Detection of heat-stable enterotoxigenic Escherichia coli by hybridization with an RNA transcript probe

Abstract

Heat-stable enterotoxigenic Escherichia coli was identified by nucleotide hybridization with RNA transcripts ot the gene encoding heat-stable A-2 enterotoxin. Radiolabeled enterotoxin gene RNA transcripts are easier to prepare and avoid the preparation of cloned DNA probes that can be nonspecific if they contain cloning vector DNA. Enterotoxigenic Escherichia coli (ETEC) is usually identified by testing isolates for enterotoxin production in bioassays or serological tests (2, 7, 11, 14, 15). Alternatively, ETEC can be identified by detecting the genes encoding these enterotoxins by DNA hybridization, a method which has been particularly useful in detecting ETEC in large numbers of specimens (3-6). Specific DNA fragments of plasmids containing the cloned enterotoxin genes have been used in DNA hybridization assays to identify ETEC (4, 5). Probes produced by endonuclease digestion must be electrophoretically separated from plasmid-cloning vector DNA to obtain a specific probe and must also be nick translated (9). Nick translation is relatively easy to perform with large DNA fragments, such as the 850-base-pair fragment used as the heat-labile enterotoxin (LT) gene probe, but is more difficult to perform on small DNA probes used to detect the genes encoding heatstable enterotoxin (STA-1 [154 base pairs] or STA-2 [216 base pairs]). Ninety-nine percent of E. coli that hybridized with the LT probe produced LT, as measured by the Y-1 adrenal cell assay, but only 72% of E. coli that hybridized with the STA-2 probe produced ST, as measured by the suckling mouse assay (5). The lack of specificity of the cloned enterotoxin gene probes was due to probes that contained cloning vector DNA. Niriety-eight percent of E. coli that hybridized with the ST oligo probe produced ST (6). To develop a more convenient probe to identify genes encoding STA-2, we clotied the genes encoding STA-2 into pSP64 and used RNA transcripts to detect ST+ ETEC by colony hybridization. Plasmid DNA was isolated from E. coli C600(pSLM004) and was digested with EcoRI, HindIII, and HpaII (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) as previously described (10). The endontclease digestion fragments were separated by electrophoresis on a 1.5% low-meltingtemperature agarose gel (FMC Corp., Marine Colloids Div., Rockland, Maine) in Tris-borate buffer, and a 216-base-pair DNA fragment encoding STA-2 was éfluted by heat, phenol extracted, and ethanol precipitated (8). This DNA fragment was blunt-end ligated with pSP64 (Promega Biotec, Madison, Wis.

Simultaneous detection of Escherichia coli heat-stable and heat-labile enterotoxin genes with a single RNA probe

“…Similar results have been obtained by other workers (20). This system has been used to detect ST-producing ETEC strains (4). In this study, we successfully synthesized and used an RNA hybrid probe to detect E. coli strains that have the genetic potential for producing ST, LT, or both.…”

“…Recently, alternative approaches have been developed for detection of the three STA alleles (estAl, estA2, and estA3; 14) or the LT genes (eltA and eltB; 6) with either RNA (4,5) or DNA (1,8,18,21,26,28) gene probes. The use of a single probe that could simultaneously detect genes of both ST and LT as an initial screening for ETEC strains could facilitate large epidemiological studies.…”

Preparation of a specific RNA probe for detection of Mycobacterium paratuberculosis and diagnosis of Johne's disease

“…Indeed, growth of mycobacteria in the stool specimen is a dispensable preliminary step, in view of the highly sensitive identification procedure, the F57 probe hybridization procedure, as preliminary work (data not shown) seems to indicate. RNA probes detected enterotoxigenic E. coli in clinical isolates (8).…”