CN106244580B - A kind of extracting method of the macro genome of bronchoalveolar lavage fluid - Google Patents
A kind of extracting method of the macro genome of bronchoalveolar lavage fluid Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
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Abstract
The present invention provide a kind of safe and simple, pollution less, the extracting method of reproducible bronchoalveolar lavage fluid macro genome DNA.The present invention including the following steps: sample liquefaction and smudge cells, free and foreign protein, salt ion the removal of target DNA, DNA absorption, DNA elution.The yield and purity of genomic DNA can be improved in the present invention, can preferably meet the various molecular biology experiments such as PCR, DNA library building, gene sequencing.
Description
Technical field
The present invention provides a kind of extracting method of macro genome of bronchoalveolar lavage fluid, belongs to field of biotechnology.
Background technique
Lower respiratory tract infection is clinical relatively conventional infection, and Species of Pathogens is more, drug resistance is strong.Etiological diagnosis is general at present
All over Sputum culturing is used, but because its is easy to pollute, the quality of liquid phlegm is difficult to ensure, seriously affects the accuracy of diagnosis.Bronchoalveolar lavage fluid
Compared with liquid phlegm, because of its diseased region that directly has drawn from, the chance for obtaining pathogen is more, and pars oralis pharyngis field planting can be greatly reduced
Pollution of the micropopulation to sample improves the separation rate of pathogen, therefore has higher spy to lower respiratory tract infection diagnosis
Anisotropic and sensibility.Accurate cause of disease can not only be obtained by carrying out etiological diagnosis using bronchoalveolar lavage fluid, while for its progress
The test of bacterial drug sensitizing effect is more valuable to clinical anti-infective therapy.
Macro genome contains all hereditary information of microbiologic population, and metagenomics handle various microorganisms in group
Classification information outside, also contain the gene information of all microorganisms；Therefore this data are conducive to our functions to group
It is analysed in depth.The fast development of two generation sequencing technologies, the genome sequencing of environmental microorganism are possibly realized in recent years,
After the data for obtaining magnanimity, biological community structure and gene function composition etc. can be analyzed comprehensively.For example, many in recent years
It learns and technique of metagenome is applied to study type II diabetes human body intestinal canal flora diversity, analyze the micro- life of human body intestinal canal
Relationship between object and type II diabetes conducts in-depth research relationship between the two.
Contacting between disease and pathogen can deeply be parsed by carrying out the research of affected part Tiny ecosystem using metagenomics means.
The key for carrying out the research of Pulmonary bacterial metagenomics is that bacteria total DNA is extracted.The cell as contained in bronchoalveolar lavage fluid
Measure less, and mainly human body cell, the content of bacterium are very low；Therefore how efficiently to be extracted from bronchoalveolar lavage fluid thin
Bacterium genomic DNA is with regard to particularly important.The extracting method of sample total DNA is broadly divided into direct extraction method and indirect DNA extraction method: directly
Extraction method is extracted after the microbial cell in first Direct Pyrolysis sample discharges its DNA；Indirect DNA extraction method then will first divide
From the microorganism in sample, after removing impurity, DNA is extracted.The present invention uses direct extraction method.Then pass through design of primers spy
Specific amplification bacterial gene fragment can exclude the interference of human genome.
Summary of the invention
The purpose of the present invention is to provide a kind of safe and simple, the macro genome of bronchoalveolar lavage fluid of pollution less, reproducible
The extracting method of DNA.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of extracting method of the macro genome of bronchoalveolar lavage fluid, including sample liquefaction and clasmatosis, target DNA dissociate
And the removal of foreign protein, salt ion, DNA absorption, DNA elution and etc..The extraction of the macro genome of bronchoalveolar lavage fluid
Steps are as follows:
(1) sample liquefaction and clasmatosis: after taking fresh irrigating solution sample, it is added 1 mol/L's of 10 times of volumes
NaOH solution stands 10 min at room temperature；After liquefaction completely, 14000 r/min are centrifuged 5 min, abandon supernatant；Going for 1ml is added
Precipitating is resuspended in ionized water, and draw solution is into 2 new ml centrifuge tubes；12000 r/min are centrifuged 5 min, stay precipitating stand-by；
The 150 μ l of lysis buffer for being incubated for 10min in 56 DEG C of water-baths in advance is added in centrifuge tube and concentration is 10 mg/ml
And the 20 μ l of lysozyme of the DTT containing 3 mmol/L is blown and beaten with pipette tips and is mixed；The Proteinase K Solution that concentration is 20mg/kg is added
40 μ l are vortexed after mixing 30 sec, 56 DEG C of 10 min of incubation, and it is uniform during which to take out primary concussion every 5 min；
(2) free and foreign protein, salt ion the removal of target DNA: the CTAB that 800 μ l of addition are preheated to 65 DEG C is mixed
Close buffer；70 DEG C of 10 min of incubation, then 12000 rpm are centrifuged 5 min, abandon supernatant, stay precipitating；It is added the 4 of 300 μ l
DEG C dehydrated alcohol of pre-cooling and the concentration of 1/2 volume are 3 mol/L, and the NaAc of pH=5.2, which is vortexed, mixes 30 sec；14000 rpm
It is centrifuged 10min under room temperature, abandons supernatant, stays precipitating；300 μ l suspension DNA of TE buffer is added；
(3) DNA is adsorbed: being taken the DNA of suspension in DNA adsorption column, is separately added into 600 μ l of the ethyl alcohol drift of 70% pH=5.5
It washes twice；Adsorption column is placed at 56 DEG C and is incubated for 5 min；
(4) DNA is eluted: be added into DNA adsorption column ingredient for the Tris-Cl of 10 mmol/L, 1 mmol/L EDTA,
The 80 μ l of eluent liquid of pH=8.0；3 min are incubated at room temperature, 14000 rpm are centrifuged 5 min, elute to obtain DNA solution.
The present invention has the advantages that
The diseased region first, present invention directly has drawn from, the chance for obtaining pathogen are more.The present invention is with bronchoalveolar lavage fluid
Sample is different from tradition using patient's sputum as the method for sample, and sample can cover microbial flora and the acquisition of lung comprehensively
The macro genomic information of pathogen；Bacterial gene fragment is expanded by late design primer specificity, and human genome can be excluded
Interference.Therefore the accuracy of diagnosis is improved.
Second, the present invention has carried out liquefaction processing to sample, sample viscosity is reduced, improves DNA elution efficiency.It compares
The conventional method directly handled after taking sample, the present invention is to the liquefaction scheme for sample design specificity.Because this
Sample is derived from the lung of patient, it is difficult to the sputum etc. containing patient in solution when avoiding drawing materials, and sputum is difficult in subsequent processing
To completely remove；This with DNA adsorption column eluted dna is brought trouble because waste liquid in this way can block adsorption column, make DNA without
Method elutes, and is trapped on pillar, reduces the DNA output extracted.The present invention liquefies sample, reduces the viscous of sample
Property, substantially increase elution efficiency.
Third, the invention enables bacteria lysis in sample is more abundant.Because of microbial flora type in bronchoalveolar lavage fluid
Diversity, some bacteriums are insensitive to lysozyme, it is difficult to broken wall；The present invention adds in lysozyme during lytic cell
DTT increases bacterium to the sensibility of lysozyme, it is made to be more easier to crack.
In addition, higher using DNA purity obtained by the present invention.CTAB cocktail buffer (0.1% used in this experiment
Beta -mercaptoethanol (v/v), the NaCl of 1.5 mol/L, 5% PVP-40 (v/v), 3% CTAB(w/v)), can be good at
Except humus and other impurities in bronchoalveolar lavage fluid, the purity of subsequent DNA is improved.
Detailed description of the invention
Fig. 1 is the area the V3-V4 PCR amplification electropherogram of the 16S rDNA of different irrigating solution sample genomes.Wherein M swimming lane is
DNA Marker；G1-G7 is respectively the PCR product of different irrigating solution sample genomic templates.
Specific embodiment
Embodiment
A kind of extracting method of the macro genome of bronchoalveolar lavage fluid, clinically takes chronic obstructive pulmonary disease patient's branch gas first
Then pipe bronchoalveolar lavage fluid extracts the macro genome of microorganism in its bronchoalveolar lavage fluid.Specific step includes:
(1) sample liquefaction and clasmatosis: after taking fresh irrigating solution sample, it is added 1 mol/L's of 10 times of volumes
NaOH solution stands 10 min at room temperature；After liquefaction completely, 14000 r/min are centrifuged 5 min, abandon supernatant, stay precipitating；It is added
Precipitating is resuspended in the deionized water of 1 ml, and draw solution is into 2 new ml centrifuge tubes；12000-r/min is centrifuged 5min, and it is heavy to stay
It forms sediment stand-by；The 150 μ l of lysis buffer and concentration for being incubated for 10 min in 56 DEG C of water-baths in advance are added in centrifuge tube
The 20 μ l pipette tips of lysozyme of DTT for 10 mg/ml and containing 3 mmol/L, which are blown and beaten, to be resuspended；It is 20 mg/kg's that concentration, which is added,
40 μ l of Proteinase K Solution is vortexed after mixing 30 sec, 56 DEG C of 10 min of incubation, during which takes out primary concussion every 5 min
Uniformly；
(2) the CTAB mixing that 800 μ l are preheated to 65 DEG C free and foreign protein, salt ion the removal of target DNA: is added
Buffer；70 DEG C of 10 min of incubation, then 12000 rpm are centrifuged 5 min, abandon supernatant, stay precipitating；4 DEG C that 300 μ l are added are pre-
The concentration of cold dehydrated alcohol and 1/2 volume is 3 mol/L, and the NaAc of pH=5.2, which is vortexed, mixes 30 sec；14000 rpm room temperature
10 min of lower centrifugation abandon supernatant, stay precipitating；300 μ l suspension DNA of TE buffer is added；
(3) DNA is adsorbed: being taken the DNA of suspension in DNA adsorption column, is separately added into 600 μ l of the ethyl alcohol drift of 70% pH=5.5
It washes twice；Adsorption column is placed at 56 DEG C and is incubated for 5 min；
(4) DNA is eluted: be added into DNA adsorption column ingredient for the Tris-Cl of 10 mmol/L, 1 mmol/L EDTA,
The 80 μ l of eluent of pH=8.0；3 min are incubated at room temperature, 14000 rpm are centrifuged 5 min, elute to obtain DNA solution.
(5) by the DNA solution of acquirement, the measurement of DNA content is carried out using Nanodrop 2000.
Using the present invention as test group, common extracting method is control group, and two methods extract the macro genome of bronchoalveolar lavage fluid
Effect difference it is as shown in table 1.Then the PCR reaction amplification area 16S rDNA V3-V4, row agarose gel electrophoresis of going forward side by side are carried out
Verifying, excludes the interference of human genome, determines the quality of microorganism total DNA.Wherein 16S rDNA V3-V4 primer are as follows:
319F:5'-ACTCCTACGGGAGGCAGCAG-3'
806R:5'-GGACTACHVGGGTWTCTAAT-3'
The Contrast on effect of 1 two methods of the table extraction macro genome of bronchoalveolar lavage fluid microorganism
It is dense to be higher than conventional method extraction gained genome according to the DNA concentration that the method for the present invention is extracted as can be seen from Table 1
Degree meets the concentration requirement of the molecular biology manipulations such as sequencing, PCR reaction；Secondly, the ratio of A260/280 is respectively positioned on 1.8-
In 2.0 ranges, illustrate that the present invention extracts gained DNA purity is high.Therefore illustrate that the present invention improves sample gene group recovery rate, mention
Take the macro genome effect of bronchoalveolar lavage fluid very ideal.
PCR amplification, gained 16S rRNA V3-V4 fragment PCR products agarose are carried out by template of above-mentioned two groups of genomes
Gel electrophoresis verification result is shown in Fig. 1.Fig. 1 display test group pcr amplification product band is single, length meets desired value and band ratio
Control group is more bright, illustrates that PCR process specificity is high, expanding effect is good, meets subsequent molecular use.Test
The PCR effect of group is also better than control group.The result further demonstrates that invention achieves ideal technical effects.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
SEQUENCE LISTING
<110>Xiamen Ji Yuan medical science and technology Co., Ltd
<120>extracting method of the macro genome of a kind of bronchoalveolar lavage fluid
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
actcctacgg gaggcagcag 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
ggactachvg ggtwtctaat 20
Claims (1)
1. a kind of extracting method of the macro genome of bronchoalveolar lavage fluid, it is characterised in that: the extracting method includes the following steps: sample
This liquefaction and clasmatosis, free and foreign protein, salt ion the removal of target DNA, DNA absorption, DNA elution；Specific step
It is rapid as follows:
(1) sample liquefaction and clasmatosis: after taking fresh irrigating solution sample, the NaOH that the 1mol/L of 10 times of volumes is added is molten
Liquid stands 10 min at room temperature；After liquefaction completely, 14000 r/min are centrifuged 5 min, abandon supernatant；The deionization of 1 ml is added
Precipitating is resuspended in water, and draw solution is into 2 new ml centrifuge tubes；12000 r/min are centrifuged 5 min, stay precipitating stand-by；From
In heart pipe be added in advance be incubated in 56 DEG C of water-baths 10 min 150 μ l of lysis buffer and concentration be 10 mg/ml simultaneously
The 20 μ l of lysozyme of DTT containing 3 mmol/L is blown and beaten with pipette tips and is mixed；The Proteinase K Solution that concentration is 20 mg/kg is added
40 μ l are vortexed after mixing 30 sec, 56 DEG C of 10 min of incubation, and it is uniform during which to take out primary concussion every 5 min；
(2) free and foreign protein, salt ion the removal of target DNA: the CTAB mixing that 800 μ l of addition are preheated to 65 DEG C is slow
Fliud flushing；70 DEG C of 10 min of incubation, then 12000 rpm are centrifuged 5min, abandon supernatant, stay precipitating；4 DEG C of pre-coolings of 300 μ l are added
Dehydrated alcohol and the concentration of 1/2 volume be 3 mol/L, the NaAc of pH=5.2, which is vortexed, mixes 30 sec；Under 14000 rpm room temperature
10 min are centrifuged, supernatant is abandoned, stays precipitating；300 μ l suspension DNA of TE buffer is added；
(3) DNA is adsorbed: being taken the DNA of suspension in DNA adsorption column, is separately added into 600 μ l of the ethyl alcohol rinsing two of 70% pH=5.5
It is secondary；Adsorption column is placed at 56 DEG C and is incubated for 5 min；
(4) DNA is eluted: be added into DNA adsorption column ingredient for the Tris-Cl of 10 mmol/L, 1 mmol/L EDTA, pH=
8.0 80 μ l of eluent；3 min are incubated at room temperature, 14000 rpm are centrifuged 5 min, elute to obtain DNA solution；
The lysis buffer ingredient are as follows: Tris, 5 mol/L of the sucrose of 8% w/v, the EDTA of 50mmol/L, 10 mmol/L
NaCl, pH=8.0；
The ingredient of the CTAB cocktail buffer is the beta -mercaptoethanol of 0.1 % vol, the NaCl of 1.5 mol/L, 5 % vol
The CTAB of PVP-40,3 %w/v.
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| CN109487345B (en) * | 2018-12-13 | 2020-03-10 | 北京先声医学检验实验室有限公司 | Nanopore sequencing platform-based metagenome sample library building method, identification method and kit |
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| CN114395615A (en) * | 2022-02-23 | 2022-04-26 | 辽宁康惠生物科技有限公司 | Extraction and library construction method of alveolar lavage fluid sample microorganism metagenome |
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| CN104560951A (en) * | 2014-12-03 | 2015-04-29 | 复旦大学泰州健康科学研究院 | Extraction method of metagenome DNA and kit for extraction method |
| CN105176969A (en) * | 2015-07-13 | 2015-12-23 | 杭州优思达生物技术有限公司 | Nucleic acid extraction method and nucleic acid extraction reagent for biological samples |
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