Development of a reverse transcription recombinase polymerase amplification assay for rapid and direct visual detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

Abstract

Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/μL RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.

Affinity Sensors for the Diagnosis of COVID-19

“…One more isothermal amplification method is based on recombinase polymerase amplification (RPA), which involves two primers that bind a double-stranded template using single-stranded binding proteins and recombinase, followed by extension with DNA polymerase [40]. Recently, reverse transcription RPA (RT-RPA) based assay for SARS-CoV-2 detection was reported characterized by test time less than 20 min and LOD 5 copies/µL [41]. Consider also one of the most recent molecular SARS-CoV-2 detection methods, which is based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based approach.…”

COVID-19 Diagnostic Methods and Detection Techniques

“…The advantages of RPA include: (1) it is the fastest nucleic acid amplification technique, requiring only 15 min for completion; (2) it requires moderate heating to only 37–42 °C, which makes the equipment's temperature control module much simpler and cost effective. A suggestion of references using RPA detection include: (1) the use of exonuclease probes with internally linked quencher (Exo-IQ) to rapidly detect SARS-CoV-2 through low-volume real-time single-tube reverse transcription recombinase polymerase amplification ( Behrmann et al, 2020 ); (2) rapid and direct visual detection of SARS-CoV-2 by a reverse transcription recombinase polymerase amplification assay ( Lau et al, 2021 ); (3) single-copy sensitive, field-deployable, and simultaneous dual-gene detection of SARS-CoV-2 RNA by modified RT-RPA ( Xia and Chen, 2020 ).…”

Current diagnostic approaches to detect two important betacoronaviruses: Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

“…For SARS-CoV-2, RT-RPA has also been widely reported to detect genetic targets of the virus such as the N gene ( Table 5 ) and the LOD of the assay could be 5 RNA copies in 1 μL of a sample with a sensitivity of 98% [119] or 7.8 RNA molecules per reaction with 100 sensitivity as compared to qRT-PCR [120] . Besides, the assay specificity could be as high as 100% [119] , [120] .…”

“…For SARS-CoV-2, RT-RPA has also been widely reported to detect genetic targets of the virus such as the N gene ( Table 5 ) and the LOD of the assay could be 5 RNA copies in 1 μL of a sample with a sensitivity of 98% [119] or 7.8 RNA molecules per reaction with 100 sensitivity as compared to qRT-PCR [120] . Besides, the assay specificity could be as high as 100% [119] , [120] . On the other hand, when other molecular targets like RdRp and ORF 1ab were used in RT-RPA to detect the presence of SARS-CoV-2, the assay sensitivity was lower [121] , [122] and the LOD of the assay could be 10-folds less sensitive than qRT-PCR [122] .…”