Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Development and Evaluation of an In-House Real-Time Rt-Pcr Targeting nsp10 Gene for SARS-CoV-2 Detection

Cyril Chik-Yan Yip
ORCID logo ,
Jane Hau-Ching Poon
,
Kit-Hang Leung
,
Wan-Mui Chan
,
Jonathan Daniel Ip
,
Allen Wing-Ho Chu
,
Vincent Chi-Chung Cheng
,
Kwok-Yung Yuen
,
Kelvin Kai-Wang To
* ORCID logo
Version 1 : Received: 19 February 2024 / Approved: 20 February 2024 / Online: 20 February 2024 (06:07:31 CET)

A peer-reviewed article of this Preprint also exists.

Yip, C.C.-Y.; Poon, J.H.-C.; Leung, K.-H.; Chan, W.-M.; Ip, J.D.; Chu, A.W.-H.; Cheng, V.C.-C.; Yuen, K.-Y.; To, K.K.-W. Development and Evaluation of an In-House Real-Time RT-PCR Targeting nsp10 Gene for SARS-CoV-2 Detection. Int. J. Mol. Sci. 2024, 25, 3552. Yip, C.C.-Y.; Poon, J.H.-C.; Leung, K.-H.; Chan, W.-M.; Ip, J.D.; Chu, A.W.-H.; Cheng, V.C.-C.; Yuen, K.-Y.; To, K.K.-W. Development and Evaluation of an In-House Real-Time RT-PCR Targeting nsp10 Gene for SARS-CoV-2 Detection. Int. J. Mol. Sci. 2024, 25, 3552.

Abstract

The emergence of SARS-CoV-2 mutations pose significant challenges to diagnostic tests as these mutations can reduce the sensitivity of commonly used RT-PCR assays. Therefore, there is a need to design diagnostic assays with multiple targets to enhance sensitivity. In this study, we identified a novel diagnostic target, nsp10 gene, using nanopore sequencing. Firstly, we determined the analytical sensitivity and specificity of our COVID-19-nsp10 assay. The COVID-19-nsp10 assay had the limit of detection of 74 copies/mL (95% confidence interval: 48-299 copies/mL) and did not show cross-reactivity with other respiratory viruses. Next, we determined the diagnostic performance of the COVID-19-nsp10 assay using 261 respiratory specimens, including 147 SARS-CoV-2-positive specimens belonging to ancestral strain and Alpha, Beta, Gamma, Delta, Mu, Eta, Kappa, Theta and Omicron lineages. Using the LightMix E-gene RT-PCR assay as the reference method, the diagnostic sensitivity and specificity of the COVID-19-nsp10 assay were 100%. The median Cp values for the LightMix E-gene RT-PCR and our COVID-19-nsp10 RT-PCR were 22.48 (range: 12.95-36.60) and 25.94 (range 16.37-36.87), respectively. The Cp values of the COVID-19-nsp10 RT-PCR assay correlated well with those of the LightMix E-gene RT-PCR assay (Spearman’s ρ = 0.968; P<0.0001). In conclusion, nsp10 is a suitable target for SARS-CoV-2 RT-PCR assay.

Keywords

SARS-CoV-2; COVID-19; nsp10; real-time RT-PCR; diagnostic

Subject

Biology and Life Sciences, Virology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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