实时定量聚合酶螺旋反应定量检测变形链球菌方法的建立

摘要

Objective:To establish the polymerase spiral reaction(PSR) real-time quantitative method for detection of Streptococcus mutans in oral cavity.Methods:The 4 sets of primers for S.mutans gtfB genes were designed and subjected to the PSR to select the optimal set,and then the result was determined by the real-time turbidity method and chromogenic method.Results:The best primer from 4 sets of primers was selected out,and the best temperature was determined to be 65℃.The specificity was indicated by the further experiments showing that there was no cross reaction with 13 other pathogen nucleic acid with the best primers.The sensitivity is up to 10 copies/μL.Conclusion:We established the PSR method of real-time quantitative detection of S.mutans.This method has the characteristic of simpleness and quickness,strong specificity and high sensitivity,which provides a new method for real-time quantitative detecting S.mutans.%目的:建立实时定量检测口腔内变形链球菌的实时定量聚合酶螺旋反应(PSR)方法.方法:针对变形链球菌的gtfB基因设计4套引物,通过实时浊度法和显色法两种方法判断结果.结果:从4套引物中筛选出最佳引物,并确定最佳温度为65℃;进一步实验表明采用最佳引物能特异性地检测变形链球菌,与13种其他病原核酸无交叉反应,敏感性达10拷贝/μL.结论:建立了实时定量检测变形链球菌的PSR方法,该方法具有简单快速、特异性强、敏感性高的特点,为实时定量检测变形链球菌提供了新技术.