实时荧光定量聚合酶链反应检测远缘链球菌的实验研究

Detection of Streptococcus Sobrinus by real-time fluorescence quantitative polymerase chain reaction method

目的 :建立一种实时荧光定量聚合酶链反应 (real-timefluorescencequantitativePCR ,FQPCR)方法 ,准确快速的定量检测唾液中的远缘链球菌。方法 :在定性PCR检测的基础上设计一对特异引物 ,以及一条TaqMan标记的寡核苷酸探针 ,对已知数量的标准远缘链球菌株 6 715以及 30名临床龋病儿童唾液样本核酸模板进行PCR扩增 ,并用培养鉴定的方法对 30例唾液样本进行对照检测。结果 :经FQPCR检测 ,10 6～ 10 2 拷贝的标准菌模板均可以用该方法检出 ,且循环阈值 (Ct值 )与起始拷贝数的对数相关性好 ,r=- 0 .9996 0 9。 30例唾液样本中 ,13例可以检出并准确定量 ,培养鉴定的方法结果有 11例阳性。结论 :实时荧光定量PCR方法可以用于快速灵敏的定量检测远缘链球菌 ,比传统的培养鉴定方法显示了更大的优越性。

AIM:To build a real-time fluorescence quantitative PCR method of detecting Streptococcus Sobrinus , which can rapidly and quantitatively detect the Streptococcus Sobrinus in comparing to the conventional cultured method. METHODS: A pair of specific primers was designed based on our normal PCR experiments, but a TaqMan oligonucleotide probe was added in the PCR reaction. Standard Streptococcus Sobrinus 6715 with different concentration and 30 saliva samples were tested by the FQ PCR method in comparing to the traditional cultured methods. RESULTS: Bacteria samples with concentration>10 2 could be detected by FQ PCR and counted accurately. The series diluted standard bacteria had a high correlation in FQ PCR (correlation =-0.999609). Results of the cultured method were similar to that of the FQ PCR methods. CONCLUSION: The QC PCR methods of what we have established in this experiment display a well specific and accurate properties. It is more timesaving and simple compared to the cultured methods and other quantitative methods to detect Streptococcus Sobrinus.