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

doi:10.7668/hbnxb.2009.04.007

Abstract

Abstract: This experiment was to establish a fast, sensitive, specific SYBR Green I fluorescent quantitative PCR as2 say 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 infected2porcine serum, then cloned into pMD218 T vector.The recombinant plasmid was sequenced and analyzed by BLAST, and the SYBR Green I fluorescent quantitative PCR assaywas established based on positive plas2 mid template. The sensitivity and specificity of the assay were performed.The recombinant plasmid was confirmed by se2 quencing and the fragment belonged to P1. The P1 real2time PCR assay had a dynamic range of detection between 101 and 108 copies/ LL,with a sensitivity of 10 copies/ LL, while the detecting results of porcine pseudorabies virus, porcine par2 vovirus 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.

Key words: Porcine circovirus2like agent, P1, Fluorescent quantitative PCR, Melting curve, Detection

CLC Number:

S82.4.2

WEN Li-bin, HE Kong-wang, YANG Han-chun, GUO Rong-li, ZHOU Jun-ming, ZHONG Shu-lin. Detection of Porcine Circovirus-like Agent P1 Using SYBR Green I Fluorescent Quantitative PCR Assay[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2009, 24(4): 31-35. doi: 10.7668/hbnxb.2009.04.007.

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http://www.hbnxb.net/EN/Y2009/V24/I4/31

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