3.2. Recombinase Polymerase Amplification (RPA)
This protocol is extracted from research article:
Nucleic Acid Testing of SARS-CoV-2
Int J Mol Sci, Jun 7, 2021; DOI: 10.3390/ijms22116150

Recombinase polymerase amplification is another isothermal amplification method that is widely used for SARS-CoV-2 detection. First described by Piepenburg et al. [152], RPA uses a complex of recombinase and two target specific primers (forward and reverse primers) to amplify the target nucleic acids [152]. Once the target nucleic acids are identified, recombinase-primer complex unwinds the target DNA and allows forward and reverse primers to hybridize [153]. The displaced DNA strand is amplified in the presence of DNA polymerase as primers elongate, and the template DNA is exponentially amplified until the reaction is completed [153]. RPA reaction is carried out at a single temperature between 37 and 42 °C, and the reaction is completed when ATPs are depleted, typically in less than an hour [154]. Amplified products are detected by gel electrophoresis, antigenic tags on primers and tag-specific antibodies, or fluorescent signals produced by a conjugated fluorophore and quencher on primers [152,153,154]. The schematic procedure of reverse transcription RPA (RT-RPA) is shown as Figure 2.

For SARS-CoV-2 detection, researchers have optimized RPA procedures by testing various experimental parameters that can now detect less than five viral copies from patient samples within 45 min from the sample collection [134]. RPA methods have also been optimized for SARS-CoV-2 detection by coupling RPA-based amplification with various CRISPR-based detection methods [155,156,157].

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