tetano
Editor, Senior Moderator
J Clin Virol
. 2022 Jun 8;153:105216.
doi: 10.1016/j.jcv.2022.105216. Online ahead of print.
A reverse-transcription droplet digital PCR assay to detect and quantify SARS-CoV-2 RNA in upper respiratory tract specimens
Dragana Milosevic[SUP] 1 [/SUP], Ann M Moyer[SUP] 1 [/SUP], Ramanath Majumdar[SUP] 1 [/SUP], Benjamin R Kipp[SUP] 1 [/SUP], Joseph D Yao[SUP] 2 [/SUP]
Affiliations
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense, single-stranded RNA virus that causes coronavirus disease 2019 (COVID-19). Symptoms are variable and range from asymptomatic or mild to severe (i.e., pneumonia) in both healthy and immunocompromised patients. We developed a reverse-transcription droplet digital PCR (RT-ddPCR) assay for quantification of SARS-CoV-2 RNA in clinical nasopharyngeal and oropharyngeal swab specimens and evaluated the assay, including reproducibility, agreement of results, analytical measurement range, linearity, analytical sensitivity, and analytical specificity. This quantitative assay had a LoD of 218 copies/mL of viral transport media, with a linear quantification range from 500 to 5,000,000 copies/mL (R[SUP]2[/SUP] of 0.9817 and 0.9853 for N1 and N2 targets, respectively). Qualitative agreement of categorical results was 90.5% (57/63) between the reference and RT-ddPCR assays. Quantitative agreement between the two assays showed correlation, with R[SUP]2[/SUP] of 0.9726 and 0.9713 for N1 and N2 targets, respectively. No cross-reactivity with common coronavirus strains was detected. This SARS-CoV-2 quantitative RT-ddPCR assay may be a useful tool for a variety of applications including identification of patients with low viral load and serial monitoring of viral load in respiratory tracts specimens of patients for evaluation of the efficacy of therapy for COVID-19.
Keywords: Droplet digital PCR; RNA quantification; SARS-CoV-2.
. 2022 Jun 8;153:105216.
doi: 10.1016/j.jcv.2022.105216. Online ahead of print.
A reverse-transcription droplet digital PCR assay to detect and quantify SARS-CoV-2 RNA in upper respiratory tract specimens
Dragana Milosevic[SUP] 1 [/SUP], Ann M Moyer[SUP] 1 [/SUP], Ramanath Majumdar[SUP] 1 [/SUP], Benjamin R Kipp[SUP] 1 [/SUP], Joseph D Yao[SUP] 2 [/SUP]
Affiliations
- PMID: 35714461
- DOI: 10.1016/j.jcv.2022.105216
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense, single-stranded RNA virus that causes coronavirus disease 2019 (COVID-19). Symptoms are variable and range from asymptomatic or mild to severe (i.e., pneumonia) in both healthy and immunocompromised patients. We developed a reverse-transcription droplet digital PCR (RT-ddPCR) assay for quantification of SARS-CoV-2 RNA in clinical nasopharyngeal and oropharyngeal swab specimens and evaluated the assay, including reproducibility, agreement of results, analytical measurement range, linearity, analytical sensitivity, and analytical specificity. This quantitative assay had a LoD of 218 copies/mL of viral transport media, with a linear quantification range from 500 to 5,000,000 copies/mL (R[SUP]2[/SUP] of 0.9817 and 0.9853 for N1 and N2 targets, respectively). Qualitative agreement of categorical results was 90.5% (57/63) between the reference and RT-ddPCR assays. Quantitative agreement between the two assays showed correlation, with R[SUP]2[/SUP] of 0.9726 and 0.9713 for N1 and N2 targets, respectively. No cross-reactivity with common coronavirus strains was detected. This SARS-CoV-2 quantitative RT-ddPCR assay may be a useful tool for a variety of applications including identification of patients with low viral load and serial monitoring of viral load in respiratory tracts specimens of patients for evaluation of the efficacy of therapy for COVID-19.
Keywords: Droplet digital PCR; RNA quantification; SARS-CoV-2.