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Kit and method for detecting lumpy skin disease viruses

A technology for nodular and skin diseases, applied in nucleic acid detection kits and detection fields for bovine nodular skin diseases, can solve problems such as poor sensitivity, and achieve short amplification time, multi-data support, and high efficiency Effect

Active Publication Date: 2021-03-09
北京弘进久安生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Virus isolation and identification takes a long time and needs to be carried out in a certain level of biosafety laboratory. Virus neutralization test is the most specific serological detection method, but because the animal body mainly shows cellular immune response to LSD virus infection, it only produces low levels of Neutralizing antibodies, therefore the method is less sensitive

Method used

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  • Kit and method for detecting lumpy skin disease viruses
  • Kit and method for detecting lumpy skin disease viruses
  • Kit and method for detecting lumpy skin disease viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: the preparation of positive template

[0063] The positive cloned plasmids were synthesized and sequenced by Beijing Liuhe Huada Gene Co., Ltd. After the sequencing results were compared with the LSDV-LD126 and LSDV001 gene sequences published on GenBank, the concentration and purity of the plasmids were measured by an automatic microplate reader, and named respectively. For LSDV-LD126, LSDV001.

[0064] The LSDV-LD126 gene sequence is shown in SEQ ID NO: 1, specifically:

[0065] ATGGGAATAGTATCTGTTGTATACGTCGTAGTACCATTTTCATTTATTGTTTTACTTTCATATATATTTTTTGAATACAAAAATGTTATTAAAAAAATGTTATTTAAAAAAAAAGGGAAAAATCAAAAAAGAACATGTGTTAGACTTAATTCAATCACTTATTCAACAAATAGTATAAAATCCACTATATCAGAAAGTACTTGGTCAAATTGTAGTAATGATACATTTGTAAAAAATGAAAAAGAAAATGTAGAGATTGTTGAAATTAAACGTTGTGATAATGAATTAATTGAAGAAAGTAATAATAACGTTTTAGAAAATGGATGTACCACAAATACAGGTGAAGAAAATTTAATTTGGGACGATAACAACGTTTATGATTTACCACCTAATGATAGTGTTTATGATTTACCACCTAACGATTTGAGTTGTAACAACGATTGTGTTTATACATTACCGGATGACAATGTTTCAAACATAGAGG...

Embodiment 2

[0070] Example 2: Design and synthesis of primers and probes

[0071] According to the LSDV genome sequence (AF325528.1) published on GenBank, the genome sequence was analyzed and compared using DNAMan software, and primers and probes were designed for the conserved region of the genome sequence. The sequence is shown in Table 1. Primers and probes were synthesized by Shanghai Shining Molecular Biotechnology Co., Ltd.

[0072] Table 1: Primers and Probes

[0073]

Embodiment 3

[0074] Example 3: Identification of Primer Specificity

[0075] LSDV-LD126 and LSDV001 positive recombinant plasmids were prepared according to the reaction system provided by the instructions of QuantiNova SYBR Green PCR Kit (CatNo.208052), that is, 10 μL of Premix Taq (2×) mixture, 0.1 μL of QN ROX ReferenceDye, 1.4 μL of each upstream primer and downstream primer ( 0.7μM), template DNA 1μL, with ddH 2 O was supplemented to 20 μL for amplification. The amplification conditions were pre-denaturation at 95°C for 2 minutes; 40 cycles of amplification at 95°C for 5s and 60°C for 10s. After the amplification, continue to do the melting curve, 95°C, 15s, 60°C, 1min; 95°C, 30s, 60°C, 15s. Verify the specificity of the primers.

[0076] The amplification results showed that the melting curve of the LD126 gene ( figure 1 ) has a single peak at the Tm value of 73.79°C, and the melting curve of the LSDV001 gene ( figure 2 ) have only a single peak at Tm of 75.79°C. From the abov...

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Abstract

The invention relates to a specific primer composition for lumpy skin disease viruses, and also relates to a kit and a method for detecting the lumpy skin disease viruses. At least two groups of specific primer pairs are designed for conserved sequences of lumpy skin disease viruses, and a detection kit and a detection method are further established. The kit and the method provided by the invention have the advantages of high sensitivity, shorter amplification time and higher efficiency, can identify whether the lumpy skin disease virus contained in the sample comes from vaccine immunity or natural infection, and are a powerful supplement to the existing lumpy skin disease virus detection method.

Description

technical field [0001] The invention relates to the field of animal virus molecular biology detection methods, in particular to a nucleic acid detection kit and detection method for bovine nodular skin disease. Background technique [0002] Bovine nodular skin disease (Lumpy skin disease, LSD), is a bovine pox disease caused by bovine nodular skin disease virus (Lumpyskin disease virus, LSDV), belongs to the poxviridae vertebrate poxvirus subfamily goat Poxvirus is a genus, which is not the same as vaccinia virus. The clinical features of the disease include fever, nodules in the skin, mucous membranes and internal organs, emaciation, enlarged lymph nodes, skin edema, and death in some cases. The disease can also cause a temporary decrease in milk production, cause temporary or permanent infertility in bulls, damage to hides, and even animal death due to secondary bacterial infection, thereby having a serious economic impact. The sickness rate of this disease is between 5%...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2561/101
Inventor 崔贝贝杨思佳李霆张现东应天翼余涛李岩松
Owner 北京弘进久安生物科技有限公司
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