Advanced One-Pot RPA-CRISPR/Cas12a Reaction with Glycerol and Betaine for High-Sensitivity Diagnosis of mecA-Carrying Strains in Clinical Samples

Abstract

The mecA gene confers methicillin resistance in both MRSA and MR-CoNS by encoding the PBP2a protein and poses a significant public health threat due to its resistance to beta-lactam antibiotics. Rapid and accurate detection of mecA is critical for timely treatment, reducing morbidity, and preventing its spread in healthcare settings. In this study, we developed an advanced one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a system, enhanced with glycerol and betaine, for ultrasensitive detection of the mecA gene. Glycerol's viscosity effect prevents premature interaction between Cas12a and early amplification products, while betaine enhances nucleic acid amplification. The assay demonstrated superior sensitivity, detecting as low as 5 copies/μL of mecA DNA within 60 min. Specificity testing against a panel of bacterial species confirmed the high selectivity of the assay for mecA-carrying strains with negligible cross-reactivity. Furthermore, this method exhibited excellent performance across various clinical samples, including blood, urine, and bronchoalveolar lavage fluid. Our findings underscore the potential of this advanced RPA-CRISPR/Cas12a assay as a powerful diagnostic tool for rapid, cost-effective, and highly sensitive mecA detection, offering a promising solution for clinical diagnostics and infection control.

Figure 1

Comparison of traditional and advanced one-pot RPA-CRISPR/Cas12a assay workflows for mecA-carrying strains detection. (A) The traditional diagnostic method results in a diagnosis within 24 h, whereas the advanced method delivers results within 1 h. (B) The advanced one-pot RPA-CRISPR/Cas12a assay utilizes betaine-enhanced RPA for efficient amplification and glycerol-stabilized Cas12a–crRNA complexes to prevent premature interaction with amplification products, ensuring high sensitivity and specificity in mecA detection.

Figure 2

Effect of betaine, DMSO, and PEG on the fluorescence intensity of the RPA-CRISPR/Cas12a assay at varying glycerol concentrations. Bar graphs depicting the change in fluorescence (ΔFluorescence) at 30 min for different concentrations of betaine (0.5%, 1%, 5%, 0%) in comparison with DMSO and PEG under 5%, 10%, and 15% glycerol conditions, respectively (A–C). The amplification curves showing the fluorescence intensity over time (up to 30 min) for 0% and 5% betaine at 5%, 10%, and 15% glycerol, respectively (D–F).

Figure 3

Comparison of advanced vs classic RPA-CRISPR/Cas12a assays for mecA detection. Fluorescence intensity over time for the detection of serially diluted mecA DNA (10⁴–10¹ copies/μL) using the advanced RPA-CRISPR/Cas12a assay (A) and the classic RPA-CRISPR/Cas12a assay (B). Sensitivity analysis comparing the advanced (C) and classic (D) assays for detecting low concentrations of mecA DNA (1 and 5 copies/μL). The inset in (C) shows a fluorescence image confirming the presence of amplified products at different DNA concentrations.

Figure 4

Specificity and detection limit of the advanced RPA-CRISPR/Cas12a assay in various spiked biological samples. (A) Specificity analysis showing fluorescence intensity over time for mecA detection in MRSA compared to various other bacterial strains, including MSSA (methicillin-sensitive Staphylococcus aureus), Enterococcus faecium, Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Detection limit of the advanced assay in spiked biological samples, including (B) blood, (C) bronchoalveolar lavage fluid, and (D) urine, was 5 copies/μL of mecA DNA.

Figure 5

Comparison of detection consistency and diagnostic accuracy between advanced and classic RPA-CRISPR/Cas12a methods. (A) Heatmap analysis showing the detection results of the advanced RPA-CRISPR/Cas12a method across different biological samples: blood, urine, and bronchoalveolar lavage fluid (BALF). A: advanced method; B: classic method; C: culture method (gold standard method). Pink indicates positive detection, while blue indicates negative detection. (B) Receiver operating characteristic (ROC) curve comparing the diagnostic accuracy of the advanced RPA-CRISPR/Cas12a method and the classic method.