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Published on: March 17, 2016
Development of a revised ICC-qPCR method used for Pseudorabies virus inactivation validation study of biologically
Yu Zhang1, Le Zhang2, Xiaojie Duan2
1School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, 610031, China; National Institutes for Food and Drug Control, Beijing, 102629, China.
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.
Insights
A revised integrated cell culture-qPCR (ICC-qPCR) method accurately assesses virus transmission risk in biological materials. This method effectively validates Pseudorabies virus (PRV) inactivation, ensuring material safety.
Area of Science:
- Veterinary Virology
- Molecular Biology
- Biotechnology
Background:
- Evaluating virus transmission risk in biological materials is crucial for safety.
- Pseudorabies virus (PRV) poses a significant risk in biologically sourced materials.
- Existing methods for virus inactivation validation may lack precision or convenience.
Purpose of the Study:
- To develop and optimize an integrated cell culture-qPCR (ICC-qPCR) method for precise virus transmission risk evaluation.
- To apply the revised ICC-qPCR method for validating PRV inactivation in biologically sourced materials.
- To compare the efficacy of the revised ICC-qPCR with traditional methods.
Main Methods:
- Establishment and optimization of an integrated cell culture-qPCR (ICC-qPCR) assay for PRV.
- Optimization of post-infection period (12-hr) for detection limit and quantitative range.
- Validation using mimic samples with specific inoculation and washing protocols to eliminate false positives.
- Virus inactivation studies on acellular porcine corneas using varying irradiation doses (12 kGy and 25 kGy).
- Comparison of results from general ICC-qPCR, revised ICC-qPCR, and standard cell culture methods.
Main Results:
- The optimized ICC-qPCR achieved a detection limit of -0.25 Log10TCID50/100 μL and a quantitative range of 0.75-3.75 Logs.
- Specific protocols for mimic samples effectively eliminated false positive signals from non-infectious viruses.
- Revised ICC-qPCR showed high PRV inactivation values (4.85 Logs at 12 & 25 kGy) consistent with cell culture (5.08 Logs).
- General ICC-qPCR yielded lower inactivation values (2.49 & 2.31 Logs) compared to revised ICC-qPCR and cell culture.
Conclusions:
- The revised ICC-qPCR method offers a precise and convenient approach for evaluating virus transmission risk.
- This method is effective in validating PRV inactivation in biologically sourced materials.
- The revised ICC-qPCR demonstrates superior accuracy and consistency compared to general ICC-qPCR, with results comparable to traditional cell culture methods.
