微滴式数字PCR定量检测鸡毒支原体方法的建立

Establishment of a Droplet Digital PCR for Quantitative Detection of Mycoplasma gallisepticum

为建立微滴式数字PCR(droplet digital PCR,ddPCR)定量检测鸡毒支原体(Mycoplasma gallisepticum,MG)方法,以MG的mgpc基因序列为目的序列,在其保守区域内设计合成了1对特异性引物和1条探针,通过各反应条件优化,建立了检测MG的ddPCR方法,并测定了建立方法的特异性、敏感性以及重复性。结果显示:建立方法的最佳引物浓度为20μmol/μL,探针浓度为10μmol/μL,退火温度为55℃;该方法只检出MG,没有检出鸡滑液囊支原体、禽衣阿华支原体、禽流感病毒、鸡新城疫病毒、鸡传染性支气管炎病毒、鸡传染性喉气管炎病毒、禽脑脊髓炎病毒、禽偏肺炎病毒、禽呼肠孤病毒、鸡沙门氏菌和鸡大肠杆菌,且相互间没有交叉反应;本方法最低检测MG重组质粒标准品的检测限为3.9拷贝/μL。重组质粒标准品浓度在3.9~41025拷贝/μL范围内,绘制的ddPCR绝对定量曲线与测得值呈良好的线性关系(R2=0.999);3次重复检测试验结果的变异系数均小于5%。对采集的60份病鸡喉拭子、气囊拭子及肺样品进行MG检测,结果ddPCR方法的阳性检出率(15.0%)高于荧光定量PCR方法(13.3%)。结果表明,本研究建立的ddPCR方法定量检测MG特异性强,灵敏度高,重复性好,为绝对定量检测MG提供了技术手段。

In order to establish a droplet digital PCR(ddPCR)for quantitative detection of Mycoplasma gallisepticum(MG),taking mgpc gene sequence of MG as a target,a pair of specific primers and a probe were designed and synthesized in its conserved region,a ddPCR was established through optimization of all reaction conditions,then its specificity,sensitivity and repeatability were detected.For the established method,its optimal primer concentration was 20μmol/μL,probe concentration was 10μmol/μL,and annealing temperature was 55℃;only MG could be detected,no Mycoplasma synoviae(MS),Mycoplasma iowae(MI),avian influenza virus(AIV),Newcastle disease virus(NDV),avian infectious bronchitis virus(IBV),avian infectious laryngotracheitis virus(ILTV),avian encephalomyelitis virus(AEV),avian reovirus(ARV),Salmonnella pullorum(S.pullorum),or Escherichia coli(E.coli)was observed,all of which failed to react with each other;the minimum limit for quantitative detection of MG recombinant plasmid DNA was 3.9 copies/μL.The concentration of recombinant plasmid standard was in the range of 3.9 to 41025 copies/μL,a good linear relation was observed between the absolute quantitative curve of ddPCR with the measured value(R2=0.999);all the variable coefficients of the results of 3 repeated tests were all less than 5%.60 throat swabs,air bag swabs and lung samples collected from sick chickens were detected for MG,and the positive detection rate of ddPCR(15.0%)was higher than that of fluorescent quantitative PCR(13.3%).In conclusion,the established method could be used as a technical tool for absolute quantitative detection of MG due to its strong specificity,high sensitivity and good repeatability.