RPB0308

Pathogen Description

Target Pathogen Pathogen Name NCBI Taxonomy ID Order Family Genus Species Pathogen type
Staphylococcus aureus Staphylococcus aureus, Micrococcus aureus, Staphylococcus pyogenes aureus 1280 Bacillales Staphylococcaceae Staphylococcus Staphylococcus aureus Bacterium

Primer Description

Primer Name Sequence(5'-3') Length(bp) Primer Final Concentration(μM） GC Content（%） Predicted Melting Temperature(℃） Molecular Weight(g/moles) Positions in GenBank accession number
F GATTATGGCTCAGGTACTGCTATCCACCCTC 31 10 µM 51.61 63.43 9422.17 \
R GCCAACCTTTACCATCGATTTTATAACTTG 30 10 μM 36.67 55.82 9090.98 \

Gene Description

Target Gene GenBank ID
mecA gene \

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
a novel, user-friendly, sensitive, and accurate diagnostic tool for the prevention and control of mecA-carrying strains, especially MRSA. RPA-CRISPR\Cas12a \ 60min 37 °C CRISPR\Cas12a 5 copies/μL 1 copy/μL \

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2025 Advanced One-Pot RPA-CRISPR\Cas12a Reaction with Glycerol and Betaine for High-Sensitivity Diagnosis of mecA-Carrying Strains in Clinical Samples Jingyuan Wang,Dan Wang,Linlin Fan,Xin Ye,Jian Hu,Xiaoqin Wang ACS Omega 39959114 10.1021/acsomega.4c09078

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

Author(s):

Jingyuan Wang,Dan Wang,Linlin Fan,Xin Ye,Jian Hu,Xiaoqin Wang

Journal:

ACS Omega

Year:

2025

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.