CN103060309A - Extraction method for metagenome - Google Patents
Extraction method for metagenome Download PDFInfo
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Abstract
The present invention discloses an extraction method for metagenome, and a method of probe hybridization-bead capture is used to reduce the host DNA content in a host metagenomic DNA sample. According to the method, the Alu repeat sequence of the host DNA sequences and conserved sequence at both ends thereof are used as templates to design one-way or two-way probes, wherein the 5 'end of each probe is modified with biotin, streptavidin coated beads are used to capture host DNA hybridized with probes, in order to achieve aims of weakening host DNA background in a metagenome, and enhancing sequencing data efficiency.
Description
Technical field
The present invention relates to extract grand genomic method, relate to particularly the method that reduces host DNA in host's macro genome DNA sample.
Background technology
Grand genome is all genetic material that appear in a certain environmental sample, comprises the genome of many individual species.Metagenomics refers to and directly from environmental sample, obtains genetic material, the science that grand genome is studied.Along with the development of high-throughput DNA sequencing and information biology, the using value of metagenomics constantly promotes.By analyzing grand genomic library, can find population characteristic and the interaction rule of microorganism in certain environment, inquire into influencing each other of microorganism and environment.To the order-checking of microbial population DNA, can screen the gene with specific function, obtain the albumen of specific function.
Human respiratory touches the microorganisms such as a large amount of virus and bacterium every day, and to respiratory tract, grand genomic research can be understood the feature of each microbial population effectively, thereby helps clinical diagnosis and treatment.In the grand genome of respiratory tract, with respect to host people's genome content, pathogen gene group content is generally smaller.Therefore, in the grand gene order-checking of respiratory tract, for reaching the sequential covering degree that the pathogen gene group is enough, the impact of eliminating host genome is very important.
In the human respiratory sample, the quality of total DNA is the key of grand gene order-checking success or not.To the transmissible disease sample, process the method that DNA pollutes and need to spend many time, or need special treatment process while collecting sample.During general extraction DNA, need to utilize the lysing cell such as washing agent SDS or N,O-Diacetylmuramidase, Proteinase K, then utilize phenol/chloroform extracting, last ethanol precipitation DNA.When extracting macro genome DNA, due to the diversity of cell type and the interference that has host DNA, so need the extraction of more effective purification process for DNA.
People's Alu repeated sequence belongs to short sequence (short interspersed elements, the SINEs) family that is dispersed in.The about 280bp of the length of each Alu sequence, have variable poly A(poly-A) tail.Alu repeated sequence surpasses 1,000,000, accounts for 10% of human genome, has been used to the analysis of some genetic diseases of the mankind.Alu repeated sequence is different, can be divided into several subfamilies, is fabulous mark, can be used to detect human genome.Alu, as special probe, is used round pcr, quantitatively human gene group DNA.In human genome, many new Alu sequences are found, and most of sequence do not exist in other primates genomes, therefore according to the analysis of special Alu repeated sequence, can be used as human DNA and identify and quantitative favorable method.
The magnetic bead that Streptavidin is coated, be widely used at present biotinylation nucleic acid, antibody or other biological elementization part and target molecule and separated and process.The superparamagnetic pearl that diameter is 2.8 μ m, have the covalently bound individual layer restructuring Streptavidin also sealed with BSA in addition to surface.Due to the Streptavidin with individual layer, most vitamin H binding sites spatially not only can be in conjunction with free biotin, but also can be in conjunction with biotinylated part.They demonstrate quick liquid phase reaction dynamics, and the definite specific surfaces of shape is convenient to carry out efficient capture, separation and downstream process.
Real-time fluorescence quantitative PCR (Real-time polymerase chain reaction, be called for short Real-time PCR, be also referred to as quantitative Real-time polymerase chain reaction, be called for short Q-PCR), be based on polymerase chain reaction (polymerase chain reaction, abbreviation PCR) Protocols in Molecular Biology, carry out quantitative analysis to target dna molecule when this technology can be increased to target fragment.Real-time fluorescence quantitative PCR is used special probe sequence or primer, increase along with DNA profiling amount in amplification procedure, the fluorescence intensity discharged is also increasing, so real-time fluorescence quantitative PCR can utilize less sample, the content of detection of dynamic initial target DNA.
Summary of the invention
The object of the invention is to provide a kind of extraction mankind grand genomic method, and the method is utilized the probe hybridization method of residues, can effectively reduce host DNA background interference in the grand genome of host.
First aspect present invention, provide a kind of grand Extraction Methods of Genome, it is characterized in that utilizing probe hybridization magnetic capture mode to reduce the impact of host DNA in host's macro genome DNA sample.
According to the embodiment of the present invention, after described probe and the hybridization of described host genome DNA sample with described magnetic bead coupling.
According to the embodiment of the present invention, provide a kind of grand Extraction Methods of Genome, comprised step:
1) will be marked with the described probe of vitamin H and described host's macro genome DNA sample of extraction is hybridized;
2) with being coated with the described magnetic capture probe of Streptavidin and the complex body of described host DNA, and separate magnetic bead-probe-target dna mixture with magnetic frame;
3) hybridization solution and the magnetic bead scavenging solution after hybridization that will remove described magnetic bead-probe-target dna mixture carry out purifying recovery DNA.
According to the embodiment of the present invention, after described probe and described magnetic bead coupling with the hybridization of described host genome.
According to another implementation of the invention, provide a kind of grand Extraction Methods of Genome, comprised step:
1) utilize the described probe coupling of the described magnetic bead and the mark vitamin H that are coated with Streptavidin;
2) by coupling, good mixture and described host's macro genome DNA sample hybridized;
3) remove magnetic bead-probe-target dna mixture, hybridization solution and hybridization scavenging solution are carried out to purifying recovery DNA.
According to the embodiment of the present invention, in described grand Extraction Methods of Genome, use multiple forward and reverse probe at the coupling temperature.Use multiple forward and reverse probe, can enlarge the scope that probe is combined when host DNA is hybridized, the Streptavidin site on simultaneously can abundant saturated magnetic bead, improve the utilising efficiency of magnetic bead.
According to the embodiment of the present invention, in described grand Extraction Methods of Genome, after hybridization completes, described magnetic bead-probe-DNA mixture is heated or the sodium hydroxide method sex change, reclaimed the magnetic bead that coupling has probe.Reclaim the magnetic bead that coupling has probe, can reduce experimental cost.
According to the embodiment of the present invention, for human genome Alu repeated sequence family designing probe, by these probe hybridizations, subdue, reach the purpose that reduces human DNA ratio in sample.Its middle probe with multiple Alu repeated sequence family, take and the conserved sequence at two ends as template, the bi-directional design probe, probe length is between 40 ~ 70nt, and 5 ' terminal modifiedly have a vitamin H.The optional people's Alu sequence from sequence table SEQ ID NO:1 ~ 139 of probe sequence.Utilize the special Alu sequence of people, eliminate the impact of host's human genome in the respiratory tract sample, the grand gene order-checking of high-throughput is had to the help of highly significant.
Fourth aspect present invention provides the respiratory tract sample treatment, for different sampling methods, sample physical properties, adopts the methods such as dilution liquefaction, centrifugal collection, the cell in the maximum efficiency enriched sample.
The method is not only applicable to study for the human respiratory metagenomics, also be applicable to other positions of human body for example oral cavity, digestive tube, skin, the research of reproductive tract metagenomics, can effectively reduce the high background interference of people DNA, thereby effective enriched microorganism DNA, reduce the sequencing data waste that the host DNA background causes, improve the quantum of output of valid data, microorganism information in the grand genome of more deep excavation.
The accompanying drawing explanation
Fig. 1 is segmental bronchus and bronchoalveolar lavage fluid and the total DNA gel electrophorogram of airway secretions.
Fig. 2 is the qPCR typical curve that plasmid standard is drawn.
Fig. 3 is that biased sample stoste in embodiment 1, hybridization solution reclaim plasmid qPCR absolute quantitation amplification curve in liquid and magnetic bead scavenging solution.3 groups of set of curves are distinguished representative simulation hybrid dna former state from left to right, hybridization solution BW, and scavenging solution W, horizontal line means threshold line.
Embodiment
experimental example bronchoalveolar lavage fluid (BAL), airway secretions and whole blood total DNA extraction
One, bronchoalveolar lavage
Bronchoalveolar lavage fluid cell is selected to middle lobe of right lung (B4 or B5) or left lung tongue section, and limitation tuberculosis becomes and in homologous bronchi lung section, carries out lavation.At first inject 2% lignocaine 1 ~ 2ml through the biopsy hole by thin silicone tube in the lung section of wanting lavation, do the toponarcosis of lavation lung section; Then by Bronchofiberscope the top closely section of wedging or inferior segmental bronchus opening part, then through the biopsy hole, by silicone tube, inject fast 37 ℃ of sterile salines.Each 25 ~ 50ml, total amount 100 ~ 250ml, generally be no more than 300ml; Use immediately 50 ~ 100mmHg vacuum suction to reclaim irrigating solution, the rate of recovery is 40% ~ 60% usually; Recovering liquid is removed to mucus by the double-layer sterile filtered through gauze immediately, and record total amount; In the silicon plastic bottle of packing into or painting silicon sterilizing Glass Containers (minimizing cell adhesion), be placed in the vacuum flask that contains ice cube.
Two, airway secretions attracts
Select suitable sputum aspirator tube, the external diameter of sputum aspirator tube should not surpass tracheal catheter internal diameter 1/2; Check that whether sputum aspirator is intact, attract should be no more than-50mmHg of negative pressure; Carry out the swollen lung of pure oxygen, air flue lavation after cleaning oral cavity.Blocking-up sputum aspirator tube negative pressure, insert tracheal catheter by sputum aspirator tube and do, exhaustion pars oralis pharyngis secretory product after suction sputum., put forward the open negative pressure of 0.5cm on while reaching the tracheal catheter end, in rotation, carry; The suction sputum action is soft, rapid, and each suction sputum time is no more than 15 seconds; Strict aseptic technique.
Three, instrument and reagent are prepared
ABI 7500 real-time fluorescence quantitative PCR instrument (Applied Biosystems, Life Technologies company).
Qubit
2.0 fluorescent quantitation instrument (Invitrogen, Life Technologies company).
Proteinase K: 100 mg Proteinase Ks are dissolved in to 5 mL distilled waters, are mixed with the solution of 20 mg/ml ,-20 ℃ of preservations.
1L PBS damping fluid (pH7.4):
Potassium primary phosphate (KH
2pO
4) (purchased from Beijing Chemical Plant), 0.27g
Sodium phosphate dibasic (Na
2hPO
4) (purchased from western Gansu Province chemical industry), 1.42g
Sodium-chlor (NaCl) (purchased from western Gansu Province chemical industry), 8g
Repone K (KCl) (purchased from Beijing Chemical Plant), 0.2g
Add the approximately abundant stirring and dissolving of 800 mL of deionized water, then add concentrated hydrochloric acid to adjust pH to 7.4, last constant volume is to 1L, room temperature preservation after autoclave sterilization.
DNA extraction test kit: QIAamp DNA Mini Kit(QIAGEN, article No. 51306).
Four, extraction step
1, get irrigating solution and collect 1 ~ 5ml, 13000rpm centrifugal concentrating 10 minutes, abandon supernatant, adds equal-volume PBS dilution, and assist in dissolving is disperseed, and for the airway secretions of thickness, need to first with 2 ~ 3 times of volume PBS, be diluted, and softly mixes and carries out homogeneity;
2, prepare some 1.5ml centrifuge tubes, every pipe adds 50 μ l Proteinase Ks in pipe;
3, every pipe adds the respiratory secretions sample after 500 μ l dilutions disperse, and less than 500 μ l supply with PBS;
4, every pipe adds 500 μ l buffer AL(QIAamp DNA Mini Kit), softly mix 15 seconds and be placed in 56 ℃ of water-baths, hatch 10 minutes;
5, after taking out, centrifugal fast, the drop that tube wall and pipe are covered is collected in return pipe, add 500 μ l dehydrated alcohols, vortex mixes 15 seconds, fast centrifugal after, will manage interior entire mixture and be transferred in centrifugal column and (can carry out at twice), centrifugal 1 minute of 8000rpm, shift centrifugal column to new centrifuge tube;
6, add 500 μ l buffer AW1, centrifugal 1 minute of 8000rpm, discard liquid in pipe;
7, add 500 μ l buffer AW2, centrifugal 3 minutes of 14000rpm, after discarding liquid in pipe, sky gets rid of 1 minute;
8, shift centrifugal column to the new centrifuge tube of 1.5ml, add 30 μ l distilled waters, room temperature was placed after 2 minutes, centrifugal one minute of 8000rpm.
The total DNA extracted, through the agarose gel electrophoresis analysis, the results are shown in Figure 1, M:DL2000, and band is descending is 2000bp, 100bp, 750bp, 500bp, 250bp, 100bp.Swimming lane 1 and swimming lane 2 extract for airway secretions the DNA obtained, and concentration is respectively 72ng/ μ l, 15ng/ μ l; Swimming lane 3 and swimming lane 4 extract for bronchoalveolar lavage fluid the DNA obtained, and concentration is 21ng/ μ l.As can be seen from Figure 1: the genomic dna band is complete.
Fig. 2 is the qPCR typical curve that plasmid standard is drawn.5 times of gradient dilutions of standard substance employing BAP plasmid (2.6 kbp) become the sample of 5 concentration gradients, by standard substance concentration and Ct value drawing standard curve, for quantitative analysis sample plasmid content.In figure, X-coordinate Log CO means template concentrations, and ordinate zou Ct means the Ct value of sample, and each square dot means standard model.Carry out thus in simulated experiment before and after hybridization plasmid in sample quantitatively.
Irrigating solution and whole blood carry out total DNA extraction with reference to above-mentioned steps.
embodiment hybridization is subdued and is reduced host DNA background in macro genome DNA
The present invention utilizes Alu sequence high characteristics of the frequency of occurrences in people DNA, for Alu family sequence and two ends conservative region designing probe, 5 ' end biotin labeling, and not homotactic multiple probe is used in conjunction with, by the coated magnetic bead of avidin together, to hybridize compound DNA and catch out, thereby reduce the background interference of host DNA in grand genome.
Required reagent and mother liquor preparation:
20 * SSPE: take 210.6g NaCl(purchased from western Gansu Province chemical industry), 27.6g NaH
2pO
4 .h
2o(is purchased from Beijing Chemical Plant), 5.845g EDTA(is purchased from western Gansu Province chemical industry), deionized water dissolving, adjust pH to 7.7, is settled to 1L, filtration sterilization packing, 4 ℃ of preservations.
100 * Denhardt ' s regent solution: take 2g ficoll (Ficoll, 400 types, purchased from Sigma), 2g polyvinylpyrrolidone (PVP-40, purchased from Sigma, P-3004), 2g BSA(is purchased from rope Bora biotechnology), adding water to cumulative volume is 100ml.Filtration sterilization, be distributed into aliquot ,-20 ℃ of storages.
20 * SSC: take 88.2g Trisodium Citrate (purchased from western Gansu Province chemical industry), 175.3g NaCl, add the 900ml water dissolution, uses NaOH(purchased from Beijing Chemical Plant) regulate pH to 7.0, deionized water is settled to 1L, filtration sterilization.10%SDS solution: get 10gSDS(and completely newly open up and reach science and technology purchased from Beijing), deionized water dissolving, be settled to 100ml.
1M Tris-HCl; Taking 72.66gTris-base(completely newly opens up and reaches science and technology purchased from Beijing), add the 500ml deionized water, use HCl(purchased from Beijing Chemical Plant) and regulate pH to 7.5, be settled to 600ml.
50mM EDTA solution: take 1.86gEDTA and be dissolved in the 100ml deionized water.
4M NaCl solution: take 23.376gNaOH and be dissolved in the 100ml deionized water.
Above mother liquor is all used the filtration method degerming.
Hybridization solution: 50ml 2 * B& W buffer: get 0.5ml 1M Tris-HCl (pH 7.5), 1ml 50mM EDTA, 25ml 4M NaCl, add water to 50ml.
50ml 2 * hybridization solution HYB buffer: get 25ml 20 * SSPE solution, 5ml 100 * Denhardt ' s solution, 10ml 50mM EDTA, 1ml 10% SDS, add water to 50ml.
50ml magnetic bead scavenging solution wash buffer: get 2.5ml 20 * SSC, 0.5ml 10%SDS, add water to 50ml
Probe sex change magnetic bead reclaims liquid (0.15M NaOH): take 0.6gNaOH, be dissolved in the 100ml deionized water, packing is preserved.
1 pair of simulation biased sample of embodiment carries out probe hybridization and subdues (first coupling after hybridization)
1, the simulation hybrid dna is got 20 μ l(and is mixed at 9: 1 according to Nanogram Amounts with the 2815bp plasmid by people's whole blood DNA, the about 100ng/ μ of total amount l) in 200 μ l pipes, be placed in 95 ℃ of preheatings of PCR instrument 5 minutes, 65 ℃ 5 minutes; Get 27 μ l 2 * hybridization solution HYB and be placed in 65 ℃ of preheatings of PCR instrument 5 minutes;
2, get each 0.1 μ l of probe that sequence number is SEQ ID NO:1 ~ 70, about 70pmol, 65 ℃ of preheatings 2 minutes;
3, mix above-mentioned system totally 54 μ l, 65 ℃, hybridize 1 hour;
4, get magnetic bead M-280(invitrogen) 30 μ l, with 2 * B& W buffer cleans 2 times, abandons supernatant, with 54 μ l 2 * B& W is resuspended;
5, will hybridize system (54 μ l) and add in magnetic bead, and mix, under room temperature, (20 ℃ ~ 25 ℃) hatch 15 minutes, during flick tube wall, mix;
6, after the magnetic bead cohesive process finishes, the 1.5ml centrifuge tube is placed on magnetic frame, room temperature is placed 1 ~ 2 minute, after treating that magnetic bead is adsorbed onto tube wall, draws supernatant and retains, and is labeled as BW;
7, with 100 μ l wash buffer, magnetic bead is cleaned in piping and druming gently, then is placed on magnetic frame staticly, and the supernatant sucking-off retains and is labeled as W, then repeats 2 times;
8, with 100 μ l wash buffer, magnetic bead is cleaned in piping and druming gently, then is placed on magnetic frame staticly, and the supernatant sucking-off retains and is labeled as W, then repeats 2 times; The hybridization solution BW and the wash buffer that return are cleaned to Qiagen PCR purification kit (QIAquick PCR Purification Kit, QIAGEN, article No. 28106) post recovery DNA for recovery liquid W afterwards, 50 μ L eluted dnas.Magnetic bead can be preserved by following method: the resuspended magnetic bead of 20 μ l 0.15M NaOH solution, under room temperature, hatch 10 minutes, and be placed on magnetic frame and remove supernatant after 1 ~ 2 minute, add 30 μ l 1 * B& W buffer solution, preserve magnetic bead for 4 ℃.Magnetic bead adds the resuspended magnetic bead of 50 μ L 1 * SSC after cleaning with 50 μ L 1 * SSC solution, hatches 5 minutes for 95 ℃, is placed on magnetic frame after standing 1 ~ 2 minute, removes supernatant, adds 30 μ L 1 * B& W solution is preserved.
2 pairs of respiratory tract irrigating solution sample total DNA of embodiment are carried out probe hybridization and are subdued (first coupling after hybridization)
Except in embodiment 1 step 1, select human respiratory irrigating solution sample DNA, get in 2 * hybridization solution HYB, 32 μ l, step 2 and get each about 120pmol of 3 μ l(of probe that sequence number is SEQ ID NO:1 ~ 4), in step 4 with 64 μ l 2 * B& Beyond W is resuspended, first hybridizes rear coupling probe hybridization with method in the same manner as in Example 1 and condition and subdue.
3 pairs of simulation biased samples of embodiment carry out probe hybridization and subdue (hybridizing after first coupling)
1, get magnetic bead M-280(invitrogen) 30 μ l, with 2 * B& W buffer cleans 2 times, abandons supernatant, adds 15 μ l 2 * B& The resuspended magnetic bead of W buffer;
2, add each about 70pmol of 0.1 μ l(of probe that sequence number is SEQ ID NO:71 ~ 139 in resuspended magnetic bead) and 8 μ l ddH
2o, hatch under room temperature 15 minutes, during gently revolve and mix; Be placed in the magnetic frame room temperature and place 1 ~ 2 minute, remove supernatant;
3, the simulation hybrid dna is got 20 μ l(and is mixed at 9: 1 according to Nanogram Amounts with about 2.6kbp plasmid by people's whole blood DNA, the about 100ng/ μ of total amount l) in 200 μ lPCR pipes, be placed in 95 ℃ of preheatings of PCR instrument 5 minutes, 65 ℃ 5 minutes; Get 26 μ l 2 * hybridization solution HYB and be placed in 65 ℃ of preheatings of PCR instrument 5 minutes;
4, hybrid dna and hybridization solution are added in magnetic bead, mix, be placed in 65 ℃, PCR instrument, hybridize 1 hour, during interval mix;
5, hybridization finish after step with reference to the step 6-8 of embodiment 1.
4 pairs of respiratory tract irrigating solution sample total DNA of embodiment are carried out probe hybridization and are subdued (hybridizing after first coupling)
Except adding each 1 μ l(120pmol of probe that 12 kinds of sequence numbers are SEQ ID NO:1 ~ 12 in step 2) and 3 μ l water, step 3 in get human respiratory irrigating solution sample DNA sample 20 μ l, carry out first coupling with method in the same manner as in Example 3 and condition after hybridization probe hybridization subdue.
test example 1 is measured the reduction that the front and back host DNA is subdued in hybridization
Utilize real-time fluorescence quantitative PCR to carry out absolute quantification analysis, measure the content difference that plasmid DNA in the biased sample of front and back is subdued in hybridization, and the proportional difference of people DNA and plasmid DNA.Real-time fluorescence quantitative PCR reagent adopts THUNDERBIRD Probe qPCR Mix(Toyobo), the primer that the sequence number of choosing particular sequence design on plasmid is SEQ ID NO:142 and SEQ ID NO:143 and the probe that sequence number is SEQ ID NO:144,5 ' end flag F AM fluorescence group of probe, 3 ' end mark TAMRA cancellation group.Carry out the Realtime-PCR experiment according to THUNDERBIRD Probe qPCR Mix specification sheets.By standard, the content of hybridizing the front and back plasmid DNA is carried out to absolute quantitation, 3, each sample repeats samples.After total DNA content of Q-bit fluorescent quantitative measurement DNA sample, by total amount, deducting the plasmid DNA amount, is people's DNA content, measures and take ng as unit, the results are shown in Table 1.
Fig. 3 is that biased sample stoste in embodiment 1, hybridization solution reclaim plasmid qPCR absolute quantitation amplification curve in liquid and magnetic bead scavenging solution.3 groups of set of curves are distinguished representative simulation hybrid dna former state, hybridization solution BW, scavenging solution W from left to right.Green horizontal line means threshold line, and the cycle number that threshold line is corresponding with the amplification curve intersection is the Ct value, by the Ct value, can relatively calculate initial DNA profiling amount.The Ct value is less, and original template concentration is larger, in conjunction with the DNA solution volume, can calculate the original template total amount.
Through real-time fluorescence quantitative PCR, the plasmid in hybrid dna stoste and hybridization recovery liquid is carried out to absolute quantification analysis, the plasmid amount that merging BW and W purifying obtain after reclaiming.In embodiment 1, the plasmid organic efficiency is about 91% of plasmid in initial mixing DNA, and after hybridization, people and plasmid DNA amount (ng) ratio are about 1: 1; In embodiment 3, the plasmid organic efficiency is about 83.5% of plasmid in initial mixing DNA, and after hybridization, people and plasmid DNA amount (ng) ratio are about 0.92: 1, can effectively reduce the content of people DNA, the results are shown in Table 1.
Plasmid DNA and people's DNA content and ratio in table 1 quantitative analysis embodiment 1 and 3 before and after hybridization.
test example 2 is measured the DNA of bacteria content
The universal primer that the conservative region implementation sequence of the 16S rDNA of directed toward bacteria and ancient bacterium number is SEQ ID NO:140 and SEQ ID NO:141, purpose is the real-time fluorescence quantitative PCR by dye method, detects the variation of the relative content of microorganism in the total DNA of hybridization front and back respiratory tract irrigating solution.Carry out life science section of Realtime-PCR(TOYOBO company limited, Osaka, Japan, article No. QPS-201 according to THUNDERBIRD SYBR qPCR Mix specification sheets) experiment.Because initial DNA usage quantity is 20 μ l, reclaiming the wash-out water consumption after hybridization is 40 μ l, thus in Real-time PCR quantitative experiment, after hybridization, reclaim DNA consumption volume for hybridization before 2 times of DNA consumption volume, to adjust the difference of the two initial cumulative volume ratio.
Through Real-time PCR quantitative experiment, in embodiment 2 and embodiment 4, the bacteria content after hybridization is subdued improves respectively approximately 3 times and 16 times, in Table 2.Use this method, can reuse coupling has the magnetic bead of probe to hybridize and subdue actual respiratory tract irrigating solution sample, reduces the cost that sample hybridization is processed.Before and after bacteria content hybridization, ratio is in Table 2.
Bacteria content before and after table 2 quantitative analysis embodiment 2 and embodiment 4 sample hybridization
* ddCt=is hybridized the front sample Ct value of rear sample Ct value-hybridization
* content ratio (DNA of bacteria content after hybridization: DNA of bacteria content before hybridization)=2
-ddCt.
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<222> (1)..(69)
<400> 15
gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60
gcagcagca 69
<210> 16
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 16
ctactactac tactactact actactacta ctactactac tactactact actactacta 60
ctactacta 69
<210> 17
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 17
ggaggaggag gaggaggagg aggaggagga ggaggaggag gaggaggagg aggaggagga 60
ggaggagga 69
<210> 18
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 18
gaagaagaag aagaagaaga agaagaagaa gaagaagaag aagaagaaga agaagaagaa 60
gaagaagaa 69
<210> 19
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 19
tcatcatcat catcatcatc atcatcatca tcatcatcat catcatcatc atcatcatca 60
tcatcatca 69
<210> 20
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 20
ccaccaccac caccaccacc accaccacca ccaccaccac caccaccacc accaccacca 60
ccaccacca 69
<210> 21
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 21
tagatagata gatagataga tagatagata gatagataga tagatagata gatagataga 60
tagatagat 69
<210> 22
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 22
caaacaaaca aacaaacaaa caaacaaaca aacaaacaaa caaacaaaca aacaaacaaa 60
caaacaaac 69
<210> 23
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 23
taaataaata aataaataaa taaataaata aataaataaa taaataaata aataaataaa 60
taaataaat 69
<210> 24
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 24
gaaagaaaga aagaaagaaa gaaagaaaga aagaaagaaa gaaagaaaga aagaaagaaa 60
gaaagaaag 69
<210> 25
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 25
tccatccatc catccatcca tccatccatc catccatcca tccatccatc catccatcca 60
tccatccat 69
<210> 26
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 26
cgaacgaacg aacgaacgaa cgaacgaacg aacgaacgaa cgaacgaacg aacgaacgaa 60
cgaacgaac 69
<210> 27
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 27
tgaatgaatg aatgaatgaa tgaatgaatg aatgaatgaa tgaatgaatg aatgaatgaa 60
tgaatgaat 69
<210> 28
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 28
cggacggacg gacggacgga cggacggacg gacggacgga cggacggacg gacggacgga 60
cggacggac 69
<210> 29
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 29
ggaaggaagg aaggaaggaa ggaaggaagg aaggaaggaa ggaaggaagg aaggaaggaa 60
ggaaggaag 69
<210> 30
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 30
tacatacata catacataca tacatacata catacataca tacatacata catacataca 60
tacatacat 69
<210> 31
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 31
tcaatcaatc aatcaatcaa tcaatcaatc aatcaatcaa tcaatcaatc aatcaatcaa 60
tcaatcaat 69
<210> 32
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 32
gacagacaga cagacagaca gacagacaga cagacagaca gacagacaga cagacagaca 60
gacagacag 69
<210> 33
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 33
cctacctacc tacctaccta cctacctacc tacctaccta cctacctacc tacctaccta 60
cctacctac 69
<210> 34
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 34
gcccgcccgc ccgcccgccc gcccgcccgc ccgcccgccc gcccgcccgc ccgcccgccc 60
gcccgcccg 69
<210> 35
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 35
ttaattaatt aattaattaa ttaattaatt aattaattaa ttaattaatt aattaattaa 60
ttaattaat 69
<210> 36
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 36
gggagggagg gagggaggga gggagggagg gagggaggga gggagggagg gagggaggga 60
gggagggag 69
<210> 37
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 37
ggcaggcagg caggcaggca ggcaggcagg caggcaggca ggcaggcagg caggcaggca 60
ggcaggcag 69
<210> 38
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 38
gccagccagc cagccagcca gccagccagc cagccagcca gccagccagc cagccagcca 60
gccagccag 69
<210> 39
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 39
caaaacaaaa caaaacaaaa caaaacaaaa caaaacaaaa caaaacaaaa caaaacaaaa 60
caaaacaaa 69
<210> 40
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 40
tggaatggaa tggaatggaa tggaatggaa tggaatggaa tggaatggaa tggaatggaa 60
tggaatgga 69
<210> 41
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 41
taaaataaaa taaaataaaa taaaataaaa taaaataaaa taaaataaaa taaaataaaa 60
taaaataaa 69
<210> 42
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 42
gcccagccca gcccagccca gcccagccca gcccagccca gcccagccca gcccagccca 60
gcccagccc 69
<210> 43
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 43
gctcagctca gctcagctca gctcagctca gctcagctca gctcagctca gctcagctca 60
gctcagctc 69
<210> 44
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 44
gaaaagaaaa gaaaagaaaa gaaaagaaaa gaaaagaaaa gaaaagaaaa gaaaagaaaa 60
gaaaagaaa 69
<210> 45
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 45
tacaatacaa tacaatacaa tacaatacaa tacaatacaa tacaatacaa tacaatacaa 60
tacaataca 69
<210> 46
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 46
ctccactcca ctccactcca ctccactcca ctccactcca ctccactcca ctccactcca 60
ctccactcc 69
<210> 47
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 47
tctaatctaa tctaatctaa tctaatctaa tctaatctaa tctaatctaa tctaatctaa 60
tctaatcta 69
<210> 48
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 48
tagaatagaa tagaatagaa tagaatagaa tagaatagaa tagaatagaa tagaatagaa 60
tagaataga 69
<210> 49
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 49
ggagaggaga ggagaggaga ggagaggaga ggagaggaga ggagaggaga ggagaggaga 60
ggagaggag 69
<210> 50
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 50
ttaaattaaa ttaaattaaa ttaaattaaa ttaaattaaa ttaaattaaa ttaaattaaa 60
ttaaattaa 69
<210> 51
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 51
gccccgcccc gccccgcccc gccccgcccc gccccgcccc gccccgcccc gccccgcccc 60
gccccgccc 69
<210> 52
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 52
gcgcagcgca gcgcagcgca gcgcagcgca gcgcagcgca gcgcagcgca gcgcagcgca 60
gcgcagcgc 69
<210> 53
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 53
ggggagggga ggggagggga ggggagggga ggggagggga ggggagggga ggggagggga 60
ggggagggg 69
<210> 54
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 54
ggcccggccc ggcccggccc ggcccggccc ggcccggccc ggcccggccc ggcccggccc 60
ggcccggcc 69
<210> 55
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 55
tcccatccca tcccatccca tcccatccca tcccatccca tcccatccca tcccatccca 60
tcccatccc 69
<210> 56
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 56
ccccacccca ccccacccca ccccacccca ccccacccca ccccacccca ccccacccca 60
ccccacccc 69
<210> 57
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 57
ccctaaccct aaccctaacc ctaaccctaa ccctaaccct aaccctaacc ctaaccctaa 60
ccctaaccc 69
<210> 58
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 58
cccgaacccg aacccgaacc cgaacccgaa cccgaacccg aacccgaacc cgaacccgaa 60
cccgaaccc 69
<210> 59
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 59
tatacatata catatacata tacatataca tatacatata catatacata tacatataca 60
tatacatat 69
<210> 60
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 60
tcaccatcac catcaccatc accatcacca tcaccatcac catcaccatc accatcacca 60
tcaccatca 69
<210> 61
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 61
ccccaacccc aaccccaacc ccaaccccaa ccccaacccc aaccccaacc ccaaccccaa 60
ccccaaccc 69
<210> 62
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 62
caaaaacaaa aacaaaaaca aaaacaaaaa caaaaacaaa aacaaaaaca aaaacaaaaa 60
caaaaacaa 69
<210> 63
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 63
ccctcaccct caccctcacc ctcaccctca ccctcaccct caccctcacc ctcaccctca 60
ccctcaccc 69
<210> 64
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 64
gcccccgccc ccgcccccgc ccccgccccc gcccccgccc ccgcccccgc ccccgccccc 60
gcccccgcc 69
<210> 65
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 65
gaaaaagaaa aagaaaaaga aaaagaaaaa gaaaaagaaa aagaaaaaga aaaagaaaaa 60
gaaaaagaa 69
<210> 66
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 66
ggagaaggag aaggagaagg agaaggagaa ggagaaggag aaggagaagg agaaggagaa 60
ggagaagga 69
<210> 67
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 67
cccccacccc cacccccacc cccaccccca cccccacccc cacccccacc cccaccccca 60
cccccaccc 69
<210> 68
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 68
taaaaataaa aataaaaata aaaataaaaa taaaaataaa aataaaaata aaaataaaaa 60
taaaaataa 69
<210> 69
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 69
gggagaggga gagggagagg gagagggaga gggagaggga gagggagagg gagagggaga 60
gggagaggg 69
<210> 70
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 70
gccccagccc cagccccagc cccagcccca gccccagccc cagccccagc cccagcccca 60
gccccagcc 69
<210> 71
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 71
gggggagggg gagggggagg gggaggggga gggggagggg gagggggagg gggaggggga 60
gggggaggg 69
<210> 72
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 72
gagacagaga cagagacaga gacagagaca gagacagaga cagagacaga gacagagaca 60
gagacagag 69
<210> 73
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 73
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60
ttttttttt 69
<210> 74
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 74
gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 60
ggggggggg 69
<210> 75
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 75
tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 60
tgtgtgtgt 69
<210> 76
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 76
tatatatata tatatatata tatatatata tatatatata tatatatata tatatatata 60
tatatatat 69
<210> 77
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 77
tctctctctc tctctctctc tctctctctc tctctctctc tctctctctc tctctctctc 60
tctctctct 69
<210> 78
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 78
gcgcgcgcgc gcgcgcgcgc gcgcgcgcgc gcgcgcgcgc gcgcgcgcgc gcgcgcgcgc 60
gcgcgcgcg 69
<210> 79
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 79
ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg gcggcggcgg cggcggcggc 60
ggcggcggc 69
<210> 80
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 80
ttattattat tattattatt attattatta ttattattat tattattatt attattatta 60
ttattatta 69
<210> 81
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 81
ttgttgttgt tgttgttgtt gttgttgttg ttgttgttgt tgttgttgtt gttgttgttg 60
ttgttgttg 69
<210> 82
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 82
tgctgctgct gctgctgctg ctgctgctgc tgctgctgct gctgctgctg ctgctgctgc 60
tgctgctgc 69
<210> 83
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 83
tagtagtagt agtagtagta gtagtagtag tagtagtagt agtagtagta gtagtagtag 60
tagtagtag 69
<210> 84
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 84
tcctcctcct cctcctcctc ctcctcctcc tcctcctcct cctcctcctc ctcctcctcc 60
tcctcctcc 69
<210> 85
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 85
ttcttcttct tcttcttctt cttcttcttc ttcttcttct tcttcttctt cttcttcttc 60
ttcttcttc 69
<210> 86
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 86
tgatgatgat gatgatgatg atgatgatga tgatgatgat gatgatgatg atgatgatga 60
tgatgatga 69
<210> 87
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 87
tggtggtggt ggtggtggtg gtggtggtgg tggtggtggt ggtggtggtg gtggtggtgg 60
tggtggtgg 69
<210> 88
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 88
atctatctat ctatctatct atctatctat ctatctatct atctatctat ctatctatct 60
atctatcta 69
<210> 89
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 89
gtttgtttgt ttgtttgttt gtttgtttgt ttgtttgttt gtttgtttgt ttgtttgttt 60
gtttgtttg 69
<210> 90
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 90
atttatttat ttatttattt atttatttat ttatttattt atttatttat ttatttattt 60
atttattta 69
<210> 91
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 91
ctttctttct ttctttcttt ctttctttct ttctttcttt ctttctttct ttctttcttt 60
ctttctttc 69
<210> 92
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 92
atggatggat ggatggatgg atggatggat ggatggatgg atggatggat ggatggatgg 60
atggatgga 69
<210> 93
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 93
gttcgttcgt tcgttcgttc gttcgttcgt tcgttcgttc gttcgttcgt tcgttcgttc 60
gttcgttcg 69
<210> 94
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 94
attcattcat tcattcattc attcattcat tcattcattc attcattcat tcattcattc 60
attcattca 69
<210> 95
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 95
gtccgtccgt ccgtccgtcc gtccgtccgt ccgtccgtcc gtccgtccgt ccgtccgtcc 60
gtccgtccg 69
<210> 96
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 96
cttccttcct tccttccttc cttccttcct tccttccttc cttccttcct tccttccttc 60
cttccttcc 69
<210> 97
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 97
atgtatgtat gtatgtatgt atgtatgtat gtatgtatgt atgtatgtat gtatgtatgt 60
atgtatgta 69
<210> 98
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 98
attgattgat tgattgattg attgattgat tgattgattg attgattgat tgattgattg 60
attgattga 69
<210> 99
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 99
ctgtctgtct gtctgtctgt ctgtctgtct gtctgtctgt ctgtctgtct gtctgtctgt 60
ctgtctgtc 69
<210> 100
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 100
gtaggtaggt aggtaggtag gtaggtaggt aggtaggtag gtaggtaggt aggtaggtag 60
gtaggtagg 69
<210> 101
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 101
cgggcgggcg ggcgggcggg cgggcgggcg ggcgggcggg cgggcgggcg ggcgggcggg 60
cgggcgggc 69
<210> 102
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 102
attaattaat taattaatta attaattaat taattaatta attaattaat taattaatta 60
attaattaa 69
<210> 103
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 103
ctccctccct ccctccctcc ctccctccct ccctccctcc ctccctccct ccctccctcc 60
ctccctccc 69
<210> 104
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 104
ctgcctgcct gcctgcctgc ctgcctgcct gcctgcctgc ctgcctgcct gcctgcctgc 60
ctgcctgcc 69
<210> 105
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 105
ctggctggct ggctggctgg ctggctggct ggctggctgg ctggctggct ggctggctgg 60
ctggctggc 69
<210> 106
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 106
tttgttttgt tttgttttgt tttgttttgt tttgttttgt tttgttttgt tttgttttgt 60
tttgttttg 69
<210> 107
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 107
tccattccat tccattccat tccattccat tccattccat tccattccat tccattccat 60
tccattcca 69
<210> 108
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 108
tttattttat tttattttat tttattttat tttattttat tttattttat tttattttat 60
tttatttta 69
<210> 109
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 109
gggctgggct gggctgggct gggctgggct gggctgggct gggctgggct gggctgggct 60
gggctgggc 69
<210> 110
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 110
gagctgagct gagctgagct gagctgagct gagctgagct gagctgagct gagctgagct 60
gagctgagc 69
<210> 111
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 111
tttcttttct tttcttttct tttcttttct tttcttttct tttcttttct tttcttttct 60
tttcttttc 69
<210> 112
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 112
tgtattgtat tgtattgtat tgtattgtat tgtattgtat tgtattgtat tgtattgtat 60
tgtattgta 69
<210> 113
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 113
ggagtggagt ggagtggagt ggagtggagt ggagtggagt ggagtggagt ggagtggagt 60
ggagtggag 69
<210> 114
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 114
tagattagat tagattagat tagattagat tagattagat tagattagat tagattagat 60
tagattaga 69
<210> 115
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 115
tctattctat tctattctat tctattctat tctattctat tctattctat tctattctat 60
tctattcta 69
<210> 116
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 116
ctcctctcct ctcctctcct ctcctctcct ctcctctcct ctcctctcct ctcctctcct 60
ctcctctcc 69
<210> 117
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 117
ttaatttaat ttaatttaat ttaatttaat ttaatttaat ttaatttaat ttaatttaat 60
ttaatttaa 69
<210> 118
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 118
gggcggggcg gggcggggcg gggcggggcg gggcggggcg gggcggggcg gggcggggcg 60
gggcggggc 69
<210> 119
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 119
gcgctgcgct gcgctgcgct gcgctgcgct gcgctgcgct gcgctgcgct gcgctgcgct 60
gcgctgcgc 69
<210> 120
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 120
cccctcccct cccctcccct cccctcccct cccctcccct cccctcccct cccctcccct 60
cccctcccc 69
<210> 121
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 121
ggccgggccg ggccgggccg ggccgggccg ggccgggccg ggccgggccg ggccgggccg 60
ggccgggcc 69
<210> 122
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 122
gggatgggat gggatgggat gggatgggat gggatgggat gggatgggat gggatgggat 60
gggatggga 69
<210> 123
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 123
ggggtggggt ggggtggggt ggggtggggt ggggtggggt ggggtggggt ggggtggggt 60
ggggtgggg 69
<210> 124
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 124
gggttagggt tagggttagg gttagggtta gggttagggt tagggttagg gttagggtta 60
gggttaggg 69
<210> 125
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 125
gggttcgggt tcgggttcgg gttcgggttc gggttcgggt tcgggttcgg gttcgggttc 60
gggttcggg 69
<210> 126
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 126
atatgtatat gtatatgtat atgtatatgt atatgtatat gtatatgtat atgtatatgt 60
atatgtata 69
<210> 127
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 127
tgatggtgat ggtgatggtg atggtgatgg tgatggtgat ggtgatggtg atggtgatgg 60
tgatggtga 69
<210> 128
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 128
gggttggggt tggggttggg gttggggttg gggttggggt tggggttggg gttggggttg 60
gggttgggg 69
<210> 129
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 129
ttgtttttgt ttttgttttt gtttttgttt ttgtttttgt ttttgttttt gtttttgttt 60
ttgtttttg 69
<210> 130
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 130
gggtgagggt gagggtgagg gtgagggtga gggtgagggt gagggtgagg gtgagggtga 60
gggtgaggg 69
<210> 131
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 131
ggcgggggcg ggggcggggg cgggggcggg ggcgggggcg ggggcggggg cgggggcggg 60
ggcgggggc 69
<210> 132
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 132
ttctttttct ttttcttttt ctttttcttt ttctttttct ttttcttttt ctttttcttt 60
ttctttttc 69
<210> 133
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 133
tccttctcct tctccttctc cttctccttc tccttctcct tctccttctc cttctccttc tccttctcc 69
<210> 134
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 134
gggtgggggt gggggtgggg gtgggggtgg gggtgggggt gggggtgggg gtgggggtgg 60
gggtggggg 69
<210> 135
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 135
ttatttttat ttttattttt atttttattt ttatttttat ttttattttt atttttattt 60
ttattttta 69
<210> 136
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 136
ccctctccct ctccctctcc ctctccctct ccctctccct ctccctctcc ctctccctct 60
ccctctccc 69
<210> 137
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 137
ggctggggct ggggctgggg ctggggctgg ggctggggct ggggctgggg ctggggctgg 60
ggctggggc 69
<210> 138
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 138
ccctccccct ccccctcccc ctccccctcc ccctccccct ccccctcccc ctccccctcc 60
ccctccccc 69
<210> 139
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 139
ctctgtctct gtctctgtct ctgtctctgt ctctgtctct gtctctgtct ctgtctctgt 60
ctctgtctc 69
<210> 140
<211> 15
<212> DNA
<213> Bacteria
<400> 140
yaacgagcgc aaccc 15
<210> 141
<211> 17
<212> DNA
<213> Bacteria
<400> 141
cggtgtgtac aagaccc 17
<210> 142
<211> 25
<212> DNA
<213> plasmid
<400> 142
ggtaccgtcg acatggctca gcgtc 25
<210> 143
<211> 20
<212> DNA
<213> plasmid
<400> 143
aagcttgtca tcgagctcga 20
<210> 144
<211> 30
<212> DNA
<213> plasmid
<400> 144
tggagaacca cctttcggac cgtgccattc 30
Claims (9)
1. a grand Extraction Methods of Genome, is characterized in that utilizing probe hybridization magnetic capture mode to reduce the impact of host DNA in host's macro genome DNA sample.
2. grand Extraction Methods of Genome according to claim 1, is characterized in that described probe and the hybridization of described host genome DNA sample afterwards and described magnetic bead coupling.
3. grand Extraction Methods of Genome according to claim 2 is characterized in that comprising step:
1) will be marked with the described probe of vitamin H and described host's macro genome DNA sample of extraction is hybridized;
2) with being coated with the described magnetic capture probe of Streptavidin and the complex body of described host DNA, and separate magnetic bead-probe-target dna mixture with magnetic frame;
3) hybridization solution and the magnetic bead scavenging solution after hybridization that will remove described magnetic bead-probe-target dna mixture carry out purifying recovery DNA.
4. grand Extraction Methods of Genome according to claim 1, is characterized in that after described probe and described magnetic bead coupling the hybridization with described host genome.
5. grand Extraction Methods of Genome according to claim 4 is characterized in that comprising step:
1) utilize the described probe coupling of the described magnetic bead and the mark vitamin H that are coated with Streptavidin;
2) by coupling, good mixture and described host's macro genome DNA sample hybridized;
3) remove magnetic bead-probe-target dna mixture, hybridization solution and hybridization scavenging solution are carried out to purifying recovery DNA.
6. according to the described grand Extraction Methods of Genome of any one of claim 1 ~ 5, it is characterized in that using multiple forward and reverse probe at the coupling temperature.
7. according to the described grand Extraction Methods of Genome of claim 3 or 5, after it is characterized in that having hybridized, described magnetic bead-probe-DNA mixture is heated or the sodium hydroxide method sex change, reclaimed the magnetic bead that coupling has probe.
8. grand Extraction Methods of Genome according to claim 1, is characterized in that described probe is selected from sequence SEQ ID NO:1 in sequence table to sequence SEQ ID NO:139.
9. grand Extraction Methods of Genome according to claim 1, is characterized in that described host's macro genome DNA sample takes from human respiratory, oral cavity, digestive tube, skin or reproductive tract.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361374A (en) * | 2013-07-23 | 2013-10-23 | 扬州日兴生物科技股份有限公司 | Chitosanase gene derived from soil metagenome library and obtaining method and application of chitosanase gene |
| CN104789687A (en) * | 2015-05-08 | 2015-07-22 | 上海允英医疗科技有限公司 | Probe preparation method for multi-gene capture sequencing |
| CN106148326A (en) * | 2016-07-27 | 2016-11-23 | 上海美吉生物医药科技有限公司 | The extracting method of macro genome DNA |
| CN106414775A (en) * | 2014-04-11 | 2017-02-15 | 宾夕法尼亚大学董事会 | Compositions and methods for metagenome biomarker detection |
| EP3256608A4 (en) * | 2015-02-13 | 2019-02-20 | Icahn School of Medicine at Mount Sinai | Rna containing compositions and methods of their use |
| CN110093409A (en) * | 2019-04-26 | 2019-08-06 | 南京世和基因生物技术有限公司 | A kind of infection line detecting method and kit based on high-flux sequence |
| CN110205318A (en) * | 2019-05-15 | 2019-09-06 | 杭州杰毅生物技术有限公司 | Macro Extraction Methods of Genome based on CRISPR-Cas removal host genome DNA |
| CN111455021A (en) * | 2019-01-18 | 2020-07-28 | 广州微远基因科技有限公司 | Method and kit for removing host DNA in metagenome |
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| CN102839168A (en) * | 2012-07-31 | 2012-12-26 | 深圳华大基因研究院 | Nucleic acid probe, and preparation method and application thereof |
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| CN102839168A (en) * | 2012-07-31 | 2012-12-26 | 深圳华大基因研究院 | Nucleic acid probe, and preparation method and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361374A (en) * | 2013-07-23 | 2013-10-23 | 扬州日兴生物科技股份有限公司 | Chitosanase gene derived from soil metagenome library and obtaining method and application of chitosanase gene |
| CN106414775A (en) * | 2014-04-11 | 2017-02-15 | 宾夕法尼亚大学董事会 | Compositions and methods for metagenome biomarker detection |
| EP3256608A4 (en) * | 2015-02-13 | 2019-02-20 | Icahn School of Medicine at Mount Sinai | Rna containing compositions and methods of their use |
| CN104789687A (en) * | 2015-05-08 | 2015-07-22 | 上海允英医疗科技有限公司 | Probe preparation method for multi-gene capture sequencing |
| CN106148326A (en) * | 2016-07-27 | 2016-11-23 | 上海美吉生物医药科技有限公司 | The extracting method of macro genome DNA |
| CN111455021A (en) * | 2019-01-18 | 2020-07-28 | 广州微远基因科技有限公司 | Method and kit for removing host DNA in metagenome |
| CN111455021B (en) * | 2019-01-18 | 2024-06-04 | 广州微远医疗器械有限公司 | Method and kit for removing host DNA in metagenome |
| CN110093409A (en) * | 2019-04-26 | 2019-08-06 | 南京世和基因生物技术有限公司 | A kind of infection line detecting method and kit based on high-flux sequence |
| CN110205318A (en) * | 2019-05-15 | 2019-09-06 | 杭州杰毅生物技术有限公司 | Macro Extraction Methods of Genome based on CRISPR-Cas removal host genome DNA |
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|---|---|
| CN103060309B (en) | 2014-12-17 |
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