Hello,
I want to identificate Cryptococcus species with qPCR in tissue samples or extracts. So the aim is to generate a high specificity and sensitivity.
A possible target could be the ribosomal or capsule sequences. This should ensure high specificity. Additionally any advice for primer design?
The next focus would be for high sensitivity to differentiate well between C.neoformans and C.gattii species. I want to work with Taqman probes.
Its my first qPCR project and i am planing the next two weeks before ordering everything.
Appreciate your help!
Greetings David
Dear David,
Try using universal primers for 16srDNA, which their sequences exist on databases and previous publications. Identification of the species should be done by using a software like MEGA and online databases. It seems that your aim is only detection and it makes your work much easier. Don't worry and follow your kits' manuals.
Good luck
Dear Arman,
how do I design a proper sequence for all known strains in cryptococcus gattii with geneious?
Should I download all rRNA sequences first and search then for the 16srDNA because i have no matches when i blast it?
Appreciate your help.
Dear David,
The sequences of rDNA universal Primers is available on database and in the previous related publications. Universal Primers can be used for a wide range of bacteria.
For example 27F and 515R are two frequently used Primers.
Dear Arman,
I cannot find universal Primers for a Taqman Real Time PCR in different databases. But I try to find out more in publications. More genomes appear so that is why I need to find a consensus sequence in a conserved part of the genome.
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