Development and Validation of a PulseNet Standardized Pulsed-Field Gel Electrophoresis Protocol for Subtyping ofVibrio cholerae

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Abstract
PulseNet is a network that utilizes standardized pulsed-field gel electrophoresis (PFGE) protocols with the purpose of conducting laboratory-based surveillance of foodborne pathogens. PulseNet standardized PFGE protocols are subject to rigorous testing during the developmental phase and careful evaluation during a validation process assessing its robustness and reproducibility in different laboratories. Here we describe the development and validation of a rapid PFGE protocol for subtyping Vibrio cholerae for use in PulseNet International activities. While the protocol was derived from the existing PulseNet protocol for Escherichia coli O157, various aspects of this protocol were optimized for use with V. cholerae, most notably a change of the primary and secondary restriction enzyme to SfiI and NotI, respectively, and the use of a two-block electrophoresis program. External validation of this protocol was undertaken through a collaboration between three PulseNet Asia Pacific laboratories (Public Health Laboratory Centre, Hong Kong, National Institute of Infectious Diseases, Japan, and International Center for Diarrhoeal Diseases Research–Bangladesh) and PulseNet USA. Comparison of PFGE patterns generated by each of the participating laboratories demonstrated that the protocol is robust and reproducible.