CN104195134A - Kit and method for extracting nucleic acids from urine - Google Patents
Kit and method for extracting nucleic acids from urine Download PDFInfo
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- CN104195134A CN104195134A CN201410479242.3A CN201410479242A CN104195134A CN 104195134 A CN104195134 A CN 104195134A CN 201410479242 A CN201410479242 A CN 201410479242A CN 104195134 A CN104195134 A CN 104195134A
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Abstract
The invention relates to a method for extracting nucleic acids with urine as a sample in the biological field, and application thereof, and provides a special kit and method for rapidly extracting the nucleic acids from a large amount of urine. The method comprises the following steps: enriching the nucleic acids in the urine onto a nucleic acid adsorbing column in a suction filtration column manner, washing, eluting and the like for extracting a trace amount of nucleic acids in the urine. The nucleic acids obtained by using the kit provided by the invention comprise free nucleic acids in the urine and nucleic acids in exfoliated cells in the urine; the operation is simple and rapid; the nucleic acids are high in yield and high in purity; the kit and the method can be directly applied to molecular biological detection.
Description
Technical field
The present invention relates to the extracting method of field of biology amplifying nucleic acid (DNA and RNA), be specifically related to a kind of method and application thereof of extracting nucleic acid from mankind's urine.
Technical background
Urine be blood flow after kidney, form through glomerular filtration, reabsorption and secretion, excrete by bladder, urethra.Urine is to keep the stable final meta-bolites of organismic internal environment, except containing water, glucose, inorganic salt, protein, urea, uric acid, also contain epidermic cell, dissociative DNA and RNA (mRNA, miRNA etc.) that erythrocyte, white corpuscle, organ come off.Free nucleic acid in urine comprises the nucleic acid discharging through the free nucleic acid of the next autologous circulation of kidney filtration and after from urethra lysis.The biological information that is richly stored with in urine, and have without extra earning collection, process the advantages such as relatively simple.The change of urine composition and content is not only subject to the impact of urinary system, reproductive system, and relevant with physiology or the pathological change of the systems such as blood circulation, internal secretion, digestion, metabolism, breathing.From urine, isolate nucleic acid, and these nucleic acid fragments are tested, can help other early stage, the effective Molecular Detection of disease of cancer, pulmonary tuberculosis, acquired immune deficiency syndrome (AIDS), malaria and potential some.
Due to the free nucleic acid in urine and somatic content very low, adopt conventional method for extracting nucleic acid to be difficult to obtain enough and high-quality nucleic acid from urine cell.The physico-chemical properties such as urine pH value, density, temperature and sample all may affect the integrity of cell in urine from the timed interval of collecting processing, and then affect the rate of recovery while extracting nucleic acid.In order to obtain high-quality nucleic acid with high-recovery from urine, reduce storage-temp except adopting, shorten sample collection and treatment time and minimizing and test the method for the maintenance cell integrities such as processing (centrifugal or concentrated) to sample, the method adopting when nucleic acid extraction is particularly important.
In the time adopting centrifugal column method to extract nucleic acid, conventionally, by the centrifugal sample adsorption column that passes through, the nucleic acid in sample is attached on the pellosil of adsorption column.But so upper quadrat method is low for nucleic acid content, sample size is compared with for large urine, required loading overlong time, is unfavorable for the extraction of nucleic acid.Also after having method first urine is centrifugal, get sedimentation cell as sample, can effectively reduce the volume of sample, but this class extracting method can lose the free nucleic acid in urine supernatant, and free nucleic acid in urine is comprising very large biological information.Therefore, low for urine Nucleic Acid, extract the larger practical situation of required sample size when nucleic acid, invent a kind of from urine fast enriching extract the method for nucleic acid, there is important using value by clinical application urine is carried out to Molecular Detection.The invention provides the special suction filtration device of enrichment urine nucleic acid, can be by the nucleic acid fast enriching in a large amount of urine specimens to nucleic acid adsorption column, then through washing, elution step, extract high-quality nucleic acid, can be used for Molecular Detection.
Summary of the invention
The object of the invention is in order to overcome laboratory the deficiencies in the prior art, a kind of dedicated kit and extracting method that can extract fast nucleic acid from large capacity urine is provided.
The contained reagent of extraction urine nucleic acid reagent box provided by the invention and consumable material device are in table 1, extraction flow process is: the lysate ULB that adds 0.5～3 times of amount in urine, after 37～55 DEG C of insulation 10～60min, by sample, all by nucleic acid adsorption column, the nucleic acid in sample is in connection with on the pellosil to adsorption column.Wash after adsorption column respectively with washings UWB1 and UWB2, then nucleic acid is eluted from adsorption column with elutriant.
Table 1: the contained reagent of urine nucleic acid rapid extraction test kit and device
The present inventor is according to the difference of target sample, such as healthy male urine sample, healthy women urine sample, diabetic subject's urine sample and Pancreas cancer patients urine sample etc., through experiment repeatedly, each factor is optimized, finds out and extract the optimal performance step of urine nucleic acid and the optimum formula of corresponding solution.
The composition of lysate ULB includes but not limited to SDS, EDTA, triton x-100, Guanidinium hydrochloride, and the concentration in formula is in table 2.
Table 2: lysate ULB formula
In lysate ULB, the concentration of SDS is 0.1g/uL～1g/uL, and optimal concentration is 0.2ng/uL～0.6ng/uL;
The concentration of EDTA is 0.5mmol/L～2mmol/L, and optimal concentration is 1mmol/L～1.5mmol/L; The concentration of triton x-100 is 0.5%～6%, and optimal concentration is 2%～3%; The concentration of Guanidinium hydrochloride is 2mol/L～8mol/L, and optimal concentration is 5mol/L～8mol/L.
The formula of washings UWB1 is in table 3.Wherein the concentration of Guanidinium hydrochloride is 2mol/L～8mol/L, and optimal concentration is 3mol/L～6mol/L; Sodium chloride concentration is 75mmol/L～1.5mol/L, and optimal concentration is 120mmol/L～200mmol/L; The concentration of EDTA is 0.5mmol/L～2mmol/L, and optimal concentration is 1 mmol/L～1.5mmol/L; Alcohol concn is 40%～80%, and optimal concentration is 55%～75%.The composition of washings UWB2 and formula are in table 4.
Elutriant can be the low salts solution that pure water or pH value are greater than 7.0.
In order not destroy urine equilibrium system, ensure the integrity of each component in urine, whole urines are passed through adsorption column by the present invention, instead of urine centrifuging and taking sedimentation cell is extracted to nucleic acid.Contriver utilize method that suction filtration crosses post by urine specimen all by adsorption column, process 30mL sample and only need the upper prop time of 5～10 minutes, greatly simplified operation steps.Suction filtration is crossed the device of post and is seen Fig. 1, its principle is: by off-gas pump, air in waste collection bottle is pumped, make external atmosphere pressure be greater than the air pressure in waste collection bottle, utilize draught head, accelerate to contain urine in sample pipe by adsorption column, reach fast nucleic acid enriching to the object on adsorption column.
Table 3: washings UWB1 formula
Table 4: washings UWB2 formula
The present invention has following advantage and using value:
1. convenient, fast: the present invention crosses the method for post the nucleic acid in urine is carried out to enrichment with suction filtration, urine specimen gets i.e. use, without concentrated, compared with the centrifugal method of crossing post, can save the operating time of 5～10 times.
2. easy and simple to handle, safety: simple to operate, whole leaching process approximately 20 minutes, is applicable to extensive sample preparation, and step is simple, easy to operate.
3. sample is few by sample amount, yield is high: this test kit only needs 10mL urine specimen, can obtain high-quality DNA and RNA sample (DNA extraction yield women is generally 50～200ng/mL urine, and the male sex is generally 3～50ng/mL urine).
4. extract gained nucleic acid complete: this test kit does not add any processing to urine sample, without centrifugal, concentrated, the nucleic acid that extraction obtains has comprised the nucleic acid in cast-off cells in free nucleic acid in urine and urine, has ensured to greatest extent the integrity of sample amplifying nucleic acid.
5. the nucleic acid purity extracted is high: the nucleic acid that this test kit extracts can be directly used in the downstream experiments such as PCR, DNA methylation qualification, cancer detection.
6. safety non-toxic: in this test kit, agents useful for same and device are nontoxic to human body, non-corrosiveness and irritating smell.
Brief description of the drawings:
Fig. 1: urine nucleic acid extraction suction filtration device.
Fig. 2: taking Healthy People urine DNA as template, GAPDH is that goal gene carries out pcr amplification.
Fig. 3: taking Pancreas cancer patients urine DNA as template, saltant type KRAS (34G>T) carries out pcr amplification for goal gene.
Embodiment
With specific embodiment, the present invention is described in further detail below.Should be understood that, specific embodiment is only that the present invention is made to clearer explanation, instead of limitation of the present invention.
Embodiment 1:
Get the freshly voided urine of 10mL Healthy People, add 15mL lysate ULB, put upside down and mix, 37 DEG C of water-baths 10 minutes.Suction filtration device is assembled on filter flask, is configured to a set of complete suction filtration system (seeing Fig. 1).Sample is poured into and contained in sample pipe, start off-gas pump, completely by after adsorption column, close off-gas pump until sample, take out adsorption column.In adsorption column, add 500 μ L UWB1, after the centrifugal 1min of 12000rpm, then add 500 μ L UWB2, the centrifugal 1min of 12000rpm, finally carries out wash-out with 35 μ L elutriants.
Pcr amplification experiment: taking people's GAPDH as goal gene, get nucleic acid solution that 2 μ L elute as pcr template, add the premixed liquid of 11 μ L deionized waters and 7 μ L, reaction system is 20 μ L.Premixed liquid compound method is in table 5, and PCR response procedures is in table 6.
Primer sequence: GAPDH-F gcgacacccactcctccaccttt; GAPDH-R tgctgtagccaaattcgttgtcata
The compound method of table 5:PCR premixed liquid:
Table 6:PCR response procedures
After PCR reaction finishes, after getting 5 μ L amplified productions and mixing with 6 × DNA Loading Buffer, add in the well of 4% sepharose, after 45 minutes, under gel imaging instrument, observe electrophoresis result with 5V/cm electrophoresis, see Fig. 2.In electrophorogram, can see clearly, the band of the 120bp obtaining that increases taking people's GAPDH as goal gene.
Embodiment 2:
Get the urine 20mL that is diagnosed as Pancreas cancer patients, do two pipe parallel laboratory tests, every pipe urine sample is 10mL.Respectively add 15mL lysate ULB, put upside down and mix, 37 DEG C of water-baths 10 minutes.Suction filtration device is assembled on filter flask, is configured to a set of complete suction filtration system (seeing Fig. 1).Sample is poured into and contained in sample pipe, start off-gas pump, completely by after adsorption column, close off-gas pump until sample, take out adsorption column.In adsorption column, add 500 μ L UWB1, after the centrifugal 1min of 12000rpm, then add 500 μ L UWB2, the centrifugal 1min of 12000rpm, finally carries out wash-out with 35 μ L elutriants.
Pcr amplification experiment: taking the KRAS after people's sudden change as goal gene, get nucleic acid solution that 2 μ L elute as pcr template, add the premixed liquid of 11 μ L deionized waters and 7 μ L, reaction system is 20 μ L, makes 2 pipe parallel controls and one group of negative control.Premixed liquid compound method is in table 7, and PCR response procedures is shown in the table 6 in embodiment 1.
Primer sequence is: KRAS-F aaacttgtggtagttggagtta; KRAS-R tatctgtatcaaagaatggtcc
The compound method of table 7:PCR premixed liquid
After PCR reaction finishes, after getting 5 μ L amplified productions and mixing with 6 × DNA Loading Buffer, add in the well of 4% sepharose, after 45 minutes, under gel imaging instrument, observe electrophoresis result with 5V/cm electrophoresis, see Fig. 3.In electrophorogram, can see clearly, the band obtaining that increases taking people's saltant type KRAS as goal gene, length is 280bp, negative control, without band, therefore can be confirmed the KRAS gene that contains saltant type in this patient's urine DNA.
Claims (8)
1. one kind is extracted test kit and the extracting method of nucleic acid from urine, can from large capacity urine, extract trace dna fast, it is characterized in that: in urine, add lysate ULB, after insulation for some time, by sample all by nucleic acid adsorption column, nucleic acid in sample is in connection with on the pellosil to adsorption column, washs after adsorption column respectively with washings UWB1 and UWB2, then nucleic acid eluted from adsorption column with elutriant.
2. a kind of test kit and extracting method that extracts nucleic acid from urine as claimed in claim 1, is characterized in that: the nucleic acid extracting has comprised the nucleic acid in cast-off cells in free nucleic acid in urine and urine.
3. a kind of test kit and extracting method that extracts nucleic acid from urine as claimed in claim 1, is characterized in that: 0.5～3 times of amount that the consumption of lysate ULB is urine specimen.
4. a kind of test kit and extracting method that extracts nucleic acid from urine as claimed in claim 1, is characterized in that: in lysate ULB, the concentration of SDS is 0.1g/uL～1g/uL, and optimal concentration is 0.2ng/uL～0.6ng/uL; The concentration of EDTA is 0.5mmol/L～2mmol/L, and optimal concentration is 1mmol/L～1.5mmol/L; The concentration of triton x-100 is 0.5%～6%, and optimal concentration is 2%～3%; The concentration of Guanidinium hydrochloride is 2mol/L～8mol/L, and optimal concentration is 5mol/L～8mol/L.
5. a kind of test kit and extracting method that extracts nucleic acid from urine as claimed in claim 1, is characterized in that: in urine specimen, add after lysate ULB, in 37～55 DEG C of insulation 10～60min.
6. a kind of test kit and extracting method that extracts nucleic acid from urine as claimed in claim 1, it is characterized in that: adopt the method for suction filtration that urine specimen is all passed through to adsorption column, by off-gas pump, air in waste collection bottle is pumped, make external atmosphere pressure be greater than the air pressure in waste collection bottle, utilize draught head, accelerate to contain urine in sample pipe by adsorption column, reach fast nucleic acid enriching to the object on adsorption column.
7. a kind of test kit and extracting method that extracts nucleic acid from urine as claimed in claim 1, is characterized in that: the concentration of Guanidinium hydrochloride is 2mol/L～8mol/L, and optimal concentration is 3mol/L～6mol/L; Sodium chloride concentration is 75mmol/L～1.5mol/L, and optimal concentration is 120mmol/L～200mmol/L; The concentration of EDTA is 0.5mmol/L～2mmol/L, and optimal concentration is 1mmol/L～1.5mmol/L; Alcohol concn is 40%～80%, and optimal concentration is 55%～75%.
8. a kind of test kit and extracting method that extracts nucleic acid from urine as claimed in claim 1, is characterized in that: elutriant UWB2 can be the low salts solution that pure water or pH value are greater than 7.0.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106947683A (en) * | 2017-05-24 | 2017-07-14 | 苏州天隆生物科技有限公司 | A kind of nucleic acid extraction purification devices and method |
| CN110684764A (en) * | 2019-10-10 | 2020-01-14 | 杭州比格飞序生物科技有限公司 | Lysis solution for nucleic acid extraction, nucleic acid extraction kit and nucleic acid extraction method |
| CN114410744A (en) * | 2022-01-27 | 2022-04-29 | 深圳安吉康尔医学检验实验室 | Method for processing sample, nucleic acid extraction method and library thereof |
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| CN101684463A (en) * | 2008-09-28 | 2010-03-31 | 杭州优思达生物技术有限公司 | Method and kit for rapidly extracting nucleic acids from trace clinical samples |
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| CN101684463A (en) * | 2008-09-28 | 2010-03-31 | 杭州优思达生物技术有限公司 | Method and kit for rapidly extracting nucleic acids from trace clinical samples |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106947683A (en) * | 2017-05-24 | 2017-07-14 | 苏州天隆生物科技有限公司 | A kind of nucleic acid extraction purification devices and method |
| CN106947683B (en) * | 2017-05-24 | 2024-02-20 | 苏州天隆生物科技有限公司 | Nucleic acid extraction and purification device and method |
| CN110684764A (en) * | 2019-10-10 | 2020-01-14 | 杭州比格飞序生物科技有限公司 | Lysis solution for nucleic acid extraction, nucleic acid extraction kit and nucleic acid extraction method |
| CN114410744A (en) * | 2022-01-27 | 2022-04-29 | 深圳安吉康尔医学检验实验室 | Method for processing sample, nucleic acid extraction method and library thereof |
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