Characterization of two DNA probes specific for Serpulina hyodysenteriae

Sotiropoulos, C; Smith, Stuart; Coloe, Peter J.

doi:10.1128/jcm.31.7.1746-1752.1993

Cited by 11 publications

(6 citation statements)

References 28 publications

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“…innocens B256 (Fig. lA, lane 2), which was consistent with earlier results obtained by using these probes in a colony blot hybridization (32).…”

supporting

confidence: 92%

“…The DNA was transferred from the agarose gel to a 0.45-jim-poresize nylon membrane (Hybond N; Amersham, Sydney, Australia) by the method of Southern (33). The filters were washed in 2 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), air dried, UV cross-linked at 312 nm for 2.5 min, and used directly for hybridization as described previously (32). All hybridizations were performed under high-stringency conditions (65°C).…”

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confidence: 99%

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Identification and characterization of Serpulina hyodysenteriae by restriction enzyme analysis and Southern blot analysis

1994

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Chromosomal DNA restriction enzyme analysis and Southern blot hybridization were used to characterize Serpulina hyodysenteriae strains. When chromosomal DNAs from selected strains (reference serotypes) of S. hyodysenteriae were digested with the restriction endonuclease Sau3A and hybridized with a 1.1-kb S. hyodysenteriae-specific DNA probe, a common 3-kb band was always detected in S. hyodysenteriae strains but was absent from Serpulina innocens strains. When the chromosomal DNA was digested with the restriction endonuclease Asp 700 and hybridized with two S. hyodysenteriae-specific DNA probes (0.75 and 1.1 kb of DNA), distinct hybridization patterns for each S. hyodysenteriae reference strain and the Australian isolate S. hyodysenteriae 5380 were detected. Neither the 1.1-kb nor the 0.75-kb DNA probe hybridized with Asp 700or Sau3A-digested S. innocens chromosomal DNA. The presence of the 3-kb Sau3A DNA fragment in S. hyodysenteriae reference strains from diverse geographical locations shows that this fragment is conserved among S. hyodysenteriae strains and can be used as a species-specific marker. Restriction endonuclease analysis and Southern blot hybridization with these well-defined DNA probes are reliable and accurate methods for species-specific and strain-specific identification of S. hyodysenteriae.

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“…innocens B256 (Fig. lA, lane 2), which was consistent with earlier results obtained by using these probes in a colony blot hybridization (32).…”

supporting

confidence: 92%

mentioning

confidence: 99%

Identification and characterization of Serpulina hyodysenteriae by restriction enzyme analysis and Southern blot analysis

1994

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“…The development of a more convenient method that replaces the hazardous and technically difficult radioactive labeling with a stable enzymatic method of detection would be of particular value for routine diagnostic testing and field research (27). Recently, a method for the nonisotopic detection of genes with DNA probes was developed as an easier and safer alternative, such as biotin, digoxigenin, and chemiluminescence (2,6,7,9,10).…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Cloned pR72H Probe for Vibrio parahaemolyticus Detection and Development of a Nonisotopic Colony Hybridization Assay

Lee

Chen

Chou

1995

Microbiology and Immunology

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Vibrio parahaemolyticus is a halophilic bacterium often found in shellfish and is an important causative agent of food poisoning in Taiwan. A rapid and efficient detection method is required to identify this foodborne pathogen. A 0.76-Kb HindIII DNA fragment was cloned from the chromosomal DNA of V parahaemolyticus strain no. 93, designated as pR72H fragment, was used as a polynucleotide probe. It was labeled with digoxigenin-11-dUTP (DIG) by the random primer-labeling method. The sensitivity and specificity of the digoxigenin-labeled 0.76-Kb DNA probe was determined by colony hybridization assay. Under stringent hybridization conditions, 122 of 124 isolates of V. parahaemolyticus showed positive hybridization reaction with DIG-0.76-Kb DNA probe; the negative strains were attributed to slow growth. The DIG-0.76-Kb probe did not hybridize with 86 isolates of other vibrios and a number of other enterics as well as nonenteric microorganisms. The sensitivity and specificity of this DIG probe are 98 % and 100%, respectively. This nonisotopic colony hybridization assay can be very useful for routine monitoring of V. parahaemolyticus in the food industry, environmental analysis and clinical laboratories.

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“…Further studies also have suggested that the 16S rRNA probe might not be specific only to S. hyodysenteriae (39). Dot blot hybridization with whole chromosomal probes and DNA probes for identification of S. hyodysenteriae has been reported (5,33). Although the sensitivity of the whole chromosomal probes was not reported, colony dot blot hybridization with DNA probes was shown to be only slightly better than culture (104 organisms per g of feces).…”

Section: Discussionmentioning

confidence: 99%

Rapid detection of Serpulina hyodysenteriae in diagnostic specimens by PCR

et al. 1994

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PCR assay for the detection of Serpulina hyodysenteriae in diagnostic specimens was developed on the basis of sequence analysis of a recombinant clone designated pRED3C6. Clone pRED3C6, which contained a 2.3-kb DNA fragment unique to S. hyodysenteriae, was identified by screening a plasmid library of S. hyodysenteriae isolate B204 genomic DNA in Escherichia coli by colony immunoblot with the mouse monoclonal antibody 10G6/G10, which was produced against cell-free supernatant antigens from the same isolate. Southern blot analysis of Hindlll-digested genomic DNA of S. hyodysenteriae serotypes 1 through 7 and of four weakly beta-hemolytic intestinal spirochetes, including Serpulina innocens, with the 23-kb DNA fragment of pRED3C6 indicated that the cloned sequence was present exclusively in the seven serotypes of S. hyodysenteriae. An oligonucleotide primer pair for PCR amplification of a 1.55-kb fragment and an internal oligonucleotide probe were designed and synthesized on the basis of sequence analysis of the 23-kb DNA fragment of pRED3C6. Purified genomic DNAs from reference isolates of S. hyodysenteriae serotypes 1 through 9, S. innocens, weakly beta-hemolytic intestinal spirochetes belonging to genotypic groups distinct from those of reference Serpulina spp., other cultivable reference isolates of the order Spirochaetales, and enteric bacteria including Escherichia coli, Salmonella spp., Campylobacter spp., and Bacteroides vulgatus were amplified with the oligonucleotide primer pair in a hot-start PCR. The 1.55-kb products were obtained only in the presence of genomic DNA from each of the nine serotypes of S. hyodysenteriae. The specificity of the 1.55-kb products for S. hyodysenteriae was confirmed on the basis of production of a restriction endonuclease pattern of the PCR products identical to the predicted restriction map analysis of pRED3C6 and positive hybridization signal with the S. hyodysenteriae-specific internal oligonucleotide probe. By using total DNA obtained from normal swine feces inoculated with decreasing concentrations of S. hyodysenterae

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Characterization of two DNA probes specific for Serpulina hyodysenteriae

Cited by 11 publications

References 28 publications

Identification and characterization of Serpulina hyodysenteriae by restriction enzyme analysis and Southern blot analysis

Identification and characterization of Serpulina hyodysenteriae by restriction enzyme analysis and Southern blot analysis

Characterization of a Cloned pR72H Probe for Vibrio parahaemolyticus Detection and Development of a Nonisotopic Colony Hybridization Assay

Rapid detection of Serpulina hyodysenteriae in diagnostic specimens by PCR

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