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O'Grady, J,Lacey, K,Glynn, B,Smith, TJ,Barry, T,Maher, M
2009
December
Fems Microbiology Letters
tmRNA - a novel high-copy-number RNA diagnostic target - its application for Staphylococcus aureus detection using real-time NASBA
Published
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Staphylococcus aureus real-time NASBA internal amplification control (IAC) milk tmRNA SEQUENCE-BASED AMPLIFICATION LISTERIA-MONOCYTOGENES PCR DETECTION FOOD SAFETY RAW-MILK PATHOGENS GENE OUTBREAK MASTITIS SAMPLES
301
218
223
A real-time nucleic acid sequence-based amplification assay, targeting tmRNA, was designed for the rapid identification of Staphylococcus aureus. The selectivity of the assay was confirmed against a panel of 76 Staphylococcus strains and species and 22 other bacterial species. A detection limit of 1 cell equivalent was determined for the assay. A chimeric in vitro transcribed internal amplification control was developed and included in the assay. Application of the assay in natural and artificially contaminated unpasteurized (raw) milk enabled detection of 1-10 CFUS. aureus mL-1 in 3-4 h, without the need for culture enrichment. Staphylococcus aureus was detected in all artificially contaminated milk samples (n=20) and none of the natural milk samples (n=20). Microbiological analysis of the natural milk samples was performed in parallel according to ISO 6888-3 and confirmed the absence of S. aureus. The method developed in this study has the potential to enable the specific detection of S. aureus in raw milk in a significantly shorter time frame than current standard methods. The assay further demonstrates the usefulness of tmRNA/ssrA as a nucleic acid diagnostic target.
DOI 10.1111/j.1574-6968.2009.01822.x
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