Kingfisher flex

Frozen mosquito samples stored in microcentrifuge tubes were allowed to thaw at room temperature for up to 15 min. Using the Zymo Direct-zol™-96 MagBead RNA kit (Zymo Research, Irvine, CA, USA), 400 μL of TRI reagent and two RNase-free 3.2 mm stainless steel beads (Next Advance, Troy, NY, USA) were added to each specimen tube. Samples were lysed using the Qiagen TissueLyser II for 7 min at a frequency of 24/s followed by centrifugation for 10 min at 14,000 rcf. Two hundred microliters of supernatant was removed from each sample tube and pipetted into a 96-deep well plate. Two hundred microliters of 99.5% ethanol, 20 μL of MagBinding beads, and 5 μL of Proteinase K were added to each sample well before adding the plate to the Thermo Scientific KingFisher Flex automated instrument. An extraction control, consisting of all reagents and no mosquito matrix, was added to the sample plate as well. The elution plate was stored at −20°C for short-term storage or used immediately for further analysis on the Applied Biosystems ABI 7500 Fast DX, MagPix, MinION and MiSeq. A second extraction was performed on the homogenized mosquitos with the addition of 50 μL DNase added to the sample prior to being placed on the Thermo Scientific KingFisher Flex automated instrument.

First, 800 µL of each patient’s urine was aliquoted into each well of a 96-deep well plates, sealed with foil, and then centrifuged. Then, 700 µL supernatant was removed from each well and 50 µL of enzyme mix for lysis was added to each well with concentrated urine and an extraction negative control (100 µL nuclease-free water) and incubated at 65 °C for 20 min on KingFisher Flex (ThermoFisher Scientific, Carlsbad, CA, USA). Subsequently, 240 µL of binding solution containing DNA bead, proteinase K, and XENO (Internal control, ThermoFisher) was added into each well and then processed on KingFisher Flex for protein digestion and DNA extraction for 30 min.

Total RNA was prepared from viral isolates using a MagMAX™ 96 Viral Isolation Kit (Thermo Fisher Scientific), using 50 μL of collected medium, with KingFisher Flex (Thermo Fisher Scientific) according to the manufacturer’s instructions. Total RNA was also prepared from 100 μL of RNase-treated samples using a MagMAX™ CORE Nucleic Acid Purification Kit (Thermo Fisher Scientific), using 60 μL of elution buffer, with KingFisher Flex (Thermo Fisher Scientific), according to the manufacturer’s instructions.

Cell pellets, corresponding to approx. 1 million cells, were dissolved in 200 µL lysis buffer (5% SDS and 50 mM Tris, pH=7.55) and homogenized using probe sonication on ice. Debris was removed by centrifugation and proteins were quantified with the Pierce BCA protein assay kit (Thermo Fisher Scientific, Germany). 50 µg of proteins were used for hydrophilic interaction liquid chromatography (HILIC, ReSyn Biosciences, South Africa) clean-up and automated protein on-bead digestion with trypsin using KingFisher Flex (Thermo Fisher Scientific, Germany) in a 96-well format.
Peptides were recovered from the plate and dried in a Speedvac (Thermo Fisher Scientific, Germany) prior to C18 desalting using BioPureSPN Mini, PROTO 300 C18 (The Nest Group, Inc., MA, USA). Cleaned peptides were dried in the Speedvac and stored at − 20 °C before quantification and injection into the mass spectrometer.

The CDPH/VRDL performed real-time reverse transcription-polymerase chain reaction (RT-qPCR) on the 203 samples described above for SARS-CoV-2. Prior to May 21, 2020 [15 (link)], samples were extracted using Qiagen DSP Viral RNA Mini Kit with carrier RNA added (Qiagen) with extracts tested for SARS-CoV-2 using the FDA EUA approved 2019-nCoV CDC Real-Time RT-PCR Diagnostic Panel assay, which targets two regions of the nucleoprotein gene (N1 and N2). After May 21, 2020 [16 (link)], samples were extracted using the KingFisher Flex (Thermo Fisher Scientific) instrument according to the manufacturer’s instructions. These later samples were tested using the FDA EUA approved the Taqpath™ Multiplex Real-time RT-PCR test, which includes nucleoprotein (N) gene, spike (S) gene, and ORF1ab gene targets.

Total RNA was isolated by using the Mag-Bind Viral RNA 96 kit (Omega Biotek; Norcross, GA, USA) on Kingfisher flex (Thermo Fisher Scientific; Waltham, MA, USA). Briefly, tick leg or mice brain or spleen tissues were placed in tubes containing 250 µl PBS-G (PBS with 0.5% gelatin, 30% rabbit serum and 1% 100× antibiotic–antimycotic [10,000 μg/ml of streptomycin and 25 μg/ml of amphotericin B]) and a stainless-steel ball bearing and macerated using a mixer mill. A 50-µl aliquot of homogenate was used for the RNA extraction following the protocol of the kit’s manufacturer (Omega Biotek). RT-qPCR was performed on a Bio-Rad C1000 Touch system with a CFX96 optical module using the Reliance One-Step Multiplex RT-qPCR Supermix (Bio-Rad Laboratories, Inc.; Hercules, CA, USA) with the following cycline parameters: reverse transcription at 55 °C, 15 min; then 95 °C, 10 min; followed by 95 °C/15 s and 60 °C/30 s for 40 cycles. The primer/probe set used to target the 3′-untranslated regions of POWV II was as previously reported [21 ]. Quantification of POWV II genome equivalents was performed using a previously developed assay with the same reagents and cycling parameters as described above [22 (link)].