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.

Microfluidic Paper-Based Analytical Devices (μPADs) and Micro Total Analysis Systems (μTAS): Development, Applications and Future Trends

“…LOC connected with micellar electrokinetic chromatography (MEKC) and electrochromatography (CEC) have been applied to the separation of various components in multidisciplinary field, but techniques based on microchip capillary electrophoresis (MCE) are the most common methods integrated with μTAS devices and many studies were based on it. Practical applications of that separation protocols are widely used in many studies in genetics and molecular biology, including polymerase chain reaction (PCR) [94] amplification of DNA in nanoliter droplets [95, 96], reverse transcription (RT)-polymerase chain reaction (PCR) [97, 98], DNA extraction and real-time PCR for rapid pathogen identification [97, 99, 100], simultaneous DNA amplification and detection [101], removal of PCR inhibitors [102], mRNA isolation and amplification [103], mRNA quality and quantity control, nucleic acid detection, amplification and purification [104, 105], single cells genetic research and manipulations [106–109], gene expression research [108, 110], forensic DNA analysis [17] and many others. In molecular biology, the ability to manipulate and analyze single cells is important to understand the molecular mechanisms underlying cellular function [111].…”

Combination of multiplex reverse-transcription loop-mediated isothermal amplification with an immunochromatographic strip for subtyping influenza A virus

“…6. Previously, our group reported an integrated RT-PCR and ICS microdevice to detect H1 gene of influenza A H1N1 virus with 1.41 pg (about 10 5 copies) LOD [27]. In this study, we improved the detection sensitivity (10 copies) and multiplexity using multiplex RT-LAMP and ICS technology [15], and will incorporate them in the advanced microdevice for on-site viral detection.…”

“…So, the high amount of biotin-dUTP can increase the detection sensitivity in the ICS, but can simultaneously decrease the isothermal amplification efficiency. Thus, the concentration ratio of dTTP and biotin-dUTP was fixed to 50:3 based on our previous report [27], and the concentration of dNTPs was tuned with 0.8, 1.2, 1.6, 2.0, and 2.4 mM in the multiplex RT-LAMP reaction. At the concentration of 0.8 and 1.2 mM dNTPs (lane 1 and 2 in Fig.…”

Enhanced DNA sensing via catalytic aggregation of gold nanoparticles

“…(Elghanian et al 1997; Ghosh and Pa 2007) Gold NPs with appropriate surface functionalization provide a simple and inexpensive method for sensing an expanding range of analytes such as nucleic acids,(Bai et al 2010; Deng et al 2012; Reynolds III et al 2000; Storhoff et al 1998) biomolecules,(Beqa et al 2011; Fu et al 2012; Kim et al 2012; Wang et al 2010; Zhao et al 2007) organic molecules,(Ai et al 2009) and metal ions. (Beqa et al 2011; Chen et al 2012; Lee et al 2007; Lou et al 2011; Luo et al 2012; Xue et al 2008) Gold NP-based detection methods have several advantages over other techniques because 1) gold NPs have high extinction coefficients, which translates to a stronger signal and high sensitivity and 2) their color change can be detected without instrumentation.…”