Detection primer set, probe set and detection reagent kit for novel coronavirus 2019
A detection kit, coronavirus technology, applied in the determination/inspection of microorganisms, recombinant DNA technology, resistance to vector-borne diseases, etc., can solve the problems that have not yet been determined, and achieve shortened detection time, high sensitivity, and effective laboratory detection. effect of the method
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Embodiment 1
[0059] This embodiment provides a primer and probe for 2019 novel coronavirus. Based on the Taqman fluorescent probe technology, the corresponding specific detection primers and Taqman probes were designed with the highly conserved regions of the S gene, ORF1ab gene and N gene of the 2019 novel coronavirus. At the same time, human ribonucleotidase P (RNase P ) as the internal standard, design the corresponding internal standard primer pair and internal standard probe. The details are shown in Table 1 below:
[0060] Table 1. Primer and Probe Information
[0061]
[0062] Among them, the 5' end of 2019-nCov S-P is marked with the fluorescent reporter group JOE, and the 3' end is marked with the fluorescent quencher group BHQ1; the 5' end of 2019-nCov ORF1ab-P is marked with the fluorescent reporter group CY5, 3' The end is labeled with a fluorescent quencher group BHQ2; the 5' end of 2019-nCov N-P is labeled with a fluorescent reporter group FAM, and the 3' end is labeled ...
Embodiment 2
[0064] This embodiment provides a 2019 novel coronavirus detection kit, including RT-PCR enzyme mixture, RT-PCR buffer, RT-PCR primer mixture, RT-PCR probe, RT-PCR positive control substance, RT-PCR PCR negative control.
[0065] Wherein, the RT-PCR enzyme mixture includes 2-3U / μL hot start Taq enzyme, 1-2U / μL reverse transcriptase, 15-30mM Tris, 10%-30% glycerol, 0.5-1.5mM DTT;
[0066] RT-PCR buffer includes 250mM Tris, 25mM MgCl 2 , 50mM KCl, 0.025% Triton X-100 buffer system and 6.5mM dNTPs;
[0067] RT-PCR primer mixture and RT-PCR probe are primers and probes in Example 1;
[0068] The RT-PCR positive control substance is the plasmid solution of the primer targeting fragments of the 2019 novel coronavirus S gene, ORF1ab gene and N gene and the internal standard gene using pUC57 as the plasmid vector.
[0069] RT-PCR negative control substance was DEPC water.
Embodiment 3
[0071] The present embodiment provides a kind of detection method of 2019 novel coronavirus, comprising the following steps:
[0072] 1. Extraction of Nucleic Acids
[0073] Obtain the sample to be tested from the throat swab, inactivate it (heat at 56°C for 30 minutes), and extract it according to the method of the extraction kit. In this example, the nucleic acid extraction or purification reagent produced by Shenzhen Zijian Biotechnology Co., Ltd. (Record No. : Guangdong Shenji No. 20180040).
[0074] 2. PCR amplification
[0075] Configure the RT-PCR reaction solution system according to Table 2:
[0076] Table 2. RT-PCR reaction solution system
[0077]
[0078] Wherein, the RT-PCR enzyme mixture and RT-PCR buffer are the RT-PCR enzyme mixture and RT-PCR buffer in Example 2.
[0079] Use the RNA extracted in step 1 as a template, add 5 μL to the above reaction solution system, and perform multiple fluorescent PCR amplification reactions on the ABI 7500 instrument. ...
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