CN106497915A - A kind of paramagnetic particle method extracts the lysate of saliva nucleic acid - Google Patents
A kind of paramagnetic particle method extracts the lysate of saliva nucleic acid Download PDFInfo
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- CN106497915A CN106497915A CN201610848181.2A CN201610848181A CN106497915A CN 106497915 A CN106497915 A CN 106497915A CN 201610848181 A CN201610848181 A CN 201610848181A CN 106497915 A CN106497915 A CN 106497915A
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Abstract
The invention discloses a kind of paramagnetic particle method extracts the lysate of saliva nucleic acid, it is characterised in that：The lysate is made up of the chelating agent of the Tris HCl of 10 ~ 150mM, the high chaotropic salt of 1 ~ 10M, 0.1 ~ 10% anion surfactant, 1 ~ 20% nonionic surfactant, 0.1 ~ 4% CTAB and 1 ~ 100mM.In the present invention, the lysate of paramagnetic particle method nucleic acid extraction can effectively remove the customary impurities such as the special impurities such as mucopolysaccharide in desalivation, mucoprotein, swill and other protein, fat fat, carbohydrate, salt while abundant, effectively cell lysis, so that nucleic acid extraction effect more preferably, nucleic acid purity higher, ensure that the detection in later stage is more efficient, testing result is more accurate.The lysate of the present invention is simultaneously suitable for the nucleic acid extraction of the biological specimens such as whole blood, cell culture, bacterial cultures, swab, tissue.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of lysate of paramagnetic particle method extraction saliva nucleic acid.
Background technology
Method for extracting nucleic acid includes traditional phenol chloroform method, salting out method, filter membrane centrifugal column method etc..Traditional nucleic acid extraction
Method respectively has its pluses and minuses, but extraction effect is not good enough.The method for extracting nucleic acid of current main-stream has pellosil adsorption column method, anti-DNA mono-
The affine in immunity method of clonal antibody, the wide variety of paramagnetic particle method method for extracting nucleic acid of automation platform etc..
Paramagnetic particle method nucleic acid extraction referred to superparamagnetism silica nano-magnetic microballon (hereinafter referred to as magnetic bead) as carrier,
Nucleic acid is adsorbed by magnetic bead in high chaotropic saline solutions and is carried out from the principle of magnetic bead surfaces desorption in low salt solutions amplifying nucleic acid
The method of nucleic acid extraction.Wherein magnetic bead has the special feature that, such as good skin effect and bulk effect, good selective magnetic ring
Ying Xing, physicochemical properties are stable etc., make paramagnetic particle method nucleic acid extraction be widely used.But different paramagnetic particle method nucleic acid is carried
Method is taken, and causes final nucleic acid extraction because of the lysate selected by which, with reference to the difference of the components such as liquid, cleaning solution and content
Efficiency difference is very big.On the other hand, the diversity based on testing sample, including cell, bacterium, serum, blood plasma, seminal fluid, hair,
Saliva, urine or Pleural effusions etc., different testing samples adopt the extraction effect of same paramagnetic particle method method for extracting nucleic acid gained
Very big difference be there is also with extraction efficiency.
Wherein saliva belongs to the nucleic acid extraction testing sample for being easier to obtain, and its composition is deposited with the composition in serum, blood plasma
In greatest differences, saliva is colourless, tasteless, odorless liquid, and slant acidity, pH value 6 ~ 7, ratio of viscosities water are big 18 ~ 35 times.In saliva
Containing materials such as amylase, lysozyme, peroxidase, mucoprotein, mucopolysaccharide, phosphatide.And mucoprotein, mucopolysaccharide are to make saliva
Sticky main matter, and its maximum from his sample type different.And existing paramagnetic particle method extracting method is generally directed to serum, blood
Slurry, when which is directly used in the nucleic acid extraction of saliva, gained nucleic acid concentration is not good enough, limits paramagnetic particle method extracting method in saliva nucleic acid
Efficient application in extraction.
Content of the invention
It is an object of the invention to, for above-mentioned the deficiencies in the prior art, there is provided a kind of paramagnetic particle method extracts saliva nucleic acid
Lysate.
The technical solution used in the present invention is：A kind of paramagnetic particle method extracts the lysate of saliva nucleic acid, and which is by 10 ~ 150mM
Tris-HCl, the high chaotropic salt of 1 ~ 10M, 0.1 ~ 10% anion surfactant, 1 ~ 20% non-ionic surface active
Agent, the chelating agent composition of 0.1 ~ 4% CTAB and 1 ~ 100mM.
Used as the further improvement of such scheme, the lysate is preferably by the Tris-HCl of 50 ~ 100mM, the height of 3 ~ 8M
Chaotropic salt, 0.1 ~ 5% anion surfactant, 1 ~ 10% nonionic surfactant, 0.5 ~ 2% CTAB and 20 ~
The chelating agent composition of 70mM.
Saliva is viscous liquid, and which contains more mucopolysaccharide, mucoprotein and swill.Wherein mucopolysaccharide is nitrogenous
Heterogeneity polysaccharide, be a kind of linear molecule with specific order of recurring unit, be also constitute iuntercellular connective tissue
Main component, chemical composition are that uronic acid and junket aminohexose are alternately present, sometimes linkage containing sulfur, also referred to as glycosaminoglycan.And glue
Albumen is mainly made up of mucopolysaccharide, is a kind of high-molecular-weight protein family of high glycosylation modification, big with complicated biology
Molecular structure, its can be assembled to form gel, play the several functions such as the lubrication of tissue and the conduction of cell signal, can be with shape
Into chemical barrier.Thus the mucopolysaccharide, mucoprotein and swill in saliva largely affects saliva nucleic acid extraction effect
Rate, is to cause saliva extraction nucleic acid efficiency low, the low key factor of purity.
In technical solution of the present invention, creatively add the CTAB of specified quantitative being effectively increased mucopolysaccharide, mucoprotein etc.
Solubility, while by the compounding of the anion surfactant and nonionic surfactant of specified quantitative, make mucopolysaccharide, viscous
The impurity such as albumen and other oroteins can fully be dissociated, settles, be stablized, and reduce between nucleic acid molecules electrostatic repulsion forces and easy
In being precipitated by alcohols, so as to reach the purpose of isolating protein and other impurities.
As the further improvement of such scheme, the high chaotropic salt selected from guanidinium isothiocyanate, guanidine hydrochloride, potassium rhodanide,
One of which in lithium chloride, sodium perchlorate.
Used as the further improvement of such scheme, the anion surfactant is selected from SDS, sodium laurate, dodecane
One of which in base benzene sulfonic acid.
Used as the further improvement of such scheme, the nonionic surfactant is selected from NP-40, polysorbas20, Triton-
One of which in X-100.
Specifically, high chaotropic salt can open the primary structure for unfolding protein so that the albumen in nucleic acid-protein compound
Matter is easier to be dissociated by surfactant.And anion surfactant can neutralize the negative electrical charge of nucleic acid, make quiet between nucleic acid molecules
Electric repulsive force reduces, and nucleic acid is easy to be precipitated by alcohols and can be effectively gone isolating protein and other impurities；CTAB can increase viscous
The solubility of polysaccharide and mucoprotein etc., so as to reach deimpurity purpose, and CTAB and anion surfactant have compared with
Good compatibility；Nonionic surfactant has emulsification, diffusion, solubilising, drop resistance, the effect such as stable, and which is difficult by forceful electric power solution
The impact that matter is present, is not easy to be affected by acid, alkali, has good compatibility with other types surfactant, its solubility
Raise with temperature and reduce, while with certain deproteinized ability.In the present invention lysate have anion surfactant,
Nonionic surfactant and the compound system of CTAB, can be such that the cracking of biological specimen and the dissociation of impurity settles more fully, enter
One step improves nucleic acid extraction efficiency and nucleic acid extraction purity.Further, when anion surfactant is selected from SDS, laurate
One of which, nonionic surfactant in sodium, DBSA is in NP-40, polysorbas20, Triton-X-100
One of which when, which compounds the cracking to biological specimen with CTAB and the sedimentation dissociation of impurity has excellent effect, especially
It is to the biological specimen with sticky nature, particularly saliva sample, so as to effectively improve the nucleic acid purity of extraction.
Used as the further improvement of such scheme, the chelating agent is selected from ethylenediamine tetra-acetic acid, aminotriacetic acid, citric acid
One of which in sodium.
Present invention also offers a kind of kit, which includes lysate as above, combines liquid and Proteinase K.Enter one
Step ground, the combination liquid is selected from ethanol or isopropanol.Specifically, lysate, be re-dubbed mixed system with reference to liquid and Proteinase K,
The mixed system contains the lysate of " N " ul（50≤“N”≤300）, " 0.1N " ul Proteinase K and " N " ul combination liquid.
Wherein selecting for " N " value can be according to needed for practical operation, and " N " value is too small or crosses the nucleic acid extraction for being mostly unfavorable for biological specimen,
In the present invention, " N " value takes 200, and which can be conducive to the automation mechanized operation of plant equipment.
Another aspect of the present invention is to provide a kind of answering for lysate of paramagnetic particle method extraction saliva nucleic acid as above
With.The lysate is particularly well-suited to extract the nucleic acid in saliva sample, and which is simultaneously applicable to whole blood, cell culture, thin
The nucleic acid extraction of the biological specimens such as bacterium culture, swab, tissue.
As the further improvement of such scheme, described extract through lysate after biological specimen nucleic acid can be used for PCR,
Digestion, molecule hybridization, library construction.
The invention has the beneficial effects as follows：In the present invention, the lysate of paramagnetic particle method nucleic acid extraction fully, is effectively being cracked carefully
The special impurities such as mucopolysaccharide in desalivation, mucoprotein, swill and other protein, fat fat, carbon can be effectively removed while born of the same parents
The customary impurities such as hydrate, salt so that nucleic acid extraction effect more preferably, nucleic acid purity higher, it is ensured that the detection in later stage is higher
Effect, testing result are more accurate.The lysate of the present invention is simultaneously suitable for whole blood, cell culture, bacterial cultures, swab, group
The nucleic acid extraction of biological specimen such as knit.
Description of the drawings
Fig. 1 is detected for 1 each sample Real-time PCR of embodiment（Detection gene RNaseP）Testing result figure.
Specific embodiment
The present invention is specifically described with reference to embodiment, in order to art personnel to the present invention
Understand.The present invention will be further described it is emphasized that embodiment is only intended to be necessary here, it is impossible to is interpreted as to this
The restriction of invention protection domain, art person skilled in the art, according to the non-intrinsically safe that foregoing invention content is made to the present invention
The modifications and adaptations of property, should still fall within protection scope of the present invention.Following mentioned raw materials are unspecified simultaneously, are
Commercially available prod；The processing step or preparation method not referred in detail be processing step known to a person skilled in the art or
Preparation method.
Embodiment 1：Nucleic acid extraction and detection are carried out as biological specimen using saliva
1st, saliva sample is processed：Saliva collection pipe preserves pipe for the saliva of Guangdong Guo Sheng medical science and technologies limited company.Take 12
Part saliva sample, without carrying out pre-treatment, turns upside down after being well mixed for several times, is applied directly to be cracked in cracking liquid system
Digestion.
2nd, reagent：
A, press raw material components proportioning prepare 12 parts of lysates, as shown in the table：
B, magnetic bead-combine liquid：The mixed liquor that the magnetic bead of 20ul is dispersed in gained in 200ul isopropanols；
C, cleaning solution A：By the potassium rhodanide of Tris-HCl, 3M of 50mM, 0.5% CTAB, 2% PVP and 40% ethanol
Composition；
D, cleaning solution B：70% ethanol.
3rd, paramagnetic particle method nucleic acid extraction is carried out to 12 parts of saliva samples respectively with mentioned reagent, step is as follows：
The saliva sample of 200ul, the lysate of sequence number corresponding with sample of 200ul, the Proteinase K of 20ul are dispensed into 1 respectively
In number nucleic acid extraction plate, it is 70 DEG C of cracking reactions 20min in temperature, adds the magnetic bead of 220ul-combine liquid, quick mixes
5min, magnetically attractive 3 times；The cleaning solution A of 500ul is dispensed in No. 2 nucleic acid extraction plates；The cleaning solution B of 500ul is dispensed into No. 3
In nucleic acid extraction plate；The TE solution of 50ul is dispensed in No. 4 nucleic acid extraction plates；Under magneticaction, bar magnet adsorbs No. 1 nucleic acid
The magnetic bead extracted in plate is transferred in No. 2 nucleic acid extraction plates, discharges magnetic bead, quickly mixes 2min, magnetically attractive 3 times；Bar magnet absorption afterwards
Magnetic bead is transferred in No. 3 nucleic acid extraction plates, discharges magnetic bead, quickly mixes 2min, magnetically attractive 3 times；Bar magnet absorption magnetic bead is transferred to 4 afterwards
In number nucleic acid extraction plate, magnetic bead is discharged, quickly mix 2min, magnetically attractive 3 times obtains final product the nucleic acid for having extracted.
4th, detection of nucleic acids：
Respectively 12 parts of saliva samples for having extracted in embodiment 1 are carried out with nucleic acid concentration detection, testing result such as table 1 below institute
Show；Respectively 1 each saliva sample Real-time PCR of embodiment are detected（Detection people house-keeping gene RNasep）Testing result figure
As shown in figure 1, can be drawn by Fig. 1 data, the lysate of the present invention does not press down for the sample of nucleic acid after paramagnetic particle method nucleic acid extraction
Thing processed, PCR effects are good.
Embodiment 2：Nucleic acid extraction and detection are carried out as biological specimen using blood
1st, blood sample is processed：Blood collection is in the blood fresh-keeping tube of Guangdong Guo Sheng medical science and technologies limited company.Collect
10 blood of human body samples, turn upside down after being well mixed, and being applied directly to crack in liquid system carries out cracking digestion.
2nd, reagent：
A, lysate：By the lithium chloride of Tris-HCl, 5M of 100mM, 3% sodium laurate, 5% Triton-X-100,1%
CTAB and the aminotriacetic acid of 50mM mix；
B, magnetic bead-combine liquid：The mixed liquor that the magnetic bead of 10ul is dispersed in gained in 100ul ethanol；
C, cleaning solution A：By the lithium chloride of Tris-HCl, 5M of 100mM, 1% CTAB, 0.2% PVP and 40% ethanol group
Into；
D, cleaning solution B：70% ethanol.
3rd, paramagnetic particle method nucleic acid extraction is carried out to 10 parts of blood of human body samples respectively with mentioned reagent, step is as follows：
Respectively the Proteinase K of the blood sample of 200ul, the lysate of 200ul, 20ul is dispensed in No. 1 nucleic acid extraction plate,
Temperature is 70 DEG C of cracking reactions 20min, adds the magnetic bead of 220ul-combine liquid, quickly mixes 5min, magnetically attractive 3 times；By 500ul
Cleaning solution A be dispensed in No. 2 nucleic acid extraction plates；The cleaning solution B of 500ul is dispensed in No. 3 nucleic acid extraction plates；By 100ul
TE solution be dispensed in No. 4 nucleic acid extraction plates；Under magneticaction, bar magnet adsorbs the magnetic bead transfer in No. 1 nucleic acid extraction plate
Into No. 2 nucleic acid extraction plates, magnetic bead is discharged, quickly mix 2min, magnetically attractive 3 times；Bar magnet absorption magnetic bead is transferred to No. 3 nucleic acid and carries afterwards
Take in plate, discharge magnetic bead, quickly mix 2min, magnetically attractive 3 times；Bar magnet absorption magnetic bead is transferred in No. 4 nucleic acid extraction plates afterwards, is discharged
Magnetic bead, quickly mixes 2min, and magnetically attractive 3 times obtains final product the nucleic acid for having extracted.
4th, detection of nucleic acids：
10 parts of blood of human body samples after to said extracted carry out nucleic acid concentration detection and detect people's pipe by Rael-time PCR
Family gene RNasep, and and comparative example, i.e., 10 parts of blood of human body samples are carried out using import reagent box invitrogen
The result of nucleic acid extraction is compared, and testing result is as shown in table 2 below.The nucleic acid extraction that lysate of the invention can be obtained by table 2
The effect of Contrast on effect import reagent box invitrogen will be got well.
Claims (10)
1. a kind of paramagnetic particle method extracts the lysate of saliva nucleic acid, it is characterised in that：Tris- of the lysate by 10 ~ 150mM
HCl, the high chaotropic salt of 1 ~ 10M, 0.1 ~ 10% anion surfactant, 1 ~ 20% nonionic surfactant, 0.1 ~
The chelating agent composition of 4% CTAB and 1 ~ 100mM.
2. a kind of paramagnetic particle method according to claim 1 extracts the lysate of saliva nucleic acid, it is characterised in that：The lysate
By the Tris-HCl of 50 ~ 100mM, the high chaotropic salt of 3 ~ 8M, 0.1 ~ 5% anion surfactant, 1 ~ 10% nonionic
Surfactant, the chelating agent composition of 0.5 ~ 2% CTAB and 20 ~ 70mM.
3. a kind of paramagnetic particle method according to claim 1 and 2 extracts the lysate of saliva nucleic acid, it is characterised in that：The height
One of which of the chaotropic salt in guanidinium isothiocyanate, guanidine hydrochloride, potassium rhodanide, lithium chloride, sodium perchlorate.
4. a kind of paramagnetic particle method according to claim 1 and 2 extracts the lysate of saliva nucleic acid, it is characterised in that：Described the moon
One of which of the ionic surface active agent in SDS, sodium laurate, DBSA.
5. a kind of paramagnetic particle method according to claim 1 and 2 extracts the lysate of saliva nucleic acid, it is characterised in that：Described non-
One of which of the ionic surface active agent in NP-40, polysorbas20, Triton-X-100.
6. a kind of paramagnetic particle method according to claim 1 and 2 extracts the lysate of saliva nucleic acid, it is characterised in that：The chela
Mixture is selected from ethylenediamine tetra-acetic acid, aminotriacetic acid, the one of which in sodium citrate.
7. a kind of kit, it is characterised in that：The kit includes lysate as described in any one of claim 1 ~ 6, knot
Close liquid and Proteinase K.
8. a kind of kit according to claim 7, it is characterised in that：The combination liquid is selected from ethanol or isopropanol.
9. a kind of paramagnetic particle method as described in any one of claim 1 ~ 6 extracts the application of the lysate of saliva nucleic acid, and its feature exists
In：The lysate is applied to the biological specimens such as extraction whole blood, cell culture, bacterial cultures, swab, tissue, saliva
Nucleic acid.
10. paramagnetic particle method according to claim 9 extracts the application of the lysate of saliva nucleic acid, it is characterised in that：The warp
The nucleic acid of the biological specimen after lysate extraction can be used for PCR, digestion, molecule hybridization, library construction.
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Cited By (5)
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| CN108315325A (en) * | 2017-03-03 | 2018-07-24 | 绍兴迅敏康生物科技有限公司 | A kind of method and reagent for extracting nucleic acid substances using magnetic bead |
| CN108531472A (en) * | 2017-03-05 | 2018-09-14 | 北京天健惠康生物科技有限公司 | A kind of lysate for nucleic acid extraction |
| CN109810974A (en) * | 2019-04-08 | 2019-05-28 | 珠海丽珠试剂股份有限公司 | Extract the reagent combination product and its method of human immunodeficiency virus HIV-1 nucleic acid |
| CN112011534A (en) * | 2019-05-30 | 2020-12-01 | 苏州海狸生物医学工程有限公司 | Saliva genome DNA extraction and purification method based on nano magnetic beads and kit |
| CN113308459A (en) * | 2020-02-26 | 2021-08-27 | 重庆中元汇吉生物技术有限公司 | Whole blood nucleic acid extraction lysis solution by magnetic bead method, kit and extraction method |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109810974A (en) * | 2019-04-08 | 2019-05-28 | 珠海丽珠试剂股份有限公司 | Extract the reagent combination product and its method of human immunodeficiency virus HIV-1 nucleic acid |
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| CN112011534A (en) * | 2019-05-30 | 2020-12-01 | 苏州海狸生物医学工程有限公司 | Saliva genome DNA extraction and purification method based on nano magnetic beads and kit |
| CN113308459A (en) * | 2020-02-26 | 2021-08-27 | 重庆中元汇吉生物技术有限公司 | Whole blood nucleic acid extraction lysis solution by magnetic bead method, kit and extraction method |
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Application publication date: 20170315 |