J Bickley

DNA from Listeria monocytogenes was used as the model system from this investigation, with PCR primers based on the listeriolysin O gene. Under standard polymerase chain reaction (PCR) conditions and with no prior treatment, amplification failed in the presence of more than 5% milk. Since inhibition of the PCR occurred at the same milk concentrations with full fat, half fat and fat-free milk, inhibition was not attributed to the fat content of the milk. Calcium ions were, however, identified as a major source of PCR inhibition. The results demonstrated that the inhibitory effects of calcium ions and milk could be partially reversed by increasing the magnesium concentration in the reaction to well above the standard levels normally required for PCR. This work has important implications for the use of the PCR in the direct detection of food pathogens.

Although a high prevalence of antibodies to Helicobacter pylori has been documented within families, culture and DNA typing of strains from infected children and their parents has not been evaluated. This study aimed to analyse H pylori infection within family groups. Endoscopy, gastric biopsy, and H pylori culture were performed on all eight parents of four children who presented with dyspepsia and who had a positive H pylori culture. All biopsy specimens were cultured on Columbia based blood agar under microaerophilic conditions for four days. The DNA from each strain was extracted and electrophoretic patterns were compared after digestion with restriction endonucleases Hae III or Hind III. Ribotyping using a biotinylated cDNA probe prepared from 16S and 23S rRNA of H pylori NCTC 11638 was also used. Seven of the parents were positive for H pylori on urease testing, histology, and on culture. DNA typing showed the same or a similar strain to be present in at least two family members in three of the four family groups. In family 1, the mother, father, and child all had an identical strain; in family 2, father and son had a similar related strain; father and mother had the same strain in family 3; and all strains were unique in family 4. These data provide evidence for either intrafamilial cross infection or a common source of infection within family groups.

A polymerase chain reaction (PCR) assay with oligonucleotide primers homologous to a portion of the urease C gene of Helicobacter pylori was evaluated for specificity with pure DNA and biopsy material. The assay was used to test for the presence of the organism in dental plaque. The species specificity of detection was confirmed by ensuring that the primers did not amplify DNA extracts from H. cinaedi, H. felis, H. fennelliae, H. mustelae and H. nemestrinae. Sixty-two gastric biopsy samples collected from 14 patients (antrum, body and duodenal sites) were cultured and PCR was performed on the samples after culture. Primer sites were conserved in genomically diverse strains. Samples prepared by single-step heat lysis of bacterial cells and biopsy material did not inhibit PCR. The overall specificity was 96% irrespective of genotype. H. pylori was not cultured from dental plaque (15 patients), neither was H. pylori DNA detected by PCR in either urea breath test-positive or -negative individuals. The results showed that primer pair sequences within the urease C gene are conserved in most strains and provide an accurate basis for detecting H. pylori. As the PCR assay was not inhibited and did not yield false positive results with crude extracts from organisms or in the presence of biopsy material, its value as a diagnostic test was confirmed.

Nine members of a family with a high incidence of duodenal ulcer disease were studied by interview, examination of hospital records, endoscopy, and antral biopsy. Helicobacter pylori was confirmed by CLO test, histology and culture. DNA extraction from pure isolates of H pylori was possible in six family members and strain typing was performed by restriction fragment length polymorphism. DNA restriction digestion was followed by vacublotting and then DNA hybridisation, using a cDNA probe complimentary to H pylori rRNA cistrons. Eight of the nine family members were H pylori positive by CLO test and histology. Five had duodenal ulcer disease. Three family members (one from each generation) harboured clonal variants of a single parent strain of H pylori but only two had duodenal disease. The other three members harboured different strains. Intrafamilial clustering of clonal variants of H pylori occurs in some duodenal ulcer disease families. Family members however, may develop duodenal disease irrespective of the colonising strain.