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

Pending Publication Date: 2018-05-29
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to factors such as the need for large-scale equipment, complex operation, and long reaction time, the common fluorescent quantitative PCR method restricts its application in rapid on-site detection.

Method used

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  • Yak coronavirus detection kit based on constant temperature isolated fluorescent PCR platform and application thereof
  • Yak coronavirus detection kit based on constant temperature isolated fluorescent PCR platform and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a yak coronavirus detection kit based on a constant temperature isolated fluorescent PCR platform and an application thereof; the yak coronavirus detection kit consists of a reaction buffer liquid, a fluorescent quantitative PCR reaction liquid freeze-dried tube, and positive control and negative control freeze-dried tubes. The fluorescent quantitative PCR reaction liquid freeze-dried tube is prepared by mixed freeze-drying of a Taq enzyme, a reverse transcriptase, a primer for detection, a probe and dNTPs. The kit has the advantages of short detection time (the reaction time is only 42 minutes), high specificity, high sensitivity and easy storage (storage at the temperature of 4 DEG C), is used with cooperation of a constant temperature isolated PCR instrument, issuitable for spot rapid detection of yak coronavirus, and can be widely popularized at a basic level.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a yak coronavirus detection kit based on a constant temperature isolation fluorescent PCR platform and its application. Background technique [0002] Bovine Coronavirus (BCV) was discovered in the feces of calves with diarrhea by Stair and Mebus in the United States in 1972, and it was proved to be one of the important pathogens causing diarrhea in calves (Jonsson E, Conway P. Probiotics for pigs[J]. Springer Netherlands, 1992.), and the yak coronavirus (Yak BCV) was first isolated from the virus in China by Song Guanglin and others in 1985, but no further research was carried out. In 1990, Yao Huochun et al. found the existence of yak coronavirus in calf yak diarrhea, and showed that yak coronavirus may be an important cause of yak diarrhea (Yao Huochun, Du Nianxing. Serological investigation of bovine coronavirus[J]. Nanjing Agriculture University Journal, 1990,13(2):1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/107C12Q2545/114C12Q2521/107Y02A50/30
Inventor 岳华汤承何琪富
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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