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. 2022 May;36(5):e24355.
doi: 10.1002/jcla.24355. Epub 2022 Mar 21.

Establishment of an in-house real-time RT-PCR assay for the detection of severe acute respiratory syndrome coronavirus 2 using the first World Health Organization international standard in a resource-limited country

Linh Tung Nguyen  1 , Phuong Minh Nguyen  1 , Duc Viet Dinh  2 , Hung Ngoc Pham  2 , Lan Anh Thi Bui  3 , Cuong Viet Vo  3 , Ben Huu Nguyen  1 , Hoan Duy Bui  1 , Cuong Xuan Hoang  1 , Nhat Minh Van Ngo  4 , Truong Tien Dang  4 , Anh Ngoc Do  5 , Dung Dinh Vu  6   7 , Linh Thuy Nguyen  6   8 , Mai Ngoc Nguyen  6   8 , Thu Hang Thi Dinh  6 , Son Anh Ho  6 , Luong Van Hoang  4   6 , Su Xuan Hoang  6 , Quyet Do  9
Affiliations

Establishment of an in-house real-time RT-PCR assay for the detection of severe acute respiratory syndrome coronavirus 2 using the first World Health Organization international standard in a resource-limited country

Linh Tung Nguyen et al. J Clin Lab Anal. 2022 May.

Abstract

Background: The COVID-19 pandemic caused by SARS-CoV-2 remains public health burdens and many unresolved issues worldwide. Molecular assays based on real-time RT-PCR are critical for the detection of SARS-CoV-2 in clinical specimens from patients suspected of COVID-19.

Objective: We aimed to establish and validate an in-house real-time RT-PCR for the detection of SARS-CoV-2.

Methodology: Primers and probes sets in our in-house real-time RT-PCR assay were designed in conserved regions of the N and E target genes. Optimized multiplex real-time RT-PCR assay was validated using the first WHO International Standard (NIBSC code: 20/146) and evaluated clinical performance.

Results: The limit of detection validated using the first WHO International Standard was 159 IU/ml for both E and N target genes. The evaluation of clinical performance on 170 clinical samples showed a positive percent agreement of 100% and the negative percent agreement of 99.08% for both target genes. The Kappa value of 0.99 was an excellent agreement, the strong correlation of Ct values observed between two tests with r2 = 0.84 for the E gene and 0.87 for the N gene. Notably, we assessed on 60 paired saliva and nasopharyngeal samples. The overall agreement was 91.66%, and Kappa value of 0.74 showed a high agreement between two types of samples. When using nasopharyngeal swabs as the reference standard, positive percent agreement, and negative percent agreement were 91.83% and 90.90%, respectively.

Conclusion: In the present study, we established and validated an in-house real-time RT-PCR for molecular detection of SARS-CoV-2 in a resource-limited country.

Keywords: SARS-CoV-2; clinical performance; real-time RT-PCR.

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Conflict of interest statement

The authors have no conflicts of interest to disclose.

Figures

FIGURE 1
FIGURE 1
Correlation analysis for the C t values of E gene between LDA and LiliF assay
FIGURE 2
FIGURE 2
Correlation analysis for the C t values of N gene between LDA and LiliF assay
FIGURE 3
FIGURE 3
Phylogenetic tree was constructed using the Spike partial gene sequences (22,798–23,522) obtained in this study, and 18 reference sequences of SARS‐CoV‐2 Wuhan strains retrieved from Genbank and GISAID (MN908947.3, NC_045512.2, EPI_ISL_402123, EPI_ISL_416428, EPI_ISL_455714, and EPI_ISL_455711) and SARS‐CoV‐2 Alpha (EPI ISL 718726 and EPI ISL 1905042), Beta (EPI ISL 1910930 and EPI ISL 1909220), Gamma (EPI ISL 1859008), Kappa (EPI ISL 1360304 and EPI ISL 1372093), Delta (EPI_ISL_3694368, EPI ISL 1544070, and EPI ISL 1519290), Omicron (EPI ISL 8048814 and EPI ISL 6590782) variants using CLUSTAL_W with Kimura's correction
FIGURE 4
FIGURE 4
Alignment of primers (qVN‐F and qVN‐R) and probe (qVN‐Pr) of our LDA assay with sequence data of N gene obtained in this study and reference sequence (NC_045512.2) retrieved from GenBank
See this image and copyright information in PMC

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