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

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.

Key words:multienzyme isothermal rapid amplification (MIRA);nucleic acid test strip;Mycoplasma pneumoniae