RNA extraction Total RNA from cell culture supernatant of viruses from the different serotypes obtained from infected cells and from mosquitoes collected and frozen in the field was obtained using Trizol LS (GIBCO BRL, Gaithersburg, Md.) according to the manufacturer’s recommendations. Total RNA from acute-phase plasma collected from patients in the epidemiologic surveillance program by the Secretaria de Salud Oaxaca, Mexico during 2000- 2001 and 2004-2006 was also obtained by using the Viral Nucleic Acid Extraction Kit from Real Genomics (Real Biotech Corporation). The RNA suspended in 50 μl of H2O treated with diethylpyrocarbonate (DEPC, SIGMA-ALDRICH) was used as a template to obtain different DNA products by Reverse Transcription with the Polymerase Chain Reaction (RT-PCR). Multiplex dengue RT-PCR (reverse transcription-polymerase chain reaction) A multiplex one step RT-PCR (SuperScriptTM One-Step RT-PCR with Platinum Taq, invitrogen) was performed to determine the serotype in each sample. Briefly, five primers targeting the NS3 gene were included in the assay, resulting in products of different sizes (DENV-1, 169 pb; DENV-2, 362 pb; DENV-3, 272 pb; DENV-4, 472 pb). Then, 5 µl of extracted RNA was used as template in 25 µl reaction volume. Essentially, the protocol of Seah et al. [12] was followed to confirm the DENV serotype. The thermocycler was set to: 10 cycles at 95 ˚C for 30 sec, annealing at 50 ˚C for 1 minute, and then a 72 ˚C temperature for 1 min with a ramp time of 1 min and 25 cycles at 95˚C for 30 sec, 50˚ C for 30 sec, and 72˚C for 30 sec with a ramp time of 30 sec. Finally, 7 min extension at 72˚ C was added. Reaction mixtures were stored at 4˚ C until further processing. The specific primers for DENV-1, DENV-2, DENV-3, and DENV-4 were synthesized with 5´ C6 amino linker by The Midland Certified reagent Company Inc. (Midland, TX, USA) as follow: 5´-(Texas Red) AGTTTCTTTTCCTAAACACCTCG-3´; DENV-1; 5´-(_6 FAM) CCGGTGTGCTCRGCYCTGAT-3´, DENV-2; 5´-(_CY5) TTAGAGTYCTTAAGCGTCTCTTG-3´, DENV-3; 5´-(_Yakima Yelow) CCTGGTTGATGACAAAAGTCTTG-3´, DENV-4. Synthesis was validated using each pair of oligonucleotides by RT-PCR and found to yield PCR products of the expected size. DNA microarray fabrication DNA (RT-PCR product) capture probe was immobilized according to the procedure described by Zammatteo, et al. [32]. Briefly, phosphorylated capture probes were denature for 10 min at 92°C and diluted to a concentration of 1.6 mM CHAPS, 1% CAPS, 50% DMSO buffer pH 6.5. The DNA was UV-cross link at 250 mJ and finally incubated at ambient temperature by 16 h as the specifications of the Schott´s protocol. Samples were spotted using the Robot Spotter GeneTAC G3 arraying system (Genomic Solutions, Harvard Bioscience, Inc., Massachusetts, USA). Each solid pin deliver a volume of around 1 nl, designed to create a spot diameter of 300 µm. The center-to-center spacing of bright spots was 500µm. DNA microarray chip was prepare for hybridization by washing extensively with 0.1% SDS, rinsed twice with double deionizer water and heated in double deionizer water for 3 min at 95° C. After blocking with prehybridization buffer (4X SSC, 1 %SDS nd 10 mg/ml BSA) for 45 min at 42° C, DNA microarray chip was washed with double deionizer water for 20 s and dried under filtered air flow. Hybridization and Detection The hybridization of specific fluorescent primers for DENV was performed onto the specific immobilized probe DNA microarrays. A volume of 5 µl containing the specific primer for each DENV-1 -4 serotype was used. The microarray substrates and coverslips were sealed in a chamber from BIO-RAD (Bio-Rad Laboratories, Inc., Ca., USA) and incubated at 42o C for 16–18 h at high humidity. The tested concentrations for each specific primer of DENV-1 to -4 were 0.1, 0.2, 0.4, 1.0, 5, 10 and 20 µM. Coverslip and supports were gently removed (4× SSC in a wash bottle) and arrays were washed by immersion into 1× SSC, 0.1% SDS for 10 min (10 slides in 250 ml), 0.1× SSC, 0.1% SDS twice for 10 min and 0.1× SSC twice for 10 min. Finally, blow the slide dry with air by pushing the liquid away from the spots and towards the outer edges. Slides were scanned using Scanner GeneTac LS IV (Genomic Solutions, Molecular Dynamics Generation III) to detect Texas Red, FAM, Yakima yellow and Cy5 fluorescence. For the detection of DENV serotypes we used the excitation lines at 583, 494, 530 and 646 nm wavelength of a laser scanner (Scanner Gene TAC LSIV from Genomic Solutions) by adding pixel intensities of each spot image. Establishment of threshold intensities To analyze the results, we needed to determine whether a particular median pixel intensity value was indicative of hybridization. For this purpose, we defined threshold intensities as the value for normalized median pixel intensities above which hybridization was judged to be positive.
Less...
More...