The samples that were positive by rRT-PCR were subjected to conventional PCR using Invitrogen SuperScript III One-Step PCR System with Platinum Taq DNA Polymerase. Serotype-specific primers targeting VP1 were selected based on the antigen ELISA results (results not shown). The primers were as described by [20 (link)] and are presented in Table 2 . For each sample, the reaction master mix was prepared by adding 9.2 µL of nuclease-free water, 1.6 µL of each primer (reverse and forward), 0.8 µL of dNTPs, 4 µL of buffer, 0.8 µL of Qiagen One–step RT-PCR enzyme and 2.0 µL of template, leading to a final 20 µL volume. The four serotype-specific PCRs included nuclease-free water as a negative control and known positive controls that were supplied by the National Animal Disease Diagnostic Centre as RNA extracts that had previously been stored at −80oC. The PCR was run in a Techne TC-412 thermocycler (Techne, USA) using the following cycle conditions; 50oC for 30 min and 95oC for 15 min, 95oC for 10 sec, followed by 35 cycles of 30 sec at 60oC for serotype O, 30 sec at 55°C for serotype A and 30 sec at 50°C for SAT 1, SAT 2 and SAT 3 with extensions of 72oC for 30 sec and 72oC for 10 min as modified from a protocol described by Knowles et al. [20 (link)].
One step rt pcr enzyme
Manufactured by Qiagen
Sourced in United States
The One-Step RT-PCR enzyme is a reagent used for the process of reverse transcription and polymerase chain reaction (RT-PCR) in a single step. It enables the conversion of RNA into complementary DNA (cDNA) and the subsequent amplification of the cDNA in a single reaction.
Automatically generated - may contain errors
Lab products found in correlation
Onestep rt pcr buffer, by Qiagen (2 mentions)
Onestep rt pcr kit, by Qiagen (2 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Agarose gel, by Thermo Fisher Scientific (1 mentions)
Redgel, by Biotium (1 mentions)
100 bp marker, by Thermo Fisher Scientific (1 mentions)
Big dye terminator reaction mix, by Thermo Fisher Scientific (1 mentions)
Cfx96 platform, by Bio-Rad (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
Generuler, by Thermo Fisher Scientific (1 mentions)
6 protocols using one step rt pcr enzyme
1
Serotype-Specific RT-PCR for FMDV
2
Cloning of Rabies Virus N Gene
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For cloning, a segment of viral genome covering a part of the N gene (positions 1–933 according to the Pasteur virus genome accession number X03673) of CVS-11 was generated using primers designed for this purpose (TWclon-F and TWclon-R, Table 2 ). Amplification was performed using the OneStep RT-PCR Kit with the master mix consisting of 29.25 μL of RNase-free water, 10 μL 5x QIAGEN OneStep RT-PCR buffer, 0.4 mM of each dNTP, 0.6 μM of the cloning primers, 10 U RNasin (40 U/μL), and 2 μL of QIAGEN OneStep RT-PCR Enzyme mixed with 5 μL of extracted RNA to a total volume of 50 μL. Amplifications were performed in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, USA) using the following cycling conditions: 30 min at 50°C, followed by 15 min at 95°C and 40 repetitive cycles of 1 min at 94°C, 1 min with a temperature gradient from 56°C to 46°C in steps of 2°C, and 1 min at 72°C. Elongation at 72°C was extended for 10 additional minutes in the last cycle. After gel electrophoresis excised PCR fragments with a length of 933 bp were eluted with the QIAquick Gel Extraction Kit (QIAGEN, Germantown, USA) and cloned into the pCR-II-TOPO vector using the TOPO TA Cloning Kit (Invitrogen, Foster City, USA) according to the manufacturer's instructions.
3
Gel-based RT-PCR VP6 Gene Detection
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The extracted RNA was subjected to VP6 gene detection by gel-based RT-PC [18 (link), 23 ], (Qiagen, Inc.,Valencia, CA, USA) using the primers:
VP6F (nt 747–766) 5′ GACGGVGCRACTACATGGT 3′ and 21.
74VP6R (nt 1126 to 1106) 5′ GTCCAATTCATNCCTGGTGG 3′ [24 (link)].
RNA sample (4 μL) was mixed with 3 μl of primer mix (20 μM) in 0.5 ml PCR tube/well, vortexed and centrifuged at 8000 rpm for 10 s, then denatured at 97 °C for 4 min and rapidly cooled for 1 min on ice. The mixture was centrifuged briefly then placed back on ice, and 23 μL of master mix (made of H2O, 16 μL; Qiagen One step RT-PCR buffer 5X, 5 μL; dNTP (10 mM), 1 μL; Qiagen One step RT-PCR enzyme, 1 μL) was added in the tube. The reaction was amplified using an ABI 9700 thermocycler under the following conditions: 42 °C, 30 min; 95 °C, 15 min; and 30 cycles of 94 °C 30s, 42 °C 30s, 72 °C 45 s; with a final cycle of 7 min at 72 °C, then 4 °C on hold [18 (link), 23 ]. The amplification products were separated on a 2% agarose gel (Invitrogen) containing 10 μL of red gel (Biotium), with a 100 bp marker (Invitrogen), and the bands were visualized using a “Gel Doc TMXR +” illuminator (BIO-RAD).
VP6F (nt 747–766) 5′ GACGGVGCRACTACATGGT 3′ and 21.
74VP6R (nt 1126 to 1106) 5′ GTCCAATTCATNCCTGGTGG 3′ [24 (link)].
RNA sample (4 μL) was mixed with 3 μl of primer mix (20 μM) in 0.5 ml PCR tube/well, vortexed and centrifuged at 8000 rpm for 10 s, then denatured at 97 °C for 4 min and rapidly cooled for 1 min on ice. The mixture was centrifuged briefly then placed back on ice, and 23 μL of master mix (made of H2O, 16 μL; Qiagen One step RT-PCR buffer 5X, 5 μL; dNTP (10 mM), 1 μL; Qiagen One step RT-PCR enzyme, 1 μL) was added in the tube. The reaction was amplified using an ABI 9700 thermocycler under the following conditions: 42 °C, 30 min; 95 °C, 15 min; and 30 cycles of 94 °C 30s, 42 °C 30s, 72 °C 45 s; with a final cycle of 7 min at 72 °C, then 4 °C on hold [18 (link), 23 ]. The amplification products were separated on a 2% agarose gel (Invitrogen) containing 10 μL of red gel (Biotium), with a 100 bp marker (Invitrogen), and the bands were visualized using a “Gel Doc TMXR +” illuminator (BIO-RAD).
4
Full Genome Sequencing of Viral Isolates
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Viral isolates were amplified for all genes and analysed for full genome sequences using the same set of primers (Supplemental Table 2). The virus from the third sample could not be isolated after two passages and was only utilized for full length sequencing of HA and NA genes due to limited sample for further analysis. Overall a total reaction volume of 50 µL contained the following reagents: 10 µL of 5× PCR buffer, 2 µL of 10 µM dNTPs, 1 µL of 10 µM forward and reverse primer, 0.25 µL RNAsin 40 U/µL, 2 µL Qiagen One-step RT–PCR enzyme and 5 µL RNA. This mixture was run using the following program: 60°C for 1 min, 42°C for 10 min, 50°C for 30 min, 95°C for 15 min, 30 cycles at (94°C for 30 s, 55°C for 30 s, 72°C for 90 s) and 72°C for 7 min. Amplicons were then sequenced by Sanger sequencing with Big Dye Terminator Reaction Mix (Applied Biosystems) on the ABI 3500XL genetic analyser. Contiguous sequences were assembled using CLC Main Workbench 5.5 (Qiagen, Hilden, Germany).
5
Surveillance of Stool Samples for Rotavirus Detection
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Surveillance stool specimens underwent total nucleic acid extraction and quantitative real-time reverse transcription polymerase chain reaction (qPCR) for stool RVA detection as previously described28 (link)–30 (link). Briefly, total nucleic extractions were performed on stool using the QIAamp stool DNA mini kit (QIAGEN, Valencia, CA) followed by qPCR using the CFX96 platform (Bio-Rad, Hercules, CA) using Ultraplex 1-step ToughMix enzyme (Quantabio, Beverly, MA) and previously described primers and probes targeting the NSP3 gene segment of RVA30 (link). Specimens were considered positive at qPCR cut-off of Ct < 34, the analytic limit of detection of the assay15 (link). We also performed a sensitivity analysis using a cut-off Ct < 36, as we anticipated that many specimens would shed very low quantities of virus. Positive specimens underwent conventional reverse transcription PCR to amplify the RVA VP8* gene segment using QIAGEN OneStep RT-PCR enzyme with VP4F and VP4R primers31 (link), followed by Sanger sequencing using VP4F or VP4R primers and BLAST analysis to confirm vaccine versus wild-type RVA, as previously described28 (link). All qPCR-positive specimens were additionally tested by stool EIA.
6
HPV16 E6/E7 Expression Analysis
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Samples were analyzed by RT-PCR according to the DNA results for each specimen. HPV16 positive samples were further subjected to amplification of the E6 and E7 transcripts [22 (link)]. RT-PCR was performed using primers for the constitutively expressed β-actin gene [22 (link)] as a positive control and subsequently for sample normalization. Details of the target genes, primer sequence, and amplicons sizes are shown in Table 1 . The RT-PCR was performed in 50 μL reactions using the adjusted amount of RNA template (1 μg) with the one-step RT-PCR Kit (Qiagen). Reactions contained 0.6 μM forward and reverse primers, 1x Qiagen one-step RT-PCR buffer, 400 μM dNTP mix, and 2 μL Qiagen one-step RT-PCR enzyme. The reaction was allowed to proceed for 30 min at 50°C. HPV16 type specific plasmids were used as positive controls. For each of the target genes, control reactions without template were performed in order to rule out contamination. Cycling protocols for all RT-PCR reactions are shown in Table 1 . The amplicons were evaluated by 1.5% gel electrophoresis, marked by a 50 pb DNA ladder (Gene Ruler, Fermentas), stained with ethidium bromide, and visualized under UV light.
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