ICC-qPCR病毒灭活验证方法的建立及其应用

目的：建立细胞培养-核酸扩增荧光定量PCR（integrated cell culture quantitative PCR，ICC-qPCR）病毒灭活验证方法；结合传统细胞病变效应（cytopathic effect，CPE）法，考察ICC-qPCR方法的适用性；利用这2种方法验证复用电生理导管环氧乙烷病毒灭活工艺的有效性。方法：通过制备指示病毒（伪狂犬病毒，PRV）质粒DNA作为定量标准品，用PRV特异性引物建立定量PCR方法；再用PRV病毒侵染宿主细胞，同时用热灭活PRV作为对照，确定PRV活病毒全部进入宿主细胞但不扩增的最佳样本收获时间，通过检测细胞中活病毒，建立ICC-qPCR活病毒检测方法；确立CPE法测定滴度与ICC-qPCR法检测DNA拷贝数之间的线性关系。最后，模拟临床使用的最坏情况对一次性电生理导管进行含指示病毒（PRV）生物污染物负载，模拟环氧乙烷病毒灭活工艺，通过ICC-qPCR和CPE法验证病毒灭活工艺的有效性。结果：本研究中建立的ICC-qPCR方法的标准曲线线性关系r²>0.99；定量下限为10⁴ copies&#183;μL^-1，约相当于2 logs&#183;mL^-1，检测下限为10³ copies&#183;μL^-1，约相当于-1 logs&#183;mL^-1；特异性结果表明只能检测到PRV的扩增曲线，无非特异性扩增；重复性试验的RSD<5%。确定ICC-qPCR方法中PRV最佳收获时间为宿主细胞染毒后1~2 h。ICC-qPCR检测拷贝数与病毒滴度间具有良好的线性关系（r²>0.99）。ICC-qPCR法病毒灭活工艺验证结果表明：经环氧乙烷灭活后病毒负载导管其病毒滴度降低>9.0 logs；阴性对照组（负载不含PRV生物污染物）和空白导管样品均未检出病毒DNA。CPE法的病毒滴定结果表明，经环氧乙烷灭活后病毒负载导管其病毒滴度降低≥ 8.5 logs&#183;mL^-1；阴性对照组和空白导管样品均未检出PRV。结论：本研究建立的ICC-qPCR方法应用于PRV病毒灭活验证，可以有效检测活病毒，比传统CPE法（-0.5 logs&#183;mL^-1）灵敏度高。将此方法应用于复用电生理导管的环氧乙烷病毒灭活工艺验证，同时与CPE法进行比较，验证了ICC-qPCR方法的适用性以及环氧乙烷灭活电生理导管负载的PRV病毒的工艺有效性。

Objective: To develop an integrated cell culture quantitative PCR (ICC-qPCR) method for validating the virus inactivation process;and by using the established ICC-qPCR method,in combination with the traditional cytopathic effect (CPE) method, to validate the inactivation of virus by ethylene oxide for the reused catheters. Methods: A quantitative PCR method using pseudorabies virus (PRV) plasmid-DNA as a quantitative standard and the PRV-specific polynucleotides as primers was established. Active PRV and the heat-inactivated control was spiked to host cells, and harvested for CPE and ICC-qPCR at the optimal post-spiking harvest time when virus have all infiltrated yet without amplification. After regression curve analyzing,the linearity between CPE and ICC-qPCR data was confirmed. To validate the inactivation of virus by ethylene oxide,virus-containing biological soil was loaded onto the disposable catheter simulating the worst-case contamination in clinical used before performing the inactivation process,and the residual live virus was determined by ICC-qPCR in combination with CPE method. Results: The established ICC-qPCR method showed a good linear relationship with that of the CPE method,with a correlation coefficient of r²>0.99.The limit of quantitation (LOQ) of the ICC-qPCR method was 10⁴ copies&#183;μL^-1 (about 2 logs&#183;mL^-1),while the limit of detection (LOD) was 10³ copies&#183;μL^-1 (about -1 logs&#183;mL^-1). The amplification curve showed a single peak only for the target PRV without non-specific amplified products. The RSD of repeatability tests was less than 5%. The optimized harvest time of cell-infiltrated live PRV was at 1 to 2 h after spiking. The established ICC-qPCR method also had a good linear relationship between virus DNA copies and virus titer (r²>0.99). The results of virus inactivation validation showed that after the ethylene oxide process the detected virus DNA was below the LOQ,suggesting that the spiked viruses were reduced by more than 9.0 logs, and no viral DNA was detected in the negative control (loading biological oil without PRV) or the blank control. Moreover, the results of CPE method showed that after the ethylene oxide process the virus titer was reduced by more than 8.5 logs&#183;mL^-1,and no PRV was detected in the negative control or the blank control. Conclusion: The ICC-qPCR method was well established,which can effectively detect live PRV within cells,and is more sensitive than traditional CPE method (-0.5 logs&#183;mL^-1) for virus inactivation validation. The results of validation showed that the application of inactivation of virus by ethylene oxide on reused catheters was effective both by ICC-qPCR method and CPE method.

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