Development of a duplex recombinase-aided amplification assay for direct detection of Mycoplasma pneumoniae and Chlamydia trachomatis in clinical samples.

Background: Pneumonia caused by Mycoplasma pneumoniae is common in the elderly and children, and pneumonia caused by Chlamydia trachomatis is prevalent in newborns. This study aimed to establish a rapid, sensitive, and simple method for the direct detection of M. pneumoniae and C. trachomatis in clinical samples without DNA extraction.

Methods: We established a duplex recombinase-aided amplification (RAA) assay with the RNAseP gene as an internal control for detecting the P1 gene of M. pneumoniae and the ORF8 gene of C. trachomatis, respectively. The results were obtained at 39 °C within 15-20 min. A total of 130 clinical samples suspected of M. pneumoniae or C. trachomatis infection were collected and tested by duplex RAA and PCR. DNA extracted via a commercial kit or treated with a nucleic acid-releasing agent was used and compared, respectively. Standard recombinant plasmids were used to test the sensitivity of the duplex RAA assay. In addition, other similar common pathogens were used to verify the specificity of the duplex RAA assay.

Results: The sensitivity of the duplex RAA assay for detecting M. pneumoniae and C. trachomatis was 10 copies/μL using recombinant plasmids. Compared with PCR, the sensitivity and specificity of duplex RAA assays for M. pneumoniae and C. trachomatis was 100% using clinical DNA samples extracted using a commercial kit and a nucleic acid-releasing agent, and the Kappa value was 1.

Conclusion: The advantages of this duplex RAA assay include high sensitivity and specificity, short duration, and simple extraction steps, with potential for use in the on-site detection of M. pneumoniae and C. trachomatis in resource-limited settings.