A multiplex fluorescent immunoassay primer, kit and method for rapidly distinguishing 5 kinds of poultry immunosuppressive pathogens

An immunosuppressive and multiple fluorescence technology, applied in the field of multiple fluorescence immunoassay primers, can solve problems such as difficulty, and achieve the effects of reducing detection cost, strong specificity and less sample consumption.

Active Publication Date: 2018-05-25
GUANGDONG LAB ANIMALS MONITORING INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with conventional PCR, fluorescent quantitative PCR has advantages in sensitivity, specificity, and speed. However, real-time fluorescent PCR technology is limited by the type of fluorescence and the instrument itself. It can only detect up to 5 targets, and the success of the experiment is extremely difficult. Big

Method used

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  • A multiplex fluorescent immunoassay primer, kit and method for rapidly distinguishing 5 kinds of poultry immunosuppressive pathogens
  • A multiplex fluorescent immunoassay primer, kit and method for rapidly distinguishing 5 kinds of poultry immunosuppressive pathogens
  • A multiplex fluorescent immunoassay primer, kit and method for rapidly distinguishing 5 kinds of poultry immunosuppressive pathogens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1 Primer Design

[0101] After screening a large number of designed primers, it was found that the primer pairs C1 and C2, M1 and M2, R1 and R2, O1 and O2, B1 and B2 were paired with multiple fluorescence for simultaneous detection of chicken infectious anemia virus and chicken Marek's disease virus , avian reticuloendotheliosis virus, avian reovirus and chicken infectious bursal virus, which are five poultry immunosuppressive pathogens, have the best effect, and their base sequences are shown below.

[0102] Primer C1: 5'-CGACATCGGAGGAGACAG-3' (SEQ ID NO: 1),

[0103] Primer C2: 5'-GGAAGCGGATAGTCATAGTAGA-3' (SEQ ID NO: 2);

[0104] Primer M1: 5'-CCCATTCCCTCTTCTGCC-3' (SEQ ID NO: 3),

[0105] Primer M2: 5'-GCTGAGCGTAAACCGTC-3' (SEQ ID NO: 4);

[0106] Primer R1: 5'-GACTGCCTTGTGACTGCT-3' (SEQ ID NO: 5),

[0107] Primer R2: 5'-ACTCCCACTGTTGTCTAAATC-3' (SEQ ID NO: 6);

[0108] Primer O1: 5'-CGTGTAACGGTGCGACTG-3' (SEQ ID NO: 7),

[0109] Primer O2: 5'-CCGCTAGA...

Embodiment 2

[0119] Example 2 Multiplex Fluorescence Immunoassay Kit for Rapidly Distinguishing 5 Poultry Immunosuppressive Pathogens

[0120] The kit includes the following components:

[0121] (1) The multiplex fluorescent immunoassay primer set designed in Example 1;

[0122] (2) 5 kinds of fluorescently encoded microspheres containing anti-tag sequences with different fluorescent colors, and the anti-tag sequences can be complementary to the tag sequences in the multiple fluorescent immunoassay primers; all five kinds of microspheres can be purchased From luminex, the numbers of fluorescently encoded microspheres corresponding to CAV, MDV, REV, REO, and IBDV are MTAG-A019, MTAG-A065, MTAG-A056, MTAG-A034, and MTAG-015.

[0123] (3) Streptavidin-phycoerythrin complex.

Embodiment 3

[0124] Example 3 Establishment of multiple fluorescence immunoassay detection method for 5 kinds of poultry immunosuppressive pathogens

[0125] (1) Construction of five avian respiratory pathogenic plasmids

[0126] Use Tiangen's automatic nucleic acid extractor to extract the nucleic acid (RNA / DNA) of CAV, MDV, REV, REO, and IBDV pathogens, respectively, and the primer pairs C1 and C2, M1 and M2, R1 and R2, and B1 designed in Example 1 and B2 were amplified by RT-PCR, 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.

[0127] (2) Plasmid PCR amplification

[0128] Use the primers designed in Examp...

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Abstract

The invention discloses a multiple fluoroimmunoassay primer, kit and method for rapidly distinguishing 5 avian immunosuppression pathogens. Operation is simple, a target amplified fragment is acquired through a PCR, an amplified product, fluorescence coded microspheres and streptavidin-phycoerythrin are hybridized, the MFI value is read through a detector, and viruses of different types are distinguished. The method can simultaneously detect the chicken infectious anemia virus, the chicken Marek's disease virus, the avian reticuloendotheliosis virus, the avian reovirus and the chicken infectious bursa disease virus accurately, specificity is high, sensitivity is high, and repeatability is good. Compared with a traditional detection method, the method achieves simultaneous detection of multiple different target molecules in the same sample, the use number of samples is small, operation is simple and rapid, and the detection cost can be greatly reduced.

Description

technical field [0001] The invention belongs to the field of virus detection in the breeding industry, and in particular relates to a multiple fluorescence immunoassay primer, a kit and a method for rapidly distinguishing five kinds of poultry immunosuppressive pathogens. Background technique [0002] Immunosuppression is the temporary or permanent insufficiency of the immune response and high susceptibility to disease in poultry due to a compromised immune system. In recent years, immunosuppressive viruses have received increasing attention. Poultry viral immunodeficiency disease is an important infectious disease that plagues the current poultry industry. The presence of immunosuppressive virus infection in chicken flocks will not only make chickens more susceptible to other viruses or bacteria, but also make them more susceptible to infection. Symptoms and lesions are not typical. In addition, it will also cause the immune response of chickens to certain vaccines to dec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6804C12N15/11
CPCC12Q1/6816C12Q1/701C12Q2600/16C12Q2537/143C12Q2563/149C12Q2563/107
Inventor 郭鹏举朱余军丛锋王静饶丹黄韧陈梅丽
Owner GUANGDONG LAB ANIMALS MONITORING INST
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