Integrated centrifugal reverse transcriptase loop-mediated isothermal amplification microdevice for influenza A virus detection
| العنوان: | Integrated centrifugal reverse transcriptase loop-mediated isothermal amplification microdevice for influenza A virus detection |
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| المؤلفون: | Jae Hwan Jung, Byung Hyun Park, Tae Seok Seo, Seung Jun Oh, Goro Choi |
| المصدر: | Biosensors & Bioelectronics |
| سنة النشر: | 2014 |
| مصطلحات موضوعية: | Reverse transcriptase loop-mediated ampliciation, Biomedical Engineering, Biophysics, Loop-mediated isothermal amplification, Hemagglutinin (influenza), Biosensing Techniques, Biology, medicine.disease_cause, Article, Birds, Integrated microdevice, Influenza A Virus, H1N1 Subtype, Centrifugal microdevice, Influenza, Human, Electrochemistry, medicine, Influenza A virus, Animals, Humans, Sample pretreatment, Influenza A Virus, H5N1 Subtype, Elution, Influenza A Virus, H3N2 Subtype, RNA, General Medicine, Molecular biology, Influenza A virus subtype H5N1, Reverse transcriptase, eye diseases, Real-time fluorescent detection, Influenza in Birds, biology.protein, RNA, Viral, RNA extraction, sense organs, Nucleic Acid Amplification Techniques, Biotechnology |
| الوصف: | 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. Highlights • An integrated microdevice cosistng of RNA purification and RT-LAMP with real-time monitoring was demonstrated. • Sequential sample transportation and purified RNA separation were automatically controlled by centrifugal force and optimized microchannels. • Influenza viruses were isothermally amplified and the products were identified by using a miniaturized fluorescence detector. • Limit of detection for influenza A H1N1 was 10 copies. • Clinical samples were successfully analyzed on the microdevice. |
| تدمد: | 1873-4235 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::b7f291db05a37693703f6cd80e62b207Test https://pubmed.ncbi.nlm.nih.gov/25569879Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....b7f291db05a37693703f6cd80e62b207 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 18734235 |
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