Rapid Pulsed-Field Gel Electrophoresis Protocol for Subtyping of
            <i>Campylobacter jejuni</i>

Ribot, Efrain M.; Fitzgerald, Collette; Kubota, Kristy; Swaminathan, Bala; Barrett, Timothy J.

doi:10.1128/jcm.39.5.1889-1894.2001

Cited by 335 publications

(250 citation statements)

References 19 publications

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“…Preparation of Campylobacter DNA was performed by the Pulsenet protocol (Ribot et al, 2001). PFGE by SmaI (Roche) was also performed by the Pulsenet protocol and by KpnI (Takara, Conda, Madrid, Spain) using the protocol of On et al (1998).…”

Section: Methodsmentioning

confidence: 99%

Drinking water as the source ofCampylobacter coliinfection in grandparent heavy breeders

Pérez-Boto

García-Peña²,

Abad-Moreno³

et al. 2010

Avian Pathology

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Section: Methodsmentioning

confidence: 99%

Drinking water as the source ofCampylobacter coliinfection in grandparent heavy breeders

Pérez-Boto

García-Peña²,

Abad-Moreno³

et al. 2010

Avian Pathology

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“…The first molecular methods to be used extensively, such as pulse field gel electrophoresis (PFGE) fingerprinting and serotyping, showed that Campylobacter in clinical samples were highly diverse comprising the two common species with numerous subtypes within them (Frost et al 1998;Ribot et al 2001). However, the most influential advances in understanding the evolutionary forces that shape this complex population arose from the development and application of multilocus sequence typing (MLST) (Maiden et al 1998), which enabled evolutionary and population genetic analyses to be applied to isolate characterization data.…”

Section: The Impact Of Sequence-based Molecular Typingmentioning

confidence: 99%

The Evolution ofCampylobacter jejuniandCampylobacter coli

Sheppard

Maiden

2015

Cold Spring Harb Perspect Biol

136

127

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The global significance of Campylobacter jejuni and Campylobacter coli as gastrointestinal human pathogens has motivated numerous studies to characterize their population biology and evolution. These bacteria are a common component of the intestinal microbiota of numerous bird and mammal species and cause disease in humans, typically via consumption of contaminated meat products, especially poultry meat. Sequence-based molecular typing methods, such as multilocus sequence typing (MLST) and whole genome sequencing (WGS), have been instructive for understanding the epidemiology and evolution of these bacteria and how phenotypic variation relates to the high degree of genetic structuring in C. coli and C. jejuni populations. Here, we describe aspects of the relatively short history of coevolution between humans and pathogenic Campylobacter, by reviewing research investigating how mutation and lateral or horizontal gene transfer (LGT or HGT, respectively) interact to create the observed population structure. These genetic changes occur in a complex fitness landscape with divergent ecologies, including multiple host species, which can lead to rapid adaptation, for example, through frame-shift mutations that alter gene expression or the acquisition of novel genetic elements by HGT. Recombination is a particularly strong evolutionary force in Campylobacter, leading to the emergence of new lineages and even large-scale genome-wide interspecies introgression between C. jejuni and C. coli. The increasing availability of large genome datasets is enhancing understanding of Campylobacter evolution through the application of methods, such as genome-wide association studies, but MLST-derived clonal complex designations remain a useful method for describing population structure.

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“…The CDC uses this method in their PulseNet system (41,46). The Dice correlation (10) considers the presence or absence of PFGE bands that have been identified in the fingerprints to create the correlation matrix used by the UPGMA algorithm (45).…”

Section: Vol 42 2004 Assessment Of Flow Cytometry and Gel Electrophmentioning

confidence: 99%

Performance Assessment of DNA Fragment Sizing by High-Sensitivity Flow Cytometry and Pulsed-Field Gel Electrophoresis

et al. 2004

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The sizing of restriction fragments is the chief analytical technique utilized in the production of DNA fingerprints. Few techniques have been able to compete with pulsed-field gel electrophoresis (PFGE), which is capable of discriminating among bacteria at species and strain levels by resolving restriction fragments. However, an ultrasensitive flow cytometer (FCM) developed in our lab has also demonstrated the ability to discriminate bacteria at species and strain levels. The abilities of FCM warrant a quantitative parallel comparison with PFGE to assess and evaluate the accuracy and precision of DNA fragment sizing by both techniques. Replicate samples of Staphylococcus aureus Mu50 were analyzed along with two clinical S. aureus isolates. The absolute fragment sizing accuracy was determined for PFGE (5% ؎ 2%) and FCM (4% ؎ 4%), with sequence-predicted Mu50 SmaI fragment sizes used as a reference. Precision was determined by simple arithmetic methods (relative standard deviation for PFGE [RSD PFGE ] ‫؍‬ 3% ؎ 2% and RSD FCM ‫؍‬ 1.2% ؎ 0.8%) as well as by the use of dendrograms derived from Dice coefficient-unweighted pair group method with arithmetic averages (UPGMA) and Pearson-UPGMA analyses. All quantitative measures of PFGE and FCM precision were equivalent, within error. The precision of both methods was not limited by any single sample preparation or analysis step that was tracked in this study. Additionally, we determined that the curve-based clustering of fingerprint data provided a more informative and useful assessment than did traditional bandbased methods.DNA fragment sizing is arguably the most widely used analytical method in molecular biology, biochemistry, and microbiology. Specific applications of DNA fragment sizing include microbe identification and discrimination, genotyping, and sequencing. Traditionally, DNA fragments are characterized via size-dependent separation methods such as gel electrophoresis.Conventional gel electrophoresis, using a solid polymer (e.g., agarose or polyacrylamide) and a static electric field, is ubiquitous in today's molecular biology and biochemistry labs. Such methods are routinely used to separate and size DNA fragments of Յ20 kb (4).Pulsed-field gel electrophoresis (PFGE), developed in 1984 by Schwartz et al. (44), extended the fragment sizing range to over 1 Mb (4,6,9,19,44). For PFGE, large DNA fragments (i.e., Ͼ10 kb) are separated in an agarose gel by a pulsed electric field. A popular application of PFGE has been strainlevel bacterial fingerprinting through the sizing of DNA fragments resulting from the digestion of whole genomic DNAs with rare-cutting restriction endonucleases (5,11,28,35,47,49). Macrorestriction-based bacterial fingerprinting has found applications primarily in the public health and food safety industries. For example, the Centers for Disease Control and Prevention (CDC) have formed PulseNet, the National Molecular Subtyping Network for Foodborne Disease Surveillance (46; http://www.cdc.gov/pulsenet). PulseNet utilizes macrorestriction fi...

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Rapid Pulsed-Field Gel Electrophoresis Protocol for Subtyping of Campylobacter jejuni

Cited by 335 publications

References 19 publications

Drinking water as the source ofCampylobacter coliinfection in grandparent heavy breeders

Drinking water as the source ofCampylobacter coliinfection in grandparent heavy breeders

The Evolution ofCampylobacter jejuniandCampylobacter coli

Performance Assessment of DNA Fragment Sizing by High-Sensitivity Flow Cytometry and Pulsed-Field Gel Electrophoresis

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