猪流行性腹泻病毒RT-PCR鉴别诊断方法的建立  

Development of a RT-PCR Method for Differentiation of the Wild-Type PEDVs and the Attenuated PEDVs

作　　者：吴玉璐[1] 程群[1] 虞凌雪[1] 侯怡轩[2] 王康[1] 刘光清[1] 童光志[1] 周艳君[1] 

机构地区：[1]中国农业科学院上海兽医研究所猪传染病研究室,上海200241 [2]上海交通大学农业与生物学院,上海200240

出　　处：《中国农业科学》2013年第20期4370-4377,共8页Scientia Agricultura Sinica

基　　金：国家生猪现代产业技术体系建设项目(NYCYTX-009)

摘　　要：【目的】利用RT-PCR技术建立一种在临床上便于区分猪流行性腹泻病毒(PEDV)自然感染与疫苗免疫毒株的方法。【方法】根据GenBank公布的PEDV ORF3基因序列,在其存在基因缺失区两端的保守区域设计特异性引物,对近两年采集的仔猪腹泻样品进行检测,分析流行毒株的ORF3基因,选择部分阳性样品利用鉴定引物进行RT-PCR扩增,优化其反应体系、Tm值等条件,进行鉴别诊断方法的特异性、敏感性试验,并对大量临床样品进行检测验证。【结果】从新发仔猪腹泻临床样品中克隆获得了11株PEDV的ORF3基因,序列分析显示新分离的9株ORF3基因不存在缺失,属于自然感染野毒,其与疫苗株的核苷酸同源性为95.8%—97.1%。建立的鉴别诊断方法可以特异性扩增PEDV的ORF3基因,其中获得的PEDV自然感染毒株基因片段大小约300 bp,而弱毒疫苗株则为250 bp左右;该鉴别诊断方法与其他猪源病毒无交叉扩增,其敏感性可达到100TCID50/0.1mL,对仔猪腹泻临床样品检测结果显示,PEDV自然感染的阳性率为65.4%。【结论】初步建立了基于PEDV ORF3基因的RT-PCR鉴别诊断方法,该方法可以用于区分自然感染的野毒和弱毒疫苗免疫毒株,为PEDV的疫情诊断和流行病学监测提供了一种特异、快速的检测方法。[ Objective ] The objective of this study is to establish an effective tool for differentiating diagnosis of the PEDVs in epidemiological investigations by the method of RT-PCR. [Method] According to the truncation in the ORF3 gene of PEDV reference strains in GenBank, the wild-type PEDV isolates and attenuated isolates, two pairs of primers were designed. The clinical samples from different farms occurred with diarrhea epidemic were tested, and their ORF3 gene was analyzed. Some representative samples were selected and amplified by the primers ORF3-JD1/2. The procedure of the RT-PCR was perfected. A large number of clinical samples were collected to carry out the specificity and sensitivity test. [Result] The sequences of ORF3 gene of 11 PEDV isolates were obtained. No deletion in ORF3 gene of 9 strains was found, and it indicated that they were wild-type PEDVs. And the results showed 95.8%-97.1% nucleotide sequence homology identity between wild isolates and attenuated ones. The RT-PCR was shown to specifically amplify a 300 bp fragment from the wild-type PEDVs or a 250 bp fragment from the attenuated PEDVs. This method showed no cross-amplification between PEDVs and other porcine virus. And the sensitivity of detection of viral was upto 100 TCIDs0. The clinical samples were tested by using this RT-PCR method, and the results showed a 65.4% PEDV positive rate. [Conclusion] The RT-PCR could be used as an effective tool for differentiating diagnosis of the PEDVs in epidemiological investigations.

关 键 词：猪流行性腹泻病毒 ORF3基因 RT-PCR 鉴别诊断 

分 类 号：S858.28[农业科学—临床兽医学]