Nested-Polymerase Chain Reaction for the Detection ofHelicobacter pyloriInfection with Novel Primers Designed by Sequence Analysis of Urease A Gene in Clinically Isolated Bacterial Strains

doi:10.1006/bbrc.1996.0216

Biochemical and Biophysical Research Communications

Volume 219, Issue 1, 6 February 1996, Pages 266-272

https://doi.org/10.1006/bbrc.1996.0216 Get rights and content

Abstract

We have established a highly sensitive semi-nested PCR assay for the detection ofH. pyloriinfection using gastric juice samples, which can be aspirated with disposable nasogastric tubes. The primers targetingH. pyloriurease A gene were designed based on the sequence conservation analysis of sixteenH. pyloristrains isolated from Japanese patients. The efficacy of the PCR assay, designated as the URA-PCR, was confirmed byin vitroandin vivoassessments. Its sensitivity was 97.5% in the gastric juice samples aspirated from forty patients with provenH. pyloriinfection, and was significantly higher than that obtained with previously described PCR assays.

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Lack of Association Between Infectious Burden and Carotid Atherosclerosis in Japanese Patients
2007, Journal of Stroke and Cerebrovascular Diseases
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Samples were amplified for 40 cycles of denaturation at 94°C for 1 minute, annealing at 58°C for 2 minutes, and primer extension at 72°C for 2 minutes. We detected H. pylori DNA using the highly sensitive seminested PCR assay of Kawamata et al26 with A-2F2 (5′-ATA TTA TGG AAG AAG CGA GAG C-3′) and A-2R (5′-AAA TCG GCT CAC ACT TCC A-3′) that target the H. pylori urease A gene as outer primers, and A-2F3 (5′-CAT GAA GTG GGT ATT GAA GC-3′) and A-2R as internal primers. The first and second PCR amplifications were composed of more than 30 and 20 cycles, respectively, of denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 1 minute.
Several infectious agents, such as Chlamydia pneumoniae, cytomegalovirus (CMV), herpes simplex virus (HSV), and Helicobacter pylori, have been implicated in the pathogenesis of atherosclerosis; however, but the contribution of infection may vary among races and geographic conditions. The present study investigates the association between the presence of these pathogens and carotid atherosclerosis and examines the relevance of an infectious burden during atherogenesis in Japanese patients undergoing carotid endarterectomy. We investigated a total of 50 carotid atherosclerotic plaques resected during carotid endarterectomy by polymerase chain reaction (PCR) for C. pneumoniae, CMV, HSV, and H. pylori and by immunocytochemistry (ICC) for C. pneumoniae. We also examined the presence of antibodies to IgG and/or IgA for each pathogen in blood samples. We detected HSV DNA in 2 specimens (4%) and positive ICC for C. pneumoniae in 8 (16%). The results of PCR, ICC, or serum antibodies, as well as the number of seropositive antibodies, did not correlate with severely stenotic, ulcerative, or symptomatic plaques. Our findings indicate that the detection rate of infectious agents within atherosclerotic plaques was significantly lower in our patients than that in other studies. Thus, an inflammatory mechanism might not correlate with the pathogenesis of carotid atherosclerosis among Japanese patients with severe carotid artery stenosis.
Evaluation of quantitative real time PCR for the measurement of Helicobacter pylori at low concentrations in drinking water
2005, Water Research
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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).
A rapid DNA extraction and quantitative, real time polymerase chain reaction (QRTPCR) analysis method targeting the ureA gene of Helicobacter pylori was evaluated for the measurement of these organisms on membrane filters at levels that might be expected to be found in drinking water samples. No interference was seen from high levels of background organisms and related, non-target species were detected at approximately 4–5 log₁₀ lower levels of sensitivity than H. pylori by this assay. A standard curve was generated for the method from analyses of filters containing known numbers of added H. pylori cells. Cell numbers on these filters were determined by staining with a species-specific fluorescent antibody and solid phase cytometry analyses. The mean detection sensitivity of the method was 10 H. pylori cells per filter with a 95% confidence sensitivity of 40 cells and a 95% confidence precision interval of ±0.57 log₁₀ based on duplicate analyses of the samples. One liter drinking water samples from several locations in the US were inoculated with the same H. pylori cell suspensions used to generate the standard curve and gave measurements that were consistent with the standard curve suggesting that these sample matrices produced no interference in the method. This method may be useful for the rapid screening of drinking water for H. pylori.
Immunochemical aspects of Hafnia alvei O antigens
2002, FEMS Immunology and Medical Microbiology
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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 virulence-associated genes ureA, encoding the urease subunit A, and nap, encoding the neutrophil activating protein, in Helicobacter pylori grown both in the stomach of C57/Bl6 mice and in Brucella broth was quantified by quantitative competitive reverse transcriptase-PCR using a homologous RNA standard (competitor) and an external standard (16S rRNA). The results showed that the ureA and nap transcripts were increased up to 15 and 80 times, respectively, in vivo compared to in vitro. The transcription of ureA and nap also differed in that ureA showed highest expression early in infection in mice whereas nap transcription was variable throughout the 18-week infection period.
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 Microbiology
Citation 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 virulence-associated genes ureA, encoding the urease subunit A, and nap, encoding the neutrophil activating protein, in Helicobacter pylori grown both in the stomach of C57/Bl6 mice and in Brucella broth was quantified by quantitative competitive reverse transcriptase-PCR using a homologous RNA standard (competitor) and an external standard (16S rRNA). The results showed that the ureA and nap transcripts were increased up to 15 and 80 times, respectively, in vivo compared to in vitro. The transcription of ureA and nap also differed in that ureA showed highest expression early in infection in mice whereas nap transcription was variable throughout the 18-week infection period.
Helicobacter pylori is not the cause of sudden infant death syndrome (SIDS)
2001, American Journal of Gastroenterology
Citation 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).
OBJECTIVES:
The cause of sudden infant death syndrome (SIDS) is unknown, but our previous hypothesis proposed that Helicobacter pylori could be a causative organism. In this study, we aimed to test this hypothesis by examining gastric and tracheal tissues from a prospective cohort of SIDS infants and re-examining previously studied paraffin-fixed tissues for H. pylori.
METHODS:
Fresh gastric antral and trachea specimens obtained at postmortem from nine consecutive new cases of SIDS in Perth, Western Australia were studied prospectively. Tissues were evaluated for H. pylori by rapid urease test (CLOtest), bacterial culture, histology (hematoxylin and eosin, Warthin-Starry Silver, and immmunoperoxidase staining), and polymerase chain reaction (PCR). The latter two tests were also used for the re-examination of paraffin-embedded specimens from infants who died from SIDS (n = 17) and other non-SIDS causes (n = 7) in Kansas City, Missouri.
RESULTS:
Specimens from nine consecutive SIDS infants in Western Australia showed no evidence of H. pylori by any analyses. In the paraffin-embedded gastric and trachea specimens from Missouri, rod and coccoid-shaped bacteria were seen histologically in 33.3% of the specimens, but these were not typical H. pylori. Upon analysis by PCR, “H. pylori DNA” was detected in 53% (9/17) of SIDS samples versus 57% (4/7) in non-SIDS samples. In all cases the immunoperoxidase stain was negative, suggesting that PCR either 1) gave false positive results in this type of potentially contaminated postmortem specimen or 2) H. pylori DNA was indeed present but not increased in prevalence in SIDS infants.
CONCLUSIONS:
H. pylori is unlikely to be an etiological agent in SIDS.
Accurate diagnosis of Helicobacter pylori: Polymerase chain reaction tests
2000, Gastroenterology Clinics of North America
Citation Excerpt :
Monteiro et al53 indicated that human error could be a problem in the detection of amplicons by agarose gel electrophoresis because of subjective evaluation, especially when faint bands are present. Various approaches have been used to increase the sensitivity of PCR (e.g., reverse transcriptase-PCR,14,60 nested PCR,30,31,55 and other post-PCR modifications). These methods can improve considerably the sensitivity of PCR, but because of the high risk for contamination linked to the manipulation of either RNA or the amplicons, the use of these procedures is problematic.

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¹: 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.

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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

Abstract

Regular Article
Nested-Polymerase Chain Reaction for the Detection ofHelicobacter pyloriInfection with Novel Primers Designed by Sequence Analysis of Urease A Gene in Clinically Isolated Bacterial Strains