Inhibition of DNAse activity in PFGE analysis of DNA from Campylobacter jejuni

Gibson, J. R.; Sutherland, K.; Owen, Robert J.

doi:10.1111/j.1472-765x.1994.tb00474.x

Cited by 108 publications

(78 citation statements)

References 7 publications

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“…The preparation of bacterial DNA for PFGE and the separation of Srma I restriction fragments using a CHEF-DR II system (Bio Rad Laboratories, USA) were as previously described [10]. In brief, the bacterial cells were incorporated into 1 % w/v agarose plugs and lysed for 48 h at 56 'C in two changes of solution containing 1 % w/v N-Lauroyl sarcosine, 0 5 mM-EDTA pH 9 5 and 0-5 mg/ml proteinase K. The plugs were washed and stored at 4 'C in Tris-EDTA buffer (10 mm-Tris, 10 mm-EDTA, pH 7 5).…”

Section: Pulsed Field Gel Electrophoresismentioning

confidence: 99%

“…The blocks were loaded onto a 1 % w/v agarose gel prepared in 0 5 x TBE (45 mM-Tris, 45 mm boric acid, 1 mM-EDTA). Electrophoretic separation of Sma I generated fragments was at 200 V, 14 C, for 22 h with ramped pulse times from 10 to 35 s; of Kpn I generated fragments, 200 V, 14°C, 23 h with ramped pulse times from 4 to 20 s. DNA degradation in Lior biotype II (DNase positive) strains was prevented by formaldehyde fixation of the bacterial cells prior to their incorporation into agarose [10].…”

Section: Pulsed Field Gel Electrophoresismentioning

confidence: 99%

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Comparison of PFGE, ribotyping and phage-typing in the epidemiological analysis ofCampylobacter jejuniserotype HS2 infections

Gibson¹,

Fitzgerald²,

Owen³

1995

Epidemiol. Infect.

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SUMMARYIn this study we have evaluated the ability of three typing methods, pulsed field gel electrophoresis (PFGE), phage-typing and ribotyping, to discriminate not only between strains of differing serotypes but also between strains within a single serotype, heat stable serotype 2 (HS2). Forty-five isolates derived from cases of campylobacter enteritis occurring in the Cardiff area were examined. These included 18, mostly HS2, strains associated with an outbreak. The typing results for these and a further 39 epidemiologically unrelated strains of serotype HS2 were compared. This is the first report documenting the use of PFGE in an epidemiological investigation of Camnpylobacter jejuni in the UK. The results presented suggest that this technique is the most discriminatory of the three subtyping methods examined.

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Section: Pulsed Field Gel Electrophoresismentioning

confidence: 99%

Section: Pulsed Field Gel Electrophoresismentioning

confidence: 99%

Comparison of PFGE, ribotyping and phage-typing in the epidemiological analysis ofCampylobacter jejuniserotype HS2 infections

Gibson¹,

Fitzgerald²,

Owen³

1995

Epidemiol. Infect.

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“…jejuni isolates were grown on sheep blood 5% agar for 48 h at 378C in a micro-aerobic atmosphere. Bacterial colonies were harvested, resuspended in 900 ìl of cold saline and treated with 100 ìl of formaldehyde on ice for 1 h to inactivate endogenous nucleases [12]; bacterial suspensions were then washed three times in 1000 ìl of cold saline by centrifugation at 12 000 rpm and resuspended in 1000 ìl of saline. The optical densities of the bacterial suspensions were then adjusted to 1.9 at 405 nm and 625-ìl samples were gently mixed with 375 ìl of InCert agarose 1.5% (FMC BioProducts, Rockland, ME, USA).…”

Section: Pfgementioning

confidence: 99%

Comparison of SmaI-defined genotypes of Campylobacter jejuni examined by KpnI: a population-based study

Michaud

Ménard

Gaudreau³

et al. 2001

Journal of Medical Microbiology

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Pulsed-®eld gel electrophoresis (PFGE) was used to analyse 147 isolates collected in two regions of Que Âbec province (Estrie and Montre Âal) between March 1998 and Feb. 1999, to determine the utility of molecular strain typing for a population-based collection of Campylobacter jejuni and to compare directly the discriminatory power of Sma I and Kpn I restriction digests. With a combination of epidemiological criteria including space and time plus molecular strain typing, 49% of isolates from Estrie and 39% of isolates from Montre Âal were identi®ed as belonging to a putative cluster. For 41% of the cases, sources were either missing or explicitly unknown; the remaining sources were subject to recall bias. Thus, the evaluation of sporadic cases of campylobacter enteritis by descriptive clinical investigation alone is neither sensitive nor reliable for identifying sources of infection. In the PFGE analysis, Kpn I digests provided appreciably greater discriminatory power than Sma I digests. When combining the PFGE analyses with basic epidemiological criteria, 30% of the putative Sma I clusters were inconsistent with the epidemiological criteria compared with 17% of the Kpn I clusters. Among the 98 isolates assigned to clusters by Sma I, only 65% gave concordant results with Kpn I. In contrast, among the 81 isolates assigned to clusters by Kpn I, 92% gave concordant results with Sma I. Finally, clusters that were epidemiologically related to ingestion of raw milk and speci®c water sources correlated better with the typing results based on Kpn I than Sma I. Thus, Kpn I is the enzyme of choice for molecular epidemiology studies of C. jejuni. The combination of continuous epidemiological surveillance and molecular strain typing may be useful for identifying new sources and mechanisms of transmission for community-acquired C. jejuni infection and ultimately for developing new approaches to prevention.

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“…Thus, PFGE typing might be suitable for epidemiological investigations of the P. pneumotropica isolates. extensive degradation of the genomic DNA (2,12,16,22,26). Evans et al (11) reported that DNA degradation was caused by free radicals in tris buffer during the electrophoresis.…”

Section: Discussionmentioning

confidence: 99%

“…Although we tried a method which prevents DNA degradation by adding a free radical scavenger such as thiourea to the PFGE buffer (18), the DNA banding patterns were not obtained in this study. We further examined the formaldehyde fixation method (12), and heat treatment (22,24). However, the DNA banding patterns were not obtained.…”

Section: Discussionmentioning

confidence: 99%

Molecular Typing of Pasteurella pneumotropica Isolated from Rodents by Amplified 16S Ribosomal DNA Restriction Analysis and Pulsed‐Field Gel Electrophoresis

Sasaki

Kawamoto

Okiyama

et al. 2006

Microbiology and Immunology

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Pasteurella pneumotropica, a ubiquitous Gram-negative bacterium, is an opportunistic pathogen for rodents (28). This agent can be frequently isolated from the upper respiratory tract, lungs, vagina, trachea and other digestive organs. P. pneumotropica does not significantly affect the health of immunocompetent animals. However, immunodeficient animals infected with P. pneumotropica develop severe or lethal pneumonia (1,8,13,25). Chapes et al. (8) reported that P. pneumotropica induced lethal pneumonia in mice lacking alleles for MHCII, Tlr4, and Nramp1. Furthermore, Hart et al. (13) reported that Toll-like receptor 4 positive macrophages were indispensable for defense against the P. pneumotropica infections in mice. Therefore, control and monitoring of P. pneumotropica infections are requisites for managing the immunodeficient animals.P. pneumotropica was first characterized by Jawetz (17). Heyl (15) then proposed the biotype of P. pneumotropica based on utilization of carbon sources, and P. pneumotropica was reclassified as P. pneumotropica biotypes Jawetz and Heyl (27). Subsequently, Boot and Bisgaard (6) reported the detailed biochemical variations of P. pneumotropica including wild type strains. Further, Boot et al. (7) reported that the P. pneumotropica isolates could be differentiated by haemagglutinating properties of the isolates. However, host variation and the some phenotypic characteristics of the P. pneu- Abstract: A total of 52 isolates of Pasteurella pneumotropica obtained from rodents were examined for their genetic heterogeneity. On the basis of DNA restriction analysis, including amplified 16S ribosomal DNA restriction analysis (ARDRA) and pulsed-field gel electrophoresis (PFGE), differences were identified among the isolates. ARDRA typing with HaeIII revealed 4 different banding patterns of the P. pneumotropica isolates. Eighty-two percent of the 23 isolates identified as a-1 were derived from mice, whereas all the isolates identified as a-3 were derived from rats. Most of the isolates, which showed hemolytic activity on blood agar, obtained from mice and rats, were identified as a-2 and a-4, respectively. By restriction analysis of genomic DNA, ApaI and NotI digestion differentiated 9 variants and an undiscriminating group. However, no close relation with regard to the phenotypic characteristics was observed among the variants. The isolates identified as a-2 and a-4 could not be distinguished by PFGE analysis. DNA restriction analysis revealed that the genetic diversity of the P. pneumotropica isolates was more complex than the phenotypic characteristics among the species, and that at least the P. pneumotropica isolates were clearly differentiated into 4 groups by ARDRA typing with HaeIII.

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Inhibition of DNAse activity in PFGE analysis of DNA from Campylobacter jejuni

Cited by 108 publications

References 7 publications

Comparison of PFGE, ribotyping and phage-typing in the epidemiological analysis ofCampylobacter jejuniserotype HS2 infections

Comparison of PFGE, ribotyping and phage-typing in the epidemiological analysis ofCampylobacter jejuniserotype HS2 infections

Comparison of SmaI-defined genotypes of Campylobacter jejuni examined by KpnI: a population-based study

Molecular Typing of Pasteurella pneumotropica Isolated from Rodents by Amplified 16S Ribosomal DNA Restriction Analysis and Pulsed‐Field Gel Electrophoresis

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