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Yak rotavirus detection kit based on constant-temperature isolation type fluorescence PCR (Polymerase Chain Reaction) platform and application

A detection kit and technology for rotavirus, which are applied in the determination/inspection of microorganisms, DNA/RNA fragments, biochemical equipment and methods, etc. , long reaction time and other problems, to achieve the effect of reducing the possibility of degradation and pollution, short detection time, and saving reagents

Inactive Publication Date: 2017-06-20
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.
[0004] At the same time, due to the special environment of the Qinghai-Tibet Plateau, the yak rotavirus gene has undergone great variation, especially the VP6 gene that is often used as a target gene. Research by Zhou Fang et al. Detection rate of PCR method (Zhou Fang, Yue Hua, Zhang Bin, et al. 2016. Sequence analysis of yak rotavirus VP6 gene and establishment and application of RT-PCR detection method[J]. Journal of Animal Husbandry and Veterinary Medicine, 47(7) , 1465-1473.)

Method used

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  • Yak rotavirus detection kit based on constant-temperature isolation type fluorescence PCR (Polymerase Chain Reaction) platform and application
  • Yak rotavirus detection kit based on constant-temperature isolation type fluorescence PCR (Polymerase Chain Reaction) platform and application

Examples

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Effect test

Embodiment 1

[0033] Example 1 Yak rotavirus detection kit based on constant temperature isolation fluorescent PCR platform

[0034] A yak rotavirus detection kit based on a constant temperature isolation fluorescent PCR platform, comprising the following components:

[0035] (1) Reaction buffer: 3ml, composed of 500mM KCl, 100mM Tris-HCl (pH 8.3), 15mM MgCl 2 and ddH 2 O (balance) composition.

[0036](2) Freeze-dried tubes of fluorescent quantitative PCR reaction solution (50 tubes):

[0037] Fluorescent quantitative PCR reaction solution including Taq enzyme 1 μL / tube (5U·μL -1 ), reverse transcriptase 1.25μL / tube (20U·μL -1 ), each 3.5 μL / tube of upstream and downstream primers for detection (10 μmol·μL -1 ) and probe 0.25μL / tube (10μmol·μL -1 ), dNTPs 4μL / 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, form a dry powder and close the tube to prepare a fluorescent quantitative PCR reaction solution Freeze-dried...

Embodiment 2

[0041] Example 2 The method of using the yak rotavirus detection kit based on the constant temperature isolation fluorescent PCR platform

[0042] The method includes the following steps:

[0043] 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;

[0044] 2) Preparation of the fluorescent PCR reaction system: the preparation of the fluorescent PCR reaction system was performed on ice;

[0045] 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 syste...

Embodiment 3

[0048] The mensuration of embodiment 3 standard substance preparation and sensitivity

[0049] 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.

[0050] The cDNA prepared above was amplified by PCR with primers of yak rotavirus 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: 2.0 μL of cDNA, 12.5 μL of 2×Taq PCR Master Mix, 1.0 μL of upstream and downstream primers, 8.5 μL of ddH2O, and a total volume of 25 μL. The PCR amplification conditions were: 95°C for 5min, 95°C for 30s, 52°C for 30s, 72°C for 30s, 30 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.),...

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Abstract

The invention discloses a yak rotavirus detection kit based on a constant-temperature isolation type fluorescence PCR (Polymerase Chain Reaction) platform and application. The yak rotavirus detection kit is composed of a reaction buffering solution, a fluorescence quantitative PCR reaction solution freeze-drying pipe, a positive reference substance freeze-drying pipe and a negative reference substance freeze-drying pipe. The fluorescence quantitative PCR reaction solution freeze-drying pipe is formed through mixing and freeze-drying a Taq enzyme, reverse transcriptase, a primer for detecting, a probe and dNTPs (deoxyribonucleoside triphosphate). The kit has the advantages of short detection time (reaction time is only 42min), high specificity, high sensitivity and convenience for storage (preservation at 4 DEG C); the kit is matched with a constant-temperature isolation type PCR instrument, is very suitable for on-situ rapid detection of yak rotaviruses and can be widely popularized in grassroots.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a yak rotavirus detection kit based on a constant temperature isolation fluorescent PCR platform and its application. Background technique [0002] Mebus et al. discovered bovine rotavirus in 1968, and yak rotavirus (Yak RV) was discovered and isolated for the first time in 1986 in Zhang Min et al.'s research on yak diarrhea, but no further research was carried out (Zhang Min, Wang Qingjiang, Liu Dengwei, et al. Research on rotavirus of Hongyuan yak [J]. Sichuan Animal Husbandry and Veterinary Medicine, 1986,3:2-4.). In 1990, Li Mingrui et al found the existence of yak rotavirus in calf yak diarrhea in Lanzhou, and proved that yak rotavirus is an important cause of yak diarrhea (Li Mingrui, Li Chunling, Li Hao, etc. 1990. Yak calf epidemic Investigation of pathogens of diarrhea [J]. Chinese Veterinary Science (2), 12-13.). The clinical symptoms of calf yaks infected by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/107C12Q2545/114C12Q2521/107
Inventor 汤承岳华李凡
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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