tetano
Editor, Senior Moderator
PLoS One
. 2021 Jan 6;16(1):e0245164.
doi: 10.1371/journal.pone.0245164. eCollection 2021.
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)
Yee Ling Lau[SUP] 1 [/SUP], Ilyiana Binti Ismail[SUP] 2 [/SUP], Nur Izati Binti Mustapa[SUP] 2 [/SUP], Meng Yee Lai[SUP] 1 [/SUP], Tuan Suhaila Tuan Soh[SUP] 2 [/SUP], Afifah Haji Hassan[SUP] 2 [/SUP], Kalaiarasu M Peariasamy[SUP] 3 [/SUP], Yee Leng Lee[SUP] 3 [/SUP], Maria Kahar Bador Abdul Kahar[SUP] 4 [/SUP], Jennifer Chong[SUP] 4 [/SUP], Pik Pin Goh[SUP] 5 [/SUP]
Affiliations
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.
. 2021 Jan 6;16(1):e0245164.
doi: 10.1371/journal.pone.0245164. eCollection 2021.
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)
Yee Ling Lau[SUP] 1 [/SUP], Ilyiana Binti Ismail[SUP] 2 [/SUP], Nur Izati Binti Mustapa[SUP] 2 [/SUP], Meng Yee Lai[SUP] 1 [/SUP], Tuan Suhaila Tuan Soh[SUP] 2 [/SUP], Afifah Haji Hassan[SUP] 2 [/SUP], Kalaiarasu M Peariasamy[SUP] 3 [/SUP], Yee Leng Lee[SUP] 3 [/SUP], Maria Kahar Bador Abdul Kahar[SUP] 4 [/SUP], Jennifer Chong[SUP] 4 [/SUP], Pik Pin Goh[SUP] 5 [/SUP]
Affiliations
- PMID: 33406112
- DOI: 10.1371/journal.pone.0245164
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.