Mycoplasma detection - (Nov/02/2010 )
I need to set up a mycoplasma detection test in my lab, Does anyone know a direct culture method protocol for mycoplasma detection?
Thank you very much
DAPI staining is very popular. you need to fix cells with Paraformaldehyde 4% and then stain with DAPI for 1-5 minute (1-5ug/ml). view under fluorescent microscope.
or you can alternatively go for multiplex PCR. Many commercial kits are available.
There is a VenorGeM Mycoplasma Detection Kit from Sigma that worked very well for me. All you need to do is to culture your cells for a couple of days without antibiotics, and harvest 100uL of the media, heat up to 95C for 5 min and spin down, your template is ready. Then you do a PCR using the kit compounds which also comes with a positive control. You may need to purchase the taq polymerase they recommend though, it worked well with that I don't know about using another enzyme. You see a higher and lower band in the agarose gel, high band meaning positive, they have the details in the product sheet.