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Primer and kit for identifying four brucella in dog and application of primer and kit

A Brucella and reaction technology, applied in the field of molecular biology, can solve the problems of high potential transmission risk and high rate of Brucella infection

Pending Publication Date: 2020-08-07
山东省健牧生物药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In pastoral areas, because dogs are mostly free-range, eating raw poultry and dead livestock is common, and the positive rate of canine serum Brucella antibodies can reach 41%; the positive rate of smooth antibodies in pet dogs in a city from 2016 to 2017 was 8.76% %, the rough type is 1.99%, indicating that the infection rate of Brucella in dogs is higher, and the potential transmission risk is greater

Method used

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  • Primer and kit for identifying four brucella in dog and application of primer and kit
  • Primer and kit for identifying four brucella in dog and application of primer and kit
  • Primer and kit for identifying four brucella in dog and application of primer and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Establishment and optimization of PCR reaction system and specificity test:

[0026] 1. Strains: Brucella bovis, Brucella melis, Brucella suis and Brucella canis inactivated bacterial fluids were donated by the China Veterinary Drug Administration, Escherichia coli, Salmonella and Listeria Bacteria were preserved by the Poultry Research Institute of Shandong Academy of Agricultural Sciences.

[0027] 2. Reagents: DL1000bp DNA Marker and 2×PCR Mix were purchased from Treasure Bio (Beijing) Co., Ltd., and other reagents were purchased from commercial companies.

[0028] 3. Bacterial genome extraction: Refer to the instructions of the TIANGEN Bacterial Genome Extraction Kit to extract the genomic DNA of the above bacterial strains, and store at -20°C for later use.

[0029] 4. Establishment of PCR reaction system and condition optimization: Establish a 25 μL reaction system:

[0030] 1) Annealing temperature: 12.5 μL of 2×PCR Mix, 2 μL of DNA template to be tested, 0.5 μ...

Embodiment 2

[0034] Validate the specificity of the established method

[0035] Using the above established Brucella PCR detection method to detect according to the DNA of Brucella bovis, Brucella ovis, Brucella suis, Brucella canis, Salmonella and Escherichia coli, Verify the specificity of the established method. PCR reaction products were electrophoresed through agarose gel, and the electrophoresis picture was figure 2 ,exist figure 2 The lanes are: M. DL1000 DNAMarker; 1: Negative control (template is DEPC); 2: Brucella bovis; 3: Brucella melis; 4: Brucella suis; 5: Brucella canis; 6: Salmonella; 7: Escherichia coli.

[0036] From figure 2 It can be seen that the specific band of 352bp has been detected from the Brucella canis sample with the established Brucella canis PCR method; to Brucella bovis, Brucella sheep and pig species A specific band of 709bp was detected for Brucella; no specific band was amplified for Escherichia coli, Salmonella and the negative control, and the ...

Embodiment 3

[0038] sensitivity test

[0039]1. After measuring the content and purity of four kinds of genomic DNA (dog breed, sheep breed, cattle breed and pig breed) samples with NanoDrop™ One / OneC ultra-micro ultraviolet spectrophotometer, 10-fold dilution into six gradients, respectively 10.7 ng / μL, 1.07 ng / μL, 107 pg / μL, 10.7 pg / μL, 1.07 pg / μL, and 107 fg / μL.

[0040] 2. Take 2 μL of templates with different contents and use diagnostic primers F and R for PCR amplification to verify the sensitivity of the detection method. The PCR reaction product is subjected to agarose electrophoresis, and the electrophoresis picture is image 3 .

[0041] exist image 3 Each lane in the middle is M. DNA DL1000 Marker; 1-6 corresponds to the gene concentration of Brucella canis: 1, 50.7 ng / μL; 2, 5.07 ng / μL; 3, 507 pg / μL; 4, 50.7 pg / μL μL; 5, 5.07 pg / μL and 6, 507 fg / μL. 7~12 correspond to the gene concentration of Brucella melis: 7, 62 ng / μL; 8, 6.2 ng / μL; 9, 620 pg / μL; 10, 62 pg / μL; 11, 6.2 p...

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Abstract

The invention belongs to the technical field of molecular biology, and relates to a single PCR detection primer, kit and method for four brucella (brucella melitensis, brucella bovis, brucella suis and brucella canis) capable of infecting dogs. The rough brucella (dog brucella lacking 357bp) and smooth brucella (cattle breed, sheep breed and pig breed) can be respectively amplified by 352 bp and 709 bp just by only one pair of primers, so that effective identification can be carried out. In addition, the primers can effectively avoid the generation of primer dimers and hairpin structures, ensure the efficient PCR amplification and enhance the specificity and sensitivity of the detection technology.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a single strain of four types of Brucella (Brucella melis, Brucella bovis, Brucella suis and Brucella canis) that can infect dogs. Re-PCR detection primers, kits and methods. Background technique [0002] In recent years, the hidden dangers of pet-derived zoonotic diseases brought about by the surge in the number of pet dogs raised in my country have been increasing day by day. It is of great practical significance to strengthen the awareness of prevention of zoonotic diseases from pets and take effective measures to control the occurrence of diseases to ensure public health safety. Brucellosis is a zoonotic disease caused by bacteria of the genus Brucella invading the body. Reproductive system diseases have great public health significance. Because Brucella is an intracellular parasite, it is difficult for even sensitive drugs to enter the cells to exert a therapeutic...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/686C12Q2565/125
Inventor 徐怀英秦卓明黄迪海刘星丽仉伟郝文娟盛晓丹郭卉刘霞
Owner 山东省健牧生物药业有限公司
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