應用染色體 DNA 的限制?圖譜鑑定弧形菌

Abstract

收集Vibrio alginolyticus, V. anguillarum, V. damsela, V. harveyi, V. ordalii, V. parahaemolyticus和V. vulnificus七種弧形菌十四株，萃取其DNA，利用Cla I, EcoR I, Hind III以及Sac I等四種限制酵素處理，再經0.8%洋菜膠體電泳，染色後照相，分析2至23kb範圍內的DNA片段，結果顯示DNA片段形成的圖譜有種的特異性，圖譜穩定，重複性良好，可做為鑑定弧形菌“種間”的藍圖。而且這種DNA圖譜也能反映“種內”的異同，如血清型(Serotype)及生物群(Biogroup)的鑒別，因為(1)同屬C血清型的三株V. anguillarum菌株，其圖譜相似程度很高，而分屬於C血清型的菌株其相似程度就較低些，(2)當比較V. vulnificus生物群的圖譜時顯示相同生命物群之草蝦株其圖譜非常相似，但屬於不同生物群之鰻魚株和草蝦株之間的相似程度則較低。為了分辨genomic DNA圖譜中是否有來自質體的片段，質體均被萃取，分離和純化，然後質體DNA圖譜再與genomic DNA圖譜相互比較，結果顯示V. anguillarum A, B, C血清型之genomic DNA圖譜並不因質體DNA的存在而有所影響。在C血清型的三株V. anguillarum均具有兩種質體，其分子量已為54.2及26.2 Kb,但由 Cla I, EcoR I, Hind III和Sac I處理得知其質體DNA的組成並不相同,弧形菌染色體DNA經Bcl I, Dpn I, Mbo I和Sau 3A I的處理結果，發現弧形菌染色體DNA在-GATC-鹽基須序中的腺嘌呤(adenine)有甲基化(methylation)的現象。

Seven species of fish pathogens, Vibrio alginolyticus, V. anguillarum, V. damsela, V. harveyi, V. ordalii, V. parahaernolyticus, and V. vulnificus were collected. Genomic DNA were extracted and cleaved by restriction enzymes such as Cla I, EcoR I, Hind III, and Sac I. The resulting DNA fragments, ranging from 2 to 23 Kb, were separated electrophoretically in agarose gel to form specific restriction patterns. The pattern of each bacterium were characteristic, stable, and reproducible as fingerprint. Despite some general similarity, the restriction pattern of different Vibrio species were heterogeneous. Fingerprint of genomic DNA also reflected intraspecific differences as shown by serotypes and biogroups. Plasmid DNA were also isolated and analyzed electrophoretically to confirm whether the fingerprint of genomic DNA were contaminated by. In comparison of DNA fingerprint of V. anguillarum strains we found the DNA pattern was not affected by the presence of plasmids no matter which serotype strain was used. Two plasmids with the same molecular weight were found among three strains of V. anguillarum serotype C. However, when these plasmids were digested with restriction endonucleases, they were shown to be the differerent species. In addition, the adenine nucleotide in the GATC sequence of Vibrio species was found methylated.