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.

翻译后摘要：9615515，PI-威廉姆斯：蛭弧菌是原核，寄生的捕食者，易感的革兰氏阴性杆菌。根据蛭弧菌在实验室容器中减少其猎物数量的观察，一个合乎逻辑的问题是，捕食性细菌在控制自然界中的微生物种群方面是否发挥了某种作用？ 尚未报道解决这一问题的充分实验。这在很大程度上是由于在技术上的限制，以检测和监测捕食者的环境。目前可用于特定检测蛭弧菌的唯一方法是培养技术。与几乎所有的文化方法一样，这种技术有很大的局限性。 已经构建了允许对许多细菌物种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，尚未开发出用于蛭弧菌的此类探针。一个蛭弧菌的探测器将允许研究监测环境中这些捕食者的数量。检测那些寄生在宿主细胞内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。 本研究的目的是研制一种可用于水体中蛭弧菌特异性检测的基因探针和PCR引物。将进行初步努力以鉴定蛭弧菌的已知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。 下文描述了开发对蛭弧菌组或这些生物体的亚组特异性的探针的目的和实验方法。 1.用作检测探针和PCR引物的特异性DNA寡核苷酸序列的鉴定和合成;用作DNA探针的候选基因组限制性片段序列的鉴定及其通过克隆和/或PCR合成的生产;候选rRNA序列的鉴定和rRNA探针的构建。 2.探讨PCR技术在实验室微环境中检测蛭弧菌的应用及敏感性。 3.研究使用属和种特异性探针确认培养基上回收的蛭弧菌噬斑（菌落）的鉴定。