Detection of Bacteroides fragilis in clinical specimens by polymerase chain reaction amplification of the neuraminidase gene

Jotwani, Ravi; Kato, Naoya; Kato, Haru; Watanabe, Kunitomo; Ueno, Kazue

doi:10.1007/bf00298376

Cited by 21 publications

(26 citation statements)

References 13 publications

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“…A segment of the toxin B gene was amplified by using primer NK104 (sequence, 5Ј-GTGTAGCAATGAAAGTCCAAGTTTACGC-3Ј; positions 2945 to 2972) (4) and primer NK105 (sequence, 5Ј-CACTTAGCTCTTTGATTGCTGCACC T-3Ј; positions 3123 to 3148) (4), which were derived from the nonrepeating sequence of the C. difficile toxin B gene. The specificity of the PCR product with primer set NK104-NK105 was confirmed by Southern blot analysis as described previously (10). Probe NK106 (sequence, 5Ј-GACTTACTTCCTACATTATCT GAAGG-3Ј; positions 3058 to 3083) (4) was used and was 3Ј end labeled with digoxigenin with a digoxigenin labeling kit (Boehringer Mannheim, Mannheim, Germany).…”

Section: Methodsmentioning

confidence: 99%

Identification of Toxin A-Negative, Toxin B-Positive Clostridium difficile by PCR

et al. 1998

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Toxigenic strains of Clostridium difficile have been reported to produce both toxins A and B nearly always, and nontoxigenic strains have been reported to produce neither of these toxins. Recent studies indicate that it is not always true. We established a PCR assay to differentiate toxin A-negative, toxin B-positive (toxin A−, toxin B+) strains from both toxin-positive (toxin A+, toxin B+) strains and both toxin-negative (toxin A−, toxin B−) strains as an alternative to cell culture assay and enzyme-linked immunosorbent assay (ELISA). By using the PCR primer set NK11 and NK9 derived from the repeating sequences of the toxin A gene, a shorter segment (ca. 700 bp) was amplified from toxin A−, toxin B+ strains compared to the size of the segment amplified from toxin A+, toxin B+ strains (ca. 1,200 bp), and no product was amplified from toxin A−, toxin B− strains. We examined a total of 421 C. difficile isolates by PCR. Of these, 48 strains showed a shorter segment by the PCR, were negative by ELISAs for the detection of toxin A, and were positive by cell culture assay. Although the cytotoxin produced by the toxin A−, toxin B+ strains was neutralized by anti-toxin B serum, the appearance of the cytotoxic effects on Vero cell monolayers was distinguishable from that of toxin A+, toxin B+ strains. By immunoblotting, the 44 toxin A−, toxin B+ strains were typed to serogroup F and the remaining four strains were serogroup X. Pulsed-field gel electrophoresis separated the 48 strains into 19 types. The PCR assay for the detection of the repeating sequences combined with PCR amplification of the nonrepeating sequences of either the toxin A or the toxin B gene is indicated to be useful for differentiating toxin A−, toxin B+ strains from toxin A+, toxin B+ and toxin A−, toxin B− strains and will contribute to elucidation of the precise role of toxin A−, toxin B+ strains in intestinal diseases.

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Section: Methodsmentioning

confidence: 99%

Identification of Toxin A-Negative, Toxin B-Positive Clostridium difficile by PCR

et al. 1998

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“…PCR amplification of the neuraminidase, nanH, and glutamine synthase genes allowed direct detection of B. fragilis from clinical specimens containing other aerobic and anaerobic bacteria (Yamashita et al, 1994;Jotwani et al, 1995). It was shown that the presence of a specific PCR amplified nanH product completely correlated with positive cultures for B. fragilis.…”

Section: Molecular Approachesmentioning

confidence: 99%

The Medically Important Bacteroides spp. in Health and Disease

Smith¹,

Rocha²,

Paster³

2006

The Prokaryotes

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“…In the past decade, molecular genetics-based techniques, including hybridization assays using a DNA probe or an RNA probe (10,17,30) and peR amplification (15,18,37), have been developed for the specific detection of B. fragilis, and some of these techniques have been shown to have adequate sensitivity and speci-Abbreviations: Ala-tRNA, alanyl transfer RNA; ATCC, American Type Culture Collection; Ile-tRNA, isoleucyl transfer RNA; ITS, internal transcribed spacer; JCM, Japanese Collection of Microorganisms; NCTC, National Collection of Type Cultures; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; rRNA, ribosomal RNA; rrn, ribosomal RNA operon.…”

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confidence: 99%

Genetic Variation in 16S‐23S rDNA Internal Transcribed Spacer Regions and the Possible Use of This Genetic Variation for Molecular Diagnosis of Bacteroides Species

Kuwahara

Nakayama

et al. 2001

Microbiology and Immunology

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Abstract:The structural variation in 16S-23S rDNA internal transcribed spacer regions (ITS) among Bacteroides species was assessed by peR amplification and sequencing analysis, and its possible use for molecular diagnosis of these species was evaluated. Ninety strains of the genus Bacteroides, including the species B. distasonis, B. eggerthii, B.fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis and B. vulgatus, produced one to three ITS amplification products with sizes ranging from 615 to 810 bp, Some Bacteroides strains could be differentiated at species level on the basis of ITS amplification patterns and restriction fragment length polymorphism (RFLP) analysis using a four-nucleotide-recognizing enzyme, Msp I. The results of sequence analysis of ITS amplification products revealed genes for I1e-tRNA and Ala-tRNA in all strains tested. The nucleotide sequence, except for that in tRNA-coding regions, was highly variable and characteristic for each species, but a common sequence among B. fragilis, B. thetaiotaomicron and B. ovatus was observed. A digoxigenin-Iabeled oligonucleotide probe (named FOT1), which was designed from this conserved sequence, specifically hybridized to the ITS amplification products from B.fragilis, B. thetaiotaomicron and B. ovatus. These results suggest that the ITS region is a useful target for the development of rapid and accurate techniques for identification of Bacteroides species.Key words: Bacteroides, 16S-23S rDNA spacer, Molecular diagnosisThe genus Bacteroides, which consists of Gram-negative, saccharolytic nonpigmenting obligate anaerobes, is frequently isolated from human infected tissues. One species, B. fragilis, is considered to be the most medically important because this species accounts for over half of the anaerobes from clinical specimens (7) and sometimes causes severe septicemia with a high mortality rate in compromised hosts (9). Various biochemical tests, including analysis of electrophoretic patterns of dehydrogenase (33), cellular fatty acid and sugar composition (4, 5, 25), lipid analysis (26, 29), serology (13,19,36) and bacteriophage typing (3), have been used to discriminate Bacteroides species. However, these analytical procedures require many troublesome steps, and in some cases it is difficult to differentiate each Bacteroides species due to their similar behavior in many bio-*Addresscorrespondenceto Dr. YoshinariOhnishi, Department of Bacteriology,School of Medicine, The University of Tokushima, Kuramoto-cho, Tokushima,Tokushima770-8503, Japan. Fax:+81-88-633-7069. E-mail: ohnishi@basic.med.tokushima-u.ac.jp 191 chemical tests (12,32). Despite the similarities in biochemical characteristics, none of the Bacteroides species has been found to share more than 30% DNA homology with any other species in DNA-DNA hybridization studies (30). These findings indicate that molecular geneticsbased techniques are more suitable for the differentiation of Bacteroides species.In the past decade, molecular genetics-based techniques, including hybridization assays...

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Detection of Bacteroides fragilis in clinical specimens by polymerase chain reaction amplification of the neuraminidase gene

Cited by 21 publications

References 13 publications

Identification of Toxin A-Negative, Toxin B-Positive Clostridium difficile by PCR

Identification of Toxin A-Negative, Toxin B-Positive Clostridium difficile by PCR

The Medically Important Bacteroides spp. in Health and Disease

Genetic Variation in 16S‐23S rDNA Internal Transcribed Spacer Regions and the Possible Use of This Genetic Variation for Molecular Diagnosis of Bacteroides Species

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