I have blood samples, and I want to detect a specific virus—whether it is present or not in these samples. I have extracted RNA and converted it to cDNA. Can Real-time PCR be used to detect the virus? I have a known sample for the virus as a refrence sample.
I think that, if you have cDNA from the sample, the fastest and pretty reliable method to detect virus is real time qPCR for a gene/transcript. If you do so, remember to check the specificity of the oligos/primers (you can verify for undesired homologies https://blast.ncbi.nlm.nih.gov/Blast.cgi @BLAST @NCBI); an amplicon of 80-90 bases of distance primer1-primer2 is enough.
If you don't have acces to a qPCR machine, you can also perform a classic PCR in a thermocycle and then an agarose gel (with Et-bromide or SyBR) to see the band with UV transilluminator. In that case I strongly recommend high number of cycles (40-45) and also that the amplicon of interest has more than 250 nucleotides so you can see a heavier and more clear band.
Last but not least, you can send your cDNA samples to sequencing, indicating the primers you want to be used and your region of interest. This is a very robust and specific method. I carely recommend to perform a previous PCR with your primers of interest and then send the PCR product to sequencing (I don't know if your institution has sequencing facility or if you have to send it to a company).
Also remember to treat samples with DNAse I when extracting blood for a good yeald, if not you'll find contamination by DNA.
By the way, what type of virus are you working with? And what gene/sequence are you planning to check for the virus identification?
I hope it helps you!
Francesc
Dear Dr Saif
You may use some simple assays at the initial phase like Immunofluorescence assay (IFA) using polyclonal immune sera (PCI) against the interested virus, are even if multiple samples needs to be screened you can optimise ELISA based assay using the same PCI for handling large sample size.Both can be used as screening assays. If you get a signal then you can confirm with genome based RTPCR assays. Best of luck
Hi, Saif. If you know which virus is, you could use specific primers to amplify the gene, either by finding them in the literature or by designing new ones.
This would be useful for Real time PCR as you propose or even for conventional PCR with an electrophoresis gel, running your positive control sample and your problem sample in both cases. In the first case, if the curve is amplified only for the positive control, the problem sample does not have the virus, but if both are amplified the virus is present in your problem sample. In the same way, if you run the electrophoresis, and there is only a band in the control sample you do not have the virus in the problem sample, if you get the same band in both samples you do have the virus.
If you want to quantify, you could clone a specific gene (the same one as the primers), make a standard curve and extrapolate to make an absolute quantification of the genomic copies of the virus.
I hope this could help you. Happy Holidays.
Hii
Realtime PCR will be the reliable appraoch, if the virus is known.
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