Some additional info from my medical research buddy (immunology) re. PCR methods and false positives.

"I posted another message to BEE-L last night re. the primers used in the Shutler, et al. research (2014) the results of which we generally take as our pathogen, pest and disease baseline on the Island of Newfoundland.  It's a fishing trip.  
 
You pointed out to me at lunch that the Shutler et al. research used different DWV primers than Desal, Kumar and Currie in "Occurrence, detection, and quantification of economically important viruses in health and unhealthy honey bee…colonies in Canada" (2016).

I note too that different DWV primers are used by Dolezal, et al. in "Honey bee viruses in wild bees" but these researchers reference primers used by Carrillo-Tripp et al. in "In vivo and in vitro infection dynamics of honey bee viruses" (2016). 

In your work with PCR methodologies, do researchers use different primers?  They must….????  What difference does it make if different primers are used as long as the virus is identified correctly?

- Yes, different labs can use different PCR primers to identify the same gene. There is software that allows one to "fish" a good primer pair from a known gene sequence.  Why do they opt to make their own primers? Sometimes, the size of the amplified gene segment in a gel is too close to another amplified gene segment they follow (they try to detect two genes simultaneously in the same PCR reaction).  There are other reasons, too, dictated by technical issues...

- False positives: It is unlikely that junk material has been amplified if the band size in a gel  is correct (as dictated by the position of the primers within the gene sequence). However, to confirm that there is absolutely no chance that "wrong" material has been amplified, one needs to sequence the amplified segment (by removing the band from the gel, dissolve the DNA and subject it to sequencing). This is not done routinely. Most labs consider a "correct" size band as strong evidence that the correct gene has been amplified."

             ***********************************************
The BEE-L mailing list is powered by L-Soft's renowned
LISTSERV(R) list management software.  For more information, go to:
http://www.lsoft.com/LISTSERV-powered.html