QIAamp DSP Viral RNA Mini Kit (50)

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Publication protocol

The Assay includes a set of oligonucleotide primers and dual-labeled 5′-fluorescent (Taqman) probes for in vitro, qualitative detection of DENV-1–4. The targeted regions of DENV RNA are transcribed into complimentary DNA (cDNA) and amplified by the polymerase chain reaction (PCR). The fluorophore-labeled probes anneal to amplified DNA fragments and the fluorescent signal intensity is monitored by the ABI 7500 Fast Dx instrument during each PCR cycle. Target amplification is recorded as increase and accumulation of fluorescence over time in contrast to background signal. The assay can be run in singleplex (each DENV serotype detected in a separate reaction) or in multiplex (the four DENV serotypes are run in the same reaction) formats facilitated by the targeting of each DENV serotype with a different colored probe, i.e. each Taqman probe targets a single DENV serotype and is conjugated to a fluorophore that emits fluorescence at different excitation wavelengths: 5′-FAM DENV-1 BHQ1-3′, 5′HEX DENV-2 BHQ1-3′, 5′-TR DENV-3 BHQ2-3′, and 5′-Cy5 DENV-4 BHQ3-3′. All validations in this study were performed using the CDC DENV-1–4 Real Time RT-PCR Assay in accordance to the manufacturer's instructions (Package Insert, KK0128 available at www.cdc.gov/dengue). Viral RNA was extracted using the QIAamp DSP Viral RNA Mini kit (Qiagen cat# 61904) following manufacturer's suggested protocol. All RT-PCR reactions were performed and validated using SuperScript III Platinum One-Step Quantitative RT-PCR System without Rox (Invitrogen, cat# 11732-088). To assemble a multiplex RT-PCR reaction, 5 µL of RNA were mixed with the following reagents: 2.2 µL of nuclease-free H2O, 12.5 µL of 2× premix, 0.5 µL of forward and reverse primers for DENV-1 and DENV-3 (final concentration 1 µM), 0.25 µL of forward and reverse primers for DENV-2 and DENV-4 (final concentration 500 nM), 0.45 µL of each Taqman probe (final concentration 180 nM), and 0.5 µL of SuperScript III RT/Platinum Taq mix to a final reaction volume of 25 µL. Individual reactions were run in either 8-tube optical strips or 96-well plates and placed in the ABI 7500 FAST Dx thermocycler (Applied Biosystems, cat# 4406985). The standard cycling method was selected and fluorescence capture set to detect emissions through the FAM, HEX, Texas Red, and CY5 channels in each well. Thermocycling parameters were as follows: reverse transcription (RT) at 50°C for 30 min, RT inactivation at 95°C for 2 min and fluorescence detection for 45 cycles of 95°C for 15 seconds and annealing at 60°C for 1 min. Amplification curves were evaluated by serotype and the threshold line placed above overt background signal, usually intersecting the initial exponential phase of the curve for each serotype individually. Amplification curves with CT values >37 render erratically and are difficult to ascertain with increasing CT values; therefore present unreliable results and were considered negative.

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