TITLE: Molecular biology test for rapid detection cagAgen EPIYA motif in H.pylori isolates
AUTHORS: Eliana Rocio Rodríguez Gómez, William Otero Regino, Pedro A. Monterrey, Alba Alicia Trespalacios Rangel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>In this study , we aimed to  determine the current circulation of </span><span style = "font-style:italic;">cagA</span><span> gene EPIYA motifs present in Colombian </span><span style = "font-style:italic;">Helicobacter pylori </span><span>isolates using a rapid molecular test. The </span><span style = "font-style:italic;">cagA </span><span>gene 3' region was amplified through conventional Polymerase Chain Reaction (PCR) and PCR products obtained were sequenced and analyzed with bioinformatics tools.Aditionally, to confirm the prediction of the number EPIYA C repeats based on the  PCR product molecular weight, reamplification and secuencing analysis were performed. </span></div></div>

[STEPS]
?. The entire 3´ repeat regions of the cagA gene was amplified by polymerase chain reaction (PCR) using the cagA primers, sense primer (CAGTF 5’-ACCCTAGTCGGTAATGGG-3’) and antisense primer (CAGTR 5’ GCTTTAGCTTCTGAYACYGC 3’)
?. PCR amplification was carried out a volume of  containing 0.3 µM concentrations of primers (Invitrogen, Carlsbad, CA, USA),   H. pylori genomic DNA and 1 U of master mix for Taq DNA polymerase (Promega, Madison, USA)
50 µl
100 ng
?. The polymerase chain reaction conditions included:  AB1Initial denaturation95°C/10 min2Denaturation95°C/30 sec,3Hybridization52.3°C/30 sec4Extension72°C/ 36 sec5Final extension72°C/5 min
AB1Initial denaturation95°C/10 min2Denaturation95°C/30 sec,3Hybridization52.3°C/30 sec4Extension72°C/ 36 sec5Final extension72°C/5 min
?. PCR products were analyzed by 2% agarose gel electrophoresis with Syber Safe and analyzed in the Gel DocXR+system (Biorad, Hércules, CA, USA).
?. Samples which presented two or more bands in agarose gels from PCR amplifications (samples with mixed infection), were visualized and cut using an UV transilluminator. Each gel band with different size was put in an eppendorf tube and then purified using the Wizard SV Gel and PCR Clean- Up System Kit (Promega, Madison, USA), according to the manufacturer’s instructions. DNA concentration was determined by spectrophotometry using NanoDrop 2000 (Thermo Scientific, Wilmington, NC).
?. The DNA amplification obtained from gel bands was subsequently amplified by conventional PCR. Sequencing for PCR products and bioinformatic analysis.
?. All PCR products from this study were sequenced by Universidad de Los Andes, Bogotá, Colombia. Sequencing reactions were carried out for both DNA strands (forward and reverse sense) by the Sanger method using as control for sequences the H. pylori reference strain (NCTC 11637)
?. Nucleotide sequences were aligned and analyzed using BLASTx, including H. pylori reference strain cagA sequences (NCTC 11637) (GenBank access AF202973).
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							The deduced peptide sequences containing EPIYA motifs were aligned by CLUSTAL W (European Bioinformatics Institute http://www.ebi.ac.uk/Tools/clustalw2/).
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