Document Type

Article

Publication Date

2011

Publication Title

The FEBS Journal

Volume

278

Publisher Name

Wiley

Abstract

PCR detection of viral pathogens is extremely useful, but suffers from thechallenge of detecting the many variant strains of a given virus that ariseover time. Here, we report the computational derivation and initial experi-mental testing of a combination of 10 PCR primers to be used in a singlehigh-sensitivity mixed PCR reaction for the detection of dengue virus. Pri-mer sequences were computed such that their probability of misprimingwith human DNA is extremely low. A ‘cocktail’ of 10 primers was shownexperimentally to be able to detect cDNA clones representing the four sero-types and dengue virus RNA spiked into total human whole blood RNA.Computationally, the primers are predicted to detect 95% of the 1688 den-gue strains analyzed (with perfect primer match). Allowing up to one mis-match and one insertion per primer, the primer set detects 99% of strains.Primer sets from three previous studies have been compared with the pres-ent set of primers and their relative sensitivity for dengue virus is discussed.These results provide the formulation and demonstration of a mixed primerPCR reagent that may enable the detection of nearly any dengue strainirrespective of serotype, in a single PCR reaction, and illustrate anapproach to the broad problem of detecting highly mutable RNA viruses.

Comments

Author Posting. © FEBS, 2011. This article is posted here by permission of the publisher Wiley for personal use, not for redistribution. The article was published in The FEBS Journal, Volume 278, 2011, http://dx.doi.org/10.1111/j.1742-4658.2011.08091.x

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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