Qiaamp rna kit

We described details of the laboratory procedures in our previous work published elsewhere [7 (link)]. We performed nucleic acid isolation from DBS using the QIAamp RNA kit (Qiagen GmbH, Germany), according to manufactures instructions. Briefly, we excised DBS samples by hand using scissors and placed two of the spots in lysis buffer and incubated at room temperature with gentle rotation for 30 min, subsequent washes and a final elution of 50μl.

Total RNA was isolated using QIAamp RNA Kit (QIAGEN catalog #52304) for blood samples or TRIzol reagent (Invitrogen™ catalog #15596-026) for solid samples. The quality of total RNA was assessed using a Bioanalyzer 2100 (Agilent Technologies, USA). The first-strand cDNA was synthesized with SMARTer PCR cDNA Synthesis Kit (Clontech catalog #634925) and the 3′ SMART CDS Primer IIA (5′-AAGCAGTGGTATCAACGCAGAGTACT(₃₀)N₋₁N-3′), according to the manufacturer’s protocol. The second-strand cDNA was synthesized and the product was further amplified with PrimeSTAR HS DNA Polymerase (TAKARA catalog #R010A) and Primer M1 (5′-AAGCAGTGGTATCAACGCAGAGT-3′) for 12–15 PCR cycles (15 s at 95 °C, 30 s at 65 °C, and 6 min at 68 °C). cDNA product was purified with TaKaRa MiniBEST DNA Fragment Purification Kit (Catalog #9761) and dissolved with sterile RNAse-free water to a final concentration of 100 ng/μl.

Orthohantavirus nucleic acids were extracted from host animal liver and lung tissues as well as from patient blood samples using the QIAamp RNA kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The viral RNA is stored in a −80 °C freezer until use. RT-PCR amplification of the L-gene fragment of orthohantavirus was performed on the extracted RNA with the orthohantavirus universal primer: HAN-L-F1: 5′-ATGTAYGTBAGTGCWGATGC-3′, HAN-L-R1: 5′-AACCADTCWGTYCCRTCAT-C-3′; HAN-L-F2: 5′-TGCWGATGCHACIAARTGGTC-3′, and HAN-L-R2: 5′-GCRTCRTCWGARTGRTGDGCAA-3′ [24 (link)], using the FastKing One-Step RT-PCR Kit (TIANGEN BIOTECH, Beijing, China) and Premix Ex Taq polymerase (TaKaRa Bio Inc, Shiga, Japan) according to the manufacturer’s instructions. Nested RT-PCR was performed with a total reaction system of 25 μL. Reaction conditions: reverse transcription at 42 °C for 30 min, pre-denaturation at 95 °C for 3 min, followed by denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, extension at 72 °C for 1 min for 35 cycles, followed by supplemental extension at 72 °C for 30 s, and finally cooling at 12 °C for 1 min to be used. PCR products were extracted on a 1.2% agarose gel for electrophoretic analysis and observed in a UV gel imager for the presence or absence of target bands of specific molecular weight size (about 412 bp), and gel images were saved.

Ribonucleic acid (RNA) was extracted from the collected concentrates using a QIAamp RNA kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions. The RNA extracts were used as templates in the RT-PCR assay for SARS-CoV-2 detection using the VERI-Q nCoV-OM detection kit. Each PCR reaction mix consisted of 10 μl of 2x One-Step RT-PCR Master mix, 1 μl Primer/Probe mixture, 1 μl internal positive control, and 8 μl of the RNA template in a total volume of 20 μl. RNA-positive and RNA-negative (nuclease-free water) controls were included in each PCR run. The PCR was performed in an ABI 7500 RT-PCR system (Life Technologies, Grand Island, NY, USA). The cycling conditions were set at 50°C for 10 minutes in 1 cycle, 95°C for 3 minutes in 1 cycle, 95°C for 9 seconds, and 58°C for 30 seconds in 45 cycles.

After RNA extraction with the Qiagen QiaAmp RNA kit, one step RT-dPCR reactions were carried out as described in [9 (link)]. For N gene quantification, a duplex assay was performed using China N and SarE genes with primers and probe concentrations of 400 nM and 200 nM, respectively, and optimized by varying the annealing temperature. The sequence of China N and SarE primers is described in [9 (link)–11 (link)], Droplet generation was conducted as described in [9 (link)]. The PCR was performed under the following conditions: 60 min reverse transcription at 50 ºC and 10 min enzyme inactivation at 95 ºC followed by 45 cycles using a two-step thermal profile of 30 s denaturation at 95 ºC and 60 s annealing and extension at 55 ºC; followed by 10 min at 98 ºC and then cooled to 4 ºC. Following thermal cycling, the PCR plates were transferred to a droplet reader (QX200 BioRad, USA) and the data analyzed using QuantaSoft Analysis Pro 1.0.596 (BioRad, USA).