CN108048529A - A kind of quality-control product of lung cancer methylated genes detection kit for stablizing preservation and application - Google Patents
A kind of quality-control product of lung cancer methylated genes detection kit for stablizing preservation and application Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q2600/00—Oligonucleotides characterized by their use
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention discloses a kind of quality-control products for the lung cancer methylated genes detection kit for stablizing preservation, and it includes following components：Positive reference product, negative reference product and detection limit reference material.The positive reference product, negative reference product and detection limit reference material include corresponding DNA and DNA preservation solution respectively.DNA preservation solution provided by the invention can effectively prevent the degradation of dissociative DNA, and stable environment is provided for DNA, extend the holding time of quality-control product；DNA quality-control products provided by the invention containing DNA preservation solution of the present invention have many advantages, such as that manufacturing cost is low, performance is stable and easy to maintain, the factors such as personnel's operation, instrument state and kit validity can be effectively monitored, drastically increase the accuracy and reliability of detection kit.
Description
Technical field
The invention belongs to technical field of gene detection, and in particular to a kind of lung cancer methylated genes detection for stablizing preservation
The quality-control product of kit and application.
Background technology
DNA methylation marker plays an increasingly important role in the early diagnosis of lung cancer in recent years, noninvasive to develop
Property technique study specificity is laid a good foundation in lung cancer early sign object.The releasable DNA of lung carcinoma cell in blood circulation system,
The Circulating DNA of abnormal higher concentration can be detected in Patients with Various Cancers and blood plasma.By comparing the study found that starting
For sub-district DNA methylation in cancerous lung tissue with having similar change pattern in corresponding serum, plasma sample, this shows blood sample
The epigenetics variation of interior related gene can be as the marker of tumour early screening.Have now been found that a series of and lung cancer
Relevant tumor suppressor gene methylates due to being abnormal and causes transcriptional inactivation.Tumor suppressor gene promoter methylation is sent out with lung cancer
The relation of hair tonic exhibition is more and more paid attention to.
Methylate most common technology restrictive enzyme cutting method, high-resolution melting curve method, bisulfite are detected at present
Methylation status of PTEN promoter of sodium PCR sequencing PCR, southern blotting technique method and classics etc..These methods respectively have advantage and disadvantage, according to Specimen origin and
The difference of molecular marker, the detection method of selection difference.But can not all meet simultaneously quantitative accuracy, hypersensitivity,
High-throughput, low false positive and false negative, the requirement of low cost and low stain.In numerous diagnostic techniques, gene sequencing is most straight
One of sight, most accurate method.Appearance the early diagnosing, effectively for lung cancer of rapid development and the gene therapy of molecular biology
Treatment provides new foundation.Lung cancer has many driving genes, and the occurrence probability that different patients methylate is different, it is therefore desirable to
Polygenes, multidigit point are detected simultaneously.The autotelic specific region for studying genome of sequencing approach is targeted using high-throughput, this
It is a kind of large-scale multiplexed PCR amplification method, can realizes thousands of or even up to ten thousand kinds of primer pair target area low frequencies or dilute
There is the detection of mutation.Include fresh using the detection sample type of lung cancer methylation detection kit or fix paraffin through formaldehyde
Embed (Formalin-Fixed and Parrffin-Embedded, FFPE) tissue samples, can provide higher flux with
Wider target area coverage can screen the several genes such as p16, RASSF1A, CDH1 and CDH13 simultaneously, while ensure to face
Bed laboratory is to the demand of data result high quality and high confidence level.
At present, there is no quality-control product pins that is economic, effective, stablizing general lung cancer methylation detection kit on the market
It sells, and to be the important method of evaluation and the performances such as a kit detection accuracy of verification and stability and quality refer to quality-control product
Mark.But conventional quality-control product is very easy to degrade, and the quality-control product to methylate as detection lung cancer, Stability and veracity are
A insoluble big problem.
The content of the invention
Based on this, a kind of DNA preservation solution is provided it is an object of the invention to overcome above-mentioned the deficiencies in the prior art part,
It can effectively prevent the degradation of dissociative DNA, stable environment is provided for DNA, extend the holding time of quality-control product.
To achieve the above object, the technical solution taken of the present invention is：A kind of DNA preservation solution, it is aurin front three acid ammonium salt, sweet
Propylhomoserin and bestatin hydrochloride；And the molar ratio of the aurin front three acid ammonium salt, glycine and bestatin hydrochloride
For (100~500)：(50000~150000)：(0.02~0.06).
Preferably, the molar ratio of the aurin front three acid ammonium salt, glycine and bestatin hydrochloride is 300：
80000：0.04.
The present invention also provides purposes of the DNA preservation solution in DNA preservations.
DNA preservation solution of the present invention can be used for the quality-control product for preparing gene methylation detection kit.
It is still a further object of the present invention is to provide a kind of matter for the lung cancer methylated genes detection kit for stablizing preservation
Control product.
To achieve the above object, the technical solution taken of the present invention is：A kind of lung cancer methylated genes for stablizing preservation
The quality-control product of detection kit, it includes following components：Positive reference product, negative reference product and detection limit reference material；
The positive reference product include target gene methylate modification DNA and the DNA preservation solution；
The negative reference product include target gene do not methylate modification DNA and the DNA preservation solution；
The detection limit reference material does not methylate comprising the methylate DNA of modification, target gene of target gene
The DNA of modification and the DNA preservation solution, and the methylate DNA of modification of the target gene accounts for the mass ratioes of DNA total amounts
For 1~5%.
Preferably, the quality-control product includes at least one of gene mass controlled product of p16, RASSF1A, CDH1 and CDH13.
Above-mentioned quality-control product, be inventor at work by being drawn after great amount of samples Analysis and Screening, the matter of type described above
As positive reference, the lung cancer that can cover most Chinese population methylates type control product, existing preferable with reference to effect,
Cost can be reduced again.
Preferably, the positive reference product, negative reference product and detection limit the component aurin three of DNA preservation solution in reference material
Formic acid ammonium salt molar concentration be 100-500 μM, glycine molar concentration be 50-150mM, bestatin hydrochloride molar concentration
For 20-60nM.
Preferably, the positive reference product, negative reference product and detection limit the component aurin three of DNA preservation solution in reference material
Formic acid ammonium salt molar concentration is 300 μM, and glycine molar concentration is 80mM, and bestatin hydrochloride molar concentration is 40nM.
The present invention also provides the preparations of the quality-control product of the lung cancer methylated genes detection kit for stablizing preservation
Method comprises the following steps：
(1) preparation of positive reference product：It methylates the cancerous lung tissue sample of modification 1. choosing and having been identified as target gene
This, extracts DNA and carries out bisulphite modified with purifying；2. the DNA is added in into the DNA solution of purifying in proportion to preserve
Liquid to get；
(2) preparation of negative reference product：It does not methylate the cancerous lung tissue of modification 1. choosing and having been identified as target gene
Sample extracts DNA and carries out bisulphite modified with purifying；2. the DNA is added in into the DNA solution of purifying in proportion to protect
Liquid storage to get；
(3) preparation of detection limit reference material：1. the positive reference product DNA and negative reference product DNA is mixed, make described
The mass ratio that positive reference product DNA accounts for total DNA is 1~5%；2. the DNA is added in into the DNA solution of purifying in proportion to preserve
Liquid to get.
The quality-control product of the lung cancer methylated genes detection kit provided by the invention for stablizing preservation can be used for monitoring lung
The correlated performances such as accuracy, precision and the stability of cancer methylated genes detection kit.
Compared with the prior art, beneficial effects of the present invention are：(1) DNA guarantors are included in quality-control product provided by the invention
Liquid storage can effectively prevent the degradation of dissociative DNA, stable environment is provided for DNA, extend the holding time of quality-control product；(2) originally
The component of invention DNA preservation solution and the usage amount of each component are that inventor draws have by numerous studies and statistical analysis
The effect of dissociative DNA is preserved well；(3) usage amount of DNA preservation solution is inventor by largely grinding in quality-control product of the present invention
Study carefully what is drawn with statistical analysis, can make quality-control product that there is good stability；(4) DNA quality-control products provided by the invention have
Manufacturing cost is low, performance is stable and it is easy to maintain the advantages that, can effectively monitor personnel operation, instrument state and kit it is effective
The factors such as property drastically increase the accuracy and reliability of detection kit.
Specific embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention
It is described further.
Embodiment 1
A kind of embodiment of DNA preservation solution of the present invention includes aurin front three acid ammonium salt, glycine and bestatin hydrochloric acid
Salt；And the molar ratio of the aurin front three acid ammonium salt, glycine and bestatin hydrochloride is 100：50000：0.02.
Embodiment 2
A kind of embodiment of DNA preservation solution of the present invention, aurin front three acid ammonium salt, glycine and bestatin hydrochloride；
And the molar ratio of the aurin front three acid ammonium salt, glycine and bestatin hydrochloride is 500：150000：0.06.
Embodiment 3
A kind of embodiment of DNA preservation solution of the present invention, aurin front three acid ammonium salt, glycine and bestatin hydrochloride；
And the molar ratio of the aurin front three acid ammonium salt, glycine and bestatin hydrochloride is 300：80000：0.04.
Embodiment 4
The present invention can stablize a kind of embodiment of the quality-control product of the lung cancer methylated genes detection kit of preservation, comprising
The gene mass controlled product of p16, RASSF1A, CDH1 and CDH13.
The gene mass controlled product of p16 include positive reference product, negative reference product and detection limit reference material；The positive reference
The DNA preservation solution that product methylate comprising p16 genes described in the DNA of modification and embodiment 3；The negative reference product include
P16 does not methylate the DNA of modification and the DNA preservation solution described in embodiment 3；The detection limit reference material includes p16 genes
DNA, the p16 of modification of methylating does not methylate the DNA of modification and the DNA preservation solution described in embodiment 3, and described
P16 genes methylate modification DNA account for DNA total amounts mass ratio be 2%.
The gene mass controlled product of RASSF1A include positive reference product, negative reference product and detection limit reference material；The positive
The DNA preservation solution that reference material methylates comprising RASSF1A genes described in the DNA of modification and embodiment 3；The negative reference
The DNA preservation solution that product do not methylate comprising RASSF1A described in the DNA of modification and embodiment 3；The detection limit reference material bag
Methylate DNA, RASSF1A of modification of gene containing RASSF1A does not methylate described in the DNA of modification and embodiment 3
DNA preservation solution, and the RASSF1A genes methylate modification DNA account for DNA total amounts mass ratio be 2%.
The gene mass controlled product of CDH1 include positive reference product, negative reference product and detection limit reference material；The positive ginseng
Examine the DNA preservation solution that product methylate comprising CDH1 genes described in the DNA of modification and embodiment 3；The negative reference product bag
Do not methylate the DNA of modification and the DNA preservation solution described in embodiment 3 containing CDH1；The detection limit reference material includes CDH1
Methylate DNA, CDH1 of modification of gene does not methylate the DNA of modification and the DNA preservation solution described in embodiment 3, and
The CDH1 genes methylate modification DNA account for DNA total amounts mass ratio be 2%.
The gene mass controlled product of CDH13 include positive reference product, negative reference product and detection limit reference material；The positive ginseng
Examine the DNA preservation solution that product methylate comprising CDH13 genes described in the DNA of modification and embodiment 3；The negative reference product bag
Do not methylate the DNA of modification and the DNA preservation solution described in embodiment 3 containing CDH13；The detection limit reference material includes
Methylate DNA, CDH13 DNA of the modification and DNA described in embodiment 3 that do not methylate of modification of CDH13 genes is preserved
Liquid, and the CDH13 genes methylate modification DNA account for DNA total amounts mass ratio be 2%.
The component aurin tricarboxylic acid ammonium of DNA preservation solution in the positive reference product, negative reference product and detection limit reference material
Salt molar concentration is 300 μM, and glycine molar concentration is 80mM, and bestatin hydrochloride molar concentration is 40nM.
Embodiment 5
The present invention can stablize a kind of embodiment of the quality-control product of the lung cancer methylated genes detection kit of preservation, comprising
The gene mass controlled product of RASSF1A and CDH13.
The gene mass controlled product of RASSF1A include positive reference product, negative reference product and detection limit reference material；The positive
The DNA preservation solution that reference material methylates comprising RASSF1A genes described in the DNA of modification and embodiment 1；The negative reference
The DNA preservation solution that product do not methylate comprising RASSF1A described in the DNA of modification and embodiment 1；The detection limit reference material bag
Methylate DNA, RASSF1A of modification of gene containing RASSF1A does not methylate described in the DNA of modification and embodiment 1
DNA preservation solution, and the RASSF1A genes methylate modification DNA account for DNA total amounts mass ratio be 1%.
The gene mass controlled product of CDH13 include positive reference product, negative reference product and detection limit reference material；The positive ginseng
Examine the DNA preservation solution that product methylate comprising CDH13 genes described in the DNA of modification and embodiment 1；The negative reference product bag
Do not methylate the DNA of modification and the DNA preservation solution described in embodiment 1 containing CDH13；The detection limit reference material includes
Methylate DNA, CDH13 DNA of the modification and DNA described in embodiment 1 that do not methylate of modification of CDH13 genes is preserved
Liquid, and the CDH13 genes methylate modification DNA account for DNA total amounts mass ratio be 1%.
The component aurin tricarboxylic acid ammonium of DNA preservation solution in the positive reference product, negative reference product and detection limit reference material
Salt molar concentration is 100 μM, and glycine molar concentration is 50mM, and bestatin hydrochloride molar concentration is 20nM.
Embodiment 6
The present invention can stablize a kind of embodiment of the quality-control product of the lung cancer methylated genes detection kit of preservation, comprising
The gene mass controlled product of p16.
The gene mass controlled product of p16 include positive reference product, negative reference product and detection limit reference material；The positive reference
The DNA preservation solution that product methylate comprising p16 genes described in the DNA of modification and embodiment 2；The negative reference product include
P16 does not methylate the DNA of modification and the DNA preservation solution described in embodiment 2；The detection limit reference material includes p16 genes
DNA, the p16 of modification of methylating does not methylate the DNA of modification and the DNA preservation solution described in embodiment 2, and described
P16 genes methylate modification DNA account for DNA total amounts mass ratio be 5%.
The component aurin tricarboxylic acid ammonium of DNA preservation solution in the positive reference product, negative reference product and detection limit reference material
Salt molar concentration is 500 μM, and glycine molar concentration is 150mM, and bestatin hydrochloride molar concentration is 60nM.
Embodiment 7
The present embodiment illustrates the preparation method of quality-control product of the present invention by taking the preparation method of the gene mass controlled product of p16 as an example.
A kind of embodiment of the gene mass controlled product preparation methods of p16, comprises the following steps：
(1) preparation of positive reference product：It methylates the cancerous lung tissue sample of modification 1. choosing and having been identified as p16 genes
This, extracts DNA and carries out bisulphite modified with purifying；2. it is added in into DNA solution after purification described in embodiment 3
DNA preservation solution makes aurin front three acid ammonium salt molar concentration for 300 μM, and glycine molar concentration is 80mM, bestatin hydrochloric acid
Salt molar concentration for 40nM to get；
(2) preparation of negative reference product：It does not methylate the cancerous lung tissue sample of modification 1. choosing and having been identified as p16 genes
This, extracts DNA and carries out bisulphite modified with purifying；2. DNA described in embodiment 3 is added in into DNA solution after purification
Liquid is preserved, makes aurin front three acid ammonium salt molar concentration for 300 μM, glycine molar concentration is 80mM, bestatin hydrochloride
Molar concentration for 40nM to get；
(3) preparation of detection limit reference material：1. the positive reference product DNA and negative reference product DNA is mixed, make described
The mass ratio that positive reference product DNA accounts for total DNA is 2%；2. it adds in DNA described in embodiment 3 into DNA solution after purification to preserve
Liquid makes aurin front three acid ammonium salt molar concentration for 300 μM, and glycine molar concentration is 80mM, bestatin hydrochloride mole
Concentration for 40nM to get.
Embodiment 8
The present embodiment studies the preservation effect of DNA to inventing the DNA preservation solution.
1st, experimental design
By taking the preservation of p16 gene methylations DNA as an example, in order to study DNA preservation solution of the present invention to p16 gene methylations
The preservation effect of DNA, the DNA preservation solution for being utilized respectively the present invention preserves p16 gene methylation DNA at different conditions, and divides
She Zhi glycine component not replaced and not add the control group of DNA preservation solution, the coke of identical mass ratio is added in the control group
Diethyl carbonate water (diethyl pyrocarbonate, DEPC), it is specific as shown in table 1：
1 experimental design of table
2nd, experimental method
It chooses and has been identified as p16 genes and methylate the cancerous lung tissue sample of modification, extraction DNA is equally divided into 10
Group adds DNA preservation solution or DEPC water by the design of table 1, each group p16 gene methylations DNA sample (i.e. p16 bases is prepared
Because of positive reference product).Experiment carries out DNA concentration measure before starting to each group p16 gene masculines reference material, and records.Then will
Each group Sample storage measures each group sample DNA respectively after 2 weeks under the conditions of corresponding by 2100 biological analysers of Agilent
Concentration detects the stability of each group p16 gene masculine reference materials and degradation situation with this.Simultaneously degradation is calculated using the following formula
Rate：DNA concentration × 100% before degradation rate %=(DNA concentration after DNA concentration-experiment before experiment)/experiment.
3rd, experimental result
After 2 weeks, each group p16 gene masculine reference materials are detected, the results are shown in Table 2 for specific experiment：
2 experimental result of table
From above-mentioned experimental result, DNA preservation solution provided by the invention is (real in -20 DEG C (experimental groups 1~3) and 37 DEG C
Test group 4~6) when can significantly improve the stability of DNA, can effectively prevent its degradation during preservation.Wherein, experimental group
3 and experimental group 6 in DNA preservation solution it is best to the preservation effect of DNA.And under identical preservation condition, the present invention is preserved into liquid
After component glycine is changed to epsilon-polylysine, the degradation rate of DNA is significantly higher than (control group 1 and 3) of the invention, while without using
The degradation rate of DNA is also significantly greater than the DNA that liquid is preserved using the present invention in the DNA solution (control group 2 and 4) of present invention preservation liquid
Solution.Especially under 37 DEG C of preservation conditions, after glycine is changed to cysteine, degradation rate is DNA solution after 2 weeks
10.3% (control group 3), at the same do not add the present invention preserve liquid DNA solution degradation rate is up to 16% (control group after 2 weeks
4) the DNA solution degradation rate for, being with the addition of present invention preservation liquid is only 6% or so (experimental group 4~6).DNA of the present invention is added in protect
The DNA of liquid storage does not occur degradation situation within 2 weeks, according to medical instrument accelerated aging tests determine the term of validity basic principle and
Correlation technique understands that the term of validity for adding in the DNA solution of preservative agent is 6 months 3 years.
Embodiment 9
The present embodiment by taking the quality-control product of lung cancer methylated genes detection kit that can stablize preservation in embodiment 4 as an example,
The accuracy of research quality-control product of the present invention.
1st, detection method
Quality-control product of the present invention is detected using high throughput targeting PCR sequencing PCR, evaluates the accuracy of the quality-control product.
2nd, testing result
Testing result is as shown in table 3：
Table 3DNA quality-control product accuracy testing results
From above-mentioned experimental result, positive reference material, negative reference product and inspection in quality-control product described in the embodiment of the present invention 4
The methylated genes surveyed in limit reference material can be detected accurately, and gene type complies fully with.Illustrate reference provided by the invention
Product accuracy rate is 100%.
Embodiment 10
The present embodiment by taking the quality-control product of lung cancer methylated genes detection kit that can stablize preservation in embodiment 4 as an example,
Study the stability of the quality-control product.
1st, detection method
It is analyzed using the quality-control product of three set different production batch of the high-throughput targeting PCR sequencing PCR to randomly selecting.
2nd, testing result
Testing result is as shown in table 4：
The DNA quality-control product Detection of Stability results of 4 three sets of different batches of table
From the above results, the testing results of the three sets of DNA quality-control products randomly selected is accurate and gene type complete one
It causes, illustrates the testing result indifference between three sets of DNA quality-control products, and testing result is completely correct.Due to three sets of DNA of detection
Quality-control product is randomly selected, and can be deduced that the testing result between other DNA quality-control products is also consistent, be illustrated offer of the present invention
DNA quality-control product performances it is very stable.And three sets of repeated reference material precision meet the coefficient of variation (CV, %) no more than 15%.
Embodiment 11
DNA preservation solution of the present invention includes following components：Aurin front three acid ammonium salt, glycine and bestatin hydrochloride.
In order to study influence of the selection of component to DNA preservation solution effect, the present embodiment grinds the selection of DNA preservation solution component
Study carefully.
1st, experimental design
By taking DNA preservation solution is to the preservation of p16 gene methylations DNA as an example, in order to which the DNA for studying different component composition is preserved
The preservation effect of liquid, contrived experiment group 1 and control group 1~7, it is specific as shown in table 5.
Table 5DNA preserves the selection of liquid component
2nd, experimental method
It chooses and has been identified as p16 genes and methylate the cancerous lung tissue sample of modification, extraction DNA is equally divided into 9
Group adds each group DNA preservation solution or DEPC water by the design of table 5, each group p16 gene methylations DNA sample is prepared (i.e.
P16 gene masculines reference material), wherein, the component aurin front three acid ammonium salt of DNA preservation solution, glycine and benzene fourth inhibit in sample
Plain hydrochloride molar concentration is the same as embodiment 4.Experiment carries out DNA concentration measure before starting to each group sample, and records.It then will be each
Group Sample storage preserves 2 weeks in -20 DEG C of conditions, and the degradation rate of DNA in each group sample is calculated using same 8 the method for embodiment.
3rd, experimental result
Experimental result is as shown in table 6：
The DNA preservation solution of 6 different component of table composition is to the experimental result of dissociative DNA preservation effect
From above-mentioned experimental result, DNA preservation solution component provided by the invention is simultaneously comprising aurin front three acid ammonium salt, sweet
(experimental group 1) can just significantly improve the stability of DNA when propylhomoserin and bestatin hydrochloride, effectively prevent it in preservation process
In degradation.The component of DNA preservation solution of the present invention is that inventor draws by numerous studies and statistical analysis, lacks and wherein appoints
Glycine is replaced with other kinds of amino acid (control group 8) not by what one or two kinds of component (control group 1~6)
It can realize the effect of the present invention.
Embodiment 12
DNA preservation solution of the present invention includes aurin front three acid ammonium salt, glycine and bestatin hydrochloride；And the gold
The molar ratio of smart front three acid ammonium salt, glycine and bestatin hydrochloride is (100~500)：(50000~150000)：
(0.02~0.06).In order to study influence of the use ratio of component to DNA preservation effects, the present embodiment is to DNA preservation solution group
The use ratio divided is studied.
1st, experimental design
In order to study influence of the component use ratio to DNA preservation solution preservation effect, contrived experiment group 2~4 and control group 8
~10, it is specific as shown in table 7.
The use ratio of 7 component of table
2nd, experimental method
It chooses and has been identified as p16 genes and methylate the cancerous lung tissue sample of modification, extraction DNA is equally divided into 6
Group adds each group DNA preservation solution or DEPC water by the design of table 7, each group p16 gene methylations DNA sample is prepared (i.e.
P16 gene masculines reference material).Experiment carries out DNA concentration measure before starting to each group sample, and records.Then by each group sample
It is stored in -20 DEG C of conditions to preserve 2 weeks, the degradation rate of DNA in each group sample is calculated using same 8 the method for embodiment.
3rd, experimental result
Experimental result is as shown in table 8：
The use ratio of 8 component of table is to the experimental result of DNA preservation solution preservation effect
From above-mentioned experimental result, aurin front three acid ammonium salt, glycine and benzene fourth in DNA preservation solution provided by the invention
The molar ratio of inhibin hydrochloride is (100~500)：(50000~150000)：DNA can be effectively prevented when (0.02~0.06)
Degradation (experimental group 2~4) during preservation.The use ratio of DNA preservation solution component of the present invention is inventor by a large amount of
What research and statistical analysis were drawn, use ratio only within the scope of the present invention can just make DNA preservation solution have guarantor well
Effect is deposited, increasing or decreasing for component use ratio can not significantly reduce DNA preservation solution of the present invention within the scope of the present invention
Preservation effect improves the degradation rate (control group 8~9) of DNA during preserving.Component does not make in proportion of the present invention for other
Influence experimental result of the dosage to DNA preservation solution preservation effect is similar with the present embodiment, and specific data are omitted.
Embodiment 13
The component aurin front three acid ammonium salt molar concentration of DNA preservation solution is 100-500 μM in reference material of the present invention, glycine
Molar concentration is 50-150mM, and bestatin hydrochloride molar concentration is 20-60nM.It is preserved to study DNA in reference material
Influence of the molar concentration of liquid component to preservation effect, the present embodiment to the molar concentration of DNA preservation solution component in reference material into
Research is gone.
1st, experimental design
By taking aurin front three acid ammonium salt as an example, researching DNA preserves the molar concentration of liquid component in the sample to preservation effect
It influences, contrived experiment group 5~8 and control group 11~13, specific as shown in table 9, glycine molar concentration and the suppression of benzene fourth in sample
The plain hydrochloride molar concentration of system is the same as embodiment 6.
The molar concentration design of 9 aurin front three acid ammonium salt of table in the sample
2nd, experimental method
It chooses and has been identified as p16 genes and methylate the cancerous lung tissue sample of modification, extraction DNA is equally divided into 7
Group adds DNA preservation solution each component or DEPC water by the design of table 9, each group p16 gene methylations DNA sample is prepared (i.e.
P16 gene masculines reference material).Experiment carries out DNA concentration measure before starting to each group sample, and records.Then by each group sample
It is stored in -20 DEG C of conditions to preserve 2 weeks, the degradation rate of DNA in each group sample is calculated using same 8 the method for embodiment.
3rd, experimental result
Experimental result is as shown in table 10：
Influence experimental result of the 10 Gold Samples essence front three acid ammonium salt mole of table to preservation effect
From above-mentioned experimental result, mole of the component aurin front three acid ammonium salt of DNA preservation solution of the present invention in the sample
Concentration can effectively improve the stability of DNA when being 100~500 μM, prevent its degradation (experimental group 5~8) during preservation,
Wherein, when the molar concentration when aurin front three acid ammonium salt in the sample is 300 μM, preservation effect is preferably (experimental group 8).The present invention
The molar concentration of DNA preservation solution each component in the sample is that inventor draws by numerous studies and statistical analysis, is only existed
The scope of the invention can just make the good preservation effect of acquirement, not DNA preservation solution component molar concentration within the scope of the present invention
The preservation effect of DNA preservation solution of the present invention can be significantly reduced by increasing or decreasing, and the degradation rate for improving DNA during preserving is (right
According to group 11~12).The influence of glycine molar concentration and bestatin hydrochloride molar concentration to DNA preservation effects in sample
Experimental result is similar with the present embodiment, and specific data are omitted.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, those of ordinary skill in the art should
Understand, technical scheme can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And scope.
Claims (10)
1. a kind of DNA preservation solution, which is characterized in that include aurin front three acid ammonium salt, glycine and bestatin hydrochloride；And
The molar ratio of the aurin front three acid ammonium salt, glycine and bestatin hydrochloride is (100~500)：(50000~
150000)：(0.02~0.06).
2. DNA preservation solution according to claim 1, which is characterized in that the aurin front three acid ammonium salt, glycine and benzene fourth
The molar ratio of inhibin hydrochloride is 300：80000：0.04.
3. according to purposes of claim 1~2 any one of them DNA preservation solution in DNA preservations.
4. purposes according to claim 3, which is characterized in that the DNA preservation solution is used to prepare methylated genes detection
The quality-control product of kit.
A kind of 5. quality-control product for the lung cancer methylated genes detection kit for stablizing preservation, which is characterized in that the quality-control product
Include following components：Positive reference product, negative reference product and detection limit reference material；
The positive reference product include target gene methylate modification DNA and DNA preservation solution described in claim 1；
The negative reference product include target gene do not methylate modification DNA and DNA described in claim 1 preserve
Liquid；
The detection limit reference material does not methylate modification comprising the methylate DNA of modification, target gene of target gene
DNA and DNA preservation solution described in claim 1, and the methylate DNA of modification of the target gene accounts for DNA total amounts
Mass ratio is 1~5%.
6. the quality-control product of the lung cancer methylated genes detection kit according to claim 5 for stablizing preservation, feature
It is, the quality-control product includes at least one of gene mass controlled product of target gene p16, RASSF1A, CDH1 and CDH13.
7. the quality-control product of the lung cancer methylated genes detection kit according to claim 5 for stablizing preservation, feature
It is, the component aurin front three acid ammonium salt of DNA preservation solution rubs in the positive reference product, negative reference product and detection limit reference material
Your concentration is 100-500 μM, and glycine molar concentration is 50-150mM, and bestatin hydrochloride molar concentration is 20-60nM.
8. the quality-control product of the lung cancer methylated genes detection kit according to claim 7 for stablizing preservation, feature
It is, the component aurin front three acid ammonium salt of DNA preservation solution rubs in the positive reference product, negative reference product and detection limit reference material
Your concentration is 300 μM, and glycine molar concentration is 80mM, and bestatin hydrochloride molar concentration is 40nM.
9. the Quality Control of the lung cancer methylated genes detection kit of preservation can be stablized according to claim 5~8 any one of them
The preparation method of product, which is characterized in that comprise the following steps：
(1) preparation of positive reference product：It methylates the cancerous lung tissue sample of modification, carries 1. choosing and having been identified as target gene
It takes DNA and carries out bisulphite modified with purifying；2. DNA described in claim 1 is added in into the DNA solution of purifying in proportion
Preserve liquid to get；
(2) preparation of negative reference product：Identify that hot spot does not occur and methylates the cancerous lung tissue sample of modification for target gene 1. choosing
This, extracts DNA and carries out bisulphite modified with purifying；2. claim 1 institute is added in into the DNA solution of purifying in proportion
State DNA preservation solution to get；
(3) preparation of detection limit reference material：1. the positive reference product DNA and negative reference product DNA is mixed, make the positive
The mass ratio that reference material DNA accounts for total DNA is 1~5%；2. it is added in proportion into the DNA solution of purifying described in claim 1
DNA preservation solution to get.
10. the Quality Control of the lung cancer methylated genes detection kit of preservation can be stablized according to claim 5~8 any one of them
Purposes of the product in monitoring lung cancer methylated genes detection kit accuracy, precision and stability.
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