Study On Rapid Detection Of Vibrio Cholerae By Loop-mediated Isothermal Amplification

ObjectiveVibrio cholerae is an important pathogen causing a severe enteric disease, Cholera, a life-threatening diarrhoea, named as No.2 disease in China. The governments annually spend a lot of manpower and material resources for the pathogen detection and disease prevention. The aims of this study were to research Loop-Mediated Isothermal Amplification(LAMP),a novel technology of nucleic acid amplification, to establish a rapid, reliable and importantly easy-to-use detection method ofⅤ. cholerae, providing references and sevices to the early diagnosis of pathogen and disease survey of Cholera.Contents and MethodsBy refering the resources from relative reports, the basic system of LAMP reaction was built, and the target genes ofⅤ. cholerae were comfirmed. The suitable target sequences of the genes(available from Genbank) were analyzed and selected for LAMP usage by some softwares, BLAST, GENtle, DNAMan and so on. The LAMP primers were designed by special software, PrimerExplorer version 4 on Eiken Genome Site. Based on the basic reaction system and the designed primers, the LAMP assay forⅤ. cholerae was developed on the realtime PCR cycler using fluorescent SYBR Green I for detection, improved through a series of optimization tests including temperature,time and component concentration of reaction, and evaluated through methodological comparision with traditional isolation and identification assays according to Cholera Prevention and Cure Manual(China Health Ministry) and a ordinary PCR assays on the basis of familiar target genes, ace, rtxA and ctxA ofⅤ. cholerae. ResultsThe LAMP detection method ofⅤ. cholerae aimed at two characteristic genes of extracellular secretion protein system M subunit (epsM) and cholera toxin A subunit (ctxA), the former having species specificity for all V. cholerae and the latter pointing at CT-producing V. cholerae strains. The assay correctly identified allⅤ. cholerae with epsM primers from 20 tested bacteria strains comprising 8 genera, and all 5 strains of CT-producing V. cholerae with ctxA primers.The detection limits of the assay in pure cultrue were determined to be 12.5cfu/μl(25 cfu per reaction) and 5cfu/μl(10 cfu per reaction) of V. cholerae cells for epsM and ctxA primers respectively, obviously lower than those two tested detection methods, especially the traditional isolation and identification assays, and the limits of the PCR assay were 100 cfu/reaction(rtxA) and 50 cfu/reaction(ctxA) respectively. Despite the results from the methodological evaluation on 75 practical samples that the LAMP assay was not obviously different to the PCR assay for the detection rate of V. cholerae(χ2=0.500, P>0.05,α2-side=0.05, Kappa=0.894), but both of them surpassed the traditional isolation and identification assays greately(P<0.05,α2-side=0.05).ConclusionsCompared with the traditional isolation and identification assays, the LAMP detection method and the PCR assay are with greatly higher specificity and sensitivity of the V. cholerae detection. But the LAMP method is faster than the latter, with a reaction in an hour, and more esay and convenient to use without specaial instruments such as PCR thermal cycler because of its isothermal amplification. So the LAMP assay is a more rapid, reliable, simple detection method ofⅤ. cholerae, worthy of popularization, with important significance to disease survey and emergency detection of Cholera.