Work

Development of a LAMP assay using hybridization-based TaqMan-style probes (HyTaq) for simultaneous detection of wild-type and macrolide-resistant Mycoplasma pneumoniae

Year: 2025

Type: article

Abstract: ABSTRACT Mycoplasma pneumoniae is a major cause of community-acquired pneumonia and increasingly exhibits macrolide resistance due to the A2063G and A2064G mutations in the 23S rRNA gene. We developed a loop-mediated isothermal amplification (LAMP) assay with hybridization-based TaqMan-style probes (HyTaq) to simultaneously detect M. pneumoniae wild-type strains and macrolide resistance mutations (A2063G and A2064G) within a single reaction (hereafter referred to as the MP-R HyTaq-LAMP assay). Analytical performance was evaluated using plasmid standards, and clinical validation was conducted with 224 nasopharyngeal swab samples. The MP-R HyTaq-LAMP assay detected wild-type strains and the A2063G and A2064G mutations with a limit of detection of 1 × 10³ copies/μL. In clinical samples, the assay demonstrated 95.79% sensitivity for A2063G and 100% sensitivity for wild-type strains, along with 100% specificity against 103 non-infection samples. Additionally, no cross-reactivity was observed with 16 other common respiratory pathogens. The MP-R HyTaq-LAMP assay provides rapid, multiplex detection of M. pneumoniae and associated macrolide resistance mutations without thermal cycling, demonstrating its strong potential as a point-of-care diagnostic tool. IMPORTANCE Macrolide-resistant Mycoplasma pneumoniae is a significant cause of treatment failure in community-acquired respiratory infections, particularly in children and adolescents. Early and accurate detection of resistance-associated mutations, such as A2063G and A2064G, is essential for guiding effective antibiotic therapy. In this study, we developed an MP-R HyTaq-based loop-mediated isothermal amplification assay capable of simultaneously detecting and discriminating wild-type strains and macrolide resistance mutations (A2063G and A2064G) in a single-tube, isothermal reaction. Its high specificity, rapid turnaround time, and minimal equipment requirements make this assay suitable for point-of-care testing in diverse clinical settings.

Source: Journal of Clinical Microbiology

DOI: 10.1128/jcm.01291-25

Authors: Eunji Lee, Woo Sang Jung, Chae Seung Lim, Woong Sik Jang

Institutions: Korea University, Korea University, Korea University Guro Hospital

Language: English

Topic: Microbial infections and disease research

Subfield: Microbiology

Field: Immunology and Microbiology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: diamond

Funders: Korea Health Industry Development Institute, National Research Foundation of Korea