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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 rapid method for the detection of Mycoplasma pneumoniae in throat swab specimens of children with acute respiratory tract infection by using self-designed primers and ring-mediated isothermal amplification (LAMP) technique. Methods Six specific LAMP primers (SMPs) were designed for the repetitive sequence SDC1 in Mycoplasma pneumoniae genome. The real-time turbidimeters were used to amplify and record the results. Sensitivity and specificity of this method were tested and compared with those reported in the literature (JMP). The method and real-time quantitative PCR (Q-PCR) were used to detect 55 cases of children’s throat swab specimens and the results were compared. Results The Mycoplasma pneumoniae-specific SMP primers designed in this study were highly sensitive. The lowest copy number of FH DNA was 6 in Mycoplasma pneumoniae standard strains. The LAMP method with SMP primers is specific and does not cross-react with other mycoplasmas and bacteria. 55 cases of clinical specimens were detected by LMP using SMP primer and JMP primer respectively, the total coincidence rate of the two methods was 98.2%. The total coincidence rate of LAMP method and Q-PCR using SMP primer was 96.4%. Conclusion The LAMP method established by this study is more sensitive and specific than Q-PCR, which is simpler and less time-consuming than Q-PCR. It can be used for the rapid laboratory detection of Mycoplasma pneumoniae in clinical children’s throat swab specimens. Good clinical application prospects.