猪繁殖与呼吸综合征病毒和猪肺炎支原体双重荧光PCR检测方法的建立

Establishment of Duplex Real-time PCR for Detection of Porcine Reproductive and Respiratory Syndrome Virus and Mycoplasma hyopneumoniae

为了建立快速检测猪繁殖与呼吸综合征病毒(PRRSV)和猪肺炎支原体(Mhp)的双重荧光PCR检测方法,本试验根据NCBI数据库中的PRRSV ORF7和Mhp P36基因,分别设计引物和TaqMan探针,通过优化扩增反应条件,建立PRRSV和Mhp的双重荧光PCR检测方法;分析所建立PCR方法的特异性、敏感性和重复性,并初步应用于临床样品检测。结果显示,25μL PCR反应体系中各引物最佳添加量:PRRSV-F 1.5μL、PRRSV-R 1.0μL、PRRSV-P 0.8μL、Mhp-F 1.2μL、Mhp-R 1.3μL、Mhp-P 0.7μL。优化后的PCR反应程序:50℃反转录20 min,95℃预变性2 min,95℃变性5 s,55℃退火10 s,72℃延伸20 s (此处收集荧光),40个循环。建立的双重荧光PCR方法特异性较好,与其他猪常见病原体无交叉反应;检测PRRSV和Mhp的敏感性分别为4.2拷贝/μL和9.6拷贝/μL;批内和批间变异系数均不高于2%,具有很好的重复性。因此,本试验建立的双重荧光PCR方法可同时检测PRRSV和Mhp,为猪繁殖与呼吸综合征(PRRS)和猪支原体肺炎(MPS)的诊断和防控工作提供支持。

In order to establish a rapid detection method for porcine reproductive and respiratory syndrome virus(PRRSV) and Mycoplasma hyopneumoniae(Mhp) using duplex real-time PCR,this study designed primers and TaqMan probes targeting the PRRSV ORF7 and Mhp P36 genes based on the NCBI database,and established a duplex real-time PCR detection method for PRRSV and Mhp by optimizing the amplification reaction conditions.The specificity,sensitivity,and repeatability of the established PCR method were analyzed,and the method was preliminarily applied to clinical sample detection.The results showed that the optimal amounts of each primer in the 25 μL PCR reaction system were as follows:PRRSV-F 1.5 μL,PRRSV-R 1.0 μL,PRRSV-P 0.8 μL,Mhp-F 1.2 μL,Mhp-R 1.3 μL,Mhp-P 0.7 μL.The optimized PCR reaction program was reverse transcription at 50 ℃ for 20 min,pre-denaturation at 95 ℃ for 2 min,denaturation at 95 ℃ for 5 s,annealing at 55 ℃ for 10 s,extension at 72 ℃ for 20 s(fluorescence collection),and a total of 40 cycles.The established duplex real-time PCR method showed good specificity without cross-reaction with other common swine pathogens.The sensitivity for detecting PRRSV and Mhp was 4.2 copies/μL and 9.6 copies/μL,respectively.The intra-batch and inter-batch coefficients of variation were both less than 2%,indicating good repeatability.Thus,the duplex real-time PCR method established in this study can simultaneously detect PRRSV and Mhp,providing support for the diagnosis and control of porcine reproductive and respiratory syndrome(PRRS) and mycoplasmal pneumonia of swine(MPS).