In order to improve detection and identification ofHelicobacter pylori in highly contaminated samples, we evaluated new specific primers based on the DNA base sequence within the isocitrate dehydrogenase (icd) gene to amplify a 1,200-bp DNA segment. The specificity of the icd primer was tested against DNA derived from various bacteria, including 7Helicobacter species and a panel of 1 gram-variable, 2 gram-positive, and 16 gram-negative bacteria, as well as DNA from houseflies and feces from H. pylori-negative patients. The primers permitted the detection of all clinical H. pyloriisolates tested, but no reactions were observed with negative controls. Several procedures for DNA extraction from feces were evaluated using PCR with icd primers. The lower limits of detection ofH. pylori DNA from two different sources containing the same number of H. pylori organisms, a pure culture and feces spiked with H. pylori, were established for each extraction method tested. The results were 8.0 × 103CFU/ml for cultures of pure H. pylori, and 8.0 × 106 CFU/ml for H. pylori from feces, using the phenol-chloroform method; 8.0 × 102 and 7.0 × 103 CFU/ml, respectively, for a glass matrix and chaotropic solution protocol; 8.0 × 102 and 7.0 × 103 CFU/ml, respectively, for the QIAamp tissue kit; and 5.0 × 102 and 5.0 × 103 CFU/ml, respectively, for the XTRAX DNA extraction kit. We conclude that the use of the icd gene as a primer for PCR represents a specific and sensitive assay for detection of H. pylori in highly contaminated samples.
Cloning and mutagenesis of the Rhizobium meliloti isocitrate dehydrogenase gene.
