Divergências in silico nos sítios de anelamento de RT-qPCR do vírus da dengue e correção do conjunto afetado

Dengue comprises four serotypes with wide genetic diversity, and mutations in the regions recognised by primers and probes may compromise the reliability of RT-qPCR assays. This study assessed the impact of such divergences on the annealing sites of a fourplex RT-qPCR assay for serotype-specific detection of DENV, published in 2005, and proposed a corrected set for the most affected serotype. This is a descriptive-analytical study with an in silico approach. Twenty-four complete genomes, six per serotype, were retrieved from NCBI and formed the main panel, expanded to 45 DENV-3 and 48 DENV-2 genomes for the detailed analyses. The twelve oligonucleotides of the assay were located in each genome by local alignment against both strands, with coordinates validated against the positions published in the original protocol. Divergences were observed in all twelve oligonucleotides. Seven events fell within the five bases of the 3′ end of primers, distributed across DENV-2, DENV-3 and DENV-4, with none in DENV-1. The sense primer D3-F proved the most compromised component, since the substitution at position 18, two bases from the 3′ end, was present in 45 of the 45 DENV-3 genomes analysed, from different countries and collection periods, so that the published oligonucleotide shows perfect pairing in none of them. Complete redesign of the DENV-3 set proved unfeasible. Of the 10,707 genome positions, 8,268 (77.2%) are conserved, yet only 23 stretches reach 18 nucleotides and merely three can host an oligonucleotide able to discriminate the remaining serotypes, separated from one another by thousands of bases. In the 3′ untranslated region, all 25 sets generated contained at least one component identical to another serotype. Correction of the existing oligonucleotides through degenerate bases was therefore adopted, raising perfect pairing of D3-F from none to 44 of the 45 genomes. It is concluded that intra-serotypic conservation and serotype specificity compete for the same genomic region and that monitoring of diagnostic sites should be incorporated into routine genomic surveillance.