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).

Author(s):

Lau, Yee Ling; Ismail, Ilyiana Binti; Mustapa, Nur Izati Binti; Lai, Meng Yee; Tuan Soh, Tuan Suhaila; Haji Hassan, Afifah; Peariasamy, Kalaiarasu M; Lee, Yee Leng; Abdul Kahar, Maria Kahar Bador; Chong, Jennifer; Goh, Pik Pin;

Journal:

PLoS One

Year:

2021

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/渭L 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.