Infection with enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhea among travelers and residents of developing countries. ETEC produces either a heat-stable toxin or a heat-labile toxin, or both, encoded by plasmid-borne ST and LT genes, respectively. Diagnosis of infection with this subclass of E. coli can be performed with oligonucleotide hybridization probes; however, the sensitivity and specificity of this method are insufficient. A nonradioactive multiplex PCR assay that provides a sensitive and specific method for detecting the presence of either or both toxin genes has been developed. A simple procedure that removed inhibitors of the PCR while efficiently releasing ETEC DNA from stool specimens for subsequent amplification was used. The results for samples from a human volunteer study of ETEC infection indicated that this method of sample preparation results in greater clinical sensitivity than conventional total nucleic acid extraction and ethanol precipitation. Detection of ETEC by a multiplex PCR assay in stool specimens directly processed with a glass matrix and chaotropic solution had greater sensitivity than culture.

A sensitive and specific PCR-based assay to detect the Helicobacter pylon 16S rRNA gene present in formalin-fixed paraffin-embedded gastric biopsy specimens has been developed. A total of 95 patients with dyspepsia were evaluated for the presence of chronic active gastritis and an infection with H. pylori through the use of diagnostic assays based on biopsy specimens and serology. The "gold standard" for the presence of the bacteria was direct detection in histological sections of biopsy specimens by Giemsa stain. The results obtained with the PCR assay performed on the biopsy specimens (94% sensitivity and 100%6 specificity) were equivalent to the detection of H. pylori immunoglobulin G antibodies by the commercially available second-generation Cobas Core anti-H. pylori immunoglobulin G enzyme immunoassay (94% sensitivity and 98% specificity) for the diagnosis of H. pylori infection. Urease testing and bacterial culture of the biopsy specimens were inferior (88 and 70%o sensitivity and 96 and 98% specificity, respectively). A Western blot (immunoblot) analysis had slightly greater sensitivity (96%), although specificity was reduced to 93%. This research prototype PCR assay was shown to be highly reliable for the detection of infection with H. pylori and the presence of chronic active

Uracil DNA glycosylases are DNA repair enzymes present in virtually every organism. These enzymes function by excising from DNA uracil residues resulting from either misincorporation of dUMP residues by a DNA polymerase or deamination of cytosine. Recently, the enzyme has been exploited in PCRs as a means for controlling carryover contamination from previously amplified DNA. When the enzyme is used in amplifications of Borrelia burgdorferi target sequences, we have observed an enhancement in signal detected by a microwell plate DNA hybridization assay. This increase in signal is dependent upon the length of the target, is titratable with enzyme concentration, and has been observed with amplifications performed with both symmetric and asymmetric PCR profiles. The enhancement is shown to occur at the level of the target genomic DNA.