Use of a rapid recombinase-aided amplification assay for Mycoplasma pneumoniae detection

Abstract

Background: Mycoplasma pneumoniae is one of the most common causative pathogens of community-acquired pneumonia (CAP), accounting for as many as 30-50% of CAP during peak years. An early and rapid diagnostic method is key for guiding clinicians in their choice of antibiotics.

Methods: The recombinase-aided amplification (RAA) assay is a recently developed, rapid detection method that has been used for the detection of several pathogens. The assays were performed in a one-step single tube reaction at 39° Celsius within 15-30 min. In this study, we established an RAA assay for M. pneumoniae using clinical specimens for validation and commercial real-time PCR as the reference method.

Results: The analytical sensitivity of the RAA assay was 2.23 copies per reaction, and no cross-reactions with any of the other 15 related respiratory bacterial pathogens were observed. Compared with the commercial real-time PCR assay used when testing 311 respiratory specimens, the RAA assay obtained 100% sensitivity and 100% specificity with a kappa value of 1.

Conclusions: These results demonstrate that the proposed RAA assay will be of benefit as a faster, sensitive, and specific alternative tool for the detection of M. pneumoniae.

Keywords: Detection; Molecular diagnostic technique; Mycoplasma pneumoniae; Recombinase; Recombinase-aided amplification.

Fig. 1

Specificity of the RAA assay for M. pneumoniae detection. Only the M. pneumoniae samples produced amplification signals, whereas the negative control (buffer only) and control bacterial samples produced negative amplification signals

Fig. 2

Sensitivity of the RAA assay for M. pneumoniae detection. A serial dilution of the recombinant plasmid was used ranging from 10⁴ to 10⁰ copies/reaction. A negative control (buffer only) was also assayed