Viral gene spin viral dna rna extraction kit

A total of 41 F. rhynchophylla plant samples from various regions of Korea were collected in different time periods (Table 1). All samples were asymptomatic and collected: Jinju (n = 4 in March 2019; n = 8 in September 2019), Busan (n = 9 in October 2019; n = 6 in May 2020), Pocheon (n = 6 in September 2019), Jeonnam (n = 2 in September 2019), Yeongdong (n = 3 in September 2019), and Daegu (n = 2 in September 2019) (Figure 1). No insects were found or collected from any of these 41 plants. All samples were stored at −20 °C until processing. All leaf samples were sterilized by using 70% ethanol for 20–30 s and allowed to dry off from the ethanol with the air flow under the fume hood. Total DNA was extracted from leaf tissue samples using either a Viral Gene-Spin Viral DNA/RNA Extraction Kit (iNtRON Biotechnology) or a cetyl trimethylammonium bromide (CTAB)-based extraction protocol, following the manufacturer’s instructions [51 (link)]. Total DNA from each sample was used in RCA reaction with the TempliPhi™ kit (GE Healthcare, Chicago, IL, USA), as described by Shepherd et al. [52 (link)].

A Viral Gene-spin^™ Viral DNA/RNA Extraction Kit (iNtRON Biotechnology; Seoul, Korea) was used for the RNA extraction from homogenized samples, following the manufacturer’s instruction. M-MLV enzyme (Invitrogen; Carlsbad, CA, United States) was used to synthesize cDNA. In total, 20 μL of reagents, consisting of 4 μL of 5X M-MLV buffer, 1 μL of dNTP, 1 μL of random primer (Invitrogen; Carlsbad, CA, United States), 1 μL of MMLV reverse transcriptase (Invitrogen; Carlsbad, CA, United States), and 9 μL of distilled water, were mixed with 4 μL of RNA. Then, the mixture was placed in the thermal machine at 25°C for 10 min, 37°C for 1 h, and 65°C for 10 min.
Identification of the TMUV genome was carried out using PCR to detect the target 400 bp gene, with the TV-3F and TV-3R primers (Table 1), as described elsewhere (13 (link)). PCR was performed using GoTag^™ Green Master Mix (Promega) at 94°C for 5 min, 40 cycles of 94°C for 30 s, then 55°C for 30 s and 72°C for 30 s, with an extension step at 72°C for 10 min. The PCR products were loaded in 1.5% agarose gels for electrophoresis and then the gel was photographed under UV light.

RNA was extracted from harvested allantoic fluid using a Viral Gene-spin™ Viral DNA/RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Gyeonggi, Republic of Korea) and cDNA was synthesized by a TOPscript™ cDNA Synthesis kit (Enzynomics, Daejeon, Republic of Korea). Full genomes were amplified using universal primer sets and sequenced as previously described [51 (link),65 (link)]. The nucleotide and amino acid sequences of SL20 were compared with other genes in the Genbank using BLAST. The BioEdit program (v7.2.5) was used for nucleotide sequence translation, amino acid comparison, and calculation of amino acid identity. The variable amino acids of HA and NA were located in the 3D structures using PyMOL program and amino acids closely located in the interfaces of polymerase was visualized using 3D view of RCSB PDB “https://www.rcsb.org/3d-view (accessed on 12 January 2023).

Viral RNA was extracted from 150 µL of OP and CL swab sample suspension using a Viral Gene-Spin viral DNA/RNA extraction kit (iNtRon Biotechnology, Korea). Real-time reverse-transcription polymerase chain reaction (rRT-PCR) targeting the Matrix gene was performed as previously described [16 (link)]. For extrapolation of cycle threshold (Ct) values to infectious units, serial dilutions of the inoculum were calculated as EID₅₀/mL. Corresponding virus doses were analyzed by rRT-PCR. The resulting calibration curves were highly predictable (R² = 0.99) and used for converting Ct values to EID₅₀/mL.

Total fecal DNA was extracted using the manufacturer’s protocol of the QIAamp DNA Feces mini kit (Qiagen, Germany) with two modifications of the optimized lysis buffer (500 mM NaCl, 50 mM Tris–HCl (pH 8.0), 50 mM EDTA (pH 8.0), and 4% sodium dodecyl sulfate) from the previous paper (Ku and Lee, 2014 (link)) and an additional 10 min boiling step. After homogenization of 0.25 g feces with 1 mL optimized lysis buffer, the fecal suspension was incubated for 10 min in boiling water to lyse the cells. In the final step, fecal DNA was eluted using 100 μL of molecular water (Welgene, South Korea). The extracted total fecal DNA was quantified using NanoDrop 2000 (Thermo Scientific, United States). For extraction of total bacteriophage DNA from collected sewage samples, sewage components and bacteria were removed by centrifugation at 11,000 × g for 30 min and subsequent filtration with Acrodisc syringe filters (Pall, United States; pore size = 0.45 μm). After their removal, the filtrate solutions (final volume, 10 mL) were used for total bacteriophage DNA extraction using the manufacturer’s protocol of the QIAamp DNA Blood Maxi Kit (Qiagen). After the food application tests, total bacteriophage DNA was extracted from homogenized food samples using the Viral Gene-spin Viral DNA/RNA Extraction Kit (Intron Biotechnology, South Korea) following the standard manual procedure.