Identification and detection method matched with attenuated live vaccines for NADC30-like PRRSV and application thereof

An attenuated live vaccine, HP-PRRSV technology, applied in the direction of microbial-based methods, biochemical equipment and methods, microbial measurement/testing, etc., can solve the problem of inducing low levels of neutralizing antibodies, increasing the complexity of strains, exacerbating and other problems, to achieve the effect of rapid diagnosis and purification

Inactive Publication Date: 2020-03-13
TIANJIN AGRICULTURE COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since 2014, the prevalence of NADC30-like strains in my country has shown an intensified trend. At present, there are a large number of research reports that this type of strain and HP-PRRSV have become the two dominant epidemic strains in pig farms, which increases the complexity of the strains and brings great harm to my country. Pig farming presents new challenges
Inactivated vaccines are relatively safe and there is no risk of strong virulence, but compared with attenuated vaccines, their immune efficacy is lower than that of attenuated vaccines, and the level of neutralizing antibodies induced is low, resulting in unreliable immune effects, large immune doses, and side effects However, attenuated vaccines play an important role in the prevention and control of PRRSV, but attenuated vaccines provide partial cross-protection against heterologous strains, especially NADC30- The emergence of like strains has produced a large number of recombinant strains due to its easy recombination characteristics. The homology between genomes of NADC30-like strains is low, and there are still large differences between different strains in the same lineage. Now It is difficult to have sufficient cross-protection with vaccines
At the same time, attenuated vaccines are not suitable for storage and transportation at room temperature. Therefore, it is imminent to develop a vaccine that is safer, more efficient, and heat-resistant, especially for the two dominant strains of NADC30-1ike and HP-PRRSV that are currently prevailing.

Method used

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  • Identification and detection method matched with attenuated live vaccines for NADC30-like PRRSV and application thereof

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Experimental program
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Effect test

Embodiment 1

[0046] The raw materials and reagents used in the present invention are commercially available. NADC30 clinical field strain (see-Isolation and Identification of Porcine Reproductive and Respiratory Syndrome Virus in Tianjin and Analysis of Genetic Evolution-Chapter 4 in Master's Thesis: Virus Isolation and Identification), and then isolated and preserved by our laboratory; also refer to the method below To separate:

[0047] (1) Take fresh PRRS-positive (NADC30 RT-PCR positive) samples, shred them, beat them with a tissue processor, and centrifuge at 4°C, 6000rpm for 10 minutes; take a small amount of supernatant and freeze-thaw it three times, and filter it out with a 0.22μm filter membrane. bacteria;

[0048] (2) Inoculate a single layer of PAMs cells, absorb at 37°C for 1 hour, discard the supernatant, add serum-containing DMEM culture solution, and place in a 37°C, 5% CO2 incubator for 48 hours;

[0049] (3) Use an inverted microscope to regularly observe the cell CPE, ...

Embodiment 2

[0052] A NADC30-like live attenuated PRRSV vaccine, the vaccine strain is natural recombinant PRRSV, the backbone is HP-PRRSVMLV, and has the molecular characteristics of 131 amino acid deletions of the NADC30 strain, and the recombination position is located at 1737nt of the non-structural protein coding region (Nsp2) -3506nt; NADC30 PRRSV live attenuated vaccine was prepared as follows:

[0053] (1) Inoculate the monolayer of MARC-145 cells with the seed liquid, incubate at 37°C for 48h-60h, and harvest the virus liquid when more than 70% of the cells appear CPE.

[0054] (2) The harvested virus liquid was serially diluted 10×, inoculated in 96-well plate, 8 wells were inoculated for each dilution, 0.1 mL / well, and a normal cell control was set at the same time, at 37°C in 5% CO 2 Cultivate for 2 days to 3 days, measure TCID 50 .

[0055] (3) Adjust the concentration of the harvested seed solution, mix it with a heat-resistant freeze-drying protective agent with gelatin as...

Embodiment 3

[0058] Reaction system preparation and reaction conditions 1

[0059] Reaction system preparation (20 μL): 2 μL of 10×PCR Buffer (with Mg2+), 2 μL of dNTP Mixture (2.5 mmol / Leach), 1 μL of each Primer, 0.5 μL of rTaq (5 U / μL), 5 μL of cDNA template, PCR Water 8.0 μL. Reaction conditions: 95°C for 5min; 94°C for 40s, 55°C for 45s, 72°C for 1.5 min, 35 cycles; 72°C for 10min

[0060] Reaction system preparation and reaction conditions 2

[0061] The optimal reaction system is (20 μL): 2 μL of 10×PCR Buffer (with Mg2+), 2 μL of dNTP Mixture (2.5 mmol / L each), 1 μL of each Primer, 0.5 μL of rTaq (5 U / μL), cDNA template 5 μL, PCR water 8.0 μL. Reaction conditions: 95°C for 5 min; 94°C for 40 s, 57°C for 45 s, 72°C for 1.5 min, 35 cycles; 72°C for 10 min.

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Abstract

The invention discloses an identification RT-PCR method matched with attenuated live vaccines for NADC30-like PRRSV and application thereof, belonging to the technical field of agricultural and veterinary biotechnology. According to the invention, through contrastive analysis of the genomes of the attenuated live vaccines for NADC30-like PRRSV, the matching identification RT-PCR method is established, three specific primers are adopted for identification, vaccine strain infection and wild virus infection can be rapidly identified according to the size of an amplified target fragment, differentsubtypes of wild viruses can be distinguished and diagnosed, and rapid diagnosis and purification of the porcine reproductive and respiratory syndrome are achieved.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to a RT-PCR method and application for matching identification of live attenuated PRRSV-like NADC30 vaccines. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease of pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV), characterized by reproductive disorders in sows and respiratory diseases in growing pigs. Huge economic loss. Especially in 2006, the highly pathogenic HP-PRRSV broke out, and its clinical morbidity and mortality were high, which brought unprecedented economic losses to my country's pig industry. Since 2012, domestic strains with high homology to the NADC30 gene in the United States have been isolated one after another. Due to the close genetic evolution relationship with the NADC30 strain, this type of virus is now collectively referred to as NADC30-like ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/686C12Q1/701C12Q2521/107
Inventor 孙英峰姜轩张奥石青青付旭彬马吉飞李留安张建斌
Owner TIANJIN AGRICULTURE COLLEGE
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