基于DPO引物检测猪传染性胃肠炎病毒real-time RT-PCR方法的建立及应用

Development and application of DPO-based real-time RT-PCR method for detection of transmissible gastroenteritis coronavirus

为建立快速、特异性检测猪传染性胃肠炎病毒(TGEV)的方法,以TGEV的N基因为靶基因,利用双启动寡核苷酸引物(dual priming oligonucleotide,DPO),建立了检测TGEV的real-time RT-PCR方法。结果显示,与常规引物相比,DPO引物的特异性强,有3个以上碱基与模板发生错配,将导致扩增效率极大降低或终止扩增反应,同时DPO引物有效退火温度范围较宽,在40~65℃均能高效地扩增靶基因,不需要对退火温度进行优化。本研究建立的方法灵敏度高,对TGEV核酸的最低检测限可达1.52×10~1 copies/μL,同时利用该方法对10种病毒株进行检测,仅TGEV为阳性,显示了良好的检测特异性。该方法的检测重复性好,以质粒标准品为模板的组内、组间重复性检测结果的变异系数均小于1.00%。利用建立的方法对采集的213份仔猪腹泻临床样品进行检测,共检出TGEV阳性样本31份,阳性率为14.55%,与常规RT-PCR方法检测结果的符合率为93.55%,与基于N基因测序结果的符合率为100.00%。本研究基于DPO引物建立的检测TGEV的real-time RT-PCR方法,为TGEV的准确检测和流行病学调查提供技术支持。

In order to establish a rapid and specific method for the accurate detection of porcine transmissible gastroenteritis coronavirus(TGEV),a dual priming oligonucleotide(DPO)-based real-time RT-PCR method was developed targeting the N gene of TGEV.Compared to the conventional primers,the DPO primer has a higher specificity,and only when more than three nucleotides mismatch occurred,the amplification efficiency would be substantially decreased,or the extension will terminate.Moreover,the DPO primer allowed a wide annealing temperature from 40℃ to 65℃ to specifically amplify the target gene.The DPO-based real-time RT-PCR method developed in this study has a strong specificity with a detection limit of 1.52×101 copies/μL.The method has a good repeatability and the coefficients of variation of intra-assay and inter-assay were both less than 1.00%.Using the method to detect 213 collected piglet diarrhea samples,39 of them were TGEV positive with the positive rate of 14.55%,which showed coincidence rates of 93.55% and 100.00% compared to conventional RT-PCR results and N gene-based sequencing results,respectively.This DPO-based real-time RT-PCR method provided technical support for accurate detection and epidemiological investigation of TGEV.