Development of Cost Effective Multiplex PCR Assay for Detection of MRSA

Methicillin Resistant Staphylococcus aureus (MRSA) is one of the leading causes of Hospital Acquired Infections (HAIs) and is notorious for its high mortality rate. Many multiplex assays to detect MRSA are currently in the market but are either expensive or have a low specificity with newer strains containing mutations in the molecular targets being discovered recently. The goal of this project was to develop a cost-effective multiplex PCR assay to detect MRSA. The entire project was done at AutoGenomics Inc. in the Department of Research and Development using their automated Infiniti Plus analyzer and is first of its kind. It uses biofilm based microarray chips which contain anti-tag sequences corresponding to the tag sequences attached to the Allele Specific Primer Extension (ASPE) primers. Sequence specific primers were designed to target 1) mecA gene which is responsible for antibiotic resistance in MRSA, 2) nuc which is specific to S. aureus and 3) mecC which is a homolog of the mecA gene. ASPE probes were also designed for the same with a tag sequence attached to their 5' end. Multi-sequence alignment was done to make sure that the primers were unique and the multiplex PCR was optimized to avoid any cross-reactivity or PCR failure. The test was developed using known DNA samples of MRSA, MSSA and other negative controls obtained from ATCC. After the optimization, the test was correctly able to identify MRSA strains and the assay was further optimized to run 8 samples on a single chip. 10 different DNA samples were tested on the MRSA-O assay and they were correctly identified showing a specificity of 100% after running the tests in triplicate. The MRSA-O test was also found to be sensitive enough to detect as low as 178 DNA copies. Optimization of the assay to process 8 samples on a single chip will reduce the cost per test drastically but further studies need to check its efficiency on clinical samples. The analytical specificity of the assay also needs to be determined to see if other bacteria in the sample may interfere with the ability of MRSA-O to detect MRSA in their presence.