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| The Development of a SYBRGreen Real-time qPCR for the Detection of Porcine Circovirus Type 2 |
| FENG Hua1, LIU Yunchao1, CHEN Yumei3, WEI Qiang1, ZHANG Gaiping1,2 |
1. Key Laboratory of Animal Immunology of Ministry of Agriculture, Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China;
2. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China;
3. School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China |
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Abstract To develop a SYBRGreen Real-time quantitative PCR (qPCR)assay for PCV2 diagnose, the recombinant plasmid pMD19T-ORF2 was constructed as template based on PCV2 ORF2 (706 bp), and the specific primers were designed. The primer concentration and annealing temperature were optimized, respectively. Besides, the sensitivity, specificity, reproducibility and clinical samples detection of this method were further tested. The results showed that the detection limit for this assay was 3.0×102 copies/mL, while it was 3.0×104 copies/mL for conventional PCR. The melt curve analysis presented a single melt peak located at 77-79℃. And no cross reaction with CSFV, PPV, PRRSV and PRV was detected. The inter-assay coefficient of variation ranged from 0.29% to 0.32% and the inter-assay coefficient of variation ranged from 0.32% to 0.36%. In addition, a higher PCV2 positive rate was detected by SYBRGreen Real-time qPCR (81.5%, 31/38)compared with conventional PCR (68.4%, 26/38)in clinical samples detection. Therefore, the PCV2 SYBRGreen Real-time qPCR is more rapid, sensitive, specific and stable, which was more suitable for PCV2 clinical infection diagnose.
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Received: 14 March 2018
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2018, Vol. 33 
