tetano
Editor, Senior Moderator
Virol J
. 2020 Dec 28;17(1):197.
doi: 10.1186/s12985-020-01467-y.
Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2
Yang Zhang[SUP] 1 [/SUP], Chunyang Dai[SUP] 1 [/SUP], Huiyan Wang[SUP] 1 [/SUP], Yong Gao[SUP] 2 [/SUP], Tuantuan Li[SUP] 2 [/SUP], Yan Fang[SUP] 2 [/SUP], Zuojun Shen[SUP] 1 [/SUP], Lichang Chen[SUP] 2 [/SUP], Zhaowu Chen[SUP] 1 [/SUP], Xuejun Ma[SUP] 3 4 [/SUP], Ming Li[SUP] 5 [/SUP]
Affiliations
Abstract
Background: Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient.
Method: In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients.
Result: The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23-1145.69) for ORF1ab and 528.1 (95% CI: 347.7-1248.7) for N, 401.8 (95% CI: 284.8-938.3) for ORF1ab and 336.8 (95% CI: 244.6-792.5) for N, and 194.74 (95% CI: 139.7-430.9) for ORF1ab and 189.1 (95% CI: 130.9-433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively.
Conclusion: In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.
Keywords: COVID-19; Highly sensitive; OSN-qRT-PCR; SARS-CoV-2; ddPCR; qRT-PCR.
. 2020 Dec 28;17(1):197.
doi: 10.1186/s12985-020-01467-y.
Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2
Yang Zhang[SUP] 1 [/SUP], Chunyang Dai[SUP] 1 [/SUP], Huiyan Wang[SUP] 1 [/SUP], Yong Gao[SUP] 2 [/SUP], Tuantuan Li[SUP] 2 [/SUP], Yan Fang[SUP] 2 [/SUP], Zuojun Shen[SUP] 1 [/SUP], Lichang Chen[SUP] 2 [/SUP], Zhaowu Chen[SUP] 1 [/SUP], Xuejun Ma[SUP] 3 4 [/SUP], Ming Li[SUP] 5 [/SUP]
Affiliations
- PMID: 33371898
- DOI: 10.1186/s12985-020-01467-y
Abstract
Background: Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient.
Method: In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients.
Result: The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23-1145.69) for ORF1ab and 528.1 (95% CI: 347.7-1248.7) for N, 401.8 (95% CI: 284.8-938.3) for ORF1ab and 336.8 (95% CI: 244.6-792.5) for N, and 194.74 (95% CI: 139.7-430.9) for ORF1ab and 189.1 (95% CI: 130.9-433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively.
Conclusion: In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.
Keywords: COVID-19; Highly sensitive; OSN-qRT-PCR; SARS-CoV-2; ddPCR; qRT-PCR.