Improved LNA probe-based assay for the detection of African and South American yellow fever virus strains

Abstract

Background: Real-time assays for Yellow fever virus (YFV) would help to improve acute diagnostics in outbreak investigations. Objectives: To develop a real-time assay for YFV able to detect African and South American strains. Study design: Three short probe (14–18 nt) formats were compared and a plasmid-transcribed RNA standard was used to test the performance of the assays. Additionally the new TaqM1 enzyme was tested. Results: A locked nucleotide probe (LNA probe) performed best with an analytical sensitivity of 10 RNA molecules detected. 44 African and 10 South American strains were detectable. One South American strain from 1984 had a one-nucleotide deviation in the hybridisation sequence for which the LNA probe had to be adapted. Comparison of enzymes revealed that not all enzymes are suitable for LNA probes. Conclusion: The developed LNA probe based YFV real-time PCR performed best in an enzyme mix and less efficient using multifunctional enzymes.