利用环介导等温扩增技术检测儿童咽拭子标本中肺炎支原体

Detection of Mycoplasma pneumoniae in nasopharyngeal swab samples collected from children with acute respiratory infections by loop-mediated isothermal amplification

目的采用自行设计的引物,利用环介导等温扩增(LAMP)技术,建立一种快速检测急性呼吸道感染患儿咽拭子标本中肺炎支原体的方法。方法针对肺炎支原体基因组内存在的重复序列SDC1设计6条特异性LAMP引物(SMP),使用实时浊度仪进行扩增反应并记录结果。对该方法进行灵敏度和特异性检测,并与文献报道的引物(JMP)进行比较。应用该方法和实时荧光定量PCR(Q-PCR)检测55例儿童咽拭子标本并对结果进行比较。结果本研究中设计的肺炎支原体特异性SMP引物灵敏度高,最低检出肺炎支原体标准株FH的DNA拷贝数为6个。采用SMP引物的LAMP方法特异性好,与其他支原体和细菌间无交叉反应。55例临床标本分别使用SMP引物和JMP引物进行LAMP检测,两种方法的总符合率为98.2%;使用SMP引物的LAMP方法与Q-PCR的总符合率为96.4%。结论采用本研究设计的SMP引物所建立的LAMP方法灵敏度更高,特异性好,较Q-PCR操作简便,耗时短,可用于临床儿童咽拭子标本中肺炎支原体的实验室快速检测,具有良好的临床应用前景。

Objective To establish a method of loop-mediated isothermal amplification（LAMP）for rapidly detecting Mycoplasma pneumonia （MP）in nasopharyngeal swab samples collected from children with acute respiratory infec-tions.Methods According to the repeat sequence S DC1 of MP genome,6 special primers for LAMP（SMP）were designed and the method for detecting MP DNA was developed.Sensitivity of the LAMP using SMP primers was tested using MP type strain FH DNA,and specificity was evaluated through cross-reaction with other Mycoplasmas and bacte-rial DNAs.The sensitivity of SMP was also compared with the reported LAMP primers（JMP）for MP.To test the reli-ability of SMP,55 nasopharyngeal swab samples were detected by the LAMP using SMP primers and real-time PCR （Q-PCR）,respectively.Results The LAMP using SMP primers could detect 6 copies of FH DNA,and no amplifica-tion was shown in DNA of other Mycoplasma or bacterials.The sensitivity of SMP was ten times higher than that of JMP.For 55 clinical specimens,the total consistency rate of LAMP methods using SMP and JMP primers,respective-ly,was 98.2%,and the total consistency rate of Q-PCR and LAMP using SMP primers was 96.4%.Conclusion LAMP method using the SMP primers designed in this research is more sensitive compared with the reported LAMP method for MP.Also,the LAMP method is more rapid and convenient for detecting MP from nasopharyngeal swab samples,so this method should be very useful for the rapid detection of MP in clinical diagnosis of MP infection.