Kuipers J G, Nietfeld L, Dreses-Werringloer U, Koehler L, Wollenhaupt J, Zeidler H, Hammer M
Department of Internal Medicine and Dermatology, Hannover Medical School, Germany.
Ann Rheum Dis. 1999 Feb;58(2):103-8. doi: 10.1136/ard.58.2.103.
To optimise sample preparation of synovial fluid for Chlamydia trachomatis (CT) specific polymerase chain reaction (PCR).
Serial dilutions of purified CT elementary bodies in synovial fluid were prepared. The synovial fluid pellet was processed by eight different methods of sample preparation. Then samples were analysed by CT specific PCR. The sensitivity of PCR was the basis of ranking of the eight different methods.
Highest sensitivity was achieved by methods including an additional step of DNA isolation. Additional extraction of protein and polysaccharides by cetyltrimethylammonium bromide (CTAB) increased sensitivity. Addition of hyaluronidase did not increase sensitivity of QIAEX-DNA extraction but was necessary, however, before phenol-chloroform-DNA extraction.
The method of synovial fluid sample preparation significantly influences the sensitivity of subsequent PCR. Additional DNA isolation and extraction of PCR inhibitors by CTAB led to higher sensitivity.
优化用于沙眼衣原体(CT)特异性聚合酶链反应(PCR)的滑液样本制备方法。
在滑液中制备纯化的CT原体的系列稀释液。通过八种不同的样本制备方法处理滑液沉淀。然后通过CT特异性PCR分析样本。PCR的灵敏度是对这八种不同方法进行排序的依据。
通过包括额外DNA分离步骤的方法可实现最高灵敏度。用十六烷基三甲基溴化铵(CTAB)额外提取蛋白质和多糖可提高灵敏度。添加透明质酸酶不会增加QIAEX-DNA提取的灵敏度,但在酚-氯仿-DNA提取之前却是必要的。
滑液样本制备方法显著影响后续PCR的灵敏度。额外的DNA分离和通过CTAB提取PCR抑制剂可提高灵敏度。