tetano
Editor, Senior Moderator
Sci Rep
. 2021 Nov 4;11(1):21658.
doi: 10.1038/s41598-021-00900-8.
High sensitivity-low cost detection of SARS-CoV-2 by two steps end point RT-PCR with agarose gel electrophoresis visualization
Solange Figueroa[SUP] #[/SUP][SUP] 1 [/SUP], Byron Freire-Paspuel[SUP] #[/SUP][SUP] 1 [/SUP], Patricio Vega-Mariño[SUP] 2 [/SUP], Alberto Velez[SUP] 2 [/SUP], Marilyn Cruz[SUP] 2 [/SUP], Washington B Cardenas[SUP] 3 [/SUP], Miguel Angel Garcia-Bereguiain[SUP] 4 [/SUP]
Affiliations
Abstract
More than one year since Coronavirus disease 2019 (COVID-19) pandemic outbreak, the gold standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is still the RT-qPCR. This is a limitation to increase testing capacities, particularly at developing countries, as expensive reagents and equipment are required. We developed a two steps end point RT-PCR reaction with SARS-CoV-2 Nucleocapsid (N) gene and Ribonuclease P (RNase P) specific primers where viral amplicons were verified by agarose gel electrophoresis. We carried out a clinical performance and analytical sensitivity evaluation for this two-steps end point RT-PCR method with 242 nasopharyngeal samples using the CDC RT-qPCR protocol as a gold standard technique. With a specificity of 95.8%, a sensitivity of 95.1%, and a limit of detection of 20 viral RNA copies/uL, this two steps end point RT-PCR assay is an affordable and reliable method for SARS-CoV-2 detection. This protocol would allow to extend COVID-19 diagnosis to basic molecular biology laboratories with a potential positive impact in surveillance programs at developing countries.
. 2021 Nov 4;11(1):21658.
doi: 10.1038/s41598-021-00900-8.
High sensitivity-low cost detection of SARS-CoV-2 by two steps end point RT-PCR with agarose gel electrophoresis visualization
Solange Figueroa[SUP] #[/SUP][SUP] 1 [/SUP], Byron Freire-Paspuel[SUP] #[/SUP][SUP] 1 [/SUP], Patricio Vega-Mariño[SUP] 2 [/SUP], Alberto Velez[SUP] 2 [/SUP], Marilyn Cruz[SUP] 2 [/SUP], Washington B Cardenas[SUP] 3 [/SUP], Miguel Angel Garcia-Bereguiain[SUP] 4 [/SUP]
Affiliations
- PMID: 34737323
- DOI: 10.1038/s41598-021-00900-8
Abstract
More than one year since Coronavirus disease 2019 (COVID-19) pandemic outbreak, the gold standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is still the RT-qPCR. This is a limitation to increase testing capacities, particularly at developing countries, as expensive reagents and equipment are required. We developed a two steps end point RT-PCR reaction with SARS-CoV-2 Nucleocapsid (N) gene and Ribonuclease P (RNase P) specific primers where viral amplicons were verified by agarose gel electrophoresis. We carried out a clinical performance and analytical sensitivity evaluation for this two-steps end point RT-PCR method with 242 nasopharyngeal samples using the CDC RT-qPCR protocol as a gold standard technique. With a specificity of 95.8%, a sensitivity of 95.1%, and a limit of detection of 20 viral RNA copies/uL, this two steps end point RT-PCR assay is an affordable and reliable method for SARS-CoV-2 detection. This protocol would allow to extend COVID-19 diagnosis to basic molecular biology laboratories with a potential positive impact in surveillance programs at developing countries.