基于颜色判定的环介导等温扩增检测人型支原体的研究

Detection of Mycoplasma hominis using colorimetric loop-mediated isothermal amplification

目的建立一种基于颜色判定的快速、灵敏、简便的人型支原体环介导等温扩增(LAMP)方法。方法使用PrimerExplorer V5软件设计针对人型支原体MHO_2540基因保守区域的4条特异等温扩增引物,用羟基萘酚蓝(HNB)作为结果判读指示剂并优化体系中dNTPs、MgSO_4浓度,建立人型支原体LAMP检测方法,进行灵敏度、特异度等的检测并与普通PCR比较。结果LAMP对标准浓度人型支原体核酸的检测限约为10^(2 )CFU,与普通PCR相同,但比实时荧光定量PCR差一个数量级。人型支原体阳性临床标本拷贝数>10^(3 )CFU者LAMP阳性率100%,与普通PCR检出率(100%)一致,拷贝数10~1~10^(3 )CFU的弱阳性标本LAMP检出率为63.6%(14/22),普通PCR检出率为31.8%(7/22),差异有统计学意义(χ~2=4.46,P<0.05)。结论LAMP检测人型支原体敏感、特异且操作简便,可在基层推广应用。

Objective To establish a rapid,sensitive,and simple technique of loop-mediated isothermal amplification（LAMP）for detection of Mycoplasma hominis based on color determination.Methods The software Primer Explorer V5was used to design four specific isothermal amplification primers for the conserved region of the MHO2540gene of M.hominis.Hydroxyl naphthol blue（HNB）was used as a reading indicator to optimize the concentration of dNTPs and MgSO4in the system to establish a LAMP technique for detection of M.hominis.A M.hominis LAMP assay was established.The sensitivity and specificity of the LAMP test were compared to those of conventional PCR and real-time PCR.Results The detection limit of LAMP for standard concentrations of M.hominis nucleic acids was approximately 102CFU.The sensitivity of LAMP was the same as that of conventional PCR but one order of magnitude worse than that of real-time PCR.According to LAMP,100%of clinical specimens tested positive for M.hominis at a concentration of more than 10^3 CFU,and this rate was consistent with the rate at which human Mycoplasma was detected with conventional PCR（100%）.LAMP detected M.hominis at a rate of 63.6%（14/22）in weakly positive samples with a copy number of 10^1-10^3 CFU while conventional PCR detected M.hominis at a rate of 31.8%（7/22）.The rates of detection differed significantly（χ^2=4.46,P〈0.05）.Conclusion LAMP is sensitive and specific at detecting M.hominis,it is easy to perform,and it could be widely used at the local level.