猪瘟病毒TaqMan实时荧光定量PCR检测方法的建立及初步应用

Development and Application of TaqMan Fluorescence Quantitative PCR Assay for Detecting Classical Swine Fever Virus

根据GenBank中猪瘟病毒(classical swine fever virus,CSFV)5′NTR的保守序列,设计1对特异性引物和TaqMan探针,以CSFV全长基因重组质粒pGEM-CSFV为标准品,建立TaqMan实时荧光定量PCR标准曲线,进行特异性、敏感性和重复性试验,并检测人工感染CSFV后不同时期猪血浆中CSFV的载量。结果表明,标准曲线循环阈值与模板浓度具有良好的线性关系,R2=0.999;敏感性高,最低检测限为1×101拷贝/μL;特异性强,与猪圆环病毒2型、猪细小病毒、猪伪狂犬病病毒和猪繁殖与呼吸综合征病毒无交叉反应性;重复性好,组内和组间变异系数均小于2%。人工感染CSFV后第7天猪血浆中CSFV载量达到峰值,之后逐渐降低。结果表明,建立的TaqMan实时荧光定量PCR方法具有特异、敏感、重复性好等优点,为CSFV的定量分析及临床诊断奠定基础。

According to the conserved sequence of 5rnontranslated region （5rNTR）of classical swine fever virus （CSFV） in GenBank, a pair of primers and a TaqMan probe were designed. The recombinant plasmid pGEM-CSFV containing the complete gene of CSFV was as a standard control to establish fluorescence quantitative PCR standard curve. Specificity, sensitivity and reproducibility of the method were determined. CSFV load in plasma was measured at different time points after experimental infection of CSFV in pigs. The results showed that standard curve established had a good linear relationship between threshold cycle and template concentration, R^2 = 0. 999. The assay had high sensitivity and could detect 1 × 10^1 eopies/μL. The speci ficity of the method was high, there was no cross reaction between CSFV and other pathogens containing porcine circovirus 2, porcine parvovirus, porcine pseudorabies virus and porcine reproductive and respiratory syndrome virus. The method showed good reproducibility, there was a coefficient of variations less than 2 % for both inter-assay and intra-assay. CSFV load in plasma increased to peak value at 7 days after experimental infection of CSFV in swine and gradually decreased later. These results indicated that the developed TaqMan fluorescence quantitative PCR assay had the advantages of good specificity, sensitivity and reproducibility. It provided a basis for quantification analysis and clinical diagnosis of CSFV.