Multi-immunofluorescence assay primers, kit and method for detecting chicken Marek's disease viruses and chicken infectious anemia viruses

Abstract

The invention discloses multi-immunofluorescence assay primers, a kit and a method for detecting chicken Marek's disease viruses and chicken infectious anemia viruses. The primers, kit and method are simple in operation. The method comprises acquiring a target amplified fragment by PCR, then hybridizing the amplified product, fluorescence-coded microspheres and streptavidin-phycoerythrin, then reading an MFI value through a detector and distinguishing the different types of pathogens. The method can simultaneously detect the chicken Marek's disease viruses and chicken infectious anemia viruses, has the advantages of strong specificity, high sensitivity and good reproducibility, and can realize simultaneous detection of many different target molecules in the same sample. The multi-immunofluorescence assay primers have good flexibility and can detect more or less types of pathogens on the basis of the demands.

Description

technical field

[0001] The invention belongs to the field of virus detection in the aquaculture industry, and in particular relates to multiple immunofluorescence analysis primers, a kit and a method for detecting chicken Marek's disease virus and chicken infectious anemia virus. Background technique

[0002] In recent years, immunosuppressive viruses have seriously affected the economic benefits of the poultry industry, and people have paid more and more attention to the harm they have brought to my country's poultry industry. Recently, there have been increasing reports of chicken Marek's disease virus (MDV) and chicken infectious anemia virus (CAV), which are significant threats to the poultry industry worldwide. Marek's disease has always been one of the major diseases threatening the poultry industry in my country. It not only causes malignant lymphoma and death clinically, but more importantly, it damages the immune organs of infected chickens, resulting in an immunos...

Examples

Embodiment 1

[0079] Example 1 Primer Design

[0080] After screening a large number of designed primers, it was found that the primer pair C1 and C2, M1 and M2 have the best effect on simultaneous detection of chicken infectious anemia virus (CAV) and chicken Marek's disease virus (MDV) for multiplex immunofluorescence analysis , whose base sequence is shown below.

[0081] Primer C1: 5'-CGACATCGGAGGAGACAG-3' (SEQ ID NO: 1),

[0082] Primer C2: 5'-GGAAGCGGATAGTCATAGTAGA-3' (SEQ ID NO: 2);

[0083] Primer M1: 5'-CCCATTCCCTCTTCTGCC-3' (SEQ ID NO: 3),

[0084] Primer M2: 5'-GCTGAGCGTAAACCGTC-3' (SEQ ID NO: 4).

[0085] The invention adopts the method of multiple immunofluorescence analysis to distinguish chicken infectious anemia virus and chicken Marek's disease virus, so the above primers are further modified to meet corresponding operation requirements. The 5' ends of primers C1 and M1 are connected with tag sequences, and the connected tag sequences are respectively:

[0086] The tag...

Embodiment 2

[0089] Example 2: The establishment of multiple fluorescent immunoassay kits for detecting CAV and MDV

[0090] The kit includes the following components:

[0091] (1) Multiplex fluorescent immunoassay primers designed in Example 1;

[0092] (2) Two kinds of fluorescently encoded microspheres containing anti-tag sequences that encode different fluorescent colors, and the anti-tag sequences can be complementary to the tag sequences in the primers for multiple fluorescent immunoassays; both types of microspheres can be purchased From luminex company, the numbers of fluorescently encoded microspheres corresponding to CAV and MDV are MTAG-A019 and MTAG-A065, respectively.

[0093] (3) Streptavidin-phycoerythrin complex.

Embodiment 3

[0094] Example 3 Establishment of Multiple Fluorescence Immunoassay Detection Method for CAV and MDV

[0095] (1) Construction of plasmids

[0096] The DNA of CAV and MDV pathogens were extracted with Tiangen’s automatic nucleic acid extractor, PCR amplification was performed with primer pairs C1 and C2, M1 and M2 respectively, and the amplified products were detected by agarose gel electrophoresis and purified by gel cutting. The purified cDNA was connected to the pMD-19T vector with a kit from TaKaRa Company, the connected product was transformed into DH5a competent cells, single clones were selected, colony PCR identification was carried out, and the colonies identified as positive bacteria were subjected to plasmid extraction. Send for sequencing.

[0097] (2) Plasmid PCR amplification

[0098] Use the primers described in Example 1 to carry out single-fold and double-fold PCR amplification of CAV and MDV primers, respectively.

[0099] Preparation of upstream primer mi...

technical field

[0001] The invention belongs to the field of virus detection in the aquaculture industry, and in particular relates to multiple immunofluorescence analysis primers, a kit and a method for detecting chicken Marek's disease virus and chicken infectious anemia virus. Background technique

[0002] In recent years, immunosuppressive viruses have seriously affected the economic benefits of the poultry industry, and people have paid more and more attention to the harm they have brought to my country's poultry industry. Recently, there have been increasing reports of chicken Marek's disease virus (MDV) and chicken infectious anemia virus (CAV), which are significant threats to the poultry industry worldwide. Marek's disease has always been one of the major diseases threatening the poultry industry in my country. It not only causes malignant lymphoma and death clinically, but more importantly, it damages the immune organs of infected chickens, resulting in an immunos...

Examples

Embodiment 1

[0079] Example 1 Primer Design

[0080] After screening a large number of designed primers, it was found that the primer pair C1 and C2, M1 and M2 have the best effect on simultaneous detection of chicken infectious anemia virus (CAV) and chicken Marek's disease virus (MDV) for multiplex immunofluorescence analysis , whose base sequence is shown below.

[0081] Primer C1: 5'-CGACATCGGAGGAGACAG-3' (SEQ ID NO: 1),

[0082] Primer C2: 5'-GGAAGCGGATAGTCATAGTAGA-3' (SEQ ID NO: 2);

[0083] Primer M1: 5'-CCCATTCCCTCTTCTGCC-3' (SEQ ID NO: 3),

[0084] Primer M2: 5'-GCTGAGCGTAAACCGTC-3' (SEQ ID NO: 4).

[0085] The invention adopts the method of multiple immunofluorescence analysis to distinguish chicken infectious anemia virus and chicken Marek's disease virus, so the above primers are further modified to meet corresponding operation requirements. The 5' ends of primers C1 and M1 are connected with tag sequences, and the connected tag sequences are respectively:

[0086] The tag...

Embodiment 2

[0089] Example 2: The establishment of multiple fluorescent immunoassay kits for detecting CAV and MDV

[0090] The kit includes the following components:

[0091] (1) Multiplex fluorescent immunoassay primers designed in Example 1;

[0092] (2) Two kinds of fluorescently encoded microspheres containing anti-tag sequences that encode different fluorescent colors, and the anti-tag sequences can be complementary to the tag sequences in the primers for multiple fluorescent immunoassays; both types of microspheres can be purchased From luminex company, the numbers of fluorescently encoded microspheres corresponding to CAV and MDV are MTAG-A019 and MTAG-A065, respectively.

[0093] (3) Streptavidin-phycoerythrin complex.

Embodiment 3

[0094] Example 3 Establishment of Multiple Fluorescence Immunoassay Detection Method for CAV and MDV

[0095] (1) Construction of plasmids

[0096] The DNA of CAV and MDV pathogens were extracted with Tiangen’s automatic nucleic acid extractor, PCR amplification was performed with primer pairs C1 and C2, M1 and M2 respectively, and the amplified products were detected by agarose gel electrophoresis and purified by gel cutting. The purified cDNA was connected to the pMD-19T vector with a kit from TaKaRa Company, the connected product was transformed into DH5a competent cells, single clones were selected, colony PCR identification was carried out, and the colonies identified as positive bacteria were subjected to plasmid extraction. Send for sequencing.

[0097] (2) Plasmid PCR amplification

[0098] Use the primers described in Example 1 to carry out single-fold and double-fold PCR amplification of CAV and MDV primers, respectively.

[0099] Preparation of upstream primer mi...

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