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 RA PD PCR results. These primers have been used in previously published R APD fingerprinting studies. As expected, the MgCl2 concentration and t emplate concentration in the reaction mixture may affect the RAPD band ing patterns. The results obtained were not comparable between runs wh en 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 direct ed against known repetitive sequences in Gram negative bacteria (REP1/ REP2 and ERIC2) were examined to compare with RAPD as a means of finge rprinting organisms. The reproducibility was excellent. The results su ggest RAPD primers can provide some useful comparative information on suspected related strains when tested on the same day and under the sa me conditions. PCR using REP1/REP2 and ERIC2 primers may provide a mor e reliable and reproducible alternative method for fingerprinting Gram negative bacteria.