title: Integrated microdevice of reverse transcription-polymerase chain reaction with colorimetric immunochromatographic detection for rapid gene expression analysis of influenza A H1N1 virus.
abstract: An integrated microdevice of a reverse transcription-polymerase chain reaction (RT-PCR) reactor and an immunochromatographic strip was constructed for colorimetric detection of gene expression of influenza A virus subtype H1N1. An RT-PCR cocktail, which included Texas Red-labeled primers, dNTP including biotin-labeled dUTP, and RNA templates of influenza A H1N1 virus, was filled in the PCR chamber through the micropump, and the RT-PCR was performed to amplify the target H1 gene (102 bp). The resultant amplicons bearing biotin moieties and Texas Red haptens were subsequently eluted to the immunochromatographic strip, in which they were first conjugated with the gold nanoparticle labeled anti-hapten antibody in the conjugation pad, and then captured on the streptavidin coated test line through the biotin-streptavidin interaction. By observing a violet color in the test line which was derived from the gold nanoparticle, we confirmed the H1N1 target virus. The entire process on the integrated microdevice consisting of a micropump, a 2 μL PCR chamber, and an immunochromatographic strip was carried out on the portable genetic analyzer within 2.5h, enabling on-site colorimetric pathogen identification with detection sensitivity of 14.1 pg RNA templates.
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 Program), Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Korea.
authors.forename: Yong Tae
authors.initials: YT
authors.lastname: Kim
authors.forename: Yuchao
authors.initials: Y
authors.lastname: Chen
authors.forename: Jong Young
authors.initials: JY
authors.lastname: Choi
authors.forename: Won-Jung
authors.initials: WJ
authors.lastname: Kim
authors.forename: Hyun-Mi
authors.initials: HM
authors.lastname: Dae
authors.forename: Jaean
authors.initials: J
authors.lastname: Jung
authors.forename: Tae Seok
authors.initials: TS
authors.lastname: Seo
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D003124:Colorimetry / Q000379:methods*; D015870:Gene Expression; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics*; D012367:RNA, Viral / Q000032:analysis*; D011934:Reagent Strips; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D013997:Time Factors
chemical_list: D012367:RNA, Viral; D011934:Reagent Strips