Rep-PCR tests for identification of bacteria
Published: Thu, 27 Dec 2012, 16:09Last updated: Mon, 11 May 2015, 16:19
This standard describes how to perform rep-PCR tests for identification of bacterial isolates. Approved in 2010-09. Rep-PCR genomic fingerprinting makes use of DNA primers complementary to naturally occurring, highly conserved, repetitive DNA sequences, present in multiple copies in the genomes of most Gram-negative and several Gram-positive bacteria (Louws et al., 1994; Rademaker et al., 1998; Watts et al., 2001). Three families of repetitive sequences have been identified, including the 35-40 bp repetitive extragenic palindromic (REP) sequences, the 124-127 bp enterobacterial repetitive intergenic consensus (ERIC) sequence, and the 154 bp BOX element (Versalovic et al., 1994). These sequences appear to be located in distinct, intergenic positions around the genome. Primers designed from these sequences can, after PCR amplification, distinguish distinct genomic regions located between REP, ERIC or BOX elements. The amplified fragments can be resolved in a gel matrix, yielding a profile referred to as a rep-PCR genomic fingerprint. The method presented here, yielding reproducible results over the years, is based on those published by Smith et al. (2001) and Versalovic et al. (1994). The procedure for performing the DNA isolation and the PCR is described in the Appendix.
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- Diagnostic protocol