Rapid colloidal gold detection test strip for pig porcine reproductive and respiratory syndrome virus

A technology for respiratory syndrome and test strips, applied in biological tests, measuring devices, material testing products, etc., can solve the problems of SN being unsuitable for early diagnosis, heavy cell culture workload, and poor sensitivity to acute infection, and achieve stable and reliable results. , the results are clear and easy to identify, easy to save the effect

Active Publication Date: 2014-08-20
WUHAN CHOPPER BIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] SN detection of PRRSV antibody has good specificity, but the SN antibody of PRRSV appears later in serum than IFA antibody, and its sensitivity to acute infection is poor. Therefore, SN is not suitable for early diagnosis, and cell culture is heavy and technical. For laboratory research use only

Method used

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  • Rapid colloidal gold detection test strip for pig porcine reproductive and respiratory syndrome virus
  • Rapid colloidal gold detection test strip for pig porcine reproductive and respiratory syndrome virus

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1 Cloning and expression of porcine reproductive and respiratory syndrome virus M protein and GP5 protein

[0045] (1) Obtaining the target gene

[0046] According to the characteristics of the pET-32a(+) expression vector, both ends were designed to contain restriction endonucleases Bam H I. xho Primers for I restriction sites:

[0047] M protein gene upstream primer: 5'-GTAGGTACCACCATGGGGTCGTCCTTAGATGACTTC-3',

[0048] M protein gene downstream primer: 5'-GACCTCGAGCCGTTGTTATTTGGCATATT-3';

[0049] The target gene fragment M was amplified. The amplification conditions were: denaturation at 94°C for 3 minutes; 35 cycles at 94°C for 45s, 60°C for 45s, and 72°C for 60s; finally, 72°C for 10 minutes.

[0050] GP5 protein gene upstream primer: 5'-CTCGAGATGTTGGGGAAGTGCTTGACC-3',

[0051] GP5 protein gene downstream primer: 5'-GGTACCCTAGAGACCCCATCGTTC-3';

[0052] The target gene fragment GP5 was amplified. Amplification conditions: 42°C for 5 min; 95°C for 10...

Embodiment 2

[0061] Example 2 Preparation of Anti-Porcine Reproductive and Respiratory Syndrome Virus M Protein and GP5 Protein Monoclonal Antibody

[0062] The recombinant porcine reproductive and respiratory syndrome virus M protein and GP5 protein purified in Example 1 were respectively immunized to Balb / C mice, and after three immunizations, mouse splenocytes and mouse myeloma cells whose Elisa titer reached 1:10000 were taken Sp2 / 0 fusion, HAT medium 37 ℃, saturated humidity, 5% CO 2 Culture confluent cells.

[0063] On the fourth day after fusion, start to observe the colonies. When the colonies grow to about 1 / 6 of the bottom of the well, change half of the HAT medium. The next day, use the recombinant PRRSV-M protein and PRRSV-GP5 protein to coat the plate for indirect Elisa detection, and select positive Wells were subcloned.

[0064] Subcloning procedure: Observe the colony size of the mother clone under a microscope, subcloning three times by limiting dilution method, and the ...

Embodiment 3

[0077] Example 3 Colloidal gold rapid detection test strip for porcine reproductive and respiratory syndrome virus

[0078] 1. Preparation of colloidal gold rapid detection test strips for porcine reproductive and respiratory syndrome virus

[0079] (1) Glass fiber membrane containing colloidal gold-labeled porcine reproductive and respiratory syndrome virus M protein monoclonal antibody

[0080] 1) Take the solution of colloidal gold and porcine reproductive and respiratory syndrome virus M protein monoclonal antibody adjusted to the optimal pH 7.6, use the ratio of 15-20μg monoclonal antibody per 1mL colloidal gold, and fully stir the colloidal gold and monoclonal antibody After 15 minutes, add stabilizer 3% PEG20000 to make the final concentration 0.05%, and stir for another 10-15 minutes.

[0081] 2) Centrifuge at 9000-11000r / min at 4°C for 60min.

[0082] 3) Aspirate the supernatant carefully, and take the loose pink precipitate at the bottom of the tube, which is the c...

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Abstract

The invention discloses a rapid colloidal gold detection test strip for a porcine reproductive and respiratory syndrome virus. The test strip comprises a sample absorbing pad, a glass fiber membrane containing a colloidal gold marked M protein monoclonal antibody of the porcine reproductive and respiratory syndrome virus, a nitrocellulose membrane with a detection line T and a control line C, a piece of water absorbing paper and a substrate, wherein the detection line T is wrapped with a GP5 protein monoclonal antibody of the porcine reproductive and respiratory syndrome virus; the control line C is wrapped with a second antibody IgG; the sample absorbing pad, a glass fiber membrane, the nitrocellulose membrane and the water absorbing paper are adhered to the substrate sequentially in a mutual lap joint manner. The test strip adopts a membrane chromatography double-antibody sandwich method for detecting the porcine reproductive and respiratory syndrome virus in a sample, is simple, convenient and rapid to operate, does not need special instruments, equipment or special training, is easy to popularize, is applicable to basic level, is applicable to large-scale on-site detection and epidemiological investigation in emergency, and plays an assistant role in diagnosis on infection of the porcine reproductive and respiratory syndrome virus, wherein the result is clear and easy.

Description

[0001] technical field [0002] The invention belongs to the field of animal viral disease monitoring and diagnostic reagents, in particular to a colloidal gold rapid detection test strip for porcine reproductive and respiratory syndrome virus. Background technique [0003] Porcine reproductive and respiratory syndrome (PRRS), commonly known as blue ear disease, is a reproductive disorder in sows and piglets caused by porcine reproductive and respiratory syndrome virus (PRRSV). An infectious disease characterized by respiratory disease. It is one of the 93 legally reportable animal diseases listed by the World Organization for Animal Health (Office International DesEpizooties, OIE). According to statistics, the disease causes an economic loss of nearly 560 million U.S. dollars to the swine industry in the United States every year (Cho and Dee, 2006). PRRSV was isolated from aborted fetuses in my country in 1996, which confirmed the existence of porcine reproductive and res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/558G01N33/56983G01N2333/08
Inventor 李建漆世华谢红玲温文生韩兴舒银辉廖园园刘洁秦红刚徐松牟林琳朱薇冯钊
Owner WUHAN CHOPPER BIOLOGY
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