一步法多重RT-PCR检测猪流行性腹泻病毒、猪传染性胃肠炎病毒和猪A群轮状病毒方法的建立和应用

摘要

根椐GenBank中登录的猪流行性腹泻病毒（PEDV）、猪传染性胃肠炎病毒（TGEV）和猪A群轮状病毒（PARV）基因序列，分别设计3对引物，在建立各病毒单项一步法RT-PCR技术的基础上，优化多重RT-PCR反应条件，建立了3种病毒的一步法多重RT-PCR检测方法，用这3对引物对同一样品中的PEDV、TGEV和PARV核酸模板进行多重RT-PCR扩增，结果可同时扩增PEDV的450 bp、TGEV的609 bp、PARV的241 bp特异性片段，而对其它4种病原的扩增结果均为阴性。敏感性测定结果表明，该多重RT-PCR技术能检出10 pg的PEDV、10 pg的TGEV和1 pg的PARV模板。用67份临床病料对建立的多重RT-PCR技术和单项RT-PCR技术进行对比验证，结果显示，两者的总符合率为100%。表明建立的一步法多重RT-PCR检测方法，具有特异、快速、准确的特点，可用于这3种病毒的同时检测和鉴别诊断。%A multiplex RT-PCR was optimized to simultaneously detect three pathogens of Porcine epidemic diarrhea virus(PEDV),Porcine transmissible gastroenteritis virus(TGEV) and Porcine group A rotavirus(PARV) in this article.Three sets of specific primers were designed according to the sequences of PEDV, TGEV and PARV at the GenBank.By using three pairs of virus specific primers, three RT-PCR assay were established to amplify the conservative regions of the three viruses,respectively.Consequently,a multiplex RT-PCR method to detect the three viruses in one tube was developed.The multiplex RT-PCR system would amplify a 450 bp fragment for PEDV, a 609 bp for TGEV and a 241 bp for PARV simultaneously or separately in the samples,depending on its infection status.But not specific band amplified from other four pig pathogenic viruses.As little as 10 pg of PEDV, 10 pg of TGEV and 1 pg of PARV RNA were detected using gel electrophoresis in the multiplex PCR.To evalu-ate the multiplex PCR, 67 clinical samples were comparatively detected.The data showed that the multiplex RT-PCR method being 100%coincidence with the single RT-PCR,and it can be used for this three viruses detection and differential diagnosis.