检测DENV-2活病毒含量RTCA标准曲线法的建立

A method of evaluating the live DNEV-2 titer using the standard curve according to RTCA

目的利用实时无标记细胞分析系统(real-time cellular analysis,RTCA)建立半数细胞指数时间(CIT50)与病毒滴度(TCID50)相关性的标准曲线,评估RTCA技术在监测2型登革病毒(DENV-2)感染BHK细胞后引发细胞病变效应(CPE)中的可行性和应用价值。方法检测不同培养条件下BHK细胞的CI值变化,筛选合适的初始细胞接种密度及血清浓度;应用RTCA分析已知不同TCID50滴度DENV-2感染BHK细胞的CIT50值,构建CIT50-TCID50标准曲线,计算回归方程;收集DENV-2感染C6/36细胞后第1~4d的培养上清(D1,D2,D3,D4),利用RTCA法和TCID50法分别检测上清中DENV-2滴度,分析CIT50-TCID50标准曲线法的灵敏度和可行性。结果 BHK细胞以初始接种密度为2×104个/孔,血清浓度为2%的培养条件为最佳;不同滴度DENV-2感染后BHK细胞的生长曲线均左移,CIT50与TCID50间呈线性负相关,线性方程为y=-7.007x+75.546(R2=0.9854);不同时间感染C6/36细胞培养上清中病毒含量(logTCID50/mL):RTCA标准曲线法为4.90±0.05(D2)、6.18±0.20(D3)、6.04±0.10(D4);传统TCID50法为0(D1)、6.25±0.49(D3)、5.87±0.35(D4)。结论利用RTCA技术建立的CIT50-TCID50标准曲线法,可用于已知DENV-2在BHK细胞的定量检测,结果较TCID50法更客观,且该法操作便捷且灵敏度高,为评估DENV-2感染性活病毒颗粒的毒力提供了新的技术手段。

Objective To use real-time cellular analysis（RTCA）to determine the standard curve for the time for the cell index to decrease 50%（CIT50）and the 50%tissue culture infectious dose（TCID 50）in order to evaluate the feasibility of using RTCA to detect the cytopathic effect（CPE）of DENV-2 in BHK cells. Methods To select the optimal cell concentration for inoculation and the optimal serum concentration,the cell index（CI）curve of BHK cell of different initial numbers of cells（1,2,and 4×10^4 cells/well）and different serum concentrations（0,2,510%FBS）was dynamically monitored with RTCA.The standard curve for CIT50-TCID50 and the equation for linear regression were determined by analyzing the CIT50 in BHK cells infected with DENV-2 at different TCID50.The live DENV-2 titers in infected C6/36 cell culture supernatant at different stages of infection（D1,D2,D3,and D4）were contrasted with the standard curve according to RTCA and conventional TCID50. Results The optimal conditions for detecting the DENV-2 titer were 2×10^4 BHK cells per well and a concentration of 2% FBS in DMEM medium.The growth curve for BHK cells infected with different titers of DENV-2 shifted to the left.The CIT50 of infected BHK cells was significantly inversely correlated with the logTCID50 of DENV-2,and the equation of linear regression was y=-7.007 x＋75.546,with R2=0.9854.The live DENV-2 titer（logTCID50/ml）was calculated using the standard curve according to RTCA.The live DENV-2 titer in C6/36 cell culture supernatant was 4.90±0.05 at D2,6.18±0.20 at D3,and 6.04±0.10 at D4.The titer was not detected at D2,it was 6.25±0.49 at D3,and it was 5.87±0.35 at D4 according to conventional TCID50.The live DENV-2 titer according to the two methods did not differ significantly（P〉0.05）,but the standard curve according to RTCA was more sensitive.Conclusion Results indicated that RTCA is a more objective,convenient,and sensitive method of detecting live DENV-2particles in BHK cells than conventional TCID 50.The standard curve according to RTCA would be a useful