Demonstration of the application of the tmRNA transcript of the bacterial ssrA gene as a molecular diagnostic target using a combination of NASBA and BiaCore technologies

Objectives: The tmRNA transcript of the bacterial ssrA gene exhibits several properties that make it suitable as a molecular target in Nucleic Acid Diagnostics (NAD). Sequence homology at the 5 and 3 ends of the gene facilitate universal PCR amplification for characterisation of tmRNA from different species, while sequence heterogeneity in the internal portions of the gene enable the design of oligonucleotide probes to identify bacteria at genus and species level. We have previously demonstrated that tmRNA is present at approximately 1000 copies cfu in a range of pathogens. The natural amplification of this target has the potential to improve the detection capability of an NAD test. The objective of this study was to demonstrate the utility of tmRNA as a molecular diagnostic target using a combination of BiaCore biosensor and Nucleic Acid Sequence Based Amplification (NASBA) technologies. Streptococcus pneumoniae was chosen as a model organism for these evaluations. S. pneumoniae is a leading cause of morbidity and mortality worldwide. Infants and young children are most at risk, presenting with meningitis, pneumonia, bacteraemia, and other infections. Methods: The ssrA gene was sequenced in geographically distinct S. pneumoniae strains and in related species of the streptococcus genus and other bacterial species that may be present in the same clinical environment as S. pneumoniae. Species-specific probes were designed by sequence alignment and by the SLICsel bioinformatics software package. Initial evaluation of probe specificity was performed on microarrays using fluorescently labelled in vitro transcribed cRNAs. Selected probes were immobilised onto Biacore sensor chips and further evaluated using a series of unlabelled tmRNA containing targets including; cRNA, total RNA, and NASBA products. Results: Using selected S. pneumoniae specific probes, 10^12 cRNA copies of the tmRNA target were detected on the BiaCore. It was not possible to detect tmRNA from total S. pneumoniae RNA using this system. Inclusion of a NASBA step enabled BiaCore detection of a single S. pneumoniae cell. Specificity was demonstrated using NASBA products from a panel of clinically relevant bacteria. Conclusion: We have demonstrated that the tmRNA transcript of the ssrA gene in S. pneumoniae is suitable for use as a molecular diagnostic target. Application of a combination of NASBA and BiaCore biosensor technologies enabled specific detection of a single S. pneumoniae cell.