Quantitative real-time PCR for the clinical detection of Helicobacter pylori

Ribeiro, Marcelo Lima; Ecclissato, C.; Mattos, Ricardo Gabriel; Mendonça, Sérgio; Pedrazzoli, José

doi:10.1590/s1415-47572007000300022

Cited by 11 publications

(15 citation statements)

References 28 publications

(44 reference statements)

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“…The importance of this has so far been highlighted only by Kobayashi et al, and Riberio et al, who normalized the counts of H. pylori to human DNA in picograms/nanograms. 9 , 15 . Another aspect of standardization in the current study was that extracts showing Ct >30 in the ACTB gene assays where not considered for further testing and DNA extraction was redone.…”

Section: Discussionmentioning

confidence: 99%

“…11 , 12 However, there have been significant differences among the described assays in terms of design, detection limit and gold standard used for comparison. 9 , 12 – 15 Furthermore, there has been no attempt to normalize bacterial counts to enable reliable comparison of results between studies. Q-PCR assays for the diagnosis of H. pylori infection remain, therefore, largely unstandardized.…”

Section: Introductionmentioning

confidence: 99%

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Normalized real-time PCR for diagnosis of H. pylori infection

Al-Moayad

Alghalibi

Al-Shamahy

et al. 2015

Qatar Medical Journal

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Objectives: There is increasing interest in the use of quantitative PCR (q-PCR) for diagnosis of H. pylori infection. However, the assay remains largely unstandardized, making comparison between studies unreliable. The objective of this study was to assess accuracy of a normalized q-PCR assay for diagnosis of the infection. Subjects and methods: Seventy-six fresh gastric biopsy specimens were collected from patients undergoing upper gastrointestinal tract endoscopy and examined by rapid urease test (RUT), culture, and a commercial TaqMan q-PCR assay targeting the ureA gene. Counts obtained from the latter assay were normalized to the human ACTB gene. A subject was considered to be infected if two or more assays were positive. Results: The detection rates were 42.1%, 52.6%, and 78.9% by culture, RUT and q-PCR, respectively. Bacterial density ranged 0.005 to 4800 bacteria per 100 human cells. Because q-PCR showed low initial specificity (45.7%), the cutoff value for the assay was recalculated as 1 bacterium per 100 human cells, using ROC curve analysis. Accordingly, the sensitivities and specificities were 79.5% and 97.3%, respectively, for culture; 94.9% and 91.9%, respectively, for RUT; and 94.9% and 94.6%, respectively, for q-PCR. By gold standard, 39 of the dyspeptic patients (51.3%) were found to be infected. Conclusions: With the identified cutoff value, the q-PCR assay diagnosed H. pylori infection with an accuracy slightly superior to that of RUT. However, the possibility that low counts detected only by q-PCR represent true infections warrants further investigation. Normalization of bacterial counts for standardization of q-PCR H. pylori assays is recommended.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Normalized real-time PCR for diagnosis of H. pylori infection

Al-Moayad

Alghalibi

Al-Shamahy

et al. 2015

Qatar Medical Journal

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“…After that, Proteinase K (Sigma Chemical CO., St. Louis, MO) was added to a final concentration of 0.5 mg/mL. The samples were then incubated at 37ºC overnight and the bacterial DNA extracted using the phenol/chloroform method (23). …”

Section: Patients Materials and Methodsmentioning

confidence: 99%

Characterization of virulence genes cagA and vacA in Helicobacter Pylori and their prevalence in gastrointestinal disorders

Cogo

Monteiro

Nogueira

et al. 2011

Braz. J. Microbiol.

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Prevalence of H. pylori infection was determined using cultures of gastric biopsy samples of patients attended at the academic hospital of the Federal University of Paraná, Curitiba, Paraná, Brazil. Molecular methods were used to characterize the cagA and vacA genes from bacterial isolates associated with different diseases presented by patients. Out of a total of 81, forty-two gastric biopsy samples tested were positive for H. pylori, with a prevalence of 51.9%. No significant difference was found with regard to the gender (p=0.793) and age (p=0.183) of the patients. Genotype s1m1 vacA gene was found in 67% of the cases of peptic ulcer investigated (p=1.0), despite the limited number of patients with this disease (n=3). A correlation between the presence of less virulent strains (s2m2) and reflux esophagitis was found in the majority of the cases (45%), but without statistical significance. An association between the prevalence of cagA gene, found in 92% of isolates, and peptic ulcer was not observed (p=1.0), suggesting that this gene cannot be considered a specific marker of severity in our environment. The results reinforce the importance of conducting regional studies and the need to characterize H. pylori virulence genes associated with different diseases.

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“…The DNA samples obtained were subjected to real-time PCR, using SYBR-Green PCR master mix (Applied Biosystem). The RT-PCR was targeted at the 26 KDa Helicobacter species-specific antigen (SSA) gene with primer sequence (forward: 5'-TGGCGTGTCTATTGACAGCGAGC-3', reverse: 5'-CCTGCTG GCATACTTCACCATG-3') [20,21]. The reaction mixture composed of the following : 20 μL of 2X SYBR Green PCR Master mix (Qiagen), 50 nM of each primer and 1 μL of extracted DNA (200 ng).The reaction was cycled with initial PCR activation step at 95ºC for 10 min, followed by 40 cycles of denaturation at 95ºC for 30 s, annealing at 60ºC for 60 s and primer extension at 60ºC for 60 s. Applied Biosystems ABI 7500 SDS real-time thermal cycler was used for PCR data acquisition and analysis.…”

Section: Pcr Analysismentioning

confidence: 99%