Detection And Identification Of Mycobacteria

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长时间。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，没有商业上可用的扩增分析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。实验已经成功地进行了一个版本，可以同时检测六种常见临床分离的分枝杆菌物种的核酸扩增产物的存在或不存在。国家卫生研究院和贝克曼库尔特公司最近提交了一份联合专利申请。由于样品制备是分子诊断分析的关键组成部分，我们已经开始了一项新的努力，研究压力循环技术（PCT）对提高分枝杆菌细胞核酸释放效率的有用性。最近，BBI生物技术公司根据CRADA将一种压力循环器（压力循环装置）引入CPD。利用现场装置，对不同分枝杆菌种类的培养物进行了进一步的压力循环实验。利用PCT作为核酸提取前的预处理，我们能够提高含有gordonae分枝杆菌和结核分枝杆菌悬浮液的平均PCR信号。

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 1 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 and isothermal nucleic acid amplification techniques have been used in assays that offer a high degree of specificity and reasonable sensitivity for detection of Mycobacterium tuberculosis in clinical samples. At present, no commercially available amplification assay systems are capable of detecting multiple Mycobacterium species while excluding cross-reactive signals from other bacteria commonly present in clinical samples. Experiments have been successfully performed with a version that simultaneously detects the presence or absence of nucleic acid amplification products from six common clinically isolated Mycobacterium species. A joint patent application between the N.I.H. and Beckman Coulter Corporation has been recently been submitted. Because sample preparation is a critical component of molecular diagnostic assays, we have initiated a new effort to investigate the usefulness of a pressure cycling technology (PCT) for improving the efficiency of nucleic acid release from mycobacterium cells. A barocycler (device for pressure cycling) has been recently brought into CPD under a CRADA with BBI Biotech. With a device on site further experiments with pressure cycling performed on cultures of different Mycobacterium species were initiated. Using PCT as a pretreatment before nucleic acid extraction we were able to increase the average PCR signals from suspensions containing Mycobacterium gordonae and suspensions of Mycobacterium tuberculosis.