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.
Highlights
The coronavirus disease 2019 (COVID-2019) outbreak was declared as a global health emergency by the World Health Organization (WHO) on 30 January 2020 [1]
Several real-time reverse transcription polymerase chain reaction (RT-qPCR) methods have been developed for the detection of SARS-like coronaviruses and the specific detection of SARS-CoV-2
The optimal detection conditions of SARS-CoV-2 RT-Recombinase polymerase amplification (RPA) assay was explored with a range of temperatures (30, 37 and 45 ̊C) and incubation time (5, 10, 15, 20, 25 and 30 mins)
Summary
The coronavirus disease 2019 (COVID-2019) outbreak was declared as a global health emergency by the World Health Organization (WHO) on 30 January 2020 [1]. Several real-time reverse transcription polymerase chain reaction (RT-qPCR) methods have been developed for the detection of SARS-like coronaviruses and the specific detection of SARS-CoV-2. The time required from transporting the sample to obtaining the results may need 2–3 days [2]. In a public health emergency, this time-consuming and expensive testing method is treated less favourably. Recombinase polymerase amplification (RPA) is a highly specific and sensitive isothermal amplification technique (37–42 ̊C) that can amplify DNA/RNA as low as 1 copy per reaction in < 30 mins. A reverse transcription (RT) RPA assay was developed and optimized for the detection of SARS-CoV-2 in less than 20 mins. To enable detection by the naked eye, we used SYBR Green I for the colorimetric detection of the amplification reaction and direct visualization via lateral flow (LF) strip
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have