Quantinova probe rt pcr kit

Viral ribonucleic acid was extracted from the oro- and naso-pharyngeal swab samples using the COVID-19 ORF1ab/N (two regions, so four sequences of primer should be presented) gene nucleic acid detection kit (manual).
Real-time RT-PCR assays for SARS-CoV-2 RNA detection were performed using Quanti Nova Probe RT-PCR Kit (Qiagen) in a Light Cycler 480 Real-Time PCR System (Roche, Basel, Switzerland) as previously described [13 (link)]. Each 20 μl reaction mixture contained 10 μl of 2 × Quanti Nova Probe RT-PCR Master Mix, 0.2 μl of QN Probe RT-Mix, 1.6 μl of each 10 μM forward and reverse primer, 0.4 μl of 10 μM probe, 1.2 μl of RNase-free water and 5 μl of RNA as the template. The thermal cycling condition was 10 min at 45 °C for reverse transcription, 5 min at 95 °C for PCR initial activation, and 40 cycles of 94 °C for 10 s and 58 °C for 30 s. According to the cycle threshold (Ct) analysis, if the Ct values of the fluorescein amidites (FAM) channel and victoria (VIC) channel were ≤ 37, and the curve is S-shaped with a significant exponential growth period, the test result specimen regarded as positive, if the Ct value of one channel was ≤ 37, the specimen tested again. If the Ct values of both channels are > 37, and the internal standard channel test result was positive, then the test result specimen regarded as negative.

RNA was extracted from infected Caco2 cells using the QIAsymphony RNA kit (Qiagen, Germany), and RNA was extracted from hamster lung tissues using the RNeasy Mini kit (Qiagen, Germany). Viral gene copies of SARS-CoV-2 was quantified by the RNA-dependent RNA polymerase (RdRp) using the QuantiNova Probe RT-PCR kit (Qiagen, Germany) as previously described 28 (link), 29 (link).

Influenza D virus was quantified in nasal swab, BAL fluid, and tissue samples using a one-step RT-qPCR as previously described (10 (link)). Briefly, the viral polymerase basic 1 (PB1) gene was amplified with specific primers and quantified by using a specific probe and the QuantiNova probe RT-PCR kit (Qiagen, Germany) on a LightCycler 96 real-time PCR system (Roche, Switzerland). Viral copy numbers in samples were determined by using a standard plasmid containing the PB1 product of influenza virus D/bovine/France/5920/2014 (18 (link)). For quantification of M. bovis genomic DNA copy numbers in nasal swab, BAL fluid, and tissue samples, qPCR was performed with specific primers, probe, and specific standard using the Bio-T Mycoplasma bovis PCR kit (Biosellal, France) on the LightCycler 96 real-time PCR system (Roche, Switzerland) according to the manufacturer’s instructions.

To set a RT-qPCR assay for a single multiplex reaction capable of simultaneously detecting three viruses, we used sets of primers and probes employed for DENV [27 (link)], ZIKV [28 (link)], and CHIKV [29 (link)] detection (Table S1). Reactions were performed using the QuantiNova Probe RT-PCR kit (Qiagen, catalog #208354, Hilden, Germany). The mixture consisted of 0.08 µL of each primer (800 nM), 0.04 µL of each probe (100 nM), 5.0 µL of QuantiNova Probe RT-PCR Master Mix (5×), 0.1 µL of QuantiNova Probe RT Mix, 0.05 µL of ROX passive reference dye, and 3.5 µL of the transcripts dilutions, in a final volume of 10 μL. Cycling conditions were 15 min at 45 °C and 5 min at 95 °C, followed by 45 cycles of 5 s at 95 °C and 45 sec at 60 °C. Multiplex RT-qPCR assays were done on a QuantStudio 5 Real-Time PCR System (Applied BioSystems, Waltham, MA, USA), with automatic baseline and threshold. The singleplex reaction (separate for each virus) was carried out using the same PCR conditions and concentrations from the multiplex reaction, only adjusting the water volume.

DNA was extracted at NIMPE from 5 mm punches of DBS using QIAamp DNA 96 Blood kit (Qiagen), following manufacturer’s instructions and a final elution in 200 μl of water. A duplex quantitative real-time polymerase chain reaction (qPCR) targeting 18S ribosomal gene of P. falciparum and P. vivax was conducted to confirm species identified by LM [13 (link)]. Briefly, a 7.5 µl mix was prepared using primers and probes from QuantiNova Probe RT-PCR Kit (Qiagen) in a 7500 Real-Time PCR System (Applied Biosystems). Ct value > 40 was considered negative. Samples with no amplification in the Pf–Pv specific qPCR were checked for presence of other Plasmodium species using the generic QMAL qPCR assay [14 (link)].