猪流行性腹泻病毒与猪伪狂犬病病毒二重RT-PCR检测方法的建立及应用

为建立猪流行性腹泻病毒（PEDV）与猪伪狂犬病病毒（PRV）的快速鉴别检测方法，本研究根据GenBank已登录的PEDV膜蛋白M基因和PRV gE基因保守区域序列设计了2对特异性引物，以PRV和PEDV混合总RNA为反转录模板，初步建立了PRV和PEDV的二重RT-PCR检测方法，并进行了特异性、敏感性、重复性验证和临床应用检测。结果显示，该方法对两种病毒的最低检测限均为10 TCID₅₀/mL病毒含量，重复性好，特异性强，可特异性地扩增PEDV和PRV细胞培养物，但对其他7种病原对照扩增不出任何条带，对26份临床疑似PEDV和PRV感染样品检测结果与测序鉴定结果完全一致。本研究成功建立了PEDV和PRV的二重RT-PCR检测方法，为临床上猪流行性腹泻和猪伪狂犬病的快速鉴别诊断提供了方法。

Establishment and Application of Duplex RT-PCR Assay for Detection of Porcine Epidemic Diarrhea Virus and Pseudorabies Virus

In order to establish an assay for detecting porcine epidemic diarrhea virus (PEDV) and pseudorabies virus (PRV),two pairs of primers were designed basing on the M gene of PEDV and gE gene of PRV,respectively.The total RNA of standard PEDV and PRV strains were used as templates to establish the duplex RT-PCR assay.The specificity,sensitivity,repetition and clinic detection of the established assay were tested.The result revealed that the threshold of duplex RT-PCR was 10 TCID₅₀/mL of PEDV and PRV,and no products were amplified from the cell or the nucleic acid of other 7 kinds of pathogenic viral or bacterial microorganism.The detection results for 26 clinical suspicious PEDV or PRV infected pigs were consistent with the results tested by sequencing.This study suggested that the duplex RT-PCR method was highly specific,repeatable and sensitive,and was suitable for clinic rapid differential diagnosis of PEDV and PRV.