Restriction site insertion-PCR (RSI-PCR) for rapid discrimination and typing of closely related microbial strains

Taking advantage of point mutations between DNA sequences of closely relate d microbial strains, PCR primers modified with respect to the target sequen ce at positions 2-5 near the 3' end were designed to obtain a fragment harb ouring an artificial restriction site specific for a given strain. The modi fied forward primer coupled with a specific reverse primer allows for the a mplification of DNA fragments which can be digested with the specific endon uclease only in those strains where the restriction site is inserted by the DNA polymerase. The effectiveness of the method, named restriction site in sertion-PCR (RSI-PCR), was tested on isolates of the 'Bacillus cereus group ' for the rapid typing and discrimination of these closely related strains. (C) 1999 Federation of European Microbiological Societies. Published by El sevier Science sly. An rights reserved.