Development and Validation of a Specific PCR Primer for the Detection of the Meca Gene in MRSA

Vollständiger Abstract

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Methicillin-Resistant Staphylococcus aureus (MRSA) is a major pathogen responsible for nosocomial infections with high prevalence in Indonesia. The mecA gene encodes beta-lactam antibiotic resistance, making its specific molecular detection crucial for rapid diagnosis and infection control. This study aimed to develop and validate specific PCR primers for detecting the mecA gene in MRSA as a marker of antibiotic resistance. Laboratory experimental research was conducted at the UPH Advanced Biology Laboratory in November 2025 using certified pure MRSA isolates. DNA extraction was performed with the QIAamp DNA Mini Kit and quantified using a spectrophotometer. Primer design was carried out in silico using NCBI PrimerBLAST and BioEdit, followed by conventional PCR optimization with varying annealing temperatures. Amplification results were validated through agarose gel electrophoresis, with qualitative assessment based on DNA band intensity and sharpness. The mecA primer successfully amplified an 818-bp fragment at an optimal annealing temperature of 55.5°C, producing a single, sharp band without smearing. The negative control (S. aureus) showed no amplification, confirming primer specificity. Despite low DNA concentrations (4.5–7.6 ng/μL), amplification was consistently successful, indicating that this method is capable of reliably detecting the target gene at low DNA levels. This validated method provides an accurate molecular diagnostic tool for rapid identification of resistant pathogens. It supports epidemiological surveillance, nosocomial infection control, and early detection programs within the Indonesian healthcare system. By enabling precise detection of mecA-mediated resistance, the approach strengthens laboratory capacity for monitoring MRSA and contributes to improved public health strategies.