CN104263721A - Phlegm paper for protecting nucleic acids (DNA and RNA) in sputum and nucleic acid extraction method - Google Patents
Phlegm paper for protecting nucleic acids (DNA and RNA) in sputum and nucleic acid extraction method Download PDFInfo
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- CN104263721A CN104263721A CN201410481817.5A CN201410481817A CN104263721A CN 104263721 A CN104263721 A CN 104263721A CN 201410481817 A CN201410481817 A CN 201410481817A CN 104263721 A CN104263721 A CN 104263721A
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Abstract
The invention relates to a method for storing integrity of nucleic acids (DNA and RNA) in a sputum sample in the field of biology. The invention provides phlegm paper, which can be used for conveniently storing nucleic acids in the sputum sample at room temperature; nucleic acid protecting liquid is infiltrated on the phlegm paper, and can be used for inactivating nuclease in the sputum sample; DNA and RNA in the sputum are effectively prevented from being degraded, so as to store and transport the sputum sample. At room temperature (25-35 DEG C), the nucleic acids in the sputum sample can be stably kept for 7-14 days. Meanwhile, the invention also provides a corresponding nucleic acid extraction method.
Description
Technical field
The present invention relates in field of biology the method for how to preserve sputum sample amplifying nucleic acid (DNA and RNA) integrity, relate to phlegm paper and the method for extracting nucleic acid of convenient storage sputum sample amplifying nucleic acid at normal temperatures particularly.
Technical background
One deck mucous membrane is all covered with, at submucosa containing more mucous gland and serous gland at person's windpipe, bronchial inwall.Under normal circumstances; goblet cell and a small amount of mucus of glandular secretion cover mucous layer surface, shield, can keep the moistening of tunica mucosa tracheae to mucous membrane; suction tracheae, intrabronchial dust granules, bacterium etc. are adhered to, stop that it enters lung tissue depths.When trachea-bronchial epithelial cell and lung are subject to the stimulation of adverse factor or pathogenic infection and are inflamed, the mucous hyperemia of respiratory tract, oedema, massive inflammatory cells infiltrated, vasodilation, oozes out increase, the goblet cell of mucous layer and submucosal glandular hyperplasia hypertrophy, mucus secretion increases in a large number, produce some degeneration necrosis histocytes, retention is in segmental bronchus, and the histocyte of mucus and these degeneration necrosis just constitutes phlegm.Normal people does not generally cough up phlegm or only has a small amount of frothy sputum or mucoid phlegm, and when respiratory tract has pathology, respiratory mucosa irriate, secretory product increases, and phlegm also increases.Pathology comprises bronchiectasis, pulmonary abscess, pulmonary tuberculosis, lung cancer etc.; Mucus, foreign matter, pathogenic micro-organism is comprised, the compositions such as the mucomembranous epithelial cell that various inflammatory cell and necrosis come off, tumour cell in sputum.
Nucleic acid (DNA and RNA) in sputum is the important sample of the molecular diagnosis of the disease such as Respiratory malignant tumor and respiratory tract infection.From Exfoliated Cells in Sputa from Patients withLung carcinoma, extract nucleic acid (DNA and RNA), carry out gene test by PCR, quantitative fluorescent PCR equimolecular Biological Detection means, can be used for generaI investigation and the diagnosis of lung cancer patient.But, containing highly active nucleic acid decomposition enzyme (DNA enzymatic and RNA enzyme) in sputum, cause DNA and RNA in sputum to be easy to be degraded.DNA and RNA degraded can cause Molecular Detection accuracy and sensitivity decrease, even molecular diagnosis can not be used for when degrading serious, this directly limit the clinical application being detected the disease such as respiratory tract neoplasms or infection by sputum, reduces the value of this important clinical sample of sputum in medical diagnosis on disease.Therefore, invention one stores sputum under being used for normal temperature, and the method that in protection sputum, DNA and RNA is not degraded, carries out Molecular Detection to clinical application sputum and have important using value.The invention provides a kind of phlegm paper and method for extracting nucleic acid of protection sputum amplifying nucleic acid (DNA and RNA).Phlegm paper infiltrates nuclease protection liquid, wherein containing sequestrant, reductive agent and damping fluid, provides the optimum environment preserving nucleic acid.The sputum of lung cancer patient is wrapped in phlegm paper, in absorption sputum while moisture, can the free nucleic acid in available protecting sputum and the nucleic acid in cast-off cells.
Summary of the invention
The invention provides a kind of disposable phlegm paper preserving sputum sample, for solving the protection problem of conveniently storage and the sputum amplifying nucleic acid of sputum.
Phlegm paper is in advance through the process of nuclease protection immersion profit, and nuclease protection liquid is become to be grouped into damping fluid etc. by sequestrant, reductive agent.Phlegm is told on phlegm paper by patient, and phlegm paper is while absorption moisture, and the composition of nuclease protection liquid acts on the nuclease in sputum, makes enzyme deactivation, DNA and RNA in available protecting sputum, from degraded, is convenient to storage and transport sputum sample.When extracting nucleic acid, phlegm paper is added in specific cleavage liquid, concussion 30min, then lysate is added on nucleic acid absorption post, after specific washings (WB1, WB2) washing, with elutriant, nucleic acid is eluted from adsorption column.The nucleic acid (DNA and RNA) extracted includes the free nucleic acid in sputum and the nucleic acid in cell/tissue.
The composition of nuclease protection liquid includes but not limited to DTT, EDTA, Tris-HCl, and the concentration in formula is in table 1:
Table 1: nuclease protection liquid formula
In nuclease protection liquid, DTT concentration is 5mmol/L ~ 500mmol/L, and wherein optimal concentration is 115mmol/L ~ 135mmol/L; EDTA concentration is 10mmol/L ~ 1.0mol/L, and wherein optimal concentration is 720mmol/L ~ 850mmol/L; Tris-HCl concentration is 10mmol/L ~ 200mmol/L, and wherein optimal concentration is 50mmol/L ~ 150mmol/L, and optimal pH is pH5.8 ~ pH7.2.
Containing DTT, tween, Triton, Guanidinium hydrochloride, EDTA in lysate, the concentration in formula is in table 2:
Table 2: lysate formula
In lysate, DTT concentration is 5mmol/L ~ 500mmol/L, and wherein optimal concentration is 215mmol/L ~ 260mmol/L; EDTA concentration is 10mmol/L ~ 1.0mol/L, and wherein optimal concentration is 180mmol/L ~ 285mmol/L; Tween 20 concentration is 0.5% ~ 10%, and wherein optimal concentration is 3.5% ~ 4.2%; Triton x-100 concentration is 0.5% ~ 10%, and wherein optimal concentration is 3.8% ~ 4.5%; Concentration of guanidine hydrochloride is 2mol/L ~ 6mol/L, and wherein optimal concentration is 4mol/L ~ 6mol/L.
The composition of washings WB1 and formula are in table 3:
Table 3: washings WB1 fills a prescription
The composition of washings WB2 and formula are in table 4:
Table 4: washings WB2 fills a prescription
The form of phlegm paper can be one of following three kinds of modes, but is not limited to this three kinds of forms:
1. square or circular paper, front infiltrates nuclease protection liquid, and reverse side is water-proof material, and patient to spit to front FOLD AND PACK voluntarily.
2. envelope type, inner infiltration nuclease protection liquid, outside is water-proof material, and patient spits in envelope, and autoadhesion seals.
3. boxlike, be placed with in the carton of flat pattern and infiltrate the scraps of paper (at the bottom of box, a slice, on lid, fully wraps up sputum to a slice) that DNA protects liquid, patient spits in box, shuts.
The present invention has following advantage and using value:
1. single use, simple to operate, easy to use, pollution-free danger;
2. the acid of available protecting sputum middle reaches freestone and the integrity of cast-off cells nucleic acid molecule and stability;
3., under room temperature (25 ~ 35 DEG C), the nucleic acid Absorbable organic halogens in sputum sample is preserved 7 ~ 14 days, does not need to take low-temperature protection measure in sample transportation;
4. the method extracting nucleic acid from sputum sample is easy and simple to handle, and the Nucleic acid quality extracted is high, can be used for Clinical Laboratory.
Embodiment
With specific embodiment, the present invention is described in further detail below.Should be understood that, specific embodiment is only make clearer explanation to the present invention, instead of limitation of the present invention.
Embodiment:
Obtain 10 parts, the sputum sample of the patient be stored in phlegm paper in hospital, sample does not save 1 ~ 14 day not etc. under room temperature (25 ~ 35 DEG C).
Phlegm paper operating scissors containing sputum are shredded, is placed in test tube, adds lysate in the ratio of 1:5, shaking table shakes 30min.Nucleic acid absorption post is placed in collection tube, is all transferred to by above-mentioned lysate in nucleic acid absorption post, room temperature centrifugal (12,000rpm, 1min), abandons filtrate.In nucleic acid absorption post, add the washings WB1 of 500 μ L, room temperature centrifugal (12,000rpm, 1min), abandons filtrate.In nucleic acid absorption post, add 500 μ L washings WB2, room temperature centrifugal (12,000rpm, 1min), abandons filtrate.Nucleic acid absorption post is placed in new 1.5mL nuclease free pollution centrifuge tube room temperature to leave standstill or the air-dry 3-5min of Bechtop, with the ethanol of thorough volatile residue.Carefully add the pure water of 50 μ L through the nuclease free pollution of 65 DEG C of temperature baths to adsorption column post film overcentre, room temperature leaves standstill 3 ~ 5min, and centrifugal (12,000rpm, 1min), elutriant is nucleic acid solution.
Detect concentration and the purity of nucleic acid solution, result is as shown in the table:
| ? | Concentration (ng/ μ L) | 260/280 | 260/230 |
| Sample 1 | 124.6 | 1.86 | 1.77 |
| Sample 2 | 203.2 | 1.75 | 1.84 |
| Sample 3 | 89.3 | 1.87 | 1.72 |
| Sample 4 | 167.3 | 1.80 | 1.76 |
| Sample 5 | 72.8 | 1.72 | 1.65 |
| Sample 6 | 267.7 | 1.83 | 1.88 |
| Sample 7 | 184.8 | 1.88 | 1.74 |
| Sample 8 | 112.5 | 1.86 | 1.78 |
| Sample 9 | 137.5 | 1.77 | 1.81 |
| Sample 10 | 218.4 | 1.96 | 1.85 |
Claims (7)
1. a phlegm paper, can protect the nucleic acid in sputum (DNA and RNA) not to be degraded after parcel sputum at normal temperatures.
2. phlegm paper as claimed in claim 1 has infiltrated nuclease protection liquid; composition comprises DTT (DL-Dithiothreitol; dithiothreitol (DTT)), EDTA (Ethylene Diamine Tetraacetic Acid, ethylenediamine tetraacetic acid (EDTA) and disodium salt thereof), Tris-HCl (tris-HCI buffer).
3. nuclease protection liquid as claimed in claim 2, DTT concentration is 5mmol/L ~ 500mmol/L, and wherein optimal concentration is 115mmol/L ~ 135mmol/L; EDTA concentration is 10mmol/L ~ 1.0mol/L, and wherein optimal concentration is 720mmol/L ~ 850mmol/L; Tris-HCl concentration is 10mmol/L ~ 200mmol/L, and wherein optimal concentration is 50mmol/L ~ 150mmol/L, and optimal pH is pH5.8 ~ pH7.2.
4. phlegm paper as claimed in claim 1, is made up of pros and cons, and front infiltrates nuclease protection liquid, and reverse side is water-proof material.
5. phlegm paper as claimed in claim 1, can be square or circular paper, envelope type or boxlike in shape.
6. one kind for extracting the lysate of the sputum sample nucleic acid be stored on phlegm paper, composition comprises DTT (DL-Dithiothreitol, dithiothreitol (DTT)), EDTA (Ethylene Diamine Tetraacetic Acid, ethylenediamine tetraacetic acid (EDTA) and disodium salt thereof), tween 20 (Tween-20, Polyethylene glycol sorbitan monolaurate), triton x-100 (Triton X-100, t-Octylphenoxypolyethoxyethanol) and Guanidinium hydrochloride (Guanidine Hydrochloride).
7. lysate as claimed in claim 6, DTT concentration is 5mmol/L ~ 500mmol/L, and wherein optimal concentration is 215mmol/L ~ 260mmol/L; EDTA concentration is 10mmol/L ~ 1.0mol/L, and wherein optimal concentration is 180mmol/L ~ 285mmol/L; Tween 20 concentration is 0.5% ~ 10%, and wherein optimal concentration is 3.5% ~ 4.2%; Triton x-100 concentration is 0.5% ~ 10%, and wherein optimal concentration is 3.8% ~ 4.5%; Concentration of guanidine hydrochloride is 2mol/L ~ 6mol/L, and wherein optimal concentration is 4mol/L ~ 6mol/L.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104673717A (en) * | 2015-02-12 | 2015-06-03 | 中国医科大学 | Biological tissue RNA (ribonucleic acid) protection reagent as well as preparation method and application thereof |
| CN107058288A (en) * | 2017-01-24 | 2017-08-18 | 南方医科大学 | A kind of kit of rapid extraction viral RNA |
| CN107119044A (en) * | 2017-05-02 | 2017-09-01 | 重庆市公安局巴南区分局 | A kind of wiping reagent for contact DNA transfers on carrier |
| CN107227306A (en) * | 2017-06-26 | 2017-10-03 | 郑州安图生物工程股份有限公司 | A kind of swab eluent with Sample preservation and inactivation function |
| CN108949748A (en) * | 2018-07-30 | 2018-12-07 | 浙江今复康生物科技有限公司 | A kind of sputum liquefaction and nuclease protection reagent |
| CN109762809A (en) * | 2019-03-22 | 2019-05-17 | 北京医院 | The extracting method of cfDNA in sputum sample |
| CN111172239A (en) * | 2020-02-28 | 2020-05-19 | 上海思路迪医学检验所有限公司 | Virus sample preserving fluid, nucleic acid extraction reagent and virus nucleic acid extraction method |
| CN111518797A (en) * | 2020-04-30 | 2020-08-11 | 上海安五生物科技有限公司 | Normal-temperature protection solution and preparation method and application thereof |
| CN112304930A (en) * | 2020-04-20 | 2021-02-02 | 浙江今复康生物科技有限公司 | Disulfide bond detection method and sputum detection kit containing disulfide bonds |
| CN112501158A (en) * | 2020-12-04 | 2021-03-16 | 麦凯(上海)生物科技有限公司 | Human liquid sample whole nucleic acid extraction kit and application thereof |
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| CN2540955Y (en) * | 2002-05-20 | 2003-03-26 | 席晓艳 | Portable sputum aspirating paper bag (case) |
| CN2652311Y (en) * | 2003-10-17 | 2004-11-03 | 何庶 | Sputum paper |
| CN101613696A (en) * | 2009-08-05 | 2009-12-30 | 公安部物证鉴定中心 | Extract the reagent of purify DNA |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104673717B (en) * | 2015-02-12 | 2017-11-28 | 中国医科大学 | A kind of biological tissue RNA protections reagent and its preparation method and application |
| CN104673717A (en) * | 2015-02-12 | 2015-06-03 | 中国医科大学 | Biological tissue RNA (ribonucleic acid) protection reagent as well as preparation method and application thereof |
| CN107058288A (en) * | 2017-01-24 | 2017-08-18 | 南方医科大学 | A kind of kit of rapid extraction viral RNA |
| CN107058288B (en) * | 2017-01-24 | 2020-06-05 | 南方医科大学 | Kit for rapidly extracting viral RNA |
| CN107119044B (en) * | 2017-05-02 | 2020-11-06 | 重庆市公安局巴南区分局 | Wiping reagent for contact DNA transfer on carrier |
| CN107119044A (en) * | 2017-05-02 | 2017-09-01 | 重庆市公安局巴南区分局 | A kind of wiping reagent for contact DNA transfers on carrier |
| CN107227306A (en) * | 2017-06-26 | 2017-10-03 | 郑州安图生物工程股份有限公司 | A kind of swab eluent with Sample preservation and inactivation function |
| CN107227306B (en) * | 2017-06-26 | 2021-01-26 | 郑州安图生物工程股份有限公司 | Swab eluent with sample preservation and inactivation functions |
| CN108949748A (en) * | 2018-07-30 | 2018-12-07 | 浙江今复康生物科技有限公司 | A kind of sputum liquefaction and nuclease protection reagent |
| CN109762809A (en) * | 2019-03-22 | 2019-05-17 | 北京医院 | The extracting method of cfDNA in sputum sample |
| CN111172239A (en) * | 2020-02-28 | 2020-05-19 | 上海思路迪医学检验所有限公司 | Virus sample preserving fluid, nucleic acid extraction reagent and virus nucleic acid extraction method |
| CN112304930A (en) * | 2020-04-20 | 2021-02-02 | 浙江今复康生物科技有限公司 | Disulfide bond detection method and sputum detection kit containing disulfide bonds |
| CN112304930B (en) * | 2020-04-20 | 2022-08-23 | 浙江今复康生物科技有限公司 | Disulfide bond detection method and sputum detection kit containing disulfide bonds |
| CN111518797A (en) * | 2020-04-30 | 2020-08-11 | 上海安五生物科技有限公司 | Normal-temperature protection solution and preparation method and application thereof |
| CN112501158A (en) * | 2020-12-04 | 2021-03-16 | 麦凯(上海)生物科技有限公司 | Human liquid sample whole nucleic acid extraction kit and application thereof |
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Application publication date: 20150107 |