链球菌SYBR Green I 荧光定量PCR检测方法的建立

Development of a SYBR Green I based real-time PCR assay for detection of Streptococcus

为建立链球菌的快速定量检测方法,本研究根据链球菌延伸因子EF-Tu基因保守序列设计一对引物,以链球菌DNA为模板通过PCR扩增其197 bp的EF-Tu基因片段,并克隆至p MD18-T载体中。以纯化的重组质粒为标准品建立标准曲线,建立了链球菌SYBR Green I荧光定量PCR检测方法。结果显示在3.98×106拷贝/m L^3.98×102拷贝/μL范围内具有较好的线性关系,相关系数为R2=0.995,最低检出量为39.8拷贝/μL。此外,该方法具有良好的特异性,对大肠杆菌、金黄色葡萄球菌、变形杆菌等其他细菌的检测结果均为阴性。重复性试验表明,该方法的组内和组间变异系数均低于1.0%。利用该方法对34份疑似链球菌感染病料进行检测,结果显示本研究所建立的方法对链球菌的检出率比常规PCR高14.7%,比细菌分离鉴定方法高29.4%。表明该方法可以用于对链球菌感染的定量研究和流行病学调查。

To develop a quantitative assay for the detection of Streptococcus,a SYBR Green Ⅰ based real-time PCR was established with a pair of specific primers designed according to the elongation factor EF-Tu gene sequences of Streptococcus.Accordingly,the standards curve of the real-time PCR displayed a linear correlations in a ranges from 3.98×106 copies/μL to 3.98× 102 copies/μL of recombinant plasmid containing the 197 bp fragment of the bacteria EF-Tu gene with a correlation coefficient of 0.995.The assay was highly specific for Streptococcus detection with a limit detection of 39.8 copies/μL,and no amplification was found for other bacteria.The coefficient of variations was less than 1％ in the replicate test.The Streptococcus detection rate of 34 clinical samples showed that the method was more sensitive than conventional PCR and bacteria isolation and identification method.This method was proved to be specific and sensitive,which could be used in the quantitative study and epidemiological investigation of Streptococcus infection.