猪传染性胃肠炎与流行性腹泻病毒二重RT-PCR 检测方法的建立及应用

为建立猪传染性胃肠炎病毒(TGEV)与流行性腹泻病毒(PEDV)的快速鉴别诊断方法,本研究根据GenBank已登录的TGEV核蛋白(N)基因和PEDV膜蛋白(M)基因保守区域序列分别设计了1对特异性引物,以TGEV和PEDV混合总RNA为反转录模板,建立了TGEV和PEDV的二重RT-PCR检测方法,并进行了特异性、敏感性和重复性试验;利用所建立的检测方法对临床疑似样品进行了应用检测,并对检测到的阳性样品进行克隆测序。结果表明,成功建立了TGEV和PEDV二重RT-PCR检测方法,该方法的检测灵敏度最低极限为10 TCID₅₀/mL病毒含量,重复性好,特异性强,可特异性地扩增TGEV和PEDV细胞培养物,但对ST细胞和其他7种病原对照扩增不出任何条带;对22份临床疑似TGEV和PEDV感染样品检测结果与测序结果完全一致。本研究成功建立了TGEV与PEDV二重RT-PCR检测方法,可适用于猪传染性胃肠炎和流行性腹泻病的快速鉴别诊断。

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

A duplex RT-PCR for detection porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) was developed using the primers designed basing on the N and M gene coding for the nucleoprotein of TGEV and membrane glycoprotein of PEDV respectively. The total RNA of standard TGEV and PEDV strains were used as the positive control to establish the duplex RT-PCR assay. The sensitivity, specificity and repetition assay of duplex RT-PCR were tested, and samples taken from clinic suspicious TGEV or PEDV infected pigs and having been testified by routine cell culture and identification were detected by the established duplex RT-PCR assay. The results indicated that the duplex RT-PCR assay was successfully established. The specificity and sensitivity of the duplex RT-PCR assay revealed that the threshold of duplex RT-PCR was 10 TCID₅₀/mL of TGEV or PEDV, and no products were amplified from the nucleic acid of ST cell or the other 7 kinds of pathogenic viral or bacterial microorganism acting as the negative control. The repetition test indicated that the duplex PCR was repeatable. The detection results for 22 clinic suspicious TGEV or PEDV infected pigs were consistent with the results tested by sequencing. The study suggested that the established duplex RT-PCR method was highly specific and sensitive,and was suitable to clinic rapid identified diagnosing of TGEV and PEDV.