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

## TL;DR

This paper introduces a new LAMP assay that can detect both wild-type and macrolide-resistant Mycoplasma pneumoniae in a single test, offering a fast and practical diagnostic tool.

## Contribution

The novel HyTaq-LAMP assay enables simultaneous detection of wild-type and resistant M. pneumoniae without thermal cycling.

## Key findings

- The assay detected wild-type and mutations with a limit of 1 × 10³ copies/μL.
- It showed 95.79% sensitivity for A2063G and 100% for wild-type in clinical samples.
- No cross-reactivity with 16 other respiratory pathogens was observed.

## 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.

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.