Multiplex logic processing isothermal diagnostic assays for an evolving virus

Sanchita Bhadra; Miguel A. Saldaña; Hannah Grace Han; Grant L. Hughes; Andrew D. Ellington

ZIKV has an ~10 kilobase RNA genome encoding a polyprotein that is cleaved into ten structural and non-structural (NS) proteins: 5'-Capsid (C)-preMembrane (prM)-Envelope (E)-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3'. ZIKV clusters into two African lineages and one Asian lineage that includes the current epidemic strains. 29 The Asian and African lineages of ZIKV show 0.2% to 10.6% intra-lineage and 4.5% to 12.1% inter-lineage nucleotide variation. 29 The epidemic strains have accumulated additional changes and have potentially undergone multiple natural recombinations. 29, 30 The strains are likely continuing to evolve, presenting a challenging target for surveillance and diagnostics. Here, we designed four reverse transcription (RT) LAMP assays to amplify relatively conserved regions in four ZIKV genes -capsid (CA), NS1, NS3, and NS5. Each individual assay included two inner primers (FIP and BIP), two outer primers (B3 and F3), and one loop primer (LP). The CA LAMP assay utilized an additional sixth primer, termed stem primer 31 (SP), complementary to the target region between F1 and B1 priming sites ( Figure 1A) . Specific nucleotide positions within these primers were substituted with degenerate nucleobases to allow pairing with variant ZIKV RNA (Supplementary Table T1 ). The NS3 LAMP primer set had the lowest degeneracy with only 10 positions varying between two nucleobases and one position varying between three nucleobases. Twelve positions in the NS1 LAMP primer set varied between two nucleobases. The CA LAMP primers had a slightly higher degeneracy with 13 polymorphic positions varying between two bases. The NS5 primer set was the most degenerate with 15, 6, and 1 positions varying between two, three, or four bases, respectively.