Computational protocol: Evaluation of a new fluorescence quantitative PCR test for diagnosing Helicobacter pylori infection in children

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Protocol publication

[…] Bacterial genomic DNA was extracted by using boiling lysis. The dental plaque sample was rinsed in 500 μl of 0.9% NaCl and centrifuged (Eppendorf 5148) at 10,000 x g for 5 min. 50 μl DNA extraction buffer was added into the precipitate. Gastric fluid was centrifuged at 10,000 x g for 5 min, and 50 μl DNA extraction buffer was added into the precipitate. One gastric biopsy specimen was homogenized after adding 50 μl DNA extraction buffer.Each sample was incubated at 100°C for 10 min in 50 μl DNA extraction buffer containing 50 mM NaOH, 10 mM Tris–HCl (pH 8.0), 1% Triton X-100, 1% NP-40, and 0.5 mM EDTA (pH 8.0). After centrifuged at 15,000 x g for 5 min, 5 microliters of the supernatant was amplified by PCR in 50 μl of 1×PCR buffer containing 200 μM of each deoxynucleoside triphosphate (dNTP), 2 U of Taq DNA polymerase, 0.12 μM of TaqMan probe and 0.2 μM of each primer. In order to prevent contamination, we replaced dTTP with dUTP and added 0.5 U of uracil-DNA glycosylase (UDG) to the PCR system. The amplification was performed by using LightCycler 480II (Roche) under the following conditions: incubation at 50°C for 3 min, followed by denaturation at 94°C for 3 min, and 40 cycles of 94°C for 10 s, 55°C for 45 s, and fluorescence collection at 55°C. A final extension was performed at 40°C for 1 min. PCR primers and probe for the target gene UreA were designed by Primer Express 2.0. Forward primer sequence is 5'-GGC TGA ATT GAT GCA AGA AG-3; reverse primers sequence is 5'-GGT ATG CAC GGT TAC GAG TT-3' and TaqMan probe sequence is FAM-5'-TCCC ATC AGG AAA CAT CGC T-3'-TAMRA. Basic Local Alignment Search Tool (BLAST) database searches were performed for primers and probes to find any sequence similarities. No matches with any human or bacterial DNA targets were found for any of the selected primers and probes. […]

Pipeline specifications

Software tools Primer Express, BLASTN
Application qPCR
Organisms Helicobacter pylori, Homo sapiens
Diseases Gastritis, Infection, Helicobacter Infections