CN107090448A - A kind of rapid extraction nuclei aoid methods detected for clinical sample PCR - Google Patents
A kind of rapid extraction nuclei aoid methods detected for clinical sample PCR Download PDFInfo
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Abstract
The present invention relates to a kind of rapid extraction nuclei aoid methods detected for clinical sample PCR.This method is applied to extract the DNA of the pathogenic microorganisms such as Gram-negative bacteria, virus, mycoplasma and Chlamydia, extracts product DNA and can be used for fluorescence quantitative PCR detection.The invention washes the sample containing pathogenic microorganisms such as Gram-negative bacteria, virus, mycoplasma and Chlamydias once with physiology salt, the solution containing Chelex 100, NaOH, SDS is added in precipitation, it is resuspended, add the liquor kalii acetici of certain pH, after centrifugation, supernatant is taken to be used for fluorescence quantitative PCR detection.Compared with existing method for extracting nucleic acid：(1) the step of Proteinase K hydrolysis is purified with post is eliminated, the time of nucleic acid extraction is substantially reduced；(2) avoid in operating process because sample shift caused by cross pollution and DNA Loss, improve the accuracy of testing result.
Description
Technical field
The invention belongs to molecular biology and medical science, it is related to a kind of for the quick of clinical sample PCR detections
Nuclei aoid methods are extracted, are specifically referred to using easy reliable method extracting clinical sample DNA, then using fluorescence quantifying PCR method
The nucleic acid of extracting is detected.
Background technology
Nucleic acid is the carrier of hereditary information, is the material base of gene expression.Either carry out nucleic acid structure or work(
It can study, it is necessary first to the genomic nucleic acids required for quickly and effectively separating and extract from complicated and diversified biological specimen,
And the Nucleic acid quality and its integrality after extracting can all directly influence subsequent experimental result.It is presently used for clinical sample
The extraction nucleic acid of PCR detections is often included with method for extracting：Boiling method, alkali density method and guanidinium isothiocyanate-post purifying extraction process.
Boiling method is under the high temperature conditions to crack cell, and core is extracted in conjunction with Chelex-100 metal ion chelation agents
Acid.Chelex-100 has very high affinity and chelation to high volence metal ion, can suppress digestion of the nuclease to DNA
Effect, it is therefore prevented that degradeds of the DNA in boiling part, such processing procedure had both reached separation and Extraction DNA purpose.But
It is that spring lid phenomenon easily occurs for boiling method, causes cross pollution, and impurity is more, influences PCR amplification.
Guanidinium isothiocyanate-post purifying extraction process is used after guanidinium isothiocyanate cell lysis, by nucleic acid absorption in centrifugal column film
On, by adding different washing reagents and being centrifuged repeatedly, to reach the purpose of nucleic acid and impurity separation, obtain the nucleic acid of purifying.
But the method reagent consumables cost is higher, and need to be centrifuged repeatedly, be not easy to high flux, automation mechanized operation.
Alkali density method is to crack cell with alkali and dodecane sulfonate, and alkali can keep virus covalently closed circular
While DNA double chain state, it is denatured HMW chromosomal DNA, dodecane sulfonate and cell protein, broken cell
Wall and the chromosomal DNA of denaturation formation compound, these complex precipitates after centrifugation, and DNA is present in supernatant.Although
This method is initially only applicable to extract DNA, with limitation.But, through continuing to optimize and improving, alkali density method is wide
The general every field for animal, plant, microorganism and legal medical expert's research.Alkaline lysis has easy to operate, extraction rate fast, logical
A kind of the characteristics of measuring big, it is considered to be effective DNA rapid extracting methods.
The content of the invention
Of the invention is to solve the defect that traditional method for extracting nucleic acid is present, and proposition is a kind of to be used for what clinical sample PCR was detected
The method of rapid extraction nucleic acid.In order to realize the goal of the invention of the present invention, the technical scheme used is used for clinical sample for one kind
The method of the rapid extraction nucleic acid of this PCR detections, described method comprises the following steps：
1. taking a small amount of biological specimen, the biological specimen is selected from bacterium, virus, mycoplasma, the swab of choamydiae infection, palace
Neck brush or liquid basal cell sample.
2. take 0.5ml samples to add in 1.5ml EP pipes, 12000rmp centrifugations 2min.
3. careful abandon supernatant, adding vibration on 1ml physiological saline, vortex oscillator, to break up precipitation (light with pipette tips if necessary
Tip-tap dissipates precipitation), 12000rpm centrifugations 2-5min.
4. careful abandon supernatant, 50-80ul NaOH, 1%-5% containing 5%-10%Chelex-100,0.2-0.4M is added
Cell is resuspended in SDS, pH=11-13 solution P1.Note：Sedimentation cell is dispelled with liquid-transfering gun tenderness, lid is covered tightly, leniently mixes
Close, should not acutely shake, solution strain is limpid.
5. adding 5-10ul pH=4.8-5.7 liquor kalii acetici P2, the mouth of pipe is covered tightly, pipe is gently overturned and mixed for several times
(EP ttom of pipe can be gently played with finger for several times), sees white flock precipitate, and 12000rmp centrifugation 1-5min, supernatant is directly used in
PCR reacts.
Wherein, the first precedence technique scheme of the invention is that centrifugation time is 2-5min after described addition physiological saline,
It is preferred that 4-5min；
The second precedence technique scheme of the present invention is that described solution P1 addition is 50-80ul, preferably 50-60ul.
The 3rd precedence technique scheme of the present invention is that Chelex-100 concentration is 5%-10% in described solution P1,
It is preferred that 5%-8%.
The 4th precedence technique scheme of the present invention is that NaOH concentration is 0.2-0.4M in described solution P1, preferably
0.2-0.3M。
The 5th precedence technique scheme of the present invention is that SDS concentration is 1%-5%, preferably 1%- in described solution P1
3%.
The 6th precedence technique scheme of the present invention is that described solution P1 pH is 11-13, preferably 12-13.
The 7th precedence technique scheme of the present invention is that described solution P2 addition is 5-10ul, preferably 5-8ul.
The 8th precedence technique scheme of the present invention is that described solution P2 pH is 4.8-5.7, preferably 5.3-5.7.
The 9th optimal technical scheme of the present invention is that centrifugation time is 1-5min after described addition solution P2, preferably
1-2min。
Wherein, biological specimen of the invention is selected from bacterium, virus, mycoplasma, the swab of choamydiae infection, Uterine neck bush and liquid
Basal cell's sample.
The present invention technical advantage be：
1. the present invention is simple to operate, the time is short.Need to use lysozyme lysis cell in conventional method, Proteinase K removes sample
Protein in this, this process need to take 1-2 hours, then carry out post purifying DNA, and centrifugation step is more.The method behaviour of the present invention
Make simple, using aqueous slkali come lysed sample, albumen in membranolysis, cell is combined with dodecyl sodium sulfate (SDS),
Sodium ion after potassium ion displacement with generating water insoluble potassium dodecanesulfonate (PDS) in SDS, so as to reach seperated nuclear acid
Purpose.Whole extraction process is only 8-10min, the time required to substantially reducing.
2. operating procedure of the present invention is few and can complete whole extraction process in same EP pipes, it is to avoid in operating process
The cross-contamination phenomena caused by sample is shifted, improves the accuracy of testing result.
3. the alkaline lysis used in the present invention is applied not only to extract DNA in bacterium, it may also be used for extract virus, Zhi Yuan
DNA in body, Chlamydia.
, can abundant cell lysis 4. the present invention uses Chelex-100 combination NaOH.Alkalescence is stronger in itself by Chelex-100,
The more preferable cell lysis of NaOH can be helped, and Chelex-100 can chelate some the factor of inhibitory action to amplified reaction
(such as heavy metal ion), improves the efficiency of PCR amplifications.
Brief description of the drawings
Fig. 1 is extracted DNA fluorescence quantitative PCR detection result by two kinds of Different Extraction Methods in embodiment.
Wherein 1 is the DNA detection knots that the precious biological broad spectrum type genomic DNA small scale purification kit of No. 1 sample is extracted
Really；2 be the DNA testing results that No. 1 sample is extracted with the inventive method；3 be the precious biological broad spectrum type genome of No. 2 samples
The DNA testing results that DNA small scale purifications kit is extracted；4 be the DNA testing results that No. 2 samples are extracted with the inventive method；5
The DNA testing results extracted for the precious biological broad spectrum type genomic DNA small scale purification kit of No. 3 samples；6 be No. 3 samples
The DNA testing results extracted with the inventive method.
Fig. 2 is extracted DNA regular-PCR product electrophoresis result by two kinds of Different Extraction Methods in embodiment.
Wherein well 1：Product after No. 1 sample PCR primer is concentrated through the inventive method；Well 2：No. 2 sample PCR productions
Product after thing is concentrated through the inventive method；Well 3：Product after No. 3 sample PCR primers are concentrated through the inventive method；Well
4：No. 1 sample PCR primer product after the common DNA product purification kit concentration of Tiangeng；Well 5：No. 2 sample PCR primers
The product after the common DNA product purification kit concentration of Tiangeng；Well 6：DL2000DNA Maker；Well 7：No. 3 samples
PCR primer product after the common DNA product purification kit concentration of Tiangeng.
Fig. 3 is the DNA sequencing collection of illustrative plates of the inventive method extraction in embodiment.
Fig. 4 is the DNA sequencing of precious biological broad spectrum type genomic DNA small scale purification kit extraction in embodiment
Collection of illustrative plates.
Embodiment
1. extract ureaplasma urealyticum sense with precious biological broad spectrum type genomic DNA small scale purification kit and the inventive method
DNA in the urethral secretions of dye, extracts 3 samples, parallel comparison simultaneously with both method for extracting respectively.
1.1 extract the DNA in urethral secretions with precious biological broad spectrum type genomic DNA small scale purification kit.
1.1.1 the cell-preservation liquid of 200ul secretion containing urethra tube is taken, 400ul buffer solutions GA is added.
1.1.2 180ul Buffer GL and 20ul Proteinase K solution is added, vortex 10sec is mixed, 56 DEG C
Place 10min.
1.1.3 200ul buffer solution GB and 200ul absolute ethyl alcohols are added, fully reverse to mix, brief centrifugation is to remove pipe
The drop of lid inwall.
1.1.4 previous step resulting solution is all added in an adsorption column (adsorption column is put into collecting pipe), 12,
000rpm (≈ 13,400 × g) centrifuges 30sec, outwells the waste liquid in collecting pipe, adsorption column is put back in collecting pipe.
1.1.5 the 500ul Buffer WA for having added absolute ethyl alcohol are added into adsorption column, 12,000rpm (≈ 13,
400 × g) centrifugation 30sec, outwells the waste liquid in collecting pipe, adsorption column is put back in collecting pipe.
1.1.6 700ul Buffer WB, 12,000rpm (≈ 13,400 × g) centrifugation 30sec are added into adsorption column,
The waste liquid in collecting pipe is outwelled, adsorption column is put back into collecting pipe.
1.1.7 repeat step 7.
1.1.8 12,000rpm (≈ 13,400 × g) centrifuges 2min, outwells waste liquid.Adsorption column room temperature is placed several minutes,
Thoroughly to dry rinsing liquid remaining in sorbing material.
1.1.9 adsorption column is transferred in a clean centrifuge tube, 50ul elutions is vacantly added dropwise to adsorbed film centre position
Buffer solution, room temperature places 5min, 12,000rpm (≈ 13,400 × g) centrifugations 2min.
1.2 extract the DNA in urethral secretions with the method for the present invention.
1.2.1. the cell-preservation liquid of 500ul secretion containing urethra tube is taken to pour into 1.5ml EP pipes, 12000rmp centrifugations
2min。
1.2.2. carefully abandon supernatant, add vibration on 1ml physiological saline, vortex oscillator and break up precipitation and (use rifle if necessary
Head gently breaks up precipitation), 12000rpm centrifugations 5min.
1.2.3. supernatant is carefully abandoned, 50ul is added containing the molten of 5%Chelex-100,0.2M NaOH, 1%SDS, pH=13
Cell is resuspended in liquid P1.Note：Sedimentation cell is dispelled with liquid-transfering gun tenderness, lid is covered tightly, is gently blended, should not acutely be shaken,
Solution strain is limpid.
1.2.4. 5ulpH=5.5 liquor kalii acetici P2 is added, the mouth of pipe is covered tightly, pipe is gently overturned to mixing for several times (available
Finger gently plays EP ttom of pipe for several times), see white flock precipitate, 12000rmp centrifugation 1min, supernatant is directly used in PCR reactions.
2.DNA authentication methods
The Taqman probes and primer of ureaplasma urealyticum gene are selected, is surveyed with fluorescence quantifying PCR method and regular-PCR product
Two methods of sequence detect that above two Different Extraction Method extracts DNA simultaneously.All primer and probes are by Primer
The Software for Design of Express 3.0, is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.Primed probe information is as follows：
For fluorescence quantifying PCR method detect the sense primer of ureaplasma urealyticum gene for 5 '-
TGTCAGGATCATCAAATCAATTCAC-3’(SEQ ID NO:1)；
For fluorescence quantifying PCR method detect the anti-sense primer of ureaplasma urealyticum gene for 5 '-
AGTGSAAATGTGATCCAACTTGG-3’(SEQ ID NO:2)；
Detect that the probe of ureaplasma urealyticum gene is 5 '-FAM- for fluorescence quantifying PCR method
CAGGAGCAATTAACTTCGCTGAAGGCG-BHQ1-3’(SEQ ID NO:3)；
For regular-PCR product sequence measurement detect the sense primer of ureaplasma urealyticum gene for 5 '-
ATGAAGAATCTGTGGGAGGTGG-3’(SEQ ID NO:4)；
For regular-PCR product sequence measurement detect the anti-sense primer of ureaplasma urealyticum gene for 5 '-
AATCGTCGCATTCCCTATGCT-3’(SEQ ID NO:5)。
2.1 fluorescence quantitative PCR detection
PCR system：10 × PCR buffer 5ul, 25mM MgCl26ul, 25mM dNTP 0.8ul, 10uM sense primers
1.25ul, 10uM anti-sense primer 1.25ul, 10uM probe 0.5ul, 5U/ul thermal starting archaeal dna polymerase 0.8ul, deionization sterilizing
Water supplies 50ul.
PCR augmentation detection reaction conditions：37℃5min；95℃5min；95 DEG C of 15S, 60 DEG C of 60S, 40cycles.
The sequencing detection of 2.2 regular-PCR products
PCR system：10 × PCR buffer 5ul, 25mM MgCl26ul, 25mM dNTP 0.8ul, 10uM sense primers
1.25ul, 10uM anti-sense primer 1.25ul, 5U/ul thermal starting archaeal dna polymerase 0.6ul, deionization aqua sterilisa supply 50ul.
PCR augmentation detection reaction conditions：37℃5min；95℃5min；95 DEG C of 15S, 60 DEG C of 30S, 72 DEG C of 30s,
40cycles；72℃7min.
PCR primer is through 1% agarose gel electrophoresis, 50V, 30 minutes.Under the uviol lamp of gel imager, sample is taken
Migration position picture on Ago-Gel, as a result as shown in Figure 2.Simultaneously Suzhou gold only intelligence biotechnology is sent by PCR primer
Co., Ltd is sequenced, and NCBI BLAST compare the homology of sequencing result and target gene online, and observe the matter of Sequencing chromatogram
Amount.The DNA sequencing result that the inventive method is extracted is shown in Fig. 3, precious biological broad spectrum type genomic DNA small scale purification kit extracting
DNA sequencing result see Fig. 4.
3. interpretation of result interpretation of result
By that can find that 2,4, the 6 Ct values respectively than 1,3,5 are small in Fig. 1 to the fluorescent PCR detection for extracting product DNA.Through
Analysis, the DNA concentration after being extracted due to this method is higher, and disturbs PCR impurity less in the DNA of this method extraction, therefore
The augmentation detection Ct values that this method extracts product are respectively less than precious biological broad spectrum type genomic DNA small scale purification kit extraction production
Thing DNA augmentation detection Ct values.It can thus be concluded that, the DNA that the inventive method is extracted can be used for the quantitative fluorescent PCR inspection of clinical sample
Survey.
The DNA regular-PCRs product electrophoretic band size and brightness extracted in Fig. 2 through two methods are similar.By Fig. 3 and figure
4 understand, the DNA sequencing graph-spectrum quality that two methods are extracted is identical, and sequence is after comparison, is all higher than with aim sequence homology
98%.Thus prove, the DNA that two kinds of DNA extraction methods are extracted could be used for the regular-PCR product sequencing inspection of clinical sample
Survey.
In this description, the present invention is described with reference to its specific embodiment.But it is clear that can still make
Various modifications and alterations are without departing from the spirit and scope of the present invention.Therefore, specification and drawings are considered as illustrative
And it is nonrestrictive.
SEQUENCE LISTING
<110>Jiangsu Rui Bo bio tech ltd
<120>A kind of simple and quick extraction nuclei aoid methods
<130> 20170321
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
<400> 1
tgtcaggatc atcaaatcaa ttcac 25
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence
<400> 2
agtgsaaatg tgatccaact tgg 23
<210> 3
<211> 27
<212> DNA
<213>Artificial sequence
<400> 3
caggagcaat taacttcgct gaaggcg 27
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence
<400> 4
atgaagaatc tgtgggaggt gg 22
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence
<400> 5
aatcgtcgca ttccctatgc t 21
Claims (9)
1. a kind of Rapid nucleic acid extracting method detected for clinical sample PCR, it is characterised in that used reagent is comprising molten
Liquid P1 and solution P2.
2. a kind of Rapid nucleic acid extracting method detected for clinical sample PCR, it is characterised in that described solution P1 contains
Chelex-100、NaOH、SDS。
3. a kind of Rapid nucleic acid extracting method detected for clinical sample PCR, it is characterised in that described solution P2 compositions are
Potassium acetate buffer solution.
4. a kind of Rapid nucleic acid extracting method detected for clinical sample PCR, it is characterised in that described method includes following
Step：
(1) a small amount of biological specimen is taken, the biological specimen is selected from bacterium, virus, mycoplasma, the swab of choamydiae infection, uterine neck
Brush and liquid basal cell sample.
(2) 0.5ml samples are taken to pour into 1.5ml EP pipes, 12000rmp centrifugations 2min.
(3) carefully abandon supernatant, add vibration on 1ml physiological saline, vortex oscillator and break up precipitation and (gently beaten with pipette tips if necessary
Dissipate precipitation), 12000rpm centrifugations 2-5min.
(4) supernatant is carefully abandoned, 50-80ul NaOH, 1%-5%SDS, pH containing 5%-10%Chelex-100,0.2-0.4M is added
Cell is resuspended in=11-13 solution P1.Note：Sedimentation cell is dispelled with liquid-transfering gun tenderness, lid is covered tightly, is gently blended, no
Acutely to shake, solution strain is limpid.
(5) 5-10ulpH=4.8-5.7 liquor kalii acetici P2 is added, the mouth of pipe is covered tightly, pipe is gently overturned to mixing for several times (available
Finger gently plays EP ttom of pipe for several times), see white flock precipitate, 12000rmp centrifuges 1-5min, and it is anti-that supernatant is directly used in PCR
Should.
5. the method according to claim 2, it is characterised in that Chelex-100 concentration is in described solution P1
5%-10%, preferably 5%-8%, NaOH concentration is 0.2-0.4M, preferably 0.2-0.3M, and SDS concentration is 1%-5%, excellent
Select 1%-3%.
6. the method according to claim 3, it is characterised in that described solution P1 pH is 11-13, preferably 12-13.
7. method according to claim 3, it is characterised in that described solution P2 pH is 4.8-5.7, preferably 5.3-
5.7。
8. method according to claim 4, it is characterised in that the described centrifugation time added after physiological saline is 2-
5min, preferential 4-5min.
9. method according to claim 4, it is characterised in that the described centrifugation time added after solution P2 is 1-
5min, preferential 1-2min.
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Cited By (3)
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| CN114426969A (en) * | 2022-03-16 | 2022-05-03 | 厦门飞朔医学检验实验室有限公司 | Method for rapidly extracting nucleic acid for clinical sample detection |
| CN114561380A (en) * | 2022-04-02 | 2022-05-31 | 予果生物科技（北京）有限公司 | Bacterial nucleic acid extraction detection reagent, kit, method and application thereof |
| CN115125238A (en) * | 2022-07-23 | 2022-09-30 | 新乡学院 | Method for separating and purifying tumor extracellular vesicle DNA |
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Cited By (5)
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| CN114426969A (en) * | 2022-03-16 | 2022-05-03 | 厦门飞朔医学检验实验室有限公司 | Method for rapidly extracting nucleic acid for clinical sample detection |
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| CN114561380B (en) * | 2022-04-02 | 2022-12-16 | 予果生物科技（北京）有限公司 | Bacterial nucleic acid extraction detection reagent, kit, method and application thereof |
| CN115125238A (en) * | 2022-07-23 | 2022-09-30 | 新乡学院 | Method for separating and purifying tumor extracellular vesicle DNA |
| CN115125238B (en) * | 2022-07-23 | 2023-11-14 | 新乡学院 | Method for separating and purifying tumor extracellular vesicle DNA |
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Application publication date: 20170825 |