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Methods for detecting bicistronic mRNA virus of scylla serrata and kits thereof

A bicistronic and mud crab technology, applied in the field of mud crab breeding biology, can solve problems that have not been reported before, and achieve high specificity, high specificity detection, and high sensitivity

Inactive Publication Date: 2010-05-19
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] At present, there are few reports on the rapid diagnosis of double-antibody sandwich ELISA immunology using high-affinity monoclonal antibodies in the detection of aquatic animal viruses, especially the research on high-affinity monoclonal antibodies for MCDV, and the use of high-affinity monoclonal antibodies for MCDV There is no report on the double-antibody sandwich ELISA immunological rapid diagnosis of MCDV with monoclonal antibody

Method used

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  • Methods for detecting bicistronic mRNA virus of scylla serrata and kits thereof
  • Methods for detecting bicistronic mRNA virus of scylla serrata and kits thereof
  • Methods for detecting bicistronic mRNA virus of scylla serrata and kits thereof

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Experimental program
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Effect test

Embodiment 1

[0060] The method of embodiment 1HRP labeling antibody

[0061] In this embodiment, the sodium periodate method is used to prepare and purify the HRP enzyme-labeled MCDV monoclonal antibody. The specific steps are as follows:

[0062] (1) Weigh 5mg HRP and dissolve it in 0.5ml distilled water, add freshly prepared 0.06mol / l NaIO 4 Aqueous solution 0.5ml, mix well and place in 4℃ refrigerator for 30min;

[0063] (2) Take out and add 0.5ml of 0.16mol / l ethylene glycol aqueous solution, room temperature for 30min, add 1ml of aqueous solution containing 5mg of purified monoclonal antibody, mix well, protect from light, slowly stir, transfer to dialysis bag, at 0.01MpH4.4 Acetic acid buffer was dialyzed overnight at 4°C; the monoclonal antibody was a monoclonal antibody to MCDV structural protein 1, a monoclonal antibody to MCDV structural protein 2 or a monoclonal antibody to MCDV structural protein 3;

[0064] (3) Mix HRP: monoclonal antibody at four mass ratios of 3:1, 2:1, 1:...

Embodiment 2

[0070] The selection of embodiment 2 enzyme-labeled antibody working concentration

[0071] In the immunoenzyme technique, the selection of the working concentration of the enzyme-labeled antibody has a great influence on the whole test. Therefore, the working concentration must be accurately titrated before the formal test.

[0072] The antigen (or antibody) is physically adsorbed on a solid-phase carrier, and then a series of diluted enzyme-labeled antibodies (or anti-Ig antibodies) react with the antigen (or antibody) adsorbed on the carrier, and the reaction between the enzyme and the substrate The degree of color reaction is used to determine the titer of the enzyme-labeled antibody, or the working concentration.

[0073] 1. Determine the optimal working concentration of the enzyme-labeled antibody

[0074] This embodiment adopts the indirect ELISA method to determine the working concentration of the enzyme-labeled antibody. The steps are: first dilute the antigen (100 n...

Embodiment 3

[0077] Example 3 Enzyme-labeled antibody immune activity assay

[0078] Enzyme-labeled antibody immune activity assay: first coat the antigen (in the hemolymph of crabs infected with MCDV), use the enzyme-labeled MCDV monoclonal antibody as the primary antibody, and dilute the enzyme-labeled antibody (1:100x2 n , n=0, 1, 2, 3...11), then add the substrate chromogenic solution TMB, after color development, use 2M H 2 SO 4 0.05ml to stop the reaction, the dual-wavelength microplate reader measures the A450 / A630 value. A450 / A630≥1.0 was judged as positive.

[0079] The results are shown in Table 1. The A450 / A630 value of the monoclonal antibody against structural protein 1 was 1.003 at 1:16400, and the immunological activity of the enzyme-labeled antibody of the monoclonal antibody against structural protein 1 reached 1:6400, while the value of the enzyme-labeled antibody against structural protein 3 was 1:6400. The A450 / A630 value of the monoclonal antibody is 1.201 at 1:128...

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Abstract

The invention discloses methods for detecting bicistronic mRNA viruses of a scylla serrata and kits thereof. A poly-anti-mono monoclonal-antibody and double-antibody sandwich ELISA method and a double-antibody and monoclonal-antibody sandwich ELISA method are adopted for detecting the bicistronic mRNA viruses of the scylla serrata by using monoclonal antibody combined with MCDV specificity. Both the poly-anti-mono monoclonal-antibody and double-antibody sandwich ELISA method and the double-antibody and monoclonal-antibody sandwich ELISA method can sensitively and specifically detect the MCDV masculine in the haemolymph of the virus-infected crabs with the detectable rate of 82 to 92 percent, while the double-antibody and monoclonal-antibody sandwich ELISA method has better sensitivity and specificity on the detection of the bicistronic mRNA viruses of the scylla serrata. The two methods of the invention have the advantages that: corresponding kits can be prepared by the two methods, so the MCDV can be detected with fast speed, high efficiency, sensitivity, specificity and stability, and the methods have instructive meaning for detection in production and future scientific researches.

Description

technical field [0001] The invention relates to the biological technology field of mud crab cultivation, in particular to a method for detecting bicistronic virus in mud crab and a kit thereof. Background technique [0002] Scylla serrata (Scylla serrata, scientific name Mud crab), commonly known as blue crab, is mainly distributed in the tropical and subtropical waters of the Indo-Pacific Ocean. It is an edible crab with high economic value and is one of the famous and excellent breeding species. [0003] Viruses are the main pathogens in the breeding process of mud crab, which cause serious economic losses in mud crab farming. In 2004, two viruses were found in mud crabs suffering from "sleeping sickness" (SD) in Zhuhai: Mud crab dicistrivirus (MCDV) and reovirus (mud crab reovirus, MCRV). [0004] MCDV is a single-strand positive-strand RNA virus ssRNA(+) found in aquatic crustaceans. Its existence alone can cause disease in Scylla serrata, and the mortality rate can re...

Claims

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Application Information

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IPC IPC(8): G01N33/577C07K16/10
Inventor 张锐翁少萍何建国董传甫邓燮雄
Owner SUN YAT SEN UNIV
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