Stark M, Reizenstein E, Uhlén M, Lundeberg J
Department of Biochemistry, KTH, Royal Institute of Technology, Stockholm, Sweden.
J Clin Microbiol. 1996 Apr;34(4):778-84. doi: 10.1128/jcm.34.4.778-784.1996.
In the present study, novel solid-phase methods were used for both sample preparation and PCR detection of Bordetella pertussis. The sample preparation was performed by immunomagnetic separation with paramagnetic beads coated with polyclonal antibodies directed toward the surface antigens of the bacteria. The precoated immunobeads were directly used on nasopharyngeal aspirates to capture the bacteria on the solid support and were subsequently transferred to the PCR tube with no further manipulations. The region encompassing the pertussis toxin promoter was analyzed to allow direct discrimination between the three major Bordetella species (B. pertussis, B. parapertussis, and B. bronchiseptica). The resulting amplicons were captured on a second magnetic solid phase, allowing detection and restriction analysis of the target sequence. A colorimetric detection system based on a DNA binding fusion protein enabled the use of standardized enzyme-linked immunosorbent format tests both for the detection of Bordetella spp. and for species evaluation. When the optimized system was evaluated on 55 clinical aspirate samples, 21 of 22 (95%) culture-positive samples were positive by the system that we developed. In addition, two samples were positive by the PCR-based assay, while the culture assay was negative. The implications of these results are discussed.
在本研究中，采用新型固相方法进行百日咳博德特氏菌的样品制备和PCR检测。样品制备通过免疫磁珠分离法进行，磁珠表面包被有针对该细菌表面抗原的多克隆抗体。预包被的免疫磁珠直接用于鼻咽抽吸物，以在固相载体上捕获细菌，随后无需进一步处理即可转移至PCR管中。对包含百日咳毒素启动子的区域进行分析，以便直接区分三种主要的博德特氏菌（百日咳博德特氏菌、副百日咳博德特氏菌和支气管败血博德特氏菌）。扩增产物在第二个磁性固相上捕获，从而实现对目标序列的检测和限制性分析。基于DNA结合融合蛋白的比色检测系统使得能够使用标准化的酶联免疫吸附试验形式来检测博德特氏菌属并进行菌种鉴定。当在55份临床抽吸物样本上评估优化后的系统时，22份培养阳性样本中有21份（95%）通过我们开发的系统检测为阳性。此外，有两份样本基于PCR的检测为阳性，而培养检测为阴性。讨论了这些结果的意义。
