Al-Soud Waleed Abu, Bennedsen Mads, On Stephen L W, Ouis Ibn-Sina, Vandamme Peter, Nilsson Hans-Olof, Ljungh Åsa, Wadström Torkel
Department of Medical Microbiology, Dermatology and Infection, Lund University, Sölvegatan 23, SE-223 62 Lund, Sweden 2Department of Clinical Microbiology 7806, National University Hospital, Righospitalet, Tagensvej 20, DK-2200, Copenhagen, Denmark 3Danish Veterinary Institute, Bülowsvej 27, DK-1790, Copenhagen, Denmark 4Laboratorium voor Microbiologie, University of Ghent, Belgium.
J Med Microbiol. 2003 Sep;52(Pt 9):765-771. doi: 10.1099/jmm.0.05314-0.
Helicobacter species are fastidious bacterial pathogens that are difficult to culture by standard methods. A PCR-denaturing gradient gel electrophoresis (PCR-DGGE) technique for detection and identification of different Helicobacter species was developed and evaluated. The method involves PCR detection of Helicobacter DNA by genus-specific primers that target 16S rDNA and subsequent differentiation of Helicobacter PCR products by use of DGGE. Strains are identified by comparing mobilities of unknown samples to those determined for reference strains; sequence analysis can also be performed on purified amplicons. Sixteen DGGE profiles were derived from 44 type and reference strains of 20 Helicobacter species, indicating the potential of this approach for resolving infection of a single host by multiple Helicobacter species. Some more highly related species were not differentiated whereas in highly heterogeneous species, sequence divergence was observed and more than one PCR-DGGE profile was obtained. Application of the PCR-DGGE method to DNA extracted from faeces of zoo animals revealed the presence of Helicobacter DNA in 13 of 16 samples; a correlation was seen between the mobility of PCR products in DGGE analysis and DNA sequencing. In combination, this indicated that zoo animals are colonized by a wide range of different Helicobacter species; seven animals appeared to be colonized by multiple Helicobacter species. By this approach, presumptive identifications were made of Helicobacter bilis and Helicobacter hepaticus in a Nile crocodile, Helicobacter cinaedi in a baboon and a red panda, and Helicobacter felis in a wolf and a Taiwan beauty snake. All of these PCR products ( approximately 400 bp) showed 100 % sequence similarity to 16S rDNA sequences of the mentioned species. These results demonstrate the potential of PCR-DGGE-based analysis for identification of Helicobacter species in complex ecosystems, such as the gastrointestinal tract, and could contribute to a better understanding of the ecology of helicobacters and other pathogens with a complex aetiology.
幽门螺杆菌属是苛求性细菌病原体，难以用标准方法培养。开发并评估了一种用于检测和鉴定不同幽门螺杆菌属物种的聚合酶链反应-变性梯度凝胶电泳（PCR-DGGE）技术。该方法包括通过靶向16S rDNA的属特异性引物对幽门螺杆菌DNA进行PCR检测，随后利用DGGE对幽门螺杆菌PCR产物进行区分。通过将未知样品的迁移率与参考菌株的迁移率进行比较来鉴定菌株；也可以对纯化的扩增子进行序列分析。从20种幽门螺杆菌属的44个模式菌株和参考菌株中获得了16个DGGE图谱，表明该方法有潜力解决单一宿主被多种幽门螺杆菌属物种感染的问题。一些亲缘关系较近的物种未被区分开来，而在高度异质的物种中，观察到了序列差异，并获得了不止一个PCR-DGGE图谱。将PCR-DGGE方法应用于从动物园动物粪便中提取的DNA，结果显示16个样品中有13个存在幽门螺杆菌DNA；在DGGE分析中PCR产物的迁移率与DNA测序之间存在相关性。综合来看，这表明动物园动物被多种不同的幽门螺杆菌属物种定植；7只动物似乎被多种幽门螺杆菌属物种定植。通过这种方法，在一条尼罗鳄中推定鉴定出了胆汁幽门螺杆菌和肝幽门螺杆菌，在一只狒狒和一只小熊猫中鉴定出了西奈幽门螺杆菌，在一只狼和一条台湾丽纹蛇中鉴定出了猫幽门螺杆菌。所有这些PCR产物（约400 bp）与上述物种的16S rDNA序列显示出100%的序列相似性。这些结果证明了基于PCR-DGGE的分析在鉴定复杂生态系统（如胃肠道）中幽门螺杆菌属物种方面的潜力，并有助于更好地理解幽门螺杆菌和其他病因复杂的病原体的生态学。
