利用荧光RT-PCR法检测环境水体中诺如病毒

Detection of norovirus in environmental water by quantitative real-time RT-PCR

目的：建立一种快速敏感的方法检测环境水体中的诺如病毒。方法：选择诺如病毒阳性的粪便标本,10倍梯度稀释,模拟被污染水体,用16%聚乙二醇8000-0.525mol/L NaCl溶液浓缩病毒,用实时定量RT-PCR检测病毒核酸。结果：重组质粒标准品做标准曲线,标准品拷贝数的对数（x）与Ct值的关系为Ct=39.886-3.485x,相关系数R2=0.995,灵敏度为100拷贝,比普通RT-PCR高1个数量级,比ELISA高3个数量级,重复性佳,回收率在14%~44%之间,随着样品中NV浓度降低而下降,。用实时定量RT-PCR、常规RT-PCR和ELISA方法检测了42份现场水样,阳性率分别为40%、24%和0。结论：为环境水体中诺如病毒的检测提供了一种灵敏、可行的定量检测方法。

Objective：To develop a quick and sensitive approach to detect norovirus（NV） in environmental water.Methods： Clinical NV positive fecal samples were tenfold serially diluted to simulate polluted water.Virus was precipitated with 16%PEG8000-0.525 mol/L NaCl solution.Viral nucleic acid RNA was detected by quantitative real-time RT-PCR.Results： Recombinant plasmids were used for generating standard curve.The relation between logarithm of the amount of plasmids（x） and cycle threshold（Ct） was Ct=39.886-3.485x,with a correlation coefficient of 0.995.With well repeatability,the limitation of detection was 100 copies of viral genome,twice less than conventional RT-PCR and four times less than ELISA.Recovery rates were 14%~44%,and decreased according to the reduction of NV concentration in simulated samples.42 field water samples were tested by quantitative real-time RT-PCR,conventional RT-PCR and ELISA,and the positive rates were 40%,24% and 0%,respectively.Conclusion： A sensitive and practicable molecular method has been developed for detecting NV in environmental water.