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J Virol Methods ; 284: 113937, 2020 10.
Article in English | MEDLINE | ID: covidwho-638504

ABSTRACT

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.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/virology , Electrophoresis, Capillary/methods , Pneumonia, Viral/virology , Polymerase Chain Reaction/methods , Base Sequence , Betacoronavirus/genetics , COVID-19 , COVID-19 Testing , Coronavirus Infections/diagnosis , DNA Primers/genetics , DNA, Complementary/genetics , Genome, Viral , Humans , Nucleic Acid Amplification Techniques/methods , Pandemics , Pneumonia, Viral/diagnosis , SARS-CoV-2 , Sensitivity and Specificity
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