Real-time fluorescence PCR assays for detection and characterization of heat-labile I and heat-stable I enterotoxin genes from enterotoxigenic Escherichia coli. 2004

Udo Reischl, and Mohammad T Youssef, and Hans Wolf, and Eija Hyytia-Trees, and Nancy A Strockbine
Institute of Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany. Udo.Reischl@klinik.uni-regensburg.de

To facilitate the diagnosis of enterotoxigenic Escherichia coli (ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161 E. coli isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC.

UI MeSH Term Description Entries
D004269 DNA, Bacterial Deoxyribonucleic acid that makes up the genetic material of bacteria. Bacterial DNA
D004768 Enterotoxins Substances that are toxic to the intestinal tract causing vomiting, diarrhea, etc.; most common enterotoxins are produced by bacteria. Staphylococcal Enterotoxin,Enterotoxin,Staphylococcal Enterotoxins,Enterotoxin, Staphylococcal,Enterotoxins, Staphylococcal
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001427 Bacterial Toxins Toxic substances formed in or elaborated by bacteria; they are usually proteins with high molecular weight and antigenicity; some are used as antibiotics and some to skin test for the presence of or susceptibility to certain diseases. Bacterial Toxin,Toxins, Bacterial,Toxin, Bacterial
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012703 Serotyping Process of determining and distinguishing species of bacteria or viruses based on antigens they share. Serotypings
D014644 Genetic Variation Genotypic differences observed among individuals in a population. Genetic Diversity,Variation, Genetic,Diversity, Genetic,Diversities, Genetic,Genetic Diversities,Genetic Variations,Variations, Genetic
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D017931 DNA Primers Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques. DNA Primer,Oligodeoxyribonucleotide Primer,Oligodeoxyribonucleotide Primers,Oligonucleotide Primer,Oligonucleotide Primers,Primer, DNA,Primer, Oligodeoxyribonucleotide,Primer, Oligonucleotide,Primers, DNA,Primers, Oligodeoxyribonucleotide,Primers, Oligonucleotide

Related Publications

Udo Reischl, and Mohammad T Youssef, and Hans Wolf, and Eija Hyytia-Trees, and Nancy A Strockbine
February 2006, Journal of food protection,
Udo Reischl, and Mohammad T Youssef, and Hans Wolf, and Eija Hyytia-Trees, and Nancy A Strockbine
April 2016, Foodborne pathogens and disease,
Udo Reischl, and Mohammad T Youssef, and Hans Wolf, and Eija Hyytia-Trees, and Nancy A Strockbine
February 2007, Journal of microbiological methods,
Udo Reischl, and Mohammad T Youssef, and Hans Wolf, and Eija Hyytia-Trees, and Nancy A Strockbine
May 1983, Infection and immunity,
Udo Reischl, and Mohammad T Youssef, and Hans Wolf, and Eija Hyytia-Trees, and Nancy A Strockbine
January 1981, Journal of clinical microbiology,
Udo Reischl, and Mohammad T Youssef, and Hans Wolf, and Eija Hyytia-Trees, and Nancy A Strockbine
December 1990, Journal of clinical microbiology,
Udo Reischl, and Mohammad T Youssef, and Hans Wolf, and Eija Hyytia-Trees, and Nancy A Strockbine
March 1976, The Journal of infectious diseases,
Udo Reischl, and Mohammad T Youssef, and Hans Wolf, and Eija Hyytia-Trees, and Nancy A Strockbine
September 1983, Nihon saikingaku zasshi. Japanese journal of bacteriology,
Udo Reischl, and Mohammad T Youssef, and Hans Wolf, and Eija Hyytia-Trees, and Nancy A Strockbine
September 1979, Infection and immunity,
Udo Reischl, and Mohammad T Youssef, and Hans Wolf, and Eija Hyytia-Trees, and Nancy A Strockbine
September 2006, Cellular microbiology,
Copied contents to your clipboard!