Detection and identification of acid-fast bacilli of Mycobacterium species by conventional procedures requires growing the organisms from patient specimens and then testing the isolates for various phenotypic characteristics. These methods may take days to one or more months. The development of a few highly specific molecular probes for testing cultures growing acid-fast bacilli has greatly reduced the time to identification of some mycobacterial isolates. Recently, the polymerase chain reaction (PCR) and isothermal nucleic acid amplification techniques have become utilized in assays that offer a high degree of specificity and reasonable sensitivity for detection of Mycobacterium tuberculosis in clinical samples. At present, there are no FDA approved amplification assay systems that are capable of detecting multiple Mycobacterium species while excluding cross-reactive signals from other bacteria commonly present in clinical samples. Protein export is an important aspect of bacterial pathogenesis since a majority of bacterial virulence factors are extracytoplasmic proteins. Although little is known about the protein export pathway in Mycobacteria, the general secretory (Sec) pathway has been extensively studied in other bacteria, in particular in E. coli. SecA1 is the mycobacterial homologue of the E. coli SecA protein, an essential preprotein translocase ATPase that provides the driving force for the export of proteins across the cytoplasmic membrane. The mycobacterial SecA pathway is unusual in that it has two SecA proteins: SecA1 is the essential housekeeping SecA protein, while SecA2 is a non-essential accessory secretion factor. The purpose of this work is to investigate the use of secA1 gene sequences for the identification of Mycobacterium spp. We decided to target a 700-base fragment of secA1 coding for 233 amino acid residues located on the N-terminal half of the protein, and which includes the substrate specificty domain, or SSD, and contiguous sequences essential for protein translocation. Because of the particular nature of mycobacterial cell walls and after comparing the available secA1 gene sequences from 5 mycobacterial species, we hypothesized that SecA1 protein would be conserved in the genus Mycobacterium while exhibiting some amino acid differences among the different species, probably reflecting particular secretion needs related to pathogenicity, intracellular survival, environmental adaptability and stress tolerance. A preliminary study has shown the potential of using secA1 gene sequences for the identification of Mycobacterium spp by testing 44 reference strains as a foundation. The procedure has also been successfully applied to 69 clinical isolates to demonstrate the feasibility of this identification method. In addition, preliminary work was initiated to evaluate the use of the secA1 assay directly on clinical samples. As far as we know, this is the first time a secretion related gene is systematically used for the detection and identification of microorganisms. A manuscript describing the initial work with the secA1 gene target of Mycobacterium spp. was published in 2005. Forther studies are underway to optimize the sensitivity of the direct detection of Mycobacterium spp. in patient materials.
