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Liquid phase chip multiple detection kit for swine PRRSV (Porcine Reproductive and Respiratory Syndrome Virus), SIV (Swine Influenza Virus) and HEV (Hepatitis E Virus) antibodies

An antibody and pig breeding technology, applied in the detection field, can solve the problems of long detection time, ELISA method cannot be used for multiple diagnosis, cumbersome detection, etc., and achieve the effect of rapid detection method

Active Publication Date: 2017-05-31
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ELISA method cannot be used for multiple diagnosis; and a new method for proofreading the ELISA method is currently lacking
[0007] Although there are some detection methods for porcine PPRSV, HEV and SIV antibodies in the prior art, the detection is relatively cumbersome and takes a long time
The prior art lacks a detection method capable of simultaneously detecting the three antibodies of PPRSV, HEV and SIV

Method used

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  • Liquid phase chip multiple detection kit for swine PRRSV (Porcine Reproductive and Respiratory Syndrome Virus), SIV (Swine Influenza Virus) and HEV (Hepatitis E Virus) antibodies
  • Liquid phase chip multiple detection kit for swine PRRSV (Porcine Reproductive and Respiratory Syndrome Virus), SIV (Swine Influenza Virus) and HEV (Hepatitis E Virus) antibodies
  • Liquid phase chip multiple detection kit for swine PRRSV (Porcine Reproductive and Respiratory Syndrome Virus), SIV (Swine Influenza Virus) and HEV (Hepatitis E Virus) antibodies

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0041] 1. Preparation of SIV HA recombinant protein

[0042] 1. Extraction of influenza virus total RNA and synthesis of cDNA

[0043] Refer to the instructions of the RNA extraction kit to extract the total viral RNA. After the total RNA was obtained, the reverse transcription operation was performed referring to the instruction manual of M-MLV reverse transcriptase of Bao Bioengineering (Dalian) Co., Ltd. The reaction system is shown in Table 1.

[0044]

[0045] The reagents in Table 1 were added and mixed thoroughly, and placed in a water bath at 42°C for 1 h, and the obtained cDNA product was stored at -20°C for future use.

[0046] 2. Amplification of SIV H1N1 HA gene

[0047] Using Oligo7.0 biological software, a pair of specific primers were designed according to the SIV H1N1 HA gene sequence (GenBank: JN375120.1, the nucleotide sequence of the expression signal peptide part was removed), provided by Yingwei Jieji (Shanghai) Trading Co., Ltd. Synthesis, the upstr...

Embodiment 1

[0100] 1. Recombinant protein

[0101] The PRRSV nsp7, SIV HA and HEV ORF2 recombinant proteins used in this experiment are all recombinant plasmids independently constructed by our laboratory and obtained by expression using the Escherichia coli expression system. See Table 2 for recombinant plasmid and recombinant protein information.

[0102]

[0103] 2. Coupling of antigens and microspheres

[0104] Referring to Luminex’s xMAP® Technology Encyclopedia, two-step amide reaction: first, phosphorylated microspheres are activated by disodium hydrogen phosphate buffer, Sulfo-NHS and EDC solution, and second, PRRSV Nsp7, SIV HA, and HEV ORF2 recombinant proteins are used as antigens Covalent amide bonds were formed with the microspheres respectively, and the microspheres coupled with the antigen were resuspended in PBS-TBN solution and stored at 4°C in the dark. The specific operation steps are as follows:

[0105] (1) Vortex the microsphere suspension for 1 min to disperse...

Embodiment 2

[0118] Example 2 Using the antigen-coupled microspheres prepared in Example 1 to establish a liquid-phase chip detection method

[0119] 1. For the single-plex detection technology of liquid-phase protein chips, refer to the xMAP® technology encyclopedia of Luminex, and the specific steps are as follows:

[0120] (1) Add 50 μL (2500 pieces) of antigen-coupled microspheres and 50 μL of the sample to be tested (mAb or serum) to each well, incubate at room temperature (500 r / min) in the dark for 1 h, and wash with PBST for 2 h. times, 200 μL / well;

[0121] (2) Add biotin-labeled chicken anti-mouse (1:1000) or rabbit anti-pig (1:5000) IgG antibody 100 μL / well, shake at room temperature (500 r / min) and incubate in the dark for 1 h, wash with PBST for 2 times, 200 μL / well;

[0122] (3) Add 100 μL / well of 1:1,000 diluted streptomycin-phycoerythrin (SA-PE), incubate at room temperature (500 r / min) in the dark for 0.5 h, wash twice with PBST, 200 μL / well;

[0123] (4) Add 125 μL of ...

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Abstract

The invention provides a liquid phase chip multiple detection kit for swine PRRSV (Porcine Reproductive and Respiratory Syndrome Virus), SIV (Swine Influenza Virus) and HEV (Hepatitis E Virus) antibodies. Different fluorescence labeling microspheres of PRRSV nsp7, SIV HA (Hemagglutinin) and HEV ORF2 (Opening Reading Frame) antibodies are used as carriers; on the basis of a single liquid phase chip detection method of the PRRSV, SIV and HEV antibodies, a multiple liquid phase chip detection method for simultaneously detecting three antibodies is established, provides a more effective and quicker detection method for monitoring the HEV, PRRSV and SIV antibodies, and provides a new clue for establishing a method of differential diagnosis of swine diseases and serum detection of other animal epidemic disease antibodies; aiming at breeding situation of quick development of pig industry in China, complex conditions of the swine diseases, serious mixed infection phenomenon and the like, accurate, quick and effective diagnosis and monitoring of epidemic disease conditions of swine herds are further enhanced.

Description

technical field [0001] The invention relates to the technical field of detection, in particular to a liquid phase chip multiple detection kit for pig PRRSV, SIV and HEV antibodies. Background technique [0002] Liquid phase chip technology, also known as xMAP technology (flexible multiple-analyte profiling), is a new type of biomolecular detection technology integrating flow cytometry, laser technology, digital signal processing technology and chemistry. Biochip technology certified by the Drug Administration (FDA) can be used for clinical diagnosis. At present, the third-generation detection technology --- Flexmap3D has been launched. Compared with the previous generation detection technology, Flexmap3D is more efficient. It can detect 500 different target molecules in one sample at the same time, and can detect 384 different samples at a time. An efficient detection method. [0003] Swine Influenza (SI) is an acute, febrile and highly contagious respiratory infectious di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/577G01N33/68C09K11/06
CPCC09K11/06G01N33/56983G01N33/577G01N33/68G01N2333/005G01N2333/11G01N2333/14G01N2333/435
Inventor 王衡张桂红纪方晓冀池海曾梦陈万里
Owner SOUTH CHINA AGRI UNIV
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