Overlapping fragments of the genome of CCoV-I strain 23/03 were obtained through RT-PCR reaction carried out using primer sets designed based on the genome sequence of other alphacoronaviruses and the kit SuperScript™ One-Step RT-PCR for Long Templates (Life Technologies srl). Additional RT-PCR assays and subsequent sequencing attempts were performed to close gaps between assembled contigs and to sequence unresolved genomic regions using primers designed on the alignment of the reference Alphacoronavirus strains. The very 5′ and 3′ ends were amplified using 5′ and 3′ RACE System for Rapid Amplification of cDNA Ends (Invitrogen), respectively, following the manufacturer's instructions. The PCR products were detected by electrophoresis through a 1.5% agarose gel and visualization under UV light after ethidium bromide staining.
Superscript one step rt pcr for long templates
Manufactured by Thermo Fisher Scientific
The SuperScript™ One-Step RT-PCR for Long Templates is a kit designed for reverse transcription and long-range PCR amplification in a single-tube reaction. The kit includes a reverse transcriptase enzyme and a high-fidelity DNA polymerase.
Automatically generated - may contain errors
Lab products found in correlation
Amplitaq gold, by Thermo Fisher Scientific (2 mentions)
Exosap it kit, by Thermo Fisher Scientific (2 mentions)
5 and 3 race system for rapid amplification of cdna ends, by Thermo Fisher Scientific (1 mentions)
Sephadex g50 resin, by Merck Group (1 mentions)
Applied biosystems 3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
6 protocols using superscript one step rt pcr for long templates
1
Comprehensive Genome Sequencing of CCoV-I
2
Pestivirus RNA Detection Protocol
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The detection of pestivirus RNA in clinical samples and RNA transcript dilutions was carried out using a nPCR protocol previously developed for the characterisation of bovine pestiviruses (Decaro et al., 2012b (link)). First- and second-step amplifications were carried out using SuperScript™ One-Step RT-PCR for Long Templates (Life Technologies Italia) and AmpliTaq Gold (Life Technologies Italia), as previously described (Decaro et al., 2012b (link)). Oligonucleotides are reported in Table 1 .
3
Characterization of Bovine Pestiviruses by Nested PCR
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Biological samples testing positive for pestivirus by real-time RT-PCR and/or by gel-based RT-PCR were submitted to a nested PCR (nPCR) protocol previously developed for the characterization of bovine pestiviruses (Decaro et al., 2012b (link)). RT-PCR was carried out using SuperScriptTM One-Step RT-PCR for Long Templates (Life Technologies Italia) and the following thermal protocol: reverse transcription at 50 °C for 30 min, inactivation of Superscript II RT at 94 °C for 2 min, 45 cycles of 94 °C for 30 s, 50 °C for 30 s, 68 °C for 1 min, with a final extension at 68 °C for 10 min. Nested PCR was performed using AmpliTaq Gold (Life Technologies Italia). The thermal conditions consisted of activation of AmpliTaq Gold polymerase at 94 °C for 10 min and 25 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s and polymerization at 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. The PCR products were detected as described for the diagnostic gel-based RT-PCR. The position and sequence of the primers used for conventional amplification are reported in Table 1 .
4
Detecting Pestivirus RNA Using Gel-Based RT-PCR
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The detection of pestivirus RNA in all biological samples, reference viruses and RNA transcript dilutions was carried out by a panpestivirus gel-based RT-PCR (Vilcek et al., 1994 (link)), using SuperScriptTM One-Step RT-PCR for Long Templates (Life Technologies Italia) and primers 324 and 326 that bind to the 5′UTR region of the pestivirus genome. The following thermal protocol was used: reverse transcription at 50 °C for 30 min, inactivation of Superscript II RT at 94 °C for 2 min, 45 cycles of 94 °C for 30 s, 55 °C for 30 s, 68 °C for 30 s, with a final extension at 68 °C for 10 min. The PCR products were detected by electrophoresis in 1.5% agarose gels and visualized under UV light after ethidium bromide staining. The position and sequence of the primers used for conventional amplification are reported in Table 1 .
5
SARS-CoV-2 Viral Amplification and Sequencing
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The protocol for SARS-CoV-2 amplification and sequencing is detailed elsewhere [9 ]. Briefly, viral RNA was reverse-transcribed and amplified using the kit One-Step Invitrogen (SuperScript One-Step for long templates RT-PCR; Foster City, CA) and 2 different primers (5’-3’); 38F (-GTC AGT GTG TTA ATC TTA CAA CCA G -) as the forward, and 1191R (-TGC ATA GAC ATT AGT AAA GCA GAG A -) as the reverse, (the given position refers to the Wuhan strain of SARS-CoV-2). For each PCR reaction, positive and negative controls were used to ensure the effectiveness of the reaction and the absence of cross-contamination, respectively. Amplification results were revealed after agarose-gel electrophoresis and positive results were kept for the sequencing process. PCR products were then purified through the ExoSAP-IT kit (Applied Biosystems, Lithuania). Sequencing was performed with four different overlapping primers: 38F (-GTC AGT GTG TTA ATC TTA CAA CCA G -), 514F (-TCT CAG CCT TTT CTT ATG GAC CT -), 655R (-CCT GAG GGA GAT CAC GCA CTA -) and 1191R (-TGC ATA GAC ATT AGT AAA GCA GAG A -) . The sequencing product was purified by gel filtration chromatography using Sephadex G-50 resin (Sigma-Aldrich) to eliminate excess primers, unincorporated dideoxynucleotides (ddNTPs) and salts. Capillary electrophoresis was performed on Applied Biosystems 3500 genetic analyzer (Applied Biosystems, Tokyo, Japan).
6
SARS-CoV-2 Viral RNA Detection by RT-PCR
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Viral RNA was retro-transcribed and amplified using the kit One-Step Invitrogen (SuperScript® One-Step for long templates RT/PCR; Foster City, CA) and 2 different sequence-specific primers (5′-3′): 38F (- GTCAGTGTGTTAATCTTACAACCAG-) as the forward, and 1191R (- TGCATAGACATTAGTAAAGCAGAGA-) as the reverse, (the given position refers to the Wuhan strain of SARS-CoV-2). The RT-PCR reaction contained for each sample 25 μl reaction mix, 8 μl MgSO 4 (5 μM), 3 μl DNAse- and RNAse-free water, 0.75 μl sense primer (10 μM stock), 0.75 μl antisense primer (10 μM stock), 1 μl RNAseOUT (5 U/μl Invitrogen), 1.5 μl RT-Taq (Superscript III RT/Platinum high fidelity) and 10 μl of extracted RNA. The RT-PCR conditions consisted of an initial step of 1 cycle at 50 °C for 30 min; 1 cycle of 94 °C for 2 min; 40 cycles (95 °C, 30 s; 52 °C, 30 s; 72 °C, 90 s); a final elongation step of 1 cycle at 72 °C for 10 min. The expected cDNA is about 1200 base pairs (bp)in length (position 38 [ORF]-1191 [ORF]). For each PCR reaction, positive and negative controls were used to ensure the effectiveness of the reaction and the absence of cross-contamination, respectively. Amplification results were revealed after agarose-gel electrophoresis and positive results were kept for the sequencing process. Then PCR products were purified through the ExoSAP-IT™ kit (Applied Biosystems™, Lithuania).
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