Dengue virus serology early diagnosis reagent

A dengue virus and diagnostic reagent technology, applied in the field of serological detection of dengue virus, can solve the problems of expensive kits, difficult to popularize and apply at the grassroots level, and not suitable for early diagnosis of dengue virus infection, and achieve good specificity and sensitivity , the effect of simple operation

Inactive Publication Date: 2011-02-02
中国疾病预防控制中心病毒病预防控制所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, foreign kits are expensive and difficult to popularize and apply at the grassroots

Method used

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  • Dengue virus serology early diagnosis reagent
  • Dengue virus serology early diagnosis reagent
  • Dengue virus serology early diagnosis reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 Expression of recombinant dengue virus E protein domain III and rEIII

[0041] Materials and methods

[0042] 1. Construction of DV type 1-4 EIII domain and rEIII fusion recombinant expression vector

[0043] 1.1 Acquisition of DV1-4 type EIII region gene fragment and rEIII coding gene fragment

[0044] In this example, the EIII gene of DV types 1-4 was obtained by extracting dengue virus RNA and reverse transcription. Of course, those skilled in the art can artificially synthesize the EIII region gene fragments of DV1-4 types and the rEIII code by using the disclosed EIII domain sequence. Gene fragment.

[0045] 1.1.1 Acquisition of cDNA

[0046] C6 / 36 cells were cultured in 1640 growth medium (containing 30% eagle's, 10% fetal bovine serum, 1% double antibody, 1% glutamine), and cultured at 28°C, 5% CO2 until the cells grew into a monolayer, Inoculate 1-4 standard strains of dengue virus (type 1 GZ01 / 95 strain, type 2 ZS01 / 01 strain, type 3 H87 strain...

Embodiment 2

[0179] Application of embodiment 2 recombinant dengue virus EIII protein and rEIII in dengue virus fever serum detection

[0180] In this example, the Mac-ELISA method was established by using the dengue virus type 1-4 EIII recombinant protein and rEIII fusion protein prepared in Example 1 to explore the application of the recombinant dengue virus EIII protein and rEIII in the detection of dengue virus fever serum.

[0181] Experimental Materials

[0182] 1. Coating antibody: goat anti-human IgM-μ chain antibody (SIGMA, USA).

[0183] 2. Serum to be tested: dengue fever monitoring serum (collected from Guangdong, Guangxi, Fujian and other places), healthy human serum, epidemic hemorrhagic fever patient serum (Virus Disease Institute, Chinese Center for Disease Control and Prevention), Japanese encephalitis patient IgM positive serum (China Institute of Virology, Academy of Preventive Medicine).

[0184] 3. Enzyme-labeled antigen: HRP-labeled recombinant EIII protein and reco...

Embodiment 3

[0259] The composition of embodiment 3 dengue virus infection early diagnosis kits

[0260] According to Example 2, this example provides a detection kit for early diagnosis of dengue virus infection. The kit consists of the following parts:

[0261] 1) A microtiter plate coated with goat anti-human IgM-μ chain antibody;

[0262] 2) HRP labeled rEIII antigen;

[0263] 3) ELISA washing solution (PBST): 0.05% Tween20, 1X PBS, pH7.4;

[0264] 4) Substrates of HRP:

[0265] Solution A: 5mg tetramethylbenzidine (TMB) is dissolved in 5mL dimethyl sulfoxide (DMSO). Dissolve in acetate buffer (0.2mol / L.pH 5.8-6.0) according to 1:4;

[0266] Liquid B: 0.03% H 2 o 2

[0267] 5) ELISA reaction termination solution: 2moI / L H 2 SO 4 .

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Abstract

The invention provides a dengue virus serology early diagnosis reagent, in particular, EIII structural domains of dengue viruses 1, 2, 3 and 4 are serially connected to obtain a recombinant fusion protein rEIII used for serodiagnosis of the dengue virus infection. In addition, the invention also provides an early diagnosis agent kit suitable for the dengue virus infection. The invention provides a new idea for the serodiagnosis of the dengue virus infection. The dengue virus infection can be effectively diagnosed by using the diagnosis reagent, especially, the Mac-ELISA (Enzyme Linked Immunosorbent Assay) diagnosis agent kit can be effectively used for the early serodiagnosis of the dengue virus infection. The diagnosis reagent has the advantages of better specificity and sensitivity and simple operation, and is suitable for being widely popularized and used.

Description

technical field [0001] The invention relates to dengue virus serological detection, in particular to a dengue virus serological early diagnosis reagent. Background technique [0002] Dengue virus (dengue virus, DV) belongs to Flaviviridae (Flaviviridae) genus Flavivirus (Flavivirus), divided into four serotypes, namely dengue 1, 2, 3, 4 viruses. It can cause dengue fever (dengue fever, DF), dengue hemorrhagic fever (dengue hemorrhagic fever, DHF) and dengue shock syndrome (dengue shock syndrome, DSS), among which dengue fever is the most common. It is mainly transmitted through the bite of Aedes aegypti or Aedes albopictus. In recent decades, the epidemic area of ​​dengue virus in the world has been expanding. According to the World Health Organization, the health of about 250 million people in the world is threatened by dengue fever, and about 51 million people are infected with dengue virus every year, of which about 500,000 people are dengue hemorrhagic fever, and about ...

Claims

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Application Information

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IPC IPC(8): C07K19/00G01N33/543C12R1/19C12P21/02G01N33/569C12N15/70C12N15/62
CPCY02A50/30
Inventor 李德新梁米芳张全福陆鹏李建东王晓芳
Owner 中国疾病预防控制中心病毒病预防控制所
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