Pitfalls in the use of random amplified polymorphic DNA (RAPD) for fingerprinting of gram negative organisms.

Two arbitrary PCR primers for random amplified polymorphic DNA (RAPD) bacterial fingerprinting were used to test factors which may affect RAPD PCR results. These primers have been used in previously published RAPD fingerprinting studies. As expected, the MgCl2 concentration and template concentration in the reaction mixture may affect the RAPD banding patterns. The results obtained were not comparable between runs when using the Hybaid thermal cycler when all other conditions were kept constant. Addition of DMSO, gelatin and repeated subculturing did not appear to affect the banding patterns. A second set of primers directed against known repetitive sequences in Gram negative bacteria (REP1/REP2 and ERIC2) were examined to compare with RAPD as a means of fingerprinting organisms. The reproducibility was excellent. The results suggest RAPD primers can provide some useful comparative information on suspected related strains when tested on the same day and under the same conditions. PCR using REP1/REP2 and ERIC2 primers may provide a more reliable and reproducible alternative method for fingerprinting Gram negative bacteria.