tetano
Editor, Senior Moderator
Biosens Bioelectron. 2014 Dec 23;68C:218-224. doi: 10.1016/j.bios.2014.12.043. [Epub ahead of print]
[h=1]Integrated centrifugal reverse transcriptase loop-mediated isothermal amplification microdevice for influenza A virus detection.[/h] Jung JH[SUP]1[/SUP], Park BH[SUP]2[/SUP], Oh SJ[SUP]3[/SUP], Choi G[SUP]4[/SUP], Seo TS[SUP]5[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] 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 47min. 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.
Copyright ? 2014 Elsevier B.V. All rights reserved.
[h=4]KEYWORDS:[/h] Centrifugal microdevice; Influenza A virus; Integrated microdevice; Real-time fluorescent detection; Reverse transcriptase loop-mediated ampliciation; Sample pretreatment
PMID: 25569879 [PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/25569879
[h=1]Integrated centrifugal reverse transcriptase loop-mediated isothermal amplification microdevice for influenza A virus detection.[/h] Jung JH[SUP]1[/SUP], Park BH[SUP]2[/SUP], Oh SJ[SUP]3[/SUP], Choi G[SUP]4[/SUP], Seo TS[SUP]5[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] 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 47min. 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.
Copyright ? 2014 Elsevier B.V. All rights reserved.
[h=4]KEYWORDS:[/h] Centrifugal microdevice; Influenza A virus; Integrated microdevice; Real-time fluorescent detection; Reverse transcriptase loop-mediated ampliciation; Sample pretreatment
PMID: 25569879 [PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/25569879