A Rapid Nucleic Acid Extraction-Free Fluorescent PCR for the Detection of <i>Mycoplasma pneumoniae</i>

Abstract

Background: Mycoplasma pneumoniae is a major respiratory pathogen. Accurate and timely diagnosis is critical for guiding treatment, as clinical symptoms overlap with those of other respiratory pathogens. Molecular assays, particularly fluorescent PCR, offer higher sensitivity and specificity but typically require time-consuming nucleic acid extraction steps, prolonging the detection process to over 1 hour. Objectives: This study aimed to develop a rapid and efficient method for direct sample analysis to overcome the limitations of conventional, time-consuming methods for M. pneumoniae detection. Methods: Primers and probe were designed based on conserved regions of the P1 gene. The specificity of primers and probe was verified by BLAST analysis. Assay conditions were optimized by adjusting primer/probe concentrations and two-stage annealing/extension parameters. Specificity was evaluated using 322 throat swab samples (including 32 M. pneumoniae-positive samples) collected from Jiaxing hospitals in 2024. Sensitivity was assessed by testing serial dilutions of M. pneumoniae positive control, with the detection limit calculated by probit analysis. Reproducibility was determined by intra- and inter-assay variability. The performance of the newly developed assay was compared with a commercial fluorescent PCR kit using 10-fold diluted M. pneumoniae-positive samples. Results: Melting curve analysis revealed that by 86°C, nearly all amplification products had completely denatured, which was used for denaturation in the second amplification stage. Optimal assay conditions included a primer concentration of 1 µM, a probe concentration of 0.6 µM, and annealing/extension conditions of 65°C for 10 s in the first stage and 60°C for 5 s in the second stage. The assay showed high specificity with no cross-reactivity to other respiratory pathogens. Sensitivity analysis indicated that the limit of detection of the assay in 95% of cases was 553.26 copies/mL (95% CI: 341.32 - 2063.47 copies/mL). The method demonstrated good reproducibility, with intra-assay variability of 1.86% and 3.17%, and inter-assay variability of 3.73% and 4.70% at 2500 and 1000 copies/mL, respectively. Head-to-head comparison using the same set of 10-fold diluted clinical samples demonstrated that the newly developed assay achieved a higher detection rate (62.5%, 20/32) than the commercial fluorescent PCR kit (53.1%, 17/32). Conclusions: The developed extraction-free fluorescent PCR offers a rapid, simple, and reliable approach for M. pneumoniae detection. It shows promise for clinical applications, particularly in outpatient settings requiring prompt diagnosis.