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Next-Generation Immunoassays
Published in Richard O’Kennedy, Caroline Murphy, Immunoassays, 2017
Valerie Fitzgerald, Paul Leonard
There are a number of characteristics of the LAMP method that make it superior to other similar methods. Firstly, unlike other PCR-based methods, LAMP does not require high-precision thermal cycling equipment due to the use of an isothermal enzyme. Secondly, the simplified reaction, which occurs in one tube, can be monitored and a positive reaction confirmed in a number of ways, either by turbidity or by fluorescence, without complicated or time-consuming post-amplification analysis [52]. These properties make LAMP a very attractive method for diagnosis of disease and infection in instances where sophisticated equipment and highly skilled personnel are not available. A very interesting modification of the LAMP method, described by Lee et al., outlines a reverse-transcription loop-mediated isothermal amplification–enzyme-linked immunosorbant hybridisation (RT-LAMP-ELISA) assay for the detection of Mycobacterium tuberculosis in patient samples. With this technique the authors detail a rapid, ‘one-tube’ method for detection of active tuberculosis in patients as well as the ability to differentiate M. tuberculosis from other Mycobacteria species from a single sputum sample [51].
Optimized expression of large fragment DNA polymerase I from Geobacillus stearothermophilus in Escherichia coli expression system
Published in Preparative Biochemistry & Biotechnology, 2023
Eva Agustriana, Isa Nuryana, Fina Amreta Laksmi, Kartika Sari Dewi, Hans Wijaya, Nanik Rahmani, Danu Risqi Yudiargo, Astadewi Ismadara, Moch Irfan Hadi, Awan Purnawan, Apridah Cameliawati Djohan
The emergence of the COVID-19 pandemic over the past two years demands the availability of a detection method. The detection is commonly performed by Real-time Polymerase Chain Reaction (RT-PCR). Despite its robustness, RT-PCR requires special equipment (e.g., thermal cyclers), highly trained personnel, and is relatively time-consuming. Alternative methods like LAMP (loop-mediated isothermal amplification), and RT-LAMP (reverse-transcriptase loop-mediated isothermal amplification) could be the solution since they enable a more feasible detection method with no need for thermal cyclers, less time-consuming, yet sensitive and specific[1–2]. LAMP employs four primers (two inner and two outer primers) and a DNA polymerase with strand displacement activity. The involvement of four primers that recognize six distinct sequences at the initial step and two primers that recognize four distinct sequences at the subsequent steps will enable the recognition of the target, thus ensuring the specificity of this procedure. Furthermore, this method is performed under isothermal conditions[3]. The amplification of the RNA template is made possible by the incorporation of a reverse transcription step (RT-LAMP). For that purpose, reverse transcriptase is added to the reaction mixture. By this means, RT-LAMP allows the synthesis and amplification of cDNA molecules from the RNA template, thus making it possible to detect them[4]. Previous studies reported the application of this method in the detection of viruses including Swine influenza virus (SIV) H1N1, Bovine viral diarrhea virus (BVDV)-type I, Porcine circovirus-2 (PVC-2)[5], MERS-CoV[6], and even SARS-CoV-2[7–8].