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Detection reagent and detection method for PRRSV

A technology for detecting reagents and samples, which is applied in the field of detection reagents for porcine reproductive and respiratory viruses, and fragments in the Nsp2 coding sequence, which can solve problems such as difficulty in distinguishing naturally infected animals, cumbersome operations, and high requirements for experimental conditions

Active Publication Date: 2012-10-17
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.

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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 (composition and preparation of the washing solution: 0.01M PBS with pH 7.4 plus 0.05% Tween20), wash each well 3 times, 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 completed, discar...

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

The invention relates to detection reagents and detection methods for porcine reproductive and respiratory syndrome virus (PRRSV). On the one hand, the invention provides immunogenic fragments in coded non-structural protein 2 (Nsp2), and in particular, the immunogenic fragments provided in the invention exist in Nsp2 protein of wild type PRRSV instead of in Nsp2 protein of attenuation PRRSV; on the other hand, the invention also provides oligonucleotide primers, and in particular, a to-be-amplified area of the oligonucleotide primers provided in the invention includes at least a part of the coding sequence of Nsp2 protein and the part of the coding sequence exists in Nsp2 coding sequence of wild type PRRSV instead of in Nsp2 coding sequence of attenuation PRRSV. The invention further provides the methods of using relevant detection reagents in detection and a method of using the above-mentioned methods to distinguish pigs immunized by attenuation PRRSV from pigs infected by wild type PRRSV.

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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IPC IPC(8): C07K7/06C07K7/08C07K14/00C07K16/10C12N15/11C12Q1/70C12Q1/68G01N33/68G01N33/569
CPCC07K7/06C07K14/082G01N2469/20C12N2770/10011C07K16/10C07K14/00G01N33/569C12Q1/701G01N2333/08C12Q1/68C12Q1/70C07K7/08G01N33/56983G01N33/68C12N15/11
Inventor 武华刘准
Owner 华威特(江苏)生物制药有限公司
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