Development of a revised ICC-qPCR method used for Pseudorabies virus inactivation validation study of biologically sourced materials

Abstract

To develop a precise and convenient method to evaluate the virus transmission risk of biologically sourced materials, an integrated cell culture-qPCR (ICC-qPCR) method for Pseudorabies virus (PRV) was established and revised for applications to this new field. The optimized post-infection period was found at 12-hr to achieve a reasonable detection limit (−0.25 Log10TCID50/100 μL, Logs) and a quantitative range (0.75–3.75 Logs). The results of mimic samples suggested that three 10-fold dilutions at the time of virus inoculation combined with three washes after virus absorption, and the sets of non-amplified samples as controls could efficiently eliminate the false positive signals caused by high levels of noninfectious viruses. The virus inactivation validation studies of acellular porcine corneas suggested that the logs inactivation of PRV at 12 kGy irradiation dose obtained by general ICC-qPCR, revised ICC-qPCR and cell culture were 2.49, 4.85 and 5.08, respectively. At 25 kGy, those were 2.31, 4.85 and 5.08, respectively. The results obtained by the revised ICC-qPCR were consistent with cell culture and more precise than general ICC-qPCR. Therefore, the revised ICC-qPCR proposed in this study has an application prospect in the PRV inactivation validation studies of biologically sourced materials.