基于颜色判定的环介导等温扩增检测生殖支原体的研究

Detection of Mycoplasma genitalium by colorimetric loop mediated isothermal amplification

目的建立一种基于颜色判定的快速、灵敏、简便的检测生殖支原体(MG)感染的环介导等温扩增(LAMP)方法。方法使用PrimerExplorer V5软件依据gap基因保守区域设计4条特异性MG等温扩增引物,通过优化体系dNTPs、MgSO4浓度,建立一种基于羟基萘酚蓝(HNB)作为结果判读指示剂的MG等温扩增方法,进行灵敏度、特异性检测并与普通PCR比较;对30份性病门诊病例标本进行LAMP、RT-PCR和普通PCR检测,并进行比较分析。结果建立的LAMP方法对8种常见支原体以及8种泌尿生殖道常见病原菌扩增均为阴性,特异性度为100%。对于MG ATCC 33530标准菌株核酸的检测限约为10~2copies,与MG普通PCR一致,比MGRT-PCR检测限(10 copies)低一个数量级。30份临床标本使用LAMP、普通PCR和RT-PCR方法的MG阳性检出率分别为16.7%,16.7%和20.0%。结论建立的MG LAMP检测方法灵敏、特异且操作简便,可在基层推广应用。

Objectives To establish a simple,rapid,and sensitive method of colorimetric loop-mediated isothermal amplification(LAMP)to detect Mycoplasma genitalium.Methods Four specific primers were designed with the online software Primer Explorer V5(http://primerexplorer.jp/e/).The concentrations of dNTPs and MgSO4 were optimized,and the specificity and sensitivity of the established LAMP method for the detection of M.genitalium were evaluated.The amplification process of LAMP was monitored with hydroxyl naphthol blue(HNB).Thirty clinical specimens were evaluated using LAMP and PCR assays.Results The novel LAMP method had exceptional sensitivity and specificity.This LAMP method yielded negative results for 8 other mycoplasmas and 8 common genital tract pathogens,and it had a specificity of 100%.Verification with standard nucleic acid concentrations indicated a detection limit of 102 CFUs,which is the same as for conventional PCR and one order of magnitude lower than that for real-time PCR.LAMP detected M.genitalium in 16.7%of 30 clinical specimens,conventional PCR detected M.genitalium in 16.7%,and real-time PCR detected M.genitalium in 20.0%.Conclusion The LAMP assay to detect M.genitalium is simple,rapid,and sensitive.This method could facilitate rapid detection of M.genitalium in primary hospitals and CDCs.