Rapid detection and identification of dengue viruses by polymerase chain reaction (PCR)
Yenchitsomanus, P.T.; Sricharoen, P.; Jaruthasana, I.; Pattanakitsakul, S.N.; Nitayaphan, S.; Mongkolsapaya, J.; Malasit, P.
Southeast Asian Journal of Tropical Medicine and Public Health 27(2): 228-236
1996
ISSN/ISBN: 0125-1562 PMID: 9279982 Document Number: 467152
A polymerase chain reaction (PCR) method using sets of newly designed primers for rapid detection and simultaneous identification of dengue (DEN) virus serotypes was developed and tested. The test is based on 2 sets of primers specific within the envelope (E) and non-structural (NSI) regions of the DEN virus genome. Two sets of universal primers that bind to 2 target sequences which are shared by all the 4 serotypes of the virus within the E and NSI regions are used. The resulting products are further amplified by another pair of inner or nested universal primers, which also bind to another set of shared sequences within the E and NSI regions, respectively. The nested PCR of both the E and NSI regions can detect DEN virus of all 4 serotypes at a sensitivity of 1 pfu or less. For the identification of serotypes, a mixture of 4 pairs of serotype-specific primers, specific to the E region, was used. The primers were designed to bind to serotype specific sequences within the regions flanked by the outer universal primers, and giving the amplified products of different sizes, each corresponds to one particular serotype (405 bp for DEN 1, 346 bp for DEN 2, 196 bp for DEN 3, and 143 bp for DEN 4). A protocol was developed and successfully applied to detect DEN virus in cell-culture supernatants and patients' sera. The technique is simple and rapid, capable of not only detecting DEN virus but also identifying the serotypes of the virus in clinical specimens.