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Kit for identifying virulent strain and attenuated vaccine strain of duck tembusu virus and use thereof

A technology for duck tambusu virus and attenuated vaccine, which can be applied in the directions of microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc. Vaccine strains and other issues to achieve high sensitivity and specificity

Active Publication Date: 2014-12-17
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims to solve the technical problem of being unable to quickly and efficiently differentiate and diagnose the virulent and attenuated vaccine strains of Duck Tembusu virus, and provides a kit for distinguishing the virulent and attenuated vaccine strains of Duck Tembusu virus. The kit can quickly, easily and accurately distinguish duck Tembusu virus natural infection virulent and attenuated vaccine immune strains, with strong specificity, high sensitivity and good repeatability

Method used

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  • Kit for identifying virulent strain and attenuated vaccine strain of duck tembusu virus and use thereof
  • Kit for identifying virulent strain and attenuated vaccine strain of duck tembusu virus and use thereof
  • Kit for identifying virulent strain and attenuated vaccine strain of duck tembusu virus and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 The determination of primer design and PCR conditions for distinguishing duck Tembusu virus virulent strain and attenuated vaccine strain

[0046] (1) Primer design and synthesis

[0047] Eight virulent duck Tembusu virus strains and duck Tembusu virus strains published in GenBank, including BYD-1 strain, FS strain, ALD strain, JM strain, JS804 strain, ZJ-6 strain and SD strain The genome sequence of the virus attenuated vaccine strain (FX2010-180P strain) was compared and analyzed, and 3 primers were designed. Among them, P1 (5′-CTGAAACATGCCGCYAGA-3′, Y=C / T.SEQ ID NO: 3) is a specific upstream primer for duck Tembusu virus virulent strain (FX2010), and P2 (5′-GTGTAGTAATACATTTTTCCCTAGCA-3′ ,SEQ ID NO: 4) is the specific upstream primer of duck Tembusu virus attenuated vaccine strain (FX2010-180P strain), the base "A" at the 3' end of the P2 primer is neither a virulent sequence nor an attenuated sequence, add " A" is to improve the specificity of the differe...

Embodiment 2

[0066] Embodiment 2RT-PCR sensitivity test

[0067] According to the annealing temperature determined by the primer pair SEQ ID NO: 3 / SEQ ID NO: 4 / SEQ ID NO: 5 in Example 1, the virulent strain of duck Tembusu virus (FX2010) and the attenuated vaccine strain of duck Tembusu virus were (FX2010-180P) were serially diluted, take 0.1ml containing 10TCID 50 、10 2 TCID 50 、10 3 TCID 50 and 10 4 TCID 50 The virus solution was tested by RT-PCR method with the primer pair.

[0068] The virus liquid with different virus content was used as the detection material, and was detected by RT-PCR method. The results showed that for the virulent strain of duck Tembusu virus (FX2010), 10TCID 50 The virus liquid can detect the specific band ( Figure 4), for duck Tembusu virus attenuated vaccine strain (FX2010-180P), 10 3.0 TCID 50 Virus fluid can detect specific bands ( Figure 4 ). Figure 4 Medium, 1-4: attenuated strain of duck Tembusu virus (FX2010-180P, virulence 10-10 4.0 TCI...

Embodiment 3

[0069] Embodiment 3RT-PCR specificity test

[0070] According to the annealing temperature determined by the primer pair of SEQ ID NO: 3 / SEQ ID NO: 4 / SEQ ID NO: 5 in Example 1, the primer pair is used to amplify duck Tambusu virus virulent strain (FX2010), duck Tambusu virus Genomes of Attenuated Suvirus Vaccine Strain (FX2010-180P), Duck Plague Virus, Muscovy Duck Parvovirus, H9 Subtype Influenza Virus, Type I Duck Hepatitis Virus, Duck Reovirus and Normal Cell Negative Control, RT-PCR Method for Testing specificity.

[0071] Results: RT-PCR was used to amplify the genomes of duck Tembusu virus virulent strain (FX2010) and duck Tembusu virus attenuated vaccine strain (FX2010-180P), and a fragment with a size of 860bp and 429bp could be amplified respectively, while The genomes of duck plague virus, muscovy duck parvovirus, H9 subtype influenza virus, type I duck hepatitis virus, duck reovirus and normal cell negative controls did not amplify any fragments, indicating that th...

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Abstract

The invention discloses a kit for identifying virulent strain and attenuated vaccine strain of duck tembusu virus and a use thereof. The kit comprises an upstream primer P1 aiming at a duck tembusu virus virulent strain sequence shown in the formula of SEQ ID NO: 1, wherein the last basic group at the tail end 3' of the P1 is the same as a 1454th basic group of the sequence shown in the formula of SEQ ID NO: 1 and the penultimate basic group at the tail end 3' of the P1 is the same as a 1453th basic group of the sequence shown in the formula of SEQ ID NO: 1, also comprises an upstream primer P2 aiming at a duck tembusu virus attenuated vaccine strain sequence shown in the formula of SEQ ID NO: 2, wherein the penultimate basic group at the tail end 3' of the P2 is the same as a 1891th basic group of the sequence shown in the formula of SEQ ID NO: 2, and also comprises an universal downstream primer P3 aiming at a 1971th-10991th nucleotide sequence shown in the formula of SEQ ID NO: 1 or SEQ ID NO: 2. The kit can fast, simply and accurately distinguish natural-infection virulent strain and attenuated vaccine strain of duck tembusu virus, has high specificity, high sensitivity and good repeatability and has a very important meaning for clinical wide application of the attenuated vaccine strain of duck tembusu virus.

Description

technical field [0001] The invention relates to the technical field of duck tembusu virus detection, in particular to a kit for distinguishing duck tembusu virus virulent strains and attenuated vaccine strains and an application thereof. Background technique [0002] Since April 2010, an infectious disease characterized by slow growth of meat ducks and a sharp drop in egg production of laying ducks has occurred in Shanghai, Zhejiang, Jiangsu and other places in my country. In just a few months, almost all eggs in southern provinces Duck farms have been attacked by the disease, causing huge economic losses to the duck industry. Domestic researchers have proved that the infectious disease is caused by a new flavivirus—Duck Tembusu virus (DTMUV) through virus isolation and identification, virus genome sequence analysis, and animal regression experiments. Belongs to the flavivirus genus Ntaya virus group. After ducks are infected with the virus, the main clinical symptoms are h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701
Inventor 李泽君李国新
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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