Nucleic acid was extracted from cell culture supernatants with an AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit (Axygen, Union City, CA, USA), which was used in accordance with the manufacturer’s instructions. The RT-PCR reactions were performed with a PrimeScript™ One Step RT-PCR Kit Ver.2 (Takara, Dalian, China) to amplify the partial VP1 gene by using VP1 gene-specific primer pairs 222 and 224 [40 (link)]. These sequences of partial VP1 were uploaded into GenBank BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ ) for direct identification of enterovirus serotypes. The primers (Additional file 1 : Table S2) used for amplification of the complete genomes were based on the prototype strain, and were designed using a “primer-walking” strategy [41 ]. An ABI 3730XL automatic sequencer (Applied Biosystems, Foster City, CA, USA) was used to sequence the resulting DNA templates at Tsingke Biological Technology Co., Ltd., Beijing.
Axyprep body fluid viral dna rna miniprep kit
Manufactured by Corning
Sourced in United States, China
The AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit is a laboratory equipment product designed for the rapid and efficient extraction of viral DNA or RNA from various body fluid samples. The kit utilizes a silica-based membrane technology to purify the target nucleic acids, providing a simple and reliable method for sample preparation.
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56 protocols using axyprep body fluid viral dna rna miniprep kit
1
Enterovirus Genome Sequencing Protocol
2
Nucleic Acid Extraction from Biological Samples
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Tissue samples (0.1 g) were mixed with 1 mL of PBS, ground with a mortar and freeze-thaw three times. Mouth swabs were infiltrated with 1 mL of PBS, and the suspension was centrifuged at 1000 × g for 5 min at 4 °C to obtain the supernatant. Nucleic acid extraction from the blood, tissue, and mouth swabs was performed using the Axyprep Body Fluid Viral DNA/RNA Miniprep kit (Axygen, HangZhou, China). The extracted nucleic acid was obtained using the reverse transcription HiScript II 1st Strand cDNA Synthesis kit (+ gDNA wiper) (Vazyme, Nanjing, China) to obtain cDNA, which was stored at−80 °C for further use.
3
Quantification of Fowl Adenovirus in Tissues
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Total DNA was extracted from the liver, spleen, and kidney using an AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit (Axygen, Union City, CA, USA) according to the manufacturer’s instructions. The fragment in the L1 region of the hexon gene of FAdV-4 was used as the target gene, and the chicken ovotransferrin gene was used as a reference gene. Real-time PCR amplification in a 20 μL reaction mixture was performed with Premix Ex Taq (probe qPCR) (TaKaRa, Shiga, Japan) and a QuantStudio 5 system (Applied Biosystems, Foster City, CA, USA). As described above, the final viral load concentration was presented as the copy number per 106 cells [20 (link),21 (link)].
4
SARS-CoV-2 RNA Quantification by qRT-PCR
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Cell culture supernatants and Vero E6 cells were harvested for RNA extraction using the AxyPrep™ body fluid viral DNA/RNA Miniprep kit (Axygen, Cat No. AP-MN-BF-VNA-250, Hangzhou, Zhejiang, China) and AxyPrep™ multisource total RNA Miniprep kit (Axygene, Cat No. AP-MN-MS-RNA-250G) according to the manufacturer's instructions. Reverse transcription was performed with a Hifair II 1st Strand cDNA synthesis kit with gDNA digester (Yeasen Biotech, Cat:11121ES60, Shanghai, China), and qRT-PCR was performed using QuantStudio 1 Real-Time PCR detection system (Applied Biosystems, Foster City, CA, USA) with Hieff qPCRSYBR Green Master Mix (Yeasen Biotech, Cat:11202ES08, Shanghai, China) or two-step Taqman probe assay. The sequence information of primers used is listed in Supplementary Table 1. And the PCR products were inserted into T vector (Ruibo Xingke Biotech, Beijing, China) to generate the standard plasmid after sequencing confirmation. The standard curve was generated by determination of copy numbers from serially dilutions (103 − 109 copies) of the plasmid. qRT-PCR amplification of SYBR Green method was performed as follows: 95°C for 5 min followed by 40 cycles consisting of 95°C for 10 s, 55°C for 20 s, and 72°C for 31 s. And the Taqman method was performed as follows: 50°C for 2 min, 95°C for 10 min followed by 40 cycles consisting of 95°C for 10 s, 60°C for 1 min.
5
Viral Pathogen Detection in Waterfowl
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The livers and spleens from the dead ducks were collected, homogenized, and suspended in phosphate-buffered saline (PBS). After centrifugation, the supernatants were filtered through 0.22 μm filters and then extracted by AxyPrep Body Fluid Viral DNA/RNA MiniPrep Kit (AxyGen, Shenzhen, China) and stored at −80 °C. Then, they were subjected to PCR or RT-PCR for the detection of potential pathogens, including Goose parvovirus (GPV), duck Tembusu virus (DTMUV), egg drop syndrome virus (EDSV), duck hepatitis A virus type 1 (DHAV-1), Baiyangdian (BYD) virus, adenovirus, infectious bronchitis virus (IBV), H9N2 avian influenza viruses (AIV) and C. psittaci. The specific primer pairs of GPV [39 (link)] and others [40 (link)] were all the same as previous studies by our laboratory.
The virus was propagated in 12-day-old specific-pathogen-free embryonated Muscovy duck eggs (Wens, Yunfu, China), as described in previous studies [26 (link)]. The embryos were incubated at 37 °C for five to seven days and monitored daily. The virus titration with EID50 was determined in the same 12-day-old eggs by the Reed–Muench method [41 (link)].
The virus was propagated in 12-day-old specific-pathogen-free embryonated Muscovy duck eggs (Wens, Yunfu, China), as described in previous studies [26 (link)]. The embryos were incubated at 37 °C for five to seven days and monitored daily. The virus titration with EID50 was determined in the same 12-day-old eggs by the Reed–Muench method [41 (link)].
6
Quantifying ILTV Viral Replication
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Plaque tests and ILTV-specific RT-qPCR assays were used to measure the amounts of viral replication, as previously described (Qiao et al., 2020 (link)). AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit was used for genomic DNA extraction (Axygen, Union City, CA, USA). Using Luna Universal qPCR Master Mix (NEB, Ipswich, MA, USA), absolute RT-qPCR was carried out in accordance with the manufacturer’s instructions. pMD18-T plasmid containing gC genes of ILTV was used as standards. Three or more independent experiments were performed for each experiment.
7
Viral RNA Purification from Cell Culture
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The viral RNA was purified from 200 µL of cell culture supernatant using the AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit (Axygen Biosciences, EUA), following manufacturer’s recommendations. The RNA was eluted with 60 µL of elution buffer and stored at − 80 °C until use.
8
Toxin Protection Against Viral Infection
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This assay was carried out to evaluate whether the toxins could protect VERO E6 cells against viral infection. VERO E6 cells were seeded (2×105 cells/well) in a 24-wellsplate and incubated at 37°C for 24 h. The medium was then removed, and the cells were incubated with several concentrations of PLA2-CB or crotoxin (0.000004, 0.04, 0.08, 0.5, 1, and 2 ng/µL). After 1 h of incubation, the supernatant was removed, and cells were washed five times with PBS at room temperature. The cells were infected with 25–100 PFU of DENV-2 in 400 µL of L-15 for 1 h. The supernatant was removed, and 1 mL of L-15m supplemented with 2% FBS was added to the cells. After 72 h of incubation at 37°C, the cell supernatants were collected for viral RNA extraction using AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit (Axygen, USA) following the manufacturer's recommendations. Virus inhibition was evaluated by qRT-PCR, as mentioned above.
9
Viral DNA/RNA Extraction from Body Fluids
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An AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit (Axygen, CA, USA) was used according to the manufacturer's instructions. The quality and quantity of purified DNA were measured using spectrophotometry (NanoDrop 2000 Spectrophotometer, Thermo Scientific, Wilmington, USA).
10
Quantification of KSHV LANA Expression
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Viral DNA was collected and prepared from culture supernatant by using the AxyPrep™ Body Fluid Viral DNA/RNA Miniprep Kit (Axygen) according to the manufacturer's protocol and then was used to amplify the KSHV LANA gene by qPCR using primers in Table 1 .
Relative expression levels were calculated using the ∆∆CT method after normalization to protrudin (protrudin plasmid was added to supernatant as a control). Individual samples were assayed in duplication.
Relative expression levels were calculated using the ∆∆CT method after normalization to protrudin (protrudin plasmid was added to supernatant as a control). Individual samples were assayed in duplication.
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