Gene chip and kit for detecting swine epidemic encephalitis B viruses (SEEBVs) and/or swine fever viruses (SFVs)

A technology of Japanese encephalitis virus and swine fever virus, which is applied in the fields of biochemical equipment and methods, measurement/testing of microorganisms, nucleotide library, etc., can solve the problem of sensitive detection of Type B without chip sensitivity test Encephalitis virus and swine fever virus and other problems have achieved good application prospects, rapid detection, and short time-consuming effects

Inactive 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 Symposium of the Animal Infectious Diseases Branch of the Chinese Society of Animal Husbandry and Veterinary Medicine, disclosed the detection of Japanese encephalitis in 2007. virus, swine fever virus and other 6 kinds of virus gene chips, but there is no chip sensitivity test in this document, which cannot prove that it can detect Japanese encephalitis virus and swine fever virus sensitively

Method used

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  • Gene chip and kit for detecting swine epidemic encephalitis B viruses (SEEBVs) and/or swine fever viruses (SFVs)
  • Gene chip and kit for detecting swine epidemic encephalitis B viruses (SEEBVs) and/or swine fever viruses (SFVs)
  • Gene chip and kit for detecting swine epidemic encephalitis B viruses (SEEBVs) and/or swine fever viruses (SFVs)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Preparation of the gene chip of the present invention

[0049] 1. Materials and instruments

[0050] The same experimental materials and instruments as mentioned above.

[0051] 2. Experimental method

[0052] 2.1 Preparation of PCR primers and detection probes

[0053] (1) Design of pathogen-specific probes: through the alignment analysis of the nucleic acid sequences of porcine epidemic encephalitis virus and classical swine fever virus included in GenBnak, the sequences of conserved regions were selected: JEV C, M, E, NS1, NS5; CSFV 5'UTR, 3'UTR, Npro, Erns, E2. Design detection probes for conserved sequences, and select specific probe sequences from them.

[0054] (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.

[0055] (3) Probe preparation: use a small amount of v...

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 t...

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, Erns, CSF1 and the forward marker primer molar ratio are 50:1, 40:1, 80:1, 80:1, the hybridization temperature is 48°C, and the hybridization time is 3h), and JEV, CSFV, 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 CSFV and JEV separately or simultaneously, but will 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 kit for detecting swine epidemic encephalitis B viruses (SEEBVs) and/or swine fever viruses (SFVs). The gene chip comprises a solid phase carrier and a probe immobilized on the solid phase carrier, wherein the probe comprises any one or two oligonucleotide fragments shown in SEQ ID NO:1 or 2 and/or any one or two oligonucleotide fragments shown in SEQ ID NO:3 or 4. The kit comprises the gene chip and reagents for amplifying genes of the SEEBVs and/or the SFVs. The gene chip and the detection kit can be used for accurately and effectively detecting the SEEBVs and the SFVs, have strong specificity, high sensitivity and good application prospects, consume short time and are fast in detection.

Description

technical field [0001] The invention relates to a gene chip and a kit for detecting porcine epidemic Japanese encephalitis virus and / or swine fever virus. Background technique [0002] Mixed infection with multiple pathogens is a common phenomenon in current pig farm diseases. The disease symptoms caused by these two pig viruses are similar and they are often mixed infections. They are two important viral diseases that currently endanger the pig industry. Therefore, early differential diagnosis and timely understanding of the epidemic situation are particularly important for controlling the prevalence 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, enzyme-linked immunosorbent assay, serum neutralization test, polymerase chain reaction, etc. have been est...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C40B40/06
CPCC12Q1/701C12Q2600/112C40B40/06
Inventor 文心田曹三杰黄小波邓静文翼平伍锐姜来生尹人杰赵松常晓霞
Owner SICHUAN AGRI UNIV
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