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

根据脆弱类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，并用套式聚合酶链式反应和杂交技术对该厌氧菌进行了特异性检测。该方法对脆弱芽孢杆菌细胞和DNA的检测下限分别为10个集落形成单位和10fg染色体DNA。为了确定第一对和第二对引物的特异性，对59株脆弱芽孢杆菌和45株其他物种的菌株进行了检测。所有的脆弱芽孢杆菌菌株均能扩增出大小正确的DNA片段。除一株普通芽孢杆菌产生的DNA片段大小与脆性芽孢杆菌的DNA片段大小相近外，其余菌株的DNA片段均为阴性。用3‘-地高辛标记的寡核苷酸探针PH1进行斑点杂交，能够排除假阳性结果。杂交…中的阳性斑点59株脆性芽孢杆菌的扩增产物中均可检测到较多的裂解产物，而其他菌种的扩增产物均未检出阳性斑点。用脆弱芽孢杆菌感染败血症模型小鼠的血液样本进行套式PCR-斑点杂交试验。该方法可在10份血液中检出20个脆弱芽胞杆菌集落形成单位。针对纳米H基因的套式聚合酶链式反应-斑点杂交法有望应用于临床标本的脆性芽孢杆菌的快速检测。另外，我们还测定了脆性芽孢杆菌的β-异丙基苹果酸脱氢酶基因(LeuB)的核苷酸序列和包括脆性芽孢杆菌在内的7种拟杆菌的16S-23S rRNA基因间隔区的大小。这些结果将为今后建立类杆菌属种的鉴别系统提供参考。较少

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