Rapid pulsed-field gel electrophoresis protocol for subtyping of Campylobacter jejuni.
We developed a rapid pulsed-field gel electrophoresis (PFGE) protocol for subtyping Campylobacter isolates based on the standardized protocols used by PulseNet laboratories for the subtyping of other food-borne bacterial pathogens. Various combinations of buffers, reagents, reaction conditions (e.g., cell suspension concentration, lysis time, lysis temperature, and restriction enzyme concentration), and electrophoretic parameters were evaluated in an effort to devise a protocol that is simple, rapid, and robust. PFGE analysis of Campylobacter isolates can be completed in 24 to 30 h using this protocol, whereas the most widely used current protocols require 3 to 4 days to complete. Comparison of PFGE patterns obtained in six laboratories showed that subtyping results obtained using this protocol are highly reproducible.
Ribot
E M
EM
Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA. eyr4@cdc.gov
Fitzgerald
C
C
Kubota
K
K
Swaminathan
B
B
Barrett
T J
TJ
D000818:Animals; D015373:Bacterial Typing Techniques; D002169:Campylobacter Infections / Q000382:microbiology*; D016123:Campylobacter jejuni / Q000145:classification* / Q000235:genetics*; D002417:Cattle; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods / Q000592:standards; D006801:Humans; D013997:Time Factors