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Detection reagent and detection method for PRRSV

A technology for detecting reagents and samples, applied in chemical instruments and methods, biochemical equipment and methods, biological testing, etc., can solve problems such as difficulty in distinguishing naturally infected animals, high requirements for experimental conditions, and danger of live virus divergence

Active Publication Date: 2015-05-06
华威特(江苏)生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current detection methods for PRRSV have many limitations, such as the need for virus isolation and identification, the risk of divergence in the use of live viruses, high requirements for experimental conditions, cumbersome operations, and difficulty in distinguishing naturally infected animals from vaccine-immunized animals, etc.

Method used

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  • Detection reagent and detection method for PRRSV
  • Detection reagent and detection method for PRRSV
  • Detection reagent and detection method for PRRSV

Examples

Experimental program
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Effect test

Embodiment 1

[0161] Embodiment 1: Application of the B cell epitope polypeptide of the present invention to establish a diagnostic method for porcine reproductive and respiratory syndrome indirect ELISA

[0162] A PRRSV TJ strain was isolated in our laboratory. After cell passage to 92 generations, it was found that the Nsp2 gene of the virus was further deleted, and the virulence of the virus was weakened. PRRSV TJM-F92 generation attenuated strain (ie: PRRSV TJM strain) has been prepared and developed into a PRRS attenuated live vaccine, which is characterized in that the full length of the Nsp2 nucleotide sequence of the virus is 2490bp, which is the same as the Nsp2 nucleotide sequence of PRRSV TJ strain Compared to a contiguous deletion of 360 nucleotides (see image 3 ), the 598-717th amino acid in the Nsp2 protein of this nucleotide encoding PRRSV TJ strain (see Figure 4 , SEQID NO: 1); compared with the Nsp2 protein sequence encoded by the VR-2332 strain, the Nsp2 protein encoded...

Embodiment 2

[0187] Embodiment 2: Preparation of the indirect ELISA diagnostic kit of the present invention

[0188] 1 Preparation of antigen-coated microtiter plates

[0189] 1.1 Coating: The final concentration of the antigen is 12.5 μg / mL, the coating volume per well is 100 μL, cover the coated microwell plate, and incubate overnight in a refrigerator at 4°C;

[0190] 1.2 Washing: After overnight incubation, discard the coating solution in the ELISA plate, and start washing (the composition and preparation of the washing solution is: 0.01M PBS with pH7.4 plus 0.05% Tween20), wash each well 3 times, and the time interval For 3 minutes each time, after washing, the coated plate was buckled dry on absorbent paper.

[0191] 1.3 Blocking: block with a blocking solution (blocking agent is PBS with 10% fetal bovine serum), after the sealing is completed, cover the wells of the coated microplate and place them in an incubator at 37°C for 2 hours.

[0192] 1.4 Washing: After the sealing is com...

Embodiment 3

[0262] Embodiment 3: B cell epitope polypeptide differential diagnosis test of the present invention

[0263] 1 test material

[0264] 1.1 Detection kit Polypeptide differential diagnosis kit (differential diagnosis procedure established by Example 1); commercial IDEXX PRRS antibody detection kit (purchased from Beijing Aideshi Yuanheng Biotechnology Co., Ltd.)

[0265] 1.2 Experimental animals and serum: porcine reproductive and respiratory syndrome virus (PRRSV TJM strain) vaccine immune serum is prepared by the inventor according to conventional methods with PRRS negative pigs; positive serum is challenged by porcine reproductive and respiratory syndrome virus (PRRSV TJ strain) PRRS Negative pigs were bled on day 20 to collect serum. As the negative serum, the serum of PRRS-negative pigs was used. The experimental animals were PRRS-negative healthy piglets aged 4-6 weeks.

[0266] 2 test method

[0267] The test method used the indirect ELISA method to detect the antibo...

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Abstract

This application relates to detection reagents and detection methods for porcine reproductive and respiratory viruses. In one aspect, the invention provides immunogenic fragments of encoded non-structural protein 2 (Nsp2). In particular, the immunogenic fragments provided herein are not present in the Nsp2 protein of attenuated PRRSV but are present in the Nsp2 protein of wild-type PRRSV. In another aspect, the invention also provides oligonucleotide primers. In particular, the region to be amplified by the oligonucleotide primers provided by the present application includes at least part of the coding sequence of the Nsp2 protein, which is not present in the Nsp2 coding sequence of attenuated PRRSV, but is present in the Nsp2 coding sequence of wild-type PRRSV. . This application also provides methods for detecting these related detection reagents, and methods for using these methods to distinguish attenuated PRRSV-immune pigs from wild-type PRRSV-infected pigs.

Description

technical field [0001] The invention relates to the field of veterinary medicine, in particular to a detection reagent and a detection method for porcine reproductive and respiratory virus (Porcine Reproductive and Respiratory Syndrome Virus, PRRSV). Specifically, the present invention relates to immunogenic fragments in the Nsp2 protein encoded by PRRSV and uses thereof, as well as fragments in the Nsp2 coding sequence in PRRSV and uses thereof. Background technique [0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is a contagious disease with high infection rate, acute onset and high fatality rate. In 2006, the disease broke out on a large scale in my country, causing a large number of abortions, stillbirths, and mummified fetuses in pregnant sows, high fever, anorexia, and death in fattening pigs, which caused serious economic losses to the pig industry in my country. After virus isolation and sequence comparison of the virus genome, it was found that the hig...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C07K7/08C07K14/00C07K16/10C12N15/11C12Q1/70C12Q1/68G01N33/68G01N33/569
CPCC07K16/10C12Q1/701G01N33/56983G01N2333/08G01N2469/20C12N2770/10011
Inventor 武华刘准
Owner 华威特(江苏)生物制药有限公司
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