tetano
Editor, Senior Moderator
J Virol Methods
. 2020 Oct 29;114007.
doi: 10.1016/j.jviromet.2020.114007. Online ahead of print.
End-point RT-PCR: a potential alternative for diagnosing coronavirus disease 2019 (COVID-19)
Jos? Valter Joaquim Silva J?nior[SUP] 1 [/SUP], Ingryd Merchioratto[SUP] 2 [/SUP], Pablo Sebastian Britto de Oliveira[SUP] 2 [/SUP], Tha?sa Regina Rocha Lopes[SUP] 3 [/SUP], Patr?cia Chaves Brites[SUP] 4 [/SUP], Elehu Moura de Oliveira[SUP] 4 [/SUP], Rudi Weiblen[SUP] 2 [/SUP], Eduardo Furtado Flores[SUP] 5 [/SUP]
Affiliations
Abstract
Real-time reverse transcription-polymerase chain reaction (RT-qPCR) is considered the "gold standard" for the direct diagnosis of SARS-CoV-2 infections. However, routine diagnosis by RT-qPCR is a limitation for many laboratories, mainly due to the infrastructure and/or disproportionate relationship between demand and supply of inputs. In this context, and to increase the diagnostic coverage of SARS-CoV-2 infections, we describe an alternative, sensitive and specific one-step end-point RT-PCR for the detection of the SARS-CoV-2 E gene. The performance of the RT-PCR was evaluated in 43 clinical samples, of which 10 and 33 were previously identified as negative and positive, respectively, by RT-qPCR. Among the positive samples, 15 and 18 were from asymptomatic and symptomatic individuals, respectively. Here, 32/33 of the positive samples in the RT-qPCR, including from asymptomatic individuals, were found positive in the RT-PCR (Ct 15.94 to 34.92). The analytical sensitivity of the assay was about 7.15-9 copies of vRNA/μL, and nonspecific amplifications were not observed in SARS-CoV-2 negative samples. Importantly, the RT-PCR reactions were performed in a 10 μL final volume. Finally, considering specificity, analytical sensitivity and cost reduction, we believe that the RT-PCR platform described here may be a viable option for the diagnostic of SARS-CoV-2 infections in laboratories in which RT-qPCR is not available and/or not possible.
Keywords: COVID-19; E gene; Molecular diagnosis; RT-PCR; SARS-CoV-2.
. 2020 Oct 29;114007.
doi: 10.1016/j.jviromet.2020.114007. Online ahead of print.
End-point RT-PCR: a potential alternative for diagnosing coronavirus disease 2019 (COVID-19)
Jos? Valter Joaquim Silva J?nior[SUP] 1 [/SUP], Ingryd Merchioratto[SUP] 2 [/SUP], Pablo Sebastian Britto de Oliveira[SUP] 2 [/SUP], Tha?sa Regina Rocha Lopes[SUP] 3 [/SUP], Patr?cia Chaves Brites[SUP] 4 [/SUP], Elehu Moura de Oliveira[SUP] 4 [/SUP], Rudi Weiblen[SUP] 2 [/SUP], Eduardo Furtado Flores[SUP] 5 [/SUP]
Affiliations
- PMID: 33130151
- DOI: 10.1016/j.jviromet.2020.114007
Abstract
Real-time reverse transcription-polymerase chain reaction (RT-qPCR) is considered the "gold standard" for the direct diagnosis of SARS-CoV-2 infections. However, routine diagnosis by RT-qPCR is a limitation for many laboratories, mainly due to the infrastructure and/or disproportionate relationship between demand and supply of inputs. In this context, and to increase the diagnostic coverage of SARS-CoV-2 infections, we describe an alternative, sensitive and specific one-step end-point RT-PCR for the detection of the SARS-CoV-2 E gene. The performance of the RT-PCR was evaluated in 43 clinical samples, of which 10 and 33 were previously identified as negative and positive, respectively, by RT-qPCR. Among the positive samples, 15 and 18 were from asymptomatic and symptomatic individuals, respectively. Here, 32/33 of the positive samples in the RT-qPCR, including from asymptomatic individuals, were found positive in the RT-PCR (Ct 15.94 to 34.92). The analytical sensitivity of the assay was about 7.15-9 copies of vRNA/μL, and nonspecific amplifications were not observed in SARS-CoV-2 negative samples. Importantly, the RT-PCR reactions were performed in a 10 μL final volume. Finally, considering specificity, analytical sensitivity and cost reduction, we believe that the RT-PCR platform described here may be a viable option for the diagnostic of SARS-CoV-2 infections in laboratories in which RT-qPCR is not available and/or not possible.
Keywords: COVID-19; E gene; Molecular diagnosis; RT-PCR; SARS-CoV-2.