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Kit for simultaneously extracting plasma and blood cell pathogenic bacterium DNA and application

A technology for blood cells and pathogenic bacteria, which is applied in the field of kits for simultaneously extracting plasma and blood cell pathogenic bacteria DNA, can solve the problem of parasitic bacteria that ignore blood cells, and achieve the effect of simple, fast and high-purity detection process.

Inactive Publication Date: 2021-11-12
微岩医学科技(北京)有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the deficiency of the above-mentioned background technology, the present invention provides a kit and application for simultaneously extracting plasma and blood cell pathogen DNA, which solves the problem of only detecting plasma free nucleic acid and ignoring blood cell parasites, thereby enabling rapid and comprehensive detection of blood cells All pathogens, providing accurate pathogen detection results for clinic

Method used

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  • Kit for simultaneously extracting plasma and blood cell pathogenic bacterium DNA and application
  • Kit for simultaneously extracting plasma and blood cell pathogenic bacterium DNA and application
  • Kit for simultaneously extracting plasma and blood cell pathogenic bacterium DNA and application

Examples

Experimental program
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Effect test

Embodiment 1

[0028] A method of plasma and blood cells while extracting embodiment pathogen DNA

[0029] (1) The separation of plasma and blood cells: blood samples 2-3 ml, was added anticoagulant; centrifuged to separate plasma and blood cells; plasma in the upper layer was aspirated tube 1.5mlEP standby;

[0030] (2) the extracted intracellular pathogen DNA: Add red blood cell lysis solution in the blood cell pellet in the lower layer, wherein the blood cells and red blood cell lysis in a mass ratio of 1: 4, after mixing, at a 3000rpm speed, centrifugal 5min, to erythrocytes complete lysis , a white precipitate was taken; white precipitate was added after the leukocyte lysate and RNase A 20μL 200μL and Proteinase K 20μL, mixing, 10min in a water bath at 37 ℃, then centrifuged, the supernatant was removed; was added to the precipitate bacterial cell lysis after the solution and Proteinase K 10μL 100μL mixing, at 56 ℃ water bath for 10min, and then at a speed of 12000rpm, centrifuged 10min, th...

Embodiment 2

[0032] Example 2 The method for simultaneously extracting plasma and blood cells of the pathogen DNA

[0033] (1) the plasma and blood cell separation: Select a blood sample 2-3 ml, was added anticoagulant; centrifuged to separate plasma and blood cells; plasma in the upper layer was aspirated tube 1.5mlEP standby;

[0034] DNA (2) Extraction with intracellular pathogens: blood cell pellet lower first added to erythrocyte lysis buffer, wherein the blood cells and red blood cell lysis in a mass ratio of 1: 3, after mixing, at a 3000rpm speed, centrifugal 5min, to red blood cells completely cleavage, taking white precipitate; added leukocyte lysate and RNase A 20μL 200μL Proteinase K20μL white precipitate and, after mixing, at 30 deg.] C water bath for 30min, then centrifuged, the supernatant was removed; bacterial cell lysis was added to the precipitate after the solution and Proteinase K 10μL 100μL mixed, at 50 deg.] C water bath for 30min, and then at a speed of 12000rpm, centrif...

Embodiment 3

[0036] The method of Example 3 for simultaneously extracting plasma and blood cells of the pathogen DNA

[0037] (1) the plasma and blood cell separation: Select a blood sample 2-3 ml, was added anticoagulant; centrifuged to separate plasma and blood cells; plasma in the upper layer was aspirated tube 1.5mlEP standby;

[0038] DNA (2) Extraction with intracellular pathogens: blood cell pellet lower first added to erythrocyte lysis buffer, wherein the blood cells and red blood cell lysis in a mass ratio of 1: 5, after mixing, at a 3000rpm speed, centrifugal 5min, to red blood cells completely cleavage, taking white precipitate; added leukocyte lysate and RNase A 20μL 200μL Proteinase K20μL white precipitate and, after mixing, in a water bath at 40 ℃ 20min, then centrifuged, the supernatant was removed; bacterial cell lysis was added to the precipitate after the solution and Proteinase K 10μL 100μL mixed, at at 60 ℃ 20min, and then at a speed of 12000rpm, centrifuged 10min, the supe...

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Abstract

The invention relates to the technical field of molecular biology, and particularly relates to a kit for simultaneously extracting plasma and blood cell pathogenic bacterium DNA and application. The kit comprises an anticoagulant, red blood cell lysis buffer, white blood cell lysis buffer, RNase A, Proteinase K, bacterial cell lysis buffer and a protein precipitator. Compared with the prior art, the kit has the advantages that plasma and blood cells in a blood sample are separated, DNA of pathogenic bacteria in the blood cells is extracted firstly, then the DNA is combined with the plasma, purification is carried out to obtain all the pathogenic bacterium DNA in the blood sample, and a comprehensive and accurate pathogen detection result is provided for clinic. The kit is suitable for blood of multiple species and whole blood pathogenic bacterium DNA including plasma free DNA and intracellular bacterium DNA; the obtained DNA is high in purity and can be directly used for PCR, enzyme digestion and other experiments, the detection process is simple and rapid, and the DNA of all bacteria in the blood can be obtained within one hour.

Description

Technical field [0001] The present invention relates to molecular biology, and in particular relates to a kit for simultaneously extraction and application of plasma and blood cells of the pathogen DNA. Background technique [0002] With the continual emergence of new pathogens abuse and antibiotics, drug-resistant pathogenic increasing common infectious pathogen spectrum changes, resulting in growing new infectious diseases and vulnerable populations. Traditional diagnostic techniques microorganisms comprising: growing the culture and isolate microorganisms, pathogen-specific antibodies (serology) or microbial detection and identification of nucleic acid molecules (DNA or RNA) antigen, is usually carried out by PCR. Microbial infections disease caused by deterioration very quickly, so in clinical diagnosis, how to quickly and accurately detect pathogenic microorganisms timely and accurately provide results for the clinical detection of pathogens, become a trend at this stage det...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 盖伟宋翠丹魏雪郑亚风马桂红
Owner 微岩医学科技(北京)有限公司
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