Detection and Identification of Mycobacteria in Clinical

用常规方法检测和鉴定分支杆菌的抗酸杆菌需要从患者标本中培养细菌，然后检测分离物的各种表型特征。这些方法可能需要几天到一个月或更多的时间。一些用于检测生长的抗酸杆菌培养物的高特异性分子探针的发展大大减少了鉴定某些分枝杆菌分离株的时间。近年来，聚合酶链反应（PCR）和等温核酸扩增技术已被用于检测临床样品中的结核分枝杆菌，提供了高度的特异性和合理的灵敏度。目前，还没有FDA批准的扩增检测系统能够检测多种分枝杆菌，同时排除临床样品中常见的其他细菌的交叉反应信号。蛋白质输出是细菌致病的一个重要方面，因为大多数细菌毒力因子是胞质外蛋白。尽管对分枝杆菌的蛋白质输出途径知之甚少，但对其他细菌，特别是大肠杆菌的总分泌（Sec）途径已经进行了广泛的研究。SecA1是大肠杆菌SecA蛋白的分支杆菌同源物，是一种重要的蛋白前转位酶atp酶，为蛋白质跨细胞质膜的输出提供动力。分枝杆菌SecA通路的不寻常之处在于它有两种SecA蛋白：SecA1是必需的内务SecA蛋白，而SecA2是非必需的辅助分泌因子。这项工作的目的是研究secA1基因序列在分枝杆菌鉴定中的应用。我们决定以secA1的一个700碱基片段为目标，该片段编码位于该蛋白n端一半的233个氨基酸残基，其中包括底物特异性结构域（SSD）和蛋白质易位所必需的连续序列。由于分枝杆菌细胞壁的特殊性，在比较了5种分枝杆菌的secA1基因序列后，我们推测secA1蛋白在分枝杆菌属中是保守的，但在不同种类中表现出一定的氨基酸差异，这可能反映了与致病性、细胞内生存、环境适应性和应激耐受性相关的特殊分泌需求。初步研究表明，以44株参考菌株为基础，利用secA1基因序列鉴定分枝杆菌具有潜力。该程序还成功地应用于69个临床分离株，以证明该鉴定方法的可行性。此外，初步工作已开始评估直接在临床样品上使用secA1检测。据我们所知，这是第一次系统地将分泌物相关基因用于微生物的检测和鉴定。2005年发表了一份描述分枝杆菌secA1基因靶点初步工作的手稿。进一步的研究正在进行中，以优化患者材料中直接检测分枝杆菌的敏感性。

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.