Journal
FEBS JOURNAL
Volume 278, Issue 10, Pages 1676-1687Publisher
WILEY
DOI: 10.1111/j.1742-4658.2011.08091.x
Keywords
cocktail PCR; dengue virus; diagnostic; PCR; primer
Categories
Funding
- NIH/NIAID
- National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services [HHSN272200900041C]
- Department of Homeland Security [HSHQDC-08-C-00183]
- Welch Foundation [E-1264, E-1451]
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PCR detection of viral pathogens is extremely useful, but suffers from the challenge of detecting the many variant strains of a given virus that arise over time. Here, we report the computational derivation and initial experimental testing of a combination of 10 PCR primers to be used in a single high-sensitivity mixed PCR reaction for the detection of dengue virus. Primer sequences were computed such that their probability of mispriming with human DNA is extremely low. A 'cocktail' of 10 primers was shown experimentally to be able to detect cDNA clones representing the four serotypes and dengue virus RNA spiked into total human whole blood RNA. Computationally, the primers are predicted to detect 95% of the 1688 dengue strains analyzed (with perfect primer match). Allowing up to one mismatch and one insertion per primer, the primer set detects 99% of strains. Primer sets from three previous studies have been compared with the present set of primers and their relative sensitivity for dengue virus is discussed. These results provide the formulation and demonstration of a mixed primer PCR reagent that may enable the detection of nearly any dengue strain irrespective of serotype, in a single PCR reaction, and illustrate an approach to the broad problem of detecting highly mutable RNA viruses.
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