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Gene chip and kit for detecting porcine epidemic diarrhea virus and/or swine rotavirus

A technology for porcine epidemic diarrhea and porcine rotavirus, applied in biochemical equipment and methods, microbiological determination/testing, nucleotide library, etc., capable of solving undisclosed sensitive detection of porcine epidemic diarrhea virus and porcine rotavirus Viruses, undisclosed gene chip detection kits and other issues have achieved good application prospects, rapid detection, and high sensitivity

Active Publication Date: 2015-01-07
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Deng Junhua et al., "Construction of TGEV-PEDV-PRV Gene Chip Probe", Proceedings of the Seventh Symposium of the Veterinary and Veterinary Biotechnology Branch of the Chinese Society of Animal Science and Veterinary Medicine and the Veterinary Immunology Branch of the Chinese Society of Immunology, July 2008, published possible Probes for the detection of porcine epidemic diarrhea virus and porcine rotavirus, but the specific gene chip and detection kit are not disclosed, nor is it disclosed whether it can specifically and sensitively detect porcine epidemic diarrhea virus and porcine rotavirus

Method used

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  • Gene chip and kit for detecting porcine epidemic diarrhea virus and/or swine rotavirus
  • Gene chip and kit for detecting porcine epidemic diarrhea virus and/or swine rotavirus
  • Gene chip and kit for detecting porcine epidemic diarrhea virus and/or swine rotavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Preparation of the gene chip of the present invention

[0054] 1. Materials and instruments

[0055] The same experimental materials and instruments as mentioned above.

[0056] 2. Experimental method

[0057] 2.1 Preparation of PCR primers and detection probes

[0058] (1) Design of pathogen-specific probes: through the alignment analysis of the nucleic acid sequences of porcine epidemic diarrhea virus and porcine rotavirus included in GenBnak, the sequences of conserved regions were selected: PEDV M, S; PoRV NSP4, VP7. Design and detect multiple pairs of probes for conserved sequences, and select the probe sequences with strong specificity.

[0059] (2) Design of specific primers for probe sequences: specific primers were designed for the above conserved probe sequences using bioinformatics software DNAman, Primer5.0, etc. Specific primers were synthesized by Shanghai Bioengineering Company.

[0060] (3) Probe preparation: Extract porcine epidemic diarr...

Embodiment 2

[0177] Embodiment 2 Detection method of the present invention

[0178] 1. Nucleic acid extraction Extraction of viral RNA: use Tiangen (Beijing) Biochemical Technology Co., Ltd. RNA extraction kit. Extract RNA according to the kit instructions:

[0179] Take 300ul of the treated disease material solution and place it in a centrifuge tube, add 500ul of RV solution, shake vigorously for 2 minutes, and then let stand at room temperature for 5 minutes;

[0180] Add 750ul isopropanol and shake gently;

[0181] Pipette 800ul into the adsorption column and centrifuge at 12000r for 30s at 4°C;

[0182] Discard the liquid in the collection tube, transfer the remaining lysate into the adsorption column, and centrifuge at 12000r for 30s at 4°C;

[0183] After discarding the collected solution, add 500ul RP solution, and centrifuge at 12000r for 30s at 4°C to remove protein;

[0184] Add 500ul W3 solution, let it stand for 1min, and then centrifuge at 12000r for 15s at 4°C;

[0185] ...

Embodiment 3

[0210] Embodiment 3 specificity test

[0211] 1. Test method

[0212] Using the gene chip prepared in Example 1, according to the method of Example 2 (the molar ratio of the forward primer and the reverse primer is 1:40, the hybridization temperature is 48°C, and the hybridization time is 3h), PoRV, PEDV, PRRSV ( Porcine reproductive and respiratory syndrome virus), CSFV (swine fever virus), JEV (porcine Japanese encephalitis virus), PRV (porcine pseudorabies virus) 7 kinds of viruses, to verify the specificity of the gene chip and the kit of the present invention.

[0213] 2. Results

[0214] Experimental results such as Image 6 Shown, adopt the inventive method to detect, the detection result of PoRV and PEDV is positive, the detection result of PRRSV, CSFV, JEV, PRV is all negative.

[0215] The method of the present invention can only detect two kinds of viruses of the present invention, and cannot detect other viruses, which shows that the specificity of the kit and the...

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Abstract

The invention discloses a gene chip and a kit for detecting porcine epidemic diarrhea virus and / or swine rotavirus. The gene chip and detection kit disclosed by the invention can accurately and effectively detect porcine epidemic diarrhea virus and / or swine rotavirus, and are strong in specificity, high in sensitivity, short in time consumption, rapid in detection, therefore, the gene chip and detection kit disclosed by the invention have a good application prospect.

Description

technical field [0001] The invention relates to a gene chip and a kit for detecting porcine epidemic diarrhea virus and porcine rotavirus. Background technique [0002] With the increasing scale of centralized pig farming, the diarrheal diseases of pigs are becoming more and more serious. The research on the pathogens causing diarrhea in piglets has confirmed that there are and are still increasing Escherichia coli, Salmonella, Clostridium welchii, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus, mild swine fever, swine dysentery , Coccidia etc. Piglet viral diarrhea diseases caused by porcine epidemic diarrhea virus and porcine rotavirus are increasing day by day. These two types of diseases can cause high morbidity and mortality of piglets, and some areas have shown mixed infection and secondary The phenomenon of infection has caused great losses to the pig industry. In view of the fact that these two viruses are extremely...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C40B40/06
CPCC12Q1/6837C12Q1/70C12Q2565/501
Inventor 文心田黄小波曹三杰尹人杰伍锐文翼平邓静赵松张仙常晓霞
Owner SICHUAN AGRI UNIV