Open AccessEstablishment 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
Author(s) -
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
Publication year - 2022
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.24355
Subject(s) - real time polymerase chain reaction , covid-19 , medicine , detection limit , multiplex , gene , kappa , virology , biology , bioinformatics , chemistry , chromatography , genetics , mathematics , disease , infectious disease (medical specialty) , geometry
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 C t values observed between two tests with r 2 = 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.