Hello everyone,
I am working on a research project that involves the generation of iPS cells. We are currently looking for a method to determine wether our primary human cells (dermal fibroblasts) are infected with one of various viruses (e.g. HIV, HepA, HepB). Does anyone use a specific method to determine contamination of cells by specific viruses?
Thank you for your help in advance.
If you know what to look for, definitively PCR (sp if you only need a Yes-No answer). If you don't know, there are diagnostic microarrays which cover a whole variety of pathogens.
We use the clinical chemistry department of our hospital to perform these analyses for HIV1/2, HCV and HBV. We send about one million cells in suspension to them and I believe the analysis is based on PCR detection of RNA or DNA, depending on the nature of the virus. Unfortunately, in our lab we do not perform these analyses ourselves and can not provide any more specific protocol.
If you know what to look for, definitively PCR (sp if you only need a Yes-No answer). If you don't know, there are diagnostic microarrays which cover a whole variety of pathogens.
Easy and cheap method: PCR, real-time PCR
Not cheap: ELISA for typical protein (mostly capsid protein), WB
for live virus: TCID50, plaque
note: collect supernatant or cell lysis depending on infectious time and whether the virus is secretory
Thank you all for your answers. I'm planning to do a real-time PCR to analyse the cells for viral infections. Does anyone have any suggestions on how to design the primers for these viruses?
My doctoral dissertation was precisely the discovery of a new syncytium-forming virus of the retroviridae family, sub-family spumavirinae. The first indications that something was wrong with my primary tissue cultures was the persistant formation of unexplained plaques.
Through my experience pcr or reverse transcriptase - pcr is the best method since your sample might contain very low concentration of a virus if it is inside the cell. Faster, less expensive, accurate and direct unless you prefer to pay more for real time pcr.
RT and then PCR should work. If you want to look for all the viruses together then a multiplex PCR approach should bring down the time spent on detecting each of the viruses individually. Here is an article describing a multiplex approach.
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