B群链球菌及其大环内酯类耐药基因LAMP快速可视化检测方法的建立和初步应用

目的 建立一种快速可视化检测B群链球菌(GBS)及其大环内酯类耐药基因的环介导恒温扩增(LAMP)方法，为临床GBS检测提供新方法。方法 针对GBS特异性基因cfb和大环内酯类耐药基因ermB、mefA/E和linB设计LAMP反应的引物；建立并优化LAMP的反应体系和反应条件，反应结果通过DNA扩增曲线和肉眼观察反应液颜色变化判断；以携带上述基因的质粒DNA为检测标准，确定LAMP方法检测目标基因的敏感性与特异性，并与PCR方法的检测结果比较；采用LAMP方法检测临床300份孕产妇阴道直肠拭子标本中GBS，与分离培养法的检出率进行统计学分析；采用LAMP方法检测80株GBS临床分离株中大环内酯类耐药基因，并与药敏试验结果进行统计学分析。结果 建立了快速可视化检测GBS及其大环内酯类耐药基因的LAMP反应体系和方法，该法在63℃、45 min内完成；该法能够特异性检测pMD-cfb、pMD-ermB、pMD-mefA/E、pMD-linB质粒DNA，其灵敏度为10-5 ng/μL，是PCR方法灵敏度的100倍；对300份孕产妇阴道直肠拭子标本进行检测，LAMP方法对GBS检出率为12....

Establishment and preliminary application of LAMP visualization method for rapid detection of group B Streptococcus and its macrolide resistance genes

Abstract Objective To establish a rapid and visual loop-mediated isothermal amplification (LAMP)method fordetection of group B Streptococcus (GBS) and its macrolide resistance genes, and to provide a newmethod for clinical detection of GBS. Methods LAMP primers were designed for GBS specific gene cfb andmacrolide resistance genes ermB, mefA/E and linB. The reaction system and reaction conditions of LAMP methodwere established and optimized. The reaction results could be determined by DNA amplification curve and visualobservation of the color change of reaction solution. The sensitivity and specificity of LAMP method for detection oftarget genes were determined by using plasmid DNA carrying the above genes as detection standard, and the detectionresults were compared with those of PCR method. LAMP method was used to detect GBS in 300 vaginal and rectalswab specimens of pregnant women, and the detection rate of GBS was statistically analyzed compared with that ofisolation and culture method. The macrolide resistance genes of 80 clinical GBS isolates were detected by LAMP, andthe results of macrolide resistance strains detected by drug sensitivity test were statistically analyzed. Results Therapid and visual LAMP reaction system and the method for detection of GBS and its macrolide resistant genes wereestablished. The method was completed within 63℃ and 45min. The sensitivity of pMD-cfb, pMD-ermB, pMDmefA/E and pMD-linB plasmid DNA was 10-5ng/μL, which was 100 times that of PCR method. The detection ratesof GBS strains in 300 vaginal and rectal swab specimens of pregnant women were 12.7% and 11.3% respectively byLAMP method and isolation culture method. There was no statistical difference between the two methods (P>0.05).The detection rates of macrolide resistant strains in 80 strains of GBS were 53.8% and 48.8% respectively by drugsensitivity test and LAMP method, and there was no statistical difference between the two methods (P>0.05).Conclusion The rapid visual detection method of GBS and its macrolide resistant genes established in this study hasthe advantages of good specificity, high sensitivity, and visual results, which is of great significance for the preventionand treatment of clinical neonatal GBS diseases.