Use of NS3 consensus primers for the polymerase chain reaction amplification and sequencing of dengue viruses and other flaviviruses.

摘要: Consensus primers for the polymerase chain reaction were designed based on conserved motifs within serine protease and RNA helicase domains encoded by NS 3 genes of dengue other flaviviruses. Target fragments 470 bp amplified cDNA templates synthesized from RNAs types 1, 2, 3, 4, Japanese encephalitis, Kunjin, yellow fever viruses using random or specific downstream primers. PCR oligo(dT)-primed cDNAs encephalitis Kunjin viral did not yield target bands. As few as 10(3) copies could be detected. Direct DNA sequencing products reference strains 2 (NGC), (MRM 61C) (17 D) demonstrated complete concurrence with published data. However, nucleotide differences observed between our data H87 strain sequence, resulting in a single amino acid disparity. Differences at 21, 16, 11 positions noted 1 Hawaii S 275/90; 4 H 241 814669; Nakayama JaOArS 982 strains, culminating only residue differences, respectively. These disparities occurred outside putative active sites enzymatic domains, emphasizing important role NS3 protein flaviviral replication. This RNA-PCR consensus primer strategy coupled represents valuable tool molecular diagnosis epidemiology infections.