PCR Detection of Nearly Any Dengue Virus Strain Using a Highly Sensitive Primer ‘Cocktail’

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.