A viral RNA extraction kit (TaKaRa Bio Inc., Shiga, Japan) was used to extract total viral RNA from infectious fluids in accordance with the manufacturer's instructions, and S1 genes from the three IBV isolates were amplified using S1Oligo5′ and S1Oligo3′ primers (Adzhar et al., 1997 (link)). Amplified PCR products were sequenced directly using sense and antisense primers and sequences were first analyzed with BLASTn (BLASTn: http://blast.ncbi.nlm.nih.gov/Blast.cgi ). S1 genes from 96 reference IBV strains were selected and used for phylogenetic analysis with those of our three strains. Phylogenetic analyses were performed using the maximum likelihood method with the general time-reversible nucleotide substitution model and bootstrap tests of 1000 replicates in the MEGA 6 program (Tamura et al., 2013 ). In addition, pairwise comparison was conducted using S1 genes from 96 references strains and our three isolates according to the results of both BLASTn and phylogenetic analysis.
Viral rna extraction kit
Manufactured by Takara Bio
Sourced in China
The Viral RNA Extraction Kit is a laboratory tool designed for the extraction and purification of viral RNA from various sample types. The kit utilizes a silica-based membrane technology to efficiently capture and isolate viral genetic material, making it suitable for applications that require high-quality viral RNA.
Automatically generated - may contain errors
Lab products found in correlation
Realuniversal sybr green premix, by Tiangen Biotech (2 mentions)
Primescript rt reagent kit with a gdna eraser, by Takara Bio (2 mentions)
One step primescript 3 rt pcr kit, by Takara Bio (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Tianscript 2 rt kit, by Tiangen Biotech (1 mentions)
Primescript rt pcr kit, by Takara Bio (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Dna extraction kit, by Tiangen Biotech (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
9 protocols using viral rna extraction kit
1
IBV S1 Gene Sequence Analysis
2
PEDV Genome Quantification by RT-qPCR
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The viral RNA from the cell suspensions was extracted using the Viral RNA Extraction Kit (#9766, TaKaRa, Shanghai, China), following the manufacturer’s instructions. The cDNA was obtained by RT-PCR with a PrimeScript™ RT Reagent Kit with a gDNA Eraser (TaKaRa, Shanghai, China). The absolute RT-qPCR assay used to quantify the PEDV genome was performed with RealUniversal SYBR Green Premix (Tiangen, Beijing, China). As an internal reference for the quantification of the PEDV copy numbers, the PEDV 186-fragment gene was cloned into the pMD19-T vector, and primers PEDV-186-F (5′-TACTAAGCGTAACATCCTGCC-3′) and PEDV-186-R (5′-GTAGTACCAATAACAACCGAAGC-3′) were employed.
3
SARS-CoV-2 Viral RNA Quantification
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Viral RNA quantification was performed using real-time qPCR targeting the SARS-CoV-2 subgenomic RNA transcript for the envelope (E) gene, as previously reported (Wölfel et al., 2020 (link)). Briefly, RNA was isolated from the tissue homogenates using Viral RNA Extraction Kit (Takara Bio, 9,766). Real-time qPCR was performed using the One Step PrimeScript III RT-PCR Kit (Takara Bio, RR600A) with the primer pairs E-Leader, E-reverse, and E-probe (Table 1 ). The cycling conditions were one cycle at 52°C for 5 min, then 95°C for 10 s, followed by 45 cycles at 95°C for 10 s and 60°C for 30 s. pUC19-2019-nCoV-E plasmid was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) as an E gene DNA standard and the inserted base sequence reported in a previous study was used (Wölfel et al., 2020 (link)). An E gene DNA sample was also run at the same time for conversion of cycle threshold value to genomic copies using the standard curve-based method.
4
RNA Extraction and PRRSV Gene Sequencing
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We extracted RNA according to the manufacturer's instructions from the isolated strain supernatant using the Viral RNA Extraction Kit (Takara). We designed the primers online according to the sequences of the complete gene sequence of PRRSV logged by Genbank (the relevant primer information is shown in the supplementary file). We generated complementary DNA (cDNA) following the supplier's instructions using Superscript III reverse transcriptase (Invitrogen, Waltham, MA, USA). We then used the cDNA in PCR amplification using twelve primers (A‐L) specific for the viral genes as listed in Table S1 . The reaction conditions were as follows: 95°C for 5 min; 30 cycles of 95°C for 30 s, 58°C for 30 s 72°C for 1 min; and a final extension at 72°C for 7 min. Furthermore, the products were Sanger sequenced in both directions by Sangon Biotech (Shanghai) Co., Ltd.
5
PRRSV Genomic Sequencing Protocol
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Approximately 5.0 g or 300 μL of each sample was homogenized separately in phosphate-buffered saline (PBS). According to the manufacturer’s instructions, viral RNA was extracted using the Viral RNA Extraction Kit (Takara, Dalian, China) and then the TIANScriptII RT Kit (TIANGEN BIOTECH (BEIJING) CO., LTD., Beijing, China) was used to acquire cDNA through reverse transcription. The primers of Nsp2 and ORF5 were designed based on the alignment of published PRRSV genome sequences obtained from the NCBI GenBank database (Supplementary Table S2 ). The cDNA was used for the PCR amplification of Nsp2 and ORF5. Then, the amplicons for each virus were submitted to Sangon Biotech Shanghai Co., Ltd., Shanghai, China for sequencing. The nucleotide sequences were determined for at least three independent cDNA clones.
6
Viral RNA Extraction and Genome Sequencing
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Viral RNA was extracted using a Viral RNA Extraction Kit (Takara, Dalian, China) according to the manufacturer’s instructions, followed by amplification using Prime ScriptTM RT-PCR Kit (Takara, Dalian, China). The complete virus genome was amplified using 10 pair primers (Supplementary Table S1 ) and sequenced by Dalian Takara Co., Ltd. The complete s genome of the isolate was assembled using DNAMAN software.
7
DNA and RNA Extraction and Preservation
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The DNA templates of MS, MG, P. multocida, E.coli and clinical samples were extracted following the instructions of the DNA extraction kit (Beijing Tiangen Biotech Co., Ltd., China). The RNA of NDV, IBDV, IBV, and ARV were extracted by viral RNA extraction kit (Takara Biotech Co., Ltd., Beijing, China), and reversely transcribed into cDNA by reverse transcription kit (Takara Biotech Co., Ltd., Beijing, China). All the DNA/cDNA samples were stored at −20°C.
8
Viral RNA Extraction and cDNA Synthesis
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Viral RNA extraction was performed using a viral RNA extraction kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. The extracted RNA was then reverse transcribed into complementary DNA (cDNA ) using a PrimeScript RT reagent kit (TaKaRa). PCR amplification was carried out using 2X Ex Taq Master Mix (Takara Bio). The amplified products were analyzed via 1.5% agarose gel electrophoresis.
9
Quantification of PEDV Genome by qRT-PCR
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Viral RNA from cell suspensions was extracted with a viral RNA extraction kit (#9766, TaKaRa, Shanghai, China) according to the instructions. The complementary DNAs (cDNA) were obtained by RT-PCR with a PrimeScript™ RT reagent Kit with a gDNA Eraser (TaKaRa, Shanghai, China). The absolute qRT-PCR for quantifying the PEDV genome was prepared with the RealUniversal SYBR Green Premix (Tiangen, Beijing, China) and The PEDV a 186-fragment gene was cloned into the pMD19-T vector and used as an internal reference for the quantification of PEDV copy numbers (copies/ul = 6.02 × 1023 × plasmids concentrations/(DNA Length × 660). Using the standard plasmid as a template, the qRT-PCR amplification after doubling dilution obtained the correlation between the Ct value and the virus copy number, and the standard curve y = −3.2511x + 39.778 (R2 = 0.9632), where y is the Ct value and x is Log10 (copy number). The primers sequences for a 186-fragment gene: PEDV-186-F: 5′-TACTAAGCGTAACATCCTGCC-3′; PEDV-186-R: 5′-GTAGTACCAATAACAACCGAAGC-3′.
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