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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.

SUBMITTER: Xue G

PROVIDER: S-EPMC6988361 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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

Xue Guanhua G Li Shaoli S Zhao Hanqing H Yan Chao C Feng Yanling Y Cui Jinghua J Jiang Tingting T Yuan Jing J

BMC infectious diseases 20200128 1

<h4>Background</h4>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.<h4>Methods</h4>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 ...[more]

PMID: 31992210

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Project description:Hypervirulent Klebsiella pneumoniae (hvKp) is a major human pathogen associated with liver abscess, pneumonia, meningitis, and endophthalmitis. It is challenging to differentiate hvKp from classical Klebsiella pneumoniae (cKp) using conventional methods, necessitating the development of a rapid, sensitive, and convenient assay for hvKp detection. In this study, we constructed a recombinase-aided amplification (RAA) method targeting hvKp genes peg344 and rmpA, and also analyzed the pathogenic characteristics of hvKp. We optimized the reaction temperature and system, and evaluated its sensitivity, specificity, and clinical application. The primer and probe sets peg344-set1 and rmpA-set2 delivered significant fluorescent signals at 39°C with the shortest gene amplification times (sensitivity: 20 copies/reaction). This RAA assay showed no cross-reactivity with 15 other common pathogenic bacteria. Its applicability was confirmed by the evaluation of 208 clinical specimens, of which 45 were confirmed to be hvKp. The sensitivity and specificity of the RAA assay were both 100% compared with real-time PCR as the reference standard. To verify the assay, we also assessed the diversity of molecular characteristics among the hvKp isolates and identified serotype K1 and sequence type ST23 as the dominant clone. Virulence factors iroN and iutA were highly associated with virulence level. In conclusion, our novel RAA assay is a powerful tool for early diagnosis and epidemiological surveillance of hvKp. IMPORTANCEKlebsiella pneumoniae is the most common opportunistic bacterial species and a major threat to public health. Since the 1990s, hvKp has received increasing attention from public health officials and infectious disease specialists. Hypervirulent strains differ from classical strains in terms of phenotypic features and clinical outcomes. It is hard to identify hvKp from cKp using the conventional methods including colony morphology analysis, serum killing assays, mouse lethality assays, string tests, and real-time PCR. In this study, we established a rapid, sensitive and convenient recombinase-aided amplification assay for hvKp detection targeting virulence genes peg344 and rmpA. Our RAA assay provides an important tool for the rapid diagnosis of infectious diseases caused by hvKp, particularly in primary laboratories.

Dataset Information

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

Publications

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

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