Molecular Typing

Pulsed Field Gel Electrophoresis (PFGE) was selected by PulseNet as the primary genotyping methodology back in the mid 1990's. As a robust and reliable technique it has surpassed all expectations, and remains today the primary method for network participants. This page includes PFGE protocols, PulseNet under the Microscope newsletters (currently covering Image acquisition, agaroses for PFGE, and DNA stains), a link to YouTube videos explaining PFGE (in Spanish only) useful information on the Salmonella serotype Braenderup (H9812) universal standard strain, and general information on equipment needed for PFGE.

PFGE Protocols

The following information provides the standard laboratory protocols for molecular subtyping of the pathogens under surveillance by PulseNet laboratories. Clicking on the name or photograph of an organism will link you to the Adobe Acrobat version of the PulseNet protocol for that organism.

photomicrograph of campylobacter jejuni link to PDF 185KB

 Campylobacter jejuni

Updated March 2013, PDF 204 KB

Clostridium botulinum

  Clostridium botulinum

March 2010, PDF 833 KB

photomicrograph of escherichia coli o157:h7 link to PDF 246KB

  Escherichia coli (O157:H7 and non O157), Salmonella, Shigella sonnei and Shigella flexneri

Updated March 2013, PDF 173 KB

photomicrograph of listeria monocytogenes PDF 57KB

  Listeria monocytogenes

Updated March 2013, PDF 225 KB

vibrio

  Vibrio cholerae and Vibrio parahemolyticus [PDF, 163 KB]

Updated April 2015, PDF 163 KB

yersinia

  Yersinia pestis

April 2006, PDF 210 KB

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PulseNet: Under the Microscope

A series of updates and hints from the PulseNet method development laboratory.

Volume 1:
 Capturing a Gel and Adjusting the Settings for Analysis

Volume 2:
 DNA stains for agarose gels (ethidium bromide, SYBR Safe, SYBR Gold, GelRed)

Volume 3:
 A comparison of agaroses for PFGE (Seakem Gold, Agarose III, Megabase, PFGE Agarose).

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PFGE YouTube videos

PulseNet Latin America have produced a series of videos explaining PFGE. These have been uploaded to YouTube (external link) where they are accessible to all - but at this stage only in Spanish! (external link)

 

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Salmonella Braenderup H9812 Universal Size Standard

For PFGE normalization and accurate comparison of gel images require the use of a well-characterized size standard in at least three lanes of each gel. PulseNet International has adopted a strain of Salmonella serotype Braenderup (H9812) restricted with XbaI was selected as a "universal" standard strain because of its even distribution of bands over the entire range of band sizes normally seen in the foodborne pathogens tracked by PulseNet. This strain was found to have a stable PFGE pattern upon subculture and was susceptible to antimicrobial agents commonly used to treat serious Salmonella infections.

This strain has been deposited with the American Type Culture Collection (BAA-664) (external link) to make it available for other laboratories to use.

<i>Salmonella</i>. braenderup H9812 Universal Size Standard

Approximate band sizes in kilobases of the Salmonella serotype Braenderup reference standard (H9812) restricted with XbaI and run under the PulseNet standardized electrophoresis conditions specific for each organism.

Details of the development of this standard have been described in the following paper:

Hunter SB, Vauterin P, Lambert-Fair MA, Van Duyne MS, Kubota K, Graves L, Wrigley D, Barrett T, Ribot E. (external link)
Establishment of a universal size standard strain for use with the PulseNet standardized pulsed-field gel electrophoresis protocols: converting the national databases to the new size standard.
J Clin Microbiol. 2005 Mar;43(3):1045-50.

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Equipment & Reagents Required for PFGE

PulseNet PFGE protocols have been developed and validated for use on Bio-Rad CHEF Mapper XA System or CHEF-DR III System.

A list of equipment and supplies needed for the molecular subtyping of foodborne bacterial pathogens by PulseNet´s Standardized PFGE Protocols can be downloaded here.

Costs given are approximate, are in US$, and were correct as of January 10, 2007

Use of trade names and commercial sources is for identification only and does not imply endorsement by CDC, the U.S. Department of Health and Human Services, or any PulseNet International participant.

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