Real-time, label-free isothermal solid-phase amplification/detection (ISAD) device for rapid detection of genetic alteration in cancers

Abstract

Here, we first present an isothermal solid-phase amplification/detection (ISAD) technique for the detection of single-point mutations that can be performed without labelling in real-time by utilizing both silicon microring-based solid-phase amplification and isothermal recombinase polymerase amplification (RPA). The ISAD technique was performed on a silicon microring device with a plastic chamber containing 10 μL of the reaction mixture, and characterized with an assay for the detection of the HRAS (Harvey RAS) gene single-point mutation. For the solid-phase amplification, the primer of the gene was directly attached to the surface of the device via an amine modification reaction. The amplified DNA was detected, without a label, by measuring the optical wavelength shift of the silicon microring resonator during the reaction. We demonstrated that the sensitivity of the ISAD technique was 100-times higher than that of RPA and conventional PCR methods. Moreover, this technique can be used to distinguish a single-point mutation of the HRAS gene via target amplification. This novel DNA amplification/detection technique will be useful for the detection of sequence alterations such as mutations and single-nucleotide polymorphisms as DNA biomarkers in human diseases.

Isothermal Recombinase Polymerase Amplification Assay Applied to the Detection of Group B Streptococci in Vaginal/Anal Samples

“…Prior studies have applied RPA to aptamer ssDNA (single-stranded DNA) (22 ), positive control DNA (23 ), purified DNA (20, 24 -28 ), human genomic DNA (20,26,29 ), double-stranded PCR product (30,31 ), plasmid DNA (27,28 ), cDNA (25,26 ), and spiked plasma (25,32 ), but none have tested RPA with human samples. In contrast, we used actual human samples in our study.…”

A rapid amplification/detection assay for analysis of Mycobacterium tuberculosis using an isothermal and silicon bio-photonic sensor complex

“…To design MTB-ISAD device, the modified protocol was used for the detailed structure and fabrication of silicon microring resonators (SMR) that have been previously described (Shin et al, 2013a(Shin et al, , 2013b. The device (2.5 cm  0.8 cm) structures such as microring structures, waveguides, and gratings, were patterned on a commercially available 200 mm SOI (Silicon-On-Insulator) wafer with a 220 nm thick top silicon layer and 2 μm thick buried oxide layer by 210 nm deep (ultraviolet) lithography.…”

“…We also demonstrated the clinical utility of the MTB-ISAD assay by detecting MTB nucleic acid in sputum samples from patients with TB. The technique is based on an isothermal solid-phase amplification/detection (ISAD) assay that combines solid-phase-based isothermal DNA amplification (recombinase polymerase amplification (RPA)) and silicon microring resonator (SMR)-based biophotonic sensors, which do not involve labeling (Shin et al, 2013a). Although the SMRs have been used for direct detection of nucleic acids based on the hybridization mechanism between the capture probe and the target (Bogaerts et al, 2012;Baaske and Vollmer, 2012;Shin et al, 2013bShin et al, , 2013cIqbal et al, 2010), however, the MTB-ISAD technique is based on the amplification and the detection mechanism of nucleic acids that was increased the sensitivity of the nucleic acid detection by the combination.…”

Recent developments in nucleic acid identification using solid-phase enzymatic assays

“…In this case the enzyme is intended to "substitute" the thermal denaturation step normally performed during PCR amplification. The enzyme along with single stranded binding proteins facilitates the annealing of shift of a silicon micro-ring optical resonator [69]. This allowed the amplification signal to be monitored in realtime providing a lower detection limit of 500 fg×µL -1 .…”

“…This is due to the fact that the helicase enzyme unwinds ds-DNA thus eliminating the need for thermal denaturation normally employed during PCR thermo-cycling. Shin et al [69] have demonstrated one of the most sophisticated and sensitive approaches for isothermal PCR-like amplification of ds-DNA using a solid-phase immobilized primer. This was carried out using recombinase polymerase amplification (RPA) [70,71] which employs a uvsX recombinase enzyme of specific activity isolated from T4 phage.…”