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

根据脆弱拟杆菌神经氨酸酶编码基因（nanH）的序列设计寡核苷酸引物和DNA探针，通过巢式PCR和杂交试验对该厌氧菌进行特异性检测。该方法对脆弱B.fragilis细胞和DNA的检测限分别为10个菌落形成单位和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B.fragilis菌株和45株其它物种菌株。所有脆弱B.fragilis菌株通过PCR使用每个引物组产生正确大小的DNA片段。除普通B.vulgatus菌株外，其他测试的细菌物种均为阴性，该菌株产生的DNA片段大小与第二引物组的脆弱B.fragilis的DNA片段大小接近。用3 '-地高辛标记的寡核苷酸探针PH 1进行斑点杂交，可排除假阳性结果。杂交种中的阳性斑点 ...更多信息 59株脆弱B.fragilis的PCR产物均检测到阳性斑点，而其他菌种均未检测到阳性斑点。巢式PCR-斑点杂交试验使用来自脆弱B.感染的脓毒症模型小鼠的血液样品进行。该方法可在10 μ l血液中检测到20个脆弱B.菌落形成单位。以nanH基因为靶点的巢式PCR-斑点杂交法有望作为一种快速检测脆弱类B杆菌的方法应用于临床菌血症患者标本的检测。蛋白酶编码基因的克隆和测序将提供另一个靶点，可以减少与nanH联合使用时的假阴性结果。此外，我们确定了脆弱拟杆菌B的β-异丙基苹果酸脱氢酶基因（leu B）的核苷酸序列和包括脆弱拟杆菌在内的7种拟杆菌的16 S-23 S rRNA基因间间隔区的大小。这些结果将有助于今后的研究，建立一个系统的区分拟杆菌属物种。少

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