猪肺炎支原体、猪圆环病毒2型和3型多重荧光定量PCR检测方法的建立与初步应用

Establishment and Preliminary Application of Multiplex Real-time PCR Detection Methods for Mhp,PCV2 and PCV3

为建立一种快速、特异的猪肺炎支原体(Mhp)、猪圆环病毒2型(PCV2)和猪圆环病毒3型(PCV3)的多重荧光定量PCR鉴别检测方法,根据Mhp、PCV2和PCV3及β-actin基因保守区域序列,设计合成4对特异性引物及4种荧光基团标记的Taq Man探针。构建含有各目的基因的重组质粒标准品,筛选引物,优化反应条件,成功建立多重荧光定量PCR后评价该方法的特异性、敏感性、重复性并将该方法初步应用于临床样品检测。结果显示,该方法能同时特异性地鉴别检测Mhp、PCV2和PCV3,检测PRRSV、CSFV、PRV、JEV、PPV、PEDV、TGEV、FMDV病毒核酸无交叉反应;对Mhp、PCV2、PCV3和β-actin标准质粒的最小检出量分别为26.4、66.7、25.6、5990 copies/μL;该方法重复性好,组间和组内试验变异系数低于1.5%;用该方法检测101份临床样本,Mhp、PCV2和PCV3阳性率分别为20.8%、36.6%和33.7%。建立的多重荧光定量PCR方法能同时快速和定量检测Mhp、PCV2和PCV3,较普通PCR更敏感,实用性强,有效避免了多次检测耗时长、费用高的问题,为Mhp、PCV2和PCV3的监测和防控提供了可靠的检测技术。

In order to establish a rapid and specific multiplex fluorescent quantitative PCR detection method for Mycoplasma hyopneumoniae(Mhp),porcine circovirus type 2(PCV2)and porcine circovirus type 3(PCV3),four pairs of specific primers and four Taq Man probes labeled with different fluorescent groups were synthesized based on gene sequences in the conserved regionsof Mhp,PCV2,PCV3 and the referenceβ-actin gene.By constructing recombinant plasmid standards containing each target gene,screening primers,and optimizing reaction conditions,multiplex fluorescent quantitative PCR was successfully established to determine the specificity,sensitivity and repeatability of the method,and the method was preliminarily applied to the detection of clinical samples.The results showed that the method can simultaneously and specifically detect and identify Mhp,PCV2 and PCV3,and has no cross-reaction with PRRSV,CSFV,PRV,JEV,PPV,PEDV,TGEV and FMDV viral nucleic acids.The method has high sensitivity.The minimum detection amounts of Mhp,PCV2,PCV3 andβ-actin are 26.5 copies/μL,66.7 copies/μL,25.7 copies/μL and 599 copies/μL,respectively.The method was reproducible,and the intra-group and inter-group coefficients of variation were less than 1.5%.101 clinical samples were detected,the positive rates of Mhp,PCV2 and PCV3 were 20.8%,36.6%and 33.7%,respectively.The established multiplex fluorescent quantitative PCR method can simultaneously detect Mhp,PCV2 and PCV3 rapidly and quantitatively,which is more sensitive and practical than ordinary PCR,and effectively solve the problem of time-consuming and high cost of multiple detections.It provides new and reliable detection technology for monitoring and prevention.