猪δ冠状病毒实时荧光RT-PCR检测方法的建立与应用

Establishment and Application of a Real-time Fluorescent RT-PCR Assay for Porcine Deltacoronavirus

为灵敏、快速检测猪δ冠状病毒(porcine deltacoronavirus,PDCoV),根据PDCoVM基因保守区域设计引物和探针,建立了一种PDCoV实时荧光RT-PCR检测方法,并评价了该方法的特异性、灵敏性和重复性。结果显示,该方法的拷贝数对数值与Ct值在1.3×10^(3)-1.3×10^(7) copies/μL拷贝数浓度范围内呈现良好的线性关系,R^(2)=0.994;最低检出限为13 copies/μL,且无非特异性扩增;批内和批间重复性试验变异系数(CV)为0.24%~2.15%,均小于3%。利用该方法和普通RT-PCR对42份临床样品同时进行检测,发现本方法比普通RT-PCR灵敏性更高,二者符合率为95.2%。以上结果表明,本试验建立的实时荧光RT-PCR方法灵敏、特异、可靠,可用于PDCoV的临床检测和流行病学调查。

In order to sensitively and rapidly detect porcine deltacoronavirus(PDCoV),probe and a pair of primers were designed based on the conserved region of PDCoV M gene to establish a real-time fluorescent RT-PCR assay for PDCoV,followed by evaluation on its specificity,sensitivity and repeatability.The results showed that a good linear relationship was observed between the logarithmic value of copy number and Ct value from 1.3 × 10^(3) to 1.3 × 10^(7) copies/μL,R^(2)=0.994;the lowest detection limit was 13 copies/μL without any nonspecific amplification;the coefficient of variation(CV) of intra and inter batch repeatability tests were from 0.24% to 2.15%,both of which were less than 3 %.42 clinical samples were detected by the established method and general RT-PCR assay,it was found that the former was more sensitive and reliable than the latter,the coincidence rate of both methods was 95.2%.In conclusion,the established method could be used for clinical diagnosis and epidemiological investigation on PDCoV with the help of its good specificity,reliability and sensitivity.