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Kit for detecting porcine epidemic diarrhea virus, porcine 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: 2014-12-24
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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  • Kit for detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus
  • Kit for detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus
  • Kit for detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 Preparation of kit of the present invention

[0050] 1. Materials and instruments

[0051] The same experimental materials and instruments as mentioned above.

[0052] 2. Experimental method

[0053] 2.1 Preparation of PCR primers and detection probes

[0054] (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.

[0055] (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.

[0056] (3)...

Embodiment 2

[0120] Embodiment 2 The usage method of kit of the present invention

[0121] 1. Nucleic acid extraction

[0122] Extraction of viral RNA: The kit method is used to extract viral RNA, and the test uses a small amount of virus / liquid sample RNA extraction kit. Extract RNA according to the instructions of the kit, the method is as follows:

[0123] a) Take 300 μL of the sample to be tested and place it in a centrifuge tube, add 500 μL of RV solution, shake vigorously for 2 minutes, and then let stand at room temperature for 5 minutes.

[0124] b) Add 750 μL of isopropanol and shake gently.

[0125] c) Pipette 800 μL into the adsorption column and centrifuge at 12000 rpm for 30 s at 4°C.

[0126] d) Discard the liquid in the collection tube, transfer the remaining lysate into an adsorption column, and centrifuge at 12000r for 30s at 4°C.

[0127] e) Add 500 μL RP solution after discarding the collected solution, and centrifuge at 12000 rpm for 30 seconds at 4°C to remove prot...

Embodiment 3

[0147] Embodiment 3 specificity test

[0148] 1. Test method

[0149] Adopt the kit of embodiment 1, according to the method for embodiment 2, detect 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 kit of the present invention.

[0150] 2. Results

[0151] Experimental results such as figure 2 As shown, the inventive method can effectively detect PoRV, TGEV, PEDV of the present invention, and can not detect other virus, as, PRRSV, CSFV, JEV, PRV, shows that the specificity of the inventive method is strong, can not amplify other viruses. Virus.

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Abstract

The invention discloses a kit for detecting a porcine epidemic diarrhea virus, a porcine transmissible gastroenteritis virus and a porcine rotavirus. The gene detection kit disclosed by the invention can accurately and effectively detect the porcine epidemic diarrhea virus, the porcine transmissible gastroenteritis virus and the porcine rotavirus, and is strong in specificity, high in sensitivity, short in time consumption, fast in detection 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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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/701
Inventor 文心田曹三杰黄小波胡中凯文翼平伍锐黄宁尹人杰邓静赵松
Owner SICHUAN AGRI UNIV
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