权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Development of the rapid identification system for Bacteroides fragilis by PCR with a DNA probe.

使用 DNA 探针通过 PCR 开发脆弱拟杆菌快速鉴定系统。

基本信息

批准号：
06557020
负责人：
OHNISHI Yoshinari
金额：
$ 2.43万
依托单位：
University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557020/
关键词：
Bacteroides fragilis DNA probe PCR Rapid detection method Neuraminidase DNA diagnosis Infection rRNA spacer region

项目摘要

Oligonucleotide primers and a DNA probe were designed on the basis of the sequence of the neuraminidase-encoding gene (nanH) of Bacteroides fragilis and used for the specific detection of this anaerobe by the nested PCR and hybridization assay. The detection limits of B.fragilis cells and DNA by the nested PCR assay were 10 colony forming units and 10 fg of chromosomal DNA,respectively. To determine the specificity of the first and second primer sets, 59 strains of B.fragilis and 45 strains of other species were tested. All strains of B.fragilis produced the DNA fragment of correct size by PCR using each primer set. Other bacterial species tested were all negative by either of the primer sets, except for a strain of B.vulgatus, which produced a DNA fragment close in size to that of B.fragilis with the second primer set. The dot blot hybridization assay using a 3'-digoxigenin-labeled oligonucleotide probe, PH1, was able to exclude the false-positive result. Positive spots in the hybridi … More zation assay were detected from the PCR products of all 59 strains of B.fragilis, while no positive spots were detected from other bacterial species. The nested PCR-dot blot hybridization assay was performed using blood samples from a sepsis-model mouse of B.fragilis infection. Twenty colony forming units of B.fragilis in 10mul of the blood could be detected by this assay. The nested PCR-dot blot hybridization assay targeting the nanH gene is expected to be applied to clinical specimens from patients with bacteremia as a rapid detection system for B.fragilis.We purified a fibrinogen-degrading protease from B.fragilis. Cloning and sequencing of the protease-encoding gene will provide another target that can reduce false negative results when used in combination with nanH.Moreover, we determined the nucleotide sequence of beta-isopropylmalate dehydrogenase gene (leuB) of B.fragilis and the sizes of 16S-23S rRNA intergenic spacer regions in seven Bacteroides species including B.fragilis. These results will be useful for future studies to develop a system for differentiation of Bacteroides species. Less

根据脆弱拟杆菌神经氨酸酶编码基因（nanH）的序列设计了寡核苷酸引物和DNA探针，并用于通过巢式PCR和杂交测定来特异性检测该厌氧菌。巢式PCR检测脆弱拟杆菌细胞和DNA的检出限分别为10个集落形成单位和10 fg染色体DNA。为了确定第一和第二引物组的特异性，测试了 59 株脆弱拟杆菌和 45 株其他物种。所有脆弱拟杆菌菌株均使用每个引物组通过 PCR 产生正确大小的 DNA 片段。所测试的其他细菌物种均对任一引物组呈阴性，但普通双歧杆菌菌株除外，该菌株使用第二个引物组产生的 DNA 片段大小与脆弱拟杆菌的 DNA 片段接近。使用 3'-地高辛标记的寡核苷酸探针 PH1 进行的斑点印迹杂交测定能够排除假阳性结果。杂交试验中，所有 59 株脆弱拟杆菌的 PCR 产物均检测到阳性斑点，而其他细菌物种未检测到阳性斑点。使用来自脆弱拟杆菌感染的脓毒症模型小鼠的血液样本进行巢式PCR点印迹杂交测定。 10μl血液中可检出20个菌落形成单位的脆弱拟杆菌。针对nanH基因的巢式PCR点印迹杂交检测有望应用于菌血症患者的临床标本，作为脆弱拟杆菌的快速检测系统。我们从脆弱拟杆菌中纯化了一种纤维蛋白原降解蛋白酶。蛋白酶编码基因的克隆和测序将提供另一个目标，当与nanH组合使用时，可以减少假阴性结果。此外，我们确定了脆弱拟杆菌的β-异丙基苹果酸脱氢酶基因（leuB）的核苷酸序列以及包括脆弱拟杆菌在内的七个拟杆菌属物种的16S-23S rRNA基因间间隔区的大小。这些结果将有助于未来研究开发拟杆菌属物种分化系统。较少的