Real-Time Nucleic Acid Sequence-Based Amplification in Nanoliter Volumes

Abstract

Real-time nucleic acid sequence-based amplification (NASBA) is an isothermal method specifically designed for amplification of RNA. Fluorescent molecular beacon probes enable real-time monitoring of the amplification process. Successful identification, utilizing the real-time NASBA technology, was performed on a microchip with oligonucleotides at a concentration of 1.0 and 0.1 microM, in 10- and 50-nL reaction chambers, respectively. The microchip was developed in a silicon-glass structure. An instrument providing thermal control and an optical detection system was built for amplification readout. Experimental results demonstrate distinct amplification processes. Miniaturized real-time NASBA in microchips makes high-throughput diagnostics of bacteria, viruses, and cancer markers possible, at reduced cost and without contamination.

Biosensor Recognition Elements

“…Although Immuno-PCR is now a well-established technique for routine applications in both fundamental and clinical applied immunological research, to date the PCR step must be carried out using a PCR machine. However, miniaturized PCR devices using microfluidic channels and reaction chambers are now described in the literature (Koh et al, 200;Gulliksen et al, 2004) making possible development of a "chip based" immuno-PCR biosensor. Additionally, DNA-DNA hybridization detection has been reported using cantilever based recognition elements which could be applicable to Immuno-PCR Wu et al, 2001).…”

Instrumentation of a PLC-Regulated Temperature Cycler with a PID Control Unit and Its Use for Miniaturized PCR Systems with Reduced Volumes of Aqueous Sample Droplets Isolated in Oil Phase in a Microwell

“…Among the DNA assay chips, a number of miniaturized instruments have been developed for polymerase chain reactions (PCR) because the PCR amplification is widely used as a molecular biological tool to replicate millions of copies of target DNA fragments by cycling through two or three temperature steps (denaturation, annealing and extension). [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] The miniaturization of PCR devices can take advantage of reduced consumption of biological sample necessary for PCR and of increased portability. In addition, the decreased cost of fabrication with a choice of polymer materials as a substrate for PCR reaction vessel allows one-time use of the chips, leading to elimination of false positive data resulting from carryover crosscontamination.…”

“…In the batch format, a sample is injected into a confined space in the chip and then repeatedly heated and cooled according to the reaction procedure. [1][2][3][4][5][6][7][8][9][10][11][12][13] In the continuous-flow format, DNA amplification can be achieved by shuttling a PCR cocktail in a microchannel repetitively through different isothermal zones. [14][15][16][17][18][19] The batch format chip is more suitable for miniaturization and high throughput operation; however, special care must be taken in the thermal management of a stationary PCR mixture to acquire quick transitions having low overshoots, together with good rejection of the disturbance.…”

Detection of infectious salmon anaemia virus by real-time nucleic acid sequence based amplification

“…Real-time NASBA can also be used to obtain quantitative data, and will be of use in studies of ISAV pathogenesis, diagnostics, and for environmental monitoring. Furthermore, the recently developed miniaturized real-time NASBA procedure utilising nanolitre-scale reaction volumes (Gulliksen et al 2004) will facilitate high throughput screening for fish viruses such as ISAV.Although the performance of the real-time ISAV-NASBA was encouraging, further characterisation of the assay should be undertaken prior to its routine use for ISAV detection. Firstly, analysis of greater numbers of clinical samples should be performed, including samples from geographic areas not studied in this report such as the United States, Ireland, and Chile.…”