盖他病毒SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立

Development of a SYBR Green Ⅰ-based real-time RT-PCR method for detection of Getahvirus

为建立检测盖他病毒(GETV)的敏感、特异且快速的方法,本研究根据GETV E1基因的保守区域设计引物,通过反应条件的优化,建立了快速检测GETV的SYBR Green Ⅰ荧光定量RT-PCR方法。利用该方法对GETV以及临床猪常见病毒(PRRSV、PCV2、PRV、JEV、PTV、PSV)进行检测,结果显示该方法仅对GETV检测为阳性,而对其他病原均无特异性扩增,特异性强;以10倍倍比稀释后的质粒标准品pMD19-CETV为模板,采用该方法进行敏感性检测,结果显示本实验建立的该方法最低检测下限为6.87拷贝/μL,是常规RT-PCR方法的10倍,敏感性高;以不同稀释度的质粒标准品作为模板进行组内和组间的重复性试验,结果显示该方法组内和组间变异系数均小于1.5%,重复性好。利用该方法对35份临床流产死胎的猪脑组织样品进行检测,结果显示所有样品均未检出GETV,与普通RT-PCR方法检测结果一致。本研究首次建立的GETV SYBR Green Ⅰ荧光定量RT-PCR方法具有特异性强、敏感性高、重复性好的特点,可应用于对GETV的检测。

To develop a sensitive, specific, and quick method for detecting Getah virus(GETV), a pair of primers, targeted to the conserved region of E1 gene of GETV, was designed. The SYBR Green Ⅰ-based real-time RT-PCR method for GETV detection was developed by optimizing the reaction conditions. The method had the advantage of high specificity, and there were no specific amplifications occurred when amplifying the common porcine viruses(PRRSV, PCV2, PRV, JEV, PTV, and PSV) except for that of GETV. The sensitivity of the method was further tested using a 10-fold series diluted plasmid as templates. The results showed that the lower limit of detection of this method is 6.87 copies/μL, which is 10 times higher than that of the conventional RT-PCR method. Parallel intra-and inter-assays were determined by using the serial diluted plasmid as templates. The variation coefficients of intra-and inter-assays were both less than 1.5%, indicating the good repeatability of the method. Brain samples from 35 clinical stillbirths were tested negative for GETV by our method, which was consistent with the results of conventional RT-PCR assay.Therefore, this study for the first time developed a GETV SYBR Green Ⅰ-based real-time RT-PCR method with the characteristics of high specificity, sensitivity, and reproducibility, which can be used for the pathogenic diagnosis of GETV.