the extraction and purification of nucleic acids. Microfluidic technologies have also been effectively utilized for chip based real-time PCR assays. However, there are few examples of microfluidic systems which have successfully integrated these two key processes. In this study, we have implemented an electro-actuation based LOC micro-device that leverages multi-frequency actuation of samples and reagents droplets for chip based nucleic acid extraction and real-time, reverse transcription (RT) PCR (qRT-PCR) amplification from clinical samples. Our prototype micro-device combines chemical lysis with electric field assisted isolation of nucleic acid in a four channel parallel processing scheme. Furthermore, a four channel parallel qRT-PCR amplification and detection assay is integrated to deliver the sample-to-detection NAT chip. The NAT chip combines dielectrophoresis and electrostatic/electrowetting actuation methods with resistive micro-heaters and temperature sensors to perform chip based integrated NATs. The two chip modules have been validated using different panels of clinical samples and their performance compared with standard platforms. This study has established that our integrated NAT chip system has a sensitivity and specificity comparable to that of the standard platforms while providing up to 10 fold reduction in sample/reagent volumes.

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

PubMed

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

2017-03-23

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

Hepatitis E Virus (HEV) Detection and Quantification by a Real-Time Reverse Transcription-PCR Assay Calibrated to the World Health Organization Standard for HEV RNA.

PubMed

Germer, Jeffrey J; Ankoudinova, Irina; Belousov, Yevgeniy S; Mahoney, Walt; Dong, Chen; Meng, Jihong; Mandrekar, Jayawant N; Yao, Joseph D

2017-05-01

Hepatitis E virus (HEV) has emerged as a cause of chronic hepatitis among immunocompromised patients. Molecular assays have become important tools for the diagnosis and management of these chronically infected patients. A real-time reverse transcription-quantitative PCR (RT-qPCR) assay utilizing Pleiades probe chemistry and an RNA internal control for the simultaneous detection and quantification of HEV RNA in human serum was developed based on an adaptation of a previously described and broadly reactive primer set targeting the overlapping open reading frame 2/3 (ORF2/3) nucleotide sequence of HEV. A chimeric bovine viral diarrhea virus construct containing an HEV RNA insert (SynTura HEV) was developed, value assigned with the first World Health Organization (WHO) international standard for HEV RNA (code 6329/10), and used to prepare working assay calibrators and controls, which supported an assay quantification range of 100 to 5,000,000 IU/ml. The analytical sensitivity (95% detection rate) of this assay was 25.2 IU/ml (95% confidence interval [CI], 19.2 to 44.1 IU/ml). The assay successfully amplified 16 different HEV sequences with significant nucleotide mismatching in primer/probe binding regions, while evaluation of a WHO international reference panel for HEV genotypes (code 8578/13) showed viral load results falling within the result ranges generated by WHO collaborative study participants for all panel members (genotypes 1 to 4). Broadly reactive RT-qPCR primers targeting HEV ORF2/3 were successfully adapted for use in an assay based on Pleiades probe chemistry. The availability of secondary standards calibrated to the WHO HEV international standard can improve the standardization and performance of assays for the detection and quantification of HEV RNA. Copyright © 2017 American Society for Microbiology.

Biological Detection System Technologies Technology and Industrial Base Study. A Primer on Biological Detection Technologies

DTIC Science & Technology

2001-02-01

a novel method for the detection of the mecA gene that confers the principle mechanism of methicillin resistance in Staphylococcus aureus . CPT is a...with the mecA gene was used to develop a culture confirmation F-33 assay for methicillin resistant Staphylococcus aureus . The CPT assay was used to...compounds. Examples of signatures would include peptides, aptamers and phage. These are being sought to serve as adjuncts and/or replacements for antibody

Insulator-based DEP with impedance measurements for analyte detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davalos, Rafael V.; Simmons, Blake A.; Crocker, Robert W.

2010-03-16

Disclosed herein are microfluidic devices for assaying at least one analyte specie in a sample comprising at least one analyte concentration area in a microchannel having insulating structures on or in at least one wall of the microchannel which provide a nonuniform electric field in the presence of an electric field provided by off-chip electrodes; and a pair of passivated sensing electrodes for impedance detection in a detection area. Also disclosed are assay methods and methods of making.

Lab-On-a-Chip for carbon nanotubes based immunoassay detection of Staphylococcal Enterotoxin B (SEB).

PubMed

Yang, Minghui; Sun, Steven; Kostov, Yordan; Rasooly, Avraham

2010-04-21

We describe a new eight channel Lab-On-a-Chip (LOC) for a Carbon Nanotube (CNT) based immunoassay with optical detection of Staphylococcal Enterotoxin B (SEB) for food safety applications. In this work, we combined four biosensing elements: (1) CNT technology for primary antibody immobilization, (2) Enhanced Chemiluminescence (ECL) for light signal generation, (3) a cooled charge-coupled device (CCD) for detection and (4) polymer lamination technology for developing a point of care immunological assay for SEB detection. Our concept for developing versatile LOCs, which can be used for many different applications, is to use a modular design with interchangeable recognition elements (e.g. various antibodies) to determine the specificity. Polymer lamination technology was used for the fabrication of a six layer, syringe operated LOC capable of analyzing eight samples simultaneously. An anti-SEB antibody-nanotube mixture was immobilized onto a polycarbonate strip, to serve as an interchangeable ligand surface that was then bonded onto the LOC. SEB samples are loaded into the device and detected by an ELISA assay using Horse Radish Peroxidase (HRP) conjugated anti-SEB IgG as a secondary antibody and ECL, with detection by a previously described portable cooled CCD detector. Eight samples of SEB in buffer or soy milk were assayed simultaneously with a limit of detection of 0.1 ng mL(-1). CNT immobilization of the antibody increased the sensitivity of detection six fold. Use of a simple interchangeable immunological surface allows this LOC to be adapted to any immunoassay by simply replacing the ligand surface. A syringe was used to move fluids for this assay so no power is needed to operate the device. Our versatile portable point-of-care CCD detector combined with the LOC immunoassay method described here can be used to reduce the exposure of users to toxins and other biohazards when working outside the lab, as well as to simplify and increase sensitivity for many other types of immunological diagnostics and detection assays.

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

NASA Astrophysics Data System (ADS)

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. 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. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr04987c

Real-time PCR-based method for the rapid detection of extended RAS mutations using bridged nucleic acids in colorectal cancer.

PubMed

Iida, Takao; Mizuno, Yukie; Kaizaki, Yasuharu

2017-10-27

Mutations in RAS and BRAF are predictors of the efficacy of anti-epidermal growth factor receptor (EGFR) therapy in patients with metastatic colorectal cancer (mCRC). Therefore, simple, rapid, cost-effective methods to detect these mutations in the clinical setting are greatly needed. In the present study, we evaluated BNA Real-time PCR Mutation Detection Kit Extended RAS (BNA Real-time PCR), a real-time PCR method that uses bridged nucleic acid clamping technology to rapidly detect mutations in RAS exons 2-4 and BRAF exon 15. Genomic DNA was extracted from 54 formalin-fixed paraffin-embedded (FFPE) tissue samples obtained from mCRC patients. Among the 54 FFPE samples, BNA Real-time PCR detected 21 RAS mutations (38.9%) and 5 BRAF mutations (9.3%), and the reference assay (KRAS Mutation Detection Kit and MEBGEN™ RASKET KIT) detected 22 RAS mutations (40.7%). The concordance rate of detected RAS mutations between the BNA Real-time PCR assay and the reference assays was 98.2% (53/54). The BNA Real-time PCR assay proved to be a more simple, rapid, and cost-effective method for detecting KRAS and RAS mutations compared with existing assays. These findings suggest that BNA Real-time PCR is a valuable tool for predicting the efficacy of early anti-EGFR therapy in mCRC patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Battery operated preconcentration-assisted lateral flow assay.

PubMed

Kim, Cheonjung; Yoo, Yong Kyoung; Han, Sung Il; Lee, Junwoo; Lee, Dohwan; Lee, Kyungjae; Hwang, Kyo Seon; Lee, Kyu Hyoung; Chung, Seok; Lee, Jeong Hoon

2017-07-11

Paper-based analytical devices (e.g. lateral flow assays) are highly advantageous as portable diagnostic systems owing to their low costs and ease of use. Because of their low sensitivity and detection limits for biomolecules, these devices have several limitations in applications for real-field diagnosis. Here, we demonstrate a paper-based preconcentration enhanced lateral flow assay using a commercial β-hCG-based test. Utilizing a simple 9 V battery operation with a low power consumption of approximately 81 μW, we acquire a 25-fold preconcentration factor, demonstrating a clear sensitivity enhancement in the colorimetric lateral flow assay; consequently, clear colors are observed in a rapid kit test line, which cannot be monitored without preconcentration. This device can also facilitate a semi-quantitative platform using the saturation value and/or color intensity in both paper-based colorimetric assays and smartphone-based diagnostics.

Paper-based solid-phase multiplexed nucleic acid hybridization assay with tunable dynamic range using immobilized quantum dots as donors in fluorescence resonance energy transfer.

PubMed

Noor, M Omair; Krull, Ulrich J

2013-08-06

A multiplexed solid-phase nucleic acid hybridization assay on a paper-based platform is presented using multicolor immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize two types of QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) and red-emitting QDs (rQDs) served as donors with Cy3 and Alexa Fluor 647 (A647) acceptors. The gQD/Cy3 FRET pair served as an internal standard, while the rQD/A647 FRET pair served as a detection channel, combining the control and analytical test zones in one physical location. Hybridization of dye-labeled oligonucleotide targets provided the proximity for FRET sensitized emission from the acceptor dyes, which served as an analytical signal. Hybridization assays in the multicolor format provided a limit of detection of 90 fmol and an upper limit of dynamic range of 3.5 pmol. The use of an array of detection zones was designed to provide improved analytical figures of merit compared to that which could be achieved on one type of array design in terms of relative concentration of multicolor QDs. The hybridization assays showed excellent resistance to nonspecific adsorption of oligonucleotides. Selectivity of the two-plex hybridization assay was demonstrated by single nucleotide polymorphism (SNP) detection at a contrast ratio of 50:1. Additionally, it is shown that the use of preformed QD-probe oligonucleotide conjugates and consideration of the relative number density of the two types of QD-probe conjugates in the two-color assay format is advantageous to maximize assay sensitivity and the upper limit of dynamic range.

An ultrasensitive colorimeter assay strategy for p53 mutation assisted by nicking endonuclease signal amplification.

PubMed

Lin, Zhenyu; Yang, Weiqiang; Zhang, Guiyun; Liu, Qida; Qiu, Bin; Cai, Zongwei; Chen, Guonan

2011-08-28

A novel catalytic colorimetric assay assisted by nicking endonuclease signal amplification (NESA) was developed. With the signal amplification, the detection limit of the p53 target gene can be as low as 1 pM, which is nearly 5 orders of magnitude lower than that of other previously reported colorimetric DNA detection strategies based on catalytic DNAzyme.

Development and validation of four one-step real-time RT-LAMP assays for specific detection of each dengue virus serotype

PubMed Central

Bekaert, Michaël; Bakheit, Mohammed; Frischmann, Sieghard; Patel, Pranav; Simon-Loriere, Etienne; Lambrechts, Louis; Duong, Veasna; Dussart, Philippe; Harold, Graham; Fall, Cheikh; Faye, Oumar; Sall, Amadou Alpha; Weidmann, Manfred

2018-01-01

Background 4 one-step, real-time, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed for the detection of dengue virus (DENV) serotypes by considering 2,056 full genome DENV sequences. DENV1 and DENV2 RT-LAMP assays were validated with 31 blood and 11 serum samples from Tanzania, Senegal, Sudan and Mauritania. DENV3 and DENV4 RT-LAMP assays were validated with 25 serum samples from Cambodia Methodology/Principal findings 4 final reaction primer mixes were obtained by using a combination of Principal Component Analysis of the full DENV genome sequences, and LAMP primer design based on sequence alignments using the LAVA software. These mixes contained 14 (DENV1), 12 (DENV2), 8 (DENV3) and 3 (DENV4) LAMP primer sets. The assays were evaluated with an External Quality Assessment panel from Quality Control for Molecular Diagnostics. The assays were serotype-specific and did not cross-detect with other flaviviruses. The limits of detection, with 95% probability, were 22 (DENV1), 542 (DENV2), 197 (DENV3) and 641 (DENV4) RNA molecules, and 100% reproducibility in the assays was obtained with up to 102 (DENV1) and 103 RNA molecules (DENV2, DENV3 and DENV4). Validation of the DENV2 assay with blood samples from Tanzania resulted in 23 samples detected by RT-LAMP, demonstrating that the assay is 100% specific and 95.8% sensitive (positive predictive value of 100% and a negative predictive value of 85.7%). All serum samples from Senegal, Sudan and Mauritania were detected and 3 untyped as DENV1. The sensitivity of RT-LAMP for DENV4 samples from Cambodia did not quite match qRT-PCR. Conclusions/Significance We have shown a novel approach to design LAMP primers that makes use of fast growing sequence databases. The DENV1 and DENV2 assays were validated with viral RNA extracted clinical samples, showing very good performance parameters. PMID:29813062

Detection of Endotoxin Contamination of Graphene Based Materials Using the TNF-α Expression Test and Guidelines for Endotoxin-Free Graphene Oxide Production

PubMed Central

Del Rio-Castillo, Antonio E.; Newman, Leon; Vázquez, Ester; Kostarelos, Kostas; Wick, Peter; Fadeel, Bengt

2016-01-01

Nanomaterials may be contaminated with bacterial endotoxin during production and handling, which may confound toxicological testing of these materials, not least when assessing for immunotoxicity. In the present study, we evaluated the conventional Limulus amebocyte lysate (LAL) assay for endotoxin detection in graphene based material (GBM) samples, including graphene oxide (GO) and few-layered graphene (FLG). Our results showed that some GO samples interfered with various formats of the LAL assay. To overcome this problem, we developed a TNF-α expression test (TET) using primary human monocyte-derived macrophages incubated in the presence or absence of the endotoxin inhibitor, polymyxin B sulfate, and found that this assay, performed with non-cytotoxic doses of the GBM samples, enabled unequivocal detection of endotoxin with a sensitivity that is comparable to the LAL assay. FLG also triggered TNF-α production in the presence of the LPS inhibitor, pointing to an intrinsic pro-inflammatory effect. Finally, we present guidelines for the preparation of endotoxin-free GO, validated by using the TET. PMID:27880838

Detection of Endotoxin Contamination of Graphene Based Materials Using the TNF-α Expression Test and Guidelines for Endotoxin-Free Graphene Oxide Production.

PubMed

Mukherjee, Sourav P; Lozano, Neus; Kucki, Melanie; Del Rio-Castillo, Antonio E; Newman, Leon; Vázquez, Ester; Kostarelos, Kostas; Wick, Peter; Fadeel, Bengt

2016-01-01

Nanomaterials may be contaminated with bacterial endotoxin during production and handling, which may confound toxicological testing of these materials, not least when assessing for immunotoxicity. In the present study, we evaluated the conventional Limulus amebocyte lysate (LAL) assay for endotoxin detection in graphene based material (GBM) samples, including graphene oxide (GO) and few-layered graphene (FLG). Our results showed that some GO samples interfered with various formats of the LAL assay. To overcome this problem, we developed a TNF-α expression test (TET) using primary human monocyte-derived macrophages incubated in the presence or absence of the endotoxin inhibitor, polymyxin B sulfate, and found that this assay, performed with non-cytotoxic doses of the GBM samples, enabled unequivocal detection of endotoxin with a sensitivity that is comparable to the LAL assay. FLG also triggered TNF-α production in the presence of the LPS inhibitor, pointing to an intrinsic pro-inflammatory effect. Finally, we present guidelines for the preparation of endotoxin-free GO, validated by using the TET.

Hypersensitive Detection and Quantitation of BoNT/A by IgY Antibody against Substrate Linear-Peptide

PubMed Central

Li, Tao; Liu, Hao; Cai, Kun; Tian, Maoren; Wang, Qin; Shi, Jing; Gao, Xiang; Wang, Hui

2013-01-01

Botulinum neurotoxin A (BoNT/A), the most acutely poisonous substance to humans known, cleave its SNAP-25 substrate with high specificity. Based on the endopeptidase activity, different methods have been developed to detect BoNT/A, but most lack ideal reproducibility or sensitivity, or suffer from long-term or unwanted interferences. In this study, we developed a simple method to detect and quantitate trace amounts of botulinum neurotoxin A using the IgY antibody against a linear-peptide substrate. The effects of reaction buffer, time, and temperature were analyzed and optimized. When the optimized assay was used to detect BoNT/A, the limit of detection of the assay was 0.01 mouse LD50 (0.04 pg), and the limit of quantitation was 0.12 mouse LD50/ml (0.48 pg). The findings also showed favorable specificity of detecting BoNT/A. When used to detect BoNT/A in milk or human serum, the proposed assay exhibited good quantitative accuracy (88% < recovery < 111%; inter- and intra-assay CVs < 18%). This method of detection took less than 3 h to complete, indicating that it can be a valuable method of detecting BoNT/A in food or clinical diagnosis. PMID:23555605

Hypersensitive detection and quantitation of BoNT/A by IgY antibody against substrate linear-peptide.

PubMed

Li, Tao; Liu, Hao; Cai, Kun; Tian, Maoren; Wang, Qin; Shi, Jing; Gao, Xiang; Wang, Hui

2013-01-01

Botulinum neurotoxin A (BoNT/A), the most acutely poisonous substance to humans known, cleave its SNAP-25 substrate with high specificity. Based on the endopeptidase activity, different methods have been developed to detect BoNT/A, but most lack ideal reproducibility or sensitivity, or suffer from long-term or unwanted interferences. In this study, we developed a simple method to detect and quantitate trace amounts of botulinum neurotoxin A using the IgY antibody against a linear-peptide substrate. The effects of reaction buffer, time, and temperature were analyzed and optimized. When the optimized assay was used to detect BoNT/A, the limit of detection of the assay was 0.01 mouse LD50 (0.04 pg), and the limit of quantitation was 0.12 mouse LD50/ml (0.48 pg). The findings also showed favorable specificity of detecting BoNT/A. When used to detect BoNT/A in milk or human serum, the proposed assay exhibited good quantitative accuracy (88% < recovery < 111%; inter- and intra-assay CVs < 18%). This method of detection took less than 3 h to complete, indicating that it can be a valuable method of detecting BoNT/A in food or clinical diagnosis.

Differentiation of herpes simplex virus types 1 and 2 in clinical samples by a real-time taqman PCR assay.

PubMed

Corey, Lawrence; Huang, Meei-Li; Selke, Stacy; Wald, Anna

2005-07-01

While the clinical manifestations of HSV-1 and -2 overlap, the site of CNS infection, complications, response to antivirals, frequency of antiviral resistance, and reactivation rate on mucosal surfaces varies between HSV-1 and -2. Detection of HSV DNA by PCR has been shown to be the most sensitive method for detecting HSV in clinical samples. As such, we developed a PCR-based assay to accurately distinguish HSV-1 from HSV-2. Our initial studies indicated the assay using type specific primers was slightly less efficient for detecting HSV-1 and -2 DNA than the high throughput quantitative PCR assay we utilize that employs type common primers to gB. We subsequently evaluated the type specific assay on 3,131 specimens that had HSV DNA detected in the type common PCR assay. The typing results of these specimens were compared with the monoclonal antibody staining results of culture isolates collected from the same patients at the same time, and the HSV serologic status of the patient. The typing assay accurately identified both HSV-1 and -2 with a specificity of >99.5% and was significantly more sensitive than typing by culture and subsequent monoclonal antibody assays. Complete concordance was seen between the typing assay and HSV serologic status of the patient. Dual (HSV-1 and -2) infection in clinical samples was recognized in 2.6% of clinical samples using the new typing assay. This assay, when used in combination with the type common assay, can now accurately type almost all mucosal and visceral HSV isolates by molecular techniques. Copyright (c) 2005 Wiley-Liss, Inc.

A New Single-Step PCR Assay for the Detection of the Zoonotic Malaria Parasite Plasmodium knowlesi

PubMed Central

Lucchi, Naomi W.; Poorak, Mitra; Oberstaller, Jenna; DeBarry, Jeremy; Srinivasamoorthy, Ganesh; Goldman, Ira; Xayavong, Maniphet; da Silva, Alexandre J.; Peterson, David S.; Barnwell, John W.; Kissinger, Jessica; Udhayakumar, Venkatachalam

2012-01-01

Background Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection. Methodology and Significant Findings We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites. Conclusions The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi. PMID:22363751

High performance of a new PCR-based urine assay for HPV-DNA detection and genotyping.

PubMed

Tanzi, Elisabetta; Bianchi, Silvia; Fasolo, Maria Michela; Frati, Elena R; Mazza, Francesca; Martinelli, Marianna; Colzani, Daniela; Beretta, Rosangela; Zappa, Alessandra; Orlando, Giovanna

2013-01-01

Human papillomavirus (HPV) testing has been proposed as a means of replacing or supporting conventional cervical screening (Pap test). However, both methods require the collection of cervical samples. Urine sample is easier and more acceptable to collect and could be helpful in facilitating cervical cancer screening. The aim of this study was to evaluate the sensitivity and specificity of urine testing compared to conventional cervical smear testing using a PCR-based method with a new, designed specifically primer set. Paired cervical and first voided urine samples collected from 107 women infected with HIV were subjected to HPV-DNA detection and genotyping using a PCR-based assay and a restriction fragment length polymorphism method. Sensitivity, specificity, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) were calculated using the McNemar's test for differences. Concordance between tests was assessed using the Cohen's unweighted Kappa (k). HPV DNA was detected in 64.5% (95% CI: 55.1-73.1%) of both cytobrush and urine samples. High concordance rates of HPV-DNA detection (k = 0.96; 95% CI: 0.90-1.0) and of high risk-clade and low-risk genotyping in paired samples (k = 0.80; 95% CI: 0.67-0.92 and k = 0.74; 95% CI: 0.60-0.88, respectively) were observed. HPV-DNA detection in urine versus cervix testing revealed a sensitivity of 98.6% (95% CI: 93.1-99.9%) and a specificity of 97.4% (95% CI: 87.7-99.9%), with a very high NPV (97.4%; 95% CI: 87.7-99.9%). The PCR-based assay utilized in this study proved highly sensitive and specific for HPV-DNA detection and genotyping in urine samples. These data suggest that a urine-based assay would be a suitable and effective tool for epidemiological surveillance and, most of all, screening programs. Copyright © 2012 Wiley Periodicals, Inc.

Development of a real-time polymerase chain reaction assay for the detection of the invasive Mediterranean fanworm, Sabella spallanzanii, in environmental samples.

PubMed

Wood, Susanna A; Zaiko, Anastasija; Richter, Ingrid; Inglis, Graeme J; Pochon, Xavier

2017-07-01

The Mediterranean fanworm, Sabella spallanzanii Gmelin 1791, was first detected in the Southern Hemisphere in the 1990s and is now abundant in many parts of southern Australia and in several locations around northern New Zealand. Once established, it can proliferate rapidly, reaching high densities with potential ecological and economic impacts. Early detection of new S. spallanzanii incursions is important to prevent its spread, guide eradication or control efforts and to increase knowledge on the species' dispersal pathways. In this study, we developed a TaqMan probe real-time polymerase chain reaction assay targeting a region of the mitochondrial cytochrome oxidase I gene. The assay was validated in silico and in vitro using DNA from New Zealand and Australian Sabellidae with no cross-reactivity detected. The assay has a linear range of detection over seven orders of magnitude with a limit of detection reached at 12.4 × 10 -4  ng/μL of DNA. We analysed 145 environmental (water, sediment and biofouling) samples and obtained positive detections only from spiked samples and those collected at a port where S. spallanzanii is known to be established. This assay has the potential to enhance current morphological and molecular-based methods, through its ability to rapidly and accurately identify S. spallanzanii in environmental samples.

Base pair mismatch recognition using plasmon resonant particle labels.

PubMed

Oldenburg, Steven J; Genick, Christine C; Clark, Keith A; Schultz, David A

2002-10-01

We demonstrate the use of silver plasmon resonant particles (PRPs), as reporter labels, in a microarray-based DNA hybridization assay in which we screen for a known polymorphic site in the breast cancer gene BRCA1. PRPs (40-100 nm in diameter) image as diffraction-limited points of colored light in a standard microscope equipped with dark-field illumination, and can be individually identified and discriminated against background scatter. Rather than overall intensity, the number of PRPs counted in a CCD image by a software algorithm serves as the signal in these assays. In a typical PRP hybridization assay, we achieve a detection sensitivity that is approximately 60 x greater than that achieved by using fluorescent labels. We conclude that single particle counting is robust, generally applicable to a wide variety of assay platforms, and can be integrated into low-cost and quantitative detection systems for single nucleotide polymorphism analysis.

Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis.

PubMed

Li, Wenbin; Teixeira, Diva C; Hartung, John S; Huang, Qi; Duan, Yongping; Zhou, Lijuan; Chen, Jianchi; Lin, Hong; Lopes, Silvio; Ayres, A Juliano; Levy, Laurene

2013-01-01

The xylem-limited, Gram-negative, fastidious plant bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease affecting approximately half of the citrus plantations in the State of São Paulo, Brazil. The disease was recently found in Central America and is threatening the multi-billion U.S. citrus industry. Many strains of X. fastidiosa are pathogens or endophytes in various plants growing in the U.S., and some strains cross infect several host plants. In this study, a TaqMan-based assay targeting the 16S rDNA signature region was developed for the identification of X. fastidiosa at the species level. Another TaqMan-based assay was developed for the specific identification of the CVC strains. Both new assays have been systematically validated in comparison with the primer/probe sets from four previously published assays on one platform and under similar PCR conditions, and shown to be superior. The species specific assay detected all X. fastidiosa strains and did not amplify any other citrus pathogen or endophyte tested. The CVC-specific assay detected all CVC strains but did not amplify any non-CVC X. fastidiosa nor any other citrus pathogen or endophyte evaluated. Both sets were multiplexed with a reliable internal control assay targeting host plant DNA, and their diagnostic specificity and sensitivity remained unchanged. This internal control provides quality assurance for DNA extraction, performance of PCR reagents, platforms and operators. The limit of detection for both assays was equivalent to 2 to 10 cells of X. fastidiosa per reaction for field citrus samples. Petioles and midribs of symptomatic leaves of sweet orange harbored the highest populations of X. fastidiosa, providing the best materials for detection of the pathogen. These new species specific assay will be invaluable for molecular identification of X. fastidiosa at the species level, and the CVC specific assay will be very powerful for the specific identification of X. fastidiosa strains that cause citrus variegated chlorosis. Published by Elsevier B.V.

Detection and quantitation of HPV in genital and oral tissues and fluids by real time PCR

PubMed Central

2010-01-01

Background Human papillomaviruses (HPVs) remain a serious world health problem due to their association with anogenital/oral cancers and warts. While over 100 HPV types have been identified, a subset is associated with malignancy. HPV16 and 18 are the most prevalent oncogenic types, while HPV6 and 11 are most commonly responsible for anogenital warts. While other quantitative PCR (qPCR) assays detect oncogenic HPV, there is no single tube assay distinguishing the most frequent oncogenic types and the most common types found in warts. Results A Sybr Green-based qPCR assay was developed utilizing degenerate primers to the highly conserved HPV E1 theoretically detecting any HPV type. A single tube multiplex qPCR assay was also developed using type-specific primer pairs and TaqMan probes that allowed for detection and quantitation of HPV6,11,16,18. Each HPV type was detected over a range from 2 × 101 to 2 × 106copies/reaction providing a reliable method of quantitating type-specific HPV in 140 anogenital/cutaneous/oral benign and malignant specimens. 35 oncogenic and low risk alpha genus HPV types were detected. Concordance was detected in previously typed specimens. Comparisons to the gold standard detected an overall sensitivity of 89% (95% CI: 77% - 96%) and specificity of 90% (95%CI: 52% - 98%). Conclusion There was good agreement between the ability of the qPCR assays described here to identify HPV types in malignancies previously typed using standard methods. These novel qPCR assays will allow rapid detection and quantitation of HPVs to assess their role in viral pathogenesis. PMID:20723234

A novel TaqMan® assay for Nosema ceranae quantification in honey bee, based on the protein coding gene Hsp70.

PubMed

Cilia, Giovanni; Cabbri, Riccardo; Maiorana, Giacomo; Cardaio, Ilaria; Dall'Olio, Raffaele; Nanetti, Antonio

2018-04-01

Nosema ceranae is now a widespread honey bee pathogen with high incidence in apiculture. Rapid and reliable detection and quantification methods are a matter of concern for research community, nowadays mainly relying on the use of biomolecular techniques such as PCR, RT-PCR or HRMA. The aim of this technical paper is to provide a new qPCR assay, based on the highly-conserved protein coding gene Hsp70, to detect and quantify the microsporidian Nosema ceranae affecting the western honey bee Apis mellifera. The validation steps to assess efficiency, sensitivity, specificity and robustness of the assay are described also. Copyright © 2018 Elsevier GmbH. All rights reserved.

A review of current and future molecular diagnostic tests for use in the microbiology laboratory.

PubMed

Jannes, Geert; De Vos, Daniel

2006-01-01

Nucleic acid-based diagnostics gradually are replacing or complementing culture-based, biochemical, and immunological assays in routine microbiology laboratories. Similar to conventional tests, the first-generation deoxyribonucleic acid assays determined only a single analyte. Recent improvements in detection technologies have paved the way for the development of multiparameter assays using macroarrays or micro-arrays, while the introduction of closed-tube real-time polymerase chain reaction systems has resulted in the development of rapid microbial diagnostics with a reduced contamination risk. The use of these new molecular technologies is not restricted to detection and identification of microbial pathogens but also can be used for genotyping, allowing one to determine antibiotic resistance or to perform microbial fingerprinting.

An eDNA Assay to Monitor a Globally Invasive Fish Species from Flowing Freshwater

PubMed Central

Adrian-Kalchhauser, Irene; Burkhardt-Holm, Patricia

2016-01-01

Ponto-Caspian gobies are a flock of five invasive fish species that have colonized freshwaters and brackish waters in Europe and North America. One of them, the round goby Neogobius melanostomus, figures among the 100 worst invaders in Europe. Current methods to detect the presence of Ponto-Caspian gobies involve catching or sighting the fish. These approaches are labor intense and not very sensitive. Consequently, populations are usually detected only when they have reached high densities and when management or containment efforts are futile. To improve monitoring, we developed an assay based on the detection of DNA traces (environmental DNA, or eDNA) of Ponto-Caspian gobies in river water. The assay specifically detects invasive goby DNA and does not react to any native fish species. We apply the assay to environmental samples and demonstrate that parameters such as sampling depth, sampling location, extraction protocol, PCR protocol and PCR inhibition greatly impact detection. We further successfully outline the invasion front of Ponto-Caspian gobies in a large river, the High Rhine in Switzerland, and thus demonstrate the applicability of the assay to lotic environments. The eDNA assay requires less time, equipment, manpower, skills, and financial resources than the conventional monitoring methods such as electrofishing, angling or diving. Samples can be taken by untrained individuals, and the assay can be performed by any molecular biologist on a conventional PCR machine. Therefore, this assay enables environment managers to map invaded areas independently of fishermen’s’ reports and fish community monitorings. PMID:26814998

Smartphone spectroscopy: three unique modalities for point-of-care testing

NASA Astrophysics Data System (ADS)

Long, Kenneth D.; Yu, Hojeong; Cunningham, Brian T.

2015-06-01

Here we demonstrate three principle modalities for a smartphone-based spectrometer: absorption, fluorescence, and photonic crystal (PC)-based label-free detection. When combined with some simple optical components, the rear-facing CMOS camera in a mobile device can provide spectrometric data that rivals that of laboratory instruments, but at a fraction of the cost. The use of a smartphone-based platform poses significant advantages based upon the rise of smartphone apps, which allow for user-interface and data-processing algorithms to be packaged and distributed within environments that are externally maintained with potential for integration with services such as cloud storage, GIS-tagging, and remote expert analysis. We demonstrate the absorption modality of our device by performing an enzyme-linked immunosorbent assay (ELISA) on both a cancer biomarker and a peanut allergen, demonstrating clinically relevant limits of detection (LOD). Second, we demonstrate the success of a molecular beacon (MB)-based assay on the smartphone platform, achieving an LOD of 1.3 pM for a specific RNA sequence, less than that of a commercial benchtop instrument. Finally, we use a PC biosensor to perform label-free detection of a representative biological interaction: Protein A and human immunoglobulin G (IgG) in the nanomolar regime. Our work represents the first demonstration of smartphone-based spectroscopy for biological assays, and the first mobile-device-enabled detection instrument that serves to measure three distinct sensing modalities (label-free biosensing, absorption spectroscopy, and fluorescence spectroscopy). The smartphone platform has the potential to expand the use of spectrometric analysis to environments assay from the laboratory, which may include rural or remote locations, low-resource settings, and consumer markets.

Evaluation of a Rapid Fecal PCR Test for Detection of Mycobacterium avium subsp. paratuberculosis in Dairy Cattle▿

PubMed Central

Wells, Scott J.; Collins, Michael T.; Faaberg, Kay S.; Wees, Carrie; Tavornpanich, Saraya; Petrini, Kristine R.; Collins, James E.; Cernicchiaro, Natalia; Whitlock, Robert H.

2006-01-01

A high-throughput TaqMan PCR assay for detection of bovine paratuberculosis was evaluated by using fecal samples from 1,808 dairy cattle in seven naturally infected herds and 347 dairy cattle in seven herds considered free of paratuberculosis. Fecal, blood, and milk samples were submitted to laboratories where the PCR-based assay, three different fecal culture procedures for Mycobacterium avium subsp. paratuberculosis (centrifugation, sedimentation, and the BACTEC filter concentration method), two serologic enzyme-linked immunosorbent assays (ELISAs), and one milk ELISA were performed. Results from testing of dairy cattle in herds free of M. avium subsp. paratuberculosis showed that the PCR assay's specificity was 99.7%. Twenty-three percent of the dairy cows that were fecal culture positive by at least one of the three methods were positive by the PCR assay. By Bayesian non-“gold standard” analysis methods, the TaqMan PCR assay had a higher specificity than the serum ELISAs (99.3%; 95% confidence interval [CI] = 98.6 to 99.7%) and a test sensitivity similar to that of the serum ELISAs (29%; 95% CI = 24 to 35%). By classical methods, the estimated relative sensitivity of the fecal PCR assay was 4% for light and moderate fecal shedders (compared to 12 to 13% for the ELISAs) and 76% for heavy fecal shedders (compared to 67% for the milk ELISA). The PCR assay has higher sensitivity for detection of heavy fecal shedders than the evaluated milk ELISA but lower sensitivity than a serum or milk ELISA for detection of light and moderate fecal shedders. This assay can be used as a quick test for detection of cattle with heavy fecal shedding, those cattle with the highest risk of transmitting infection to susceptible cattle. PMID:16928884

Comparison of Detection Limits of 4th Generation Combination HIV Antigen/Antibody, p24 Antigen and Viral Load Assays on Diverse HIV Isolates.

PubMed

Stone, Mars; Bainbridge, John; Sanchez, Ana M; Keating, Sheila M; Pappas, Andrea; Rountree, Wes; Todd, Chris; Bakkour, Sonia; Manak, Mark; Peel, Sheila A; Coombs, Robert W; Ramos, Eric M; Shriver, M Kathleen; Contestable, Paul; Nair, Sangeetha Vijaysri; Wilson, David H; Stengelin, Martin; Murphy, Gary; Hewlett, Indira; Denny, Thomas N; Busch, Michael P

2018-05-23

Detection of acute HIV infection is critical for HIV public health and diagnostics. Clinical 4 th generation antigen-antibody (Ag/Ab) combination (combo) and p24 Ag immunoassays have enhanced detection of acute infection compared to Ab alone assays, but require ongoing evaluation with currently circulating diverse subtypes. Genetically and geographically diverse HIV clinical isolates were used to assess clinical HIV diagnostic, blood screening and next generation assays. Blinded 300 member panels of 20 serially diluted well-characterized antibody negative HIV isolates were distributed to manufacturers and end-user labs to assess relative analytic sensitivity of currently approved and pre-approved clinical HIV 4 th generation Ag/Ab combo or p24 Ag alone immunoassays across diverse subtypes. The limits of virus detection (LODs) were estimated for different subtypes relative to confirmed viral loads. Analysis of immunoassay sensitivity was benchmarked against confirmed viral load measurements on the blinded panel. Based on the proportion of positive results on 300 observations all Ag/Ab combo and standard sensitivity p24 Ag assays performed similarly and within half log LODs, illustrating similar breadth of reactivity and diagnostic utility. Ultrasensitive p24 Ag assays achieved dramatically increased sensitivities, while the rapid combo-assays performed poorly. Similar performance of the different commercially available 4 th gen assays on diverse subtypes supports their use in broad geographic settings with locally circulating HIV clades and recombinant strains. Next generation pre-clinical ultrasensitive p24 Ag assays achieved dramatically improved sensitivity, while p24 Ag detection by rapid 4 th gen assays performed poorly. Copyright © 2018 American Society for Microbiology.

Development and validation of a novel hydrolysis probe real-time polymerase chain reaction for agamid adenovirus 1 in the central bearded dragon (Pogona vitticeps).

PubMed

Fredholm, Daniel V; Coleman, James K; Childress, April L; Wellehan, James F X

2015-03-01

Agamid adenovirus 1 (AgAdv-1) is a significant cause of disease in bearded dragons (Pogona sp.). Clinical manifestations of AgAdv-1 infection are variable and often nonspecific; the manifestations range from lethargy, weight loss, and inappetence, to severe enteritis, hepatitis, and sudden death. Currently, diagnosis of AgAdv-1 infection is achieved through a single published method: standard nested polymerase chain reaction (nPCR) and sequencing. Standard nPCR with sequencing provides reliable sensitivity, specificity, and validation of PCR products. However, this process is comparatively expensive, laborious, and slow. Probe hybridization, as used in a TaqMan assay, represents the best option for validating PCR products aside from the time-consuming process of sequencing. This study developed a real-time PCR (qPCR) assay using a TaqMan probe-based assay, targeting a highly conserved region of the AgAdv-1 genome. Standard curves were generated, detection results were compared with the gold standard conventional PCR and sequencing assay, and limits of detection were determined. Additionally, the qPCR assay was run on samples known to be positive for AgAdv-1 and samples known to be positive for other adenoviruses. Based on the results of these evaluations, this assay allows for a less expensive, rapid, quantitative detection of AgAdv-1 in bearded dragons. © 2015 The Author(s).

A HRM assay for identification of low level BRAF V600E and V600K mutations using the CADMA principle in FFPE specimens.

PubMed

Huebner, Claudia; Weber, Remeny; Lloydd, Richard

2017-12-01

Melanoma patients with BRAF V600E and V600K mutations show complete or partial response to vemurafenib. Detection assays often scan for the common V600E mutation rather than the rare V600K variant, although this mutation can be found in a high proportion of melanoma patients in the South Pacific. Herein, we describe a BRAF high resolution melting (HRM) assay that can differentiate low level of V600E and V600K mutations using formalin fixed, paraffin embedded (FFPE) reference standards for assay validation. The assay is based on the competitive amplification of differentially melting amplicons (CADMA principle) and has a limit of detection of 0.8% mutant allele for V600K and 1.4% mutant allele for V600E. A differentiation between the two mutations based on the melting profile is possible even at low mutation level. Sixty FFPE specimens were scanned and mutations could be scored correctly as confirmed by castPCR. In summary, the developed HRM assay is suitable for detection of V600K and V600E mutations and proved to be reliable and cost effective in a diagnostic environment. Copyright © 2017 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.

Event-specific qualitative and quantitative PCR detection of the GMO carnation (Dianthus caryophyllus) variety Moonlite based upon the 5'-transgene integration sequence.

PubMed

Li, P; Jia, J W; Jiang, L X; Zhu, H; Bai, L; Wang, J B; Tang, X M; Pan, A H

2012-04-27

To ensure the implementation of genetically modified organism (GMO)-labeling regulations, an event-specific detection method was developed based on the junction sequence of an exogenous integrant in the transgenic carnation variety Moonlite. The 5'-transgene integration sequence was isolated by thermal asymmetric interlaced PCR. Based upon the 5'-transgene integration sequence, the event-specific primers and TaqMan probe were designed to amplify the fragments, which spanned the exogenous DNA and carnation genomic DNA. Qualitative and quantitative PCR assays were developed employing the designed primers and probe. The detection limit of the qualitative PCR assay was 0.05% for Moonlite in 100 ng total carnation genomic DNA, corresponding to about 79 copies of the carnation haploid genome; the limit of detection and quantification of the quantitative PCR assay were estimated to be 38 and 190 copies of haploid carnation genomic DNA, respectively. Carnation samples with different contents of genetically modified components were quantified and the bias between the observed and true values of three samples were lower than the acceptance criterion (<25%) of the GMO detection method. These results indicated that these event-specific methods would be useful for the identification and quantification of the GMO carnation Moonlite.

Gold nanoparticles-based electrochemical method for the detection of protein kinase with a peptide-like inhibitor as the bioreceptor

PubMed Central

Sun, Kai; Chang, Yong; Zhou, Binbin; Wang, Xiaojin; Liu, Lin

2017-01-01

This article presents a general method for the detection of protein kinase with a peptide-like kinase inhibitor as the bioreceptor, and it was done by converting gold nanoparticles (AuNPs)-based colorimetric assay into sensitive electrochemical analysis. In the colorimetric assay, the kinase-specific aptameric peptide triggered the aggregation of AuNPs in solution. However, the specific binding of peptide to the target protein (kinase) inhibited its ability to trigger the assembly of AuNPs. In the electrochemical analysis, peptides immobilized on a gold electrode and presented as solution triggered together the in situ formation of AuNPs-based network architecture on the electrode surface. Nevertheless, the formation of peptide–kinase complex on the electrode surface made the peptide-triggered AuNPs assembly difficult. Electrochemical impedance spectroscopy was used to measure the change in surface property in the binding events. When a ferrocene-labeled peptide (Fc-peptide) was used in this design, the network of AuNPs/Fc-peptide produced a good voltammetric signal. The competitive assay allowed for the detection of protein kinase A with a detection limit of 20 mU/mL. This work should be valuable for designing novel optical or electronic biosensors and likely lead to many detection applications. PMID:28331314

Surface enhanced Raman spectroscopy based nanoparticle assays for rapid, point-of-care diagnostics

NASA Astrophysics Data System (ADS)

Driscoll, Ashley J.

Nucleotide and immunoassays are important tools for disease diagnostics. Many of the current laboratory-based analytical diagnostic techniques require multiple assay steps and long incubation times before results are acquired. In the development of bioassays designed for detecting the emergence and spread of diseases in point-of-care (POC) and remote settings, more rapid and portable analytical methods are necessary. Nanoparticles provide simple and reproducible synthetic methods for the preparation of substrates that can be applied in colloidal assays, providing gains in kinetics due to miniaturization and plasmonic substrates for surface enhanced spectroscopies. Specifically, surface enhanced Raman spectroscopy (SERS) is finding broad application as a signal transduction method in immunological and nucleotide assays due to the production of narrow spectral peaks from the scattering molecules and the potential for simultaneous multiple analyte detection. The application of SERS to a no-wash, magnetic capture assay for the detection of West Nile Virus Envelope and Rift Valley Fever Virus N antigens is described. The platform utilizes colloid based capture of the target antigen in solution, magnetic collection of the immunocomplexes and acquisition of SERS spectra by a handheld Raman spectrometer. The reagents for a core-shell nanoparticle, SERS based assay designed for the capture of target microRNA implicated in acute myocardial infarction are also characterized. Several new, small molecule Raman scatterers are introduced and used to analyze the enhancing properties of the synthesized gold coated-magnetic nanoparticles. Nucleotide and immunoassay platforms have shown improvements in speed and analyte capture through the miniaturization of the capture surface and particle-based capture systems can provide a route to further surface miniaturization. A reaction-diffusion model of the colloidal assay platform is presented to understand the interplay of system parameters such as particle diameter, initial analyte concentration and dissociation constants. The projected sensitivities over a broad range of assay conditions are examined and the governing regime of particle systems reported. The results provide metrics in the design of more robust analytics that are of particular interest for POC diagnostics.

Base-driven sunlight oxidation of silver nanoprisms for label-free visual colorimetric detection of hexahydro-1,3,5-trinitro-1,3,5-triazine explosive.

PubMed

He, Yi; Wang, Li

2017-05-05

Here we report a label-free method for visual colorimetric detection of hexahydro-1,3,5-trinitro-1,3,5-triazine (HTT) explosive based on base-driven sunlight oxidation of silver nanoprisms (AgNPRs). Under natural sunlight illumination, the surface plasmon of AgNPRs is excited, which populates O 2 antibonding orbitals to generate negative-ion state (O 2 - ). The resultant O 2 - with a strong oxidation activity can etch AgNPRs to smaller nanodisks with the aid of NaOH aqueous solution, leading to a blue shift of the absorption peak and color change from blue to pink. However, when HTT is introduced, the resultant O 2 - will be consumed by the nitrite and formaldehyde that are produced from the alkaline hydrolysis of HTT. Under this condition, the etching of AgNPRs does not occur, and the detection solution remains blue. This assay can sensitively detect as low as 1nM HTT, a level which is three orders of magnitude lower than that of gold nanoparticle-based colorimetric assays (2.6μM), and shows linearity in the range of 0.003-3.3μM. The lowest detectable concentration with the naked eye is 0.1μM. Additionally, the present assay exhibits good selectivity, and can be applied in the detection of HTT in natural water and soil samples with recoveries ranging from 90% to 100%. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative and discriminative analysis of nucleic acid samples using luminometric nonspecific nanoparticle methods

NASA Astrophysics Data System (ADS)

Pihlasalo, S.; Mariani, L.; Härmä, H.

2016-03-01

Homogeneous simple assays utilizing luminescence quenching and time-resolved luminescence resonance energy transfer (TR-LRET) were developed for the quantification of nucleic acids without sequence information. Nucleic acids prevent the adsorption of a protein to europium nanoparticles which is detected as a luminescence quenching of europium nanoparticles with a soluble quencher or as a decrease of TR-LRET from europium nanoparticles to the acceptor dye. Contrary to the existing methods based on fluorescent dye binding to nucleic acids, equal sensitivities for both single- (ssDNA) and double-stranded DNA (dsDNA) were measured and a detection limit of 60 pg was calculated for the quenching assay. The average coefficient of variation was 5% for the quenching assay and 8% for the TR-LRET assay. The TR-LRET assay was also combined with a nucleic acid dye selective to dsDNA in a single tube assay to measure the total concentration of DNA and the ratio of ssDNA and dsDNA in the mixture. To our knowledge, such a multiplexed assay is not accomplished with commercially available assays.Homogeneous simple assays utilizing luminescence quenching and time-resolved luminescence resonance energy transfer (TR-LRET) were developed for the quantification of nucleic acids without sequence information. Nucleic acids prevent the adsorption of a protein to europium nanoparticles which is detected as a luminescence quenching of europium nanoparticles with a soluble quencher or as a decrease of TR-LRET from europium nanoparticles to the acceptor dye. Contrary to the existing methods based on fluorescent dye binding to nucleic acids, equal sensitivities for both single- (ssDNA) and double-stranded DNA (dsDNA) were measured and a detection limit of 60 pg was calculated for the quenching assay. The average coefficient of variation was 5% for the quenching assay and 8% for the TR-LRET assay. The TR-LRET assay was also combined with a nucleic acid dye selective to dsDNA in a single tube assay to measure the total concentration of DNA and the ratio of ssDNA and dsDNA in the mixture. To our knowledge, such a multiplexed assay is not accomplished with commercially available assays. Electronic supplementary information (ESI) available: The labeling of amino modified polystyrene nanoparticles with Eu3+ chelate and the experimental details and results for the optimization of nucleic acid binding protein and for the ratiometric measurement of DNA and RNA with quenching assay. See DOI: 10.1039/c5nr09252c

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

PubMed Central

Euler, Milena; Wang, Yongjie; Heidenreich, Doris; Patel, Pranav; Strohmeier, Oliver; Hakenberg, Sydney; Niedrig, Matthias; Hufert, Frank T.

2013-01-01

Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms. PMID:23345286

Measurement of O-GlcNAcylated endothelial nitric oxide synthase by using 2',5'-ADP-Sepharose pull-down assay.

PubMed

Long, Yang; Yan, Jianghong; Luo, Suxin; Liu, Zhenguo; Xia, Yong

2017-11-15

Endothelial nitric oxide synthase (eNOS) plays central roles in cardiovascular regulation and disease. eNOS function is critically affected by O-linked N-acetylglucosamine (O-GlcNAc) modification. The present method for measuring O-GlcNAcylated eNOS relies on immunoprecipitation. Such method exhibits low detection efficiency and is also costly. We here report a simplified assay by employing the high binding affinity of eNOS with the 2',5'-ADP-Sepharose resins. Together with the O-GlcNAc antibody, this assay readily allows the detection of O-GlcNAcylated eNOS in both cultured endothelial cells and rat vascular tissues. By using this assay, we demonstrate that eNOS O-GlcNAcylation is markedly elevated in the vessels of diabetic rats. Thus, a 2',5'-ADP-Sepharose-based pull-down assay is developed to measure O-GlcNAcylated eNOS. This assay is simple and efficient in detecting O-GlcNAcylated eNOS in cultured cells and animal tissues under both normal and disease conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

Loop-mediated isothermal amplification (LAMP) assay for the identification of Echinococcus multilocularis infections in canine definitive hosts.

PubMed

Ni, Xingwei; McManus, Donald P; Yan, Hongbin; Yang, Jifei; Lou, Zhongzi; Li, Hongmin; Li, Li; Lei, Mengtong; Cai, Jinzhong; Fan, Yanlei; Li, Chunhua; Liu, Quanyuan; Shi, Wangui; Liu, Xu; Zheng, Yadong; Fu, Baoquan; Yang, Yurong; Jia, Wanzhong

2014-05-30

Alveolar echinococcosis, caused by the metacestode larval stage of Echinococcus multilocularis, is a zoonosis of public health significance and is highly prevalent in northwest China. To effectively monitor its transmission, we developed a new rapid and cheap diagnostic assay, based on loop-mediated isothermal amplification (LAMP), to identify canine definitive hosts infected with E. multilocularis. The primers used in the LAMP assay were based on the mitochondrial nad5 gene of E. multilocularis and were designed using Primer Explorer V4 software. The developed LAMP assay was compared with a conventional PCR assay, using DNA extracted from the feces of dogs experimentally infected with E. multilocularis, on 189 dog fecal samples collected from three E. multilocularis-endemic regions in Qinghai province, the People's Republic of China, and 30 negative control copro-samples from dogs from an area in Gansu province that had been subjected to an intensive de-worming program. Light microscopy was also used to examine the experimentally obtained and field collected dog copro-samples for the presence of E. multilocularis eggs. The E. multilocularis-positivity rates obtained for the field-collected fecal samples were 16.4% and 5.3% by the LAMP and PCR assays, respectively, and all samples obtained from the control dogs were negative. The LAMP assay was able to detect E. multilocularis DNA in the feces of experimentally infected dogs at 12 days post-infection, whereas the PCR assay was positive on the 17th day and eggs were first detectable by light microscopy at day 44 post-challenge. The earlier specific detection of an E. multilocularis infection in dog copro-samples indicates that the LAMP assay we developed is a realistic alternative method for the field surveillance of canines in echinococcosis-endemic areas.

Photonic Crystal Enhanced Fluorescence for Early Breast Cancer Biomarker Detection

PubMed Central

Cunningham, Brian T.; Zangar, Richard C.

2013-01-01

Photonic crystal surfaces offer a compelling platform for improving the sensitivity of surface-based fluorescent assays used in disease diagnostics. Through the complementary processes of photonic crystal enhanced excitation and enhanced extraction, a periodic dielectric-based nanostructured surface can simultaneously increase the electric field intensity experienced by surface-bound fluorophores and increase the collection efficiency of emitted fluorescent photons. Through the ability to inexpensively fabricate photonic crystal surfaces over substantial surface areas, they are amenable to single-use applications in biological sensing, such as disease biomarker detection in serum. In this review, we will describe the motivation for implementing high-sensitivity, multiplexed biomarker detection in the context of breast cancer diagnosis. We will summarize recent efforts to improve the detection limits of such assays though the use of photonic crystal surfaces. Reduction of detection limits is driven by low autofluorescent substrates for photonic crystal fabrication, and detection instruments that take advantage of their unique features. PMID:22736539

Quantitative Fissile Assay In Used Fuel Using LSDS System

NASA Astrophysics Data System (ADS)

Lee, YongDeok; Jeon, Ju Young; Park, Chang-Je

2017-09-01

A quantitative assay of isotopic fissile materials (U235, Pu239, Pu241) was done at Korea Atomic Energy Research Institute (KAERI), using lead slowing down spectrometer (LSDS). The optimum design of LSDS was performed based on economics, easy maintenance and assay effectiveness. LSDS system consists of spectrometer, neutron source, detection and control. LSDS system induces fissile fission and fast neutrons are collected at fission chamber. The detected signal has a direct relation to the mass of existing fissile isotopes. Many current commercial assay technologies have a limitation in direct application on isotopic fissile assay of spent fuel, except chemical analysis. In the designed system, the fissile assay model was setup and the correction factor for self-shield was obtained. The isotopic fissile content assay was performed by changing the content of Pu239. Based on the fuel rod, the isotopic content was consistent with 2% uncertainty for Pu239. By applying the covering (neutron absorber), the effective shielding was obtained and the activation was calculated on the target. From the assay evaluation, LSDS technique is very powerful and direct to analyze the isotopic fissile content. LSDS is applicable for nuclear fuel cycle and spent fuel management for safety and economics. Additionally, an accurate fissile content will contribute to the international transparency and credibility on spent fuel.

A CE-FL based method for real-time detection of in-capillary self-assembly of the nanoconjugates of polycysteine ligand and quantum dots.

PubMed

Wang, Jianhao; Zhu, Zhilan; Qiu, Lin; Wang, Jianpeng; Wang, Xiang; Xiao, Qicai; Xia, Jiang; Liu, Li; Liu, Xiaoqian; Feng, Wei; Wang, Jinmei; Miao, Peng; Gao, Liqian

2018-07-06

Small molecules with free thiol groups always show high binding affinity to quantum dots (QDs). However, it is still highly challenging to detect the binding capacity between thiol-containing molecules and QDs inside a capillary. To conquer this limitation, a capillary electrophoresis with fluorescence detection (CE-FL) based assay was proposed and established to investigate the binding capacity between QDs and a poly-thiolated peptide (ATTO 590-DDSSGGCCPGCC, ATTO-C4). Interestingly, the results showed that interval time had a great influence on QDs and ATTO-C4 self-assembly, which can be attributed to longer interval time benefitting the binding of QDs to ATTO-C4. The stability assays on ATTO-C4-QD assembly indicated that high concentration of imidazole or GSH had a high capability of competing with the bound ATTO-C4, evidenced by dramatically dropping of S 625 /S 565 ratio from 0.78 to 0.30 or 0.29. Therefore, all these results above suggested that this novel CE-FL based detection assay could be successfully applied to the binding studies between QDs and thiol-containing biomolecules.

A CE-FL based method for real-time detection of in-capillary self-assembly of the nanoconjugates of polycysteine ligand and quantum dots

NASA Astrophysics Data System (ADS)

Wang, Jianhao; Zhu, Zhilan; Qiu, Lin; Wang, Jianpeng; Wang, Xiang; Xiao, Qicai; Xia, Jiang; Liu, Li; Liu, Xiaoqian; Feng, Wei; Wang, Jinmei; Miao, Peng; Gao, Liqian

2018-07-01

Small molecules with free thiol groups always show high binding affinity to quantum dots (QDs). However, it is still highly challenging to detect the binding capacity between thiol-containing molecules and QDs inside a capillary. To conquer this limitation, a capillary electrophoresis with fluorescence detection (CE-FL) based assay was proposed and established to investigate the binding capacity between QDs and a poly-thiolated peptide (ATTO 590-DDSSGGCCPGCC, ATTO-C4). Interestingly, the results showed that interval time had a great influence on QDs and ATTO-C4 self-assembly, which can be attributed to longer interval time benefitting the binding of QDs to ATTO-C4. The stability assays on ATTO-C4-QD assembly indicated that high concentration of imidazole or GSH had a high capability of competing with the bound ATTO-C4, evidenced by dramatically dropping of S 625/S 565 ratio from 0.78 to 0.30 or 0.29. Therefore, all these results above suggested that this novel CE-FL based detection assay could be successfully applied to the binding studies between QDs and thiol-containing biomolecules.