Multiplex RT-PCR used for detection and typifying of Dengue viruses serotypes 1 and 2

Abstract

Dengue Virus is a mosquito-borne flavivirus with four antigenically distinct serotypes. Intratypic genetic variation among the virus serotypes, even in the same epidemic, is well established. The development of the polymerase chain reaction (PCR) has facilitated the diagnostic assays in detecting dengue viruses. In order to detect dengue viruses circulating in Brazil, two new pairs of type-specific primers were, without the aid of any computer program, manually designed. We used as model genomic sequence of the DEN-l (Western Pacific strain), Nauru Tsland and DEN-2, New Guinea C were used as model. The genomicsense, 5' ACAAAAAGT GGA GAC CTG GGC TC 3', oligonucleotide primers (D1S) and the 5' GTC TAT TCC AAG TCT CTT GGG 3' (DIA) oligonucleotide complementar to the plusstrand corresponded respectively to the positions 769 to 791 within 5' non coding region ofthe DEN-l virus genome and 1607 to 1587. These primer sequences bracketed at 838 nucleotides base sequence in the M and E gene. The sequence sense primers of DEN-2 was 5' TGAAGG GGA CGG TTC TCC ATG T 3' (D2S) homologous to nucleotidides 1838 to 1859 and the 5' GAC TCC CAC CAA TAC TAG TGA CAC 3' (D2A) oligonucleotide used for the first-strand cDNA synthesis was designed to contain complementary sequences to the original RNA at 3' end, corresponding to positions 2312 to 2288. The amplification product has a 474bp size corresponding to E gene portion. The primer specificity were avaluated by the multiplex RT-PCR.