Multiple fluorescence immunoassay primer, kit and method capable of fast distinguishing CAV, MDV, REV and IBDV

A multiple fluorescence and immunoassay technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve difficult problems, reduce detection costs, avoid cross-hybridization, and simple operation

Active Publication Date: 2017-01-04
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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  • Multiple fluorescence immunoassay primer, kit and method capable of fast distinguishing CAV, MDV, REV and IBDV
  • Multiple fluorescence immunoassay primer, kit and method capable of fast distinguishing CAV, MDV, REV and IBDV
  • Multiple fluorescence immunoassay primer, kit and method capable of fast distinguishing CAV, MDV, REV and IBDV

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1 Primer Design

[0095] After screening a large number of designed primers, it was found that the primer pairs C1 and C2, M1 and M2, R1 and R2, B1 and B2 had the best effect on multiple fluorescence simultaneous detection of CAV, MDV, REV, and IBDV. As follows.

[0096] Primer C1: 5'-CGACATCGGAGGAGACAG-3' (SEQ ID NO: 1),

[0097] Primer C2: 5'-GGAAGCGGATAGTCATAGTAGA-3' (SEQ ID NO: 2);

[0098] Primer M1: 5'-CCCATTCCCTCTTCTGCC-3' (SEQ ID NO: 3),

[0099] Primer M2: 5'-GCTGAGCGTAAACCGTC-3' (SEQ ID NO: 4);

[0100] Primer R1: 5'-GACTGCCTTGTGACTGCT-3' (SEQ ID NO: 5),

[0101] Primer R2: 5'-ACTCCCACTGTTGTCTAAATC-3' (SEQ ID NO: 6);

[0102] Primer B1: 5'-ATGCGGAGCCTTCTGA-3' (SEQ ID NO: 7),

[0103] Primer B2: 5'-ATTAGCCCTGACCCTGTG-3' (SEQ ID NO: 8).

[0104] The present invention adopts the method of multiple fluorescence analysis to distinguish CAV, MDV, REV, and IBDV, so the above primers are further modified to meet the corresponding operation requirements....

Embodiment 2

[0110] Example 2 Multiplex Fluorescence Immunoassay Kit for Rapidly Distinguishing Chicken Infectious Anemia Virus, Chicken Marek's Disease Virus, Avian Reticuloendotheliosis Virus and Infectious Bursal Virus

[0111] The kit includes the following components:

[0112] (1) The multiplex fluorescent immunoassay primer set designed in Example 1;

[0113] (2) Four kinds of fluorescence-encoded microspheres containing anti-tag sequences encoding different fluorescent colors, and the anti-tag sequences can be complementary to the tag sequences in multiple fluorescence analysis primers; luminex company, where CAV, MDV, REV, and IBDV correspond to the fluorescently encoded microsphere numbers MTAG-A019, MTAG-A065, MTAG-A056, and MTAG-015, respectively.

[0114] (3) Streptavidin-phycoerythrin complex.

Embodiment 3

[0115] Example 3 Establishment of multiple fluorescent immunoassay detection method for rapidly distinguishing chicken infectious anemia virus, chicken Marek's disease virus, avian reticuloendotheliosis virus and infectious bursal virus

[0116](1) Construction of CAV, MDV, REV and IBDV plasmids

[0117] Extract the nucleic acid (RNA / DNA) of CAV, MDV, REV, and IBDV pathogens with Tiangen’s automatic nucleic acid extractor, and use the primer pairs C1 and C2, M1 and M2, R1 and R2, B1 and B2 respectively designed in Example 1 RT-PCR amplification was carried out, 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 se...

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Abstract

The invention discloses a multiple fluorescence immunoassay primer, kit and method capable of fast distinguishing CAV, MDV, REV and IBDV. The multiple fluorescence immunoassay primer, kit and method has the advantages that target amplification fragments are obtained through PCR, \ amplification products, fluorescence encoded microspheres and streptavidin-phycoerythrin are hybridized, MFI values are read through a detector, and different types of pathogens are distinguished; the method can simultaneously and accurately detect chicken infectious anemia viruses, chicken Marek's disease viruses, chicken reticuloendotheliosis viruses and chicken infectious bursal disease viruses and is high in specificity, high in sensitivity and good in repeatability; compared with a traditional method, the method can simultaneously detect various molecules, with different purposes, in the same sample and is low in sample use amount, simple and fast to operate, capable of greatly lowering detecting cost, good in flexibility and capable of increasing and decreasing to-be-detected pathogen types on the basis.

Description

technical field [0001] The invention belongs to the field of pathogen detection in the breeding industry, and in particular relates to a method for rapidly distinguishing chicken infectious anemia virus (CAV, Chicken infectious anemia virus), chicken Marek's disease virus (MDV, Marek's diseasevirus) and avian reticuloendotheliosis virus (REV, Reticuloendotheliosis virus) and infectious bursal virus (IBDV, Infections Bursal Disease virus) multiplex fluorescent immunoassay primers, kits and methods. Background technique [0002] The immune response or function of the poultry immune system is the main mechanism for the body to resist pathogen invasion, infection and reproduction, and is an important part of the body's defense system. The immune organs of the chicken body include the bursa of Fabricius, bone marrow, spleen, thymus, and cecal tonsil. Immunosuppressive disease is a general term for diseases caused by a variety of infectious and non-infectious factors caused by si...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6816C12Q1/701C12Q2600/16C12Q2537/143C12Q2563/149C12Q2563/107
Inventor 丛锋朱余军郭鹏举练月晓黄碧洪黄韧陈梅丽
Owner GUANGDONG LAB ANIMALS MONITORING INST
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