Development of an In-house Multiplex RT-PCR (MRTPCR) Method for Detection and Simultaneous Serotyping of Dengue Virus Infection

Abstract

An in-house multiplex RT-PCR (MRT-PCR) method for detection and simultaneous serotyping of dengue virus infection was developed using selected Philippine strains. The one-step one-tube MRT-PCR in the present study adopts the primers and cycling profile of the previously reported C-prM heminested protocol of Chien and colleagues (2006) which involves two sequential amplification on separate tubes for detection and serotyping using universal dengue primers and serotype specific primers, respectively. Modification of the two-tube into a single reaction vessel assay, where all primers are mixed in one microtube, reduced amplicon contamination and assay time. The MRT-PCR in this study was optimized against 20 previously serotyped RNA extracted from diagnosed de-identified human serum samples from a tertiary hospital. Using a clinically validated MRT-PCR protocol of The Medical City hospital as a reference standard, the developed MRT-PCR showed 90% (n=18 of 20) accuracy. The false negative Type 2 error rate was 5% (n=l of 20) diagnosing one positive sample with a negative result. A second discrepant result was Type 2 dengue using MRT-PCR and Type 3 dengue by reference method. The overall error rate was 10% (n= 2 of 20). The 90% accuracy rate with a three to four hour turnaround-time suggest its strong potential for clinical and sero-epidemiological use therefore further evaluation and clinical validation using larger number of samples are needed to obtain more reliable estimate of the assay’s accuracy.