QuantiTect Multiplex RT-PCR NR Kit (1000)

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Publication protocol

Quantitative one-step RT-PCR.: Based on the nucleotide sequence of the Japanese strain, SFTSV YG1 (GenBank accession no. AB817995, AB817987, and AB817979), the specific quantitative one-step PCR (qPCR) primer and probe sets that were targeted to the RNA-dependent RNA polymerase (RdRp), glycoprotein (GPC), or nucleoprotein (NP) genes were designed using the DNADynamo sequence analysis software program (Table 1). The primer and probe specificities and cross-reactivities were checked by Primer-BLAST, as described above in the cvPCR primer selection procedure. For the qPCR assay, an aliquot of the extracted RNA solution was added to a reaction mixture for the QuantiTect probe RT-PCR kit (Qiagen, Hilden, Germany), which contains 2× QuantiTect probe RT-PCR master mix, QuantiTect RT mix, H2O, and 10× primer-probe mix, which contains 4 μM each specific primer, 2 μM TaqMan probe(s), and 2 μM contamplicon probe. After PCR activation at 95°C for 15 min, the reverse transcription reaction was carried out at 50°C for 30 min, followed by 45 cycles of amplification under the following conditions: 94°C for 15 s and 60°C for 60 s in a LightCycler Nano (Roche).
The qPCR method was performed according to a previous report (6), with the PCR products amplified from an SFTSV strain, HB29, for the standards and using the QuantiTect multiplex RT-PCR NoROX (NR) kit (Qiagen) instead of synthetic viral RNA transcripts of HB29 and AgPath-ID with the one-step RT-PCR kit (Life Technologies).

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