猪流行性腹泻病毒巢式RT-PCR检测方法的建立及应用

Establishment and Application of Nested RT-PCR for Detecting Porcine Epidemic Diarrhea Virus

为建立一种快速、敏感的猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)检测方法,试验据GenBank中PEDV基因组序列,设计并合成针对M基因的内、外2对特异性引物,通过优化反应条件,建立了检测PEDV的巢式RT-PCR方法。结果显示,单一使用外引物或内引物进行常规RT-PCR时检测限量均为50pg PEDV RNA模板,而使用巢式RT-PCR可检测到50fg PEDV RNA模板。使用该方法对猪传染性胃肠炎病毒、猪轮状病毒、猪繁殖与呼吸综合征病毒及猪瘟病毒的扩增结果均为阴性。结果表明,建立的巢式RT-PCR方法特异性、敏感性好,可快速准确地检测出样本中微量PEDV核酸。

To detect porcine epidemic diarrhea virus （PEDV） accurately and sensitively, two pairs of specific primers amplifying M gene were designed and the nested RT-PCR method was established. The lowest detective value of the nested RT-PCR as proved by the sensitivity test was 50 fg of PEDV RNA template, however,that of the conventional RT-PCR was 50 pg. The nested RT-PCR primers designed in the study could not be used to amplify the PRRSV, CSFV, TGEV and GARV RNA templates in the specificity test. The results showed that the nested RT-PCR was a rapid, sensitive and cost-effective method for detecting trace amounts of PEDV nucleic acid.