title: Integrated centrifugal reverse transcriptase loop-mediated isothermal amplification microdevice for influenza A virus detection.
abstract: An integrated reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) microdevice which consists of microbead-assisted RNA purification and RT-LAMP with real-time monitoring by a miniaturized optical detector was demonstrated. The integrated RT-LAMP microdevice includes four reservoirs for a viral RNA sample (purified influenza A viral RNA or lysates), a washing solution (70% ethanol), an elution solution (RNase-free water), and an RT-LAMP cocktail, and two chambers (a waste chamber and an RT-LAMP reaction chamber). The separate reservoirs for a washing solution, an elution solution, and an RT-LAMP cocktail were designed with capillary valves for stable storage. Three influenza A virus strains (A/H1N1, A/H3N2, and A/H5N1) were used for RNA templates, and RT-LAMP primer sets were designed to detect hemagglutinin (HA) and conserved M gene. Sequential sample flow to the microbeads for RNA purification was achieved by centrifugal force with optimization of capillary valves and a siphon channel. Furthermore, the purified RNA solution was successfully isolated from the waste solution by changing the rotational direction, and combined with the RT-LAMP cocktail in the RT-LAMP reaction chamber for target gene amplification. Total process from the sample injection to the result was completed in 47 min. Influenza A H1N1 virus was confirmed on the integrated RT-LAMP microdevice even with 10 copies of viral RNAs, which revealed 10-fold higher sensitivity than that of a conventional RT-PCR. Subtyping and specificity test of influenza A H1N1 viral lysates were also performed and clinical samples were successfully genotyped to confirm influenza A virus on our proposed integrated microdevice.
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 PLUS Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea. Electronic address: jjaehwan@kaist.ac.kr.
authors.forename: Jae Hwan
authors.initials: JH
authors.lastname: Jung
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 PLUS Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea. Electronic address: ilypbh@kaist.ac.kr.
authors.forename: Byung Hyun
authors.initials: BH
authors.lastname: Park
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 PLUS Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea. Electronic address: osj237@kaist.ac.kr.
authors.forename: Seung Jun
authors.initials: SJ
authors.lastname: Oh
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 PLUS Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea. Electronic address: goro0123@kaist.ac.kr.
authors.forename: Goro
authors.initials: G
authors.lastname: Choi
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 PLUS Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea. Electronic address: seots@kaist.ac.kr.
authors.forename: Tae Seok
authors.initials: TS
authors.lastname: Seo
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D001717:Birds; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics / Q000302:isolation & purification; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000302:isolation & purification; D005585:Influenza in Birds / Q000175:diagnosis; D007251:Influenza, Human / Q000175:diagnosis*; D021141:Nucleic Acid Amplification Techniques; D012367:RNA, Viral / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D012367:RNA, Viral
keywords: Centrifugal microdevice; Influenza A virus; Integrated microdevice; Real-time fluorescent detection; Reverse transcriptase loop-mediated ampliciation; Sample pretreatment