Molecular biology in diagnosis and epidemiology of Helicobacter pylori: PCR for the detection and AP-PCR for characterization of patient isolates.
Infection with Helicobacter pylori causes chronic active gastritis and has been associated with gastric and duodenal ulcer disease. In biopsy samples of 110 patients with clinical symptoms of active gastritis, H. pylori was detected by means of the polymerase chain reaction (PCR), using species-specific primers defining a 858 bp DNA fragment of H. pylori urease beta-subunit. Sensitivity and specificity of the PCR was compared with culture, histology and Warthin-Starry stain (WSs), detection of H. pylori urease antibodies in serum and urease testing with the Campylobacter-like organism (CLO) test. PCR yielded specific amplification products in 53 cases, whereas culture of the organisms was positive in a subset of 50 cases. Only direct detection in histological sections of biopsy specimens had a higher sensitivity, with 65 positive samples. In contrast, the CLO test was negative in eleven culture-positive and PCR-positive cases. Significant urease antibody titres were found in 39 patients with histologically confirmed diagnosis. These results placed the sensitivity of PCR between tat of the Warthin-Starry stain (WSs) and that of culture. Therefore, PCR can be proposed as a useful rapid and time-saving technique for the detection of H.pylori in gastritis. For epidemiological purposes, fingerprinting with arbitrarily chosen primers by AP-PCR was evaluated. Strain-specific patterns with up to 13 fragments were achieved with 10-nucleotide or longer primers (21-nt) with a G + C content > or = 55%. Thirty-five of 40 strains investigated by this method were distinguishable with a single primer. These results suggest a high level of DNA sequence diversity within this species with the possibility of confirming the clonality in consecutive isolates from a single individual. Alternatively, an increased in-vivo mutation rate could be responsible for DNA divergence, resulting in specific strains for each individual patient.
Schwarz
E
E
Institut für Medizinische Mikrobiologie und Hygiene, Universität zu Köln, Germany.
Plum
G
G
Mauff
G
G
Hasbach
H
H
Eidt
S
S
Schrappe
M
M
Kruis
W
W
D000907:Antibodies, Bacterial / Q000032:analysis; D001431:Bacteriological Techniques; D001482:Base Composition; D015139:Blotting, Southern; D002167:Campylobacter / Q000276:immunology; D016172:DNA Fingerprinting; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay; D016481:Helicobacter Infections / Q000175:diagnosis* / Q000453:epidemiology* / Q000276:immunology; D016480:Helicobacter pylori / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007249:Inflammation / Q000175:diagnosis / Q000382:microbiology; D007413:Intestinal Mucosa / Q000276:immunology / Q000382:microbiology; D010437:Peptic Ulcer / Q000175:diagnosis / Q000453:epidemiology; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013272:Stomach Diseases / Q000453:epidemiology / Q000276:immunology / Q000382:microbiology; D014510:Urease / Q000235:genetics / Q000276:immunology
D000907:Antibodies, Bacterial; D017931:DNA Primers; D004269:DNA, Bacterial; D014510:Urease