Use of inosine-containing oligonucleotide primers for enzymatic amplification of different alleles of the gene coding for heat-stable toxin type I of enterotoxigenic Escherichia coli

Abstract

A method which employs the polymerase chain reaction (PCR) to identify Escherichia coli strains containing the estA gene was developed. This gene codes for heat-stable enterotoxin type I. The use of an inosine-containing pair of amplification primers allowed the amplification of a specific 175-bp DNA fragment from several different estA alleles. The amplified fragments were identified and distinguished by allele-specific oligonucleotide hybridization and characterized by restriction endonuclease analysis. An extension of the classical two-primer PCR proved to be a very simple and rapid method to identify and characterize the estA alleles. Besides the inosine-containing pair of primers, which recognized all described alleles, additional oligonucleotides were used as primers. The sequence of each of these primers was allele specific, and each was amplification compatible with one of the inosine-containing primers. Thus, in one PCR the 175-bp fragment typical for all estA alleles and an allele-specific fragment of different size were produced. These fragments could be separated by agarose gel electrophoresis and were recognized by ethidium bromide staining. Twenty-seven E. coli strains were tested with this amplification system. The presence or lack of the genetic information for production of heat-stable enterotoxin type I was perfectly consistent with the ability of these strains to produce this enterotoxin, as determined by enzyme-linked immunosorbent assay.