Standardization of Pulsed-Field Gel Electrophoresis Protocols for the Subtyping ofEscherichia coliO157:H7,Salmonella, andShigellafor PulseNet

Abstract

Standardized rapid pulsed-field gel electrophoresis (PFGE) protocols for the subtyping of Escherichia coli O157:H7, Salmonella serotypes, and Shigella species are described. These protocols are used by laboratories in PulseNet, a network of state and local health departments, and other public health laboratories that perform real-time PFGE subtyping of these bacterial foodborne pathogens for surveillance and outbreak investigations. Development and standardization of these protocols consisted of a thorough optimization of reagents and reaction conditions to ensure that the protocols yielded consistent results and high-quality PFGE pattern data in all the PulseNet participating laboratories. These rapid PFGE protocols are based on the original 3-4-day standardized procedure developed at Centers for Disease Control and Prevention that was validated in 1996 and 1997 by eight independent laboratories. By using these rapid standardized PFGE protocols, PulseNet laboratories are able to subtype foodborne pathogens in approximately 24 h, allowing for the early detection of foodborne disease case clusters and often aiding in the identification of the source responsible for the infections. 59

Genotypic diversity, pathogenic potential and the resistance profile of Salmonella Typhimurium strains isolated from humans and food from 1983 to 2013 in Brazil

“…PFGE typing was performed using the standard CDC PulseNet protocol for Salmonella (Ribot et al, 2006). Genomic DNA of the 92 strains listed in Table 1 was digested with 40 U Xba I (Life Technologies).…”

Use of multilocus variable-number tandem repeat analysis in molecular subtyping of Salmonella enterica serovar Typhi isolates

“…Isolates that had no epidemiological record identifying country of origin were considered as having originated in Taiwan. PFGE. The PulseNet PFGE protocol for Salmonella and other enterobacteria (Ribot et al, 2006) was used for PFGE analysis, except that a smaller amount of XbaI (10 U) and BlnI (16 U) was used for the digestion of each slice.…”

Genetic characteristics and changing antimicrobial resistance among Shigella spp. isolated from hospitalized diarrhoeal patients in Kolkata, India

“…PFGE of NotI-or XbaI-digested genomic DNA was performed using a CHEF-Mapper (Bio-Rad Laboratories) according to the PulseNet standardized protocol for subtyping of Shigella species (Ribot et al, 2006). PFGE images were captured by using a Gel Doc XR system (Bio-Rad).…”