利用菌落雜交技術偵測魚蝦病菌Vibrio spp.

Abstract

要由自然界中篩選及鑑定微生物是一件相當花時間的工作，此外自然界和實驗室的環境差異頗大，譬如自然環境較稀釋，營養較缺乏，物理狀況較極端，因此在自然界所發現的細菌在不同的生理狀況下會和其他的菌種共存。最近的報告顯示有些病源細菌在稀釋且受壓制的環境下，只是存活而不能培養，因此發展出一套專門用來鑑定自然界中微生物的方法是非常需要的。現在由於分子生物學的進展，可以將這方面的技巧應用到微生物生態方面的研究。在短時間之內，可以瞭解微生物社會中，微生物的種類及特性，且準確度相當高。利用基因探針分析環境中的微生物，比以往用利用微生物鑑定的方法，可以減少一半的時間。本實驗先以蝦病菌Vibrio harveyi?實驗菌種，此菌會產生螢光，控制螢光產生的基因（Lux gene）已經被研究出來且已選殖到質體上，我們已向國外索取到一個帶LuxA和LuxB兩個基因的質體pBR322，將其轉型到Escherichia coli RR1上，大量培養後，將質體抽取出來，經限制?HindIII切割後，用非放射性標定做探針，與相關菌株進行菌落雜交，結果顯示Lux gene的專一性並不是很好。本實驗再以魚蝦病菌Vibrio alginolyticus和Vibrio anguillarum?實驗菌種，利用生物技術的方法，以內核酸限制?HindIII和EcoRI將菌種的基因體DNA加以切割，由V. alginolyticus和V. anguillarum的切割片段選殖到載體pUC19上，轉型到寄主細胞E. coli TG1中，分別由前者選出160株，後者選出211株，加以純化分析載體上插入的DNA片段大小，由500-2000 bp之適當片段，前者選出102個，後者選出94個片段製成非放射性探針與相關菌株進行菌落雜交，其中前者有9個，而後者有14個探針與實驗菌株有較好的專一性。

The recent application of nucleic acid probes to detect bacteria found in environmental samples shows great promise. The applications of preexcisting DNA hybridization techniques in determining populations in environmental samples were investigated. In this study, first we use shrimp pathogenic bacterium Vibrio harveyi as tested organism. Since this organism is a luminescent bacterium and the structural genes controlling of luminescent system has been studied and cloned to several plasmids. Having obtained a plasmid containing Lux A and Lux B genes, we have transformed the plasmid pBR322 into Escherichia coli RR1. After growing in LB broth, the plasmid was isolated and cut by restriction endonuclease Hind III to prepare probe. Colony hybridization experiment was performed by the probe with Vibrio spp., However the specificity of Lux gene was not good for V. harveyi. We also use fish pathogenic bacteria Vibrio alginolyticus and Vibrio anguillarum as tested organism. Randomly cloned fragments of DNA from V. alginolyticus and V. anguillarum were used as colony hybridization probes for Vibrio spp. and other bacteria. Restriction endonuclease Hind III and EcoRI digestion fragments of DNA from V. alginolyticus and V. anguillarum were inserted into vector pUP19. Purified recombinant DNA from host cell E. coli TG1 were digested by same restriction endonuclease and subjected to electrophoresis on 1% agarose gel. Among 0.5 K pb?2 K pb size of insered DNA were recovered from agarose gel with DEAE membrane to prepare non-radioactive Dig-labeled probes. Nine out of 102 cloned fragments could hybridize only to V. alginolyticus and 14 out of 94 cloned fragments only to V. anguillarum.