9.3.1. Primer design

Designing RT-(q)PCR assays for detecting (honey bee) RNA viruses poses some unique challenges. The most critical components of a PCR assay are the two amplification primers. RNA viruses are genetically highly variable while PCR is very sensitive to nucleotide mismatches between primer and target, particularly at the 3’ primer termini where extension occurs (Onodera, 2007). A mismatch at the 3’ terminus of just one of the primers will result in non-amplification. Mismatches further away from the 3’ terminus have increasingly less influence on the success of amplification and generally only the last two 3’ nucleotides are critical for amplification specificity. The 3’ mismatch issue is therefore crucial to the specificity, accuracy, reliability and sensitivity of a PCR-based virus assay. Here we outline how to use this to our benefit, and how to avoid it when needed.