yeloblastosis Virus reverse transcriptase (Bangalore Genei, India) and 13.75µL RNA. The RT reaction mixture was incubated at 37°C for 90 minutes followed by enzyme inactivation at 65°C for 15 minutes. First round (external) PCR was carried out in a 25µL reaction using 1X Taq buffer, 200µM dNTPs (Promega, USA), 0.4µM primers (D1 and D2)12, 1.5U of Taq DNA polymerase (Bangalore Genei, India) and 3 µL cDNA. The PCR was carried out as follows: 94°C for 2 minutes followed by 35 cycles of 94°C for 30s, 52°C for 30s and 72°C for 60s and a final extension at 72°C for 10 minutes. A 511bp segment specific to all 4 dengue serotypes was amplified in this external PCR.
External PCR was followed by semi nested PCR using D1 primer and 4 serotype-specific primers; TS1, TS2, TS3 and TS412. Nested PCR was carried out in a 25µL reaction using, 1X Taq buffer, 200µM dNTPs (Promega, USA), 0.4µM primers (D1, TS1, TS2, TS3 and TS4), 1.5U of Taq DNA polymerase (Bangalore Genei, India) and 1 µL of the diluted (1:5) external PCR product. The nested PCR was run as follows: 94°C for 1 minute followed by 25 cycles of 94°C for 30s, 54°C for 30s and 72°C for 60s and a final extension at 72°C for 10 minutes. The sizes of the nested PCR products were specific for each dengue serotype i.e. 482-bp for DENV-1, 119-bp for DENV-2, 290-bp, for DENV-3 and 392-bp for DENV-4. PCR products were electrophoresed through 2% agarose gel, stained with ethidium bromide and examined under ultraviolet light using a gel documentation system (Wealtec, USA). Singleplex nested PCR reactions with D1 and one of the type specific primers (as applicable) were carried out for confirmation of the co-infection of serotypes using same conditions as mentioned above.
Partial segments (448bp, 574bp and 582 bp) of envelope protein gene were amplified using published primers for DENV-113, DENV-214 and DENV-315 strains respectively. The respective amplicons were visualized on 2% agarose gel. The specific bands were cut from the gel and DNA was extracted from the gel using QIAquick Gel Extraction Kit (Qiagen, Germany). The amplicons were sequenced in both forward and reverse directions by commercial sequencing services (Xceleris Labs, India). The identity of the sequences were checked and confirmed by BLAST. The forward and reverse sequences were aligned and manually edited in Genedoc (v2.7.000) software to obtain the consensus sequence. The sequences obtained in the present study and other sequences retrieved from the GenBank, were aligned in ClustalW (2.1). Phylogenetic trees were constructed using Maximum Likelihood method in MEGA 6.06 software. Genetic distances were calculated using Tamura- Nei model of nucleotide substitution. The robustness of the tree was assessed with 1000 bootstrap replicates. Nauru strain (GenBank Accession Number: U88535) was used as prototype strain for DENV-1, NGC 44 strain (GenBank Accession Number: AF038403) for DENV-2 and H87 strain (GenBank Accession Number: M93130) for DENV-3.
A total of 378 acute phase blood samples were collected from suspected dengue patients attending the Out Patient Department (OPD) of Dr. M. A. Ansari Health Centre, Jamia Millia Islamia from 29 August, 2013 to 19 November, 2013. Patients included in the study showed symptoms of fever, headache, body-ache, nausea, vomiting and weakness. Information regarding age was available for 326 patients, regarding sex for 334 patients and regarding duration of illness for 315 patients. The duration of acute phase of illness ranged from 1-15 days (mean ± SD: 3.4± 2.4). The mean age of patients was 20.68 years (SD ± 12.3years) with male to female ratio of 1.8: 1. Dengue virus specific RT-PCR was conducted on all samples. DENV was detected in 269 (71.16%) samples out of 378 samples tested. The male to female ratio among the dengue virus positive samples was 1.59:1. DENV-2 was detected in highest number of patients (232, 86.24%) followed by DENV-1 (52, 19.4%) and DENV-3 (23, 8.58%) among the DENV positive samples. Co-infection with more than one DENV serotype was detected in 37 (13.75%) samples. Ma