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  • 1
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A polymerase chain reaction assay (PCR) for the diagnosis of Helicobacter pylori in human gastric biopsies was developed. To prevent false-negative results while performing PCR on human tissues, an internal control is necessary. Primer set ACT1-ACT2 which specifically amplifies a 542-bp fragment of the 16S rRNA gene of H. pylori was used. dUTP and hot-start were used to prevent false-positives from carryover of previous products and avoid non-specific extension products. A competitive internal control DNA fragment was constructed to detect the presence of inhibitors. Biopsies from 101 unselected patients with gastric symptoms were tested. PCR results were compared with results from microscopy of histological sections and conventional culturing for H. pylori. Forty-two percent of the biopsies were found to contain compounds inhibiting the PCR. The addition of the internal control assures the performance of the PCR assay and is an important quality control parameter.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 29 (2000), S. 0 
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In a human gastric biopsy specimen, 30% of adhering Helicobacter pylori strain AF4 (cagA and VacA positive) was associated with adhesion pedestals. In an AGS cell assay, only a few percent of this type I strain was found to be associated with adhesion pedestals. Nevertheless, a larger proportion of the type I strain was found to invade AGS cells (P〈0.03) and to attach with depressions in the AGS cell membrane (P〈0.03) than a type II strain (cagA and VacA negative). Incubation of AGS cells and H. pylori without adding fetal calf serum (FCS) to the culture medium increased actin accumulations (FITC-phalloidin stained) beneath adhering H. pylori, and decreased H. pylori invasion of AGS cells significantly (P〈0.01). However, no increase in the number of adhesion pedestals was observed by electron microscopy. Proteinase K treatment of FCS eliminated the H. pylori invasion promoting effect (P〈0.01). Our results suggest differences in the ability of H. pylori to induce adhesion pedestals in human gastric epithelial cells and in AGS cells, but a correlation between adhesion pedestal formation in vivo and H. pylori invasion in vitro can be speculated. In addition, H. pylori invasion into AGS cells was found to be mediated by proteins in FCS.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 10 (1995), S. 0 
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Abstract A primer-set was designed for specific detection of genes that encode for 16S rRNA of Helicobacter pylori, using direct polymerase chain reaction (PCR). The primers were selected in the hypervariable regions, derived from a complete small subunit 16S rRNA sequence of the reference strain H. pylori CCUG 17874. The primer-set amplified a 537 base pair (bp) sequence specifically from chromosomal H. pylori DNA. Amplification of purified chromosomal H. pylori DNA was achieved at concentrations as low as 1 femto gram (fg), equivalent to 5 bacteria. Furthermore, as few as 1 lysed H. pylori cell was detected by this PCR technique. The specificity of the primers was 100%, since purified chromosomal DNA was detected from all 32 various H. pylori isolates, whereas no other bacteria species were detected, whether related to Helicobacter or not. The 16S rDNA primers successfully detected H. pylori in antral biopsy specimens collected from infected patients.
    Type of Medium: Electronic Resource
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