Total RNA was extracted from 100 μL of the swab sample, which was collected in RLT buffer from the RNA easy mini kit (Qiagen, Germany) as per manufacturer instructions. Real time, one step RT‐PCR was carried out with the BioRad CFX‐96 Real‐Time system (BioRad, USA) using the Superscript III/Platinum Taq One‐step qRT‐PCR kit (Invitrogen) for detection of PPRV according to procedures described elsewhere (Bao et al. 2008 ). Ten per cent of PCR positive samples were randomly selected for genetic characterization by sequencing nucleocapsid (N) and haemagglutinin (H) genes fragments. These genes fragments were amplified using the primer sets Nad1/Pad1 for N gene and hrf3/hre4 for H gene followed by direct amplicon sequencing (Couacy‐Hymann et al. 2002 ), (Balamurugan et al. 2006 ). The amplified products were analysed by electrophoresis through a 1% agarose gel and staining with ethidium bromide. The PCR products were purified by ExoSAP (Affymetrix, CA, USA) treatment for nucleotide sequencing using an automated Genetic Analyzer ABI 3500 XL (Applied Biosystem, Foster City, CA) and Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystem). The nucleotide sequences were deposited in GenBank under the accession numbers KT253989–KT253999 for the N gene and accession numbers KT254000–KT254005 for the H gene.
Superscript 3 platinum taq one step qrt pcr kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The Superscript III/Platinum Taq One‐step qRT‐PCR kit is a product designed for the reverse transcription and real-time PCR amplification of RNA targets in a single reaction. It combines the Superscript III reverse transcriptase and Platinum Taq DNA polymerase for efficient and sensitive detection of RNA.
Automatically generated - may contain errors
Lab products found in correlation
Exosap, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Rnaeasy mini kit, by Qiagen (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Cfx opus, by Bio-Rad (1 mentions)
Cfx maestro qpcr software, by Bio-Rad (1 mentions)
5 protocols using superscript 3 platinum taq one step qrt pcr kit
1
PPRV Detection and Genetic Characterization
2
Optimized RT-PCR Protocol for Genome Amplification
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RT-PCR reactions were performed as described previously by [56 (link)]. Briefly, a fragment of the targeted genome segment was amplified with different primers-probe sets using SuperScript III/ Platinum Taq One-Step qRT-PCR Kit (Invitrogen, UK). RNA samples were always heat denaturated (95°C for 3 min) prior to addition to the reaction mix [37 (link)]. Amplification was carried out in MX3005p (Stratagene, UK) using the conditions: 55°C for 30 min, 95°C for 10 min followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. Fluorescence was measured during a 60°C annealing/extension step. Cycle threshold (Ct) values were measured as the point at which the sample fluorescence signal crossed a threshold value (the background level). Negative results (for assays that did not exceed this level of signal) are reported as ‘No Ct’. All precautions were taken to avoid accidental contamination. The composition of the individual optimised assays using either one or two sets of primers and probes is presented in Table 3 .
3
RT-qPCR Assay Optimization and Validation
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Different primers–probe sets were used to amplify a fragment of each of the targeted genome segments, using SuperScript III/Platinum Taq One‐Step qRT‐PCR Kit (Invitrogen). To achieve high efficiency, the individual steps of the RT‐PCR reaction were optimized for each assay, including primers concentrations, primers–probe ratios, magnesium concentration, reverse transcription temperature and period, annealing temperature and period (Table 4 ). dsRNA samples were heat‐denatured prior to addition to the reaction mix (Shaw et al., 2007 ). To avoid accidental contamination, the reaction mix was prepared in a ‘clean work station’ and template was added in a separate ‘work station’. Amplification for all of the assays was carried out in Mx3005p (Stratagene, UK) using the same conditions as follows: 55°C for 30 min, 95°C for 10 min followed by 50 cycles of 95°C for 30 s and 60°C for 1 min. Fluorescence was measured during annealing/extension phase of the reaction. Cycle threshold (Ct) values were measured at the point at which the sample fluorescence signal crossed a threshold value (the background level). Negative results (for assays that did not exceed this level of signal) are reported as ‘No Ct’.
4
Optimized qRT-PCR Assay for Genome Detection
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The different primers-probe sets were used to amplify a fragment of the targeted genome segment, using SuperScript III/Platinum Taq One-Step qRT-PCR Kit (Invitrogen, UK). In order to achieve low cycle threshold (Ct) values and high fluorescence signals, the individual steps of the method were optimised for each of the assays, including: primers concentrations, primers-probe ratios, magnesium concentration, reverse transcription temperature, reverse transcription period, annealing temperature and annealing period. The composition of the individual assays, after optimisation, is presented in Table 4 . dsRNA samples were heat denaturated prior to addition to the reaction mix [69] (link). To avoid accidental contamination, assembly of the reaction mix was conducted in a ‘clean work’ dedicated laminar flow hood, separate to an RT-PCR assembly hood. Amplification for all of the assays was carried out in MX3005p (Stratagene, UK) using the same conditions: 55°C for 30 min, 95°C for 10 min followed by 45 cycles of 95°C for 30 s and 55°C for 1 min. Fluorescence was measured during the 55°C annealing/extension step. Cycle threshold (Ct) values were measured at the point at which the sample fluorescence signal crossed a threshold value (the background level). Negative results (for assays that did not exceed this level of signal) are reported as ‘No Ct’.
5
EHDV Serotyping by One-Step qRT-PCR
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The samples that were positive during the initial EHDV detection were then tested for serotypes 1, 2, and 6. The reactions were performed in technical triplicates using the Superscript III/ Platinum Taq One-Step qRT-PCR Kit (Invitrogen, Waltham, MA, USA). The primers and probes (LGC Biosearch Technologies, Petaluma, CA, USA) were specific to the VP2, seg-2 region of each virus serotype [24 (link)] (Supplementary Table S1 ). The samples were amplified on a Bio-Rad CFX OPUS real-time PCR machine under the following conditions: 55 °C for 30 min, 95 °C for 10 min, and 50 cycles at 95 °C for 30 s and 60 °C for 1 min. The data were analyzed using Bio-Rad CFX Maestro qPCR software.
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