Development of Loop-mediated Isothermal Amplification (LAMP) for rapid detection of Methicillin-resistance Staphylococcus aureus (MRSA) from dairy cattle
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Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of mastitis in dairy cattle, resulting in the loss of economic value in livestock. The gene mecA encodes penicillin-binding protein 2A (PB2A), which only binds weakly to β-lactam antibiotics such as penicillin and methicillin, thus conferring the antibiotic resistant property in MRSA. Currently, detection of MRSA in laboratory setting is commonly performed using bacterial culture or PCR assay targeting mecA gene, which is time-consuming, labor-intensive, and requires specialized personnel and equipment. Therefore, a more rapid detection of MRSA is needed to effectively prevent the spread of MRSA. In this study, a novel Loop-mediated Isothermal Amplification (LAMP) assay that can detect MRSA more conveniently, quickly, and accurately was developed. Bacterial culture were isolated from milk samples from dairy cattle with mastitis and PCR primers were designed to target femA, blaZ, and mecA gene to detect Staphylococcus aureus (S. aureus), penicillin-resistant S. aureus, and methicillin-resistance S. aureus, respectively. We found that all isolates possessed femA and blaZ genes, and 30% of the isolates possessed mecA gene. For LAMP assay development, primers were designed to target the coding region of mecA gene, and LAMP reactions were monitored in real-time using the fluorescence labelling of the amplified products. We were able to detect the presence of MRSA quickly, and the results were consistent with those obtained by PCR and by bacterial culture. These results indicated that the LAMP assay successfully detected mecA gene in MRSA strains isolated from dairy cattle milk, and this assay could be further developed into a test kit to be used in the field
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