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 inosinecontaining 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.

Multiplex PCR assay and simple preparation method for stool specimens detect enterotoxigenic Escherichia coli DNA during course of infection

“…The selection of oligonucleotide primers located in regions of homology that contained mixed bases at sites of variation between alleles of the estA gene resulted in the detection of a wide range of ST-containing clinical ETEC isolates. An alternative to using ST primers with degeneracies is to incorporate deoxyinosine into locations where several bases are expected, as used by Candrian et al (5). Their upstream primer EC01 is located in the same region as JW14 and performs equivalently (data not shown).…”

“…When ST primers located in the same general regions as JW7 and JW14 were chosen to be homologous to only the ST h gene sequence, amplification of DNAs from isolates producing ST p was absent (2, 7). Several nested PCR assays that used an initial set of primers to amplify both ST types and then amplification of an aliquot of the first reaction mixture with primer sets specific to either ST h or ST p have been used to type clinical ETEC isolates (5,9,17). Nested PCR assays are very sensitive; however, they are subject to contamination with carryover amplicon.…”

Universal primer cocktails for fish DNA barcoding

“…In the event that taxon-specific primer mismatches are detected, decisions on the nucleotide composition of new primers will be aided by the very large number of complete mitochondrial genomes available for fishes (Inoue et al 2001;Miya et al 2001;Miya et al 2003). Cases of failed PCR amplification may also be resolved by employing degenerate or inosine-containing primers to overcome 3′-end mismatches (Batzer et al 1991;Candrian et al 1991;Christopherson et al 1997;Sorenson et al 1999). However, such primers may increase the chance of co-amplifying other gene regions (Zhang & Hewitt 1996) or segments of mitochondrial DNA that reside in the nucleus (NUMTs) (Lorenz et al 2005).…”

Development and use of 16S rRNA gene targeted PCR primers for the identification of Escherichia coli cells in water

“…The presence of Escherichia coli in water or food can be used as a microbiological indicator for faecal contamination and as a measurement for sanitary quality (Geldreich 1983 ;Bej et al 1990Bej et al , 1991Hsu et al 1991). The E. coli cells present in water or food are mainly non-pathogenic E. coli although in some cases, pathogenic strains, such as enterotoxigenic and shiga-toxin-producing E. coli (STEC) cells may also be present (Echeverria et al 1984 ;Candrian et al 1991a ;Lee Lang et al 1994).…”