PCV2a、PCV2b和PCV2d多重PCR检测方法的建立及应用

Establishment and application of multiplex PCR assay for PCV2a,PCV2b and PCV2d

为了建立一种能够快速鉴别PCV2a、PCV2b和PCV2d的方法,本研究针对PCV2a、PCV2b和PCV2d这3个基因型分别设计3组特异性引物,通过构建阳性标准质粒,筛选出最适引物组合,建立了一种PCV2分型的多重PCR检测方法,并对该方法进行优化后,开展特异性、灵敏度、重复性试验以及临床样品检测。结果显示,利用筛选出的最适引物组合建立了多重PCR检测方法,能特异性扩增出3个目的条带:PCV2a(321bp)、PCV2b(190 bp)和PCV2d(561 bp),探索出最佳退火温度和循环数分别为60℃和25个,与其他病原(PPV、PRV、PEDV、TGEV、PRRSV、CSFV)无交叉反应;对构建的阳性标准质粒的最低检测拷贝数为103copies;同一模板的3次重复性试验结果一致;检测2个猪场流行的PCV2基因型毒株,并通过测序和进化分析进一步验证了该检测方法的准确性。结果表明,本研究建立的多重PCR检测方法有助于开展PCV2a、PCV2b和PCV2d的快速鉴别诊断,为PCV2的防控以及流行病学研究提供了技术支持。

The purpose of this study was to develop a rapid detection method for identification of PCV2a,PCV2b and PCV2d,three sets of specific primers were designed for the three genotypes of PCV2a,PCV2b and PCV2d,respectively.The optimized sets of primers were selected by constructed positive standard plasmids,and a multiplex PCR method for identification of PCV2 genotypes was established.The reaction conditions were optimized and the specificity experiments,sensitivity experiments and repeatability experiments were verified,and clinical samples were used for preliminary application by this method.The results showed that a multiplex PCR detection method was established by using the optimal sets of primers,which could specifically amplify three target fragments:PCV2a(321 bp),PCV2b(190 bp)and PCV2d(561 bp).Furthermore,the optimum annealing temperature was 60℃and cycle number was 25.This method had no cross-reaction with other pathogens(PPV,PRV,PEDV,TGEV,PRRSV,CSFV),and the limit of detection was 103copies by using the positive standard plasmid.Besides,this method had good repeatability with coefficients of three repeatability tests using the same template.The PCV2 strains prevalent in 2 pig farms were detected by the method,the main genotype was determined,and the accuracy of the detection method was further verified by sequencing and evolutionary analysis.The multiplex PCR detection method developed in this study was conducive to the rapid identified diagnosis of PCV2a,PCV2b and PCV2d,and provided technical support for the control of PCV2 and epidemiological studies.