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- W2016356113 abstract "Background: Due to the high range of viral diversity no universal, highly conserved primer set exists that is able to amplify all sequenced viral targets. We have performed a computational study that predicted that approximately 3,700 18-mers would be necessary to produce amplicons across the sequenced viral database. However, by decreasing the length of the primer from the traditional 18-mer to a 10-mer the number of primers necessary significantly decreases to approximately 1,000. Shortmers show promise for acting as universal primers that can discriminate both DNA and RNA viruses at the serotype level. As a demonstration of the specificity of shortmers for viral identification we designed 10 and 11- mers that were capable of amplifying three serotypes of Blue Tongue Virus in traditional Reverse Transcription PCR (RT-PCR). Methods: An in-house program, the Multiplex Primer Prediction (MPP) algorithm, was used to identify a primer set capable of amplifying various serotypes of the Blue Tongue Virus (BTV). The RNA of BTV strains 2, 13 and 17 was extracted in-house and selected as the template for Reverse Transcription PCR (RT-PCR) reactions. The primer set was predicted to amplify a 231 bp product from BTV 2, 13 and 17. Results: Using RT-PCR and gel electrophoresis we visually verified the presence of ∼231 bp products from the singleplex reactions containing the shortmer primer set and individual templates BTV 2, 13 and 17. The target amplicons were then gel extracted and analyzed with using Sanger sequencing, using the forward 11-mer primer. Results indicated that the BTV2 amplicon was 95% homologous to its predicted amplicon, the BTV13 amplicon was 97% homologous for its predicted amplicon sequence and the BTV 17 amplicon was 97% homologous for its predicted amplicon. The percent homology of amplicon to predicted sequence was less than 100% due to gaps in the sequence reads. Conclusion: We have succesfully predicted short primers that are capable of serotype-level viral detection. As a streamlined version of this analysis, we are currently adapting this assay to run on Luminex platform. Abstracts for SupplementInternational Journal of Infectious DiseasesVol. 14Preview Full-Text PDF Open Archive" @default.
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- W2016356113 date "2010-03-01" @default.
- W2016356113 modified "2023-10-14" @default.
- W2016356113 title "Short primers for amplification of diverse virus strains" @default.
- W2016356113 doi "https://doi.org/10.1016/j.ijid.2010.02.440" @default.
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