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Tiger-derived pseudorabies virus detection kit and tiger-derived pseudorabies virus detection method

A technology of pseudorabies virus and detection kit, which is applied in biochemical equipment and methods, measurement/testing of microbes, DNA/RNA fragments, etc., can solve the problems of detection technology of pseudorabies virus from tiger sources and achieve prevention and control the spread of the epidemic, the effect of simple operation and complex operation

Pending Publication Date: 2020-08-21
LONGYAN UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many inspection methods for porcine pseudorabies virus, but there is no detection technology for tiger-originated pseudorabies virus in the literature

Method used

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  • Tiger-derived pseudorabies virus detection kit and tiger-derived pseudorabies virus detection method
  • Tiger-derived pseudorabies virus detection kit and tiger-derived pseudorabies virus detection method
  • Tiger-derived pseudorabies virus detection kit and tiger-derived pseudorabies virus detection method

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Experimental program
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Effect test

Embodiment 1

[0028] A detection kit for tiger-derived pseudorabies virus, comprising an enzyme liquid and a reaction buffer, the enzyme liquid is composed of DNA polymerase and RNase H, and also includes a negative control and a positive control; the negative control is used 0.01mol / L, PBS buffered saline at pH7.2; the positive control is to connect the tiger-derived pseudorabies virus PCR amplification product with the cloning vector pMD-18-Tvector, transform Escherichia coli competent DH5α, and obtain a positive clone strain, And prepare the plasmid pMD18-tiger-RPV as a positive control. Also included are the following primer pairs and probes:

[0029] Detect the primer pair and Cycling probe of tiger-derived pseudorabies virus, the primer pair of described detection tiger-derived pseudorabies virus is the upstream primer Tiger-RPV-F shown in SEQIDNO:1 and the downstream primer Tiger-RPV-F shown in SEQIDNO:2 RPV-R, the Cycling probe is a probe Tiger-RPV-Pro and its sequence is shown in ...

Embodiment 2

[0042] A specificity experiment of the detection kit of Tiger-origin pseudorabies virus based on CycleavePCR technology:

[0043] Using the Pseudorabies virus from Huyuan and Porcine Pseudorabies virus verified by sequencing as templates, take 3 μL templates and add them to 17 μL fluorescent quantitative CycleavePCR reaction system, place them on a fluorescent quantitative PCR instrument, and run the program as 95°C pre-denaturation for 30s; 95 Denaturation at ℃ for 10 s, annealing at 50 ℃ for 30 s, and extension at 72 ℃ for 30 s, a total of 40 cycles; fluorescence signal collection was performed at the stage of 30 s at 72 ℃. Fluorescence curve as figure 1 As shown, curve A is tiger-derived pseudorabies virus, and curve B is porcine pseudorabies virus. The results show that only tiger-derived pseudorabies virus is used as a template to generate S-type fluorescent signals, indicating that the kit has good specificity.

Embodiment 3

[0045] A kind of detection method of the detection kit of tiger source pseudorabies virus based on CycleavePCR technology

[0046] For the biological detection of 4 strains of pseudorabies virus from Huyuan, the specific process is as follows:

[0047] 1) Sample nucleic acid extraction: 4 samples to be extracted, including A1, A2, A3, and A4, were extracted with commercial virus nucleic acid extraction kits respectively. figure 2 Electropherograms of nucleic acid extraction for 4 samples.

[0048] 2) Take 3 μL of the nucleic acid extracted in the previous step and 17 μL of the PCR reaction solution in the detection kit, add them into the PCR reaction tube, blow and mix them with a pipette, and place them on a fluorescent quantitative PCR instrument for reaction. The program of the fluorescent quantitative PCR instrument was pre-denaturation at 95°C for 30s; denaturation at 95°C for 10s, annealing at 50°C for 30s, and extension at 72°C for 30s, a total of 40 cycles.

[0049]...

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Abstract

The invention belongs to the field of animal epidemic disease molecular biological detection methods and detection reagents. The invention relates to a detection kit and a detection method for tiger-derived pseudorabies virus. A preparation method comprises the following steps: performing high-throughput sequencing on five tiger-derived pseudorabies viruses, and assembling to obtain a whole genomesequence of tiger-derived pseudorabies; comparing with a plurality of porcine pseudorabies viruses to find that a tiger-derived pseudorabies virus genome has a plurality of single nucleic acid mutations. The tiger-derived pseudorabies virus is used for detection aiming at one mononucleotide mutation; a pair of detection primers of the pseudorabies virus and a Cycling probe for detecting the tiger-derived pseudorabies virus are designed to form an inspection kit for detecting the tiger-derived pseudorabies virus, the detection method has the advantages of simplicity and convenience in operation, high sensitivity, strong specificity, good repeatability and the like, and a result can be judged according to an amplification curve immediately after the reaction is finished.

Description

technical field [0001] The invention belongs to the field of animal epidemic molecular biology testing methods and testing reagents, in particular to a testing kit and testing method for tiger-derived pseudorabies virus. Background technique [0002] Tiger-origin pseudorabies virus (Pseudorabies virus, PRV) infection is an acute infectious disease of livestock and wild animals, and it is one of the pathogens that cause major economic losses in the world's pig industry. Pigs are the storage host and main source of infection of the disease, and the clinical symptoms are mainly characterized by fever, nervous system and respiratory disturbance. Lactating piglets and young pigs are the most sensitive to the virus. After infection, body weight and appetite decrease, and the mortality rate is 50-100%. PRV, also known as porcine herpesvirus type 1, belongs to the family Herpesviridae. The rabies virus can also infect tigers at the same time, endangering the lives of endangered an...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/705C12Q1/6851C12Q2600/166C12Q2531/113C12Q2563/107C12Q2521/327
Inventor 范克伟林开雄包银莉郑琳傅文源黄翠琴杨守深
Owner LONGYAN UNIV
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