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.

Development of Recombinase Aided Amplification Combined With Disposable Nucleic Acid Test Strip for Rapid Detection of Porcine Circovirus Type 2

“…RAA is a new technology developed by China that accords with rapid detection demands in our country (36). It has significant advantages, and has been widely used in detecting pathogens (37)(38)(39)(40)(41). In this study, we developed the PCV2 RAA-strip which is simple, sensitive, specific for rapid detection of PCV2 within 30 min.…”

Development of a Rapid and Efficient RPA-CRISPR/Cas12a Assay for Mycoplasma pneumoniae Detection

“…MP strains harbor multiple genotypes ( Xiao et al, 2015 ; Meyer Sauteur et al, 2021b ), and genetic variants within the target region may lead to false negative results for molecular diagnosis ( Unckless et al, 2017 ; Merida-Vieyra et al, 2019 ; Xue et al, 2020 ). In previous studies, P1 , CARDS toxin , ATPase , 16S rRNA , 23S rRNA , RepMp1 and RepMP4 have been reported as target sequence ( Dumke et al, 2007 ; Winchell et al, 2008 ; Zhou et al, 2015 ; Leal et al, 2020 ).…”

“…In previous studies, P1 , CARDS toxin , ATPase , 16S rRNA , 23S rRNA , RepMp1 and RepMP4 have been reported as target sequence ( Dumke et al, 2007 ; Winchell et al, 2008 ; Zhou et al, 2015 ; Leal et al, 2020 ). Among them, P1 gene has been demonstrated to be high selective and efficient for sequence detection ( Merida-Vieyra et al, 2019 ; Xue et al, 2020 ). P1 gene encodes an important 170-kDa protein as the major adhesion protein, which is essential for MP infection and successful colonization.…”

“…Mycoplasma pneumoniae (MP) is a major causative agent of community-acquired pneumonia in humans, especially children and adolescents, accounting for up to 40% of cases ( Leal et al, 2020 ; Xue et al, 2020 ; Kakiuchi et al, 2021 ). MP infections occur throughout the year with epidemic peaks at intervals of 3-7 years ( Atkinson et al, 2008 ; Rivaya et al, 2020 ; Jiang et al, 2021 ).…”

Real-time recombinase-aided amplification with improved propidium monoazide for the rapid detection of viable Escherichia coli O157:H7 in milk

“…Recombinase-aided amplification (RAA), a kind of isothermal nucleic acid amplification method, can be performed at constant temperatures (37-42°C; Qin et al, 2021) and has a short detection time (less than 20 min) and high sensitivity (Shen et al, 2019). Recombinase-aided amplification does not require a complex instrument to provide cyclic temperature (Xue et al, 2020), which makes it more convenient in resource-poor settings than PCR. In this study, the specific fluorescence probe was added in the RAA system to achieve real-time fluorescence analysis.…”