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Gene chip and kit for detecting pig epidemic type B encephalitis virus and/or pig porcine reproductive and respiratory syndrome virus

A Japanese encephalitis virus, respiratory syndrome technology, applied in combinatorial chemistry, organic compound library, biochemical equipment and methods, etc., can solve the problem of sensitive detection of Japanese encephalitis virus porcine reproductive and respiratory syndrome Viruses, no chip sensitivity test, etc., to achieve good application prospects, fast detection, and short time-consuming effects

Active Publication Date: 2015-01-21
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, Hao Shiyou et al., "Establishment of Gene Chip Diagnosis Method for Porcine Viral Reproductive Disorder Syndrome", the 12th Academic Symposium of the Animal Infectious Diseases Branch of the Chinese Society of Animal Science and Veterinary Medicine, disclosed the detection of Japanese encephalitis in 2007. Gene chip of 6 kinds of virus including virus, porcine reproductive and respiratory syndrome virus, etc., but there is no chip sensitivity test in this document, which cannot prove that it can detect Japanese encephalitis virus and porcine reproductive and respiratory syndrome virus sensitively

Method used

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  • Gene chip and kit for detecting pig epidemic type B encephalitis virus and/or pig porcine reproductive and respiratory syndrome virus
  • Gene chip and kit for detecting pig epidemic type B encephalitis virus and/or pig porcine reproductive and respiratory syndrome virus
  • Gene chip and kit for detecting pig epidemic type B encephalitis virus and/or pig porcine reproductive and respiratory syndrome virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Preparation of the gene chip of the present invention

[0048] 1. Materials and instruments

[0049] The same experimental materials and instruments as mentioned above.

[0050] 2. Experimental method

[0051] 2.1 Preparation of PCR primers and detection probes

[0052] (1) Design of pathogen-specific probes: through the alignment analysis of the nucleic acid sequences of porcine epidemic encephalitis virus and porcine reproductive and respiratory syndrome virus included in GenBnak, the sequences of conserved regions were selected: JEV C, M, E, NS1, NS5; PRRSV 5'UTR, ORF1b, ORF5, ORF6, ORF7. Design detection probes for conserved sequences, and select specific probe sequences from them.

[0053] (2) Designing specific primers for the probe sequences: specific primers were designed using the bioinformatics software Primer5 for the above conserved probe sequences. Specific primers were synthesized by Dalian Bao Biological Company.

[0054] (3) Probe prepara...

Embodiment 2

[0162] Embodiment 2 detection method of the present invention

[0163] 1. Nucleic acid extraction

[0164] Tiangen’s Total RNA Extraction Kit, refer to the instructions for operation, the specific steps are as follows:

[0165] 1) Use scissors to cut tissue disease material of appropriate size into a mortar, add an appropriate amount of RZ lysate, grind until homogenized, take 300μl in a 1.5ml EP tube, add 1ml of RZ lyse solution for every 50-100mg of tissue, the sample volume is not It should be more than one-tenth of the RZ volume of the lysate.

[0166] 2) Place the homogenized sample at 15-30°C for 5 minutes;

[0167] 3) Centrifuge at 12000r / min for 5min at 4°C, take the supernatant, and transfer it to a new RNase-free centrifuge tube;

[0168] 4) Add 200 μL of chloroform, shake vigorously up and down for 15 sec, and let stand at room temperature for 3 min;

[0169] 5) Centrifuge at 12000r / min for 10min at 4°C, the sample will be divided into three layers, and gently tra...

Embodiment 3

[0199] Embodiment 3 specificity test

[0200] 1. Test method

[0201] Using the gene chip prepared in Example 1, according to the method of Example 2 (the reverse primer of Prm / M, JEV-C, PS9, Y11 and the forward marker primer molar ratio are 50:1, 40:1, 20:1, 10:1, the hybridization temperature is 48°C, and the hybridization time is 3h), and JEV, PRRSV, PPV, and PCV-2 are detected to verify the specificity of the gene chip and the kit of the present invention.

[0202] 2. Results

[0203] Experimental results such as Figure 6 As shown, the method of the present invention can effectively detect PRRSV and JEV separately or simultaneously, but not detect other viruses, such as PPV and PCV-2.

[0204] The experimental results show that the specificity of the kit and the gene chip of the present invention is strong, and other viruses cannot be detected.

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Abstract

The invention discloses a gene chip and a kit for detecting a pig epidemic type B encephalitis virus and / or pig porcine reproductive and respiratory syndrome virus. The gene chip and the detection kit disclosed by the invention can accurately and effectively detect the pig epidemic type B encephalitis virus and the pig porcine reproductive and respiratory syndrome virus and are high in specificity, high in sensitivity, short in time, fast in detection speed and good in application prospect.

Description

technical field [0001] The invention relates to a gene chip and a kit for detecting porcine epidemic Japanese encephalitis virus and / or porcine reproductive and respiratory syndrome virus. Background technique [0002] Mixed infection with multiple pathogens is a common phenomenon in current pig farm diseases. Porcine reproductive and respiratory syndrome, porcine epidemic encephalitis, and porcine epidemic encephalitis have similar disease symptoms and are often mixed infections. They are the two currently endangering the pig industry. It is an important viral disease, so early differential diagnosis and timely understanding of the epidemic situation are particularly important to control the epidemic of these two diseases. At present, the isolation of pathogens and conventional serological methods are mostly used in the diagnosis of these two diseases, but it takes a long time. Recently, the isolation and identification of pathogens, immune colloidal gold technology, enzym...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C40B40/06
CPCC12Q1/701
Inventor 文心田黄小波曹三杰邓静伍锐文翼平姜来生尹人杰赵松张仙
Owner SICHUAN AGRI UNIV
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