Virus strains of the four standard dengue serotypes were obtained from the National Institute of Virology, Pune, India (Den-1, Hawaii; Den-2, P-23085; Den-3, 633798 and Den-4, 642069). The dengue viruses isolated during this study from dengue outbreak in India since 2001 were also included. To check the cross reactivity, related flaviviruses viz., Japanese encephalitis (JaOArS982), West Nile (G22886), Yellow fever (17D vaccine strain) virus and one alphavirus (Chikungunya (S27) were also included in this study. These viruses were propagated in C6/36 cells.
A total of 620 human serum samples from febrile patients clinically suspected of having dengue fever were collected within 0 to 4 days from the time of onset of symptoms. These samples were collected from different parts of India during various outbreaks from 2001–2007 [7, 8]. In addition, a panel of 40 serum samples collected from healthy volunteers from the same area were included as negative control in this study.
C6/36 cells  were grown in Eagle's minimum essential medium (EMEM) (Sigma, USA) supplemented with 10% Tryptose Phosphate Broth (TPB) (DIFCO, USA), 10% Fetal bovine serum (FBS) (Sigma, USA), 3% L-glutamine (Sigma, USA) and gentamicin (80 mg/l) (Nicholas-Piramal, India) at 28°C. Isolation of viruses from acute phase dengue suspected samples were attempted following the standard virus adsorption technique . Briefly, preformed manolayer of cells were washed with plain medium prior to infection. The virus/suspected serum was allowed to adsorb to the cells for 1 hr at 37°C. Following adsorption, the inoculum was replenished with 2 ml of maintenance medium (EMEM with 2% FBS). Suitable cell controls were also kept along side. The cells were harvested on appearance of cytopathic effects or on 6th day post inoculation (dpi), whichever is earlier. The identification of the virus isolates obtained from the clinical samples was carried out by RT-PCR as described below.
RNA was extracted from standard viruses, virus isolates, sera of suspected dengue patients and healthy volunteers using QIAquick viral RNA mini Kit, following the manufacturer's protocol. Finally, the RNA was eluted in 60 μl of diethyl pyrocarbonate (DEPC) treated water (Sigma, USA).
Dengue complex Reverse transcription polymerase chain reaction (RT-PCR)
Conventional Dengue complex RT-PCR assays were performed according to the protocol  with slight modifications. Briefly, the RT-PCR was performed with RNA from standard dengue viruses and confirmed dengue virus-infected patient serum samples initially in a 50 ul reaction volume using Access quick RT-PCR kit (Promega, USA) with dengue virus group-specific consensus primers (D1: 5' TCAATATGCTAAAACGCGCGAGAAACCG 3' and D2: 5' TTGCACCAACAGTCAATGTCTTCAGGTTC 3').
Dengue Nested PCR
The nested PCR assay was performed according to the protocol  with slight modifications. Briefly, the 1: 10 dilution of RT-PCR amplicon was used as template in the nested PCR in a 50 μl reaction volume using master mix of Access quick RT-PCR kit, with dengue virus group-specific consensus forward primer (D1), and four serotype specific reverse primers (Ts1: 5' CGTCTCAGTGATCCGGGGG 3', Ts2: 5'CGCCACAAGGGCCATGAACAG 3', Ts3: 5' TAACATCATCATGAGACAGAGC 3' and Ts4: 5'TGTTGTCTTAAACAAGAGAGGTC3'), as reported earlier .
Single-step Dengue multiplex RT-PCR (M-RT-PCR)
A one-step single tube serotype-specific multiplex PCR was performed with RNA from standard dengue viruses and confirmed dengue virus-infected patient serum samples using a multiplex RT-PCR protocol. The amplification was carried out in a 50 μl total reaction volume with Access quick RT-PCR kit according to the manufacturer's protocol, along with five primers viz., forward D1 and four serotype specific reverse primers (Ts1, Ts2, Ts3 and Ts4).
Evaluation of multiplex RT-PCR
The evaluation of the multiplex RT-PCR assay was carried out with 620 serum samples collected over a period of six years from India.
Preparation of RNA standard
The detection limit of this assay was determined using RNA standards. The RNA standard was produced using T7 transcription kit (MBI Fermentas, USA) following the manufacturer's protocol. Initially PCR amplicons of respective dengue serotypes were generated using a modified D1 primer (T7 promoter sequence (TAATACGACTCACTATAGG) was added at the 5' end of D1 primer) and normal D2 primer. These amplicons (530 bp) of all the four serotypes were gel purified, quantitated, before being used as template in transcription reaction. The purified template was subjected to in vitro transcription (IVT) at 37°C for 1 h. The IVT products were then treated with 1 U of DNase I and incubated at 37°C for 15 min to remove the remaining DNA followed by inactivation of DNase I at 70°C for 15 min. The IVT products were ethanol precipitated and resuspended in DEPC treated water. The amount of respective dengue serotype specific RNA transcripts were determined spectrophotometrically and converted to molecular copies by using the following formula 21.
Y (molecules/μl) = [X(g/μl)/transcript length (nucleotides) × 340] × 6.023 × 1023
Sensitivity and specificity of the assay
The sensitivity of both the dengue complex and serotype specific multiplex RT-PCR assay was determined through serial dilutions of the RNA transcripts. The specificity was determined on comparison with related Flaviviruses (JE, West Nile and Yellow fever viruses), alphavirus (Chikungunya virus) and a panel of 40 serum samples from healthy volunteers.