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

• Published in: Journal of clinical laboratory analysis Vol. 36; no. 5; pp. e24355 - n/a
• Main Authors: Nguyen, Linh Tung, Nguyen, Phuong Minh, Dinh, Duc Viet, Pham, Hung Ngoc, Bui, Lan Anh Thi, Vo, Cuong Viet, Nguyen, Ben Huu, Bui, Hoan Duy, Hoang, Cuong Xuan, Ngo, Nhat Minh Van, Dang, Truong Tien, Do, Anh Ngoc, Vu, Dung Dinh, Nguyen, Linh Thuy, Nguyen, Mai Ngoc, Dinh, Thu Hang Thi, Ho, Son Anh, Hoang, Luong Van, Hoang, Su Xuan, Do, Quyet
• Format: Journal Article
• Language: English
• Published: United States John Wiley & Sons, Inc 01.05.2022; John Wiley and Sons Inc
• Subjects: Agreements; Archives & records; Brief Report; clinical performance; Coronaviruses; COVID-19; COVID-19 vaccines; Design; Disease transmission; E protein; Genes; Infections; Medical laboratories; N gene; Phylogenetics; Public health; real‐time RT‐PCR; Reproducibility; RNA polymerase; Saliva; SARS‐CoV‐2; Severe acute respiratory syndrome coronavirus 2; Thermal cycling; United States > US; Vietnam; real-time RT-PCR; SARS-CoV-2; clinical performance
• ISSN: 0887-8013; 1098-2825
• DOI: 10.1002/jcla.24355

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.