猪Delta冠状病毒、猪传染性胃肠炎病毒和猪流行性腹泻病毒多重RT-PCR检测方法的建立及应用

Establishment and application of a multiplex RT-PCR assay for simultaneous detection of porcine delta coronavirus,porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus

猪Delta冠状病毒（PDcoV）、猪流行性腹泻病毒（PEDV）和猪传染性胃肠炎病毒（TGEV）均是能引起仔猪腹泻的冠状病毒，通过临床症状和剖检很难鉴别诊断，建立同时检测且能够鉴别诊断这3种疾病的快速检测方法对于临床诊断具有重要的意义。参照GenBank中登录的PDCoV，NPEDVM和TGEVN基因的基因序列，利用Primer5．0设计合成3对能扩增特异性目的片段的引物，构建重组质粒，并对RT-PCR反应条件进行优化，建立了检测PDCoV，PEDV和TGEV的多重RT—PCR诊断方法，并对所建立的方法进行敏感性、特异性和重复性分析。利用该方法对河南176份临床样品进行检测，并与普通RT-PCR检测方法对该检测方法进行比较分析。结果表明，所建立的三重RT-PCR方法对PDCoV的最低检测量是3．14×10 3拷贝／uL；PEDV的最低检测量是3．88×10 4拷贝／uL和TGEV的最低检测量是3．68×10 4拷贝／uL。所建立的方法具有较好的特异性，对猪博卡病毒（PboV）、猪伪狂犬病病毒（PRV）、猪繁殖与呼吸综合征病毒（PRRSV）、猪瘟（CSFV）和猪圆环病毒（PCV）等猪常见病毒的扩增结果均为阴性。应用所建立的三重RT-PCR方法对176份临床收集的仔猪腹泻样品进行PEDV，PDCoV和TGEV检测，并将检测结果和单重RT-PCR检测方法比较，结果显示，多重RT—PCR检测结果与常规单一RT～PCR的结果符合率均为100％。综上，本试验建立了能同时检测PDCoV，PEDV和TGEV的三重RT—PCR方法，为临床上这3种疫病的快速检测和流行病学调查奠定了基础。

Piglet diarrhea and vomiting in clinic could be caused by porcine delta coronavirus（PD- Coy）, porcine epidemic diarrhea virus （PEDV） and porcine transmissible gastroenteritis virus （TGEV）. It is difficult to distinguish them from each other through clinical symptoms or necrop- sy. Establishment of a multiplex RT-PCR assay is important for clinical diagnosis. Three pairs of primers targeting the N gene of PDCoV, M gene of PEDV and N gene of TGEV were designed, respectively,according to the corresponding gene sequences in GenBank. After being optimized with the primers,the sensitivity,repeatability and specificity of the multiplex RT-PCR were evaluated. Results showed that the sensitivity of the multiplex RT-PCR was 3.14 × 10 3 copies/uL for PD- CoV,3.68 × 10 4 copies/uL for TGEV and 3. 88 × 10 4 copies/uL for PEDV, and no non-specific amplifications such as porcine bocavirus（PBoV）, porcine respiratory and reproductive syndrome vi- rus（PRRSV） ,porcine pseudorabies virus （PRV）, classical swine fever virus （CSFV）, porcine cir- covirus （PCV） ,were found. In 176 clinical samples collected from diarrhea pigs in Henan,the coincidence between the multiplex RT-PCR and the conventional RT-PCR was 100%. The multiplex RT-PCR could be used for epidemioligical investigation of the three diseases.