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The flaviviruses have high sequence diversity and therefore, designing primers and probes for a pan assay can be a major challenge. However, Patel and co-workers designed degenerate primers and probes for the simultaneous detection of a broad range of flaviviruses. The probes were modified to contain LNA nucleotides to match all flaviviruses (58).
The LNA nucleotides are analogous of nucleic acids. LNAs are modified RNA nucleotides in which the ribose ring is locked by the methylene bridge connecting the 2`-O atoms and the 4`-C atoms. Locking of the methylene bridge with the LNA molecules basically locks the structure into a tight conformation (71). LNA probes have higher melting temperatures because of the locked nucleotides, it is more stable and the probe length is shorter compared to TaqMan probes. LNA has increased sensitivity and specificity (72). PCR primers or probes modified with LNA nucleotides show very accurate mismatch discrimination and high specificity (73).

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