猪嵴病毒和猪星状病毒双重RT-PCR检测方法的建立  

Establishment and a duplex RT-PCR assay for identification of porcine Kobuvirus and porcine astrovirus

作　　者：顾凡[1] 覃娟娟 蔡雨函[1] 李新琼[1] 黄剑波[1] 朱玲[1] 刘书亮[2] 徐志文[1,3] 

机构地区：[1]四川农业大学动物生物技术中心,四川雅安625014 [2]四川农业大学食品学院,四川雅安625014 [3]动物疫病与人类健康四川省重点实验室,四川雅安625014

出　　处：《中国预防兽医学报》2015年第4期279-281,290,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：教育部长江学者和创新团队发展计划(IRT13083)资助;教育部新世纪优秀人才支持计划(NCET 11-1059)~~

摘　　要：为建立同时检测猪嵴病毒(PKV)和猪星状病毒(Ast V)的检测方法,本研究根据PKV 3D基因和Ast V ORF2基因设计了两对特异性引物,通过优化反应条件,首次建立了PKV和Ast V双重RT-PCR检测方法。该方法可以同时扩增出PKV的358 bp和Ast V的938 bp特异性片段,而对猪伪狂犬病病毒、猪流行性腹泻病毒、猪传染性胃肠炎病毒及大肠杆菌等病原核酸扩增结果均为阴性,具有较强的特异性;对PKV和Ast V的最低检出量分别为1.51×106拷贝/μL和1.19×106拷贝/μL;利用该方法和单项RT-PCR方法对35份来自四川省部分猪场的仔猪腹泻病料样品进行检测。结果显示,PKV和Ast V的阳性检出率分别为80%和31.4%,两者符合率为100%。本研究建立的方法对PKV和Ast V的鉴别诊断和混合感染检测具有重要的应用价值。To establish a protocol for simultaneous detections of porcine Kobuvirus （PKV） and porcine astrovirus （AstV）, a duplex RT-PCR assay was developed with two pair of primers designed according to the PKV 3D gene and AstV ORF2 gene available in GenBank. The assay was highly specific to amplify the DNA fragments of 358 bp for PKV and 938 bp for AstV, respectively, and had no amplification for other related viruses. In addition, the sensitivity of the assay was 1.51 ×10^6 copies/μL for PKV and 1.19× 10^6 copies/μL for AstV DNA, which was 10 time more sensitive than that of single RT-PCR detections. Moreover, thlrty-five samples collected from piglets with diarrhea in Sichuan province were detected by the assay, and the positive rates of PKV and AstV were 80% and 31.4%, respectively, which were consistent with the single RT-PCR detection. Therefore, the duplex RT-PCR assay has a potential application in diagnosis and differential diagnosis in PKV and AstV mixed infections.

关 键 词：猪嵴病毒 猪星状病毒 双重RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学]