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

蛭弧菌特异性检测和分化的分子方法初探

• 批准号: 9615515
• 负责人: Henry Williams
• 金额: $ 7.5万
• 依托单位: University of Maryland at Baltimore
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9615515&HistoricalAwards=false
• 关键词: Preliminary; Exploration; Molecular; Methods; Specific

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.

摘要：9615515，PI-Williams：蛭弧菌是原核寄生捕食者，捕食敏感的革兰氏阴性杆菌。根据对蛭弧菌减少实验室容器中猎物数量的观察，一个合乎逻辑的问题是，捕食性细菌在控制自然界微生物种群方面是否发挥了某种作用？ 尚未报道解决该问题的充分实验。这主要是由于检测和监测环境中捕食者的技术有限。目前唯一可用于特异性检测蛭弧菌的方法是培养技术。与几乎所有培养方法一样，该技术具有很大的局限性。 已经构建了能够特异性检测多种细菌物种的血清学和分子探针以及聚合酶链反应（PCR）引物。然而，尚未开发出针对蛭弧菌的此类探针。对蛭弧菌的探测将有助于研究监测环境中这些捕食者的数量。检测寄生在宿主细胞内的细菌的能力将允许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群体的影响。 本研究计划的目的是开发一种基因探针和PCR引物，可用于水生环境中蛭弧菌的特异性检测。我们将首先努力鉴定已知的蛭弧菌核酸序列中的独特序列，并将其作为探针和 PCR 引物进行测试。 下面描述了开发针对蛭弧菌组或这些生物体亚组的特异性探针的目标和实验方法。 1. 鉴定并合成特定的DNA寡核苷酸序列，用作检测探针和PCR引物；鉴定用作DNA探针的候选基因组限制性片段序列并通过克隆和/或PCR合成生产它们；候选 rRNA 序列的鉴定和 rRNA 探针的构建。 2. 检验PCR在实验室微观世界中检测蛭弧菌的用途和灵敏度。 3. 检查属和种特异性探针的使用，以确认培养基上回收的蛭弧菌噬菌斑（菌落）的鉴定。