A multiple fluorescence immunoassay method and reagents for rapidly distinguishing aiv, ndv, mg and ms

A multiple fluorescence and immunoassay technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of higher requirements and increased difficulty of experiments, so as to reduce the cost of detection and reduce the use of samples. The effect of small amount and high sensitivity

Active Publication Date: 2018-05-04
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 terms of sensitivity, specificity, and speed. However, in practical applications, when the sample volume is very large, single-plex fluorescent PCR has certain disadvantages in terms of cost and time. A multiplex fluorescent PCR method is much more complicated than a single-plex one, and it has higher requirements on reagents and primers. At the same time, it is necessary to ensure that there is no mutual interference between the fluorescent groups labeled by different probes. The fluorescent quantitative PCR instrument used has corresponding Multiple detection channels make the experiment more difficult

Method used

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  • A multiple fluorescence immunoassay method and reagents for rapidly distinguishing aiv, ndv, mg and ms
  • A multiple fluorescence immunoassay method and reagents for rapidly distinguishing aiv, ndv, mg and ms
  • A multiple fluorescence immunoassay method and reagents for rapidly distinguishing aiv, ndv, mg and ms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1 Primers

[0096] After screening a large number of designed primers, it was found that the primer pairs A1 and A2, N1 and N2, G1 and G2, S1 and S2 were used for rapid detection of avian influenza (AIV), Newcastle disease virus (NDV), and Mycoplasma gallisepticum (MG) simultaneously. ) and Mycoplasma gallinarum (MS), the base sequences of which are shown below.

[0097] Primer A1: 5'-ATGAGTCTTTCTACCCGAGG-3' (SEQ ID NO: 1),

[0098] Primer A2: 5'-TCAGAGGTGACAGGATTG-3' (SEQ ID NO: 2);

[0099] Primer N1: 5'-CTCATCTCAGACAGGGTCAATC-3' (SEQ ID NO: 3),

[0100] Primer N2: 5'-ATGGAGTCACCAAGGGG-3' (SEQ ID NO: 4);

[0101] Primer G1: 5'-CTTACAGATTACAGGAGCAG-3' (SEQ ID NO: 5),

[0102] Primer G2: 5'-AAGCATTTCATTTGTTTTAC-3' (SEQ ID NO: 6);

[0103] Primer S1: 5'-ATTAGCAGCTAGTGCAGTGG-3' (SEQ ID NO: 7),

[0104] Primer S2: 5'-TTTGAGGATTATCAGTATTTG-3' (SEQ ID NO: 8).

[0105] The present invention adopts the method of multiple fluorescence immunoassay to distinguish 4...

Embodiment 2

[0111] Example 2 Multiplex fluorescent immunoassay reagents for rapidly distinguishing avian influenza (AIV), Newcastle disease virus (NDV), Mycoplasma gallisepticum (MG) and Mycoplasma gallisepticum (MS)

[0112] This reagent includes the following components:

[0113] (1) The primers designed in Example 1 for multiplex fluorescent immunoassay;

[0114] (2) 4 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 4 kinds of microspheres can be purchased From luminex company, the numbers of fluorescently encoded microspheres corresponding to AIV, NDV, MS and MG are MTAG-A029, MTAG-A034, MTAG-A025 and MTAG-042, respectively.

[0115] (3) Streptavidin-phycoerythrin complex.

Embodiment 3

[0116] Example 3 Establishment of Multiplex Fluorescence Immunoassay Detection Method for Avian Influenza (AIV), Newcastle Disease Virus (NDV), Mycoplasma Gallisepticum (MG) and Mycoplasma Gallisepticum (MS)

[0117] (1) Construction of AIV, NDV, MG and MS plasmids

[0118]Extract the RNA / DNA of AIV, NDV, MG, and MS pathogens with Tiangen's automatic nucleic acid extractor, respectively, and perform RT-PCR amplification with the corresponding primers designed in Example 1, and perform agarose gelation on the amplified products respectively. Gel electrophoresis detection and gel cutting purification. Use TaKaRa’s kit to purify, connect the purified cDNA to the pMD-19T vector respectively, transform the ligated products into DH5a competent cells, select single clones, carry out colony PCR identification, and carry out the colonies identified as positive bacteria. Plasmids were extracted and sent for sequencing.

[0119] (2) Plasmid PCR amplification

[0120] AIV, NDV, MS, and...

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Abstract

The invention discloses a multiple fluorescent immunoassay method and reagent for quick distinguishing of avian influenza virus, Newcastle disease virus, Mycoplasma gallisepticum and Mycoplasma synoviae. The method is simple to perform, a target amplification fragment is acquired through PCR (polymerase chain reaction), an amplification product, fluorescence coded microspheres and streptavidin-phycoerythrin are hybridized, and MFI (mean fluorescence intensity) value is read through a detector to distinguish different types of pathogens. The method enables accurate detection for avian influenza virus, Newcastle disease virus, Mycoplasma gallisepticum and Mycoplasma synoviae, and is high in specificity, high in sensitivity and good in repeatability; compared with traditional detection methods, the method implements simultaneous detection for various different target molecules in a same sample, fewer samples are used, the operation is simple, the operation speed is high, and detection cost can be greatly reduced; the method is good in flexibility and allows addition and deduction of the types of pathogen on such basis as required.

Description

technical field [0001] The invention belongs to the field of pathogen detection in the breeding industry, and in particular relates to a multiple fluorescence immunoassay method for rapidly distinguishing avian influenza (AIV), Newcastle disease virus (NDV), mycoplasma gallisepticum (MG) and mycoplasma gallisepticum (MS). Background technique [0002] As the market demand for poultry continues to increase, the poultry industry has developed rapidly, the degree of intensification of the poultry industry has continued to increase, and the infection of poultry respiratory pathogens has been on the rise year by year. Respiratory diseases of poultry have been a major factor causing significant economic losses in the poultry industry. The pathogenesis of chicken respiratory disease has its complexity. It can be caused by viruses, mycoplasma and bacteria, and various mixed infections will appear clinically. The occurrence of these diseases cannot be ignored in chicken production. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6804C12N15/11C12R1/93
CPCC12Q1/6804C12Q1/689C12Q1/701C12Q2600/16C12Q2537/143C12Q2563/107C12Q2531/113
Inventor 郭鹏举朱余军丛峰黄韧陈梅丽
Owner GUANGDONG LAB ANIMALS MONITORING INST
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