生殖道病原菌LAMP检测的环引物设计与优化

Designing and optimization of loop primer for LAMP detection of reproductive tract pathogens

孕期生殖道病原菌感染是威胁妇女和胎儿健康的常见疾病之一,为改进现有检测方法耗时长的缺陷,对基于环介导等温扩增(loop mediated isothermal amplification,LAMP)的生殖道病原菌快速检测方法进行优化。在Gardnerella vaginalis的LAMP检测体系中,通过对不同环引物组扩增速率(Ct值)进行统计学分析,验证了环引物在LAMP反应中提升扩增速率的能力,同时探究了环引物长度和GC含量对扩增速率的影响,并将其作为环引物筛选参数应用于3种生殖道病原菌(G.vaginalis、Streptococcus agalactiae、Enterococcus faecalis)检测的环引物设计。结果表明,不同GC含量的环引物扩增速率差异不显著,而环引物的长度是影响其扩增效果的关键因素,碱基数较少的环引物有利于提高LAMP反应速率(p<0.05),设计出的引物组GV-1、GBS-132和EF-3可以在105 CFU/mL扩增浓度下在30 min内呈现完整“S”扩增曲线,具有较好的临床病原菌筛查应用潜力。

Reproductive tract infection(RTI)during pregnancy is one of the common diseases threatening the health of women and fetal.To shorten the long duration of existing methods,the rapid detection of reproductive tract pathogens based on loop mediated isothermal amplification(LAMP)was optimized.By LAMP detection system of G.vaginalis,length and GC content were used as parameters for loop primer screening,and the amplification rate(Ct value)of different loop primers were statistically analyzed.The positive effect of loop primer was verified,and the effects of length and GC contents on the amplification rate were explored,which was applied as a screening parameter for rapid detection of G.vaginalis,S.agalactiae and E.faecalis.The results showed that the amplification was not affected by GC content,while the length of the loop primer was a key factor to improve amplification efficiency,and loop primer with less bases significantly accelerate the LAMP amplification(p<0.05).The designed primer sets GV-1,GBS-132 and EF-3 present a complete"S"amplification curve within 30 minutes for pathogens at 105 CFU/mL concentration,which have good potential for clinical screening.