The Evaluation Of Virus Inactivation Effect By Using The Technique Of Long-fragment DNA Amplification

Objectives: Blood-transmitted viruses, such as HBV, HCV and HIV are great harmful to human being and more than 120 million people in our country were infected with HBV. Some virus diseases which were transmitted through blood or blood products occurred from time to time because of window periods on virus infection and emerging pathogens, as well as the flaw of detecting method. To improve the safety of blood or blood products, it is necessary to do virus inactivation treatment for them. Cell infection method was often used to test the results of virus inactivation in the research work. But for HBV and HCV, it is still difficult to evaluate their inactive effectiveness directly and objectively in vitro because no cell line is fit for culture them now. In this study, the technique of long fragment DNA amplification (Long-PCR) was investigated for its feasibility in the estimation of virus inactivation.Methods: Plasmids (pTAL-Seap) were used as test model in this experiment. After treated with special enzyme(BSPH-1) or ⁶⁰Co-γ radiation(30kGy), the plasmids were amplified in different fragments (0.3kb, 0.8kb, 1.7kb, 2.7kb, 4.8kb) by PCR method, and then Pseudorabie viruses (PRV), which are a model virus of HBV and have cell pathological effect (CPE) in vitro, were used as the target viruses. After replicated in vero cell line and treated by ⁶⁰Co-γ radiation, low pH(4.0±0.1), pasteurization (60±1.0℃), solvent/detergent(S/D, 0.3%TNBP + 1%TWEEN-80) and methylene blue /light methods (MB/L, 1.0uM MB, 40000 Lux) respectively, PRV nucleic acids were extracted and analyzed with Long-PCR and nest PCR methods. 5 pieces of primer and one common inner up primer were designed according to the conservative region of gD gene in PRV, which will be used to amplify the DNA fragments from 0.28kb to 3.9kb. At same time, the treated PRV was inoculated onto the vero cell. The CPE was observed and the virus titre was counted according to the Karber method.Results: The PCR results showed that amplification of DNA fragments within the breaking points of enzyme-digested Plasmids (pTAL-Seap) were positive and a...