牛结核病荧光定量PCR引物探针的组合筛选比较

Comparison of Combinations of Fluorescent Quantitative PCR Primers and Probe for Bovine Tuberculosis

为比较牛结核病荧光定量PCR(qPCR)不同引物探针组合的特异性和敏感性,验证其临床检测效果,选取国际、国家、行业、地方标准及文献报道的11组qPCR鉴定引物探针组合,采用牛分枝杆菌基因组DNA国家二级标准物质、参考菌株核酸和临床奶样对其进行比较、验证。结果显示:11组引物探针均无非特异性扩增,表现出良好的特异性;国标IS1081引物检测灵敏度最高,最低检测限可达1.4×10^(-1);除国标Mtb(结核分枝杆菌)引物外,其他引物探针组合重复性良好(CV<10%);30份临床样品中国标IS1081引物阳性样品检出率明显高于其他引物。结果表明,国标IS1081引物探针组合特异、灵敏、临床检测效果更优,推荐用于实验室qPCR检测牛结核病。

In order to compare the specificity and sensitivity of different combinations of fluorescent quantitative PCR(qPCR)primers and probe for bovine tuberculosis,and to verify their clinical effects,11 combinations proposed by international,national,industrial and local standards and reported by literatures were compared and verified using national secondary standard materials of Mycobacterium bovis genomic DNA,nucleic acids of reference strain as well as clinical milk samples.The results showed that the 11 combinations all had no non-specific amplification,indicating good specificity;the primers originated from national standard IS1081 could reach a minimum detection limit of 1.4×10^(-1)with the highest sensitivity;the reproducibility of other combinations was good(CV<10%),except for the national standard primers(Mtb);for 30 clinical samples,the detection rate of the positive samples detected by the primers originated from the national standard IS1081 was significantly higher than that by other combinations.In conclusion,the combination of primers and probe in national standard IS1081 was specific and sensitive,which could receive better clinical effects and was proposed to detect bovine tuberculosis by qPCR in laboratory.