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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。
Three kinds of streptococcal gene sequence analysis, designed three Taq Man probe, and select three non-interfering fluorescent groups for labeling. The real-time fluorescent PCR method was established and the specificity, sensitivity, stability and reproducibility of the established method were evaluated and compared with the traditional culture method. The results showed that the established method can specifically amplify three kinds of standard strains of Streptococcus; three kinds of Streptococcus have no cross reaction; the detection sensitivity of S.agalactiae is about 1.52 × 10 ~ 1 CFU / mL; The detection sensitivity of dysgalactiae was about 1.33 × 10 ~ 2 CFU / mL. The detection sensitivity of S.milleri was about 1.76 × 10 ~ 1 CFU / mL. The CV of the three kinds of streptococci was less than 5% A total of 5 S.agalactiae positive samples, 3 S.dysgalactiae positive samples and 1 S.milleri positive sample were detected, which were consistent with the results of traditional bacterial isolation and identification. The detection method has high sensitivity, strong specificity and good practicability.