The Development of a SYBRGreen Real-time qPCR for the Detection of Porcine Circovirus Type 2

doi:10.7668/hbnxb.2018.04.014

Abstract

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.

Key words: Porcine circovirus type 2, PCV2, ORF2, SYBRGreen Real-time qPCR

CLC Number:

S852.65

FENG Hua, LIU Yunchao, CHEN Yumei, WEI Qiang, ZHANG Gaiping. The Development of a SYBRGreen Real-time qPCR for the Detection of Porcine Circovirus Type 2[J]. Acta Agriculturae Boreali-Sinica, 2018, 33(4): 98-103. doi: 10.7668/hbnxb.2018.04.014.

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