Virus dilutions and RT-PCR

Anton Andonov aandonov at HPB.HWC.CA
Sun Feb 25 11:44:19 EST 1996


Why not try diluting the extracted viral nucleic acid.It's semiquantative
but at least you will get rid of virus aggregation.
A.Andonov
Lab Centre for Disease Control
Ottawa


On Sun, 25 Feb 1996, Tom Unruh wrote:

> 
> We have been using RT-PCR as a diagnostic for a luteovirus transmitted by an 
> insect.  When we create dilution series with purified virus to estimate the 
> sensitivity of our technique we encounter tremendous variation.  Specifically, 
> in one dilution series replicate we may get detectable amplification into the 
> attogram range of virus but only in the femtogram range in the next.  We 
> suspect that the virus forms large aggregations which prevents accurate 
> pipetting of small volumes (<10ul) but we are unaware of any studies that have 
> made similar observations.  Electron microscopy of many viruses show them to 
> be clumped.  Can you suggest some citations refering to this problem using 
> RT-PCR and can you offer suggestions to improve repeatability of our 
> sensitivity estimates.
> 
> 



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