长链核酸扩增技术评价病毒灭活效果的研究

Evaluation of virus inactivation effect by using technique of long-fragment DNA amplification

目的:探讨用长链核酸扩增技术评价病毒灭活效果的可行性。方法:针对伪狂犬病毒(pseudorabies virus,PRV)糖蛋白gD基因前后的保守区设计预计产物长短不一的5对引物,用半巢式PCR技术扩增经低pH法(4.0±0.1)、巴氏消毒法(60±1.0)℃和S/D法(有机溶剂/洗涤剂)处理后的PRV核酸,同时以细胞感染法做平行对照。结果:低pH对PRV核酸有破坏作用,处理时间越长,核酸损伤程度越明显,处理60min时,6.62lgTCID50的PRV完全被灭活。5条不同长度PCR扩增产物中,只有3.9kb的长片段检出与细胞培养结果一致。7.25lgTCID50的PRV经(60±1.0)℃处理20min后即被完全灭活,7.13lgTCID50的PRV经S/D法处理1h后被完全灭活,但各长度核酸片段扩增均为阳性,与细胞感染试验结果不符。结论:低pH对PRV核酸的损伤程度随处理时间的延长而增加;用长链PCR(3.9kb)技术来评价经低pH法灭活病毒的效果是可行的,而该法不适合评价巴氏消毒法和S/D法灭活病毒的效果。

Objective：In order to investigate the feasibility of using long fragment DNA amplification （long-PCR）to estimate virus inactivation. Methods： Five pairs of primers were designed according to conservative region of the gD gene of pseudorabies virus（PRV） and the extracted nucleic acid was amplified after the PRV was treated by low pH（4, 0±0. 1 ）, pasteurization （60 ± 1.0） ℃, solvent/detergent （ S/D, 0. 3% TNBP ± 1% Tween-80）, at the same time cell infection method was used as parallel control. Results：The longer the time of low pH treatment, the mare severe the damage on virus nucleic acids. 6.62 lgTCID50 PRV was inactivated completely when it was treated for 60 minutes, and only detection of 3.9 kb fragment was coincident with the result of cell culture. The 7.25 lgTCID50 PRV was inactivated completely when it was treated by pasteurization for 20 minutes, and the 7.13 lgTCID50 PRV was inactivated completely when it was treated by solvent/detergent for 1 h, both of the PCR results were not coincident with the result of cell infection test. Conclusion：The degree of destruction of PRV nucleic acid depend on the time of low pH treatment. It is feasible to use the technique of long-strand PCR（3.9 kb） to evaluats the virus inactivation efficacy of low pH treatment, but it is not suitable to pasteurization and S/D methods.