Biochemical and Biophysical Research Communications
Regular ArticleNested-Polymerase Chain Reaction for the Detection ofHelicobacter pyloriInfection with Novel Primers Designed by Sequence Analysis of Urease A Gene in Clinically Isolated Bacterial Strains
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Cited by (48)
Lack of Association Between Infectious Burden and Carotid Atherosclerosis in Japanese Patients
2007, Journal of Stroke and Cerebrovascular DiseasesCitation Excerpt :Specific primers were synthesized according to the published DNA sequences corresponding to highly conserved target gene regions. The primer sequences for detection and other details of the method have been reported previously.25-27 The PCR reaction proceeded in 50 μL of 10 mmol of Tris-HCl (pH 8.3) containing 1.5 mmol of MgCl2, 50 mmol of KCl, 0.01% gelatin, 0.5 μmol of each primer, 200 μmol of deoxynucleotide triphosphates (dNTPs), 1.25 U of Taq DNA polymerase, and 5 μL of template DNA isolated from patient samples.
Evaluation of quantitative real time PCR for the measurement of Helicobacter pylori at low concentrations in drinking water
2005, Water ResearchCitation Excerpt :The probe and primer sequences used in the H. pylori assay included HpyP1: 5′-6FAM-AAACTCGTAACCGTGCATACCCCTATTGAG-TAMRA for the probe; HpyF1: 5′-GGGTATTGAAGCGATGTTTCCT and HpyR1: 5′-GCTTTTTTGCCTTCGTTGATAGT for the primers. This assay targets the ureA gene of H. pylori and the primer and probe binding sequences are highly conserved in the large number of strains of this organism that are currently reported in the literature (Labigne et al., 1991; Kawamata et al., 1996; Chu and Ou, 2001; O’Rourke et al., 2004). Analyses of the primer and probe sequences in silico with the Oligo 6.0 PCR primer analysis software program (Molecular Insights Inc., Cascade CO) as previously described (Brinkman et al., 2003; Haugland et al., 2004), predicted that they would neither amplify nor detect the corresponding ureA gene region from currently sequenced strains of other Helicobacter and urease-positive Campylobacter species (Ferrero and Labigne, 1993; Solnick et al., 1994, 1995; Shen et al., 1998; O’Rourke et al., 2004; Usui et al., 2004).
Immunochemical aspects of Hafnia alvei O antigens
2002, FEMS Immunology and Medical MicrobiologyCitation Excerpt :This had to be done at 0–4°C to slow down the enzymatic degradation of the RNA long enough to allow purification of the RNA. The specificity of the different primers had already been tested by others [9,10,14]. However, since no data were available for reactivity of the primers with the normal flora of mice, we tested the primers for reactivity with samples isolated from H. hepaticus and also from control mice that had not been infected with H. pylori.
The expression of the Helicobacter pylori genes ureA and nap is higher in vivo than in vitro as measured by quantitative competitive reverse transcriptase-PCR
2002, FEMS Immunology and Medical MicrobiologyCitation Excerpt :This had to be done at 0–4°C to slow down the enzymatic degradation of the RNA long enough to allow purification of the RNA. The specificity of the different primers had already been tested by others [9,10,14]. However, since no data were available for reactivity of the primers with the normal flora of mice, we tested the primers for reactivity with samples isolated from H. hepaticus and also from control mice that had not been infected with H. pylori.
Helicobacter pylori is not the cause of sudden infant death syndrome (SIDS)
2001, American Journal of GastroenterologyCitation Excerpt :The main limitation of PCR on paraffin-sectioned tissue is that DNA is likely to be degraded and fragmented so that the method has limited sensitivity for gene products greater than 1000 bp (31). Hence, we chose the amplification of the ureA gene of H. pylori, which yields a PCR product of 314 bp (13). Although only a single round of PCR was used, we have often used this method to detect H. pylori in paraffin sections where the histologist reported “very few” or “no organisms” but where H. pylori culture was positive (unpublished data).
Accurate diagnosis of Helicobacter pylori: Polymerase chain reaction tests
2000, Gastroenterology Clinics of North AmericaCitation Excerpt :As mentioned previously, an important aspect of the PCR protocol lies in the choice of primers. Several primers have been proposed that are specific for the following Helicobacter genes: DNA sequence of the 16S ribosomal RNA (rRNA),26 23S rRNA,28 urease genes (ureA 8,31 and ureB 1,48), 26 kDa species-specific antigen of H. pylori,23 polymorphic random sequences of the chromosomal DNA,73 ureC gene (phosphoglucosamine mutase gene),54 cagA (cytotoxin-associated gene),72 and hpaA (adhesin gene).17 Other primers have been designed specifically to target the RNA (e.g., the 16S rRNA).14
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Corresponding Author: Haruhiko YOSHIDA, M.D., 2nd Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan. FAX: +81-3-3814-0021.