Specific primers, probes and rapid detection kits for detecting yellow catfish calicivirus-1
A technology of calicivirus and yellow catfish, applied in biochemical equipment and methods, measurement/testing of microorganisms, and resistance to vector-borne diseases, etc., can solve problems that hinder early warning and prevention of diseases, and achieve low cost and high detection sensitivity High and efficient effect
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Embodiment 1
[0028] A primer-probe mixture for detecting yellow catfish calicivirus-1, which consists of primer set A and Taqman fluorescent probe B, the 5' end of probe B is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescence quencher group.
[0029] The primer set A comprises primer YCCV-1-q183F and primer YCCV-1-q183R;
[0030] The sequence of the primer YCCV-1-q183F is shown in SEQ ID NO: 1;
[0031] The sequence of the primer YCCV-1-q183R is shown in SEQ ID NO: 2;
[0032] The nucleotide sequence of the probe B is shown in SEQ ID NO:3.
[0033] Wherein, the fluorescent reporting group is selected from 6-carboxyfluorescein, hexachloro-6-methylfluorescein, VIC fluorescent dye, tetrachloro-6-carboxyfluorescein, carboxy-X-rhodamine, 6-carboxytetramethyl Rhodamine, sulforhodamine, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl ester, cyanine 3, cyanine 3.5, cyanine 5 and one or more of cyanine 5.5; the fluorescence quenching group ...
Embodiment 2
[0039] Yellow catfish calicivirus-1 fluorescent quantitative detection kit, including individually packaged virus nucleic acid extraction solution, one-step RT-qPCR reaction solution, positive quality control, negative quality control and primer-probe mixture.
[0040]The primer-probe mixture is composed of primer set A and Taqman fluorescent probe B, wherein the final concentration of upstream and downstream primers of primer set A is 0.15 μM, and the final concentration of probe is 0.1 μM.
[0041] Taqman fluorescent probe B, whose nucleotide sequence is marked with HEX at the 5' end and BHQ1 at the 3' end;
[0042] The negative quality control product is sterilized normal saline without RNase; the positive quality control product is based on the purified yellow catfish calicivirus-1 genome as a template, and the primer set A is used for RT-PCR amplification, and the amplified product is connected to the carrier , the pseudovirus that is packaged after being confirmed to be ...
Embodiment 3
[0046] For the application of the yellow catfish calicivirus-1 fluorescent quantitative detection kit, take 0.05g of the sample to be tested and add 1mL of virus nucleic acid extraction solution to fully grind, centrifuge at 12000r / min for 10min, take 800uL of the supernatant, add 400uL of ethanol, and wait for 5min at room temperature , centrifuge at 12000r / min for 5min, wash the precipitate once with 75% ethanol, dry at room temperature for 5min, add 50uL DEPC water to dissolve RNA, take 5uL and add it to the reaction tube, then add 2uL of primer-probe mixture, and then add one-step RT -10uL of qPCR reaction solution, add 3uL of DEPC water, mix well and perform fluorescent quantitative PCR. The reaction conditions are 42°C for 20 minutes, 95°C for 5-10 minutes; -45 seconds, a total of 45 cycles; the fluorescence signal collection is set to HEX, and the fluorescence signal collection is set to 60°C;
[0047] Result judgment:
[0048] If there is no S-type amplification curve...
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