猪繁殖与呼吸障碍综合征病毒与猪圆环病毒2型双重荧光定量PCR检测方法的建立与应用

Establishment and Application of Double Fluorescent Quantitative PCR for Detection of Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus Type 2

为建立猪繁殖与呼吸障碍综合征(PRRSV)和猪圆环病毒2型(PCV2)TaqMan双重荧光定量PCR检测方法,研究根据Gen Bank中已登录的PRRSV-M基因序列和PCV2cap基因序列,设计了2对特异性引物和2条Taq Man探针。结果显示以含有PRRSV-M基因和PCV2-cap基因的重组质粒为模板绘制的标准曲线,其相关系数(R^(2))均大于0.99,线性关系较好。该方法仅对PRRSV和PCV2检测为阳性,而对PRV、CSFV、PEDV、TGEV检测结果均为阴性,表明其特异性较好。检测下限约为10拷贝/μL,重复性试验结果显示该方法的组内和组间变异系数均小于1.5%,利用国标荧光定量PCR方法与本实验建立的方法分别对42份临床样品进行检测,二者对PRRSV和PCV2检测的符合率分别可达96.4%和100%。研究表明所建立的荧光定量PCR方法具有快速、敏感、准确等优点,可以用于PRRSV和PCV2的双重定量检测。

In order to establish a TaqMan duplex fluorescent quantitative PCR method for detection of porcine reproductive and respiratory syndrome(PRRSV)and porcine circovirus type 2(PCV2),two pairs of specific primers and two TaqMan probes were designed according to the prrsv-m gene sequence and PCV2 cap gene sequence registered in GenBank.The results showed that the correlation coefficient(R^(2))of the standard curve drawn with the recombinant plasmid containing PRRSV-M gene and PCV2 cap gene as the template was greater than 0.990,and the linear relationship was good.The results of specificity test showed that the method was only positive for PRRSV and PCV2,but negative for PRV,CSFV,PEDV and TGEV,indicating its strong specificity.The detection limit of this method was about 10 copies/μL.The results of repeatability test showed that the intra group and inter group coefficients of variation of this method were less than 2%.42 clinical samples were detected by the national standard fluorescent quantitative PCR method and the established method.The coincidence rates of PRRSV and PCV2 detection were 96.4%and 100%respectively.The results showed that the method was rapid,sensitive and accurate,and could be used for the dual quantitative detection of PRRSV and PCV2.