Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Abstract

Nucleic acid amplification technologies (NAATs) offer the most sensitive tests in the clinical laboratory. These techniques are used as a powerful tool for screening and diagnosis of infectious diseases. Isothermal methods, as an alternative to polymerase chain reaction (PCR), require no thermocycling machine and can mostly be performed with reduced time, high throughput, and accurate and reliable results.However, current molecular diagnostic approaches generally need manual analysis by qualified and experienced personal which is a highly complex, time-consuming and labor-intensive task. Thus, the demand for simpler, miniaturized systems and assays for pathogen detection is steadily increasing. Microfluidic platforms and lab-on-a-chip devices have many advantages such as small sample volume, portability and rapid detection time and enable point-of-care diagnosis.In this article, we review several isothermal amplification methods and their implementation in microsystems in relation to quantification of nucleic acids. miniaturized systems can be diverse. The majority of publications are focussed on clinical diagnostics including foodborne organisms for environmental monitoring [10][11][12]. In case of an infectious disease, cultivation and phenotypic characterization are still the standard methods of microbiologists which need days up to weeks to obtain a diagnostic result. Hence, scientists started to make use of nucleic acid amplification methods to accelerate the analysis. The most widespread and well known technique for this purpose is the polymerase chain reaction (PCR) [10,13], which exploits the activity of the DNA polymerase. The method is based on a thermal cycling process consisting of repeated cycles of heating and cooling steps allowing for DNA melting, sequence specific primer binding and enzymatic amplification of the target nucleic acid. The quantitative assessment of target copies is possible by using a real-time PCR approach, where a concentration dependent signal (e.g. fluorescence) increases over time [14]. The latter enables the user to easily assess the pathogen load of patient samples [15][16][17][18]. Since the first example of a PCR on a miniaturized system in 1994 [19,20], numerous devices were presented, which integrate not just the amplification, but also sample preparation and detection steps [9,[21][22][23][24][25][26][27][28]. Just a few of those microsystems have reached the market so far, but some have shown a fascinating potential to replace the classical laboratory-oriented state-of-the art [29][30][31][32][33]. setups has been shown to obtain a similar result [34]. A smaller heat capacity allows for rapid changes in temperature beneficial for the PCR time as well as a higher parallelism of multiple genetic samples [34].However, a major drawback of the integration of PCR to microsystems is the necessity of sophisticated instrumentation. The reaction requires a thermal cycling instrumentation, space and considerable expertise [35]. Therefore, isothermal reactions for the ta...

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“…It may not be a viable option for the screening of COVID-19 at locations where well-equipped To circumvent the demanding thermal cycling of the RT-PCR-based amplification, new methods have been developed for enabling exponential NA amplification at constant and relatively low temperatures. [97][98][99] A practical alternative for cost-effective and rapid RNA amplification is reverse transcription loop-mediated isothermal amplification (RT-LAMP). 100,101 This method (Figure 2E) was designed to directly amplify specific reversely transcripted cDNA through incubation at a fixed temperature (65 C) through only one-step reverse transcription and achieved an efficiency of 10 9 amplicons of genomic DNA in less than 1 h. It has been demonstrated that RT-LAMP can provide a sensitivity 10-fold higher than that of qRT-PCR.…”

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“…Microfluidic systems can be used to miniaturize and automate NAATs, enabling the rapid and efficient analysis of nucleic acids with reduced reagent consumption. Microfluidic devices can integrate the various steps of NAATs, such as sample preparation, nucleic acid extraction, amplification, and detection, into a single chip or platform [ 29 ].…”

“…This combination with NAATs results in greater specificity and sensitivity, which are required for reliably detecting low viral loads and reducing the time to obtain results. Moreover, NAATs can assist in overcoming the drawback of poor signaling from a biosensor by amplifying it through a NAATs reaction [ 29 , 86 ].…”

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“…(II) A newly synthesized double-stranded T7 (dsT7) RNA polymerase promoter region is formed, allowing for RNA polymerase to start transcription. 91 (III) RNA polymerase transcribes multiple RNA transcripts containing the detectable signal. (IV) For greater sensitivity, a third probe can be added with a region complementary to step III RNA transcripts, allowing for the RNA transcripts to bind.…”

“…(I) An extension probe and a template probe bind to the target DNA, allowing DNA polymerase to extend the extension probe. (II) A newly synthesized double-stranded T7 (dsT7) RNA polymerase promoter region is formed, allowing for RNA polymerase to start transcription . (III) RNA polymerase transcribes multiple RNA transcripts containing the detectable signal.…”