Viral rna dna extraction kit

Growth curve analysis was performed as described previously (Ma et al., 2018 (link)). Briefly, the growth kinetics of US1 mutant viruses were compared to that of the parental strain. Cell cultures were infected at an MOI of 0.01 (multi-step assay) or 2 (single-step assay). After 2 h of adsorption, the cells were washed and then overlaid with DMEM containing 2% NBS. Supernatants and infected cells were separately harvested at 24, 48, 72, 96 h (multi-step assay) or 6, 12, 18, 24, 36 h (single-step assay) at successive intervals, and the amount of infectious virus was determined by plaque assay using DEF cells.
The viral dosage and sampling time points were the same as for the growth curve detection described above. First, the UL30 DNA fragment was amplified with primer P8, and the fragment diluted in a 10-fold gradient was used as a template followed by the addition of primer P9 and probe P10 for qPCR to construct a standard curve: Y = −4.262X+43.675. Primers P8, 9, and 10 used in this part are listed in Table 1. The viral DNA in the samples was extracted using a Viral RNA/DNA Extraction Kit (Takara, China). qPCR was performed after the addition of primer P9 and probe P10, and viral DNA copies were estimated at various time points using the standard curve. All of the analyses were performed independently in triplicate with the standard error.

Phage DNA was extracted and purified from phage lysates using the Viral RNA/DNA Extraction Kit (Ver.5.0, TaKaRa MiniBEST), and the genome was verified by 1% agarose gel electrophoresis and sent to Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China) for sequencing, and the results were analysed using GeneMarkS¹ and DIAMOND² to predict and annotate the protein-coding genes of the phage genome. The phage whole-genome sequence was uploaded to National Center for Biotechnology Information (NCBI) to obtain the serial number. According to the similarity of the genome sequence of MK54 to known sequences, then, create the map with genomic comparisons between analysed phage and reference phages selected from NCBI database as the most similar. We used CG View³ (Grant and Stothard, 2008 (link)) to construct the phage whole-genome map. The amino acid sequence of the main capsid protein and the large terminase subunit were used to construct a genetic phylogenetic tree using Molecular Evolutionary Genetics Analysis Version 7.0 (MEGA 7; Kumar et al., 2016 (link)).

Cervical samples from 114 adult women (18–65 of age), including 36 HPV16 positive, 18 HPV18 positive, and 60 negative controls (14 patients positive for other HR-HPVs and 46 healthy individuals) were collected from Nanjing Maternal and Child Health Hospital in 2022. All samples were analyzed and diagnosed using real-time quantitative PCR. The entire procedure was approved by the Medical Ethics Committee of Nanjing Maternal and Child Health Care Hospital (2022KY-006). DNA was extracted from clinical samples using a viral RNA/DNA extraction kit (Takara Bio.) according to the manufacturer's protocol. The DNA extracts were stored at −20°C until further use.

The culture supernatant from the BHK-21cells of rSVA-p16 at passage 3, 6, 10, and 15 (P3, 6, 10, and 15) was harvested for extracting viral RNA by a Viral RNA/DNA Extraction Kit (Takara, Dalian, China). The viral RNAs of P3, P6, P10, and P15 virus stocks were extracted and reverse transcribed into cDNA. The extracted RNA was used as a template for RT-PCR analysis using a High Fidelity One-Step RT-PCR Kit (Takara, Dalian, China). The forward/reverse primers, F1/R1 (Table 1), were designed for amplifying a 530-bp fragment. The RT-PCR reaction underwent 50 °C for 30 min, 94 °C for 2 min and then 30 cycles at 98 °C (10 s), 55 °C (5 s), and 72 °C (35 s). The plasmids p15A-SVA-p16, cDNA of wild-type SVA (wt-SVA-cDNA) and ddH2O were simultaneously subjected to PCR analysis as control groups using the F1/R1 primer pair. The PCR reaction contained 2 × PrimeSTAR Max Premix (Takara, Dalian, China) and underwent 30 cycles at 98 °C (10 s), 55 °C (5 s), and 72 °C (10 s). RT-PCR and PCR products were detected by agarose gel electrophoresis, followed by Sanger sequencing for analyzing the RT-PCR product. SVA-iLOV only needs to be detected by fluorescence observation.

Phenyl methane sulfonyl fluoride (PMSF; #ST505), proteinase K (#ST533), tritonX-100 (#P0096), WB and IP lysis buffer (#P0013), RIPA lysis buffer (#P0013D), DAPI staining solution (#C1005), 4% paraformaldehyde (PFA; #P0099), BeyoECL plus (#P0018S), enhanced BCA protein assay kit (#P0010S), and enhanced cell counting kit-8 (CCK-8; #C0041) were produced from Beyotime Biotechnology (Shanghai, China). TurboFect transfection reagent (#R0533), GST protein interaction pull-down kit (#21516), and OPP protein synthesis assay kits (#C10457) were obtained from Thermo Fisher Scientific. RNAiso plus reagent (#9108Q), viral RNA/DNA extraction kit (#9766), Prime Script RT reagent kit (#RR047B), and TB Green fast qPCR mix (#RR430A) were obtained from TAKARA (Dalian, China). Dual-luciferase reporter assay system (#E1910) was obtained from Promega (Madison, USA). Puromycin (#P8833), cholesterol (#C4951), cycloheximide (#239763-M), hexadimethrine bromide (#H9268), and L-α-Phosphatidylcholine (#P3556) were obtained from Sigma Aldrich. Chemically defined lipid concentrate (#11905-031) and advanced DMEM (#12491-015) were obtained from Invitrogen (Carlsbad, USA).

The pathogens used in this study, which included different AIV subtype reference strains, AIV field isolates and other avian pathogens, are listed in Table S1. All clinical swab samples were collected from the cloacae, larynges and tracheae of healthy chickens, geese and ducks. The sample treatment method was described previously^{34 (link)}. A viral RNA/DNA Extraction Kit (TaKaRa, Dalian, China) was used to extract genomic RNA/DNA from samples (200 μL of chicken embryo allantoic fluid or liquid cultures) per the manufacturer’s protocol. The extracted RNA was reverse transcribed to synthesise cDNA, and DNA was stored at −80 °C. All samples were manipulated inside a class-II biosafety cabinet in a biosafety level-2 laboratory.