非洲猪瘟病毒p30基因纳米PCR检测方法的建立  

Establishment of detection method of African swine fever virus p30 gene by nano-PCR

作　　者：魏宇 王海璐 孙飞雁 王楠[3] 孙亮[3] 程悦宁[1] 郭利[1] WEI Yu;WANG Hai-lu;SUN Fei-yan;WANG Nan;SUN Liang;CHENG Yue-ning;GUO Li(Key Laboratory of Special,Animal,Epidemic Disease,Ministry of Agriculture and Rural Affairs/Institute of Special,Animal,and Plant Sciences,Chinese Academy of Agricultural,Sciences,Changchun 130112,China;College of Chinese Herbal,Medicine,Jilin Agricultural,University,Changchun 130118,China;Animal Disease Prevention and Control Center of Jilin Province,Changchun 130000,China)

机构地区：[1]中国农业科学院特产研究所农业农村部经济动物疫病重点实验室,吉林长春130112 [2]吉林农业大学中药材学院,吉林长春130118 [3]吉林省动物疫病预防控制中心,吉林长春130000

出　　处：《中国兽医科学》2022年第5期547-553,共7页Chinese Veterinary Science

基　　金：吉林省科技发展计划项目(20200301016RQ,20200402036NC)。

摘　　要：为了建立准确、敏感的早期快速检测非洲猪瘟病毒(ASFV)的分子生物学方法,根据ASFV中p30蛋白在感染机体中早期表达的特点,设计出1对ASFV中p30基因保守序列的特异性引物,应用纳米PCR建立一种适用于早期检测ASFV的方法,并对其敏感性、特异性和临床样品检测进行研究和分析。结果显示:该纳米PCR的最低检出限为2.47×10^(3)copies/μL,而普通PCR的最低检出限为2.47×10^(4)copies/μL,表明纳米PCR的敏感性是普通PCR的10倍;同时,与猪瘟病毒、猪圆环病毒2型、猪繁殖与呼吸综合征病毒、伪狂犬病病毒均未发生交叉反应。此外,分别应用所建立的ASFV p30基因纳米PCR和普通PCR对18份临床样品进行检测,纳米PCR的阳性率为33.33%,普通PCR的阳性率为22.22%。上述结果表明,建立的纳米PCR检测方法可以对早期感染ASFV的病猪进行快速临床检测,具有良好的敏感性和特异性,为非洲猪瘟的诊断和防控提供了一定的技术保障。In order to establish an accurate and sensitive molecular method for early rapid detection of African swine fever virus(ASFV),a pair of specific primers for the conserved sequence of p30 gene in ASFV was designed according to the characteristics of the early expression of p30 protein in ASFV in infected organisms.To establish a nano-PCR diagnostic method for early diagnosis of ASFV,its sensitivity,specificity and clinical sample detection were studied and analyzed.The results showed that the lowest detection limit of nano-PCR was 2.47×10^(3) copies/μL,and the lowest detection limit of ordinary PCR was 2.47×10^(4) copies/μL,so the nano-PCR was 10 times more sensitive than ordinary PCR.There was no cross reaction with classical swine fever virus,porcine circovirus type 2,porcine reproductive and respiratory syndrome virus,and pseudorabies virus.In addition,18 clinical samples were detected by nano-PCR and ordinary PCR of ASFV p30 gene,respectively.The positive rate of nano-PCR was 33.33%,and that of ordinary PCR was 22.22%.The established nano-PCR detection method was a rapid clinical detection with good specificity and sensitivity that can be performed on pigs infected with ASFV in the early stage,providing technical support for the diagnosis,prevention and control of ASFV.

关 键 词：非洲猪瘟病毒 P30基因 纳米PCR检测技术 早期检测 

分 类 号：S852.659.1[农业科学—基础兽医学]