Goat pox virus Taqman-MGB (minor groove binder) probe real-time fluorescence quantitative PCR (polymerase chain reaction) detection primer, kit and detection method
A technology for real-time fluorescence quantification and goat pox virus, applied in biochemical equipment and methods, methods based on microbes, microbe determination/inspection, etc., can solve time-consuming and labor-intensive detection methods for identifying goat pox and inability to distinguish detection Viruses and other problems, to achieve cost reduction, high-throughput rapid detection, and overcome the effect of long detection time
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Embodiment 1
[0033] Embodiment 1, design and screening of primers
[0034] Goat poxvirus fluorescent quantitative PCR amplification primers and probes were designed based on the goat poxvirus reference sequence published by GenBank, compared with MEGA5, the sequences were analyzed, and primers and probes were designed in the conserved regions. The probe design software Primer Express 3.0 was used to design 4 sets of fluorescent quantitative primers, which were synthesized by Yingjun (Shanghai) Co., Ltd. ABI 7500 FAST PCR instrument was used to monitor the amplification in the reaction process in real time, and the amplification of different primer sets The parameters such as the starting time of starting time, the time of entering the maximum amplification rate, the maximum amplification rate and the time required to reach the plateau stage were analyzed, and a set of fluorescent quantitative PCR amplification primers with the highest amplification rate and good specificity were screened out,...
Embodiment 2
[0040] Embodiment 2, the preparation of positive control substance
[0041] Use the kit to extract the nucleic acid of the goat pox virus cell culture, identify the nucleic acid by PCR and electrophoresis, use PCR upstream primer SEQ ID NO.4 and PCR downstream primer SEQ ID NO.5 to amplify, and use gel recovery reagent The cassette recovered the amplified band. According to the ratio of 1:10 and the PMD19-T carrier for ligation reaction, ligated overnight at 4°C, transformed into DH5α bacteria, after resistance selection and PCR identification positive, re-sequencing verification, using a spectrophotometer to measure the OD value of its nucleic acid, so that Its 260 / 280 ratio is between 1.8 and 2.0.
Embodiment 3
[0042] Embodiment 3, the preparation of negative control substance
[0043] The kit was used to extract the DNA of sheep tissue without goatpox virus infection, and then identified by PCR and electrophoresis.
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