Accurate, rapid and low-cost diagnosis of Mycoplasma pneumoniae via fast narrow-thermal-cycling denaturation bubble-mediated strand exchange amplification.

ABSTRACT: Mycoplasma pneumoniae is a strong infectious pathogen that may cause severe respiratory infections. Since this pathogen may possess a latent period after infection, which sometimes leads to misdiagnosis by traditional diagnosis methods, the establishment of a rapid and sensitive diagnostic method is crucial for transmission prevention and timely treatment. Herein, a novel detection method was established for M. pneumoniae detection. The method, which improves upon a denaturation bubble-mediated strand exchange amplification (SEA) that we developed in 2016, is called accelerated SEA (ASEA). The established ASEA achieved detection of 1% M. pneumoniae genomic DNA in a DNA mixture from multiple pathogens, and the limit of detection (LOD) of ASEA was as low as 1.0?×?10-17 M (approximately 6.0?×?103 copies/mL). Considering that the threshold of an asymptomatic carriage is normally recommended as 1.0?×?104 copies/mL, this method was able to satisfy the requirement for practical diagnosis of M. pneumoniae. Moreover, the detection process was finished within 20.4 min, significantly shorter than real-time PCR and SEA. Furthermore, ASEA exhibited excellent performance in clinical specimen analysis, with sensitivity and specificity of 96.2% and 100%, respectively, compared with the "gold standard" real-time PCR. More importantly, similar to real-time PCR, ASEA requires only one pair of primers and ordinary commercial polymerase, and can be carried out using a conventional fluorescence real-time PCR instrument, which makes this method low-cost and easy to accomplish. Therefore, ASEA has the potential for wide use in the rapid detection of M. pneumoniae or other pathogens in large numbers of specimens. Graphical abstract.

SUBMITTER: Yang C 

PROVIDER: S-EPMC7548028 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature