Study period: August 2009–April 2010.
Ethical approval was obtained from NIMR-IRB, while informed consent was obtained from patients before stool samples were taken.
Inclusion Criteria: patients not on antibiotics or PPI’s/histamine- 2- receptor blockers at least a month before the study as well as presenting with dyspepsia symptoms.
Exclusion Criteria: those currently on antibiotics and or PPI’s/histamine 2 receptor blockers.
Clinical samples
Sampling method: Convenient sampling. Stool samples from 97 dyspeptic patients at the LUTH were used for this study. The stool samples were collected using sterile toothpicks into eppendorf tubes containing 700 μl absolute ethanol at room temperature. The samples was considered H. pylori- positive when glmM gene or both genes (glmM and cagA) were detected by PCR, and the results compared with those from UBT as the gold standard and confirmatory test.
UBT
To screen for UBT, H. pylori testing was performed using a validated Heliprobe system (Noster AB Sweden). The heliprobe UBT is a 14C-based urea breath test for the detection of H. pylori infection. It comprises the Helicap TM capsule which contains the 14C-labelled urea which is swallowed and metabolized to carbon dioxide and ammonia by the urease enzyme produced by H. pylori. Labeled 14 C exhaled in the breath is captured by a breathcard and analysed by the automatic heliprobe analyser. Levels above 50 counts/min were considered positive of H. pylori infection.
Stool-PCR
16SrRNA gene (399 bp)
Briefly, DNA from the stool samples were purified using the QIAamp® DNA Stool Mini kit (Hilden, Germany) containing an inhibitEX (removes all DNA damaging substances and PCR inhibitors in stool), and detected using a PCR assay targeting a 399 bp fragment of the 16S rRNA gene of Helicobacter spp. with two specific primers, Heli F (AAC GAT GAA GCT TCT AGC TTG CTA G) and Heli R (GTG CTT ATT CST NAG ATA CCG TCA T) (Germani et al. 1997). PCR was performed using the Ready To-Go PCR beads kit by GE Healthcare (Buckinghamshire, UK). Amplification was carried out in an Eppendorf Mastercycler gradient (Hamburg, Germany) using the following cycling parameters: An initial denaturation at 94°C for 5 min and 35 cycles of 94°C for 30 s, 56°C for 1 min and 72°C for 1 min. This was followed by a final extension of 72°C for 10 min.
The PCR product was separated on a 2% Agarose gel and 50 bp ladder was used as DNA molecular weight standard.
cag A gene (128 bp)
PCR amplification using this primer was carried out using the primer set cagA-F : 5′-ATAATGCTAAATTAGACAACTTGAGCGA-3′ and 5′-AGAAACAAAAGCAATACGATCATTC-3′ Rugge et al. (1999). The 25 μl reaction mixture consisted of x1 PCR buffer, 1.5 mM Magnesium Chloride, 200 μM of each dNTP, 20pmol of each primer and 1U Taq DNA polymerase (Promega).
Amplification was carried out in an Eppendorf Mastercycler gradient using the following cycling parameters: an initial denaturation at 94°C for 5 min and 40 cycles of 94°C for 1 min, 54°C for 1 min and 72°C for 1 min. This was followed by a final extension of 72°C for 5 min. The PCR product was separated on a 2% Agarose gel and 50 bp ladder was used as DNA molecular weight standard.
glmM gene (294 bp)
The following primers were used: F : 5′-GGATAAGCTTTTAGGGGTGTTAGGGG-3′ (738–763) and R : 5′-GCTTACTTTCTAACACTAACGCGC-3′(1010–1033) Kansau et al. (1996). The 25 μl reaction mixture consisted of x1 PCR buffer, 1.5 mM Magnesium Chloride, 200 μM of each dNTP, 20pmol of each primer and 1U Taq DNA polymerase (Promega).
The following were the conditions for amplification: one cycle of denaturation at 94°C × 5 min; 35 cycles at 94°C × 1 min, annealing at 56°C × 1 min, and elongation at 72°C × 2 min, followed by a final elongation step by 1 cycle at 72°C × 7 min. 15°C and amplification was carried out in an Eppendorf Master cycler gradient (Hamburg). The PCR product was separated on a 2% Agarose gel and 50 bp ladder was used as DNA molecular weight standard.
Sensitivity of the primers was determined by testing other bacterial strains from related genus e.g. Salmonella Typhimurium and E. coli. The results of the glm M gene was compared with the gold standard (UBT) using the positive and negative predictive values and also the sensitivity and specificity.
The glm M and cag A genes are genes commonly used for the diagnosis of H. pylori directly from biopsy specimens as well as isolates and was therefore adapted for use for stool PCR in this study.