Recombinase polymerase amplification in the molecular diagnosis of microbiological targets and its applications

Abstract

Since the introduction of the polymerase chain reaction (PCR) technique in 1983, nucleic acid amplification has permeated all fields of biological science, particularly clinical research. Despite its importance, PCR has been restricted to specialized centers and its use in laboratories with few resources is limited. In recent decades, there has been a notable increase in the development of new isothermal technologies for molecular diagnosis with the hope of overcoming the traditional limitations of the laboratory. Among these technologies, recombinase polymerase amplification (RPA) has a wide application potential because it does not require thermocyclers and has high sensitivity, specificity, simplicity, and detection speed. This technique has been used for DNA and RNA amplification in various pathogenic organisms such as viruses, bacteria, and parasites. In addition, RPA has been successfully implemented in different detection strategies, making it a promising alternative for performing diagnoses in environments with scarce resources and a high burden of infectious diseases. In this study, we present a review of the use of RPA in clinical settings and its implementation in various research areas.

Unlocking Genetic Profiles with a Programmable DNA‐Powered Decoding Circuit

“…We leveraged this strength and successfully realized the full integration of both genetic detection and data interpretation into a single tube, thereby eliminating additional in silico computation over genetic data acquired by specialized equipment. Though the entire workflow used in this study relies on a PCR machine for pre‐amplification and one‐pot LDR and a microplate reader for the fluorescence readout, however, isothermal amplification methods, for example, recombinase polymerase amplification (RPA), utilize engineered recombinase enzyme instead of thermal denaturing to help primers invade into double‐stranded DNA, [ 36 ] therefore stand as a promising alternative for the isothermal implementation of pre‐amplification and LDR. While fluorescence spectroscopy is conventionally used for the readout of DNA computing systems, there are many emerging alternatives for point‐of‐care readout, [ 37 ] for example, a DNA‐based motor can convert computational results into mechanical motions that can be readily visualized by smartphone camera, [ 37b ] thereby largely reducing the instrument required for the readout of molecular computing platforms.…”

Novel DNA Biosensing Platform for Detecting HIV Integrase for Highly Sensitive and Quantitative HIV Detection, Diagnosis, and Therapeutic Monitoring

“…Enzyme-linked immune sorbent assay (ELISA)-based techniques employing Au nanoparticles , and DNA barcodes have significantly improved the HIV-1 p24 detection limit to less than 0.5 pg mL –1 . Even better results have recently been acquired with pathogen-specific enzyme activity-based DNA biosensors. , These methods involve directly measuring the activity of microbe-expressed enzymes using various DNA nanosensors. Examples of real-time optical or electrochemical systems for a variety of enzymes relevant to the detection of human diseases such as tuberculosis and cancer , have been presented.…”

“…Even better results have recently been acquired with pathogen-specific enzyme activity-based DNA biosensors. 14 , 15 These methods involve directly measuring the activity of microbe-expressed enzymes using various DNA nanosensors. Examples of real-time optical or electrochemical systems for a variety of enzymes relevant to the detection of human diseases such as tuberculosis and cancer 16 , 17 have been presented.…”

Development and application of recombinase polymerase amplification assay for rapid detection of Blastocystis sp.

“…Upon recognition of a specific homologous sequence, the RPA technique initiates the template's synthesis and then amplifies the reverse strand through isothermal chain replacement by polymerase, thus allowing amplification of double-stranded DNA without thermal or chemical denaturation. The RPA test can be done in an hour or less with a similar reagent cost to cPCR but with a lower labour cost and acceptable sensitivity and specificity levels (Mota et al ., 2022 ). In recent years, this technique has been successfully applied to parasite diagnosis in many cases, including Giardia duodenalis (Crannell et al ., 2015 ), Entamoeba histolytica (Nair et al ., 2015 ) and Leishmania Viannia spp.…”