PCR based Re-amplification Step for Detection and Characterization of Fluorescent Pseudomonads by ARDRA

Abstract

The objective of the present study is to develop a rapid and precise method for monitoring and characterization of bacterial strain(s) at taxonomic level, using universal primers specific for 16S rRNA gene in a PCR based protocol. Authors have introduced an extra re-amplification step of PCR to study wild type fluorescent psuedomonads strains and their cold resistant mutants. Using normal PCR amplification when genomic DNA was amplified only a short fragment of 500 bp was obtained. However, when a re-amplification step (20 cycles) was done using the small volume (2μl)<i> </i>of first cycle aliquot, authors observed two bands of ~1500 and 500 bp. The same was utilized for restriction digestion using restriction enzymes, Alu I, Pst I, Xba I, Hae III, Hind III, BgI II and EcoRI separately. The restriction pattern indicating amplified ribosomal DNA restriction analysis (ARDRA) and re-amplification step used by authors seems to be a precise-technique for characterization.