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Gene chip and kit for detecting porcine epidemic diarrhea virus, transmissible gastroenteritis virus and porcine rotavirus

A porcine epidemic diarrhea and porcine rotavirus technology, applied in the direction of microbe-based methods, biochemical equipment and methods, and microbiological determination/testing, can solve the problem of undisclosed gene chips and undisclosed detection of porcine epidemic diarrhea virus For issues such as transmissible gastroenteritis virus and porcine rotavirus, it has achieved strong specificity, good application prospects, and rapid detection results

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

[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 detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus, but did not disclose the specific gene chip and detection kit, nor did it disclose whether it can specifically and sensitively detect porcine epidemic Porcine diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus

Method used

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  • Gene chip and kit for detecting porcine epidemic diarrhea virus, transmissible gastroenteritis virus and porcine rotavirus
  • Gene chip and kit for detecting porcine epidemic diarrhea virus, transmissible gastroenteritis virus and porcine rotavirus
  • Gene chip and kit for detecting porcine epidemic diarrhea virus, transmissible gastroenteritis virus and porcine rotavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of the gene chip of the present invention

[0052] 1. Materials and instruments

[0053] The same experimental materials and instruments as mentioned above.

[0054] 2. Experimental method

[0055] 2.1 Preparation of PCR primers and detection probes

[0056] (1) Design pathogen-specific probes: through the alignment analysis of the nucleic acid sequences of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and porcine rotavirus included in GenBnak, the sequence of the conserved region was selected: PEDV M , S; TGEV N, S; PoRV NSP4, VP7. Design and detect multiple pairs of probes for conserved sequences, and select the probe sequences with strong specificity.

[0057] (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.

[00...

Embodiment 2

[0182] Embodiment 2 Detection method of the present invention

[0183] 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:

[0184] 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;

[0185] Add 750ul isopropanol and shake gently;

[0186] Pipette 800ul into the adsorption column and centrifuge at 12000r for 30s at 4°C;

[0187] Discard the liquid in the collection tube, transfer the remaining lysate into the adsorption column, and centrifuge at 12000r for 30s at 4°C;

[0188] After discarding the collected solution, add 500ul RP solution, and centrifuge at 12000r for 30s at 4°C to remove protein;

[0189] Add 500ul W3 solution, let it stand for 1min, and then centrifuge at 12000r for 15s at 4°C;

[0190] ...

Embodiment 3

[0211] Embodiment 3 specificity test

[0212] 1. Test method

[0213] 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, TGEV, 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 sex.

[0214] 2. Results

[0215] Experimental results such as Image 6 Shown, adopt the method of the present invention to detect, and the detection result of PoRV, TGEV and PEDV is positive, and the detection result of PRRSV, CSFV, JEV, PRV is all negative.

[0216] The method of the present invention can only detect 3 kinds of viruses of the present invention, and cannot detect other viruses, indicating t...

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Abstract

The invention discloses a gene chip and kit for detecting porcine epidemic diarrhea virus, transmissible gastroenteritis virus and porcine rotavirus. The gene chip disclosed by the invention comprises a solid phase carrier and a probe fixed on the solid phase carrier, wherein the probe comprises any one or two oligonucleotide fragments shown in SEQ ID NO.1 or 2, any one or two oligonucleotide fragments shown in SEQ ID NO.3 or 4, and any one or two of oligonucleotide fragments shown in SEQ ID NO.5 or 6. The kit disclosed by the invention comprises the gene chip and reagents for amplifying genes of porcine epidemic diarrhea virus, transmissible gastroenteritis virus and porcine rotavirus. The gene chip and detection kit disclosed by the invention can accurately and effectively detect the porcine epidemic diarrhea virus, transmissible gastroenteritis virus and porcine rotavirus and are good in specificity, high in sensitivity, low in time consumption, high in detection speed and good in application prospects.

Description

technical field [0001] The invention relates to a gene chip and a kit for detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis 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, porcine transmissible gastroenteritis virus, and porcine rotavirus are increasing day by day. These three types of diseases can cause high morbidity and mortality in piglets, and in some areas The phenomenon of mixed infection and secondary infection has appeared, whi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C40B40/06C12R1/93
CPCC12Q1/6837C12Q1/70C12Q2565/501
Inventor 文心田曹三杰黄小波尹人杰文翼平伍锐赵松邓静常晓霞张仙
Owner SICHUAN AGRI UNIV
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