qPCR – Test Australian OsHV-1 ORF117 Primers

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).

qPCR Report (PDF): Sam_2017-12-21 15-09-49_CC009827.pdf
qPCR Data File (CFX): Sam_2017-12-21 15-09-49_CC009827.pcrd

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 amplify the French variant, but will amplify Australian and Californian variants.

See labeled amplification plots below.