It is a kind of for by swab cell elution, cracking release nucleic acid elution lysate,Kit and methodTechnical field
The present invention relates to a kind of for cell elution, the cracking on swab to be discharged to elution lysate, the kit of nucleic acidAnd method.
Background technique
Cell is to be found by British scientist Robert Hooke (Robert Hooke, 1635~1703) in 1665's.Cell is the basic structure and function unit of organism.And cell biology widely utilizes the achievement of neighboring discipline, in skillIt is to learn wildly from other's strong points in art method, it is all to be able to solve being used for problem.Such as the technique study base with molecular biologyThe structure of cause, with biochemistry, molecular biology technique study chromosome on it is various nonhistones and they are living to geneDynamic regulation and control is (tubulin, microfilament protein, each using the various albumen of immunologic technique study cytoskeletonKind of moderate fiber albumen) distribution in cell and the variation in vital movement.Recombinant DNA originating from molecular geneticsTechnology and originating from it is immunologic generate monoclonal antibody hybridoma technology, also at the powerful of cell biology.
And culture, proliferation, the preservation of cell require stringent condition.The environment of cell culture have to meet it is nontoxic andSterile is the most important condition of cultured cell in vitro, and the preference temperature that mammality and avian cells are cultivated in vitro is 37~38 DEG C,Osmotic pressure is one of essential condition of cultured cell in vitro, and the in vitro culture of cell needs ideal gaseous environment, oxygen, dioxyChange the necessary condition that carbon is cells survival.And need to provide immobilized-cell culture base and include: various battalion needed for cell growthSupport substance, including carbohydrate, amino acid, inorganic salts, vitamin etc..In order to save cell, especially it is not easy to obtain prominentModification cell or cell strain, by cell cryopreservation.The temperature frozen generally uses the temperature--196 DEG C of liquid nitrogen, and cell is collectedThe culture medium for containing protective agent (generally dimethyl sulfoxide or glycerol) is added into cryopreservation tube, is frozen with certain cooling velocity, mostIt is stored in liquid nitrogen eventually.At very low temperature, the time that cell saves is almost unlimited.Recovery is generally using the side of melting fastlyMethod after being removed from liquid nitrogen cryopreservation tube, is immediately placed in 37 DEG C of water, is allowed to melt rapidly in one minute.Then by cellIt is transferred in culture vessel and is cultivated.Temperature when protectant selection, cell density, cooling rate and recovery in frozen storage process,Melt speed etc. all to have an impact to cell viability.
And we are applied to health of masses genetic test at present, inborn genetic test, the detections such as genetic screening are all mainAfter acquiring mouth epithelial cells using dry swab, because it acquires, transport, saves more convenient, and largely used.But makeThe cell concentration collected with swab is very limited, and the condition of growth and the preservation of cell is all extremely harsh, by transport, room temperatureStorage, causes largely to damage to the cell in swab, and downstream experiment is caused to easily cause false positive or false negative.
We are efficiently eluted and are cracked release nucleic acid by many experiments development cell in swab of sening as an envoy to, can be effectiveSolution cell because transport, improper storage bring damage after caused by false positive or false negative, while raising recall rate, withIn subsequent Molecular Detection.Compared with current method for extracting nucleic acid, because cell adherence is on swab, product base on the market at presentCell on Direct Pyrolysis swab, subsequently through purifying, nucleic acid is recycled in washing after the meeting, and often the rate of recovery is relatively low, extraction costHeight, extraction efficiency are relatively low.Therefore a kind of reagent for allowing cell high-efficient elution to crack is found, without nucleic acid extraction Direct PCRMethod be of great significance to detection efficiency and quality is improved.
Summary of the invention
The purpose of the present invention is to provide a kind of for cell elution, the cracking in swab sample to be discharged to the elution of nucleic acidLysate first elutes the cell on dry swab using the elution lysate, discharges subsequently through high temperature cracking methodNucleic acid carries out real-time quantitative PCR detection SNP, directly to optimize existing detection technique after cracking.It is provided for dry swab sampleOne kind is quickly, easy, efficiently, accurately detection method, omission nucleic acid extraction process simplify operating process, saved the timeAnd cost, it is often more important that improve recall rate, operation can prevent from polluting when detecting.
To achieve the above object, the technical solution taken: a kind of for releasing cell elution, the cracking in swab samplePut the elution lysate of nucleic acid, the elution lysate includes the component of following concentration: the Tris-HCl of 25~50mM, 3~The MgCl of 11mM2, 3~6 μM EDTA, 0.1~1 μM of SDS, the Proteinase K of 5~50 μ g/ml, 0.1~0.5 μM of BSA withAnd 1~10% polysorbas20, the pH value of Tris-HCl is 7~8.5.Elution lysate of the present invention can be not only used for elutingCell on dry swab, additionally it is possible to for cracking the cell.Here polysorbas20 is used as flexible decomposition agent in cracking process,It is used as stabilizer in subsequent PCR amplification, a stable buffer system is provided.Polysorbas20 is more some than Tween 80 milder, afterContinuous PCR amplification provides stabilising system, cannot be replaced with Tween 80 or 60.
Preferably, the swab is the swab without protection liquid.Existing swab is divided to two kinds；Without the swab and band of protection liquidThe swab of liquid is protected, elution lysate of the invention is developed for the swab without protection liquid, elution cracking of the inventionLiquid is suitble to elute the swab without protection liquid, is not suitable for eluting the swab with protection liquid.
Preferably, the swab sample is buccal swab sample.
The present invention provides a kind of for cell elution, the cracking in swab sample to be discharged to the kit of nucleic acid, describedKit includes elution lysate described above.
The present invention provides a kind of for cell elution, the cracking in swab sample to be discharged to the method for nucleic acid, the sideMethod is that cell elution, cracking in swab sample are discharged nucleic acid using elution lysate described above.
The present invention provides a kind of for cell elution, the cracking in swab sample to be discharged to the method for nucleic acid, the sideMethod the following steps are included:
(1a) prepares elution lysate described above；
The elution lysate that step (1a) obtains is added in centrifuge tube by (2a), and swab sample is soaked in the elutionIt in lysate, is vortexed and mixes, be incubated for 60min at 37 DEG C；
(3a) takes out swab, in 65 DEG C of water-baths 5min, 95 DEG C of water-bath 10min.
Preferably, which is characterized in that being incubated in the step (2a) is carried out in shaking table, and revolving speed is 300 turns/min.
The present invention provides a kind of methods for carrying out SNP detection to the cell in swab sample, comprising the following steps:
(1b) prepares elution lysate described above；
The elution lysate that step (1b) obtains is added in centrifuge tube by (2b), and swab sample is soaked in the elutionIt in lysate, is vortexed and mixes, be incubated for 60min at 37 DEG C；
(3b) takes out the swab in centrifuge tube, the water-bath 5min at 65 DEG C of the centrifuge tube after swab will be taken out, at 95 DEG CWater-bath 10min takes supernatant as liquid to be detected；
Liquid to be detected is added in SNP site detection reaction solution by (4b), is expanded on real-time fluorescence quantitative PCR instrumentIncrease, SNP testing result is obtained according to amplification.
It is to crack the cell eluted in 65 DEG C of water-baths.Water-bath is the master in order to inactivate PCR inhibitor at 95 DEG CWant inactivated proteases K.
Preferably, amplification program in the step (4b) are as follows: 95 DEG C of 10min, carry out 40 circulation 95 DEG C of 15s, 65 DEG C1min, in 65 DEG C of detection fluorescence signals.
The method of the present invention can effectively first elute cell from dry swab, then discharge nucleic acid by flexible cracking,And pass through high-temperature inactivation PCR inhibitor, it is succinct easy to operate, and nucleic acid extraction process is omitted, operating process is simplified, is mentioned significantlyHigh detection rate and efficiency.
The utility model has the advantages that
Compared with prior art, present invention has the advantage that
1, for the present invention by being cracked again after elution, detector efficiency is higher than the efficiency that commercial kit is extracted.
2, present invention elution lysate does not influence downstream PCR amplification.
3, the method for the present invention is easy to operate, cumbersome purifying, washing, elution step in removal extraction.
4, the method for the present invention whole process is only uncapped once, avoids the cross contamination introduced by a large amount of operations.
5, the method for the present invention simplifies operating process, has saved time and cost.
Detailed description of the invention
Fig. 1 is to carry out SNP parting to No. 1 sample (wild type sample) using the method for the present invention in the embodiment of the present invention 1Testing result figure.
Fig. 2 is to carry out SNP parting to No. 2 samples (wild type sample) using the method for the present invention in the embodiment of the present invention 1Testing result figure.
Fig. 3 is to carry out SNP parting to No. 3 samples (heterozygous sample) using the method for the present invention in the embodiment of the present invention 1Testing result figure.
Fig. 4 is to carry out conventional nucleic acid extraction and the method for the present invention to No. 1 sample (wild type sample) in the embodiment of the present invention 2The SNP genotyping result figure compared.
Fig. 5 is to carry out conventional nucleic acid extraction and the method for the present invention to No. 2 samples (wild type sample) in the embodiment of the present invention 2The SNP genotyping result figure compared.
Fig. 6 is to carry out conventional nucleic acid extraction and the method for the present invention to No. 3 samples (heterozygous sample) in the embodiment of the present invention 2The SNP genotyping result figure compared.
Specific embodiment
The contents of the present invention are further illustrated below in conjunction with Detailed description of the invention and specific embodiment, but should not be construed as to thisThe limitation of invention.In the case where without departing substantially from spirit of that invention and essence, to made by the method for the present invention, step, condition modification orReplacement, all belongs to the scope of the present invention.Unless otherwise noted, experimental method used in embodiment is those skilled in the artKnown conventional method and technology, reagent or material are to be obtained by commercial sources.
Below in conjunction with the drawings and specific embodiments, invention is further described in detail, and the scheme that embodiment provides isPreferred embodiment, but not as the restriction to the application, PCR amplification instrument used in embodiment is ABI 7500.Pass through designThe primed probe in the site ALDH2 gene rs671 carries out Genotyping.
Upstream primer: CCCCCAGCAGGTCCCACAC (SEQ ID NO:1)；
Downstream primer: GCTACAAGATGTCGGGGAGTGG (SEQ ID NO:2)；
Wild-type probe PW:CACAGTTTTCACTTCAGTGTATGCCT (SEQ ID NO:3)；
Saltant type probe PM:CACAGTTTTCACTTTAGTGTATGCCTG (SEQ ID NO:4).
Embodiment 1
Dry swab sample is taken, real-time fluorescence quantitative PCR detection is carried out to its SNP site.
(1) elution lysate is prepared, the elution lysate includes the component of following concentration: the Tris- of 25~50mMThe MgCl of HCl, 3~11mM2, 3~6 μM EDTA, 0.1~1 μM of SDS, the Proteinase K of 5~50 μ g/ml, 0.1~0.5 μMBSA and 1~10% polysorbas20, the pH value of Tris-HCl is 7~8.5.
(2) the drying swab sample for taking 3 preservations is added in the centrifuge tube of the elution lysate containing 300 μ L steps (1),It respectively as No. 1 sample, No. 2 samples, No. 3 samples, is vortexed and mixes, in 37 DEG C of shaking tables, in the case where revolving speed is 300 turns/min, be incubated for60min。
(3) swab in centrifuge tube is taken out, by the centrifuge tube after taking-up swab in 65 DEG C of water-bath 5min, 95 DEG C of water-baths10min takes supernatant as liquid to be detected.
(4) liquid to be detected for 3 samples that above-mentioned steps (3) obtain respectively is taken into 2uL, is added to SNP site detection reactionIn liquid, being expanded on real-time fluorescence quantitative PCR instrument (ABI 7500), amplification program is 95 DEG C of 15min, carry out 40 and follow95 DEG C of 15s of ring, 65 DEG C of 1min, in 65 DEG C of detection fluorescence signals.
Experimental result is as shown in Figure 1, 2, 3, and Fig. 1,2,3 are respectively the experimental result of No. 1 sample, No. 2 samples, No. 3 samples,The result shows that the method according to the present embodiment is operated, effectively accurate parting clearly can be carried out to SNP.An and and generationSequencing result compares, and genotyping result is consistent.
Embodiment 2
Conventional nucleic acid extraction is carried out to two parts of swab samples that the same person acquires simultaneously to compare with the method for the present invention.Buccal swab sample is taken, real-time fluorescence quantitative PCR detection is carried out to its SNP site using the method for the present invention, the specific steps are as follows:
(1) elution lysate is prepared, the elution lysate includes the component of following concentration: the Tris- of 25~50mMThe MgCl of HCl, 3~11mM2, 3~6 μM EDTA, 0.1~1 μM of SDS, the Proteinase K of 5~50 μ g/ml, 0.1~0.5 μMBSA and 1~10% polysorbas20, the pH value of Tris-HCl is 7~8.5.
(2) the drying swab sample for taking 3 preservations is added in the centrifuge tube of the elution lysate containing 300 μ L steps (1),It respectively as No. 1 sample, No. 2 samples, No. 3 samples, is vortexed and mixes, in 37 DEG C of shaking tables, in the case where revolving speed is 300 turns/min, be incubated for60min。
(3) swab in centrifuge tube is taken out, by the centrifuge tube after taking-up swab in 65 DEG C of water-bath 5min, 95 DEG C of water-baths10min takes supernatant as liquid to be detected.
(4) liquid to be detected for 3 samples that above-mentioned steps (3) obtain respectively is taken into 2uL, is added to SNP site detection reactionIn liquid, being expanded on real-time fluorescence quantitative PCR instrument (ABI 7500), amplification program is 95 DEG C of 15min, carry out 40 and follow95 DEG C of 15s of ring, 65 DEG C of 1min, in 65 DEG C of detection fluorescence signals.
Buccal swab sample is taken, using the cell DNA used in commercialized kit extraction swab sample.
Experimental result is as shown in Figure 4,5, 6, and Fig. 4,5,6 are respectively the experimental result of No. 1 sample, No. 2 samples, No. 3 samples,In Fig. 4,5,6 number 1,2,3 for according to the method for the present invention operated as a result, accordingly, number 1`, 2`, 3` in Fig. 4,5,6To use commercialized kit to extract the testing result of DNA.The result shows that being operated according to the method for the present invention, pass through heightIt is cracked after effect elution, recall rate and efficiency are better than commercialized extracts kit, can substitute nucleic acid, omit nucleic acid extraction stepSuddenly, can be effectively accurate, parting efficiently is carried out to SNP.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present inventionThe limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art shouldUnderstand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present inventionAnd range.