Preliminary Exploration of Molecular Methods for the Specific Detection and Differentiation of the Bdellovibrios

摘要：9615515，PI-Williams: Bdellovibrios是原核生物，寄生捕食易感革兰氏阴性杆菌。根据观察到的蛭弧菌减少了实验室容器中猎物的数量，一个合乎逻辑的问题是，这种掠食性细菌是否在控制自然界微生物数量方面发挥了某种作用？没有足够的实验来解决这个问题。这主要是由于在环境中探测和监测捕食者的技术的限制。目前唯一可用于特异性检测的方法是培养技术。和几乎所有的培养方法一样，这种技术有很大的局限性。已经构建了能够对多种细菌进行特异性检测的血清学和分子探针以及聚合酶链反应（PCR）引物。然而，这种探针还没有开发出来用于蛭弧菌。对蛭弧菌的探测将使监测环境中这些捕食者数量的研究成为可能。如果能够检测宿主细胞内的寄生菌，就可以估计环境中受感染细胞的数量，从而确定蛭弧菌对易感细菌种群的影响。本研究的目的是开发一种可用于水生环境中蛭弧菌特异性检测的基因探针和PCR引物。最初的努力将是在已知的蛭弧菌核酸序列中识别出独特的序列，并将这些序列作为探针和PCR引物进行测试。开发针对蛭弧菌群或这些生物亚群的探针的目的和实验方法如下所述。1. 鉴定和合成用于检测探针和PCR引物的特定DNA寡核苷酸序列；鉴定用于DNA探针的候选基因组限制性内切片段序列及其克隆和/或聚合酶链反应合成；候选rRNA序列的鉴定和rRNA探针的构建。2. 目的：探讨PCR检测实验室微生物中蛭弧菌的应用及灵敏度。3. 目的探讨利用属和种特异性探针确定在培养基上恢复的蛭弧菌斑块（菌落）的鉴定。

Abstract: 9615515, PI-Williams: Bdellovibrios are procaryotic, parasitic predators which prey on susceptible gram-negative bacilli. Based on the observation that bdellovibrios reduce the population of its prey in laboratory vessels, a logical question is, does the predatory bacterium play some role in the control of microbial populations in nature? Adequate experimentation to resolve this question has not been reported. This is largely due to the limitation in technology to detect and monitor the predator in the environment. The only method currently available for the specific detection of bdellovibrios is the culture technique. As with nearly all cultural methods this technique has substantial limitations. Serological and molecular probes and polymerase chain reaction (PCR) primers which allow for the specific detection of a number of bacterial species have been constructed. However such probes have not been developed for the bdellovibrios. A probe for the bdellovibrios willould allow studies which monitor the numbers of these predators in the environment. The ability to detect those which are parasitic within host cells would allow an estimation of the number of infected cells in the environment and hence define the impact of bdellovibrios on populations of susceptible bacteria. The aim of this researchproposal is to develop a genetic probe and PCR primers which can be used for the specific detection of bdellovibrios in aquatic environments. Initial efforts will be made to identify unique sequences among known nucleic acid sequences for bdellovibrios and to test these as probes and as PCR primers. The objectives and experimental approaches to developing a probe specific for the bdellovibrio group or subgroups of these organisms are described below. 1. Identification and synthesis of specific DNA oligonucleotide sequences for use as detection probes and PCR primers; identification of candidate genomic restriction fragment sequences for use as DNA probes and their production by cloning and/or PCR synthesis; identification of candidate rRNA sequences and construction of rRNA probes. 2. To examine the use and sensitivity of PCR to detect bdellovibrios in laboratory microcosms. 3. To examine the use of genus and species specific probes to confirm the identification of plaques (colonies) of bdellovibrios recovered on culture medium.