DETECTION OF HELICOBACTER PYLORI IN STOOL SAMPLES OF CHILDREN USING REAL-TIME POLYMERASE CHAIN REACTION (q-PCR) ASSAY
2Molecular Diagnostic Unit, Israel Central Virology Laboratory, Chaim Sheba Medical Center, Tel-Hashomer, Ramat-Gan
Background
Non-invasive detection methods of Helicobacter pylori are needed for pediatric epidemiological studies. To enhance the detection accuracy of H. pylori, utilization of multiple detection methodologies is recommended.
Aim
To develop and validate a q-PCR detection assay of H. pylori in stool samples of healthy children.
Methods
Stool samples obtained from 189 children, aged 6-9 years were tested for the presence of H. pylori antigens using EIA (Premier HpSA, Meridian Bioscience Inc). These archived fecal samples were utilized to validate a multiplex q-PCR assay that was designed to detect H. pylori Urease and human RNase P genes. Validation was performed on a quantitated ATCC H. pylori extracted DNA, limit of detection (LOD) was calculated and cross-reactivity was determined using human bacterial pathogens. The q-PCR assay was applied to extracted DNA(QIAamp® Fast DNA Stool Minikit) from fecal samples of the children Convergent validity was assessed by comparing results obtained by q-PCR to those obtained EIA and calculating Kappa coefficient.
Results
The prevalence of H. pylori infection was 58.7% by EIA. LOD of the q-PCR assay was 1 CFU/reaction. No cross-reactivity with other pathogens was noted. Stool samples with cyclic threshold H. pylori positive by q-PCR (50.8%). Comparing results obtained by EIA and qPCR showed a Kappa coefficient of 0.78 (p<0.001), suggesting strong agreement beyond chance between the two methods.
Conclusions
The developed q-PCR can be used as a co-technique to enhance the accuracy of H. pylori detection in epidemiological studies, as well as in clinical settings.
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