PrimerHunter is a tool for selecting highly sensitive and specific primers for virus subtyping. It takes as input sets of both target and non-target sequences and ensures that selected primers amplify all target sequences and none of the non-target sequences by relying on accurate melting temperature computations based on the nearest-neighbor model of Santalucia and the fractional programming algorithm of Leber et al.
Results on Haemagglutinin (HA) and Neuraminidase (NA) avian influenza sequences from the NCBI flu database show that PrimerHunter has a high design success rate, being able to identify specific primer pairs for each subtype represented in the database. Validation experiments confirm that selected primers work well in practice under the computationally predicted range of PCR conditions
PrimerHunter source code
Primers for Avian Influenza Subtypes
Primer selection was performed on all complete different Avian influenza HA and NA sequences from North America available in the NCBI flu database using the following parameters:
- Primer length between 20 and 25
- Amplicon length between 75 and 200
- GC content between 25% and 75%
- Maximum mononucleotide repeat of 5
- 3′-end match mask M=11
- No required 3′ GC Clamp
- Primer concentration of 0.8uM
- Salt concentration of 50mM
- Tmintarget =Tmaxnontarget=40°C
The output files with primers for each subtype can be browsed below.
Acknowledgment and Disclaimer
This material is based upon work supported in part by the National Science Foundation under Grants No. IIS-0546457 and DBI-0543365. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.