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.
ZHANG Yu, GUO Jia-hua, QU Shu-xin, DUAN Xiao-jie, XU Li-ming
. Establishment of ICC-qPCR alternative method for assessing porcine parvovirus*[J]. Chinese Journal of Pharmaceutical Analysis, 2021
, 41(1)
: 97
-103
.
DOI: 10.16155/j.0254-1793.2021.01.11
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