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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(4.4×10-1~4.4×103TCID50/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1.0×103~3.2×104copies/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。
According to the conserved region of 5’UTR of enterovirus, a combination of cell culture and real-time fluorescence quantitative PCR (ICC-RT-q PCR) was developed for the detection of enterovirus in Hep-2 cells. Road virus approach. The sensitivity and correlation analysis of three detection methods of RT-q PCR, TCID50 and ICC-RT-q PCR were used to evaluate the feasibility of RT-q PCR and ICC-RT-q PCR in estimating waterborne infectious virus. The results showed that the RT-q PCR method overestimated the infectivity of the virus in water. The linear relationship between TCID50 and ICC-RT-q PCR was good at low concentrations of enterovirus (4.4 × 10-1 ~ 4.4 × 103TCID50 / m L) with a correlation coefficient of R2 = 0.99. The concentration of virus was 1.0 × 103 ~ 3.2 × 104copies / L by ICC-RT-q PCR and the estimated content was 3 ~ 30 TCID50 / L, which was similar to the result of virus infection of 11 ~ 29 TCID50 / L The rate was 83.3%. Higher than the TCID50 test results (33.3%). Therefore, the ICC-RT-q PCR method is rapid and sensitive, and can accurately quantify waterborne enterovirus.