Harmless brucella detecting method

A Brucella and detection method technology, applied in the field of harmless Brucella detection, can solve the problems of prevention and control, false positives in serological diagnosis, infection risks, etc., and achieves low environmental risk and fast detection speed. , the effect of high sensitivity

Inactive Publication Date: 2018-11-06
董武 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are a large number of false positives in serological diagnosis, especially the false positives caused by antibodies produced by vaccination have brought great trou

Method used

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  • Harmless brucella detecting method
  • Harmless brucella detecting method
  • Harmless brucella detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Take samples from (Agula Village, Tongliao City, Inner Mongolia) health (that is, no infection with Brucella after testing, number: sample 1), suspected infection with Brucella (number: sample 2) and confirmed infection with Brucella (number: sample 1). 1g of the sheep genital secretion of sample 3); after dripping 10ul of 4% paraformaldehyde solution respectively, the possible Brucella in the sample was completely killed, and it was used for detection.

[0044] The above three samples to be tested were respectively carried out as follows in sequence:

[0045] (1) 1×PBS 200ul+90% absolute ethanol 200ul, 5min;

[0046] (2) centrifuge at 12000rpm for 10min, and take the supernatant;

[0047] (3) For DNA purification, add the same amount of Sequencing Binding Buffer, add it to the DNA purification kit (Bao Biology) and centrifuge at 12000rpm for 30s;

[0048] (4) Add the same amount of Wash Buffer 12000rpm, 30s;

[0049] (5) Repeat step (4) once;

[0050] (6) Add the s...

Embodiment 2

[0057] Take (Tongliao Agula) healthy (that is, confirmed no Brucella infection after testing, number: sample 1), confirmed infection with Brucella (number: sample 5) and three sheep suspected of being infected with Brucella ( Respectively numbered: sample 2, sample 3, sample 4) genital secretion 1g; after treatment with 10ul of 4% paraformaldehyde solution dropwise, the possible Brucella in the sample was completely killed and used for diagnosis.

[0058] Genomic total DNA was extracted from the above five samples to be tested in the same manner as in Example 1. PCR is then performed.

[0059] The DNA concentrations of samples 1-5 were 96ng / ul, 110ng / ul, 100ng / ul, 120ng / ul and 130ng / ul, respectively, measured by Nano Drop.

[0060] Set up 20ng / ul sample group, 20ng / ul positive control group and blank negative control group.

[0061] The total CR reaction is 20ul system, 2×Taq enzyme 10ul, upstream and downstream Brucella OMP primer 2ul, purified DNA 2ul, add pure water to a ...

Embodiment 3

[0066] Take the genital secretions of sheep (Tongliao Agula) confirmed to be infected with Brucella, and divide them into 3 parts, each 1g. The above three test samples were treated with 500 mg / L 84 disinfectant, 4% paraformaldehyde and 10% formalin (the numbers are samples 1, 2 and 3)) respectively. Probable Brucella, back up for testing.

[0067] Genomic total DNA was extracted from the above three samples to be tested in the same manner as in Example 1. PCR is then performed.

[0068] The DNA concentrations of samples 1-3 were 29ng / ul, 106ng / ul, and 99ng / ul, respectively, measured by Nano Drop.

[0069] Set up 20ng / ul sample group, 20ng / ul positive control group and blank negative control group.

[0070] The total CR reaction is 20ul system, 2×Taq enzyme 10ul, upstream and downstream Brucella OMP primer 2ul, purified DNA 2ul, add pure water to a total of 20ul. 95°C for 5min, enter the cycle, denature at 95°C for 30s, anneal at 60°C for 30s, extend at 72°C for 1min, a to...

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Abstract

The invention relates to a harmless brucella detecting method. The method includes that genital secretions, eye secretions, feces or tissues and organs of diseased animals are obtained by utilizing the bacteria killing effect of a fungicide, possible brucella is killed with the fungicide, but possible brucella DNA still remains, the purified brucella DNA can be obtained by laboratory cleaning, andharmless detection of the brucella is carried out by the steps of conventional PCR, electrophoresis and the like. The method has the greatest advantage of harmlessness. On the basis of the advantagesof high detection accuracy and detection speed, the detection of the brucella is safer and non-toxic, and personal safety risks of breeders, veterinarians, samplers, laboratory personnel, epidemic prevention personnel, butchers and citizens are reduced.

Description

technical field [0001] The invention relates to a detection method for harmless Brucella, which belongs to the technical field of biological detection. Background technique [0002] Brucellosis (also known as Brucellosis, referred to as brucellosis) is a common infectious disease caused by bacteria of the genus Brucella. my country lists it as a second-class animal disease. Its pathogen is Gram-negative bacteria parasitic in the cells—Brucella. It mainly causes undulating fever and chronic infection in humans, orchitis and abortion in ruminants. Brucella bacteria are divided into 6 species and 20 biotypes according to antigenicity and main host. In more than 200 countries and regions in the world, human and animal brucellosis exists and is prevalent in more than 170 countries and regions. About 1 / 5 to 1 / 6 of the world's people are threatened by brucellosis. There are about 5 to 6 million people in the world suffering from brucellosis, and there are about 500,000 new case...

Claims

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Application Information

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IPC IPC(8): A61L2/18C12Q1/689C12Q1/04C12R1/01A61L101/32A61L101/46
CPCA61L2/18C12Q1/689
Inventor 董武杨景峰楚文庆陈浩董文静王丰
Owner 董武
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