Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: June 19, 2008
Publication Date: January 11, 2009
Citation: Hiett, K.L., Seal, B.S. 2009. Use of Repetitive Element Palindromic-PCR (rep-PCR) for the Epidemiologic Discrimination of Food-Borne Pathogens. Methods in Molecular Biology. Volume number 551 pages 49-58. Interpretive Summary: The rep-PCR technology was developed for DNA fingerprinting and anslysis to track the spread and identify sources of microbial infection, contamination, or epidemics. This is a class of techniques for uniquely identifying an individual among a population based on an organism's DNA. The polymerase chain reaction (PCR) primers are designed complementary to interspersed palindromic repetitive sequences. Palindromes aer perfect or nearly perfect inverted repeat sequences located in the genomes of most all bacteria and many eukaryotic microorganisms such as fungi. Consequently, the PCR reaction is utilized to amplify differently sized DNA fragments consisting of unique DNA sequences lying between these palindromic repeats. The amplified DNA fragments can be separated via electrophoresis and visualized by staining the DNA or separation can be performed in a semi-automated microfluidics chip and analyzed via a computer-based system. The system has proven to be a highly accurate and reproducible technology now with standardized protocols and reagents. In addition to semi-automated technique allow for same-day strain typing of microorganisms that avoids the tedious and time-consuming technique of pulse-field gel electrophoresis (PFGE).
Technical Abstract: The use of defined primers for polymerase chain reactions (PCR) amplicifcations of interspersed repetitive DNA elements present at distinct locations in prokaryotic genomes is referred to as Repetitive Element Palindromic Sequences Based-Polymerase Chain Reactions, rep-PCR. The initial discovery of Repetitive Extragenic Palindromic (REP) elements occurred in the genomes of Escherichia coli and Salmonella. The family of REP elements is generally between 33 to 40 bp in length, have 500 to 1000 copies per genome, and comprise about 1% of the bacterial genomes of E. coli of Salmonella. Another family of interspersed repetitive elements common to E. coli and Salmonella are Enterobacterial Repetitive Intergenic Consensus (ERIC) elements. ERIC elements range from 124 to 127 bp in size and have 30 to 150 copies per genome. ERIC primer sets result in more complex profiles relative to REP primer sets, however ERIC primer sets appear more sensitive to possible contaminants. The first repetitive intergenic sequence fround in a Gram-positive species (Streptococcus pneumoniae) was the BOX element, approximately 154 bp in length. Use of the BOX primer generates highly complex profiles and is often sufficient for characterization and differentiation of bacterial isolates. The application of rep-PCR to microbes has proven a discriminatory and reproducible tool for microbial subtype analyses and for microbial ecology investigations.