Dataset Information

Development of a multiplexed bead-based suspension array for the detection and discrimination of pospiviroid plant pathogens.

ABSTRACT: Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for establishing thresholds for positivity for individual assays within multiplexed arrays. When applied to the multiplexed array data generated in this study, the new method was shown to have better control of false positives and false negative results than two other commonly used approaches for setting thresholds. The 11-plex Luminex MagPlex-TAG pospiviroid array described here has a unique hierarchical assay design, incorporating a near-universal assay in addition to nine species-specific assays, and a co-amplified plant internal control assay for quality assurance purposes. All assays of the multiplexed array were shown to be 100% specific, sensitive and reproducible. The multiplexed array described herein is robust, easy to use, displays unambiguous results and has strong potential for use in routine pospiviroid indexing to improve disease management strategies.

SUBMITTER: van Brunschot SL

PROVIDER: S-EPMC3880322 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Development of a multiplexed bead-based suspension array for the detection and discrimination of pospiviroid plant pathogens.

van Brunschot Sharon L SL Bergervoet Jan H W JH Pagendam Daniel E DE de Weerdt Marjanne M Geering Andrew D W AD Drenth André A van der Vlugt René A A RA

PloS one 20140103 1

Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for esta ...[more]

PMID: 24404188

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Project description:Slaughter pigs are monitored for the presence of the zoonotic pathogen Salmonella, using both serology and bacteriology. ELISAs used to investigate pig herds are based on the detection of antibodies against components of the Salmonella cell envelope. Nearly all Salmonella isolates in food-producing animals are serovars of Salmonella enterica subspecies enterica, distributed over various serogroups as determined by the composition of their lipopolysaccharide (LPS). ELISAs for Salmonella serology are usually based on serogroup B and C1 LPS, often combined with serogroup D or E LPS. Although C2 LPS may improve serology, use of C2 LPS in a broad ELISA was never achieved.To enable detection of serum antibodies against Salmonella in pigs, a bead-based suspension array was developed with five LPS variants (B, 2× C1, C2, D1), each conjugated to a different bead set using triazine chemistry. Reactivity of the beads was confirmed with rabbit agglutination sera and with experimental pig sera. With a mixture of bead sets, 175 sera from slaughter pigs were investigated for the presence of antibodies against Salmonella. With a combination of ROC analysis (B and D LPS) and a prevalence estimation based on historic data (C LPS), individual cut-offs were defined for each LPS-conjugated bead set, and assay performance was evaluated. Results of the suspension array (BC1C1C2D) suggest that more pigs are seroconverted than indicated by a commercial BC1D1-ELISA, and that most of these extra seropositive samples give a signal on one of the beads with C LPS. These results show that expansion of a standard panel with more C LPS variants improves antibody detection.A suspension array for Salmonella serology in pigs was developed, that detects more seropositive sera than ELISA, which is achieved by expanding the panel of Salmonella LPS variants, including C2 LPS. The results demonstrate that bead-based suspension arrays allow for testing of pig sera, with the advantage of being able to set cut-offs per antigen. Ultimately, this type of assay can be applied in routine veterinary serology to test for antibodies against multiple Salmonella serovars (or other pathogens) in one single serum sample, using up-to-date antigen panels.

Project description:BACKGROUND:Insect-borne diseases could induce severe symptoms in human and clinical signs in animals, such as febrility, erythra, arthralgia and hemorrhagic fever, and cause significant economic losses and pose public health threat all over the world. The significant advantages of Luminex xMAP technology are high-throughput, high parallel and automation. This study aimed to establish a liquid bead array based on Luminex xMAP technology that was able to simultaneously detect multiple insect-borne pathogens. METHODS:Specific probes and primers to detect the nucleic acid of 10 insect-borne pathogens were designed. Probes were coupled with fluorescent carboxylated microspheres. The parameters of the system were optimized, including ratio of forward/reverse primers (1:2), hybridization temperature (50 °C) and duration (30 min) and quantity of PCR product (2 ?l). The sensitivity and specificity of the system were also evaluated. Moreover mixed nucleic acid of 10 insect-borne pathogens, including Bluetongue virus, Epizootic hemorrhagic disease virus of deer, Coxiella burnetii, African swine fever virus, West Nile fever virus, Borrelia burgdorferi, vesicular stomatitis virus, Rift Valley fever virus, Ebola virus and Schmalenberg's disease virus, and 3000 clinical samples were tested for practicability. RESULTS:The optimized detection system showed high sensitivity, specificity and reproducibility. Each probe showed specific fluorescence signal intensity without any cross-hybridization for the other insect-borne pathogens tested, which included dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus, Xinjiang hemorrhagic fever virus, spotted fever group rickettsiae, ehrlichiae and chikungunya virus. The limit of detection was 10 copies of target gene. Insect-borne pathogens were successfully detected among the 3000 clinical samples, and the results were consistent with those obtained using gold-standard assays or commercial nucleic acid detection kits. CONCLUSIONS:This optimized liquid array detection system was high-throughput and highly specific and sensitive in screening of the insect-borne pathogens. It was promising in detection of these pathogens for molecular epidemiological studies.

Dataset Information

Development of a multiplexed bead-based suspension array for the detection and discrimination of pospiviroid plant pathogens.

Publications

Development of a multiplexed bead-based suspension array for the detection and discrimination of pospiviroid plant pathogens.

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