IMMUNOMAGNETIC BEAD ENRICHMENT AND PCR FOR DETECTION OF HELICOBACTER-PYLORI IN HUMAN STOOLS

An immunomagnetic bead-based polymerase chain reaction assay (IMS-PCR) was developed for the detection of Helicobacter pylori in experimenta lly inoculated human stools and human clinical stool samples. Magnetic beads coated with anti-H. pylori rabbit antibodies were used for enri chment and concentration of H. pylori from faecal samples. Tag polymer ase inhibitors, found in human faeces, are efficiently removed by the immunomagnetic separation (IMS) and subsequent washing of the magnetic beads. Conditions of the assay were developed and optimised with faec es from a healthy, H. pylori seronegative, individual. Faeces was inoc ulated with serial dilutions of either the spiral or the coccoid form of H. pylori. These two morphologic forms could be detected at similar concentrations when inoculated in faeces using an optimised IMS-PCR m ethod. In 1 g of faeces less than 2.5X10(4) H. pylori cells were detec ted as measured with two separate sets of PCR-primers, based on a urea se A subunit genesequence and a genesequence encoding a 26 kDa surface protein of H. pylori. Previously no report has shown a sensitivity be low 10(6) H. pylori in faeces PCR. Preliminary analysis of stool sampl es from 17 patients with symptoms of gastritis and esophagitis by IMS- PCR showed a good correlation with EIA-analysis of H. pylori serum-ant ibodies from these patients. The results indicate that H. pylori cells are shed in faeces of infected patients and that immunomagnetic bead PCR might be an appropriate method for clinical diagnosis and studies involving immunoprophylaxis, antibiotic treatment as well as vaccine c andidates.