Superscript 3 platinum one step qrt pcr kit

Universal transport medium (Copan Italia, Brescia, Italy) was used to preserve the environmental samples, which were stored in a box with ice packs at 4°C and transported to the laboratory within 4 hours. A QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) was used to extract viral RNA. Influenza A virus M gene and H7N9 virus hemagglutinin (HA) RNA were detected as described previously (12 ) by using a real-time reverse transcription PCR (rRT-PCR) (SuperScript III Platinum One-Step qRT-PCR Kit; Invitrogen, Carlsbad, CA, USA) and H7-specific primers and probe provided by the Chinese National Influenza Center. Samples positive for H7N9 virus by rRT-PCR were inoculated into the allantoic sac of 10-day-old specific pathogen free embryonated chicken eggs and incubated for 48–72 h at 35°C for virus isolation (13 (link)).

Nucleic acid extraction was performed using QIAamp Viral RNA Mini Kit (Qiagen; Valencia, CA) and carried out according to the manufacturer’s instructions. Extractions were performed using 140μl of serum, or plasma where serum was unavailable. Nucleic acid samples were screened and serotyped for DENV-1-4 using the DENV multiplex RT-qPCR assay previously described by Waggoner [22 (link)]. Screening for CHIKV was carried out using the RT-qPCR general assay previously described by Panning [23 (link)]. All RT-qPCR screening was done using the SuperScript III Platinum One-Step qRT-PCR kit (Invitrogen; Carlsbad, CA) with final reaction volumes amended to 25μl instead of the manufacturer’s recommended 50μl.

For the detection of dengue RNA after extraction, the pan-dengue Taqman real-time RT-PCR system (DENV All RT-PCR) developed by Leparc-Goffard et al. [24 (link)] was used with four serotype-specific RT-PCRs. The SuperScript III Platinum One-Step qRT-PCR kit (Invitrogen) with 200nM of each primer and 100nM of probe on 5μl of RNA extract was used. Synthetic RNA control was prepared as described by Ninove et al. [25 (link)]. Three serial dilutions, 2.5 x10⁶, 2.5 x10⁴ and 2.5 x10² copies/μl of positive control were prepared and aliquoted at -80°C and used as standards in each RT-PCR run. All samples and standards were tested by DENV All RT-PCR in duplicate. Means of Ct values of the duplicates were used for the quantification of dengue RNA copies in tested samples (supporting information, S2 Table).

Suspected SARS-CoV-2 clinical samples were tested for positivity by qRT-PCR. Briefly, RNA was extracted from Viral Transport Media using the QIAamp Viral RNA Mini kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. The qRT-PCR was conducted using the SuperScript III Platinum One-Step qRT-PCR Kit as per the manufacturer’s protocol (Invitrogen Carlsbad, CA, USA) in the CFX96 Touch Real-Time PCR Detection System (BioRad Laboratories, Watford, UK), according to the cycling protocol. The reaction was performed using the specific primer set RdRpF, RdRpR and FAM-labelled probe or NP-F and NP-R and ROX labelled probes designed to detect SARS-CoV2. The 25-µL PCR reaction consists of 12.5 µL 2X Reaction Mix, 0.2 µM of each primer and 0.1 µM probe, 0.5 µL of SuperScript^® III RT/Platinum^® Taq Mix, 5 µL of RNA sample and nuclear free water. The cycling program was performed in the CFX96 Touch Real-Time PCR Detection System (Applied Biosystems, Madison, WI, USA), according to the cycling protocol. The amount of viral RNA in each sample was estimated by comparing the cycle threshold values (Ct) to the standard curve made by 10-fold dilutions of previously titrated in vitro transcribed RdRP gene.

A registered modified live vaccine (Cevac IBird^®, Ceva Animal Health Ltd., Libourne, France) for infectious bronchitis, based on 1/96 strain, was chosen for the experiment. Two 1000-dose titrated vials (LOT. 028J2S2KGD; 4.2 Log₁₀ Embryo Infectious Dose EID₅₀/dose), one for each group, were used after resuspension in 100 mL of deionized water. A 200 µL aliquot was processed for nucleic acid extraction with High Pure Viral Nucleic Acid^® Kit (Roche, Basel, Switzerland) and used for a standard curve reconstruction by real time RT-PCR following the method published by Tucciarone et al. (2018) [35 (link)]. The vaccine was ten-fold diluted and serial dilutions were tested with SuperScript^® III Platinum^® One-Step qRT-PCR Kit (Invitrogen, Waltham, MA, USA) on LightCycler^® 96 Instrument (Roche, Basel, Switzerland).