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Development of a loop-mediated isothermal amplification assay for rapid detection of Yersinia enterocolitica via targeting a conserved locus.

ABSTRACT: BACKGROUND AND OBJECTIVES:Loop-mediated isothermal amplification is a novel nucleic acid amplification assay providing as a simple diagnostic tool for rapid identification of microbial diseases in developing countries. In this study, a LAMP assay was established for Yersinia enterocolitica, a leading cause of acute enterocolitis in young children. MATERIALS AND METHODS:LAMP assay was established with four primers targeting a specific locus for the detection of Y. enterocolitica. The assay was conducted at 65°C in thermo block for 90min. The sensitivity of LAMP was evaluated in comparison to conventional PCR using pTZ57R containing the target locus. Finally, specificity was assessed using DNA from common enteropathogenic bacteria. RESULTS:Results showed that the sensitivity of LAMP assay was 44-copy number, which was 10-fold higher than that of PCR. No cross-reactivity was observed when testing against other enteropathogenic pathogens. CONCLUSION:This study showed that LAMP assay is an alternative molecular diagnostic tool for infections with Y. enterocolitica. In addition, this method may be useful in diagnosis at field or in laboratories without PCR machine.

SUBMITTER: Ranjbar R

PROVIDER: S-EPMC4685161 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Development of a loop-mediated isothermal amplification assay for rapid detection of Yersinia enterocolitica via targeting a conserved locus.

Ranjbar Reza R Afshar Davoud D

Iranian journal of microbiology 20150801 4

<h4>Background and objectives</h4>Loop-mediated isothermal amplification is a novel nucleic acid amplification assay providing as a simple diagnostic tool for rapid identification of microbial diseases in developing countries. In this study, a LAMP assay was established for Yersinia enterocolitica, a leading cause of acute enterocolitis in young children.<h4>Materials and methods</h4>LAMP assay was established with four primers targeting a specific locus for the detection of Y. enterocolitica. T ...[more]

PMID: 26697156

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Project description:Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae) causes soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai. Multiple genomes of D. fangzhongdai, and other species of Dickeya, Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with tenfold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18-20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies.

Dataset Information

Development of a loop-mediated isothermal amplification assay for rapid detection of Yersinia enterocolitica via targeting a conserved locus.

Publications

Development of a loop-mediated isothermal amplification assay for rapid detection of Yersinia enterocolitica via targeting a conserved locus.

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