羊口疮病毒PMA-PCR检测方法的建立

doi:10.16431/j.cnki.1671-7236.2022.04.035

Abstract

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

CLC Number:

S852.65^＋4

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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Establishment of PMA-PCR Method for Detection of Orf Virus

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