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Open AccessArticle

Development and Evaluation of Novel and Highly Sensitive Single-Tube Nested Real-Time RT-PCR Assays for SARS-CoV-2 Detection

1
Department of Microbiology, Queen Mary Hospital, Hong Kong, China
2
Department of Microbiology, The University of Hong Kong, Hong Kong, China
3
State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China
4
Carol Yu Centre for Infection, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
5
Department of Medicine and Geriatrics, Princess Margaret Hospital, Hong Kong, China
6
Department of Medicine, The University of Hong Kong, Hong Kong, China
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2020, 21(16), 5674; https://doi.org/10.3390/ijms21165674
Received: 30 June 2020 / Revised: 28 July 2020 / Accepted: 5 August 2020 / Published: 7 August 2020
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)
Sensitive molecular assays are critical for coronavirus disease 2019 (COVID-19) diagnosis. Here, we designed and evaluated two single-tube nested (STN) real-time RT-PCR assays, targeting SARS-CoV-2 RdRp/Hel and N genes. Both STN assays had a low limit of detection and did not cross react with other human coronaviruses and respiratory viruses. Using 213 initial respiratory specimens from suspected COVID-19 patients, the sensitivity of both the STN COVID-19-RdRp/Hel and the STN COVID-19-N assays was 100% (99/99), while that of the comparator non-nested N assay was 95% (94/99). Among 108 follow-up specimens from confirmed COVID-19 patients who tested negative by the non-nested COVID-19-RdRp/Hel assay, 28 (25.9%) were positive for SARS-CoV-2 by the STN COVID-19-RdRp/Hel or the STN COVID-19-N assay. To evaluate the performance of our novel STN assays in pooled specimens, we created four sample pools, with each pool consisting of one low positive specimen and 49 negative specimens. While the non-nested COVID-19-RdRp/Hel assay was positive in only one of four sample pools (25%), both of the STN assays were positive in two of four samples pools (50%). In conclusion, the STN assays are highly sensitive and specific for SARS-CoV-2 detection. Their boosted sensitivity offers advantages in non-traditional COVID-19 testing algorithms such as saliva screening and pooled sample screening during massive screening. View Full-Text
Keywords: SARS-CoV-2; COVID-19; single-tube nested; RT-PCR; evaluation SARS-CoV-2; COVID-19; single-tube nested; RT-PCR; evaluation
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MDPI and ACS Style

Yip, C.C.-Y.; Sridhar, S.; Leung, K.-H.; Ng, A.C.-K.; Chan, K.-H.; Chan, J.F.-W.; Tsang, O.T.-Y.; Hung, I.F.-N.; Cheng, V.C.-C.; Yuen, K.-Y.; To, K.K.-W. Development and Evaluation of Novel and Highly Sensitive Single-Tube Nested Real-Time RT-PCR Assays for SARS-CoV-2 Detection. Int. J. Mol. Sci. 2020, 21, 5674. https://doi.org/10.3390/ijms21165674

AMA Style

Yip CC-Y, Sridhar S, Leung K-H, Ng AC-K, Chan K-H, Chan JF-W, Tsang OT-Y, Hung IF-N, Cheng VC-C, Yuen K-Y, To KK-W. Development and Evaluation of Novel and Highly Sensitive Single-Tube Nested Real-Time RT-PCR Assays for SARS-CoV-2 Detection. International Journal of Molecular Sciences. 2020; 21(16):5674. https://doi.org/10.3390/ijms21165674

Chicago/Turabian Style

Yip, Cyril C.-Y.; Sridhar, Siddharth; Leung, Kit-Hang; Ng, Anthony C.-K.; Chan, Kwok-Hung; Chan, Jasper F.-W.; Tsang, Owen T.-Y.; Hung, Ivan F.-N.; Cheng, Vincent C.-C.; Yuen, Kwok-Yung; To, Kelvin K.-W. 2020. "Development and Evaluation of Novel and Highly Sensitive Single-Tube Nested Real-Time RT-PCR Assays for SARS-CoV-2 Detection" Int. J. Mol. Sci. 21, no. 16: 5674. https://doi.org/10.3390/ijms21165674

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