摘要： 为了建立一种基于SYBRGreen的猪圆环病毒2型（PCV2）荧光定量PCR诊断方法，以PCV2 ORF2基因序列为研究对象，构建pMD19T-ORF2重组质粒作为阳性标准品，设计特异性引物，对该方法的最适引物浓度、退火进行了优化，并对该方法的灵敏度、特异性、可重复性以及临床样品检出率进行了评价。结果表明，该方法引物的最佳终浓度为0.2 mol/L，最适退火温度为55℃；检测限可达到3.0×10²个拷贝/mL，而普通PCR检测限在3.0×10⁴个拷贝/mL；溶解曲线分析显示，在77~79℃有单一的溶解峰出现，并与CSFV、PPV、PRRSV、PRV没有交叉反应；组内重复性变异系数为0.29%～0.32%，组间变异系数为0.32%～0.36%；此外，该方法对临床样品的检出率为81.5%（31/38），而常规PCR为68.4%（26/38）。因此，相比常规PCR，该方法更为快速、灵敏、特异、稳定，更适合PCV2临床样品的检测，可为该PCV2早期感染的快速检测提供新方法。

关键词: 猪圆环病毒2型, PCV2, ORF2, SYBRGreen Real-time qPCR

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

中图分类号: 

• S852.65