Zr viral rna kit

Viral RNA was isolated from mouse serum and Huh-7.5 supernatant using the ZR Viral RNA Kit (Zymo) according to manufacturer's instructions. Total RNA was extracted from Huh7.5 cell pellets and mouse tissues (spleen, brain, liver and kidney) using the RNeasy Mini Kit (Qiagen) following manufacturer's instructions. Isolated tissues (20–30 mg) were suspended in Buffer RLT-1% β-mercaptoethanol (Qiagen), lysed using TissueLyser (Qiagen; 20 cycles per s for 2 min, 1 min wait, 20 cycles per s for 2 min) and centrifuged at high-speed. The resulting supernatant was used for extracting viral RNA.

Reference RNA for this work was prepared by the Viral Pathogenesis Laboratory, Microbiology and Immunology Department, University of Otago, using a sample obtained from an infected patient in Dunedin, NZ (7 (link)). Briefly, the positive clinical specimen was inoculated into VERO cells and incubated for 3–7 days at 37°C, with 5% CO₂. Culture material was inactivated using the Zymo ZR Viral RNA Kit™ (catalog number: R1035) and the RNA stored at −80°C. In addition, the Institute of Environmental Science and Research (ESR) laboratory has now successfully grown over 80 SARS-CoV-2 isolates for research with three of these isolates grown in substantial amounts to serve as reference material for NZ researchers.
A synthetic E-gene reference RNA was made for this work. A genome region downstream of the ORF3a gene stop codon, through the E gene, to the M gene start codon (302 bp) was synthetically generated and cloned into a pBluescript II KS(+) vector by Genscript (Piscataway, NJ, USA). RNA template was generated after linearising the plasmid by XhoI digest and subsequent in vitro transcription, from the T7 promoter, using the Invitrogen Maxiscript in-vitro transcription kit (ThermoFisher) according to the manufacturer's instructions. RNA quantity was measured using the Qubit RNA HS Assay kit (ThermoFisher).

RNA was extracted from viral supernatants using ZR Viral RNA Kit (Zymo Research). The viral genomic RNA was reverse transcribed in three fragments using the following sequence specific plus-strand primers: 5′-CCATTATTATCATTAAAAGGCTC-3′ (sites 16–38), 5′-GGAAAGCATTGAACAAACG-3′ (sites 3,404–3,422), and 5′-GCTTGCACAGTTCTACTTTC-3′ (sites 8,093–8,112). Reverse transcription was performed from 2 μl purified RNA using SuperScript IV First-strand Synthesis System (ThermoFisher Scientific) following manufacturer’s recommendations. Each of the three cDNA products was subject to 35 cycles of PCR using the plus-strand primers 5′-CCATTATTATCATTAAAAGGCTC-3′ (sites 16–38), 5′-CTACCACAGAAAGGGAACTG-3′ (sites 4,174–4,193), and 5′-CAGATCCCGTAACAGAAAGT-3′ (sites 8,195–8,214), and the minus-strand primers 5′-AGCTAAGATGAAGATCGGAG-3′ (sites 4,323–4,304), 5′-GTCTTTAACAAGTTCGCTGG-3′ (sites 8,393–8,374), 5′-ACGAAGACCACAAAACCAG-3′ (sites 11,922–11,904). PCR was done with Phusion High-Fidelity DNA Polymerase (ThermoFisher Scientific) following manufacturer’s instructions. PCR products were verified by agarose gel electrophoresis, purified with the DNA Clean and Concentrator kit (Zymo Research), and quantified by spectrometry. The PCR products of each sample were mixed equimolarly for Illumina sequencing in an MiSeq machine with paired-end libraries.

Viremia was determined by TCID₅₀ assay. Briefly, serum was serially diluted ten-fold in microtiter plates and added to duplicate wells of Vero cells in 96-well plates, incubated at 37°C for 5 days, then fixed and stained with 10% (W/V) crystal violet in 10% (V/V) formalin. Plates were observed under a light microscope to determine the 50% tissue culture infective doses (TCID₅₀s). Serum samples were also tested for viral RNA copies by qRT-PCR. RNA was extracted from 0.02ml of serum using the ZR Viral RNA Kit (Zymo Research, Irvine, CA). Viral RNA was quantified by qRT-PCR using the primers and probe designed by Lanciotti et al [52 (link)]. The qRT-PCR was performed using the iTaq Universal Probes One-Step Kit (BioRad, Hercules, CA) on an iCycler instrument (BioRad, Hercules, CA). Primers and probe were used at final concentrations of 500 nM and 250 nM respectively. Cycling conditions were as follows: 50°C for 10 min and 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec. Virus concentration was determined by interpolation onto an internal standard curve made up of a 5-point dilution series of in vitro transcribed RNA, with the lowest copies per reaction being 100.

RNA was isolated from liver and kidney using RNeasy mini kit (Qiagen, Hilden, Germany) and from 200 μl of sera using ZR viral RNA kit (Zymo Research Corp., Irvine, CA, USA), respectively. RNA was also isolated from pellets obtained by ultracentrifugation of 1.5 ml of sera diluted (1:3) in PBS (2.5 h at 32000 rpm, Ti50 rotor, Beckman, USA). The RNA samples were used in real-time RT-PCR immediately or were kept frozen at -80°C before processing. DNA from the liver was isolated using DNeasy Blood and Tissue kit (Qiagen GmbH, Hilden, Germany). The RNA and DNA were quantified on NanoDrop spectrophotometer.

Virus suspensions were placed into TRIzol (Invitrogen, Carlsbad, CA) and RNA extracted using the ZR Viral RNA Kit (Zymo Research, Irvine, CA) as per the manufacturer’s protocol. Real time RT-PCR was carried out using the ABI 7900HT Fast Real-Time PCR system (ABI, Carlsbad, CA). Each reaction was performed using the TaqMan RNA-to-C_T 1-Step kit (ABI) as per the manufacturer’s instructions in a 10 μl reaction. The primers and probes were identical to those used in previously published work [6 (link)]. Each probe had a corresponding primer set that was designed to anneal flanking the polymorphic region of each variant. Every sample, run in duplicate, was tested for each variant. Positive and negative controls were run on each plate and all 8 clones were included as controls to ensure no cross-detection of the other clones by an individual probe. Additionally, serial dilutions with titers from 10⁵−10¹ pfu/ml of the individual clones were used to create standard curves.