tmRNA--a novel high-copy-number RNA diagnostic target--its application for Staphylococcus aureus detection using real-time NASBA.
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.
O'Grady
Justin
J
The Biomedical Diagnostics Institute Program, National University of Ireland Galway, Galway, Ireland. justin.ogrady@nuigalway.ie
Lacey
Katrina
K
Glynn
Barry
B
Smith
Terry J
TJ
Barry
Thomas
T
Maher
Majella
M
D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D008892:Milk / Q000382:microbiology*; D012329:RNA, Bacterial / Q000235:genetics*; D021161:Self-Sustained Sequence Replication / Q000379:methods*; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification*
D012329:RNA, Bacterial; C096222:tmRNA