Virus dilutions and RT-PCR

Tom Unruh unruh at yarl.gov
Sun Feb 25 03:26:16 EST 1996


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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