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

Cyril Chik-Yan Yip; 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

doi:10.3390/ijms25063552

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

Int J Mol Sci. 2024 Mar 21;25(6):3552. doi: 10.3390/ijms25063552.

Authors

Cyril Chik-Yan Yip¹, Jane Hau-Ching Poon², Kit-Hang Leung², Wan-Mui Chan², Jonathan Daniel Ip², Allen Wing-Ho Chu², Vincent Chi-Chung Cheng¹, Kwok-Yung Yuen², Kelvin Kai-Wang To²

Affiliations

¹ Department of Microbiology, Queen Mary Hospital, Hong Kong SAR, China.
² State Key Laboratory for Emerging Infectious Diseases, Carol Yu Centre for Infection, Department of Microbiology, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China.

Abstract

The emergence of SARS-CoV-2 mutations poses 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, the 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 a 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 the ancestral strain and Alpha, Beta, Gamma, Delta, Mu, Eta, Kappa, Theta and Omicron lineages. Using a LightMix E-gene RT-PCR assay as the reference method, the diagnostic sensitivity and specificity of the COVID-19-nsp10 assay were found to be 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 a SARS-CoV-2 RT-PCR assay.

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

MeSH terms

COVID-19 Testing
COVID-19* / diagnosis
Humans
Reverse Transcriptase Polymerase Chain Reaction
SARS-CoV-2* / genetics
Sensitivity and Specificity

Grants and funding

This research was funded by the Health and Medical Research Fund (HMRF) Commissioned Research on Control of Infectious Disease (Phase IV), CID-HKU1-2. The funding source had no role in the design and conduct of this study; in the collection, analysis and interpretation of data; or in the preparation, review or approval of the manuscript.