PEDV、TGEV、RV多重RT-PCR检测方法的建立与应用

Establishment and Application of Multiple RT-PCR Method for Diagnosis of PEDV, TGEV and RV Infection

关键词:  猪流行性腹泻病毒  猪传染性胃肠炎病毒  猪轮状病毒  多重RT-PCR

Key words:  PEDV  TGEV  RV  multiple RT-PCR

摘要:

为了快速准确诊断猪流行性腹泻病毒 (PEDV)、猪传染性胃肠炎病毒 (TGEV)、猪轮状病毒(RV)引起的猪腹泻病，通过设计三对引物，建立了扩增PEDV、TGEV、RV的多重RT-PCR方法，用于临床上大量腹泻病料的鉴定。该方法特异敏感，能同时检测PEDV、TGEV、RV，而对猪瘟病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病毒、猪圆环病毒2型病毒、乙型脑炎病毒、猪细小病毒等无扩增，检测的抗原稀释极限为107。用于35个猪场120份临床样品的检测，PEDV阳性率占37.5%，TGEV阳性率占0.75%，RV阳性率占1.25%。PEDV阳性病料测序结果分析表明，与近几年分离毒株亲缘关系较近，与经典毒株和疫苗株亲缘关系较远。结果表明建立的多重PCR方法特异性强、敏感度高，能用于临床诊断及流行病学调查。

Abstract:

In order to establish a rapid and accurate method for diagnosis of porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine rotavirus (RV), three pairs of primers were designed and a multiple RT-PCR method to amplify PEDV, TGEV and RV was developed. The method is specific and sensitive and can be used simultaneously to detect PEDV, TGEV, RV, and can, tdetect CSFV, PRRSV, PRV, PCV2, JEV and PPV. The detection limit of antigen dilution was 107. For 120 clinical samples from 35 pig farms, PEDV positive rate with diarrhea accounted for 37.5% of diarrheal diseases material. TGEV positive rate was 0.75% and RV positive rate was 1.25%. Sequencing analysis of PEDV positive disease material shows that, it has closer relationship with isolates strains in recent years, than with classical strain and vaccine strain. The result showed that this multiple RT-PCR method was high sensitive, high specific and can be used in clinical diagnosis and epidemiological investigation.