猪流行性腹泻病毒、猪δ冠状病毒和猪急性腹泻综合征冠状病毒多重荧光定量RT-PCR检测方法的建立及应用

Development and application of a multiplex fluorescent quantitative RT-PCR for detection of porcine epidemic diarrhea virus,porcine δcoronavirus and porcine acute diarrhea syndrome coronavirus

为建立检测猪流行性腹泻病毒(PEDV)、猪δ冠状病毒(PDCoV)和猪急性腹泻综合征冠状病毒(SADS-CoV)的多重荧光定量RT-PCR方法,本研究针对PEDV、PDCoV、SADS-CoV的M基因、M基因、N基因序列保守区分别设计特异性引物/探针,经各反应条件优化后,建立了检测PEDV、PDCoV、SADS-CoV的多重荧光定量RT-PCR方法。特异性试验结果显示,该方法仅对PEDV、PDCoV、SADS-CoV检测为阳性,对猪传染性胃肠炎病毒、猪轮状病毒、猪繁殖与呼吸障碍综合征病毒、猪伪狂犬病毒、猪圆环病毒2型、猪细小病毒、猪瘟病毒和口蹄疫病毒等的核酸检测均为阴性。敏感性试验结果显示,该方法对PEDV、PDCoV、SADS-CoV重组质粒标准品的检测下限均为1.0×10^(1)拷贝/μL,且在10^(1)拷贝/μL~10^(6)拷贝/μL范围内各重组质粒均与各自的Ct值有良好的线性关系。重复性试验结果显示,批内、批间重复性试验的变异系数均在0.33%~2.53%,重复性和稳定性均较好。利用建立的多重荧光定量RT-PCR方法对采自河南各地的100份临床样品进行检测,并与3种病毒的单一RT-PCR检测方法、相应病毒的标准检测方法(由于SADS-CoV无标准检测方法,则采用RT-PCR扩增后测序并经NCBI BLAST比较分析测序结果)进行比较分析,以评估本实验建立检测方法的临床应用效果。多重荧光定量RT-PCR结果显示,PEDV、PDCoV、SADS-CoV的阳性率分别为38%(38/100)、14%(14/100)、5%(5/100),不存在混合感染现象,与PEDV、PDCoV标准检测方法的符合率为100%,且敏感性高于单一RT-PCR方法。本研究首次建立了一种特异、敏感、快速的多重荧光定量RT-PCR方法,可以用于PEDV、PDCoV、SADS-CoV的鉴别检测,为猪腹泻类疫病鉴别诊断及防控奠定基础。

To develop a multiplex fluorescent quantitative RT-PCR for the detection of porcine epidemic diarrhea virus(PEDV),porcine deltacoronavirus (PDCoV) and swine acute diarrhea syndrome coronavirus (SADS-CoV),in this study,specific primers/probes were designed based on the conserved regions of M,M and N gene sequences of PEDV,PDCoV and SADS-CoV,respectively.After optimization of the reaction conditions,a multiplex fluorescent quantitative RT-PCR for PEDV,PDCoV and SADS-CoV was established.The results of specificity assay showed that the method was positive for detection of PEDV,PDCoV and SADS-CoV,and negative for detection of porcine transmissible gastroenteritis virus,porcine rotavirus,porcine reproductive and respiratory syndrome virus,porcine pseudorabies virus,porcine circovirus type 2,porcine parvovirus,classical swine fever virus and foot-and-mouth disease virus.The results of sensitivity assay showed that the detection limit of this method for PEDV,PDCoV,and SADS-CoV plasmids standard was 1.0×10^(1)copies/μL,and had a good linear relationship with their Ct values in the range of 10~1copies/μL to 10~6copies/μL.The results of repeatability assay showed that the coefficients of variation (CVs) of intra-and inter-assay reproducibility ranged from 0.33%to 2.53%,indicating good repeatability and stability.To evaluate the effects of the developed method,100 clinical samples collected from different parts of Henan province were used for detection of these three viruses and compared with those of single RT-PCR and standard methods.The results of multiplex fluorescent quantitative RT-PCR showed that the positive rates of PEDV,PDCoV and SADS-CoV were 38%(38/100),14%(14/100) and 5%(5/100),respectively.There was no mixed infection.The coincidence rate with the standard detection methods of PEDV and PDCoV was 100%,and the sensitivity was higher than that of single RT-PCR.In this study,a specific,sensitive and rapid multiplex fluorescent quantitative RT-PCR method was established for the first time,which could be used for the differential detection of PEDV,PDCoV and SADS-CoV,and laid a foundation for the differential diagnosis and control of porcine diarrheal diseases.