多酶恒温扩增核酸试纸条法快速检测肺炎支原体方法的建立及应用

Establishment and application of multienzyme isothermal rapid amplification (MIRA) nucleic acid test strip method for rapid detection of Mycoplasma pneumoniae

摘要:

【背景】肺炎支原体是导致儿童和青少年呼吸道感染的重要病原体，长期以来由于其临床表现不特异而容易错过最佳治疗时期。【目的】结合多酶恒温扩增(multienzyme isothermal rapid amplification, MIRA)技术和核酸试纸条建立一种快速检测肺炎支原体的方法。【方法】以肺炎支原体社区获得性肺炎呼吸窘迫综合征(community acquired respiratory distress syndrome, CARDS)毒素编码基因为靶基因设计引物和探针，对反应体系的温度、时间等进行优化，评估其敏感性，通过检测肺炎支原体和其余7种病原体分析其特异性，并对35份临床样本进行验证。【结果】MIRA核酸试纸条法在37 ℃条件下，15 min内便可完成对肺炎支原体的检测，最低检出限为10 copies/μL；除肺炎支原体外，其余7种病原体均不能扩增，特异性较好。以实时荧光PCR检测为标准，MIRA核酸试纸条法对35份临床样本检测后的诊断特异度为100.00%、灵敏度为96.15%、阴性预测值为90.00%、阳性预测值为100.00%。【结论】本研究建立了MIRA核酸试纸条法检测肺炎支原体的方法，具有快速便携、灵敏度高、特异性好等优势，易于在基层推广使用。

Abstract:

[Background] Mycoplasma pneumoniae is an important pathogen causing respiratory tract infections in children and adolescents. It is easy to miss the optimal treatment period due to its nonspecific clinical manifestations for a long time.[Objective] To develop a method for rapidly detecting M. pneumoniae based on the multienzyme isothermal rapid amplification (MIRA) and nucleic acid test strip. [Methods] Primers and probes were designed using the community acquired respiratory distress syndrome (CARDS) toxin gene of M. pneumoniae as the target gene to optimize the temperature and time of the reaction system and evaluate the sensitivity. The specificity was analyzed by testing M. pneumoniae as well as the remaining 7 pathogens, and 35 clinical samples were validated. [Results] The MIRA nucleic acid test strip method completed the detection of M. pneumoniae within 15 minutes at 37℃, and the limit of detection was 10 copies/μL. Except for M. pneumoniae, the other 7 pathogens could not be amplified, showing good specificity. Using real-time fluorescent PCR detection as the standard, the diagnostic specificity, sensitivity, negative predictive value, and positive predictive value of MIRA nucleic acid test strip method for 35 clinical samples were 100.00%, 96.15%, 90.00%, and 100.00%, respectively. [Conclusion] In this study, the MIRA nucleic acid test strip method has been established for the detection of M. pneumoniae, which is fast, portable, sensitive, and specific, and is suitable for promotion at the grassroots level.