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

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

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 <i>N</i> genes. Both STN assays had a low limit of detection and did not cros...

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Bibliographic Details
Main Authors: Cyril Chik-Yan Yip, Siddharth Sridhar, Kit-Hang Leung, Anthony Chin-Ki Ng, Kwok-Hung Chan, Jasper Fuk-Woo Chan, Owen Tak-Yin Tsang, Ivan Fan-Ngai Hung, Vincent Chi-Chung Cheng, Kwok-Yung Yuen, Kelvin Kai-Wang To
Format: Article
Language:English
Published: MDPI AG 2020-08-01
Series:International Journal of Molecular Sciences
Subjects:
SARS-CoV-2
COVID-19
single-tube nested
RT-PCR
evaluation
Online Access:https://www.mdpi.com/1422-0067/21/16/5674
Description
Summary: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 <i>N</i> 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 <i>N</i> 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.
ISSN:1661-6596
1422-0067

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