目的:建立猪细小病毒（PPV）的细胞培养结合荧光定量PCR（ICC-qPCR）方法并进行优化,结合传统的病毒滴定（细胞培养法）及ICC-qPCR 方法,考察将其应用于病毒灭活验证研究的可行性。方法:将系列10 倍稀释的PPV 病毒接种于猪睾丸（ST）细胞,分别扩增培养0、12、18、24 h,考察理想的病毒扩增培养时间,确定ICC-qPCR 定量检测区间及获得病毒扩增倍数（K）;将病毒平行接种于病毒扩增组及非扩增的对照组,通过扩增组和对照组的系列病毒接种量与PCR 反应周期（Ct）值的拟合曲线,分别获得病毒扩增组及对照组的ICC-qPCR 检测病毒滴度（Ta、Tc）,结合扩增倍数（K）,根据公式[Log10（10Ta-10Tc）/（K-1）]直接计算样本中的感染性病毒滴度。最后,将ICC-qPCR、优化ICC-qPCR 方法和细胞培养法分别用于模拟样本（由不同比例感染性病毒和非感染性病毒组成）中的感染性病毒滴度测定,考察将其应用于病毒灭活验证研究的可行性。结果:PPV 接种后的最佳扩增培养时间为24 h,检测下限为-1 Log10TCID50·100 μL-1（Logs）,定量区间为0~5.00 Logs,扩增倍数为83.11。当模拟样本中的非感染性病毒含量较低或与感染性病毒等量时,ICC-qPCR、优化ICC-qPCR 方法和细胞培养法测得的感染性病毒滴度没有显著性差异;然而,当模拟样本中的非感染性病毒含量较高时,由优化的ICC-qPCR 方法和细胞培养法测得的感染性病毒滴度分别是1.46、1.50 Logs,二者高度一致,而常规ICC-qPCR 方法测得的结果（1.75 Logs）存在极显著性差异。结论:本研究所优化的ICC-qPCR 方法作为细胞培养法的替代方法,具有用于病毒灭活验证研究的前景。
Objective: To establish and optimize an integrated cell culture-qPCR（ICC-qPCR） alternative method for assessing porcine parvovirus（PPV）,and evaluate its feasibility when applied to the virus inactivation validation study by comparing with the paralleling carried out general ICC-qPCR and cell culture method for virus titration. Methods: A serial of 10-fold PPV dilutions were inoculated to swine testis（ST） cells and incubated for 0 h,12 h,18 h and 24 h,respectively,to find a reasonable virus amplification period,identify quantitative range of ICC-qPCR and calculate the virus amplification times（K）．The viruses in different titers were inoculated in ST cells in virus amplification group（with an amplification period） and control group（without amplification period）, respectively．And ICC-qPCR detected virus titers from virus amplification group and control group（Ta,Tc） were obtained according to the fitting curve of virus inoculation levels and corresponding PCR detected cycle threshold （Ct）,respectively．And incorporated to virus amplification time（K）,the infectious virus titer was directly calculated by a formulation[ Log10（10Ta-10Tc）/（K-1）]．The ICC-qPCR,optimized ICC-qPCR and cell culture method were performed to assess the infectious virus titers in the mimic samples（composed of infectious virus and noninfectious virus in different ratios）,respectively,in order to evaluate the feasibility of the optimized ICC-qPCR to be applied to the virus inactivation study. Results: The reasonable PPV amplification incubation period was identified at 24 h post-inoculation,achieving a reasonable detection limit（-1 Log10TCID50·100 μL-1,Logs） and quantification range（0 to 5.00 Logs）,and the virus amplification times was 83.11．When the level of noninfectious virus in the mimic samples was lower or equivalent with infectious virus,there were no significant differences among the infectious virus titers detected by the ICC-qPCR,optimized ICC-qPCR and cell culture method． However,when the level of noninfectious virus in the mimic samples was higher,the infectious virus titers detected by the optimized ICC-qPCR and cell culture were 1.46 and 1.50 Logs,respectively,which were highly consistent with each other,and there was a very significant difference from the result（1.75 Logs） detected by the general ICC-qPCR method. Conclusion: The optimized ICC-qPCR,as an alternative method to cell culture method for PPV titer assessment,has a prospect to be applied to the virus inactivation validation study.
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