Applying High-quality DNA Melting Curve Analysis in Identifying Staphylococcus aureus and Methicillin-resistant Strains

Abstract

Background and purpose: High Resolution Melting curve analysis DNA (HRM) is one of the most sensitive and precise methods for detecting Staphylococcus aureus and resistance to Methicillin. The aim of this study was to analyze the HRM for detection of methicillin-resistant S. aureus strains. Materials and methods: In this experimental study, standard strains of S.aureus ATCC25923 and ATCC 33592 were used. To identify S.aureus from ITS gene and methicillin resistance, the mecA gene was used. Analysis was performed using StepOne v2.3 and HRM v3.0.1. Sequencing results were used as gold standard. Results: The analytical sensitivity of the PCR method by ITS primer was capable of detecting 104 CFU bacteria and detecting bacteria for the mecA gene up to 103 CFU. The analytical sensitivity of the HRM method was also valid for ITS gene primer to dilute 10-2 CFU and the mecA gene primer up to a dilution of 10-5 CFU to detect bacteria. In HRM analysis, the lowest error rate was observed in the melting curves of DNA. Thus, considering the closest temperature range for analysis, the melting temperature for the ITS gene was 86 ± 0.5°C and for the mecA gene was 81± 0.5°C. The results of temperature and sequence determination proved the specificity of the HRM. Conclusion: The HRM has high sensitivity and specificity for detecting low levels of bacteria.

Keywords

• staphylococcus aureus
• methicillin resistance
• dna melting curve
• hrm