PulseNets Step-by-Step Laboratory Protocol for Molecular Subtyping of Listeria monocytogenes by Macrorestriction and Pulsed-Field Gel Electrophoresis

Subtyping Listeria monocytogenes by macrorestriction and pulsed-field gel electrophoresis (PFGE) provides sensitive and epidemiologically relevant discrimination between strains and allows public health officials to detect potential common source outbreaks of listeriosis. Fundamental to the method is the delicate process of isolating intact genomic DNA from bacterial cells embedded in a gel matrix within a reasonable time period (3–4 h) and results are available within 24 to 48 h. The intact DNA is digested with an infrequently cutting restriction endonuclease (Asc I and Apa I). PFGE technology is based on separation of large fragments (20–1000 kb) of microbial chromosomal DNA. The digested DNA is incorporated into a gel matrix and allowed to migrate by alternating the electric field between spatially distinct pairs of electrodes. This causes the DNA fragments to reorient and migrate through the pores in the agarose gel at rates proportional to their size.