The LCR project was designed to detect single base mutations in the M. tuberculosis rpoB gene, that confer resistance to the antibiotic rifampicin.  The model system consisted of nine pGEM-T Easy plasmid stocks, 8 of which carried different mutations in a 750 bp fragment of the rpoB gene from M. tuberculosis, and one of which carried a 257 bp fragment of the wild-type rpoB gene, covering the highly polymorphic region of interest.  Probes flanking the mutation junction were designed to detect the various point mutations.  Detection of ligation products was performed using agarose gel electrophoresis and ethidium bromide staining.  Using this model system, we were unable to prevent template-independent ligation.  As a result, false positive signals were consistently obtained from wild-type template DNA.  A number of strategies were undertaken to overcome this problem, none of which proved successful in our hands (see appendix 3).  As a result a decision was taken to draw a line under the LCR project and concentrate effort into the second alternative amplification technique chosen for study, NASBA.

The aim of the NASBA project was revised to explore the potential applicability of NASBA, as compared to RT-PCR and PCR for the differentiation of viable and non-viable bacteria.  This revision was discussed and fully endorsed by Professor Sandy Primrose.  Verotoxigenic E. coli (VTEC) is a food and water borne pathogen of increasing public health significance, and the study was designed to investigate the specific detection of E. coli O157, currently the most commonly isolated verotoxigenic serotype.  This work forms the main body of the report.  Two genes were chosen for amplification studies, that were specific for the E. coli O157:H7 serogroup, and thus may be utilised to detect all such isolates.  The target loci were fliC, a flagella gene encoding the H antigen and rfbE, which encodes a cell wall lipopolysaccharide gene.  The sensitivity and specificity of the three amplification techniques were first assessed.  All three techniques were demonstrated to be specific.  Of the three techniques, we found NASBA to be the most sensitive, followed by PCR and then RT-PCR.  To determine the applicability of each method for measuring cell viability, E. coli O157 were killed by three different methods; (1) by heat killing and (2) by metabolic inhibition utilising sodium azide and (3) by reduction in water activity using sodium chloride treatment.  The persistence of mRNA and DNA in these cells was then investigated by using NASBA, RT-PCR and PCR.  We found that the correlation between NASBA detection and cell viability depends on the mRNA target, the method of cell death, post-treatment storage conditions and other environmental factors.  The results demonstrated that nucleic acid detection methods may be used as an indicator of potential cell viability, although the presence of a positive signal should be confirmed by culture methods as unequivocal interpretation is not possible.