Quote:
Originally Posted by frankiecd
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LOL some people?
Procedure on how to culture COVID.
"Nasopharyngeal samples were collected with flocked swabs in universal transport medium (Copan Diagnostics, Brescia, Italy). A previously published rRT-PCR protocol for detecting the E gene [3] was adapted for processing on the Panther Fusion Hologic (San Diego, CA, USA) automated molecular diagnostic platform, using its open access functionality [11]. The Ct value obtained in this assay was employed as a measure of relative quantification throughout the study.
For the cell culture, an aliquot (250 μL) of the residual sample was decontaminated using gentamicin and amphotericin B, inoculated into 24-well plates on Vero E6 cells (ATCC CCL-81) and cultured in Medium 199 supplemented with L-glutamine and 10% foetal bovine serum. The plates were incubated in a 5% carbon dioxide atmosphere for 5 days. The development of a cytopathic effect (CPE) was examined daily. SARS-CoV-2 CPE specificity was confirmed by immunofluorescence (shell-vial technique) by using a commercial anti-SARS-CoV-2 N protein (Rockland Immunochemicals, Inc., Limerick, PA, USA) as the primary antibody and a goat anti-rabbit IgG labelled with Alexafluor 488 (Abcam, Cambridge, UK) as the secondary antibody. Upon CPE observation and at the end of the cell culture incubation period, culture supernatants were collected from each well and an rRT-PCR was performed, which was confirmed positive if it was at least 3 Ct lower than the original sample. All cell culture-related procedures were performed at a biosafety level 3 facility."
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898982/
The cell line is kidney I believe
"Vero 76 (ATCC No. CRL-1587)
Isolated from Vero in 1968, it grows to a lower saturation density (cells per unit area) than the original Vero. It is useful for detecting and counting hemorrhagic fever viruses by plaque assays.
Vero E6, also known as Vero C1008 (ATCC No. CRL-1586)
This line is a clone from Vero 76. Vero E6 cells show some contact inhibition, so are suitable for propagating viruses that replicate slowly."
Briefly what one does is take a sample of respiratory secretions and a swab and puts it in pink viral transport media cone tube. One take aliquots of the liquid and antibiotics are added to kill off bacteria and fungi and hopefully all that is left is any viruses. One then takes this treated sample and puts a little into a cell culture which is a monolayer of cells and in this case kidney cells. The tricky part is to select the proper cell lines because not all viruses infect all cells. In this case that cell line was good for COVID and hemorrhagic fever viruses which is something you don't really want to do without a biosafety level 3 setup. Biosafety level 4 is for extraterrestrial samples and very bad bugs. One checks every day with an inverted microscope the bottom of a flat bottom bottle flask for cellular disruption and destruction called the cytopathic effect. The virus was identified with the use of antibodies that react to the virus and a positive will give immunofluorescence. They also did a PCR on the culture to confirm COVID.
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