Detection of Porcine Circovirus-like Agent P1 Using SYBR Green I Fluorescent Quantitative PCR Assay

This experiment was to establish a fast,sensitive,specific SYBR Green Ⅰ fluorescent quantitative PCR assay for early diagnosis of P1 infection.A 101 bp fragment of P1 gene was amplified by PCR based on genomic DNA of P1 obtained from the P1 infected-porcine serum,then cloned into pMD-18 T vector.The recombinant plasmid was sequenced and analyzed by BLAST,and the SYBR Green Ⅰ fluorescent quantitative PCR assay was established based on positive plasmid template.The sensitivity and specificity of the assay were performed.The recombinant plasmid was confirmed by sequencing and the fragment belonged to P1.The P1 real-time PCR assay had a dynamic range of detection between 101 and 108 copies/μL,with a sensitivity of 10 copies/μL,while the detecting results of porcine pseudorabies virus,porcine parvovirus and porcine reproductive and respiratory syndrome virus were negative,which indicated this assay was sensitive and specific for P1 detection.We successfully established a SYBR Green Ⅰ Fluorescent Quantitative PCR Assay for the quantification of P1 DNA,and it has potential application for investigating the pathogenesis of P1 and the protective me chanism of animal body.