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目的建立活的非可培养(viable but nonculturable state,VBNC)状态痢疾志贺菌逆转录荧光定量PCR(reverse transcription quantitative PCR,RT-qPCR)检测方法。方法低温寡营养诱导痢疾志贺菌进入VBNC状态,用平板计数法和荧光显微镜检法确定细菌的可培养数和活菌数,以痢疾志贺菌的志贺毒素基因stx(GenBank:M19437)、侵袭性毒力基因ipaH(GenBank:NC_007607)为目的基因设计引物,提取RNA进行RT-qPCR检测,研究检出限并建立标准曲线,用加标于环境水体中的志贺菌水样进行方法验证。结果建立处于VBNC状态的痢疾志贺菌RT-qPCR检测技术,检测时间少于6 h,复杂环境水体中每500μl水样可培养的痢疾志贺菌检出限为1~5 cfu,VBNC状态的痢疾志贺菌3~25 cfu。结论该方法检测快速,可实现对VBNC状态志贺菌的检测,可用于复杂水样的直接检测,适用于水体污染调查和应急反应时快速筛查志贺菌。
Objective To establish a reverse transcription quantitative PCR (RT-qPCR) method for the detection of viable but nonculturable state (VBNC) in Shigella dysenteriae. Methods Low temperature oligotrophic induction of Shigella dysenteriae into VBNC state, the number of viable bacteria and count of viable bacteria were determined by plate counting and fluorescence microscopy. The Shiga toxin gene stx (ShiBank: M19437) Primers were designed for the target gene ipaH (GenBank: NC_007607). RNA was extracted for RT-qPCR. The detection limit was established and a standard curve was established. The method was validated by using water samples of Shigella spiked in water . Results The detection of Shigella dysenteriae in the VBNC state by RT-qPCR was established in less than 6 hours. The detection limit of Shigella dysenteriae per 500μl of water in complex environment water was 1 ~ 5 cfu, Shigella dysentery 3 ~ 25 cfu. Conclusion The method is rapid and can be used for the detection of Shigella flexneri in VBNC. It can be used for the direct detection of complex water samples and is suitable for rapid screening of Shigella in water pollution investigation and emergency response.