SensiFAST™ Probe No-ROX One-Step Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Use RNase-free plasticware and reagents		It is essential that any tubes containing amplified DNA product are not opened in the reaction set-up area.

Upstream tips
Use RNase-free plasticware and reagents
Downstream tips
It is essential that any tubes containing amplified DNA product are not opened in the reaction set-up area.

Publication protocol

The rtRT-PCR was performed by using two different real time RT-PCR platforms. The first thermal cycling was implemented in LightCycler 480 Instrument II (Roche, Meylan, France). Briefly, 4 µL of extracted RNA were added in 16 µL of a reaction mixture containing 0.4 µM of each primer, 0.1 µM of probe, 1× Master Mix, 0.2 µL of mixture of Reverse Transcriptase and hot-start DNA polymerase, 0.4 µL of Rnase inhibitor (SensiFAST Probe No-ROX One-Step kit, Bioline, London, UK). The RT-PCR program consisted in a reverse transcription step at 45 °C for 20 min followed by a denaturation at 95 °C for 10 min and 45 cycles of denaturation at 95 °C for 5 s followed by annealing/extension at 55 °C for 30 s. The second thermal cycling was implemented in Applied Biosystems 7500 Real Time PCR System (Applied-Biosystems, Thermo Fisher Scientific, Waltham, MA USA). In this assay, four µL of extracted RNA were added in 21 µL of a reaction mixture containing 0.4 µM of each primer, 0.25 µM of probe, 1 × Master Mix, 1.5 mM of MgSO4, and 0.6 µL of mixture of Reverse/Taq polymerase (SuperScript III Platinum One-Step qRT-PCR System Kit, Invitrogen, Thermo Fisher Scientific, Waltham, MA USA). RT- PCR program consisted in a reverse transcription step at 50 °C for 20 min followed by a denaturation at 95 °C for 10 min and 45 cycles of denaturation at 95 °C for 15 s followed by annealing/extension at 57 °C for 30 s. The published pan-hantavirus nested RT-PCR protocol from Klempa et al. [10] was used in comparison of method and as reference test (Supplementary Table S1).

The limit of detection (LOD) of the rtRT-PCR methods was determined using THAIV RNA 10-fold dilutions tested in duplicate (from 3.45E + 05 to 3.45E - 02 FFU/mL of viral stock) and repeated three times.

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Manufacturer protocol

Download the product protocol from Bioline for SensiFAST™ Probe No-ROX One-Step Kit below.

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