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Genes and genomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Helicase-dependent amplification is a method for in vitro DNA amplification like PCR, but that works at constant temperature. Strands of double-stranded DNA are first separated by a DNA helicase and coated by ssDNA-binding proteins. In the second step, two sequence-specific primers hybridize to each border of the DNA template. DNA polymerases are then used to extend the primers annealed to the templates to produce a double-stranded DNA, and the two newly synthesized DNA products are then used as substrates by DNA helicases, entering the next round of the reaction. Thus, a simultaneous chain reaction develops, resulting in exponential amplification of the selected target sequence (see Vincent et al., 2004 [3], for a schematic diagram).
Lateral Flow Assays
Published in Sibel A. Ozkan, Bengi Uslu, Mustafa Kemal Sezgintürk, Biosensors, 2023
Kamil Żukowski, Marcin Drozd, Robert Ziółkowski, Mariusz Pietrzak, Katarzyna Tokarska, Adam Nowiński, Elżbieta Malinowska
In real life applications the NALFA test are used for detection of DNA fragments amplified during PCR (45–47). It should be stressed, that in the case of classic, three temperature-based PCR reactions, the nucleic acids amplification step still is limited only to well-equipped laboratories with skilled staff. However, the reports dedicated to the use of LFA tests as detection element in the case isothermal nucleic acids amplification methods (48–56), show the technological approach which truly could be appropriate for the point-of-need, thus fulfilling the ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end-users). That is mainly because theoretically for the nucleic acid amplification or its detection no energy source is needed and the whole assay could be performed by unskilled persons. Importantly, this approach still offers high selectivity, low cost and satisfactory time of the analysis. Among different methods of isothermal DNA amplification the nucleic acid sequence based amplification (NASBA), transcription mediated amplification (TMA), self-sustained sequence replication (3SR), helicase dependent amplification (HAD), loop mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA) should be mentioned. The latter seems to offer the most user friendly approach (57). This is because it requires conventionally designed primers and leads to exponential amplification with no need for pretreatment of DNA sample. Moreover, the reactions are sensitive, specific and rapid and the whole system works at constant low temperature (optimum in the range between 37 and 40o C) (57). It is a reason for multiple examples of LFAs dedicated to determination/detection of RPA products, in a form of both NALFIA and NALFT (35, 57–60) (see Fig. 10.7).
Role of Microfluidics-Based Point-of-Care Testing (POCT) for Clinical Applications
Published in Raju Khan, Chetna Dhand, S. K. Sanghi, Shabi Thankaraj Salammal, A. B. P. Mishra, Advanced Microfluidics-Based Point-of-Care Diagnostics, 2022
Arpana Parihar, Dipesh Singh Parihar, Pushpesh Ranjan, Raju Khan
Huh et al. also developed a microfluidic platform with a magnetic force-activated micromixer and functionalized surfaces for cell lysis, intracellular protein purification, and SARS-CoV detection [63]. Similarly, Ramalingan and colleagues used an integrated microfluidic PCR chip to detect SARS virus DNA using an isothermal helicase-dependent amplification method [64].
The lavatory lens: Tracking the global movement of pathogens via aircraft wastewater
Published in Critical Reviews in Environmental Science and Technology, 2023
Aaron Bivins, Robert Morfino, Andrew Franklin, Stuart Simpson, Warish Ahmed
Target specific methods include techniques typically used for diagnostic assays including PCR-based techniques (qPCR, RT-qPCR, dPCR, etc.). These techniques have been widely used for wastewater surveillance. While they can be very sensitive and specific, they require the design of reagents specific to the target of interest a priori, which precludes their usefulness for measuring unknown targets such as an entirely novel pathogen. Additionally, many qPCR platforms only allow testing for up to six targets simultaneously in a single experimental run. Nonetheless, PCR-based techniques could be very useful for sensitive screening of aircraft wastewater for known pathogens, especially in highly parallel and multi-target formats such as TaqMan array cards (TAC), Fluidigm BioMark HD real-time PCR or microarrays (Capone et al., 2020; Wang et al., 2002). Other analytical techniques that warrant further investigation include loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA) which can return results in shorter time frames and have been implemented on lateral flow test strips in clinical and environmental settings (Bivins et al., 2022; Kolm et al., 2019; Zasada et al., 2022). More novel techniques that could become relevant include mass spectrometry for the detection of proteins relevant to specific pathogens (Lara-Jacobo et al., 2022), enzyme-linked immunosorbent assays (ELISA) to detect specific antibodies (Agan et al., 2022), or clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic assays (Broughton et al., 2020; Kaminski et al., 2021).