Department of Anatomy, Physiology, and Pharmacology, College of Veterinary Medicine, Auburn University, AL 36849, USA.
J Microbiol Methods. 2011 Feb;84(2):194-201. doi: 10.1016/j.mimet.2010.11.016. Epub 2010 Dec 2.
We analyzed 100 Campylobacter spp. isolates (C. jejuni and C. coli) from Grenada, Puerto Rico and Alabama, which were collected from live broilers or retail broiler meat. We analyzed these isolates with four molecular typing methods: restriction fragment length polymorphism of the flaA gene (flaA-RFLP), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and automated repetitive extragenic palindromic polymerase chain reaction (REP-PCR) using the DiversiLab system. All methods performed similarly for the typing of C. jejuni and C. coli. The DNA extraction method appears to influence the results obtained with REP-PCR. This method was better for the typing of C. jejuni than C. coli, however both REP-PCR and flaA-RFLP generated types that were indistinguishable between C. jejuni and C. coli and appeared to be random, without any relationship to species, location, or source of isolates. PFGE and MLST generated typing results that had a better correlation with the geographic location of the isolates and showed higher concordance with the Wallace coefficient. The adjusted Rand coefficient did not show higher concordance among the methods, although the PFGE/MLST combination exhibited the highest concordance. PFGE and MLST revealed a better discriminatory power for C. coli isolates than REP-PCR or flaA-RFLP. The use of readily available online tools to calculate the confidence interval of the Simpson's index of diversity and the adjusted Rand and Wallace coefficients helped estimate the discriminatory power of typing methods. Further studies using different C. jejuni and C. coli strains may expand our understanding of the benefits and limitations of each of these typing methods for epidemiological studies of Campylobacter spp.
我们分析了从格林纳达、波多黎各和阿拉巴马州采集的 100 株弯曲杆菌属分离株（空肠弯曲菌和结肠弯曲菌），这些分离株来自活肉鸡或零售鸡肉。我们使用四种分子分型方法分析了这些分离株：flaA 基因的限制性片段长度多态性（flaA-RFLP）、多位点序列分型（MLST）、脉冲场凝胶电泳（PFGE）和自动重复外回文重复聚合酶链反应（REP-PCR）使用 DiversiLab 系统。所有方法对空肠弯曲菌和结肠弯曲菌的分型都表现出相似的效果。DNA 提取方法似乎会影响 REP-PCR 的结果。该方法更适合空肠弯曲菌的分型，而不适合结肠弯曲菌的分型，但 REP-PCR 和 flaA-RFLP 产生的类型在空肠弯曲菌和结肠弯曲菌之间无法区分，似乎是随机的，与物种、位置或分离株的来源无关。PFGE 和 MLST 产生的分型结果与分离株的地理位置相关性更好，与 Wallace 系数的一致性更高。调整后的 Rand 系数并没有显示出这些方法之间更高的一致性，尽管 PFGE/MLST 组合表现出了最高的一致性。PFGE 和 MLST 显示出对结肠弯曲菌分离株的鉴别能力优于 REP-PCR 或 flaA-RFLP。使用易于获得的在线工具计算 Simpson 多样性指数的置信区间以及调整后的 Rand 和 Wallace 系数有助于估计分型方法的鉴别能力。使用不同的空肠弯曲菌和结肠弯曲菌菌株进行进一步研究可能会扩展我们对这些分型方法在弯曲杆菌属流行病学研究中的优势和局限性的理解。
