Establishment and application of a SYBR Green I- based fluorescent quantitative PCR assay for detection of porcine circovirus type 2

To investigate the dynamic Proliferation of Porcine circovirus type 2( PCV- 2) in porcine kidney 15 cell line( PK15),a pair of specific primers was designed based on PCV- 2 open reading frame 2( ORF2). The target gene was amplified using PCR,and then cloned into pMD19- T vector and indentified to prepare the positive plasmid. The fluorescent quantitative PCR assay for detection of PCV- 2 was established based on SYBR GreenⅠ,and then was used to detect the dynamic proliferation of PCV- 2 in PK15 cell line. The results showed that the standard curve of the assay had a good liner relationship between threshold cycle values and template concentrations of 10 fold serially diluted positive plasmids from 2. 0 × 102to 2. 0 × 109DNA copies per microliter. Its correlation coefficient was 0. 998 9,and the amplification efficiency was 99. 50%. The detection limit of the assay was 20 DNA copies per microliter,and the classical swine fever virus,porcine reproductive and respiratory syndrome virus and porcine parvovirus could not be detected. The highest amount of the PCV- 2 DNA with 1. 2 × 107DNA copies per microliter was detected when the PCV- 2 virus had been cultivated in PK15 cell line for 84 hours,which became the best time to harvest the virus. The results indicate that the developed SYBR Green Ⅰfluorescent quantitative PCR assay is stable and reliable with good specificity and high sensitivity.