Reagent kit special for testing high pathogenicity pig replication and syndrome virus variation strain

A syndrome virus, highly pathogenic technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problem of high technical requirements, high requirements of laboratory personnel, and difficult to be applied to on-site inspection on a large scale and other problems to achieve a strong specific effect

Inactive Publication Date: 2008-07-16
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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AI Technical Summary

Problems solved by technology

Separation and identification and peroxidase monolayer assay (IPMA) have high technical requirements, which can only be carried out in the laboratory, and have high requirements for experimenters, so it is difficult to apply them to large-scale field inspections

Method used

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  • Reagent kit special for testing high pathogenicity pig replication and syndrome virus variation strain

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Embodiment Construction

[0033] 1) Primers

[0034] According to the characteristic of deletion in the Nsp2 region in the highly pathogenic porcine reproductive and respiratory syndrome that occurred after 2006, a pair of specific primers were designed. The amplified fragment size is 404bp, and the amplified traditional PRRSV amplified fragment is 494bp. Synthesized by TaKaRa Biotechnology Co., Ltd.

[0035] PSX1: 5'-TGGTCCTAACGGTTCGGAAGAAAC-3'

[0036] PSX2: 5'-GTGAGCTGAGTATTTTGGGCGTGT-3'

[0037] 2) Treatment of sick materials

[0038] Cut the disease material into pieces and grind them in a sterilized grinder, dilute them 1:4 times with sterile cell culture medium, take the virus suspension at -20°C for 3 times, centrifuge at 8000rpm for 10min, and take the supernatant- Store at 80°C for later use.

[0039] 3) Extraction of viral RNA

[0040] ① Take 300L of grinding solution and add 600μL RNAout, shake and mix well and let stand at room temperature for 5min;

[0041] ② Add 200 μL of chlorofo...

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Abstract

The invention provides a special kit for detecting highly pathogenic porcine reproductive and syndrome virus mutation strain. The highly pathogenic porcine reproductive and syndrome virus mutation strain is detected by self-designing specific primers PSX1/PSX2, extracting virus RNA and carrying out reverse transcription into cDNA and utilizing a PCR method; the size of the estimated amplification segment of an amplification mutation strain is 404bp and an amplification traditional PRRSV amplification segment is 494bp. Compared with the prior art, the special kit has strong specificity and sensitivity, and the coincidence rate can achieve more than 90 percent compared with a virus separation and IPMA method. The amplification results of the primers to hogcholera virus, pseudorabies virus and porcine parvovirus are negative, which can distinguish a NSP2 mutation strain and a traditional strain; the method can detect the PRRSV with 12.8ng/L, thus the invention can be widely used for the clinical detection of the highly pathogenic porcine reproductive and syndrome virus mutation strain.

Description

1. Technical field [0001] The patent of the present invention relates to diagnostic technology in the field of veterinary biotechnology, specifically a diagnostic method and a special kit for detecting variant strains of highly pathogenic porcine reproductive and syndrome virus. 2. Background technology [0002] In the summer of 2006, pig "high fever" broke out in my country. At present, the Ministry of Agriculture has determined that the highly pathogenic PRRS virus variant strain is the primary cause of the disease. Many domestic scholars have done a lot of work on highly pathogenic porcine reproductive and syndrome virus mutant strains. At present, the methods for detecting variant strains of highly pathogenic porcine reproductive syndrome virus mainly include virus isolation and identification, peroxidase monolayer assay (IPMA) and RT-PCR diagnostic methods. Separation and identification and peroxidase monolayer assay (IPMA) have high technical requirements, which can o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 王金宝任慧英李俊吴家强张秀美周顺温建新时建立刘玉庆
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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