羊口疮病毒PMA-PCR检测方法的建立

doi:10.16431/j.cnki.1671-7236.2022.04.035

摘要/Abstract

摘要： 【目的】为快速有效评估宿主动物体内及周围环境中的羊口疮病毒(Orf virus,OrfV)是否失活,本研究应用核酸染料叠氮溴化丙锭(propidium monoazide,PMA)联合PCR扩增技术,以F1L基因为靶向检测对象,旨在建立一种针对具有感染活性OrfV的PMA-PCR检测方法。【方法】将病毒悬液经不同温度相同时间的水浴处理,通过PCR确定病毒样本最佳的热灭活温度;分别设置PMA曝光时间梯度及浓度梯度,进一步对PMA的曝光时间和工作浓度等因素进行优化,确定有效区分具有感染活性OrfV和失活OrfV的PMA最佳处理条件;对所建立PMA-PCR方法进行特异性试验验证,并通过对不同数量比例的热灭活OrfV、活OrfV混合病毒悬液样本进行检测来验证该方法的敏感性。【结果】 OrfV样本经60 ℃热水浴处理10 min即可被完全灭活;PMA与失活OrfV的DNA共价结合且溶液中游离PMA光解的最佳曝光时间为15 min;热灭活OrfV基因组扩增被完全抑制的最小PMA浓度为20 μmol/L,活OrfV基因组扩增不受抑制的最大PMA浓度为35 μmol/L;特异性试验结果表明,该方法检测活OrfV时出现阳性条带,而口蹄疫病毒(FMDV)、绵羊痘病毒(SPPV)、山羊痘病毒(GTPV)的扩增结果均呈阴性;经PMA处理后,在不同数量比例的活、热灭活OrfV病毒悬液中检测到活OrfV的灵敏度为10^-1.68 TCID₅₀/0.1 mL。【结论】本试验成功建立了针对活OrfV的PMA-PCR检测技术,该方法特异性强、敏感度高,可为相关工作领域评估OrfV致病性提供技术参考。

关键词: 羊口疮病毒(OrfV); PMA-PCR; 活性检测

Abstract: 【Objective】 In order to quickly and effectively evaluate whether Orf virus (OrfV) in the host animal’s body and surrounding environments were pathogenic,the nucleic acid dye propidium bromide (PMA) combined with PCR amplification technology was used to target F1L gene,so as to establish a PMA-PCR detection method for OrfV with infectious activity.【Method】 The virus suspension was treated with water bath at different temperatures for the same time,and the optimal heat inactivation temperature of virus samples was determined by PCR.The exposure time gradient and concentration gradient of PMA were set respectively,and the exposure time and working concentration of PMA were further optimized to determine the best treatment conditions for PMA to effectively distinguish between infectious active OrfV and inactivated OrfV.The specificity of the established PMA-PCR method was verified by specificity test,and the sensitivity of the method was verified by detecting different proportion of heat inactivated OrfV and active OrfV mixed virus suspension samples.【Result】 OrfV samples could be completely inactivated after 10 min of treatment in a hot water bath at 60 ℃.PMA was covalently bound to the DNA of inactivated OrfV and the best exposure time for photolysis of free PMA in the solution was 15 min.The minimum PMA concentration at which the amplification of heat inactivated OrfV genome was completely inhibited was 20 μmol/L,the maximum PMA concentration of uninhibited amplification of active OrfV genome was 35 μmol／L.Specific test result showed that this method had a positive band when detecting active OrfV.The amplification results of the FMDV,SPPV and GTPV groups were all negative.After PMA treatment,the sensitivity of detecting active OrfV in different ratios of live and heat-inactivated OrfV suspensions was 10^-1.68 TCID₅₀/0.1 mL.【Conclusion】 This experiment had successfully established a PMA-PCR detection technology for active OrfV,which had strong specificity and high sensitivity,and could provide technical reference for evaluating the pathogenicity of OrfV in related work areas.

Key words: Orf virus (OrfV); PMA-PCR; activity detection

中图分类号:

S852.65^＋4

梁倩, 包涛涛, 鲜思美, 杨倩, 李鹏飞, 顾庆林, 郑维豪, 杜鹏, 青成欣. 羊口疮病毒PMA-PCR检测方法的建立[J]. 中国畜牧兽医, 2022, 49(4): 1524-1531.

LIANG Qian, BAO Taotao, XIAN Simei, YANG Qian, LI Pengfei, GU Qinglin, ZHENG Weihao, DU Peng, QING Chengxin. Establishment of PMA-PCR Method for Detection of Orf Virus[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1524-1531.

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