The aim of this research is to develop a locus-specific DNA amplification process suitable for highly multiplexed detection of pathogenic mycobacteria in environmental samples. Specifically, this project aims to develop an assay to identify the presence of Mycobacterium ulcerans (MU) in environmental samples to determine the transmission pathway from the environmental to humans. MU is the causative agent responsible for Buruli ulcer, a devastating skin disease present in several countries. The proposed approach is to create a polymerase chain reaction (PCR) template that contains PCR primer sites that are not present in the target genome but contains a specific sequence in the target genome. This is done by creating two site-specific probes, each one containing one of the PCR primer sites or a complement thereof. These probes anneal to the target DNA at each end of a sequence in the genome that contains only three of the four possible DNA bases. Polymerase is used to extend one of the probes across this void region so that the complement of the void region is created. Then ligase is used to connect this extension product to the other probe, creating the PCR template. This void-extension-ligation (VEL) reaction can be repeated many times making several copies of the template. PCR is then performed, amplifying the targeted region of the DNA. Because the PCR primers are introduced in the probes, a single set of PCR primers can be used for a large number of targeted sequences. Other researchers have shown that a similar process called MARA, which also can use a single set of PCR primers, can be multiplexed successfully for 750 separate targeted sequences across nine DNA samples. The proposed technique is somewhat simpler than MARA and should be even more specific. Specificity and multiplex capability are key features in targeting regions that are putatively unique to M. ulcerans but which, in fact, may exist in related mycobacteria in an environmental sample. In Phase I, we plan to develop the VEL-PCR method and test it on environmental samples inoculated with M. ulcerans and others inoculated with its closes relative, M. marinum. After sensitivity and specificity have been demonstrated in Phase I, actual samples from highly endemic areas will be analyzed. Further development of VEL-PCR will be performed in Phase II to adapt it to the detection of other important pathogens.
