Bacillus Anthracis And Vibrio Cholerae Real-time Quantitative Pcr For Rapid Detection Of The Establishment And Evaluation Of The System

Background:Anthrax is a zoonotic disease caused by the spore-forming bacterium Bacillus anthracis.The term anthrax is derived from the Greek word for coal,anthrakis, because of the black skin lesions characteristic of the disease.An epidemic of anthrax in 17^th century Europe caused an estimated 60,000 human deaths.The role of toxin in the pathogenesis of anthrax was demonstrated in 1954.Anthrax was first used effectively as a bioterrorist agent in 2001.There are three clinical forms of anthrax:cutaneous, gastrointestinal,and inhalation.Cutaneous anthrax can be diagnosed by the clinical feature and be confirmed by culture,but it's difficult for gastrointestinal and inhalation anthrax because of its similarity to other diseases.So the other laboratory tests that may assist in the diagnosis are polymerase chain reaction(PCR),serology(PA-based ELISA), and tissue immunohistochemistry.More and more means were used in detection of various bacteria along with the advance of test technology.The rapid,currency,high effect,obturated real-time PCR was extensive applied in scientific research and clinical diagnosis,especially in the guard of the terror,for it can rapid detect Bacillus anthracis and aviod the spread of the anthrax.In this study,we developed single and multiplex real-time PCR assays for the rapid identification of Bacillus anthracis isolates and evaluated this assay for specificity against related species and detected in spiked human faecal samples.Objective:To develop rapid,sensitive and specific TanMan real-time quantitative polymerase chain reaction(RQ-PCR) assays for the detection of the region(GS-group specific gene) on the Bacillus anthracis complete genome and Bacillus anthracis cryptic protein and protective antigen precursor(pagA) genes on the virulence plasmid pXO1. The assays were used to detect the human faecal samples that be spiked with human vaccine of Bacillus anthracis for ulterior more in-service use.Methods:we designed the specific primers and probes for the region(GS-group specific gene) on the Bacillus anthracis complete genome and Bacillus anthracis cryptic protein and protective antigen precursor(pagA) genes on the virulence plasmid pXO1, constructed the control plasmid DNA,used the TaqMan probes and the Smart CyclerⅡof Cepheid to develop real-time PCR assays for the rapid detection of Bacillus anthracis. The assays were evaluated by the human vaccine of Bacillus anthracis and used to detect the human faecal samples that be spiked with human vaccine of Bacillus anthracis. Results:The sensitivity of the single real-time PCR assays were 10¹ copies,100fg, 10²CFU per reaction for detection of the control plasmid DNA,the human vaccine DNA, DNA extracted from the in spiked human faecal samples.The sensitivity of the double real-time PCR assays were 10² copies,1pg,10²CFU per reaction for detection of the control plasmid DNA,the human vaccine DNA,DNA extracted from the in spiked human faecal samples.The assays,single and double real-time PCR assays,had high sensitivity and specificity.The other bacteria belong to Bacillus cereus group were negative with real-time PCR.All time to complete the real-time PCR was within 1hour. So the assays enables very rapid,specific,quantitative detection of Bacillus anthracis and be easily adaptable for diagnostic purposes in laboratory or wild.Conclusion:This single and multiplex TaqMan real-time PCR assays,we developed in the study,enables very rapid,sensitive and specific,quantitative detection of Bacillus anthracis and should be applied in versatile territory for instance clinical lab test,food sanitation,disease control and customs inspection and so on. Background:Vibrio cholerae is recognized as a leading human waterbome pathogen, and cause 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 O1 rfb region(a unique sequence from rfb gene cluster of V.cholerae serotypes O1,chosen and designated by author),a copy of O139 rfb region (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.cholerae serotypes O1 and O139,and were used to evalute the real-time PCR assays that we developed.The sensitivity of the assay is assessed by both standard templates and V.cholerae genomic DNA 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 single real-time PCR assays were 10¹ copies,100fg and 1pg, 10²CFU and 10³CFU per reaction for detection of the control plasmid DNA,Vibrio cholerae O1 and O139 strains vaccine DNA,DNA extracted from the human faecal samples that were spiked with Vibrio cholerae O1 and O139 strains vaccine.The sensitivity of the double real-time PCR assays were 10² copies,100fg and 1pg,10³CFU per reaction for detection of the control plasmid DNA,Vibrio cholerae O1 and O139 strains vaccine DNA,DNA extracted from the human faecal samples that were spiked with Vibrio cholerae O1 and O139 strains vaccine.The sensitivity of the triplex real-time PCR assays were same with the double real-time PCR assays.The TaqMan PCR assay was negative for all other species tested.The assays,single,double and triplex real-time PCR assays,had high sensitivity and specificity.The total assay could be completed in 1h.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.cholerae,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.