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Multiplex fluoroimmunoassay method for rapidly distinguishing RHV (rabbit hemorrhagic disease), SV (sendai virus) and LRV (lapine rotavirus) and reagent

A multiple fluorescence and immunoassay technology, applied in the field of multiple fluorescence immunoassay methods and reagents, can solve the problems of different virus amplification efficiencies, large fragment size differences, false positive reaction products, etc., and achieve less sample consumption and higher accuracy High, high-sensitivity effects

Active Publication Date: 2017-11-07
GUANGDONG LAB ANIMALS MONITORING INST
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Multiplex RT-PCR is widely used in the mixed infection of animal viruses due to its sensitivity, specificity and simplicity of operation, but the determination of the results requires electrophoresis, which is time-consuming and laborious, and the reaction products are prone to contamination, resulting in false positives, while multiplex PCR is It is distinguished by the size of the fragments. Generally, the size of the fragments is triple or above. The size of the fragments varies greatly. The amplification efficiency of each virus is different, resulting in deviations in the results.

Method used

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  • Multiplex fluoroimmunoassay method for rapidly distinguishing RHV (rabbit hemorrhagic disease), SV (sendai virus) and LRV (lapine rotavirus) and reagent
  • Multiplex fluoroimmunoassay method for rapidly distinguishing RHV (rabbit hemorrhagic disease), SV (sendai virus) and LRV (lapine rotavirus) and reagent
  • Multiplex fluoroimmunoassay method for rapidly distinguishing RHV (rabbit hemorrhagic disease), SV (sendai virus) and LRV (lapine rotavirus) and reagent

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1 Primers for a Multiplex Fluorescence Immunoassay Method for Rapidly Differentiating Rabbit Distemper Virus, Sendai Virus and Rabbit Rotavirus

[0046] After screening a large number of designed primers, it was found that the primer pairs R1 and R2, S1 and S2, L1 and L2 were paired for rapid detection of rabbit plague virus (RHV), Sendai virus (SV) and rabbit rotavirus (LRV) at the same time. Works best with the following nucleotide sequence:

[0047] RHV primer R1: CTCTCCACAAAATAACCCATTCACA (SEQ ID NO: 1);

[0048] RHV primer R2: CCAACCCTGGTCCAATCTCG (SEQ ID NO: 2);

[0049] SV primer S1: TGACAACAAACGGAGTAAACGC (SEQ ID NO: 3);

[0050] SV primer S2: ACCATAGGTCCAAACAGCCATTC (SEQ ID NO: 4);

[0051] LRV primer L1: ATGGTTCGCTTGTGTCTTAGTTG (SEQ ID NO: 5);

[0052] LRV Primer L2: ATGCGTTGGGTGTAGTTCCTGTA (SEQ ID NO: 6).

[0053] The present invention adopts the method of multiple fluorescent immunoassays to distinguish three kinds of RHV, SV, and LRV pathogens, s...

Embodiment 2

[0058] Example 2 A Reagent for Rapidly Distinguishing Rabbit Distemper Virus, Sendai Virus and Rabbit Rotavirus by Multiplex Fluorescence Immunoassay

[0059] This reagent includes the following components:

[0060] (1) The primers designed in Example 1 for multiple fluorescent immunoassays;

[0061] (2) Three kinds of fluorescently coded microspheres containing anti-tag sequences that encode different fluorescent colors, and the anti-tag sequences can be complementary to the tag sequences in multiple fluorescent immunoassay primers; all three kinds of microspheres can be purchased From luminex company, the fluorescent coded microsphere numbers corresponding to RHV, SV, and LRV are MTAG-A056, MTAG-A062, and MTAG-029;

[0062] (3) Streptavidin-phycoerythrin complex.

Embodiment 3

[0063] Example 3 Establishment of Multiple Fluorescence Immunoassay Detection Method for Rabbit Distemper Virus, Sendai Virus and Rabbit Rotavirus

[0064] 1. Construction of rabbit plague virus, Sendai virus and rabbit rotavirus plasmids

[0065] Use the kit MiniBEST Viral RNA / DNA Extraction Kit Ver.4.0 to extract the RNA of RHV, SV, and LRV viruses, reverse transcribe into cDNA, and use the above primers to perform PCR amplification on R1 and R2, S1 and S2, L1 and L2, respectively. 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.

[0066] 2. Plasmid PCR amplification

[0067] The primer pairs R1 and ...

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Abstract

The invention discloses a multiplex fluoroimmunoassay method for rapidly distinguishing RHV (rabbit hemorrhagic disease), SV (sendai virus) and LRV (lapine rotavirus) and a reagent. The fluoroimmunoassay method is simple in operation, a target amplification fragment is obtained through PCR, then an amplification product, fluorescence coded microspheres and streptavidin-phycoerythrin are subjected to hybridization, an MFI value is read through a detector, and different types of viruses are distinguished. The method can be used for accurately detecting the RHV, the SV and the LRV simultaneously and has high specificity and sensitivity and good repeatability; compared with a traditional detection method, the method can be used for detecting different target molecules in the same sample simultaneously, few samples are required, operation is simple and rapid, and the detection cost can be reduced greatly.

Description

technical field [0001] The invention belongs to the field of virus detection, in particular to a multiple fluorescence immunoassay method and reagents for rapidly distinguishing rabbit plague virus (RHV), Sendai virus (SV) and rabbit rotavirus (LRV). Background technique [0002] Rabbit plague virus, also known as rabbit hemorrhagic disease virus (RHV), can cause a severe infectious disease in rabbits characterized by respiratory system hemorrhage, parenchyma edema, congestion and bleeding changes. The rate and death rate are extremely high, and it is listed as a B-type animal infectious disease by the World Organization for Animal Health, and is listed as a B-type animal disease by the Veterinary Bureau of the Ministry of Agriculture of my country. Rabbit rotavirus (Lapine rotavirus, LRV) is a member of the genus Rotavirus in the Reoviridae family, and is the main cause of acute viral gastroenteritis in rabbits. The mixed infection of bacteria and parasites can easily cause...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6816C12Q1/701C12Q2600/16C12Q2563/107C12Q2521/107
Inventor 伍妙梨丛峰郭鹏举黄韧张钰陈梅丽
Owner GUANGDONG LAB ANIMALS MONITORING INST
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