Capillary electrophoresis of PCR fragments with 5´-labelled primers for testing the SARS-Cov-2

•We designated an alternative to qtPCR for the identification of SARS-Cov-2.•The method was a PCR with 5-end fluorescent primers and capillary electrophoresis.•Two viral fragments amplified in several SARS-Cov-2 positive and negative specimens.•This approach would increase the testing capacity of di...

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Veröffentlicht in:Journal of virological methods 2020-10, Vol.284, p.113937-113937, Article 113937
Hauptverfasser: Gómez, Juan, Melón, Santiago, Boga, José A., Alvarez-Argüelles, Marta E., Rojo-Alba, Susana, Leal-Negredo, Alvaro, Castello-Abietar, Cristian, Alvarez, Victoria, Cuesta-Llavona, Elías, Coto, Eliecer
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Sprache:eng
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Zusammenfassung:•We designated an alternative to qtPCR for the identification of SARS-Cov-2.•The method was a PCR with 5-end fluorescent primers and capillary electrophoresis.•Two viral fragments amplified in several SARS-Cov-2 positive and negative specimens.•This approach would increase the testing capacity of diagnostic labs. Due to the huge demand for SARS-Cov-2 determination,alternatives to the standard qtPCRtestsare potentially useful for increasing the number of samples screened. Our aim was to develop a direct fluorescent PCR capillary-electrophoresis detection of the viral genome. We validated this approach on several SARS-Cov-2 positive and negative samples.We isolated the naso-pharingealRNA from 20 positive and 10 negative samples. The cDNA was synthesised and two fragments of the SARS-Cov-2 were amplified. One of the primers for each pair was 5´-end fluorochrome labelled. The amplifications were subjected to capillary electrophoresis in ABI3130 sequencers to visualize the fluorescent peaks.The two SARS-Cov-2 fragments were successfully amplified in the positive samples, while the negative samples did not render fluorescent peaks. In conclusion, we describe and alternative method to identify the SARS-Cov-2 genome that could be scaled to the analysis of approximately 100 samples in less than 5 h. By combining a standard PCR with capillary electrophoresis our approach would overcome the limits imposed to many labs by the qtPCR and increase the testing capacity.
ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2020.113937