The Establishment Of Rapid Detection System Of Vibrio Cholerae And The Anthrax Bacterium Gene

Background: Vibrio cholerae is recognized as a leading human waterborne pathogen, and causes severe diarrheal disease affecting thousands of people each year in developing countries.Traditional diagnostic testing for Vibrio is not always reliable, because this bacterium can enter a viable but nonculturable state. Therefore, nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing.Methods:In this article, A triplex, taqman real-time detection assay was developed for quantitative detection of V. cholerae. Primers and probes were designed targeting three nucleic acid sequences of two genes of potentially toxigenic strains of V. cholerae, encoding: cholera toxin subunit A (ctxA), O-antigen (rfb).Three kind of plasmids respectively carrying a copy of lrfb (a unique sequence from rib gene cluster of V. cholerae serotypes O1,chosen and designated by author), a copy of 9rib ((a unique sequence from rfb gene cluster of V. cholerae serotypes O139, chosen and designated by author) and a copy of ctx (a unique sequence from ctxA gene of V. cholerae serotypes O1 and O139, chosen and designated by author) were constructed, These plasmids were quantified by UV spectrophotometer and then seriesly diluted as as standard templates. Simulative clinic samples were also synthesized by stool from healthy volunteers spiked with vaccine cells of v.cholera serotypes O1 and O139. The sensitivity of the assay is assessed by both standard templates and simulative clinic samples. The assay was also applied to 23 species of other bacterial strains of vibrio or other genera for evaluating its specificity.Results: The sensitivity of the assay was 10 CFU per reactivation for plasmid templates and about 100 CFU per reactivation in simulative clinic samples respectively. The TaqMan PCR assay was negative for all other species tested. Quantification of the Vibrio cells was linear over at least 6 log units. The total assay could be completed in 3 h. Conclusion: The TaqMan assay developed in this study can be used as a rapid screening tool for the presence of V. cholerae in oysters, seawater and stool from clinic patients without prior isolation and characterization of the bacteria by traditional microbiological methods. It not only can be applied to quantification of v.cholera, but also can be used to differentiating V. cholerae serotype O139 from cholerae serotype O1 and detection of cholera toxin. This multiplex real-time PCR assay allows for a more reliable, rapid detection and identification of V. cholerae which is considerably faster than current conventional detection assays. Objective To establish a rapid assay with high sensitivity and specificity based on the sequences for group specific gene(GS) and pathogenicity island pag A gene.Methods The PCR primers and probes were designed after the whole sequence was systemically analyzed with bio-information tools and blasted with Genebank database. The amplicons were inserted into plasmids so that they could be used as the standard templates to evaluate the sensitivity of the diagnostic system. This assay was based on TaqMan probes and portable Smartcycle PCR machine.Result The detection level was approximately 100 copies per reaction. There was no cross-reaction with other species of Bacillus. This assay could be completed in one hour in laboratory.Conclusion The duplex TaqMan PCR assay could be used to detect Bacillus anthracis rapidly with high sensitivity and specificity.