I am detecting PRRS virus in lung samples using a diagnostic PRRS virus assay which is very reliable, it targets ORF 7 and amplifies a 80bp product.
With any positive samples I detect, I am then trying to amplify a larger, 780bp fragment of ORF 5 for phylogenetic analysis. I return to the original sample before trying to amplify with ORF 5 primers.
Samples which are positive by ORF 7 diagnostic primers are not then amplified by the phylogenetic ORF 5 primers. Any suggestions as to why these might be happening? Control material from a PRRS +ve field strain is used and is detected and amplified by both ORF 7 and ORF 5 primer sets.
Any positive samples that have been detected using the ORF 7 primer set are weak and range in ct values from 31-36.
Any help would be appreciated.
This may be do to genetic diversity of PRRS virus, please try with different set of primer. Check your primer in NCBI, BLAST analysis to determine to detect different diversity of PRRS virus. Best of luck.
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