水中肠道病毒检测方法的分析评价  

Analysis and evaluation of detection methods for enteroviruses in water environments

作　　者：徐丽梅[1] 王晓昌[1] 周进宏[1] 徐丽华 吉铮[1] 张崇淼[1,3] 

机构地区：[1]西安建筑科技大学环境与市政工程学院,西安710055 [2]济南市环境保护规划设计研究院,济南250100 [3]清华大学国家环境保护环境微生物利用与安全控制重点实验室,北京100084

出　　处：《环境工程学报》2016年第2期978-984,共7页Chinese Journal of Environmental Engineering

基　　金：国家"水体污染控制与治理"科技重大专项(2013ZX07310-001)

摘　　要：采用Hep-2细胞分离培养水中的肠道病毒,根据肠道病毒5’-UTR核酸的保守区,建立了一种细胞培养与实时荧光定量PCR（ICC-RT-q PCR）联合检测感染性肠道病毒的方法。对RT-q PCR、TCID50和ICC-RT-q PCR 3种检测方法进行灵敏度、相关性分析,评价RT-q PCR、ICC-RT-q PCR用于估计水中感染性病毒含量的可行性。结果表明,RT-q PCR方法高估了水中病毒的感染性。肠道病毒低浓度（4.4×10-1-4.4×10^3TCID50/m L）时TCID50与ICC-RT-q PCR线性关系良好,相关系数R2=0.99。水样经浓缩,ICC-RT-q PCR检测病毒含量为1.0×10^3-3.2×104copies/L,估计含量为3-30 TCID50/L,与病毒感染性11-29 TCID50/L结果相近,检出率为83.3%。高于TCID50的检测结果（33.3%）。因此,ICC-RT-q PCR方法快速、灵敏,可对水中感染性肠道病毒进行准确的定量分析。Hep-2 cells were used to isolate and cultivate the enteroviruses in water environments. Based on the 5＇untranscribed region（ 5＇-UTR） gene of enteroviruses,a newly developed integrated cell culture and reverse transcription quantitative PCR（ ICC-RT-q PCR） assay was established to detect infectious enteroviruses. In this study,the sensitivity and correlation of three detection methods including the quantitative real-time q PCR（ RT-q PCR） method,50% tissue culture infective dose（ TCID50） and ICC-RT-q PCR method were compared and analyzed. The feasibility of RT-q PCR and ICC-RT-q PCR techniques to predict viral infectivity was evaluated. Results showed that naturally occurring infectious viruses were overestimated with the RT-q PCR method. The copies of 5＇-UTR gene of enteroviruses were linearly correlated（ with a coefficient（ R2） of 0. 99） with the initial inoculum concentrations ranging from 0. 44 to 4 400 TCID50/ m L. The concentration of entroviruses quantified by ICC-RT-q PCR was 1. 0 × 10^3 to 3. 2 × 104 copies / L,and was estimated at 3—30 TCID50/ L. It was similar to the concentrations of enteroviruses in water samples determined by TCID50 method. Infectious enteroviruses were detected to be positive in 83. 3%（ 5 /6） of samples by ICC-RT-q PCR. The positive rate was higher than 33. 3% detected by TCID50. Therefore,ICC-RT-q PCR assay was considered as a rapid and sensitive method to detect and quantify infectious viruses.

关 键 词：肠道病毒 感染性 TCID50 RT-QPCR ICC-RT-qPCR 

分 类 号：X832[环境科学与工程—环境工程]