Detection method of brucella, kit and applications of kit

A technology of Brucella and detection method, which is applied in the field of Brucella detection, can solve problems such as unrealizable, and achieve the effects of pollution prevention, high sensitivity, effective and accurate quantitative detection

Inactive Publication Date: 2016-12-07
北京旌准医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] The main problem that this application solves is to provide a kind of dete

Method used

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  • Detection method of brucella, kit and applications of kit
  • Detection method of brucella, kit and applications of kit
  • Detection method of brucella, kit and applications of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] This embodiment provides a method for obtaining the sample to be tested. The nucleic acid of the sample to be tested can be extracted by a commercial spin column extraction method, a commercial magnetic bead extraction method or a commercial boiling cracking method.

[0050] The invention provides a method for obtaining Brucella DNA from a serum sample by adopting a spin column method to obtain a sample to be tested. The specific method is as follows:

[0051] 1) Before use, add 17 mL of absolute ethanol to buffer GD, add 60 mL of absolute ethanol to rinse solution PW, and shake well.

[0052] 2) Take 1ml of serum sample to be tested, centrifuge at 13,000rpm for 10min, and aspirate the supernatant as much as possible.

[0053] 3) Add 200 μl buffer GA to the cell pellet, shake until the cell is completely suspended.

[0054] 4) Add 20 μl Proteinase K and 5 μl Brucella internal standard quality control solution to the tube, and mix well.

[0055] 5) Add 220 μl buffer G...

Embodiment 2

[0064] Qualitative detection of Brucella

[0065] Present embodiment 2 provides a kind of qualitative detection method of Brucella:

[0066] 1. PCR amplification reaction of Brucella

[0067] 1) Reagents and components used in the PCR amplification reaction of Brucella DNA

[0068] (1) Brucella primer probe mixture:

[0069] The specific components of the Brucella primer-probe mixture are upstream primer Bcsp-F (50 μM) 0.6 μL, downstream primer Bcsp-R (50 μM) 0.6 μL, Taqman fluorescent probe Bcsp-FP (20 μM) 0.5 μL, purified water 3.3 μL.

[0070] The preferred components of the Brucella primer-probe mixture are: upstream primer Bcsp-F (50 μM) 0.6 μL, downstream primer Bcsp-R (50 μM) 0.6 μL, Taqman fluorescent probe Bcsp-FP (20 μM) 0.5 μL, Internal standard upstream primer (20 μM) 0.5 μL, internal standard downstream primer (20 μM) 0.5 μL, internal standard probe (10 μM) 0.375 μL, purified water 1.925 μL.

[0071] The upstream primer Bcsp-F comprises the base sequence show...

Embodiment 3

[0094] Present embodiment 2 provides a kind of quantitative detection method of Brucella:

[0095] 1. PCR amplification reaction of Brucella DNA (sample to be tested)

[0096] 1) Reagents and components used in the PCR amplification reaction of Brucella DNA

[0097] (1) Brucella primer probe mixture:

[0098] The components of the Brucella primer-probe mixture are: upstream primer Bcsp-F (50 μM) 0.6 μL, downstream primer Bcsp-R (50 μM) 0.6 μL, Taqman fluorescent probe Bcsp-FP (20 μM) 0.5 μL, internal Standard upstream primer (20 μM) 0.5 μL, internal standard downstream primer (20 μM) 0.5 μL, internal standard probe (10 μM) 0.375 μL, purified water 1.925 μL.

[0099] The upstream primer Bcsp-F comprises the base sequence shown in SEQ ID NO:1;

[0100] The downstream primer Bcsp-R comprises the base sequence shown in SEQ ID NO:2;

[0101] The Taqman fluorescent probe Bcsp-FP comprises a base sequence as shown in SEQ ID NO: 3, and the Taqman fluorescent probe Bcsp-FP has a fl...

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Abstract

The invention discloses a detection method of brucella. The detection method comprises the following steps: acquiring brucella DNA from a serum sample by adopting a centrifugal column method, thus obtaining a to-be-detected sample; carrying out PCR amplification reaction; detecting a reaction result by adopting a fluorescence quantitative PCR instrument, wherein during fluorescence signal collection, fluorescein corresponding to fluorophore at the BKV-FP 5' end of a Taqman fluorescence probe is set, and the fluorescence signals are collected at 60 DEG C; and analyzing the result. The invention further provides a kit for detecting the brucella. The detection method and the kit are high in sensitivity, and the minimal detection can achieve 1000 copies/mL. Meanwhile, the detection method is good in specificity, and the cross reaction with other bacteria, such as escherichia coli, salmonella enteritidis, hepatitis B virus, human herpes virus 4, and human cytomegalovirus can be avoided.

Description

technical field [0001] The present invention relates to a detection method, kit and application of bacteria, in particular to a detection method, kit and application of Brucella. Background technique [0002] Brucellosis, referred to as brucellosis, also known as geothermal relaxation fever, Malta fever or wave fever, commonly known as "lazy man's disease", is a disease caused by intracellular parasitic bacteria of the genus Brucella. A zoonotic infection—allergic disease, brucellosis is widely distributed all over the world. [0003] More than 60 kinds of livestock, poultry, and wild animals are currently known to be hosts of Brucella. The sources of infection related to humans are mainly sheep, cattle and pigs, followed by dogs. The secretions, excreta, streams and milk of sick animals contain a large number of germs, which spread among animals, causing infection or disease. Humans can cause brucellosis infection through contact with animals infected with brucellosis, c...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06C12R1/01
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2561/101C12Q2545/101C12Q2545/113
Inventor 叶锋刘明坤余荣
Owner 北京旌准医疗科技有限公司
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