Yak coronavirus detection kit based on constant temperature isolated fluorescent PCR platform and application thereof
A detection kit, coronavirus technology, applied in the determination/inspection of microorganisms, resistance to vector-borne diseases, DNA/RNA fragments, etc., can solve the problems of long reaction time, large equipment, complicated operation, etc., and achieve short detection time. , Simplified operation, high sensitivity effect
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Embodiment 1
[0030] Example 1 Yak coronavirus detection kit based on constant temperature isolation fluorescent PCR platform
[0031] A yak coronavirus detection kit based on a constant temperature isolation fluorescent PCR platform, comprising the following components:
[0032] (1) Reaction buffer: 3ml, composed of 500mM KCl, 100mM Tris-HCl (pH 8.3), 15mM MgCl 2 and ddH 2 O (balance) composition.
[0033] (2) Freeze-dried tubes of fluorescent quantitative PCR reaction solution (50 tubes):
[0034] Fluorescent quantitative PCR reaction solution includes Taq enzyme 1.25 μL / tube (5U·μL -1 ), reverse transcriptase 1.5μL / tube (20U·μL -1 ), each 3.0 μL / tube of upstream and downstream primers for detection (10 μmol·μL -1 ) and probe 0.3μL / tube (10μmol·μL -1), dNTPs 3μL / tube (2.5mM); add the above mixture into a 0.5ml PCR tube to drop to -50°C, then freeze-dry at 10pa pressure for 24h, close the tube after forming a dry powder, and obtain a fluorescent quantitative PCR reaction solution Fr...
Embodiment 2
[0039] Example 2 The method of using the yak coronavirus detection kit based on the constant temperature isolation fluorescent PCR platform
[0040] The method includes the following steps:
[0041] 1) RNA template: Take 200 μL of the supernatant of the stool sample, and refer to the PetNAD kit extraction manual of Jinrui Hongjie (Xiamen) Biotechnology Co., Ltd. to extract the total RNA from the supernatant of the tested sample to prepare the RNA template;
[0042] 2) Preparation of the fluorescent PCR reaction system: the preparation of the fluorescent PCR reaction system was performed on ice;
[0043] Take 100 μL of reaction buffer and add it to the freeze-dried tube of the positive control substance and mix it to prepare the positive control substance, then take 50 μL of the reaction buffer solution and 5 μL of the positive control substance and add it to the freeze-dried tube of the fluorescent quantitative PCR reaction solution to prepare the positive control reaction sys...
Embodiment 3
[0046] Embodiment 3 standard substance preparation and the mensuration of sensitivity
[0047] Take 200 μL of the supernatant of the feces sample, refer to the PetNAD kit extraction manual of Jinrui Hongjie (Xiamen) Biotechnology Co., Ltd. Total RNA was reverse transcribed into cDNA.
[0048] The cDNA prepared above was amplified by PCR with primers of yak coronavirus respectively, and the corresponding pathogenic cDNA was used as a positive control, and no template was used as a negative control. The reaction system is: cDNA 2.5 μL, 2×Taq PCR Master Mix 12.5 μL, upstream and downstream primers 1.0 μL each, ddH 2 O 8.0 μL for a total volume of 25 μL. The PCR amplification conditions were: 94°C for 5min, 95°C for 30s, 49°C for 30s, 72°C for 30s, 35 cycles, 72°C for 10min. The PCR product was identified by 3% agarose gel electrophoresis, and the target fragment was recovered with a gel recovery kit, and cloned into the pMD19-T vector (Bao Biological Engineering Co., Ltd.), an...
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