CVB4-positive isolates and GZ-HFM01 were cultured and harvested for genomic sequencing. Viral RNA was extracted using a TaKaRa Mini BEST Viral RNA/DNA Extraction Kit Ver. 5.0 (TaKaRa, Dalian, China), in accordance with the manufacturer’s instructions. Genomes were analyzed by next-generation sequencing using an Illumina NovaSeq 6000 sequencer in accordance with a protocol from Synbio-Technologies (paired-end, 2 × 150 bp). The complete genomes of CVB4 isolates were assembled using CLC Genomics Workbench 11.0 (Qiagen, Germantown, MD, USA). The complete genomes of the CVB4 isolates and GZ-HFM01 were annotated and uploaded to the GenBank database.
Takara minibest viral rna dna extraction kit ver 5
Manufactured by Takara Bio
Sourced in China, Japan
The TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 is a laboratory equipment product designed to extract viral RNA and DNA from various sample types. It utilizes a silica-based membrane technology to efficiently capture and purify viral nucleic acids.
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Lab products found in correlation
Clc genomics workbench 11, by Qiagen (3 mentions)
Novaseq 6000 sequencer, by Illumina (2 mentions)
Rna easy fast plant tissue kit, by Tiangen Biotech (2 mentions)
Cdna synthesis kit, by Vazyme (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Icycler thermal cycler, by Bio-Rad (1 mentions)
Ex taq probe q pcr, by Takara Bio (1 mentions)
Rnase if, by New England Biolabs (1 mentions)
Dnase 1, by New England Biolabs (1 mentions)
Dnase buffer, by New England Biolabs (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
Prism 9, by GraphPad (1 mentions)
11 protocols using takara minibest viral rna dna extraction kit ver 5
1
CVB4 Genome Sequencing and Annotation
2
Quantitative PCR for Gene Expression
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Total RNA from the cells was isolated with RNAiso Plus (#9109, TaKaRa) and reverse transcription of 1 μg of RNA was conducted with the cDNA synthesis kit (#R212, Vazyme) according to the manufacturer’s instructions. For extraction of viral RNA in cell culture supernatant, TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 was used (#9766, TaKaRa). Quantitative PCR was performed as previously described.60 (link) The threshold cycle (Ct) for the indicated genes was normalized to that of the housekeeping gene GAPDH and shown as the relative mRNA level. Gene-specific primers used in this study are listed in Supplementary information, Table S2 .
3
Adenovirus Infection in Tree Shrew Cells
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Two healthy tree shrews were sedated and humanely euthanized to collect their lung, trachea, and kidney tissues. Then the primary cells from the fresh tissue samples were isolated and cultured as described previously [30 (link)]. The primary cell monolayers in 24-well plates were infected with 100 TCID50 of HAdV-55, -7, -14, or rAd3-EGFP, for 1 h at 37°C. Then the monolayers were washed twice with MEM and incubated for 7 d. The infected cells were then observed daily under microscopy to check the cytopathic effect (CPE) or green fluorescent signals.
To detect viral replication, the infected cells and the culture medium were harvested at 24, 48, 96, and 144 h post-infection (h.p.i). The viral genomic DNA was extracted with a TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver. 5.0 (TaKaRa, Dalian, China) according to the manufacturer’s instructions, and the viral genomic DNA copies were determined by quantitative PCR (qPCR), as described below.
To check whether the primary cells produced infectious progeny viruses after adenovirus infection, cells were harvested at 48 or 72 h.p.i and subjected to three freeze–thaw cycles and centrifuged at 10,000 × g for 30 min to remove cell debris. The virus supernatant was serially diluted to infect HEp-2 and the CPE was analyzed between 2 and 7 d by microscopy to calculate TCID50 according to the Spearman & Kärber algorithm method [45 (link)].
To detect viral replication, the infected cells and the culture medium were harvested at 24, 48, 96, and 144 h post-infection (h.p.i). The viral genomic DNA was extracted with a TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver. 5.0 (TaKaRa, Dalian, China) according to the manufacturer’s instructions, and the viral genomic DNA copies were determined by quantitative PCR (qPCR), as described below.
To check whether the primary cells produced infectious progeny viruses after adenovirus infection, cells were harvested at 48 or 72 h.p.i and subjected to three freeze–thaw cycles and centrifuged at 10,000 × g for 30 min to remove cell debris. The virus supernatant was serially diluted to infect HEp-2 and the CPE was analyzed between 2 and 7 d by microscopy to calculate TCID50 according to the Spearman & Kärber algorithm method [45 (link)].
4
Genomic Characterization of HAdV-7 Isolates
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HAdV-7-positive samples were cultured and harvested for genomic or capsid protein genes (hexon/penton base/fiber) sequencing. Viral DNA was extracted using a TaKaRa Mini BEST Viral RNA/DNA Extraction Kit Ver. 5.0 (TaKaRa), in accordance with the manufacturer's instructions. Genomes were analysed by next-generation sequencing using an Illumina NovaSeq 6000 sequencer, in accordance with a protocol from Synbio-Technologies (paired-end, 2 × 150 bp). The complete genomes of HAdV-7 isolates were assembled using CLC Genomics Workbench 11.0 (Qiagen, Germantown, MD, USA). Capsid protein genes (hexon/penton base/fiber) sequencing analyses were performed via Sanger sequencing using primers shown in Supplementary Table S2 . The complete genomes and hexon/penton base/fiber genes of the HAdV-7 isolates were annotated and uploaded into the GenBank database.
5
Robust RNA Extraction from Seed Samples
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Total RNA extraction from large seed batches (150 mg of powdered seed material) was performed using the RNA Easy Fast Plant Tissue Kit (Code DP452, TianGen BiotechCO., LTD., Beijing, China) following the manufacturer’s instructions. Total RNA extraction from single seed powder sample (3 mg) was performed using the TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver. 5.0 (Code 9766, TaKaRa Co., Ltd, Dalian, China)), following the manufacturer's instructions.
6
Molecular Characterization of Severe Pediatric Pneumonia
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Throat swab samples or bronchoalveolar lavage fluid (BAF) were collected from hospitalized pediatric patients with severe or critical pneumonia. The samples were collected and refrigerated at 2–8 °C in a viral transport medium before being transported on ice and analyzed immediately, or stored at −80 °C until analysis. Viral genomic DNA was extracted using a TaKaRa Mini BEST Viral RNA/DNA Extraction Kit Ver.5.0 (TaKaRa, Dalian, China), according to the manufacturer’s instructions and then tested for HAdV using the TaqMan real-time PCR kit (Guangzhou HuYanSuo Medical Technology Co., Ltd., Guangzhou, China) as previously reported (13 (link)). HAdV-positive samples were further characterized at the molecular level by PCR amplification of the hypervariable regions of the hexon gene (14 (link), 15 (link)). Mycoplasma pneumoniae and respiratory viruses detected by a real-time PCR assay on throat swab sample or BAF. Bacteria and fungi detected by blood, sputum, BAF or bone marrow culture.
7
Viral DNA Extraction and qPCR Quantification
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The viral DNA from various samples was extracted using the TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 (TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions. q-PCR quantification was performed using pre-mix Ex Taq (probe q-PCR) (TaKaRa, Tokyo, Japan) with a BIO-RAD iCycler Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primers and probe for the detection of UL30 in this study were previously reported [27 (link)]. The primers and probe were as follows: forward primer 5′-CATCACCGACCCGGAGAGGGAC-3′, reverse primer, 5′-GGGCCAGGCGCTTGTTGGTGTA-3′, and probe 5′-CCGCCGAACTGAGCAGACACCCGCGC-3′. Samples that read five or fewer copy numbers were reported as negative.
8
Viral Nucleic Acid Extraction from Fecal Samples
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Tubes with fecal samples in maintenance medium were vigorously vortexed to homogenize the samples. Samples of each species from the same site were then pooled by adding 1 ml from each maintenance medium sample into a fresh sample tube. Six pooled samples, classified by species and collection site, were then centrifuged twice at 13,000×g for 20 min at 4 °C. The supernatant was then filtered through a 0.22 μm filter (Millipore Inc., USA) twice to remove eukaryotic cell- and bacterium-sized particles. Filtrates were concentrated in a 100-kDa Pellicon II filter (Millipore Inc., USA). To remove the remaining extra-cellular nucleic acids, the filtrates were treated with DNaseI (NEB, USA) and RNaseIf (NEB). 375 μl of the filtrate from each pooled sample was then digested in a mixture of 3 U DNase and 25 U RNase at 37 °C for 90 min in 10× DNase buffer (NEB). The viral DNA and RNA were simultaneously extracted using a TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 (TaKaRa Inc., Japan).
9
Genomic Analysis of Adenovirus Type 21
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HAdV-21-positive samples were cultured and harvested. Viral genomic DNA was extracted using a TaKaRa Mini BEST Viral RNA/DNA Extraction Kit Ver.5.0 (TaKaRa) according to the manufacturer’s instructions. Next-generation sequencing was conducted with Illumina NovaSeq 6000 sequencer following a protocol from Synbio-Technologies (paired-end, 2 × 150 bp). The complete genome of HAdV-21 was assembled using CLC Genomics Workbench 11.0 (Qiagen, Redwood City, CA, United States). The complete genomes of the HAdV-21 isolates were annotated based on the annotation of HAdV-21 strain BB/201903 (accession no. MN686206 ) (Ye et al., 2020 (link)). Complete genome sequences were logged in the GenBank database.
10
Kinetics of Adenovirus Replication in A549 Cells
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The virus growth kinetics were observed in A549 cells. In brief, 24 h before infection with HAdV-55, rAd55, and rAd55-dE3-EGFP, A549 cells were seeded in 24-well plates at a density of 2 × 105 cells per well. Cells were washed three times with PBS and inoculated with viruses (MOI = 0.05 TCID50 units/cell). After 2 h incubation, the supernatant was removed and cells were washed three times with PBS and supplied with the medium as described above. Supernatants and cells were collected at 2, 24, 48, 72, 96, and 120 h post-infection separately. Viral genomic DNA was extracted with a TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 (Takara, Dalian, China) according to the manufacturer’s instructions. The replication of viral DNA in cells was measured by qPCR with adenovirus-specific primers qHAdV-UniF/-UniR and probes qHAdV-UniProbe (Table 1 ). The viral genome copy number based on the plasmid standard curve method was calculated and plotted using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). The TCID50 mL−1 titer in A549 cells was determined using the Reed–Muench method.
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