一种猪伪狂犬病病毒gE基因TaqMan荧光定量PCR检测方法的建立

Development of a TaqMan Fluorescent Quantitative PCR Detection Method for Porcine Pseudorabies Virus gE Gene

研究旨在建立一种猪伪狂犬病病毒(PRV)的探针法荧光定量检测方法。选用PRV gE基因为靶基因,比对不同野毒在该基因的保守区域设计特异性引物和探针,构建PRV gE基因片段的重组质粒作为标准品,对该方法的反应体系和条件进行优化,并进一步评估该方法的敏感性、特异性和稳定性,最后与商品化试剂盒同时检测临床样本,比较符合率。结果表明,该研究建立的T a q M a n荧光定量P C R检测方法的最佳反应条件为上下游引物和探针量分别为0.2μm o l/L和0.05μm o l/L,最佳退火温度为58℃。该方法具有良好的特异性,对PRV Bartha-k61疫苗株、猪流行性腹泻病毒(PEDV)、猪细小病毒(PPV)、猪A群轮状病毒(PoRV)、猪德尔塔冠状病毒(PDCoV)、猪传染性胃肠炎病毒(TGEV)、猪链球菌(S.suis)、猪源大肠杆菌(E.coli)、猪源巴氏杆菌(P.multocida)、猪霍乱沙门氏菌(S.choleraesuis)和葡萄球菌(S.aureus)等11个菌毒种均无非特异性扩增。该方法的最低检测限为3.92 copies/μL。重复性试验结果显示,组内和组间变异系数均≤2%。与商品化的荧光定量PCR检测试剂盒比较,符合率均为100%(n=50)。综上所述,该研究建立了一种灵敏高效的检测PRV野毒株的TaqMan荧光定量PCR方法,该方法可以成为猪伪狂犬病早期快速诊断和流行病学调查的一种可靠的技术手段。

In order to develop a TaqMan quantitative PCR method to detect field strains of PRV,specific primers and probes were designed by comparing the conserved regions of the gE gene between different field strains of PRV.The recombinant plasmid of PRV gE gene was constructed to be as a standard for the optimization of reaction conditions of the TaqMan quantitative PCR in different concentrations of primers,probes and annealing temperature,respectively.Furthermore,the sensitivity,specificity and repeatability of the TaqMan quantitative PCR method for PRV were tested,respectively.Then,a number of clinical samples were used to evaluate the coincidence rate between commercial kits and this new detection method.The results showed that optimum concentrations of primers,probe,annealing temperature were 0.2μmol/L,0.05μmol/L,58℃,respectively.There were no nonspecific productions when the TaqMan quantitative PCR method were used to detect the nucleic acids of other 11 kinds of swine common pathogens(including PRV Bartha-k61 vaccine,PEDV,PPV,PoRV,PDCoV,TGEV,S.suis,E.coli,P.multocida,S.choleraesuis and S.aureus).It is indicated that this method is so specific.And the lower detection limit is 3.92 copies/μL.The repetitive testing results showed that variation coefficients of intra-group and inter-group were both less than 2%.Furthermore,the coincidence rate for the detection of clinical samples was 100%(n=50)between commercial kits and this new method.In conclusion,a sensitive and efficient TaqMan quantitative PCR detection method of the field strains of PRV was development.It can be a reliable tool for rapid diagnosis of pseudorabies in the early days of the pandemic.