Application of RAA-CRISPR dual-mode platform in rapid detection of Mycoplasma pneumoniae
10.3969/j.issn.1673-4130.2025.22.002
- VernacularTitle:RAA-CRISPR双模式平台在肺炎支原体快速检测中的应用
- Author:
Hongzhao YANG
1
;
Jie LUO
;
Kai CHANG
Author Information
1. 陆军军医大学第一附属医院检验科,重庆 400038
- Keywords:
Mycoplasma pneumoniae;
recombinase aided amplification;
CRISPR/Cas12a;
lateral flow immunochromatographic assay;
visual detection
- From:
International Journal of Laboratory Medicine
2025;46(22):2698-2703,2709
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a dual-mode detection platform for Mycoplasma pneumoniae(MP)based on recombinase aided amplification(RAA),CRISPR/Cas12a,and lateral flow immunochromatographic assay(LFIA).Methods RAA primers and crRNA were designed to target the conserved region of the MP P1 gene.Key parameters including RAA reaction time,core component concentrations of the CRISPR/Cas12a system,and CRISPR reaction time were systematically optimized.The analytical performance of the platform was evaluated via dual-mode of real-time fluorescence monitoring and LFIA naked-eye visual interpretation.Sensitivity was assessed using serially diluted MP plasmid templates(10-1-105 copy/μL),specificity against seven respiratory pathogens,including Mycoplasma hominis and Chlamydia pneumoniae,and clinical concord-ance with 25 throat swab samples validated by real-time fluorescence quantitative PCR(qPCR)as the gold standard.Results The optimal reaction conditions of the established RAA-CRISPR dual-mode platform were determined as follows:RAA reaction time of 15 min,Cas12a concentration of 80 nmol/L,crRNA concentration of 90 nmol/L,and CRISPR reaction time of 40 min.The limit of detection(LOD)for the fluorescence mode was 1 copy/μL with a detection rate of 98.3％(59/60,95％CI:94.7％-99.8％).For the LFIA mode,the LOD was 1 copy/μL with a detection rate of 96.7％(58/60,95％CI:88.4％-99.5％).No cross-reactivity was observed with any tested pathogens.Clinical validation results showed that the platform's detection re-suits were consistent with qPCR,with sensitivity,specificity,and overall agreement all reaching 100.0％.The detection time was relatively short,with the total detection time lasting no more than 55 minutes.Conclusion The established RAA-CRISPR dual-mode platform combines ultrasensitivity,visual interpretation,and operational simplicity,can provide an efficient solution for point-of-care testing(POCT)of MP infections at primary healthcare settings.
