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Method and special kit for detection of porcine cytomegalovirus antibody

A kit, antigen protein technology, applied in the direction of viruses/phages, viruses, viral peptides, etc., can solve the problems of limited detection sensitivity, inability to perform high-throughput testing, and time-consuming serology and fluorescent antibody detection.

Inactive Publication Date: 2016-04-20
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The current methods for detecting porcine cytomegalovirus are mainly based on clinical symptoms, serological tests, fluorescent antibody detection, PCR detection, ELISA, and light microscopy methods such as the detection of eagle-eye-shaped inclusion bodies in giant cells, but serology and fluorescent antibody detection are time-consuming , Laborious, requires a lot of equipment, detection sensitivity is also limited, and high-throughput inspection is not possible; PCR detection cannot detect antibodies against pathogens in serum, especially after pathogens have large mutations; ELISA uses antigen-antibody interactions, and The signal is amplified by the marker, and the sensitivity is greatly improved. It is a common method for detecting porcine cytomegalovirus at present.

Method used

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  • Method and special kit for detection of porcine cytomegalovirus antibody
  • Method and special kit for detection of porcine cytomegalovirus antibody
  • Method and special kit for detection of porcine cytomegalovirus antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Embodiment 1, the preparation of antigenic protein

[0089] 1. Carrier Construction

[0090] The double-stranded DNA molecule shown in sequence 1 of the artificially synthesized sequence listing was inserted between the BamHI recognition sequence and the HindIII recognition sequence of plasmid pET-32a(+), to obtain recombinant plasmid pET-32a-K.

[0091] The double-stranded DNA molecule shown in sequence 1 of the sequence listing encodes the protein shown in sequence 2 of the sequence listing. The protein shown in Sequence 2 in the sequence listing is the antigenic protein.

[0092] In the recombinant plasmid pET-32a-K, the double-stranded DNA molecule shown in sequence 1 of the sequence listing is fused with part of the nucleotides on the vector backbone to form a fusion gene shown in sequence 4 of the sequence listing, expressing sequence 3 of the sequence listing Antigenic proteins with His tags are indicated.

[0093] 2. Preparation of antigenic protein

[0094]...

Embodiment 2

[0101] Embodiment 2, establishment of the method for detecting porcine cytomegalovirus antibody in the serum to be tested

[0102] The method for detecting the porcine cytomegalovirus antibody in the serum to be tested is as follows:

[0103] 1. Take the microtiter plate and coat it with the antigenic protein prepared in Example 1;

[0104] 2. Add the serum to be tested or porcine cytomegalovirus negative serum and incubate;

[0105] 3. Add HRP-labeled goat anti-pig IgG antibody and incubate;

[0106] 4. Add luminescence solution and incubate;

[0107] 5. Detection by chemiluminescence detector.

[0108] Then judge as follows: if the absorbance value of the serum to be tested is greater than 2.1 with the absorbance value of the porcine cytomegalovirus negative serum, the serum to be tested contains porcine cytomegalovirus antibody; The absorbance ratio is below 2.1, and the serum to be tested does not contain porcine cytomegalovirus antibody.

Embodiment 3

[0109] Embodiment 3, the optimization of the optimal detection condition of the method for detecting the porcine cytomegalovirus antibody in the serum to be tested

[0110] In order to save reagents and workload, the single factor variable was used to determine the optimal value, and then the optimal detection conditions were determined.

[0111] 1. Determination of antigenic protein coating concentration

[0112] The steps to determine the coating concentration of antigenic protein are as follows:

[0113] 1. Take the microtiter plate, and use the antigenic protein prepared in Example 1 as the coating source to coat (dilute the antigenic protein solution prepared in Example 1 with the coating liquid, and set the following coating concentrations respectively: 3.050 μg / ml, 1.525 μg / ml, 0.610μg / ml, 0.305μg / ml, 0.203μg / ml, 0.153μg / ml, 0.122μg / ml, 0.102μg / ml, add 100μl to each well), seal with parafilm, and coat at 4°C 20h, then discard the supernatant, wash with washing solutio...

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Abstract

The invention discloses a method and a special kit for detection of a porcine cytomegalovirus antibody. The method for detection of the porcine cytomegalovirus antibody in a serum to be detected comprises the steps that antigen protein is used as a coating antigen, and the porcine cytomegalovirus antibody in the serum to be detected is detected through a chemiluminescence enzyme immunoassay method. The antigen protein comprises N-end elements and C-end elements in sequence from the tail end N to the tail end C; the amino acid sequence of the N-end elements is shown from the site 1 to the site 141 starting from the tail end N of the sequence 2 in a sequence table; the amino acid sequence of the C-end elements is shown from the site 147 to the site 244 starting from the tail end N of the sequence 2 in the sequence table. Experiments prove that the method has a high accuracy rate and has the advantages that sensitivity is high, the analysis method is simple and rapid, detection time is short, the diagnosis range is wide, and specificity is good, and therefore the method plays an important role in detection of the porcine cytomegalovirus antibody.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting porcine cytomegalovirus antibody and a special kit thereof. Background technique [0002] Porcine cytomegalovirus (porcinecytomegalovirus, PCMV) belongs to Herpesviridae, Herpesviridae, Betaherpesvirinae, and Cytomegalovirus genus. It can cause rhinitis in newborn pigs, manifested as dysplasia, rhinitis, pneumonia, weight loss and other symptoms. Fatal diseases occur. When sows are exposed to the virus for the first time during pregnancy, mummies, stillbirths, and weak offspring will occur. Porcine cytomegalovirus almost infects swine populations all over the world, but most of them are subclinical symptoms. After being cured, the virus can be transmitted for life. Transmission can be transmitted vertically and through mating through nose, eye secretions, urine and delivery fluid, and the mortality rate reaches 20-30%. Poor environment, large temperature fluc...

Claims

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Application Information

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IPC IPC(8): C07K14/045C12N15/38C07K16/08G01N33/569
CPCC07K14/005C07K16/088C12N2710/16131G01N33/56994
Inventor 刘文军孙西军杨利敏李晶范文辉郑伟楠孙蕾
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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