Superscript 3 platinium one step qrt pcr kit

Viral RNA was extracted from 72 CSF samples using 400μl of clinical specimen by the iPrep™ PureLink® Virus kit according to manufacturer’s instructions and resuspended in 100μl DEPC water. Ten microliters of the extracted viral RNA was used as a template using specific primers with the SuperScript® III Platinium One-Step qRT-PCR kit (Invitrogen, USA), according to the manufacturer’s instructions. For Phlebovirus detection and quantification, the specific primers used amplified the 3′ end of the small segment N gene and were based on published protocols (26 (link), 27 (link)). For Flavivirus detection and quantification, the specific primers targeted the NS5 gene and were based on a previously published protocol (28 (link)).

Real-time RT-PCR was performed to test for CHIKV, ZIKV and DENV as previously described [18 (link), 61 (link)]. Nucleic acid extraction was performed using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA) and carried out according to the manufacturer’s instructions. Molecular detection of DENV, CHIKV and ZIKV was performed by using the SuperScript III Platinium One-Step qRT-PCR kit (Invitrogen). We performed the samples’ amplification of the RNAse P in parallel to evaluate the extracted RNA quality. RNA Real-Time RT-PCR reactions consisted of a step of reverse transcription at 50 °C for 15 minutes of the enzyme activation at 95 °C for 2 min, and 45 cycles at 95 °C for 15 s and 60 °C for 1 min for hybridization and extension with the use of ABI 7500 equipment (Applied Biosystems). A cut-off value of Ct (Cycle Threshold) 37 was chosen as the CDC reference assay.

Viral RNAs were extracted using the QIamp Viral RNA kit (Qiagen) from 140 μL of viral stocks. Viral RNAs were quantified by RT-qPCR using the SuperScript III Platinium One-Step qRT-PCR kit (Invitrogen). For IAV, the M segment was detected using the following primers and probe: 5′-CTTCTAACCGAGGTCGAAACGTA-3′, 5′-GGTGA CAGGATTGGTCTTGTCTTTA-3′ and 5′(HEX)-TCAGGCCCC CTCAAAGCCGAG-(BHQ-1)3′. For IBV, the HA segment was detected using the following primers and probes: 5′-AC CCTACARAMTTGGAACYTCAGG-3′, 5′-ACAGCCCAAGCC CAAGCCATTGTTG-3′, 5′(FAM)-AAATCCAATTTTRCTGGT AG-(BHQ-1)3′ and 5′(FAM)-AATCCGATTTTRCTGGTAG-(BHQ-1)3′. Standard curves were obtained by subjecting in vitro-transcribed IAV-M or IBV-HA vRNAs to RT-qPCR in parallel. The following program was used on a Light Cycler480 instrument (Roche): 45°C for 15 min, 95°C for 3 min, 50 cycles of 95°C for 10 s, 55°C for 10 s, and 72°C for 20 s, then 40°C for 30 s.