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

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.

Phenotypic and genetic characteristics of Vibrio cholerae O1 carrying Haitian ctxB and attributes of classical and El Tor biotypes isolated from Silvassa, India

“…Isolates were analysed using a PulseNet standardized PFGE protocol for V. cholerae (Cooper et al, 2006) with NotI restriction enzymes (NEB). PFGE was performed using a Clamped Homogeneous Electric Fields (CHEF) Mapper (Bio-Rad).…”

A Molecular Surveillance Reveals the Prevalence of Vibrio cholerae O139 Isolates in China from 1993 to 2012

“…We used the PulseNet 1-day standardized PFGE protocol for V. cholerae (24). Cell suspensions were placed in polystyrene tubes (Falcon; 12 by 75 mm), and their optical density was adjusted to 3.8 to 4.2 using a Densimat photometer (bioMérieux, France).…”

Cost-effective procedure for Streptococcus pyogenes immobilized DNA preparation and miniPFGE subtyping

“…The time consumed by the original procedure for GAS DNA preparation was up to two to three days [17]. In recent years, optimized PFGE protocols for bacterial subtyping have reduced the bacterial DNA preparation time to 2-4 hours, but they still involve the use of solutions that contain cell-wall-disrupting enzymes and proteases or huge amounts of restriction enzymes, and some of them do not generate reproducible results of good quality, probably due to inefficient bacterial lysis [18][19][20][21][22][23][24]. As well, the separation of DNA macrorestriction fragments takes between 18 and 24 hours in the CHEF chamber [18][19][20][21][22][23][24].…”

“…In recent years, optimized PFGE protocols for bacterial subtyping have reduced the bacterial DNA preparation time to 2-4 hours, but they still involve the use of solutions that contain cell-wall-disrupting enzymes and proteases or huge amounts of restriction enzymes, and some of them do not generate reproducible results of good quality, probably due to inefficient bacterial lysis [18][19][20][21][22][23][24]. As well, the separation of DNA macrorestriction fragments takes between 18 and 24 hours in the CHEF chamber [18][19][20][21][22][23][24]. Procedures for GAS subtyping have been reported based on these optimized protocols, but in general, they have the same mentioned drawbacks in relation to the DNA preparation and the long electrophoresis times [25][26][27][28][29][30].…”