Minibest viral rna dna extraction kit

Manufactured by Takara Bio

Sourced in China, Japan

The MiniBEST Viral RNA/DNA Extraction Kit is a laboratory product designed for the extraction and purification of viral RNA and DNA from various sample types. It utilizes a silica-based membrane technology to efficiently capture and concentrate viral nucleic acids.

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RNA extraction kit (351 citations) DNA extraction kit (50 citations) MiniBEST RNA extraction kit (8 citations)

69 protocols using minibest viral rna dna extraction kit

Tick-Borne Pathogen Identification Protocol

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Nucleic acid extraction methods have been previously reported [16 (link)]. For NGS, 20 ticks from each location were pooled. The remaining
184 ticks were ground separately for further virus identification and isolation. All ticks
were flushed with phosphate buffered solution (PBS) before extraction to remove pathogens
on their surface. One ml of PBS was added to the pooled ticks, which were ground using a
glass pestle. The resulting homogenate was centrifuged at 12,000 g for 10
min at 4°C. The sediment was used to identify the tick type, while the supernatant was
filtered through 0.22 μm Pellicon II filters (Millipore, Billerica, MA, USA) to remove
cell debris and bacteria. Two hundred microliters of the filtered supernatant was used to
extract nucleic acids using the MiniBEST viral RNA/DNA extraction kit (TaKaRa Bio, Dalian,
China) following the manufacturer’s instructions. The remaining filtered supernatant was
stored at −80°C. The sediment of ticks and whole blood of host animals were lysed and
nucleic acids were extracted using a MiniBEST viral RNA/DNA extraction kit (TaKaRa Bio,
Dalian, China). Finally, 30 μl of the extracted RNA mixture was obtained for cDNA library
preparation or reverse transcription and 30 μl of the extracted DNA was obtained for
identification of tick species.

HE T., ZHU C., LI Z., AI L., HU D., WANG C., LI F., YANG X., LV H., CHEN W., QIAN H., TAN W, & WANG C. (2022). Virome analysis of ticks in Zhoushan Archipelago, China. The Journal of Veterinary Medical Science, 84(6), 847-854.

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Comprehensive Hantavirus Detection Protocol

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Total RNA and DNA were extracted from ~20 mg of liver tissue samples or 200 μL aliquot of serum samples using the MiniBEST Viral RNA/DNA Extraction Kit (TaKaRa, Kusatsu, Japan). Given the previous report that lung was the most preferred organ for the detection of SEOV (1 (link)), total RNA and DNA were also extracted from ~20 mg of lung tissue samples from animals with SEOV-positive liver tissue samples using the MiniBEST Viral RNA/DNA Extraction Kit (TaKaRa).
A nested PCR assay designed based on a conserved region within the L segment of hantaviruses was used to detect currently known and possible novel members within the genus Orthohantavirus (23 (link)). The amplified products were separated on a 1.5% agarose gel, and positive samples were sent to the Beijing Genomics Institute (Shenzhen, China) for sequencing. All the PCR screening sequences were aligned with the sequences in the NCBI nucleotide sequence (NCBI NT) database (https://www.ncbi.nlm.nih.gov/nucleotide) by using BLASTN.

He W., Fu J., Wen Y., Cheng M., Mo Y, & Chen Q. (2021). Detection and Genetic Characterization of Seoul Virus in Liver Tissue Samples From Rattus norvegicus and Rattus tanezumi in Urban Areas of Southern China. Frontiers in Veterinary Science, 8, 748232.

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SARS-CoV-2 Antiviral Efficacy of FB2001

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FB2001 (batch #: D20071301) was provided by Frontier Biotechnologies Inc. (Nanjing, Jiangsu, China). To test the antiviral effect of FB2001 on SARS-CoV-2 in the presence of different concentrations of human serum, Vero E6 cells were inoculated with SARS-CoV-2 (2019-nCoV-WIV04) at MOI of 0.01 in a BSL-3 laboratory. The medium was removed at 1 h after incubation. Cells were washed with PBS, followed by additional 24-h incubation with different concentrations of FB2001 (16.60, 5.55, 1.85, 0.62, and 0.21 μM) diluted in DMEM (200 μL/well) containing different concentrations of human serum (10%, 20%, 30%, 40% and 50%; H3667; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C. DMSO was used as a negative control. The cell supernatant was collected at 24h after incubation. The viral RNA in the supernatant was extracted using a MiniBEST viral RNA/DNA extraction kit (#9766; Takara, Japan).
The content of DMSO in the final testing concentration did not exceed 0.2%. This concentration of DMSO had no effect on the replication of SARS-CoV-2.

Shang W., Dai W., Yao C., Xu L., Tao X., Su H., Li J., Xie X., Xu Y., Hu M., Xie D., Jiang H., Zhang L, & Liu H. (2022). In vitro and in vivo evaluation of the main protease inhibitor FB2001 against SARS-CoV-2. Antiviral Research, 208, 105450.

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Viral RNA Extraction from Plasma

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HIV-1 RNA was extracted from 200 ul of plasma using the MiniBEST Viral RNA/DNA Extraction Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions.

Bao Y., Tian D., Zheng Y.Y., Xi H.L., Liu D., Yu M, & Xu X.Y. (2014). Characteristics of HIV-1 Natural Drug Resistance-Associated Mutations in Former Paid Blood Donors in Henan Province, China. PLoS ONE, 9(2), e89291.

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SARS-CoV-2 Neutralization Assay

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Neutralization assay for live SARS-CoV-2 virus was performed at the Biosafety Level 3 Laboratory at Wuhan Institute of Virology, CAS. SARS-CoV-2 virus (nCoV-2019BetaCoV/Wuhan/WIV04/2019)²⁸ (link) with MOI = 0.01 (about 400 PFU) was incubated with serial dilutions of mouse serum or macaque serum at 37°C for 1 h before being added to 4x10⁴ Vero-E6 cells that have been seeded in a 48-well plate the night before. The infection was maintained for 1 h at 37°C, and the supernatants containing the virus were removed and replaced with fresh medium. The supernatants were then collected 24 h after infection and examined for viral loads. Viral RNA was isolated with MiniBEST Viral RNA/DNA Extraction Kit (Takara, Japan) as described in the instruction, and cDNA was transcribed with PrimeScript RT reagent Kit with gDNA Eraser (Takara). Viral copies were quantified by qPCR from viral cDNA with a pair of primers targeting the S gene of SARS-CoV-2. (5′-CAATGGTTTAACAGGCACAGG-3′, 5′-CTCAAGTGTCTGTGGATCACG-3′). Half-maximal inhibitory concentrations (IC₅₀) of the serum was determined by curve fitting using GraphPad Prism and used to represent the neutralization titer.

Guo C., Peng Y., Lin L., Pan X., Fang M., Zhao Y., Bao K., Li R., Han J., Chen J., Song T.Z., Feng X.L., Zhou Y., Zhao G., Zhang L., Zheng Y., Zhu P., Hang H., Zhang L., Hua Z., Deng H, & Hou B. (2021). A pathogen-like antigen-based vaccine confers immune protection against SARS-CoV-2 in non-human primates. Cell Reports Medicine, 2(11), 100448.

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SARS-CoV-2 Inhibition Assay in Vero-E6 Cells

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Live SARS-CoV-2 (isolate Wuhan-Hu-1) inhibition assays were performed by the Wuhan Institute of Virology, Chinese Academy of Sciences, as described [26] (link). All infection experiments were performed in a biosafety level-3 laboratory. Briefly, Vero-E6 cells were pretreated with the test compounds for 1 h and then infected with SARS-CoV-2 (MOI = 0.001) for 1 h. Subsequently, fresh medium with the test compounds was added, and viral total RNA in the supernatants was extracted using a MiniBEST Viral RNA/DNA Extraction Kit (Takara, Tokyo, Japan) after 24 h of incubation. S gene copies were quantified from viral cDNA by a standard curve using an ABI 7500 (Takara TB Green Premix Ex Taq II, Tokyo, Japan) after reverse transcription. DMSO and CQ were used as negative and positive controls, respectively. IC₅₀ values are expressed as the mean ± SD from independent experiments and calculated using GraphPad Prism 8 software.

Li Y., Wu Y., Li S., Li Y., Zhang X., Shou Z., Gu S., Zhou C., Xu D., Zhao K., Tan S., Qiu J., Pan X, & Li L. (2022). Identification of phytochemicals in Qingfei Paidu decoction for the treatment of coronavirus disease 2019 by targeting the virus-host interactome. Biomedicine & Pharmacotherapy, 156, 113946.

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Viral Nucleic Acid Extraction and cDNA Synthesis

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Viral RNA and DNA were isolated using the MiniBEST Viral RNA/DNA Extraction Kit (TaKaRa, Dalian, China) following respective instructions. The amounts of viral RNA was measured using a Biophotometer plus (Eppendorf, USA). The extracted RNA was template used to synthesized cDNA with reverse transcription using random primers in a total volume of 10 μL according to the instructions of the PrimeScript™ RT Master Mix (Takara, Dalian, China). All viral DNA and cDNA were stored at − 70 °C for further employment.

Wang H., Hou P., Zhao G., Yu L., Gao Y.W, & He H. (2018). Development and evaluation of serotype-specific recombinase polymerase amplification combined with lateral flow dipstick assays for the diagnosis of foot-and-mouth disease virus serotype A, O and Asia1. BMC Veterinary Research, 14, 359.

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Dual PCR Detection of Hepatitis E Virus

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Total RNA and DNA were extracted from ~ 20 mg of liver tissue samples or 200 μL aliquot of fecal samples by using the MiniBEST Viral RNA/DNA Extraction Kit (TaKaRa, Kusatsu, Japan). We simultaneously used two polymerase chain reaction (PCR) methods for detecting HEV, one of which was a nested broad-spectrum PCR to amplify a 334 bp ORF1 fragment of all known HEV strains [8 (link)], while the other was a nested PCR to amplify the ORF1–ORF2 region of rat HEV [23 (link)]. The amplified products were separated on a 1.5% agarose gel, and the positive samples were sent to the Beijing Genomics Institute (Shenzhen, China) for sequencing.

He W., Wen Y., Xiong Y., Zhang M., Cheng M, & Chen Q. (2018). The prevalence and genomic characteristics of hepatitis E virus in murine rodents and house shrews from several regions in China. BMC Veterinary Research, 14, 414.

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Viral Nucleic Acid Extraction and PCR Detection

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RNA and DNA were extracted from the supernatants of fecal samples using the MiniBEST Viral RNA/DNA Extraction Kit, according to the manufacturer’s instructions (TaKaRa, Kusatsu, Japan). RT-PCR was conducted using a pair of previously described primers (UNIV-kobu-F and R) to amplify a 216-nt fragment located in the conserved 3D region, encoding the RNA-dependent RNA polymerase [18 (link)]. To amplify a longer 3D region, nucleotide primer sequences were designed as follows: 3D-F (5′-CTCCGGTTGTGTGSCCACTTCC-3′; 7492 nt) and 3D-R (5′-AGCACTGCTCGCGCACTTTCAT-3′; 7952 nt), to amplify a 461 bp region. For 3D gene-positive samples, a 831 bp region of the VP1 gene was amplified by PCR using the VP1-F/VP1-R primer pair [13 (link)]. All PCR conditions were 94 °C, 3 min; 94 °C,30s, 56 °C,1.5 min, 72 °C,1 min, 40 cycles; 72 °C, 10 min. Amplicons were analyzed on 1% agarose gels followed by UV light trans-illumination.

You F.F., Zhang M.Y., He H., He W.Q., Li Y.Z, & Chen Q. (2020). Kobuviruses carried by Rattus norvegicus in Guangdong, China. BMC Microbiology, 20, 94.

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Viral RNA Extraction and Sequencing Protocol

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The detailed procedures of RNA extraction, amplification and sequencing were performed as previously described.²⁴ (link) Briefly, total viral RNA was extracted from 140 μL serum samples using the MiniBest Viral RNA/DNA Extraction Kit (TaKaRa Bio, Kusatsu, Japan); reverse transcription was performed using a PrimeScript RT Reagent Kit with Genomic DNA Eraser (TaKaRa) following the manufacturer's instructions; portions of the HCV NS3, NS5A and NS5B genes were amplified using PrimeSTAR HS DNA Polymerase (TaKaRa) in a nested PCR. The primers used in this study are shown in Table S1. The extension time in the PCR cycle was modified by the length of the target gene fragment. The DNA product was sequenced by direct Sanger sequencing using an automatic sequencer (ABI Prism 3100 genetic analyser; Thermo Fisher Scientific, Waltham, MA, USA).

Liu X., Chen Z., Tang Q, & Hu P. (2022). Phylogenetic signature and prevalence of natural resistance-associated substitutions for hepatitis C virus genotypes 3a and 3b in southwestern China. Journal of Virus Eradication, 8(2), 100071.

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