CN109022417A - A kind of paramagnetic particle method nucleic acid extraction conversion reagent box and its application method - Google Patents
A kind of paramagnetic particle method nucleic acid extraction conversion reagent box and its application method Download PDFInfo
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Abstract
The present invention provides a kind of paramagnetic particle method nucleic acid extraction conversion reagent box and its application methods.The kit include lysate, Proteinase K, magnetic bead liquid, conversion reagent, nuclease protection agent, in conjunction with liquid, cleaning solution I, cleaning solution II and eluant, eluent.The application method of the kit is comprising steps of (1) cracks；(2) it converts；(3) it is layered；(4) it combines；(5) primary washing；(6) secondary wash；(7) dry；(8) it elutes.Kit of the present invention and its application method are able to achieve sample and complete conversion while carrying out automatic nucleic acid extraction.Sample just starts to convert after being cleaved release nucleic acid, and after the conversion was complete by combining liquid to make nucleic acid and Separation of Proteins, then purified with nucleic acid of the paramagnetic particle method to methylation, initial denaturation step when being detected again with the subsequent qPCR to sample in test procedure is entirely extracted conversion journey and eliminates the purification process in protokaryon acid extraction step come the de- sulfo group step for replacing front to omit.
Description
Technical field
The present invention relates to field of biotechnology, in particular to a kind of paramagnetic particle method nucleic acid extraction conversion reagent box, and use
The method of the kit.
Background technique
Epigenetics is to study in the case where the nucleotide sequence of gene does not change, the expression and regulation of gene
Heritable variation a science of heredity subdiscipline, there are many phenomenon that epigenetic, such as the gene silencing that RNA is mediated, group
It is protein modified etc..Methylation is one of the epigenetic of biology, and closely related with the generation of a variety of diseases.The study found that DNA
The generation of abnormal methylation and tumour, development, cell carcinogenesis have it is close contact, in recent years using methylation analysis as representative
Epigenetics and epigenomics are more active.
DNA methylation (DNA methylation) refers under DNA methylation transferase (DNMT) catalysis, with S- adenosine
Methionine is methyl donor, chemical modification process active methyl being transferred in DNA chain in particular bases.DNA methylation
Typically occurring in the site CpG, (cytosine-phosphate-guanine site, i.e., be close to the position of guanine after cytimidine in DNA sequence dna
Point).It is 5-methylcytosine through dnmt rna catalysis Cytosines.About 80%~90% position CpG in human gene
Point has been methylated, but in certain specific regions, is not methylated if the island CpG rich in cytimidine and guanine.This with
It is related comprising the promoter in 56% mammalian genes including all wide expression genes.1%~2% human gene
Group is CpG groups, and CpG methylation is inversely proportional with transcriptional activity.DNA methylation is repaired as a kind of apparent (epigenetic)
Decorations, in the case where not changing DNA sequence dna, to the stability of individual growth and development, gene expression pattern and genome
Important regulating and controlling effect is played, and this modification can stable delivery during development and cell Proliferation.
The mechanisms such as many hospitals, inspection body, institute regard DNA methylation assay and analysis as research emphasis now,
But since sample size to be analyzed is larger, the time-consuming and laborious and traditional bisulfite method for transformation of manual operations is taken a long time, and is grasped
It is cumbersome to make step, conversion condition is very harsh, needs to provide the bisulfite of high concentration in reaction system to complete non-first
The conversion of base cytimidine, but also it is effective in order to carry out to need to guarantee that double chain DNA molecule is in unwinding state for a long time
Conversion.Therefore, it is necessary to high salt, the system environment of high temperature and low ph value.But this environment is also easy to produce a large amount of active oxygen in simultaneously
Group, this active oxygen can make DNA that fragmentation occur and degrade, therefore will lead to PCR and subsequent analysis technology after conversion
Sensitivity decrease, while also resulting in the rate of recovery decline of DNA.Thus it is badly in need of developing a kind of conversion of high-efficient automatic at present
Equipment and matched reagent box.
Summary of the invention
To solve the above problems, the present invention provides a kind of paramagnetic particle method nucleic acid extraction conversion reagent box: the kit can be mating
Commercially available nucleic acid automatic extracting instrument uses, to enormously simplify, sample nucleic acid is extracted and the operating procedure of conversion, realization sample exist
While carrying out automatic nucleic acid extraction, dexterously sample nucleic acid is extracted and conversion process is combined into one.It is described to be combined into one
It is not that simply will extract and convert two step simple superposition, but the mode taken while carried out, i.e. sample are cleaved
Just start to convert after release nucleic acid, and after the conversion was complete, omit highly basic and take off sulfo group step, by making nucleic acid and egg in conjunction with liquid
White matter separation, is then purified with nucleic acid of the paramagnetic particle method to methylation, then while being detected with the subsequent qPCR to sample tests journey
Initial denaturation step in sequence entirely extracts conversion journey and eliminates protokaryon acid extraction step come the de- sulfo group step for replacing front to omit
In purification process, time and materials are greatly saved, while decreasing the loss of methylated nucleic acid, reduce human error
And experimental error.
Paramagnetic particle method nucleic acid extraction conversion reagent box disclosed in this invention, the kit include lysate, Proteinase K,
Magnetic bead liquid, conversion reagent, nuclease protection agent, in conjunction with liquid, cleaning solution I, cleaning solution II and eluant, eluent.
Wherein, the lysate include Tris-HCl, it is guanidinium isothiocyanate, sodium hydroxide, NonidetP40, Sodium azide, different
Propyl alcohol, Triton X-100, catechin and Carrier RNA.
The magnetic bead liquid includes Tris-HCl, EDTA-2Na and magnetic bead.The content of magnetic bead is 50mg/ in the magnetic bead liquid
Ml, the magnetic bead is nano magnetic particle made of super suitable ferroso-ferric oxide or super suitable di-iron trioxide, and appearance is repaired by surface
It is decorated with the silicon dioxide coated of hydroxy or carboxy.
The conversion reagent includes bisulfite and/or bisulfites and urea and hydroquinone；The heavy Asia
Sulfate includes sodium bisulfite, and the bisulfites includes sodium hydrogensulfite, magnesium bisulfite, ammonium bisulfite, partially Asia
One of potassium acid sulfate, sodium metabisulfite are a variety of.The molar concentration of bisulfite and/or bisulfites be 4~
9mol/L, preferably 7mol/L；The molar concentration of the urea is 4~7mol/L, preferably 6mol/L；The hydroquinone
Molar concentration is 10mmol/L.
The cleaning solution I includes Tris-HCl, ethyl alcohol, dithiothreitol (DTT) (DTT).
The cleaning solution II includes sodium acetate and ethyl alcohol.
The nuclease protection agent includes Trolox, tetrahydrofuran chaff
Alcohol, tetrahydropyrimidine, dithiothreitol (DTT), imidazolidinyl urea, vitamin, BSA, glycerol, one of glucose or a variety of.The core
The protectant mass percentage concentration of acid is 5~10%, preferably 8%.
The eluant, eluent includes Tris-HCl, EDTA-2Na and Carrier RNA.
The combination liquid is the mixed liquor of different sulfuric acid cyanoguanidine, guanidine hydrochloride, sodium perchlorate, isopropanol and dehydrated alcohol.
Present invention simultaneously discloses the application methods of above-mentioned paramagnetic particle method nucleic acid extraction conversion reagent box, comprising the following steps:
(1) it cracks: lysate and Proteinase K being added in plasma sample, 1000~1400rpm concussion mixes 15 seconds, blood
The volume ratio of slurry and lysate is 1:(0.1~0.4), the volume ratio of blood plasma and Proteinase K is 1:(0.08~0.125)；
(2) it converts: conversion reagent and nuclease protection agent, the blood relative to 1000 μ L being added into the mixed liquor of step (1)
Slurry, the additional amount of the conversion reagent are 90 μ L, and the additional amount of the nuclease protection agent is 10 μ L, vortex oscillation solution, 55 DEG C
60min is converted under conditions of~70 DEG C；
(3) it combines: being added into step (2) conversion fluid and combine liquid, 20~40s of vortex oscillation adds magnetic bead liquid, is vortexed
Oscillation stands 8~15min after mixing, and sucks supernatant, retains subnatant, the volume ratio of the combination liquid and subnatant be (2~
4): 1, the volume of the magnetic bead and the volume ratio of blood plasma add as (1~2): 50, it is placed in magnetic separator and is clarified up to solution, inhaled
Remove supernatant；
(4) primary washing: cleaning solution I is added in magnetic bead, 1~3min of vortex oscillation is placed in magnetic separator up to molten
Liquid clarification, sucks supernatant, and the number of rinsing is 1~3 time；Preferably, I additional amount of cleaning solution and blood of the first time washing
The ratio of liquid is (2~4): 1, I additional amount of cleaning solution of second of washing and the ratio of blood are (0.5~1.5): 1；
(5) secondary wash: being added 600 μ L cleaning solutions II and magnetic bead be resuspended, and centrifuge tube is placed on magnetic frame by of short duration centrifugation, to
Magnetic bead discards waste liquid after adsorbing completely；
(6) dry: most liquid is removed in of short duration centrifugation as far as possible, and drying at room temperature 5 minutes；
(7) elute: into the solution after drying be added 30~80 μ L eluant, eluents, shaken at room temperature mix 5 minutes, it is of short duration from
Centrifuge tube is placed on magnetic frame by the heart, and supernatant is transferred in new centrifuge tube after magnetic bead adsorbs completely, and marker samples are related
Information is saved in~20 DEG C.
Include in lysate described in step (1): the concentration of Tris-HCl is 15mmol/L, pH 3.3；Isothiocyanic acid
The concentration of guanidine is 10mmol/L；Naoh concentration is 5mmol/L；The concentration of Nonidet P 40 is 5%；The concentration of Sodium azide
It is 0.01%；The concentration of isopropanol is 85%；The concentration of Triton X-100 is 4%；The concentration of catechin is 2%；Carrier
The concentration of RNA is 0.5 μ g/ μ l.
Wherein, include in conversion reagent described in step (2): sodium bisulfite, sodium hydrogensulfite, magnesium bisulfite,
At least one of ammonium bisulfite, inclined potassium bisulfite, sodium metabisulfite or a variety of 4~9M of concentration (adjustment pH is 5~6), 4
~6M urea, hydroquinone 10mM.
Include in nuclease protection agent described in step (2): 6- hydroxyl -2,5,7,8- tetramethyl benzodihydropyran -2-
Carboxylic acid, tetrahydrofuran furfuryl alcohol, tetrahydropyrimidine, dithiothreitol (DTT), to biphenol, imidazolidinyl urea, vitamin, BSA, glycerol, Portugal
Grape sugar is one or more.Protectant mass percent concentration is that 5-10% (reduces drop of the DNA in bisulfite environment
Solution).
Combination liquid described in step (3) is different sulfuric acid cyanoguanidine, guanidine hydrochloride, sodium perchlorate (0.5~6M), isopropanol and anhydrous
The mixed liquor of ethyl alcohol (10~500mM).
Step (4) cleaning solution I is the ethyl alcohol that mass percent concentration is 70~80%, and the concentration of Tris-HCl is
65mmol/L, the concentration of dithiothreitol (DTT) (DTT) are 0.5%
Step (5) cleaning solution II is the ethyl alcohol that mass percent concentration is 75% and the acetic acid that concentration is 30mmol/L
Sodium solution.
Compared with prior art, kit provided by the invention has the advantage that
1, methylation conversion reagent of the present invention is different from existing method for transformation, does not need before carrying out to nucleic acid
The sodium hydroxide of phase is denaturalized de- sulfo group processing, and is different from the alternating temperature conversion condition that commercial reagents use on the market, can be in perseverance
It is converted under the conditions of temperature；And desulfonate step is carried out without using the sodium hydroxide being all made of currently on the market in purification process
Suddenly, it replaces taking off the step for initial denaturation 10 minutes in test procedure when but detecting additionally by the subsequent qPCR to sample
The step of sulfo group, thus can save the conversion process time, keep away while obtaining high conversion, high quality, high-purity nucleic acid
Exempt from the de- damage of sulfo group reagent (generally strong base solution such as sodium hydroxide solution etc.) to nucleic acid, the waste of detection reagent and to ring
The pollution in border.Entire transformation time only needs 40~60min；Transformation time is short, conversion temperature is low, operates easier.
2, paramagnetic particle method purified reagent of the present invention utilizes height suitable for the nucleic acid purification after methylated nucleic acid conversion
Salt low ph value carries out isolating and purifying for nucleic acid, then is eluted by less salt high ph-values, the spy with high-purity, high recovery efficiency
Point, and whole process of purification does not need carrier RNA yet, and can carry out purification process at normal temperature, is not necessarily to any high temperature
It is incubated for, a kind of cleaning solution is only combined in the same time, only carries out once washing process, to obtain the nucleic acid of high-purity, finally
Elution can also be eluted at normal temperature；Have the advantages that easy to operate, raising extraction efficiency and DNA purity.
3, the method that DNA of the present invention is extracted, sulphite conversion and purify, extracts constant temperature from upstream for nucleic acid
It converts and Beads enrichment purifying downstream effectively combines, to enormously simplify sample nucleic acid extraction and bisulfite
The operating procedure of conversion realizes that sample nucleic acid while carrying out automatic nucleic acid extraction, is dexterously extracted and converted by sample
Process is combined into one, after the completion of conversion, by combine liquid nucleic acid and Separation of Proteins, then with paramagnetic particle method to methylated nucleic acid into
Row purifying, this process eliminate the purification process in protokaryon acid extraction step, time and materials are greatly saved, while also reducing
The loss of methylated nucleic acid, reduces human error and experimental error is conducive to the full-automation and standardization of methylation research
Operation；
4, the kit significantly improves operation convenience compared with existing similar product, convenient for automatic in the market
Change instrument integration, is suitable for Molecular Detection.
Detailed description of the invention
Fig. 1 is the detection curve figure in embodiment 2.
Fig. 2 be embodiment 5 paramagnetic particle method nucleic acid extraction conversion reagent box of the present invention cooperation fluorescence quantitative PCR detection and
The amplification comparison diagram of commercially available nucleic acid extraction kit, nucleic acid methylation conversion reagent box cooperation fluorescence quantitative PCR detection.
Specific embodiment
The following describes the present invention further through the description of specific embodiments, but it is to limit of the invention that this, which is not,
System, those skilled in the art's basic thought according to the present invention, various modifications may be made and improves, but without departing from this
The basic thought of invention, is all within the scope of the present invention.
Embodiment 1, paramagnetic particle method nucleic acid extraction conversion reagent box and its application method
The paramagnetic particle method nucleic acid extraction conversion reagent box includes: lysate, Proteinase K, magnetic bead liquid, conversion reagent, nucleic acid
Protective agent, in conjunction with liquid, cleaning solution I, cleaning solution II and eluant, eluent.The lysate includes Tris-HCl, guanidinium isothiocyanate, hydrogen-oxygen
Change sodium, Nonidet P 40, Sodium azide, isopropanol, Triton X-100, catechin and Carrier RNA.The magnetic bead liquid packet
Include Tris-HCl, EDTA-2Na and magnetic bead.The content of magnetic bead is 50mg/ml in the magnetic bead liquid, and the magnetic bead is super suitable four oxygen
Change nano magnetic particle made of three-iron or super suitable di-iron trioxide, and appearance is had the titanium dioxide of hydroxy or carboxy by surface modification
Silicon coating.The conversion reagent includes bisulfite and/or bisulfites and urea and hydroquinone；The heavy Asia
Sulfate includes sodium bisulfite, and the bisulfites includes sodium hydrogensulfite, magnesium bisulfite, ammonium bisulfite, partially Asia
One of potassium acid sulfate, sodium metabisulfite are a variety of.The molar concentration of bisulfite and/or bisulfites be 4~
9mol/L, preferably 7mol/L；The molar concentration of the urea is 4~7mol/L, preferably 6mol/L；The hydroquinone
Molar concentration is 10mmol/L.The cleaning solution I includes Tris-HCl, ethyl alcohol, dithiothreitol (DTT) (DTT).The cleaning solution II
Including sodium acetate and ethyl alcohol.The nuclease protection agent includes Trolox,
Tetrahydrofuran furfuryl alcohol, tetrahydropyrimidine, dithiothreitol (DTT), imidazolidinyl urea, vitamin, BSA, glycerol, one of glucose or
It is a variety of.The mass percentage concentration of the nuclease protection agent is 5~10%, preferably 8%.The eluant, eluent include Tris-HCl,
EDTA-2Na and Carrier RNA.
The application method of the kit includes the following steps:
1, DNA nucleic acid extraction converts:
(1) it cracks: lysis buffer and Proteinase K being added in 1mL plasma sample, 1200rpm concussion mixes 15 seconds, institute
The volume ratio of blood plasma and the lysis buffer is stated as 10:1, the volume ratio of the blood plasma and the Proteinase K is 10:1.
(2) it converts: 90 μ L of conversion reagent and 10 μ L of nuclease protection agent, vortex oscillation being added into the mixed liquor of step (1)
Solution, is converted under the conditions of 60min by 64 DEG C.
(3) it combines: being added and combine liquid 2.5mL, vortex oscillation 30s, add 60 μ L of magnetic bead liquid, vortex oscillation is quiet after mixing
10min is set, magnetic separator is placed in and is clarified up to solution, suck supernatant.
(4) primary washing: I 1mL of cleaning solution is added into step (3) remaining subnatant, vortex oscillation 2min is placed in magnetic
Property separator clarified up to solution, suck supernatant, the number of rinsing is 2 times.
(5) secondary wash: being added 600 μ L cleaning solutions II and magnetic bead be resuspended, and centrifuge tube is placed on magnetic frame by of short duration centrifugation, to
Magnetic bead discards waste liquid after adsorbing completely.
(6) dry: most liquid is removed in of short duration centrifugation as far as possible, and drying at room temperature 5 minutes；
(7) it elutes: being added 40 μ L eluant, eluents into the solution after drying, shaken at room temperature mixes 5min, of short duration centrifugation, will be from
Heart pipe is placed on magnetic frame, supernatant is transferred in new centrifuge tube after magnetic bead adsorbs completely, marker samples relevant information,
It is saved in -20 DEG C.
Embodiment 2, step of converting optimization
6 identical samples are taken to be divided into two groups of A, B respectively, A group sample is converted through kit described in embodiment 1, B group sample
This carries out nucleic acid extraction by kit step of the present invention, and is added at the de- sulfo group in routine transformation process in its step of converting
Step conversion is managed, i.e., highly basic room temperature is added after primary washing and is incubated for 15 minutes to carry out de- sulfo group processing, other steps
It operates identical as step of converting in kit of the present invention.Two groups of sample of nucleic acid after inverted, are detected according to the following steps:
A) following reagent is mixed in PCR pipe:
| 2×PCR Mix | 20μl |
| Taq enzyme | 0.5μl |
| Convert DNA solution | 10μl |
| NF water | 9.5μl |
| It is total | 40μl |
B) following procedure is executed in ABI7500 fluorescence quantitative PCR instrument:
Testing result is as shown in Figure 1, two groups of samples of A, B are identical, except B group sample increases highly basic room temperature incubation in 15 minutes with de-
Sulfo group processing is outer, other experiment conditions are identical, by experiment map analysis it is found that conventional highly basic is incubated for de- to sample
Sulfo group processing is expected kit of the present invention in use, the effect that testing result optimizes can not be obtained, and is not carried out instead strong
The pattern detection figure that alkali is incubated for has obvious S type curve, and fluorescent value is obvious, and amplification is more preferable.Illustrate using reagent of the present invention
When box, the step for sample after conversion takes off sulfo group, does not simultaneously have in addition to increase highly basic and is incubated at room temperature step, through subsequent analysis it is found that
In 95 DEG C of reactions in 10 minutes of qPCR thermal starting, it is not only to carry out sample the initial denaturation of qPCR reaction, while can also rise
To the treatment effect for taking off sulfo group to sample.It is incubated for take off the reaction time of sulfo group, simultaneously to save highly basic in conversion process
Also obtain good reaction effect.
Embodiment 3, different conversion temperatures and transformation time influence the changing effect of sample of nucleic acid
Implementation is all made of by different groups of other conversion temperatures of setting and transformation time (being specifically shown in the following table 1), remaining step
The application method of example 1 carries out nucleic acid extraction conversion to same batch sample.12 groups pass through different conversion temperatures and transformation time
Method obtains DNA (being successively denoted as A1-A4, B1-B4, C1-C4) and carries out DNA methylation assay, testing result using real-time fluorescence PCR
It is shown in Table 2.
1 different experiments group conversion condition facilities of table
Table 2 is through different experiments group conversion condition treated methylate DNA testing result
As can be seen that, using real time fluorescent PCR method, using MSP primer detection methyl in different conversion temperature methods
Change situation, the Ct value of each group is substantially suitable, it is possible to which conversion can be normally carried out at 55~70 DEG C by verifying this conversion reagent, together
When, by being incubated for different under the conditions of identical temperature often to the impact analysis of changing effect it is found that using conversion of the invention
Reagent carries out constant temperature conversion, it is only necessary to good effect can be obtained within 30 minutes, transformation time is greatly saved.Relative to traditional
For Temp change method, the present invention is simpler using the method for constant temperature, easy to operate, and cheap, easy thermostatical instrument can be used to carry out phase
Close experimental study.
Embodiment 4, adjustment bisulfite use concentration
By the way that different groups of other conversion reagent concentration (1M~9M) are arranged, remaining step is all made of the user of embodiment 1
Method carries out nucleic acid extraction conversion to same batch sample.Five groups obtain DNA by different conversion temperatures and transformation time method
(being successively denoted as A-H) carries out DNA methylation assay using real-time fluorescence PCR, and testing result is shown in Table 3.
Table 3 is through various concentration conversion reagent treated methylate DNA testing result
As can be seen from Table III, DNA purifying is carried out using different purified reagents using after same transform mode, then adopted
With real time fluorescent PCR method, using MSP primer detection methylation status, the Ct value of each group is substantially suitable.Turn relative to traditional
For conversion reagent used in change method, the conversion reagent that the present invention uses combines the nucleic acid at concentration lower (1~5M)
Changing effect is not substantially reduced, and illustrates the conversion reagent high conversion efficiency in the present invention, and in practical applications, can be used low
Concentration Reagent carries out conversion reaction, while save the cost, can avoid reagent abuse to the adverse effect of tester's bring and ring
Border pollution.
Embodiment 5, the detection of nucleic acids after conversion
It for the quality for verifying DNA after purification, needs to detect by methylation status of PTEN promoter reaction, take with a batch totally 3
Sample and a negative control are respectively adopted 1 step of embodiment and carry out nucleic acid extraction conversion and commercial product (Bo Er is sincere) core
Sour extracts kit, nucleic acid methylation conversion reagent box are handled, and specific steps are operated in strict accordance with product description.Through this
Invention and commercial product transformed nucleic acid are uniformly diluted to 10ng/ μ L after ultraviolet specrophotometer measures concentration, then carry out fluorescence
Quantitative PCR detection, detailed process are as follows:
Reaction system is prepared: 32 μ l+ enzyme of PCR mix, 1 μ l+BisDNA30 μ l；
Response procedures are as follows:
Initial denaturation 10min or 20min can not only activate thermal starting enzyme, can also dispose what step of converting was not handled
Sulfo group (sulfo group can reduce enzymatic activity, influence detection sensitivity).
Commercial reagents comparison: Septin9 gene methylation detection kit；
Product batch number: 201711003 (Bo Er is sincere)；
Reaction system is prepared: 32 1.6 μ l+BisDNA of μ l+ enzyme of PCR mix, 30 μ l；
Response procedures are as follows:
Testing result is determined by following principle:
This technology inventor does comparative experiments with common kit in the market and vulcanization process provided by the invention, as a result
As shown in Fig. 2, negative shadow will not be generated to subsequent reactions by demonstrating DNA methylation vulcanization conversion modification reaction provided by the invention
It rings, and from the point of view of testing result, 1-8 is respectively the knot of 3 samples and 1 negative control using the detection of different reagents in figure
Fruit, wherein 1,3,5,8 is respectively the result figure detected using kit of the present invention；2,4,6,7 is using contrast agent boxes
Testing result, the comparative analysis from figure is it is found that when to same pattern detection, the testing result accuracy of kit of the present invention and city
It is consistent to sell kit test result, and curve fluorescent value is higher, detection figure S type more standard is better than commercial reagent box effect；It is logical
It crosses and comparative analysis is detected to negative sample it is found that detecting using kit of the present invention and contrast agent box without S type curve, it is comprehensive
It closes and considers, illustrate that present invention conversion and purified reagent compared with the box testing result of commercial reagent, are better than commercial product testing result.
And reagent of the invention can be converted under thermostatic effect, and paramagnetic particle method is combined to extract, and the extracting method is easy to operate
Detection, fast, the features such as extraction efficiency is high, using integrating automatic operation.
Claims (10)
1. a kind of paramagnetic particle method nucleic acid extraction conversion reagent box, it is characterised in that: the kit includes lysate, Proteinase K, magnetic
Pearl liquid, conversion reagent, nuclease protection agent, in conjunction with liquid, cleaning solution I, cleaning solution II and eluant, eluent.
2. paramagnetic particle method nucleic acid extraction conversion reagent box according to claim 1, it is characterised in that: the lysate includes
Tris-HCl, guanidinium isothiocyanate, sodium hydroxide, Nonidet P 40, Sodium azide, isopropanol, Triton X-100, catechin and
Carrier RNA；Preferably, the molar concentration of Tris-HCl is 15mmol/L, pH 3.3；Preferably, guanidinium isothiocyanate rubs
Your concentration is 10mmol/L；Preferably, the molar concentration of sodium hydroxide is 5mmol/L；Preferably, the quality hundred of NonidetP40
Dividing specific concentration is 5%；Preferably, the mass percent concentration of Sodium azide is 0.01%；Preferably, the mass percent of isopropanol
Concentration is 85%；Preferably, the mass percent concentration of Triton X-100 is 4%；Preferably, the mass percent of catechin
Concentration is 2%；Preferably, the concentration of Carrier RNA is 0.5 μ g/ μ l.
3. paramagnetic particle method nucleic acid extraction conversion reagent box according to claim 1, it is characterised in that: the magnetic bead liquid includes
Tris-HCl, EDTA-2Na and magnetic bead；Preferably, the content of magnetic bead is 50mg/ml in the magnetic bead liquid, and the magnetic bead is super suitable
Nano magnetic particle made of ferroso-ferric oxide or super suitable di-iron trioxide, and appearance has the two of hydroxy or carboxy by surface modification
Silica coating.
4. paramagnetic particle method nucleic acid extraction conversion reagent box according to claim 1, it is characterised in that: the conversion reagent includes
Bisulfite and/or bisulfites and urea and hydroquinone；The bisulfite includes sodium bisulfite, institute
State bisulfites include sodium hydrogensulfite, magnesium bisulfite, ammonium bisulfite, inclined potassium bisulfite, in sodium metabisulfite
It is one or more.
5. paramagnetic particle method nucleic acid extraction conversion reagent box according to claim 1, it is characterised in that: the cleaning solution I includes
Tris-HCl, ethyl alcohol, dithiothreitol (DTT)；Preferably, the molar concentration of Tris-HCl is 65mmol/L；Preferably, the matter of ethyl alcohol
Measuring percent concentration is 70~80%；Preferably, the mass percent concentration of dithiothreitol (DTT) is 0.5%.
6. paramagnetic particle method nucleic acid extraction conversion reagent box according to claim 1, it is characterised in that: the cleaning solution II includes
Sodium acetate and ethyl alcohol；Preferably, the molar concentration of the sodium acetate is 30mmol/L；Preferably, the quality percentage of the ethyl alcohol
Specific concentration is 75%.
7. paramagnetic particle method nucleic acid extraction conversion reagent box according to claim 1, it is characterised in that: the nuclease protection agent packet
Include Trolox, tetrahydrofuran furfuryl alcohol, tetrahydropyrimidine, dithiothreitol (DTT),
Imidazolidinyl urea, vitamin, BSA, glycerol, one of glucose or a variety of；The mass percentage concentration of the nuclease protection agent
It is 5~10%, preferably 8%.
8. paramagnetic particle method nucleic acid extraction conversion reagent box according to claim 1, it is characterised in that: the eluant, eluent includes
Tris-HCl, EDTA-2Na and Carrier RNA.
9. paramagnetic particle method nucleic acid extraction conversion reagent box according to claim 1, it is characterised in that: the combination liquid is different sulphur
Sour cyanoguanidine, guanidine hydrochloride, sodium perchlorate, isopropanol and dehydrated alcohol mixed liquor.
10. a kind of application method of magnetic bead nucleic acid extraction conversion reagent box, comprising the following steps:
(1) it cracks: lysate and Proteinase K being added in plasma sample, concussion mixes, the volume ratio of plasma sample and lysate
For 1:(0.1~0.4), the volume ratio of plasma sample and Proteinase K is 1:(0.08~0.125)；The condition that the concussion mixes
Preferably 1000~1400rpm shakes 15 seconds；
(2) it converts: conversion reagent and nuclease protection agent, the body of mixed liquor and conversion reagent being added into the mixed liquor of step (1)
For product than being 1:0.09, the volume ratio of mixed liquor and nuclease protection agent is 1:0.010, vortex oscillation solution, 55 DEG C~70 DEG C of item
It is converted 60 minutes under part；
(3) it combines: being added and combine liquid, shaken well adds magnetic bead liquid, and oscillation is stood after mixing, to magnetic bead in conjunction with nucleic acid
It is placed on magnetic separator to clarify up to solution, sucks supernatant, retain subnatant；The volume ratio of the combination liquid and subnatant is
(2~4): 1, the volume of the magnetic bead liquid and the volume ratio of plasma sample are (1~2): 50；
(4) primary washing: cleaning solution I is added in magnetic bead, vortex oscillation 1~3 minute, it is clear up to solution to be placed in magnetic separator
Clearly, supernatant is sucked, the number of rinsing is 1~3 time, it is preferable that I additional amount of cleaning solution of the first time washing and blood
Than for (2~4): 1, I additional amount of cleaning solution of subsequent wash and the ratio of blood are (0.5~1.5): 1；
(5) secondary wash: being added the resuspension magnetic bead of cleaning solution II and be then centrifuged for, centrifuge tube is placed on magnetic frame, complete to magnetic bead
Waste liquid is discarded after absorption；
(6) dry: drying at room temperature after centrifugation；
(7) it elutes: eluant, eluent being added into the solution after drying, shaken at room temperature mixes, and removes magnetic by magnetic-adsorption after centrifugation
Pearl retains supernatant.
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