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Method for enriching water body hepatitis A viruses based on immunomagnetic beads

A technology of hepatitis A virus and immune magnetic beads, which is applied in the field of biotechnology applications, can solve the problems of low content of hepatitis A virus, interference with experimental results, and high cost, and achieve shortened steps for amplifying and culturing microorganisms, good repeatability, and separation speed fast effect

Inactive Publication Date: 2014-05-21
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The difficulty in the detection of hepatitis A virus in water is that the content is low and the matrix components that interfere with the determination are complex, which has caused great difficulties for accurate determination.
Most water bodies need to concentrate the volume of water samples to enrich the virus before detection, but unnecessary pollutants in the water are also enriched, which interferes with the experimental results
For example, the flocculation and sedimentation method is usually used to concentrate water samples, but the residual organic substances may affect the detection results of PCR
In addition, traditional virus enrichment detection methods, such as cell culture virus identification, are time-consuming in operation and expensive in terms of technical costs, and hepatitis A virus is difficult to culture in vitro (Brooks, Gersberg et al.2005)

Method used

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  • Method for enriching water body hepatitis A viruses based on immunomagnetic beads
  • Method for enriching water body hepatitis A viruses based on immunomagnetic beads
  • Method for enriching water body hepatitis A viruses based on immunomagnetic beads

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1. Efficiency of Enriching Viruses with Activated Magnetic Beads Coupled with Optimal Antibody Amounts

[0029] 1. Preparation of specific immunomagnetic beads for enrichment of hepatitis A virus:

[0030] (1) Activated carboxyl magnetic beads:

[0031] Take 2 mg (25 mg / ml) carboxyl magnetic beads that are well shaken to a 2 ml centrifuge tube, add 1 ml of 2-(4-morpholine) ethanesulfonic acid (MEST, pH 5.0, 0.05% Tween- 20) Mix the activation buffer evenly on a vortex mixer, place the centrifuge tube on a magnetic separator, discard the supernatant after the magnetic beads are completely absorbed; then add 1ml MEST activation buffer to wash the magnetic beads for 2 After each pass, 300 μl of 5 mg / ml activation reagent carbodiimide (EDC, prepared from 0.01 M MES buffer at pH 5.0) and 300 μl of 5 mg / ml N-hydroxysuccinimide (NHS , prepared from 0.01M MES buffer at pH 5.0) solution, mixed by vortex, fixed on a silent mixer, and activated at 37°C for 45min. Separa...

Embodiment 2

[0051] Example 2. The ability of unit immunomagnetic beads to adsorb hepatitis A virus

[0052] 1. Adsorption of hepatitis A virus by unit immunomagnetic beads:

[0053] The concentration is 1.52×10 6 For Hepatitis A virus copies / μl, dilute at ratios of 1:10, 1:100, and 1:1000. Take 4 parts of 1 mg immunomagnetic beads (immune magnetic beads coupled with 75 μg hepatitis A virus monoclonal antibody) and respectively absorb the original concentration, 1:10, 1:100, 1:1000 diluted 300 μl virus, and incubate at room temperature for 2 hours. Separate the magnetic beads with a magnetic separator, discard the supernatant, add 500 μl PBS (or use 500 μl PBS+0.1%BSA) buffer, shake on the shaker for 5 minutes, separate the magnetic beads with a magnetic separator, and discard the supernatant; Add 500μl PBS (or 500μl PBS+0.1%BSA) buffer to wash twice to remove unadsorbed virus.

[0054] 2. Quantitative detection of the enrichment efficiency of hepatitis A virus

[0055] (1) Trizol meth...

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Abstract

The invention relates to a method for enriching water body hepatitis A viruses based on immunomagnetic beads. The method comprises the following steps: preparing specific immunomagnetic beads for enriching hepatitis A viruses; enriching the hepatitis A viruses by using the immunomagnetic beads; quantitatively detecting the enriching efficiency of the hepatitis A viruses. By enriching the hepatitis A viruses in water bodies through the immunomagnetic beads, the method has the advantages of reasonable concept, easiness and convenience in operation, high specificity, high separating speed, high repeatability and no need of expensive instrument equipment. Moreover, when 1mg of activated magnetic beads are coupled with 75 micrograms of hepatitis A virus monoclonal antibodies, the enriching efficiency of 300 microliters of hepatitis A virus water sample of which the virus concentration is 1.52*10<3> copies / microliter can be up to 64.54 percent in maximum, and the enriching efficiency of 300 microliters of hepatitis A virus water sample of which the virus concentration is 1.52*10<6> copies / microliter can be up to 91.45 percent in maximum. The method has important significance to enriching and detection of the hepatitis A viruses in water bodies.

Description

technical field [0001] The invention belongs to the application field of biotechnology and the field of environmental monitoring technology, and specifically adopts the immunological reaction principle of immune magnetic beads and the characteristics of magnetic field force to enrich the hepatitis A virus polluted in water bodies. technical background [0002] Hepatitis A Virus (HAV) is an RNA virus belonging to the family Picornaviridae and the genus Hepadovirus, and is one of the common enteroviruses. High concentrations of hepatitis A virus exist in the feces of infected patients. As early as 1973, Feinslone used immunoelectron microscopy to observe hepatitis A virus in the feces samples of sick patients (Martin and Lemon2006). They are mainly transmitted through the fecal-oral route, regardless of Outbreaks can be caused by direct person-to-person transmission or by exposure to contaminated food or water. Compared with common enteroviruses, hepatitis A virus has stronge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02C12Q1/70C12Q1/68C12R1/93
Inventor 胡国良乔煜婷曹华斌郭小权张彩英
Owner JIANGXI AGRICULTURAL UNIVERSITY
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