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...
Gespeichert in:
Veröffentlicht in: | Journal of virological methods 2020-10, Vol.284, p.113937-113937, Article 113937 |
---|---|
Hauptverfasser: | , , , , , , , , , |
Format: | Artikel |
Sprache: | eng |
Schlagworte: | |
Online-Zugang: | Volltext |
Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
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 |