Porcine reproductive and respiratory syndrome virus antibody competitive AlphaLISA detection kit and detection method thereof

A technology for respiratory syndrome and detection kits, applied in measurement devices, immunoassays, instruments, etc., can solve the problems of large differences in specificity and high requirements, and achieve small sample volume, strong anti-quenching ability, and serum dosage. less effect

Inactive Publication Date: 2014-01-01
INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing detection methods mainly include virus isolation and identification, immunoperoxidase technology, immunocolloidal gold technology, immunofluorescence antibody staining technology, nucleic acid probe hybridization technology (ISH), ELISA, PCR and real time PCR, etc. Advantages and disadvantages, and large differences in specificity
At the same time, the method has higher requirements on samples and personnel
However, there is no report on the kit and detection method for detecting porcine reproductive and respiratory syndrome virus antibody using AlphaLISA technology at present

Method used

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  • Porcine reproductive and respiratory syndrome virus antibody competitive AlphaLISA detection kit and detection method thereof
  • Porcine reproductive and respiratory syndrome virus antibody competitive AlphaLISA detection kit and detection method thereof
  • Porcine reproductive and respiratory syndrome virus antibody competitive AlphaLISA detection kit and detection method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment

[0028] 1. Construction and identification of PRRSV N recombinant protein prokaryotic expression plasmid

[0029] 1. Target fragment amplification

[0030] 1.1 RT-PCR amplification

[0031] Using the genome of strain PRRSV VR2332 (GenBank No.: EF536003.1) as a template, carry out reverse transcription polymerase chain reaction (RT-PCR) to amplify the desired fragment PRRSV N gene, its nucleotide sequence is shown in SEQ As shown in ID No.2, the encoded amino acid sequence is shown in SEQ ID No.1.

[0032] Kpn I and Xho I restriction sites were introduced into the upstream and downstream primers respectively. The primers were designed using Primer Preier5 (a primer design software), and the primers were synthesized by Baobiology (Dalian) Co., Ltd.

[0033] Table 1 Primer sequences

[0034]

[0035] The RT-PCR reaction system is shown in Table 2 (total volume 25 μL)

[0036] Table 2 RT-PCR reaction system

[0037] reaction system Volume (μL) PrimeScript 1 S...

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PUM

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Abstract

The invention discloses a porcine reproductive and respiratory syndrome virus (PRRSV) antibody competitive AlphaLISA detection kit and a detection method thereof. The PRRSV antibody competitive AlphaLISA detection kit comprises a donor bead, an acceptor bead, a PRRSV N protein monoclonal antibody and an N protein antibody having a His label. The competitive AlphaLISA detection method for detecting a PRRSV antibody is established by optimizing test reaction conditions comprising the donor bead, the acceptor bead, the monoclonal antibody, the antigen and serum is established in the invention. The PRRSV antibody detection kit has the advantages of good specificity, high sensitivity, less serum consumption, no washing, no hemolysis influence, low detection cost and short detection time.

Description

technical field [0001] The invention relates to immunological detection of porcine reproductive and respiratory syndrome virus antibody, in particular to a porcine reproductive and respiratory syndrome virus antibody competition AlphaLISA detection kit and a method thereof. Background technique [0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is the main pathogen that causes porcine reproductive and respiratory syndrome (PRRS). The disease mainly causes reproductive disorders such as abortion, premature birth, stillbirth and mummified fetuses in pregnant sows, respiratory symptoms in piglets and finishing pigs, immunosuppression and subclinical infection in infected pigs, and increased susceptibility to other pathogenic factors. Since porcine reproductive and respiratory syndrome was first reported in the United States in 1987, the disease has become a worldwide epidemic, causing huge losses to the pig industry. Since the virus was first isolated in my c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569
CPCG01N33/56983G01N33/54313G01N33/561G01N2333/135G01N2469/20
Inventor 聂福平王昱杨俊向海洋王国民肖进文
Owner INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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