Rapid detection and serotyping of dengue virus by multiplex RT-PCR and real-time SYBR green RT-PCR

Abstract

Introduction: Dengue fever and dengue haemorrhagic fever currently rank highly among the newly-emerging infectious diseases, and are considered to be the most important arboviral disease worldwide. The definitive diagnosis is culture analysis, but practical considerations limit its use. Also, the period for viral detection is limited. Within a day or two after fever subsides, rising levels of antibodies interfere with viral cultures. An alternative to this quandary is the use of viral RNA detection assays. In our laboratory, a reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed using a set of degenerate primers.

Methods: This multiplex RT-PCR assay was evaluated with 280 samples collected during the year 2003. These groups include prototype dengue virus (serotypes 1-4), acute serum from which the dengue virus was isolated, seronegative acute samples (culture negative) but whose convalescent samples seroconverted, and sera positive for other microbial diseases. This assay was then modified into a real-time SYBR Green RT-PCR assay. Sensitivity and specificity of both assays were compared.

Results: The multiplex RT-PCR assay was able to detect 134 samples whereas SYBR Green RT-PCR assay was able to detect 178 out of 306 samples. Both assays were 100 percent specific. Further analysis of 53 samples showed that the virus could be amplified at IgM positive/negative values of up to 4.2, and up to six days after onset of fever. The viral detection rate was inversely proportional to the day of fever onset as well as IgM values.

Conclusion: The sensitivity and specificity of the conventional multiplex RT-PCR assay are 98.18 percent and 100 percent, respectively, and for the real-time SYBR Green assay, 99.09 percent and 100 percent, respectively. The melting curve analysis allows all four dengue serotypes to be discriminated based on distinct melting temperature value. The accuracy and speed of this multiplex RTPCR assay makes it a suitable test for the diagnosis of dengue and for epidemiological surveillance.