title: Restriction Endonuclease Analysis Typing of Clostridium difficile Isolates.
abstract: Restriction endonuclease analysis (REA) typing using HindIII enzyme is a highly discriminatory, reproducible, and consistent method of genetic typing of Clostridium difficile (CD) isolates. REA typing analyzes CD whole cellular DNA on two levels of discrimination: REA Group designation and REA Type designation, which distinguishes specific subtypes within the REA Group. This methodology has enabled the tracking of epidemiologically significant CD strains over time and in some cases has allowed documentation of the evolution of previously rare REA Group strains that have subsequently become epidemic. The chapter details the methods used to isolate and purify CD colonies from stool samples, to obtain intact, full-length whole cellular DNA from CD isolates by use of guanidine-EDTA solution, and to analyze the HindIII-digested DNA after electrophoretic separation on agarose gels.
authors: Research Service, Hines VA Hospital, Infectious Diseases, Loyola University Medical Center, Chicago, IL, USA. susan.sambol@va.gov.
authors: Susan P
authors: SP
authors: Sambol
authors: Research Service, Hines VA Hospital, Infectious Diseases, Loyola University Medical Center, Chicago, IL, USA.
authors: Stuart
authors: S
authors: Johnson
authors: Research Service, Hines VA Hospital, Infectious Diseases, Loyola University Medical Center, Chicago, IL, USA.
authors: Dale N
authors: DN
authors: Gerding
mesh_terms: D015373:Bacterial Typing Techniques; D016360:Clostridioides difficile / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D004270:DNA, Circular / Q000235:genetics / Q000302:isolation & purification; D015247:Deoxyribonuclease HindIII / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel / Q000295:instrumentation / Q000379:methods; D004761:Enterocolitis, Pseudomembranous / Q000175:diagnosis / Q000382:microbiology; D005243:Feces / Q000382:microbiology; D006801:Humans; D000091402:Prohibitins; D015183:Restriction Mapping / Q000295:instrumentation / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D004270:DNA, Circular; C000717331:PHB2 protein, human; D000091402:Prohibitins; D015247:Deoxyribonuclease HindIII
keywords: Agarose gel electrophoresis; Clostridium difficile; Guanidine thiocyanate; HindIII; Phenol-chloroform; REA; whole cellular DNA