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Protocol publication

[…] The PCR mixture (20-µL final volume) contained HotStar Taq master mix (Qiagen, Hilden, Germany) and 10 pmol of forward DP1 (5’- GTAAAACGACGGCCAGT ACGGCGGCCGTAACTATA-3’) and reverse ZGE23 (5’- TATTTAGGTGACACTATAG ACAGGCCAGTTAGCTA-3’) primers. These primers contain sequences (written in bold-faced type) that are specific for SP6 (DP1) and M13 (ZGE23), and underlined sequences indicate 23S rRNA amplicon of 308 bp comprising of 2142, 2143 and 2182 positions. H. pylori colonies on selective medium was added to 1× TE buffer (10 mM Tris-HCL, 1 mM EDTA, pH 7.6) and heated up to 100 °C for 5 min, followed by centrifugation at 8000 rpm. 1 µL of supernatant was added to the PCR mix to amplify 23S rRNA gene. The PCR cycling conditions were 95 °C for 15 min to activate HotStart Taq DNA polymerase, followed by 40 cycles of 94 °C for 15 sec, 57 °C for 30 sec, 72 °C for 30 sec, and final extension at 72 °C for 5 min. The PCR products were purified prior to sequencing by Illustra ExoStar 1-Step (GE Healthcare Life Sciences, Buckinghamshire, United Kingdom) according to manufacturer’s instructions, and followed by Big-Dye (Perkin-Elmer Applied Biosystems, Foster City, USA) amplification using SP6 and M13 primers. Sequencing was then performed using ABI 3130XL sequencer. In total, 193 sequences were obtained and analysed using MEGA version 5.0 against wild-type 23S rRNA gene available in the GenBank database (Accession number: U27270). Sequence data can be downloaded from GenBank database (Accession numbers: KU904824-KU905015). [...] Mann-Whitney t-test, unpaired two-tailed was used to compare resistance rate between different patient groups. All analyses were performed using SPSS Statistics version 20 (SPSS, Chicago, USA) and Prism version 5.0 (GraphPad, San Diego, USA). […]

Pipeline specifications

Software tools MEGA, SPSS
Applications Miscellaneous, 16S rRNA-seq analysis
Organisms Helicobacter pylori, Homo sapiens
Diseases Infection, Peptic Ulcer, Stomach Neoplasms, Genetic Diseases, Inborn
Chemicals Amoxicillin, Metronidazole, Tetracycline, Clarithromycin, Levofloxacin