For qRT–PCR, an “Anchor SARS-CoV-2 PCR Kit” (Anchor Diagnostics GmbH, Hamburg, Germany), “Xpert ® X-Press SARS-CoV-2” (Cepehid Inc., Sunnyvale, CA, USA) and “Alinity m SARS-CoV-2 Kit” (Abbott Molecular Inc., Des Plaines, IL, USA) were used. For the preparation of tissue samples, an “RTP DNA/RNA Virus Mini Kit” (Stratec Molecular GmbH, Berlin, Germany) and “RTP Pathogen Kit” (Invitek Molecular GmbH, Berlin, Germany (case 4)) were used. For antibody tests, an “Anti-SARS-CoV-2-Elisa” (Euroimmun AG, Lübeck, Germany) and “SARS-CoV-2 IgG-Assay” (Abbott Molecular Inc., Des Plaines, IL, USA) were used. All tests were performed according to the manufacturers’ instructions.
Rtp dna rna virus mini kit
Manufactured by Stratec
Sourced in Germany
The RTP DNA/RNA Virus Mini Kit is a compact and efficient laboratory equipment designed for the extraction and purification of viral nucleic acids (DNA and RNA) from various sample types. It provides a straightforward and reliable solution for the preparatory steps in viral detection and analysis workflows.
Automatically generated - may contain errors
Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Sars cov 2 igg assay, by Abbott (1 mentions)
Anti sars cov 2 elisa, by EUROIMMUN (1 mentions)
Abi 7500 qpcr machine, by Thermo Fisher Scientific (1 mentions)
Firepol master mix, by Solis BioDyne (1 mentions)
Abi stepone software, by Thermo Fisher Scientific (1 mentions)
Nanophotometer np80, by Implen (1 mentions)
Premix ex taq probe qpcr, by Takara Bio (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Innuprep virus dna rna kit, by Analytik Jena (1 mentions)
Neb luna universal probe one step rt qpcr, by New England Biolabs (1 mentions)
Tissuelyser lt, by Qiagen (1 mentions)
Steel bead, by Qiagen (1 mentions)
High pure viral nucleic acid kit, by Roche (1 mentions)
18 protocols using rtp dna rna virus mini kit
1
SARS-CoV-2 Detection Protocols
2
Stool RNA Extraction Protocol
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To extract RNA, approximately 10% (w/v) suspension of each stool sample was prepared. Briefly, 1 g (pea-sized) or 100 μl stool from each patient was dissolved in 1000 µl phosphate buffered saline and centrifuged at 400 ×g for 20 minutes. Next, 200 μl supernatant from each sample was collected for RNA extraction using the RTP® DNA/RNA Virus Mini Kit (Stratec Biomedical AG, Germany) according to the manufacturer’s instructions. RNA extracts were stored at -70°C until use.
3
Viral DNA Extraction and qPCR Detection
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DNA was isolated from samples using the RTP DNA/RNA Virus Mini Kit (Stratec; Edmonton, AB, Canada) following the manufacturer’s guidelines. The qPCR was performed with FIREpol master mix (Solis BioDyne; Tartu, Estonia) on 1 µL of isolated DNA using the primers and TaqMan probes in Table 1 . qPCR was performed on an ABI 7500 qPCR machine and analyzed using ABI StepOne software (Applied Biosystems; Foster City, CA, USA).
4
Quantitative Assessment of Virus Replication
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Replication properties of the recombinant viruses were assessed by quantitative PCR (qPCR)-based multi-step growth kinetics as previously described [15 (link),22 (link)]. Briefly, one million CECs were infected with 100 pfu of the indicated viruses. Cells were harvested at the indicated time points over the course of five days, and viral DNA was extracted using the RTP DNA/RNA Virus Mini kit (Stratec; Berlin, Germany). MDV genome copies of three independent experiments were evaluated by qPCR. Primers and probes specific to the MDV-infected cell protein 4 (ICP4) and chicken inducible nitric oxide synthase (iNOS) are listed in Table 1 . Virus genome copies were normalized against the chicken iNOS gene.
5
Multiplex RT-PCR for Arboviral Detection
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RNA extraction was performed using the RTP® DNA/RNA Virus Mini Kit (Stratec, Birkenfeld, Germany) based on the protocol provided by the manufacturer, and quantified using a NanoPhotometer® NP80 (Implen, Munich, Germany). Viral RNA retro-transcription and amplification were performed using the Luna® Universal kit One-Step RT-PCR (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A), with 400 ng of purified RNA, and a multiplex nested RT-PCR procedure was performed. For YFV, we used the primers designed in our lab YFUTRF- GCTAATTGAGGTGYATTGGTC and VFAR-CGAACTCCTCGTCGTACCATA (outer primers) and VFAF-CAAATCGAGTTGCTAGGCAAT and VFAR-CGAACTCCTCGTCGTACCATA (inner primers), to amplify a 145 bp fragment between positions 36 and 181 in the YFV genome. For DENV, CHIKV, and ZIKV, we used the protocol and primers previously described [21 (link)]. Culture-harvested viruses were used as controls.
The final amplification products were separated on 2% agarose gel stained with ethidium bromide. Positive YFV samples were sequenced and aligned using Mega X [22 (link)], with representative sequences from BLAST® analysis (NCBI,https://blast.ncbi.nlm.nih.gov/ ). The final alignment involved 29 nucleotide sequences of 134 bp. Genetic distance was calculated using the Kimura 2-parameter method, with an unrooted phylogenetic tree constructed using neighbor-joining.
The final amplification products were separated on 2% agarose gel stained with ethidium bromide. Positive YFV samples were sequenced and aligned using Mega X [22 (link)], with representative sequences from BLAST® analysis (NCBI,
6
Quantifying MDV Genome Copies by qPCR
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To assess MDV genome copies by qPCR, DNA of infected cells was extracted with the RTP DNA/RNA Virus Mini Kit (Stratec Molecular, Germany). MDV copy numbers were determined as previously described47 (link). Briefly, MDV genome copies were measured using a set of specific primers and a probe that target ICP4. The inducible nitric oxide synthase (iNOS) was used to normalize the MDV ICP4 copy numbers as previously published47 (link).
7
Assessing Recombinant Virus Replication
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The replication properties of the recombinant viruses were assessed using plaque size assays and multi-step growth kinetics as described previously (27 (link)). Briefly, 106 CECs were infected with 100 PFU of either wt, v∆vTR, or vhTR virus. Infected cells were fixed at 6 days post-infection (dpi), and plaque areas were measured using the Bioreader System (Bio-Sys; Karben, Germany), from which plaque diameters were calculated. For multi-step growth kinetics, infected cells were harvested daily for 6 days; viral DNA was extracted using the RTP DNA/RNA virus Mini kit (Stratec; Berlin, Germany), and viral copies were quantified by qPCR. The viral genome copies were normalized to the chicken inducible nitric oxide synthase (iNOS) gene. Primers and probes specific for the MDV immediate-early gene ICP4 and the cellular iNOS gene are shown in Table 1 .
8
Assessing Recombinant Virus Replication
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The replication properties of the recombinant viruses were assessed using plaque size assays and multi-step growth kinetics as described previously (27 (link)). Briefly, 106 CECs were infected with 100 PFU of either wt, v∆vTR, or vhTR virus. Infected cells were fixed at 6 days post-infection (dpi), and plaque areas were measured using the Bioreader System (Bio-Sys; Karben, Germany), from which plaque diameters were calculated. For multi-step growth kinetics, infected cells were harvested daily for 6 days; viral DNA was extracted using the RTP DNA/RNA virus Mini kit (Stratec; Berlin, Germany), and viral copies were quantified by qPCR. The viral genome copies were normalized to the chicken inducible nitric oxide synthase (iNOS) gene. Primers and probes specific for the MDV immediate-early gene ICP4 and the cellular iNOS gene are shown in Table 1 .
9
Quantification of Viral DNA by qPCR
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Viral DNA was extracted from samples using the RTP DNA/RNA virus minikit (Stratec, Berlin, Germany) and measured by qPCR. Primers and probes specific for the DPV UL30 designed previously in our laboratory are shown in Table 3 . Ex Taq premix (probe qPCR) (TaKaRa, Dalian, China) was used to determine viral DNA copies. qPCR amplifications were performed under the following conditions: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. Then, the qPCR products were quantified by comparison with the established standard curve of the laboratory. All reactions were performed in triplicate and at least three independent experiments.
10
Fecal Viral RNA Extraction and cDNA Synthesis
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For each stool sample, 1 mL of Eagle’s minimum essential medium (E-MEM, Nutricell;
pH7.4) was added to 1 g of feces. The solution was homogenized by vortex for 1 min
and then centrifuged at 16,000 × g for 10 min. The supernatant was
used for DNA/RNA extraction. The nucleic acid were extracted from 400 μL of fecal
suspensions by the commercial kit RTP DNA/RNA Virus Mini Kit(Stratec®, Germany), following manufacturer’s recommendations. The
nucleic acids obtained were eluted in 60 μL of TE buffer and stored until −80 °C
until use. Putative viral RNA molecules were submitted to cDNA synthesis using
High-Capacity cDNA Reverse Transcription (RT) kit [Applied
Biosystems®, USA] following manufacturer’s instructions. The cDNA was
synthesized in 20 μL of total solution containing the following: 2 μL of RNA
(approximately 100 ng), 100 ng of random primers, 10X buffer from RT, 25 mM
MgCl2, 10 mM dNTPs, 0.1 Mm DTT, 40 U RNaseOUT (Invitrogen®,
USA) and 200U reverse transcriptase enzyme. The solution was incubated at 25 °C for
10 min, after 120 min at 37 °C and then left to a final step of 5 min at 85 °C. The
cDNA was stored at −80 °C until further processing.
pH7.4) was added to 1 g of feces. The solution was homogenized by vortex for 1 min
and then centrifuged at 16,000 × g for 10 min. The supernatant was
used for DNA/RNA extraction. The nucleic acid were extracted from 400 μL of fecal
suspensions by the commercial kit RTP DNA/RNA Virus Mini Kit(Stratec®, Germany), following manufacturer’s recommendations. The
nucleic acids obtained were eluted in 60 μL of TE buffer and stored until −80 °C
until use. Putative viral RNA molecules were submitted to cDNA synthesis using
High-Capacity cDNA Reverse Transcription (RT) kit [Applied
Biosystems®, USA] following manufacturer’s instructions. The cDNA was
synthesized in 20 μL of total solution containing the following: 2 μL of RNA
(approximately 100 ng), 100 ng of random primers, 10X buffer from RT, 25 mM
MgCl2, 10 mM dNTPs, 0.1 Mm DTT, 40 U RNaseOUT (Invitrogen®,
USA) and 200U reverse transcriptase enzyme. The solution was incubated at 25 °C for
10 min, after 120 min at 37 °C and then left to a final step of 5 min at 85 °C. The
cDNA was stored at −80 °C until further processing.
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