tetano
Editor, Senior Moderator
Rev Soc Bras Med Trop
. 2021 Dec 17;54:e03192020.
doi: 10.1590/0037-8682-0319-2020. eCollection 2021.
RT-qPCR half-reaction optimization for the detection of SARS-CoV-2
Priscila Lamb Wink[SUP] 1 2 3 [/SUP], Fabiana Volpato[SUP] 1 2 4 [/SUP], Daiana de Lima-Morales[SUP] 1 2 [/SUP], Rodrigo Minuto Paiva[SUP] 2 [/SUP], Julia Biz Willig[SUP] 2 5 [/SUP], Hugo Bock[SUP] 2 [/SUP], Fernanda de Paris[SUP] 2 [/SUP], Afonso Luís Barth[SUP] 1 2 3 4 [/SUP]
Affiliations
Abstract
Introduction: The main laboratory test for the diagnosis of coronavirus disease 2019 (COVID-19) is the reverse transcription real-time polymerase chain reaction (RT-qPCR). However, RT-qPCR is expensive because of the number of tests required. This study aimed to evaluate an alternative to the RT-qPCR approach for the detection of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is half of the total volume currently recommended by the US Centers for Disease Control and Prevention.
Methods: The analytical limit of detection (LoD) and the reaction efficiency using half volumes of the RT-qPCR assay were evaluated for the N1 and N2 regions using a synthetic control RNA. A panel of 76 SARS-CoV-2-positive and 26 SARS-CoV-2-negative clinical samples was evaluated to establish clinical sensitivity and specificity.
Results: The RT-qPCR assay efficiency was 105% for the half and standard reactions considering the N2 target and 84% (standard) and 101% (half) for N1. The RT-qPCR half-reaction LoD for N1 and N2 were 20 and 80 copies/µL, respectively. The clinical sensitivity and specificity were 100%. The half reaction presented a decrease of up to 5.5 cycle thresholds compared with standard RT-qPCR.
Conclusions: The use of the RT-qPCR half-reaction proved feasible and economic for the detection of SARS-CoV-2 RNA.
. 2021 Dec 17;54:e03192020.
doi: 10.1590/0037-8682-0319-2020. eCollection 2021.
RT-qPCR half-reaction optimization for the detection of SARS-CoV-2
Priscila Lamb Wink[SUP] 1 2 3 [/SUP], Fabiana Volpato[SUP] 1 2 4 [/SUP], Daiana de Lima-Morales[SUP] 1 2 [/SUP], Rodrigo Minuto Paiva[SUP] 2 [/SUP], Julia Biz Willig[SUP] 2 5 [/SUP], Hugo Bock[SUP] 2 [/SUP], Fernanda de Paris[SUP] 2 [/SUP], Afonso Luís Barth[SUP] 1 2 3 4 [/SUP]
Affiliations
- PMID: 34932760
- DOI: 10.1590/0037-8682-0319-2020
Abstract
Introduction: The main laboratory test for the diagnosis of coronavirus disease 2019 (COVID-19) is the reverse transcription real-time polymerase chain reaction (RT-qPCR). However, RT-qPCR is expensive because of the number of tests required. This study aimed to evaluate an alternative to the RT-qPCR approach for the detection of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is half of the total volume currently recommended by the US Centers for Disease Control and Prevention.
Methods: The analytical limit of detection (LoD) and the reaction efficiency using half volumes of the RT-qPCR assay were evaluated for the N1 and N2 regions using a synthetic control RNA. A panel of 76 SARS-CoV-2-positive and 26 SARS-CoV-2-negative clinical samples was evaluated to establish clinical sensitivity and specificity.
Results: The RT-qPCR assay efficiency was 105% for the half and standard reactions considering the N2 target and 84% (standard) and 101% (half) for N1. The RT-qPCR half-reaction LoD for N1 and N2 were 20 and 80 copies/µL, respectively. The clinical sensitivity and specificity were 100%. The half reaction presented a decrease of up to 5.5 cycle thresholds compared with standard RT-qPCR.
Conclusions: The use of the RT-qPCR half-reaction proved feasible and economic for the detection of SARS-CoV-2 RNA.