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.

Effect of buffer composition on PNA–RNA hybridization studied in the microfluidic microarray chip

“…The influenza A virus is the causative agent of yearly epidemics and occasional pandemics 4 and numerous microfluidic technologies have been developed to detect the presence of influenza A viral RNAs in clinical samples. Devices employed in these technologies include microfluidic chips, [5][6][7][8][9][10][11][12][13][14][15] integrated Microsystems, [16][17][18][19][20] and lateral flow strips. 8,[21][22] The detection methodologies include RT-qPCR, 15, 17, 20, 23-26 electrophoretic immunoassays, 27 PCR-capillary electrophoresis, 11-14, 18, 29 immunochromatography, 8,19,[21][22] microarray, 5,9,16,[30][31][32] and electrochemical approaches.…”

Reversible thermo-pneumatic valves on centrifugal microfluidic platforms

“…18,60 On the other hand, aliquoting is popular for multiplexing of the heat-based assays, in which the integration of the gas-impermeable valves is considered a great advantage to prevent the evaporation of miniaturized reagents and the risk of cross-contamination. 24,26,50,61 To demonstrate the ability of the new valving system to overcome those challenges, an array of RTPVs is embedded in microfluidic Design C, which is created to automate the fluidic procedures commonly required for the multiplexing of heat-based assays. As a pilot study, Design C was used for the conversion of Dengue viral RNA to cDNA, and for the preparation of PCR reaction mixtures.…”

High-throughput sample-to-answer detection of DNA/RNA in crude samples within functionalized micro-pipette tips

“…For nucleic acid amplification, loop-mediated isothermal amplification (LAMP) can bypass thermal cycling steps required by PCR while retaining a high sensitivity and specificity (Asiello and Baeumner 2011;Jung et al 2015;Lin et al 2015;Luo et al 2014;Niemz et al 2011). The results of LAMP could be directly detected by naked eyes (Tomita et al 2008).…”