基于重组酶介导等温扩增技术的羊口疮病毒核酸检测方法的建立及初步应用  

Establishment and preliminary application of a nucleic acid detection method for Orf virus based on recombinase-mediated isothermal amplification technology

作　　者：张学勇[1,2] 简莹娜 李志 郭志宏 严永盛[1,2] 朵红 ZHANG Xue-yong;JIAN Ying-na;LI Zhi;GUO Zhi-hong;YAN Yong-sheng;DUO Hong(The Academy of Animal and Veterinary Sciences,Qinghai University,Xining 810016,China;Qinghai Provincial Key Laboratory of Pathogen Diagnosis for Animal Disease and Green Technical Research for Prevention and Control,Xining 810016,China)

机构地区：[1]青海大学畜牧兽医科学院,青海西宁810016 [2]青海省动物疾病病原诊断与绿色防控技术研究重点实验室,青海西宁810016

出　　处：《中国预防兽医学报》2023年第3期271-275,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：青海省科学技术厅重点研发与转化计划项目:羊主要病毒性疫病防控新技术研究示范(2022-QY-207);青海省“昆仑英才·高端创新创业人才”项目。

摘　　要：为建立羊口疮病毒(Orf virus,ORFV)核酸的快速检测方法,本研究根据ORFV B2L基因保守序列设计特异性引物,通过优化反应温度与时间初步建立了基于重组酶介导等温扩增技术(RAA)的ORFV检测方法。优化试验结果显示:该方法在37℃反应40 min检测效果最佳。采用该方法对ORFV、山羊痘病毒、O型和A型口蹄疫病毒、小反刍兽疫病毒、山羊副流感病毒3型等临床常见症状相似的病毒核酸检测,结果显示:该方法除对ORFV的检测结果为阳性外,对羊的其他病毒的检测结果均为阴性,特异性较强。将质粒标准品10倍倍比稀释(1×10^(9)拷贝/μL~1×10^(0)拷贝/μL)后为模板,利用本研究建立的RAA方法检测,结果显示:该方法对ORFV质粒标准品的最低检测限为1×10^(4)拷贝/μL,敏感性较高。利用本研究建立的方法对95份临床样品(15份组织样品和80份血液样品)检测,结果显示:该RAA方法能够对临床样品快速检测,组织样品和血液样品的阳性率分别为33.33%(5/15)和6.25%(5/80),该检测结果与普通PCR检测方法的符合率均为100%,表明本实验建立的RAA方法能够满足临床样品的检测需求。本研究建立的ORFV RAA方法具有操作简单、特异性强、反应快速等特点,为ORFV的快速检测提供新的技术选择。To establish a rapid nucleic acid detection method for Orf virus(ORFV),we designed specific primers based on the conserved sequence of ORFV B2L gene.The ORFV detection method based on recombinase-mediated isothermal amplification(RAA)was established by optimizing the reaction time and temperature.The optimized test results showed that the method had the best detection efficiency at 37℃for 40 minues.We further used the method to detect common clinically viral pathogens with similar symptoms,such as ORFV,goat pox virus,O and A type foot-and-mouth disease virus,peste des petits ruminant virus,caprine parainfluenza virus type 3 and the results showed that this RAA method was specific for ORFV detection.The plasmid standard was diluted 10 times(1×10^(9) copies/μL-1×10^(0) copies/μL)as template,the results of sensitivity test showed that the detection limit of the RAA method using ORFV plasmid standard was 1×10^(4) copies/μL,indicating a high sensitivity of the method.In addition,95 clinical samples(15 tissue samples and 80 blood samples)were detected by the established RAA detection method and the common PCR method at the same time.The results showed that the positive rates of tissue samples were 33.33%(5/15)and blood samples were 6.25%(5/80)by using both methods,however,the RAA method was more efficient.The RAA nucleic acid detection method for detection of ORFV established in this study has the characteristics of simple operation,strong specificity and rapid response,providing a new choice for ORFV detection.

关 键 词：羊口疮病毒 B2L基因 重组酶介导的等温扩增技术 快速检测 

分 类 号：S855.3[农业科学—临床兽医学]