Duim B, Wassenaar T M, Rigter A, Wagenaar J
Department of Bacteriology, Institute for Animal Science and Health, 8200 AB Lelystad, The Netherlands.
Appl Environ Microbiol. 1999 Jun;65(6):2369-75. doi: 10.1128/AEM.65.6.2369-2375.1999.
For epidemiological studies of Campylobacter infections, molecular typing methods that can differentiate campylobacters at the strain level are needed. In this study we used a recently developed genotyping method, amplified fragment length polymorphism (AFLP), which is based on selective amplification of restriction fragments of chromosomal DNA, for genetic typing of Campylobacter jejuni and Campylobacter coli strains derived from humans and poultry. We developed an automated AFLP fingerprinting method in which restriction endonucleases HindIII and HhaI were used in combination with one set of selective PCR primers. This method resulted in evenly distributed band patterns for amplified fragments ranging from 50 to 500 bp long. The discriminatory power of AFLP was assessed with a C. jejuni strain, an isogenic flagellin mutant, and distinct C. jejuni strains having known pulsed-field gel electrophoresis and fla PCR-restriction fragment length polymorphism genotypes. Unrelated C. jejuni strains produced heterogeneous patterns, whereas genetically related strains produced similar AFLP patterns. Twenty-five Campylobacter strains obtained from poultry farms in The Netherlands grouped in three C. jejuni clusters that were separate from a C. coli cluster. The band patterns of 10 C. jejuni strains isolated from humans were heterogeneous, and most of these strains grouped with poultry strains. Our results show that AFLP analysis can distinguish genetically unrelated strains from genetically related strains of Campylobacter species. However, desirable genetically related strains can be differentiated by using other genotyping methods. We concluded that automated AFLP analysis is an attractive tool which can be used as a primary method for subtyping large numbers of Campylobacter strains and is extremely useful for epidemiological investigations.
对于弯曲杆菌感染的流行病学研究，需要能够在菌株水平区分弯曲杆菌的分子分型方法。在本研究中，我们使用了一种最近开发的基因分型方法，即扩增片段长度多态性（AFLP），它基于染色体DNA限制性片段的选择性扩增，用于对来自人和家禽的空肠弯曲杆菌和结肠弯曲杆菌菌株进行基因分型。我们开发了一种自动化AFLP指纹图谱方法，其中限制性内切酶HindIII和HhaI与一组选择性PCR引物联合使用。该方法产生了长度在50至500 bp之间的扩增片段均匀分布的条带模式。用一株空肠弯曲杆菌菌株、一个同基因鞭毛蛋白突变体以及具有已知脉冲场凝胶电泳和fla PCR-限制性片段长度多态性基因型的不同空肠弯曲杆菌菌株评估了AFLP的鉴别能力。不相关的空肠弯曲杆菌菌株产生异质模式，而遗传相关的菌株产生相似的AFLP模式。从荷兰家禽养殖场获得的25株弯曲杆菌菌株分为三个空肠弯曲杆菌簇，它们与一个结肠弯曲杆菌簇分开。从人类分离的10株空肠弯曲杆菌菌株的条带模式是异质的，并且这些菌株中的大多数与家禽菌株归为一组。我们的结果表明，AFLP分析可以区分弯曲杆菌属遗传不相关的菌株和遗传相关的菌株。然而，理想的遗传相关菌株可以通过使用其他基因分型方法来区分。我们得出结论，自动化AFLP分析是一种有吸引力的工具，可作为对大量弯曲杆菌菌株进行亚型分型的主要方法，并且对流行病学调查极为有用。
