Ribot E M, Fitzgerald C, Kubota K, Swaminathan B, Barrett T J
Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA.
J Clin Microbiol. 2001 May;39(5):1889-94. doi: 10.1128/JCM.39.5.1889-1894.2001.
We developed a rapid pulsed-field gel electrophoresis (PFGE) protocol for subtyping Campylobacter isolates based on the standardized protocols used by PulseNet laboratories for the subtyping of other food-borne bacterial pathogens. Various combinations of buffers, reagents, reaction conditions (e.g., cell suspension concentration, lysis time, lysis temperature, and restriction enzyme concentration), and electrophoretic parameters were evaluated in an effort to devise a protocol that is simple, rapid, and robust. PFGE analysis of Campylobacter isolates can be completed in 24 to 30 h using this protocol, whereas the most widely used current protocols require 3 to 4 days to complete. Comparison of PFGE patterns obtained in six laboratories showed that subtyping results obtained using this protocol are highly reproducible.
我们基于脉冲网络实验室用于其他食源细菌性病原体分型的标准化方案，开发了一种用于弯曲杆菌分离株分型的快速脉冲场凝胶电泳（PFGE）方案。我们评估了缓冲液、试剂、反应条件（如细胞悬浮液浓度、裂解时间、裂解温度和限制酶浓度）以及电泳参数的各种组合，以设计出一种简单、快速且稳健的方案。使用该方案，弯曲杆菌分离株的PFGE分析可在24至30小时内完成，而目前使用最广泛的方案则需要3至4天才能完成。六个实验室获得的PFGE图谱比较表明，使用该方案获得的分型结果具有高度可重复性。
