Multiplex fluorescence immunoassay primer, kit and method for rapidly distinguishing four respiratory pathogens

A multiple fluorescence and immunoassay technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of higher requirements and increased difficulty of experiments, and achieve lower detection costs, less sample consumption, Guaranteed the effect of annealing temperature and hybridization efficiency

Active Publication Date: 2016-11-09
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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  • Multiplex fluorescence immunoassay primer, kit and method for rapidly distinguishing four respiratory pathogens
  • Multiplex fluorescence immunoassay primer, kit and method for rapidly distinguishing four respiratory pathogens
  • Multiplex fluorescence immunoassay primer, kit and method for rapidly distinguishing four respiratory pathogens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1 Primers

[0090] After screening a large number of designed primers, it was found that the primer pairs N1 and N2, A1 and A2, B1 and B2, L1 and L2 can quickly distinguish four kinds of avian respiratory pathogens simultaneously (chicken Newcastle disease virus, avian influenza virus, chicken infectious bronchitis virus and avian infectious laryngotracheitis virus), the base sequences of which are shown below.

[0091] N1: 5'-CTCATCTCAGACAGGGTCAATC-3' (SEQ ID NO: 1),

[0092] N2: 5'-ATGGAGTCACCAAGGGG-3' (SEQ ID NO: 2);

[0093] A1: 5'-ATGAGTCTTCTACCCGAGG-3' (SEQ ID NO: 3),

[0094] A2: 5'-TCAGAGGTGACAGGATTG-3' (SEQ ID NO: 4);

[0095] B1: 5'-AATCACGCTCAAGTTCAAGGC-3' (SEQ ID NO: 5),

[0096] B2: 5'-GCATTGTTCCTCTCTCTCATC-3' (SEQ ID NO: 6);

[0097] L1: 5'-GCAGCGAAAAGAAGGC-3' (SEQ ID NO: 7),

[0098] L2: 5'-TCATCGTGCTCGGTGTCC-3' (SEQ ID NO: 8).

[0099] The present invention adopts the method of multiple fluorescence immunoassay to distinguish four kinds of...

Embodiment 2

[0105] Example 2 The kit for multiple fluorescence immunoassays for rapidly distinguishing 4 kinds of poultry respiratory pathogens

[0106] The kit includes the following components:

[0107] (1) The primers designed in Example 1 for multiplex fluorescent immunoassay;

[0108] (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 number of fluorescently encoded microspheres corresponding to Newcastle disease virus (NDV), avian influenza virus (AIV), chicken infectious bronchitis virus (IBV) and chicken infectious laryngotracheitis virus (ILTV) respectively is MTAG-A034 , MTAG-A029, MTAG-A036 and MTAG-A067.

[0109] (3) Streptavidin-phycoerythrin complex.

Embodiment 3

[0110] Example 3 Establishment of Multiple Fluorescence Immunoassay Detection Method for 4 Kinds of Poultry Respiratory Tract Pathogens

[0111] (1) Construction of four avian respiratory pathogenic plasmids

[0112] The RNA of NDV, AIV, IBV, and ILTV pathogens were extracted with Tiangen’s automatic nucleic acid extractor, and the primer pairs N1 and N2, A1 and A2, B1 and B2, L1 and L2 were used for RT-PCR amplification, and the amplified The products were detected by agarose gel electrophoresis and purified by gel cutting. The purified cDNAs were respectively connected to the pMD-19T vector, and the ligated products were transformed into DH5a competent cells. Single clones were selected and identified by colony PCR. Colonies of positive bacteria were subjected to plasmid extraction and sent for sequencing.

[0113] (2) Plasmid PCR amplification

[0114] The primers described in Example 1 were used to amplify NDV, AIV, IBV, and ILTV single, double, and quadruple PCR respect...

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Abstract

The invention discloses a multiplex fluorescence immunoassay primer, kit and method for rapidly distinguishing four respiratory pathogens. Operation is easy, a target amplified fragment is obtained through a PCR, then an amplified product, fluorescence coded microspheres and streptavidin-phycoerythrin are hybridized, an MFI value is read through a detector, and viruses of different types are distinguished. The method can be used for accurately detecting chicken new castle disease viruses, avian influenza viruses, chicken infectious bronchitis viruses and chicken infectious laryngotracheitis viruses at the same time, and is high in specificity, high in sensitivity and good in repeatability. Compared with a traditional detection method, the method can detect multiple different target molecules in the same sample at the same time, the use quantity of samples is small, operation is easy and rapid, and the detection cost can be greatly reduced. The flexibility is good, and the varieties of detected pathogens can be increased or decreased on the basis according to requirements.

Description

technical field [0001] The invention belongs to the field of virus detection in the breeding industry, and in particular relates to a primer, a kit and a method for rapidly distinguishing four kinds of poultry respiratory pathogens. 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. Respiratory diseases of chickens often occur. Chickens of various ages can be infected or even die. Adult chickens will drop eggs. At the same time, the incidence of respiratory diseases in chickens is high, which is easy to cause secondary infections of various diseases. Timely treatment of respiratory diseases is of great significance. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6804C12Q1/701C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 郭鹏举朱余军丛峰黄韧陈梅丽
Owner GUANGDONG LAB ANIMALS MONITORING INST
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