The presence of viral RNA DENV from samples with positive dengue antibodies from COVID-19 patients was confirmed by RT-PCR (Sansure Biotech Inc, Changsha, China). The RNA extraction was done using the QIAamp Viral Mini Spin Kit (Qiagen, Hilden, Germany). Briefly, the viral RNA was reversely transcribed to cDNA by using the SuperScript III First-Stand Synthesis Kit (Invitrogen, Carlsbad, CA, USA) with the reverse primer, D2 (5′-TTGCACCAACAGTCAATGTCTTCAGGTTC-3′). DENV-1, DENV-2, DENV-3, and DENV-4 genomes were amplified by a multiplex PCR using GoTaq Green master mix (Promega, Madison, WI, USA), along with the sense primer D1 (5′-TCAATATGCTGAAACGCGCGAGAAACCG-3′) and the serotype-specific reverse primers (TS1: 5′-CGTCTCAGTGATCCGGGGG-3′; TS2: 5′-CGCCACAAGGGCCATGAACAG-3′; TS3: 5′-TAACATCATCATGAGACAGAGC-3′; TS4: 5′-CTCTGTTGTCTTAAACAAGAGA-3′) to generate 482, 118, 290, and 392-bp fragments, respectively. The DENV serotype was determined by the size of the amplified fragments [11 (link)].
Superscript 3 first stand synthesis kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SuperScript III First-Strand Synthesis kit is a laboratory tool used for the reverse transcription of RNA into complementary DNA (cDNA). The kit contains the necessary components, including the SuperScript III reverse transcriptase enzyme, to perform this fundamental step in gene expression analysis and other molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Gotaq green master mix, by Promega (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Ex taq, by Takara Bio (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 3500 l genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
4 protocols using superscript 3 first stand synthesis kit
1
Detecting DENV in COVID-19 Patients
2
Quantifying Murine PRG4 Expression
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mRNA was isolated from whole liver extracts, reverse transcribed, and quantitated by qRT-PCR using the following primers for murine PRG4: 5′-CAG GAC AGC ACT CCA TGT AGT-3′ (reverse) and 5′-GGG TGG AAA ATA CTT CCC GTC-5′ (forward). mRNA was extracted using the RNeasy Kit (Qiagen). cDNA was synthetized using SuperScript III First-Stand Synthesis kit (Invitrogen). Expression was quantified using the 2−ΔΔCT method and TBP was used to normalize the expression of the target genes between samples.
3
Viral Genome Sequencing and Analysis
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Viral RNA was reverse transcribed to cDNA using the SuperScript III First-Stand Synthesis kit (Invitrogen, Carlsbad, CA, USA). Generated cDNA was subjected to amplification of the viral gene for both the PR and RT encoding regions in a separate reaction by nested PCR using Ex Taq (Takara Bio, Shiga, Japan). Primer information is shown in Additional file 3 . If a viral gene fragment failed to be amplified from the cDNA even after multiple attempts, it was amplified instead from DNA extracted from PBMC. In order to examine the genomic fragment of the major viral population in a sample, PCR products amplified at the end-point dilution of DNA templates were subjected to sequencing analysis.
Sequencing analysis of the amplified fragment was performed using the BigDye Terminator v3.1 Cycle Sequencing kit with an ABI PRISM 3500 × l genetic analyzer (Applied Biosystems, Foster City, CA, USA). Data were assembled and aligned using Genetyx ver. 10 software (Genetyx, Tokyo, Japan). Nearly the full length of the PR gene [(280 bp; corresponding to nucleotides 2262 to 2541 of a HIV-1 reference strain, HXB2 (GenBank accession no. K03455)] and part of the RT gene (762 bp; nucleotides 2550 to 3311) were sequenced and subjected to subsequent analysis.
Sequencing analysis of the amplified fragment was performed using the BigDye Terminator v3.1 Cycle Sequencing kit with an ABI PRISM 3500 × l genetic analyzer (Applied Biosystems, Foster City, CA, USA). Data were assembled and aligned using Genetyx ver. 10 software (Genetyx, Tokyo, Japan). Nearly the full length of the PR gene [(280 bp; corresponding to nucleotides 2262 to 2541 of a HIV-1 reference strain, HXB2 (GenBank accession no. K03455)] and part of the RT gene (762 bp; nucleotides 2550 to 3311) were sequenced and subjected to subsequent analysis.
4
Quantifying Interferon α2 Gene Expression
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The gene expressions of interferon α2 were quantified by amplification of specific mRNA using qPCR. Total RNA was isolated from culture fluid using QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Viral RNA was reversely transcribed into cDNA using the SuperScript III First- Stand Synthesis kit (Invitrogen, Carlsbad, CA) with the reverse primer, K-env-R1, 5’- CCAATCAGGGAAGAAGCCTTG-3’ [corresponding to nucleotide 9158 to 9138 of an HIV-1 subtype B reference strain, pNL4-3 (GenBank accession number AF324493)].The purity and concentration of cDNA were measured by nanodrop (Thermoscientific, USA). qPCRreaction was carried in a 10 μL reactionmixture containing 5 μL SsoFastTMEvaGreensupermix (Bio Rad), 1 μL each primer, 2 μltemplate and 1μL nuclease free water. For the amplification of viral interferon of viral forward: 5 ‘GCA AGT CTG CAA GCT GCT TG 3’, and reverse: 3 ‘GAT GGT CTT TTC AGC TTG GA 5’.The qPCR condition detailed werefor 40 cycles with annealing temperature 58 0C.For normalizing the gene expression data, we used GADPH as a housekeeping gene. The primers sequence were5 ‘TTT AAC AGG GCT GCT TCT GGT 3’ for forward and 3 ‘CCC ATT TTG TTG CAC GAG GGA 5’ for reverse.
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