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Method for preparing reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction kit for detecting bovine viral diarrhea viruses

A bovine viral diarrhea, ring-mediated isothermal technology, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and microorganism determination/inspection, etc. Cost, high specificity effect

Inactive Publication Date: 2012-01-25
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to solve the problem that the existing method for detecting bovine viral diarrhea virus is expensive, requires professional equipment and professionals to participate in the detection and is not suitable for the development of animal husbandry in China at the present stage, and provides a method for detecting bovine viral diarrhea virus Preparation of Reverse Transcription Loop Mediated Isothermal Amplification Reaction Kit and Its Application Method

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  • Method for preparing reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction kit for detecting bovine viral diarrhea viruses
  • Method for preparing reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction kit for detecting bovine viral diarrhea viruses

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specific Embodiment approach 1

[0025] Embodiment 1: The preparation method of the reverse transcription loop-mediated isothermal amplification reaction kit for detecting bovine viral diarrhea virus in this embodiment is realized in the following steps: 1. Retrieve the gene sequences of all bovine viral diarrhea viruses from the gene database , through Clustalw software analysis to find out the specific conserved target DNA sequence, and then use the Primer Explorer 4 software to design 6 primers; 2. Prepare the reverse transcription loop-mediated isothermal amplification reaction solution according to the obtained 6 primers, and then complete the detection of cattle 病毒性腹泻病毒的逆转录环介导等温扩增反应试剂盒的制备;其中步骤一中保守靶DNA序列为:TGAGGGACAAATCCTCCTTAGCGAAGGCCGAAAAGAGGCTAGCCATGCCCTTAGTAGGACTAGCAAAACAAGGAGGGTAGCAACAGTGGTGAGTTCGTTGGATGGCTGAAGCCCTGAGTACAGGGTAGTCGTCAGTGGTTCGACGCTTTGTGCGACAAGCCTCGAGATGCCACGTGGACGAGGGCATGCCCACAGCACATCTTAACCTGAGCGGGGGTCGTTCAGGTGAAAACGGTTTAACCAACCGCTACGAATACAGCCTGATAGGGTGCTGCAGAGGCCCACTGTATCGCTACTAAAAATCTCT...

specific Embodiment approach 2

[0036] Specific embodiment two: the use method of the reverse transcription loop-mediated isothermal amplification reaction kit for detection of bovine viral diarrhea virus in this embodiment: one, get 5 μ l of the RNA extract containing bovine viral diarrhea virus sample, add 20 μ l reverse transcription The loop-mediated isothermal amplification reaction solution was evenly mixed and placed at 62°C for reverse transcription loop-mediated isothermal amplification reaction for 1 hour; 2. After the amplification reaction, centrifuged at 10000r / min for 1min, there was precipitation at the bottom of the reaction tube , then there is bovine viral diarrhea virus in the sample to be tested, and there is no precipitation at the bottom of the reaction tube, then there is no bovine viral diarrhea virus in the sample to be tested.

[0037] In this embodiment, a negative control group can be set up for detection, and the negative control group uses water instead of RNA extract, and the re...

specific Embodiment approach 3

[0038] Specific embodiment three: the use method of the reverse transcription loop-mediated isothermal amplification reaction kit for detection of bovine viral diarrhea virus in this embodiment: 1. Take 5 μl of the RNA extract containing bovine viral diarrhea virus sample, add 20 μl reverse transcription Loop-mediated isothermal amplification reaction solution, mixed evenly and placed at 62°C for reverse transcription loop-mediated isothermal amplification reaction for 1 hour; 2. After the amplification reaction, add 5 μl of 10000×SYBR

[0039] GREEN I, then placed under ultraviolet light, the reaction solution turns green, then there is bovine viral diarrhea virus in the sample to be tested, and the reaction solution is orange, then there is no bovine viral diarrhea virus in the sample to be tested.

[0040] In this embodiment, a negative control group can be set up for detection, and the negative control group uses water instead of RNA extract, and the rest of the components ...

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Abstract

The invention provides a method for detecting bovine viral diarrhea viruses as well as a preparation method and a use method of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction kit, relating to a preparation method and a use method of a reaction kit. The invention solves the problems that the traditional method for detecting bovine viral diarrhea viruses has expensive cost, needs professional equipment and professional staff to participate in detection, and is not suitable for the development state of the animal husbandry at the present stage of China. The preparation method comprises the following steps: firstly, designing a primer, preparing RT-LAMP reaction liquid, and then, finishing the preparation. The use method comprises the following steps: adding RNA extract containing bovine viral diarrhea virus samples into the reaction liquid, ungergoing RT-LAMP reaction after mixing, and then, centrifugally detecting; or adding 10,000*SYBR GREEN I, and putting below an ultraviolet lamp for detecting; or carrying out electrophoresis detection; or carrying out insulation reaction detection by adopting a Real-time PCR instrument. The reaction kit has convenient detection, does not need expensive instruments in routine tests and reduces the cost.

Description

technical field [0001] The invention relates to the preparation of the reaction kit and its use method. Background technique [0002] Bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV), also known as bovine viral diarrhea-mucosal disease virus (Bovine viral diarrhea-mucosal disease virus, BVD-MDV), is a single-stranded positive-sense RNA virus; bovine contact infection, Symptoms are mainly diarrhea, acute and chronic mucosal diseases, persistent infection and immune tolerance, immunosuppression, abortion, stillbirth and teratogenicity in female animals; the disease mainly occurs in cattle, and can also infect pigs, sheep, deer, camels and other wild ruminants. BVDV has few characteristic clinical symptoms, and it is often difficult to be diagnosed in actual production. In addition, the fatality rate of this disease is not high, and people have not paid enough attention to the existence of this disease. monitoring and control measures. BVDV can cause persisten...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 王君伟李岩高明春马波
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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