基于cfb和scpb基因的无乳链球菌PCR检测方法比较及临床应用

目的比较基于cfb和scpb基因检测无乳链球菌(GBS)的两种PCR方法,建立合理的实时荧光PCR检测体系,实现快速检测生殖道无乳链球菌的目的。方法通过对60株临床分离的无乳链球菌和15株非无乳链球菌进行PCR扩增,对比和验证cfb和scpb基因的敏感性和特异性,选择检测性能更好的基因为靶标,建立实时荧光定量PCR(real-time PCR)检测体系。对100份宫颈拭子、精液等常见临床标本进行检测,检测结果与传统的培养法进行比较。阳性率的比较采用卡方检验,P<0.05为差异有统计学意义。结果cfb和scpb基因的特异性均为100%,敏感性分别为98.3%、66.7%,差异有统计学意义(P<0.005)。基于cfb基因的real-time PCR检测100份GBS临床样本的阳性率为35%,高于培养法(30%),差异无统计学意义,但这100份标本中有7份real-time PCR法扩增阳性但未培养出无乳链球菌。结论基于cfb基因的real-time PCR法检测GBS具有直接、快速、简便、高特异性等特点,且敏感性高于常规培养法,可应用于多种临床标本的检测,在临床上具有较大的应用前景。

Comparison and clinical application of cfb and scpb genes in PCR assays to detect Streptococcus agalactiae

Objective To compare two PCR assays based on cfb and scpb genes for the detection of Streptococcus agalactiae,and establish a rational real-time quantitative PCR(qPCR)detection system to detect S.agalactiae rapidly.Methods The sensitivity and specificity of cfb and scpb genes were compared and verified by PCR amplification of 60 clinical isolated strains of S.agalactiae and 15 strains of non-S.agalactiae,and the genes with better detection performance were selected as targets to establish a realtime quantitative PCR detection system.Then,100 clinical specimens,such as cervical swabs and semen,were detected by qPCR and compared with the traditional culture method.Chi-square test was used to compare the positive rates,and P<0.05 was considered statistically significant.Results The specificity of both cfb and scpb genes were 100%,and the sensitivity were 98.3%and 66.7%,respectively.The difference was statistically significant(P<0.005).The positive rate of GBS detected by real-time PCR and culture were 35%and 30%respectively,which has no statistical difference.However,7 samples of the 100 samples were amplified positive by real-time PCR positive,but the S.agalactiae was not isolated.Conclusion The detection of GBS by real-time PCR method targeting cfb gene was direct,rapid,simple,high specificity.And the sensitivity of the real-time PCR was higher than conventional culture method,so that the method can be applied to the detection of various clinical samples,and has great application prospect in clinic.