Using primers I previously designed, I tested them out for functionality (using the clone #1 plasmid prep DNA I made previously) and specificity (using the Australian, California, & French variants recently received)
Created a working 1:100 dilution of ALL DNA tested here.
All samples were run in duplicate.
Master mix calcs are here (Google Sheet): 20171221 – qPCR Austrailian OsHV-1 ORF117 Primer Test
Cycling params, plate layout, etc. can be viewed in the qPCR Report (see Results below).
Firstly, the primers work and generate a single melt curve peak (see melt curve plot below); so that proves functionality.
Results are interesting.
Australian samples (plasmid and DNA) amplify.
French samples (M1 & M2) do not amplify.
California samples: 3 of 4 samples amplify.
It’s possible that the California sample that did not amplify is due to too little DNA present in the 1:100 dilution I used (or, possibly no DNA is present at all). I have not quantified the DNA in these samples – went off assumption that the samples had previously been confirmed to have DNA in them by the source laboratories.
Regardless, the primers used here will
See labeled amplification plots below.