title: Modified primers for rapid and direct electrochemical analysis of coeliac disease associated HLA alleles.
abstract: Direct detection of PCR product via hybridisation assay, would facilitate the development of rapid tools for genetic analysis. Here, a PCR primer designed to generate a PCR amplicon tagged with single stranded DNA tails at each end of the duplex, which can be used for direct hybridisation with a surface immobilised probe and an enzyme labelled reporter probe is presented. Four modified sequence specific primers (SSP) pairs were designed for the selective amplification of coeliac disease associated alleles (DQA1*05, DQB1*02, DQB1*03:02 alleles), and human growth hormone (positive control). Multiplex PCR products were electrochemically detected in less than 5 min at 37 °C via direct hybridisation to short probes immobilised on individual electrodes of a genosensor array, and subsequent hybridisation to an enzyme labelled reporter probe. The developed electrochemical genosensor array exploiting the modified primers for the direct detection of PCR products was applied to the genotyping of real patient samples.
authors.affiliation: Departament d'Enginyeria Quimica, Universitat Rovira i Virgili, Avinguda Països Catalans, 26, 43007 Tarragona, Spain.
authors.forename: Hamdi
authors.initials: H
authors.lastname: Joda
authors.affiliation: Departament d'Enginyeria Quimica, Universitat Rovira i Virgili, Avinguda Països Catalans, 26, 43007 Tarragona, Spain. Electronic address: valerio.beni@liu.se.
authors.forename: Valerio
authors.initials: V
authors.lastname: Beni
authors.affiliation: Inno-Train Diagnostik GmbH, Niederhöchstädter Strasse, 62, 61476 Kronberg/Taunus, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Willems
authors.affiliation: Inno-Train Diagnostik GmbH, Niederhöchstädter Strasse, 62, 61476 Kronberg/Taunus, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Frank
authors.affiliation: Fraunhofer ICT-IMM, Carl-Zeiss Strasse, 18-20, 55129 Mainz, Germany.
authors.forename: Julian
authors.initials: J
authors.lastname: Höth
authors.affiliation: TATAA Biocenter AB, Odinsgatan 28, 03, Göteborg, Sweden.
authors.forename: Kristina
authors.initials: K
authors.lastname: Lind
authors.affiliation: TATAA Biocenter AB, Odinsgatan 28, 03, Göteborg, Sweden.
authors.forename: Linda
authors.initials: L
authors.lastname: Strömbom
authors.affiliation: Departament d'Enginyeria Quimica, Universitat Rovira i Virgili, Avinguda Països Catalans, 26, 43007 Tarragona, Spain.
authors.forename: Ioanis
authors.initials: I
authors.lastname: Katakis
authors.affiliation: Departament d'Enginyeria Quimica, Universitat Rovira i Virgili, Avinguda Països Catalans, 26, 43007 Tarragona, Spain; Institucio Catalana de Recerca i Estudis Avançats, Passeig Lluis Companys 23, 08010 Barcelona, Spain. Electronic address: ciara.osullivan@urv.cat.
authors.forename: Ciara K
authors.initials: CK
authors.lastname: O Sullivan
mesh_terms: D000483:Alleles; D001483:Base Sequence; D015374:Biosensing Techniques; D002446:Celiac Disease / Q000235:genetics* / Q000276:immunology*; D017931:DNA Primers / Q000235:genetics; D055664:Electrochemical Techniques / Q000379:methods*; D060005:Genotyping Techniques; D006680:HLA Antigens / Q000235:genetics*; D006801:Humans; D056656:Lab-On-A-Chip Devices; D060885:Multiplex Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D006680:HLA Antigens
keywords: Coeliac disease; Direct detection; Electrochemical genosensor; Human leucocyte antigens (HLA); Modified primers; Sequence specific primer (SSP)