SRB would be identified by genetic fingerprint and detected by molecular hybridization and/or polymerase chain reaction (PCR). Furthermore, incubation time before analysis could be canceled or signifcantly reduced. rRNAs and related genes play an important role in micro-organisms identification because they combine many advantages. Analysis of rRNAs sequences is one of the main concept in bacterial populations studies. rRNAs are abundant in each bacteria (e.g.: 10,000 copies in E. coli). rRNAs sequences present regions with high variability useful for determination of specific probes for each microbial species. They also present more conserved region leading to determination of specific probes for groups or bacterial species. rRNAs are easily used as targets for PCR amplification, which could allow detection of viable but non cultivable species. Furthermore PCR amplifications of variables regions lead to a synthesis of new probes. Many studies have been realized in many laboratories (more than 4,000 sequences of 16S rRNAs specific of each bacterial species). Each bacterial cell contains important quantities of rRNAs which could be detected by in situ hybridization. We will try to develop fluorescent probes species-specific or taxon-specific for enumeration of diffent SRB. Membranes treatment will be developed and associated with computing associated images analysis systems. PCR is a very powerful tool to obtain and to analyze high quantities of target DNA sequences. Its high sensitivity is important when there are few bateria and/or when biological samples are not cultivable. Furthermore, its high quickness is particularly interesting in the case of emergency diagnosis and when a short delay is allowed (traditional microbiological analysis require a long time before results can be read). PCR is perfectly adapted to rRNAs structures with variable and conserved sequences. We will have to identify the most perfroming sequences (specificity, sensitivity and relativity) to develop the most efficient molecular diagnosis tool. So we will have to test these sequences, individually and in multiplex PCR reactions, on control samples and contaminated samples.
Funding SchemeEAW - Exploratory awards
AB22 8GU Aberdeen