RPA-Based Method For The Detection Of SARS-COV2

Abstract

Background: Coronavirus disease 2019 (COVID-19) is a highly infectious disease with significant mortality, morbidity, and far-reaching economic and social disruptions. Testing is key in the fight against COVID-19 disease. The gold standard for COVID-19 testing is the reverse transcription polymerase chain reaction (RT-PCR) test. RT-PCR requires highly specialized, expensive, and advanced bulky equipment that is difficult to use in the field or in a point of care setting. There is need for a simpler, inexpensive, convenient, portable and accurate test. Our aims were to: (i) design primer-probe pairs for use in isothermal amplification of the S1, ORF3 and ORF8 regions of the SARS-CoV2 virus; (ii) optimize the recombinase polymerase amplification (RPA) assay for the isothermal amplification of the named SARS-COV2 regions; (iii) detect amplification products on a lateral flow device. and (ii) perform a pilot field validation of RPA on RNA extracted from nasopharyngeal swabs. Results: Assay validation was done at the National Reference Lab (NRL) at the Rwanda Biomedical Center (RBC) in Rwanda. Results were compared to an established, WHO-approved rRT-PCR laboratory protocol. The assay provides a faster and cheaper alternative to rRT-PCR with 100% sensitivity, 93% specificity, and positive and negative predictive agreements of 100% and 93% respectively. Conclusion: To the best of our knowledge, this is the first in-field and comparative laboratory validation of RPA for COVID-19 disease in low resource settings. Further standardization will be required for deployment of the RPA assay in field settings. Keywords: Recombinase Polymerase Amplification, COVID-19

Current trends in COVID-19 diagnosis and its new variants in physiological fluids: Surface antigens, antibodies, nucleic acids, and RNA sequencing

“…Similarly, the collateral activity of Cas13 was used to increase fluorescence after cutting RNA labeled with a fluorophore quenching reporter [ 146 ]. Gootenberg et al [ 147 ] established the highly sensitive enzymatic reporter unlocking system (SHERLOCK) by modifying the CRISPR-Cas13 method by adding an isothermal amplification step using recombinase polymerase amplification (RPA), which showed sensitivity up to 100% and specificity of ∼93% with a negative predictive value of ∼92.9% when it was used to detect S1, ORF3 and ORF8 regions of the SARS-CoV-2 virus from the nasopharyngeal swabs [ 147 ]. While the T7 RNA polymerase step facilitates the transcription of amplified DNA into RNA [ 143 ].…”

Science's Response to CoVID‐19

“…LAMP [151] is a multiple primer/multi‐homology strategy to perform PCR to generate multi‐concatenated amplicons that can be detected by several simple methods (Figure 3C). This isothermal amplification method has been applied to detection of numerous viruses, including MERS‐CoV [152] . It is noteworthy that other isothermal amplification techniques (some of which will be discussed below) are also in existence and have been used for viral detection, for instance: recombinase polymerase amplification (RPA); [153] rolling cycle amplification (RCA); [154] and nucleic acid sequence‐based amplification (NASBA) [155] .…”

Portable Tools for COVID-19 Point-of-Care Detection: A Review

“…For the RPA assay optimization, TwistAmp® nfo Kit (TANFO02KIT; TwistDX, Maidenhead, UK) was used to undertake the final reactions. The result was reported to have excellent sensitivity of 93% and a specificity of 100% [46]. Nonetheless, the requirement of RPA assay optimization to each SARS-CoV-2 region and the inability to typically distinguish the differences of single base pair in target sequences were the limitations of the recombinase polymerase amplification (RPA) technique [47].…”