Establishment and application of multiplex PCR assay for PCV2a,PCV2b and PCV2d
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 re-peatability 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 opti-mal 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 103 copies 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.
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