Rapid Subtyping of Dengue Viruses by Restriction Site-Specific (RSS)–PCR

Abstract

Dengue is a major public health problem worldwide. It is caused by four dengue virus serotypes, each further divided into distinct genetic subtypes. Strain typing is important for understanding the epidemiology and viral factors associated with disease transmission. However, most of the existing subtyping methods are expensive and technically unwieldy for timely, practical applications in developing countries. Here we describe a simple, rapid, PCR-based subtyping method, restriction site-specific (RSS)-PCR, which we used to analyze dengue virus serotypes 2 and 3. For each serotype, four primers targeted to sequences spanning polymorphic endonuclease restriction sites in the envelope gene were used to reverse transcribe and amplify viral RNA. These RT-PCR products generated distinct electrophoretic band patterns for different strains. Analysis of 73 dengue-2 strains and 54 dengue-3 strains representing a broad geographic distribution over several decades revealed that the RSS-PCR fingerprints reproducibly fell into 7 and 3 groups, respectively. These groups correlated well with previous phylogenetic classifications. This one-step assay should be widely accessible and allow more detailed epidemiologic investigations in dengue-endemic countries. This novel PCR approach to subtyping organisms based on restriction site polymorphisms should be applicable to other pathogens.

Genetic characterization of dengue virus type 3 isolates in the State of Rio de Janeiro, 2001

“…Recently, a new PCR-based approach has been used to rapidly subtype dengue viruses. The method consists of a single reverse transcriptasepolymerase chain reaction (RT-PCR) amplification using four primers that target regions spanning restriction site-specific polymorphic endonuclease (RSS-PCR) within the envelope gene (9).…”

Genotyping of Dengue Virus 2 Using Restriction Site-Specific (Rss)-PCR

“…The patterns of RSS-PCR for DENV-2 can be consulted in the Figure 1. Harris et al 1999. (4), (5), (6), (7) and (8) …”

“…Genetic variability occurs with DENV and could be demonstrated with the amplification of the reference pattern of RSS-PCR for DENV-2 using the primers described by Harris et al (1999). Only one sample after amplification showed the reference pattern of DENV-2 RSS-PCR, being a sample isolated in 1992.…”

“…Only one sample after amplification showed the reference pattern of DENV-2 RSS-PCR, being a sample isolated in 1992. The samples used by Harris et al (1999) were from the American region isolated between 1969 and 1986. The samples isolated in the decade of the 90's seem to show the pattern described by Harris et al (1999).…”

“…(ii) Primers -Four primers were used in a RSS-PCR assay according to Harris et al (1999) in order to detect the type of DENV-2 samples (Table 1). (iii) RT-PCR assay -"One-step kit" (Eppendorf) was used; 7µl of the RNA was used in a reaction of 20µl containing 25 pmol of each primer.…”