微滴式数字PCR定量检测禽偏肺病毒方法的建立

Establishment and application of droplet digital PCR for quantitative detection of avian metapneumovirus

为建立禽偏肺病毒(aMPV)微滴式数字PCR(ddPCR)检测方法,本研究以aMPV F基因为靶基因,根据其保守区域设计特异性引物和探针,通过各反应条件的优化,初步建立检测aMPV的dd PCR方法。反应条件优化结果显示,最佳引物终浓度1μmol/μL,探针终浓度0.5μmol/μL,退火温度56℃。绘制的标准曲线显示,重组质粒标准品稀释倍数的对数与相应质粒检出拷贝数的对数呈良好的线性关系(R^(2)=0.9998)。采用该方法同时检测aMPV、禽流感病毒、鸡新城疫病毒、鸡传染性支气管炎病毒、禽呼肠孤病毒、禽脑脊髓炎病毒、鸡传染性喉气管炎病毒和禽腺病毒,结果显示,仅aMPV检测为阳性,其他病原均为阴性。特异性较强。将p MD18-aMPV重组质粒标准品10倍倍比稀释(10^(5)~10^(9),4.3×10^(3)拷贝/μL~4.3×10^(-1)拷贝/μL)后作为模板,采用该方法检测,结果显示该方法对重组质粒标准品的检测限为4.3拷贝/μL,比荧光定量PCR(qPCR)检测限(43拷贝/μL)敏感。对3个稀释度质粒标准品的批内和批间重复性试验结果显示,批内和批间试验的变异系数均小于5%。对采集的82份病鸡气管和脾组织样品同时采用dd PCR和荧光定量PCR检测,结果显示,两种方法对aMPV的阳性检出率均为4.88%(4/82),阴性样品均为78份,二者的符合率达100%,dd PCR检出的4份阳性样品中aMPV的拷贝数为78拷贝/μL~920拷贝/μL。上述结果表明,本研究建立的dd PCR方法特异性强、敏感性高和重复性好,为aMPV检测提供新的技术手段,对其早期感染的防控有重要意义。

In order to establish a microdroplet digital PCR(ddPCR)for quantitative detection of avian metapneumovirus(aMPV).A pair of specific primer and probe were designed and synthesized based on the conserved region of the aMPV F gene,and a ddPCR was preliminarily established through optimization of various reaction conditions.The optimization results of reaction conditions showed that the optimal final concentration of primer and probes were 1μmol/μL and 0.5μmol/μL.The annealing temperature was 56℃.The standard curve plotted shows a good linear relationship between the logarithm of the dilution ratio of the recombinant plasmid standard and the logarithm of the detected copy number of the corresponding plasmid(R^(2)=0.9998).Using this method to simultaneously detect aMPV,avian influenza virus,Newcastle disease virus,infectious bronchitis virus,avian reovirus,avian encephalomyelitis virus,infectious laryngotracheitis virus,and avian adenovirus,the results showed that only aMPV was detected positive,while other pathogens were all negative,with strong specificity.The minimum limit for quantitative detection was 4.3 copies/μL of pMD18-aMPV plasmid standard which was diluted 10 times(10^(5)-10^(9),4.3×10^(3)copies/μL to 4.3×10^(-1)copies/μL)as templates.It was more sensitive than the 43 copies/μL detection limit of fluorescence quantitative PCR(qPCR).The results of intra-and inter batch repeatability tests on three dilution plasmid standards showed that the coefficients of variation in both intra-and inter batch tests were less than 5%.A total of 82 tracheal and spleen samples collected from infected chickens were detected by ddPCR and qPCR.The results showed that the positive detection rate of aMPV was 4.88%(4/82)by both methods and the coincidence rate reached 100%.The aMPV copy number were 78 copies/μL to 920 copies/μL.78 of 82 samples were negative.The above results indicate that the ddPCR method established in this study has strong specificity,high sensitivity,and good repeatability,providing a new technical means for the detection of aMPV and is of great significance for the prevention and control of its early infection.