三种鱼类链球菌三重实时荧光PCR方法的建立

Triplex Real-time PCR Rapid Detection for Three Fish Streptococcus

对3种链球菌基因序列的分析,设计了3条Taq Man探针,并选择3种无相互干扰的荧光基团进行标记。建立了三重实时荧光PCR的检测方法,同时对建立的方法进行了特异性、灵敏度、稳定性和重复性评价,并且与传统培养法进行了比较。结果显示,试验建立的方法能特异扩增出3种链球菌的标准阳性菌株;3种链球菌之间没有交叉反应;S.agalactiae的检测灵敏度约为1.52×10^1 CFU/mL;S.dysgalactiae的检测灵敏度约为1.33×10^2 CFU/mL;S.milleri的检测灵敏度约为1.76×10^1 CFU/mL;3种链球菌检测反应的CV值均小于5%;对采集的287份样品进行检测,共计检出5份S.agalactiae阳性样品、3份S.dysgalactiae阳性样品和1份S.milleri阳性样品与传统细菌分离鉴定的方法检测结果一致。该检测方法灵敏度高、特异性强,具有良好的实用性。

By the gene sequence analysis of three kinds of Streptococcus, which were S. agalactiae, S. dysgalactiae and S. milleri, respectively. Three TaqMan probes, which could differentiate and detect the three Streptococcus respectively without interference, were designed from highly conserved regions, and labeled with three dyes. Triplex real-time PCR method was developed, while the specificity, sensitivity, stability and reproducibility of the established detecting method were evaluate, d, and the established detecting method was compared with the traditional culture method. The results showed that three kinds of Streptococcus were specifically amplified, other strains were not amplified, which had no cross-reaction with three kinds of Streptococcus. In addition, the sensitivity of S. agalactiae was 1.52 × 10^1 CFU/mL; The sensitivity of S. agalactiae was 1.33 ×10^2 CFU/mL; The sensitivity of S. milleri was 1.76× 10^1 CFU/mL. Stability and reproducibility of the test results showed that the coefficient of variation of the detection reaction for three kinds of Streptococcus was less than 5%. Furthermore, 5 positive samples for S. agalactiae, 3 positive samples for S. dysgalactiae were detected from 287 clinical samples by the triplex real-time PCR method, which was in accordance with the testing result by the traditional culture method. Therefore, the aiplex real-time method provided a novel rapid, sensitive and good repeatability detection method for the infection of S. agalactiae, S. dysgalactiae and S. milleri.