tmRNA – a novel high-copy-number RNA diagnostic target – its application forStaphylococcus aureusdetection using real-time NASBA

Abstract

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.

Development of internally controlled duplex real-time NASBA diagnostics assays for the detection of microorganisms associated with bacterial meningitis

“…The diagnostic target used in this study for the species specific identification of N. meningitidis is the tmRNA transcript encoded for by the ssrA gene (Keiler, 2015). This diagnostics gene target has previously been demonstrated as suitable for use in nucleic acid based diagnostics for bacterial detection and identification (O'Grady et al, 2009;Wernecke et al, 2009;O'Grady et al, 2008). Furthermore, tmRNA, has also been established as a useful target in a real-time NASBA diagnostics assay (O'Grady et al, 2009).…”

“…This diagnostics gene target has previously been demonstrated as suitable for use in nucleic acid based diagnostics for bacterial detection and identification (O'Grady et al, 2009;Wernecke et al, 2009;O'Grady et al, 2008). Furthermore, tmRNA, has also been established as a useful target in a real-time NASBA diagnostics assay (O'Grady et al, 2009). Finally the diagnostic mRNA transcript target evaluated is the lepA mRNA and enables for the species specific identification of S. pneumoniae.…”

“…Incorporation of an IAC verifies the accuracy of the real-time NASBA results obtained, that the real-time NASBA reaction is functioning correctly and prevents reporting of false negative results which may occur as a result of problems with the assay reaction mixture, equipment malfunction, operator error, poor enzyme activity or inhibitory substances in the NASBA sample (Rodríguez-Lázaro et al, 2004).In recent years, a number of real-time NASBA assays have been described for the simultaneous detection of a target microorganism incorporating an IAC (O'Grady et al, 2009;Rodríguez-Lázaro et al, 2004;Tsaloglou et al, 2013;Sidoti et al, 2012;Nadal et al, 2007;Rodriguez-Lazaro et al, 2004;Ulrich et al, 2010;Telles et al, 2009;Casper et al, 2005;Patterson et al, 2005;Deiman et al, 2007;Yoo et al, 2008). However, these internally controlled real-time NASBA assays use in vitro synthesized RNAs spiked into the real-time NASBA reaction and some share a common set of primers for both the target species and the IAC which can result in competition for the primary target.…”

Bacterialtrans-Translation: From Functions to Applications

“…For example, Listeria monocytogenes was identified in foods by combining culture enrichment and real-time polymerase chain reaction (PCR) [139]; a real-time PCR test (RiboSEQ) was used on vaginal swabs from pregnant women to identify group B streptococci [140]; a rapid identification of Staphylococcus aureus was made in raw milk [141]; and Streptococcus pneumoniae was found in total RNA templates [142].…”

Detection of NASBA amplified bacterial tmRNA molecules on SLICSel designed microarray probes

“…All those characteristics make the tmRNA transcript (and its ssrA gene) a suitable tool as a target marker molecule for phylogenetical analysis and species identification in microbial diagnostics. Over the last 10 years tmRNA and its corresponding gene have been used for species identification in several methods including fluorescence in situ hybridization (FISH) detection of specific bacteria [4], real-time PCR [5] and real-time NASBA [6] analysis of food and dairy contaminants and pathogen detection using biosensors [7]. Combining the capabilities of tmRNA for species identification with DNA microarray technology offers the potential to investigate samples simultaneously for large numbers of different target tmRNA molecules.…”