Simultaneous detection and serotyping of dengue infection using single tube multiplex CDC Dengue Real-Time RT-PCR from India

Abstract

Four antigenically different dengue virus serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) are known to cause infections in humans. Some of these are known to cause more severe disease than the others. Chances for developing Dengue hemorrhagic fever-dengue shock syndrome (DHF-DSS) increases significantly with history of previous infection with one of the four serotypes. Therefore, early diagnosis, serotyping and providing early warning of dengue fever epidemics to concerned authorities becomes very important for better patient outcome and to curb the rapid spread in the community. During the 2014 outbreak, a total of 100 samples from suspected cases of dengue were collected. NS1 antigen based rapid test was used for serological diagnosis. Dengue complex one step reverse transcription-polymerase chain reaction was performed to look for presence of viral RNA. Single tube multiplex RT-PCR was also performed to look for infecting serotype. CDC Dengue Multiplex Real Time PCR assay was performed for rapid diagnosis and simultaneous serotyping of the dengue virus. Out of the 100 samples screened, 69 were found to be positive by NS1Ag Rapid test. 34 samples were found positive by dengue consensus RT-PCR assay. 22 samples were found to be positive by single tube Dengue multiplex RT-PCR assay. Serotype DEN-2 was present in maximum numbers followed by DEN-3. 44 samples were found positive by DENV CDC Multiplex Real time PCR assay. DEN-2 was found in maximum numbers followed by DEN-1. Dengue remains to be an important health problem in India and across the globe. Few serotypes of dengue are more dangerous than the others. Rapid diagnosis and serotyping remains the key for better patient management and prevention of disease spreading in the community. Highly sensitive, specific and rapid CDC real time RT-PCR assay was found to be most promising tool among all available molecular diagnostic methods. This will serve a rapid and reliable simultaneous dengue virus detection as well serotyping assay in near future for rapid identification of dengue suspected sample screening.

Keywords: Antigen; Dengue; Infection; Virus.

Fig. 1

a Agarose gel electrophoretic analysis of DEN samples by RT-PCR: Lane 1—O′ gene ruler™ Express 1 Kb DNA ladder (Fermantas Cat # SM 1163), Lane 2–16: amplified products of clinical samples, positive samples showing 511 bp. b Agarose gel electrophoretic analysis of DEN samples by conventional Multiplex RT-PCR: Lane 1—O′ gene ruler™ Express 100 bp DNA ladder (Fermantas Cat # SM 1143), Lane 2–9: positive samples showing different serotype of DENV-D1—483 bp, D2—119 bp, D3—288 bp, D4—392 bp