tetano
Editor, Senior Moderator
J Clin Lab Anal
. 2022 Mar 21;e24355.
doi: 10.1002/jcla.24355. Online ahead of print.
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[SUP] 1 [/SUP], Phuong Minh Nguyen[SUP] 1 [/SUP], Duc Viet Dinh[SUP] 2 [/SUP], Hung Ngoc Pham[SUP] 2 [/SUP], Lan Anh Thi Bui[SUP] 3 [/SUP], Cuong Viet Vo[SUP] 3 [/SUP], Ben Huu Nguyen[SUP] 1 [/SUP], Hoan Duy Bui[SUP] 1 [/SUP], Cuong Xuan Hoang[SUP] 1 [/SUP], Nhat Minh Van Ngo[SUP] 4 [/SUP], Truong Tien Dang[SUP] 4 [/SUP], Anh Ngoc Do[SUP] 5 [/SUP], Dung Dinh Vu[SUP] 6 7 [/SUP], Linh Thuy Nguyen[SUP] 6 8 [/SUP], Mai Ngoc Nguyen[SUP] 6 8 [/SUP], Thu Hang Thi Dinh[SUP] 6 [/SUP], Son Anh Ho[SUP] 6 [/SUP], Luong Van Hoang[SUP] 4 6 [/SUP], Su Xuan Hoang[SUP] 6 [/SUP], Quyet Do[SUP] 9 [/SUP]
Affiliations
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 C[SUB]t[/SUB] values observed between two tests with r[SUP]2[/SUP] = 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.
. 2022 Mar 21;e24355.
doi: 10.1002/jcla.24355. Online ahead of print.
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[SUP] 1 [/SUP], Phuong Minh Nguyen[SUP] 1 [/SUP], Duc Viet Dinh[SUP] 2 [/SUP], Hung Ngoc Pham[SUP] 2 [/SUP], Lan Anh Thi Bui[SUP] 3 [/SUP], Cuong Viet Vo[SUP] 3 [/SUP], Ben Huu Nguyen[SUP] 1 [/SUP], Hoan Duy Bui[SUP] 1 [/SUP], Cuong Xuan Hoang[SUP] 1 [/SUP], Nhat Minh Van Ngo[SUP] 4 [/SUP], Truong Tien Dang[SUP] 4 [/SUP], Anh Ngoc Do[SUP] 5 [/SUP], Dung Dinh Vu[SUP] 6 7 [/SUP], Linh Thuy Nguyen[SUP] 6 8 [/SUP], Mai Ngoc Nguyen[SUP] 6 8 [/SUP], Thu Hang Thi Dinh[SUP] 6 [/SUP], Son Anh Ho[SUP] 6 [/SUP], Luong Van Hoang[SUP] 4 6 [/SUP], Su Xuan Hoang[SUP] 6 [/SUP], Quyet Do[SUP] 9 [/SUP]
Affiliations
- PMID: 35312118
- DOI: 10.1002/jcla.24355
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 C[SUB]t[/SUB] values observed between two tests with r[SUP]2[/SUP] = 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.