CN107904152A - Sputum collecting pipe and sputum store method for detection of nucleic acids - Google Patents
Sputum collecting pipe and sputum store method for detection of nucleic acids Download PDFInfo
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- CN107904152A CN107904152A CN201711190683.1A CN201711190683A CN107904152A CN 107904152 A CN107904152 A CN 107904152A CN 201711190683 A CN201711190683 A CN 201711190683A CN 107904152 A CN107904152 A CN 107904152A
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- sputum
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The present invention provides a kind of sputum collecting pipe and sputum store method for detection of nucleic acids, nucleic acid inhibitor built in sputum collecting pipe, pH value maintains agent and dehydrating agent.The present invention is directed to sputum sample amplifying nucleic acid problem of easy degradation; design a kind of collecting pipe containing nuclease protection agent; the person under inspection for cooking sputum detection of nucleic acids is conveniently needed to collect sputum at any time using portable collecting pipe when there is phlegm; the nucleic acid in sputum sample can be protected not to be degraded at ambient temperature at the same time, time when small (72) for giving person under inspection's abundance send sample to (or express delivery) hospital or other inspection bodies progress detection of nucleic acids.
Description
Technical field
This method belongs to the Sample preservation technical field for medicine detection, is related to a kind of sputum for detection of nucleic acids and protects
Method and collecting pipe are deposited, key problem in technology is not degraded in the nucleic acid in sputum sample is protected.
Background technology
Phlegm is the secretion of human respiratory tract, it be by the movement of bronchus ciliary movement epithelial cilia, from lung to
The upper respiratory tract promotes, and finally, is excreted by the normal cough reflex of people from tracheal strips expectoration, and normal person's phlegm is seldom, simply
The a small amount of mucus for keeping respiratory tract moistening and secreting.When people's suction irritative gas, dust, pathogenic bacteria, virus etc. are harmful micro-
Biochron, the upper respiratory tract may be inflamed；Or disease occurs for lung, such as bronchiectasis, pulmonary abscess, lung cancer, are exhaled
Increase will be secreted by inhaling road, and amount of expectoration will increase, and the property of phlegm can also change, and can become cheese phlegm, blood by viscous phlegm
Phlegm, yellowish purulent sputum etc..According to sputum produce and discharge mechanism understand, in sputum include multiple pathogenic microorganisms, all kinds of inflammatory cells,
Come off downright bad mucosal epithelial cells and tumour cell etc..
At present clinically, sputum sample is mainly used for expectorative cytology detection and respiratory pathogen detection.Wherein,
Identify that the species of pathogen is most accurate at present and most quick respiratory tract by detecting the nucleic acid of pathogen in sputum sample
Pathogen diagnosis method.In the pathogen that acute respiratory infection can be caused, in the highest flight, next is only bacterium, branch to virus
Substance, mould, protozoon etc..In primary acute upper respiratory infection, virus infection up to more than 90%.Common respiratory tract
Pathogen has：Mycobacterium tuberculosis, Respiratory Syncytial Virus(RSV), adenovirus, Epstein-Barr virus, influenza virus, mycoplasma pneumoniae, rubella
Poison, streptococcus, staphylococcus, chlamydia pneumoniae, assays for parvovirus B 19, cytomegalovirus and coronavirus etc..Traditional cause of disease
Body diagnostic method is to be separately cultured or Serologic detection, is separately cultured that time-consuming, complicated, is not suitable for clinical acute disease
Diagnosis；Serologic detection is difficult to accomplish to early diagnose in disease.As quantitative fluorescent PCR and multiple PCR technique are in pathogen
The fast development of detection field, has the characteristics that high specificity, high sensitivity, easy to operate, detection is rapid, recall rate is high, should
Technology gradually occupies critical role in clinical diagnosis field.In addition, from High Risk of Lung Cancer crowd and the sputum sample of patients with lung cancer
Middle extraction nucleic acid, available for the prediction of the early stage of lung cancer and the detection in Gene Mutation of patients with lung cancer and Treatment monitoring.To sum up, sputum
The detection of sample amplifying nucleic acid is clinically of great significance.But in practical operation, the nucleic acid in sputum sample is (especially
RNA) extremely unstable, usually a few hours are just degraded under greenhouse experiment.Cause nucleolysis extraneous physical factor such as temperature,
Humidity, ultraviolet etc.；Chemical factor such as strong acid or strong alkali environment, hydrolysis etc.；Biological factor such as enzymolysis and microbial infection
Deng.Wherein, in sputum cell rupture discharge nuclease and operating process in because pollution introduce nuclease be caused by nucleic acid
The main reason for degraded.In addition, general person under inspection, even the patient in period of disease, nor at any time can expectoration sufficient amount
Sputum, most patients from morning after sputum amount it is more, but can not in time collect and preserve sputum.
Chinese patent application file CN104263721A, discloses a kind of phlegm paper of protection sputum amplifying nucleic acid (DNA and RNA)
And method for extracting nucleic acid, it is primarily present following deficiency：(1) the phlegm paper described in its patent for square or circular paper, envelope type or
Boxlike, such a phlegm paper are easily lost, and do not tolerate external force extruding, and sputum is easy excessive (especially when sputum is more), after collection sputum not
Easy to carry with and transport；(2) described in its patent, it is DTT (dithiothreitol (DTT)), EDTA that main component has been infiltrated on phlegm paper
The nuclease protection liquid of (ethylenediamine tetra-acetic acid and its disodium salt), Tris-HCl (trihydroxy aminomethane-hydrochloride buffer), infiltration
Protection liquid hold-up it is less, it is impossible to ensure that sputum sample comes into full contact with nuclease protection liquid, to the protecting effect of sputum amplifying nucleic acid
Generally.
In addition, the related patents for being used to protect nucleic acid or cell published at present, such as Chinese patent application file
Disclosed in CN105145545A《Nuclease protection liquid for long-term storage and transport tissue samples under normal temperature condition》, Chinese patent
Disclosed in application documents 106065400A《A kind of ribonucleic acid protective agent, kit, application and store method》And China is specially
Disclosed in sharp 104041484 A of application documents CN《A kind of cell-preservation liquid》Deng the technology that these patents are related to is used equally for protecting
Protect the nucleic acid of tissue or cell.However, the technology that these patents are related to is poor for the practicality of sputum sample, main cause
Have：(1) sputum sample has the characteristics that viscosity is big, albumen is more, is not easy to liquefy, is easily agglomerating, and sputum is added conventional liq protection
After agent, sputum is easily layered with protective agent, and protective agent and sputum sample contact face are limited；(2) protective agent that some patents are related into
It is point more complicated, it is more than protective agent component and of high cost such as containing preservative, buffer, chelating agent, enzyme inhibitor, fixative,
Unsuitable clinic widely uses.
The content of the invention
In order to solve the problems, such as that at present clinically sputum sample amplifying nucleic acid is easily degraded and cannot collect sputum at any time, this
Invention, which provides one kind and is convenient for carrying, can collect sputum at any time, and the side that the nucleic acid in sputum sample can be protected not to be degraded
Method.
The technical scheme is that：
It is a kind of to protect the sputum store method of nucleic acid and be convenient for carrying the sputum collecting pipe that can be collected at any time.Main bag
Collecting pipe and protective agent are included, collecting pipe can accommodate sputum sample and protective agent, it can close and can carry with；Sputum collecting
Protective agent is pre-placed in pipe, protective agent includes nucleic acid inhibitor, pH value maintains agent and dehydrating agent, plays protection sample center
The effect of the integrality of acid.
(1) collecting pipe used in is 50ml swivelling covers conical centrifuge tube, 50ml swivelling cover round bottom centrifuge tube 50ml swivelling covers are put down
Bottom centrifuge tube (can be vertical), is not limited to 3 kinds of the above, and what other can accommodate protective agent and sputum can closed and portable appearance
Device.
(2) nucleic acid inhibitor is guanidine hydrochloride, guanidinium isothiocyanate, lauryl sodium sulfate, NaTDC, 4- amino water
One or more combination in poplar acid sodium, naphthalene -1,5- sodium disulfonates, sodium triisopropyl naphthalene sulfonate.The quality of nucleic acid inhibitor
For 0.5-4.0g.
(3) pH value maintains agent as trishydroxymethylaminomethane (Tris), 3- (N- morpholines) propane sulfonic acid (MOPS), double (2-
(methylol) amino-three (methylol) methane (Bis Tris), two ethyl sulfonic acids of piperazine -1,4- (PIPES), N-2- ethoxy piperazines
One or more combination in piperazine-N-2- ethyl sulfonic acids (HEPES).It is 1.5-4.0mg that pH value, which maintains the quality of agent,.
(3) dehydrating agent for polyethylene glycol (PEG), n-butanol, sec-butyl alcohol, the one or more combination of the tert-butyl alcohol.Dehydrating agent
Quality be 0.5-2.5g or volume is 1.0-3.0ml.
Preferably, the nucleic acid inhibitor and collecting pipe are：The poly- second two of 1.0g guanidine hydrochlorides, 2.5mg Tris and 1.0g
Alcohol is placed in 50ml swivelling cover conical centrifuge tubes.
The present invention also provides a kind of sputum store method for detection of nucleic acids, comprise the following steps
(1) the sputum sample collected, the volume of sputum sample is 1-5ml；Wherein sputum sample does not limit, the phlegm of collection
Liquid sample can be the sputum of the various characters such as viscous phlegm, bloody sputum, phlegm, stiff phlegm, yellow/grey/rusty expectoration.
(2) sputum for detection of nucleic acids that sputum sample is placed directly within as described in claim 1-9 any one is received
In collector, sputum sample is set to be mixed with the protective agent in sputum collecting pipe, the storage temperature of collecting pipe is less than or equal to 25 DEG C.
The principle of the present invention is as follows：The effect of nucleic acid inhibitor is the nuclease in inhibition system, prevents sputum
Nucleic acid in sample is degraded；PH value maintains the effect of agent to be to maintain sputum sample as neutral or alkalescent, is in sputum sample
Nucleic acid metastable pH environment is provided；The effect of dehydrating agent is the moisture reduced in sputum sample, prevents sputum sample
In nucleic acid be hydrolyzed, dehydrating agent only siphons away moisture, will not dissolve nucleic acid.Sealable collecting pipe with cover is easy to carry with,
Person under inspection can be facilitated to collect sputum, and the effectively integrality of protection sputum amplifying nucleic acid at any time when having expectoration reaction suddenly.
To verify the validity of this method, contrast is carried out to this method using fluorescence quantitative PCR method and electrophoresis respectively and is tested
Card, experimental procedure and result are as follows：
(1) 1.0g guanidine hydrochlorides are weighed respectively, 2.5mg Tris and 1.0g polyethylene glycol be placed in 50ml swivelling cover point bottoms from
In heart pipe, fully mix and sputum collecting pipe is made, prepare 4 altogether and contain protectant collecting pipe, numbering 1-4；In addition 4 are prepared
The collecting pipe of unprotect agent, numbering 5-8；
(2) the patients with lung cancer sputum through clinical definite provided by Zhejiang University Medical College The First Affiliated Hospital's Respiratory Medicine
Sample.The Telomerase contained in lung carcinoma cell imparts the ability of lung carcinoma cell infinite multiplication, and Telomerase is by lung carcinoma cell
HTERT mRNA translation synthesis, therefore hTERT mRNA are the characteristic indication things of lung carcinoma cell.In the generation and expectoration of sputum
Cheng Zhong, the lung carcinoma cell to come off are blended in sputum by expectoration.Therefore it is able to detect that hTERT in the sputum of patients with lung cancer
mRNA.The sputum sample of 10 patients with lung cancer is fully mixed to the positive mixing sputum sample of lung cancer for being prepared into 16ml, is taken respectively
2ml sputums sample is into 1-8 collecting pipes；
(3) collecting pipe is placed under the conditions of room temperature (20 DEG C) and placed, different collecting pipes and standing time are as shown in the table：
| Collecting pipe is numbered | 1、3、5、7 | 2、4、6、8 |
| Standing time | 5 it is small when | 48 it is small when |
(4) mRNA in fluorescence quantitative PCR method detection sputum sample：Opened using Zhejiang Jin Fukang bio tech ltd
" reverse transcriptase of telomere subunit (hTERT) mRNA detection kits " (the state's tool note standard 20173404247) of hair, which can
Detect the hTERT mRNA in sputum sample.When PCR amplification result is Ct value ＜ 33, show that testing result represents to be positive
Contain hTERT mRNA in sample；When PCR amplification result is Ct value >=33 or No Ct, show testing result for negative i.e. table
Without hTERT mRNA in sample sheet.Product description operating procedure above-mentioned (3) are obtained No. 1 with reference to the kit, No. 2,5
Number, the sample in No. 6 collecting pipes be detected, dissolve, hybridize by sputum, washing, eluting, digestion and PCR amplification,
The experimental results are shown inthe following table：
Test result indicates that：Preserved respectively under the conditions of room temperature (20 DEG C) using this method lung cancer positive sputum 5 it is small when and
48 it is small when, the hTERT mRNA in sample are enough detected, it was demonstrated that its integrality is good；And inapplicable this method is in room temperature (20
DEG C) under the conditions of place lung cancer positive sputum 5 it is small when and 48 it is small when after,
HTERT mRNA are not detected, illustrate that it has been degraded.Therefore in this method can effectively protect sputum sample
MRNA is not degraded.
(5) nucleic acid in electrophoresis detection sputum sample：Obtain to above-mentioned (3) No. 3, add 100 μ l in No. 4 collecting pipes
Guanidine hydrochloride solution (2.0g/ml), adds 600 μ l guanidine hydrochloride solutions (2.0g/ in No. 7 obtained to above-mentioned (3), No. 8 collecting pipes
Ml 100 μ l beta -mercaptoethanols, 30 μ l 20%NP-40), then are respectively added into No. 3, No. 4, No. 7, No. 8 pipes；60 DEG C of insulations
5min, acutely concussion mix, 12000rpm centrifugation 10min, take supernatant coker acid separation column, 12000rpm centrifugations 1min；Take 700
μ l cleaning solutions (10mM MES pH6.0,35% ethanol) wash 2 times；Pillar is placed on clean 1.5ml centrifuge tubes, to pillar
Film centre position adds 30 μ l TE solution, 60 DEG C of insulation 3min；12000rpm centrifuges 1min, collects efflux, takes 15 μ l effluxes
Gel electrophoresis is carried out, electrophoresis result is shown in Fig. 1.
Test result indicates that：Preserved respectively under the conditions of room temperature (20 DEG C) using this method lung cancer positive sputum 5 it is small when and
48 it is small when, corresponding sample (being respectively 3, No. 4) visible clearly nucleic acid bands after electrophoresis, it was demonstrated that sputum sample amplifying nucleic acid is complete
Whole property is good；Without the lung cancer positive sputum using this method directly when placement 5 is small under the conditions of room temperature (20 DEG C) and when 48 is small
Sample (being respectively 7, No. 8), has no nucleic acid bands, it was demonstrated that it has been degraded after electrophoresis.Therefore this method can effectively protect phlegm
Nucleic acid in liquid sample is not degraded.
The beneficial effects of the invention are as follows：Sputum can be collected at any time the present invention provides one kind and protects sputum amplifying nucleic acid not
The method being degraded.Nuclease protection agent is placed in a kind of collecting pipe being convenient for carrying by this method, can be when person under inspection has phlegm
Sputum is collected at any time, and protective agent therein can avoid nucleic acid from being degraded well.Protective agent of the present invention is powdered
Solid, can preferably penetrate into sputum sample, so as to preferably be come into full contact with sputum sample.It is in addition, of the present invention
Protective agent component is relatively easy and cost is low, is highly suitable for clinic and widely uses, conveniently need to cook sputum detection of nucleic acids by
Inspection person collects sputum at any time when there is phlegm using portable collecting pipe, while can protect sputum sample at ambient temperature
In nucleic acid be not degraded, the time for giving person under inspection's abundance send sample to (or express delivery) hospital or other inspection bodies.
Brief description of the drawings
Fig. 1 is the electrophoresis pattern that electrophoresis detects the nucleic acid in sputum sample in the content of the invention, is followed successively by 3 from left to right
Number, No. 7, No. 4, No. 8, Marker；
Fig. 2 be embodiment 4 in electrophoresis detect sputum sample in nucleic acid electrophoresis pattern, be followed successively by from left to right No. 5,
No. 6, No. 7, No. 8, Marker.
Embodiment
Used sputum sample is by commission Zhejiang University of Zhejiang Jin Fukang bio tech ltd medicine in embodiment
The attached First Hospital Respiratory Medicine of institute takes phlegm programmed acquisition, reference by clinical criteria《Chinese tuberculosis control program Sputum smears
Microscopic criteria operation and quality control (assurrance) manual》, pass through range estimation, yellow, grey, rust, courage and uprightness, purulence, stiff, the group of presentation
Block sample is reception standard.Lung cancer positive sputum is collected by the patients with lung cancer of clinical definite.Contain in lung carcinoma cell
Telomerase impart the ability of lung carcinoma cell infinite multiplication, and Telomerase is synthesized by the hTERT mRNA translations of lung carcinoma cell,
Therefore hTERT mRNA are the characteristic indication things of lung carcinoma cell.During the generation and expectoration of sputum, the lung carcinoma cell that comes off
It is blended in sputum by expectoration.Therefore hTERT mRNA are able to detect that in the sputum of patients with lung cancer.Detection of nucleic acids uses Zhejiang
" reverse transcriptase of telomere subunit (hTERT) mRNA detection kits " (state's tool of Jiang Jinfu health bio tech ltd exploitation
Note is accurate 20173404247), which can detect the hTERT mRNA in sputum sample.When PCR amplification result is Ct values ＜ 33
When, show that testing result represents to contain hTERT mRNA in sample to be positive；When PCR amplification result is Ct value >=33 or No
During Ct, show that testing result is represented in sample without hTERT mRNA to be negative.
Embodiment 1：Sputum sample can be preserved in guanidine hydrochloride-Tris- polyethylene glycol protective agents 72 it is small when
Step 1：1.0g guanidine hydrochlorides are weighed respectively, 2.5mg Tris and 1.0g polyethylene glycol is placed in 50ml swivelling cover points bottom
In centrifuge tube, fully mix and sputum protective agent and collecting pipe is made, prepare 4 altogether and contain protectant collecting pipe, numbering 1-4；
In addition the collecting pipe of 4 unprotect agent, numbering 5-8 are prepared；Some lung cancer positive sputums are taken fully to mix the lung for being prepared into 16ml
Cancer positive sputum sample, takes 2ml sputums sample into each collecting pipe respectively；
Step 2：Collecting pipe is placed under the conditions of room temperature (20 DEG C) and is placed, different collecting pipes and standing time such as following table institute
Show：
| Collecting pipe is numbered | 1、5 | 2、6 | 3、7 | 4、8 |
| Standing time | 24 it is small when | 48 it is small when | 72 it is small when | 96 it is small when |
Step 3：Utilize reverse transcriptase of telomere subunit (hTERT) mRNA detection kits " (state's tool note is accurate
20173404247), the Sputum samples that step 2 obtains are detected with reference to product description operating procedure, it is molten by sputum
Solution, hybridization, washing, elution, digestion and PCR amplification and etc., experimental result see the table below.The results show is preserved using this method
Sputum sample room temperature place 24-72 it is small when after remain able to detect mRNA, without the sputum directly placed using this method
Sample can not just detect mRNA when room temperature placement 24 is small, therefore this method can protect sputum under the conditions of room temperature (20 DEG C)
The integrality of sample amplifying nucleic acid is when 72 is small.
| Sample number into spectrum | No. 1 | No. 2 | No. 3 | No. 4 | No. 5 | No. 6 | No. 7 | No. 8 |
| Ct values | 25.97 | 27.03 | 30.11 | 35.76 | No Ct | No Ct | No Ct | No Ct |
| Experimental result | It is positive | It is positive | It is positive | It is negative | It is negative | It is negative | It is negative | It is negative |
Embodiment 2：This method is suitable for viscous phlegm, bloody sputum, phlegm, stiff phlegm, yellow phlegm, grey phlegm, rusty expectoration
Step 1：1.0g guanidine hydrochlorides are weighed respectively, 2.5mg Tris and 1.0g polyethylene glycol is placed in 50ml swivelling cover points bottom
In centrifuge tube, fully mix and sputum collecting pipe is made, prepare 7 altogether and contain protectant collecting pipe, numbering 1-7；
Step 2：Take lung cancer positive and character is respectively viscous phlegm, bloody sputum, phlegm, stiff phlegm, yellow phlegm, grey phlegm, iron rust
Color phlegm is a case each, is respectively placed in 1-7 collecting pipes, collecting pipe is placed under the conditions of room temperature (20 DEG C) place 72 it is small when；
Step 3：Utilize reverse transcriptase of telomere subunit (hTERT) mRNA detection kits " (state's tool note is accurate
20173404247), the Sputum samples that step 2 obtains are detected with reference to product description operating procedure, it is molten by sputum
Solution, hybridization, washing, elution, digestion and PCR amplification and etc., experimental result see the table below.The results show this method is to viscous phlegm, blood
Nucleic acid in phlegm, phlegm, stiff phlegm, yellow/grey/rusty expectoration can play preferable protective effect：
| Sample number into spectrum | No. 1 | No. 2 | No. 3 | No. 4 | No. 5 | No. 6 | No. 7 |
| Ct values | 22.67 | 25.44 | 29.05 | 31.11 | 25.64 | 28.92 | 27.49 |
| Experimental result | It is positive | It is positive | It is positive | It is positive | It is positive | It is positive | It is positive |
Embodiment 3：This method is suitable for 1-5ml sputum samples
Step 1：1.0g guanidine hydrochlorides are weighed respectively, 2.5mg Tris and 1.0g polyethylene glycol is placed in 50ml swivelling cover points bottom
In centrifuge tube, fully mix and sputum protective agent and collecting pipe is made, prepare 5 altogether and contain protectant collecting pipe, numbering 1-5；
Step 2：Take some lung cancer positive sputums fully to mix the lung cancer positive sputum sample for being prepared into 10ml, take respectively
Collecting pipe is placed under the conditions of room temperature (20 DEG C) into 1-5 collecting pipes and places 72 by 1ml, 2ml, 3ml, 4ml, 5ml sputum sample
Hour；
Step 3：Utilize reverse transcriptase of telomere subunit (hTERT) mRNA detection kits " (state's tool note is accurate
20173404247), the Sputum samples that step 2 obtains are detected with reference to product description operating procedure, it is molten by sputum
Solution, hybridization, washing, elution, digestion and PCR amplification and etc., experimental result see the table below.The results show this method is to 1-5ml phlegm
Nucleic acid in liquid sample can play preferable protective effect：
| Sample number into spectrum | No. 1 | No. 2 | No. 3 | No. 4 | No. 5 |
| Ct values | 29.79 | 26.74 | 25.03 | 21.21 | 20.65 |
| Experimental result | It is positive | It is positive | It is positive | It is positive | It is positive |
Embodiment 4：Sputum sample can be preserved in guanidinium isothiocyanate-Bis Tris-n-butanol protective agent 72 it is small when
Step 1：1.2g guanidinium isothiocyanates are weighed respectively, 2.0mg Bis Tris and the 2ml tert-butyl alcohols are placed in 50ml rotations
In lid round bottom centrifuge tube, fully mix and sputum protective agent and collecting pipe is made, prepare 8 altogether and contain protectant collecting pipe, compile
Number 1-8；
Step 2：Take some lung cancer positive sputums fully to mix the lung cancer positive sputum sample for being prepared into 16ml, take respectively
2ml sputums sample into 1-8 collecting pipes, collecting pipe is placed under the conditions of room temperature (20 DEG C) place 72 it is small when；
Step 3：Utilize reverse transcriptase of telomere subunit (hTERT) mRNA detection kits " (state's tool note is accurate
20173404247) mRNA in sputum sample, the 1-4 sputums obtained with reference to product description operating procedure to step 2 are detected
Sample is detected, and is dissolved, is hybridized by sputum, washing, eluting, digestion and PCR amplification, experimental result see the table below.
The results show sputum sample can be preserved in guanidinium isothiocyanate-Bis Tris- tert-butyl alcohol protective agents 72 it is small when：
Step 4：The nucleic acid in sputum sample is detected using electrophoresis.Add respectively in the 5-8 collecting pipes obtained to step 2
Enter 600 μ l guanidine hydrochloride solutions (2.0g/ml), then add 100 μ l beta -mercaptoethanols, 30 μ into No. 3, No. 4, No. 7, No. 8 pipes respectively
The NP-40 of l 20%；60 DEG C of insulation 5min, acutely concussion mix, and 12000rpm centrifugation 10min, take supernatant coker acid separation column,
12000rpm centrifuges 1min；700 μ l cleaning solutions (10mM MES pH6.0,35% ethanol) are taken to wash 2 times；Pillar is placed in totally
1.5ml centrifuge tubes on, add 30 μ l TE solution to pillar film centre position, 60 DEG C of insulation 3min；12000rpm centrifuges 1min,
Efflux is collected, takes 15 μ l effluxes to carry out gel electrophoresis, electrophoresis result is shown in Fig. 2.The results show that sputum sample is in isothiocyanic acid
In guanidine-Bis Tris- tert-butyl alcohol protective agents preserve 72 it is small when after, corresponding sample (No. 5-8) after electrophoresis it is visible clearly
Nucleic acid bands, it was demonstrated that the nucleic acid integrality in sputum sample is good.
Embodiment 5：Guanidine hydrochloride in protective agent has in 0.5-4.0g and can play good nuclease protection effect
Step 1：Weigh 2.5mg Tris and 1.0g polyethylene glycol respectively to be placed in 50ml swivelling cover conical centrifuge tubes, altogether
8 are prepared, numbering 1-8；Respectively into 1-8 collecting pipes add 0.5g, 1.0g, 1.5g, 2.0g, 2.5g, 3.0g, 3.5g,
4.0g guanidine hydrochlorides, fully mix and sputum protective agent and collecting pipe are made；
Step 2：Take some lung cancer positive sputums fully to mix the lung cancer positive sputum sample for being prepared into 16ml, take respectively
2ml sputums sample into 1-8 collecting pipes, collecting pipe is placed under the conditions of room temperature (20 DEG C) place 72 it is small when；
Step 3：Utilize reverse transcriptase of telomere subunit (hTERT) mRNA detection kits " (state's tool note is accurate
20173404247), the Sputum samples that step 2 obtains are detected with reference to product description operating procedure, it is molten by sputum
Solution, hybridization, washing, elution, digestion and PCR amplification and etc., experimental result see the table below.Guanidine hydrochloride in the results show protective agent
Good nuclease protection effect can be played by having in 0.5-4.0g：
Embodiment 6：Tris in protective agent has in 1.5-4.0mg and can play good nuclease protection effect
Step 1：1.0g guanidine hydrochlorides are weighed respectively and 1.0g polyethylene glycol is placed in 50ml swivelling cover conical centrifuge tubes, altogether
6 are prepared, numbering 1-6；1.5mg, 2.0mg, 2.5mg, 3.0mg, 3.5mg, 4.0mg are added into 1-6 collecting pipes respectively
Tris, fully mixes and sputum protective agent and collecting pipe is made；
Step 2：Take some lung cancer positive sputums fully to mix the lung cancer positive sputum sample for being prepared into 12ml, take respectively
2ml sputums sample into 1-6 collecting pipes, collecting pipe is placed under the conditions of room temperature (20 DEG C) place 72 it is small when；
Step 3：Utilize reverse transcriptase of telomere subunit (hTERT) mRNA detection kits " (state's tool note is accurate
20173404247), the Sputum samples that step 2 obtains are detected with reference to product description operating procedure, it is molten by sputum
Solution, hybridization, washing, elution, digestion and PCR amplification and etc., experimental result see the table below.Tris in the results show protective agent exists
1.5-4.0mg has and can play good nuclease protection effect：
| Sample number into spectrum | No. 1 | No. 2 | No. 3 | No. 4 | No. 5 | No. 6 |
| Ct values | 21.79 | 22.35 | 25.73 | 22.22 | 21.75 | 20.95 |
| Experimental result | It is positive | It is positive | It is positive | It is positive | It is positive | It is positive |
Embodiment 7：Polyethylene glycol in protective agent has in 0.5-2.5g and can play good nuclease protection effect
Step 1：1.0g guanidine hydrochlorides are weighed respectively and 2.5mg Tris are placed in 50ml swivelling cover conical centrifuge tubes, altogether system
It is 5 standby, numbering 1-5；0.5g, 1.0g, 1.5g, 2.0g, 2.5g polyethylene glycol are added into 1-5 collecting pipes respectively, it is fully mixed
It is even that sputum protective agent and collecting pipe is made；
Step 2：Take some lung cancer positive sputums fully to mix the lung cancer positive sputum sample for being prepared into 10ml, take respectively
2ml sputums sample into 1-5 collecting pipes, collecting pipe is placed under the conditions of room temperature (20 DEG C) place 72 it is small when；
Step 3：Utilize reverse transcriptase of telomere subunit (hTERT) mRNA detection kits " (state's tool note is accurate
20173404247), the Sputum samples that step 2 obtains are detected with reference to product description operating procedure, it is molten by sputum
Solution, hybridization, washing, elution, digestion and PCR amplification and etc., experimental result see the table below.Poly- second two in the results show protective agent
Alcohol has in 0.5-2.5g and can play good nuclease protection effect：
| Sample number into spectrum | No. 1 | No. 2 | No. 3 | No. 4 | No. 5 |
| Ct values | 20.66 | 21.47 | 21.53 | 20.26 | 23.67 |
| Experimental result | It is positive | It is positive | It is positive | It is positive | It is positive |
Claims (10)
1. the sputum collecting pipe for detection of nucleic acids, it is characterised in that sputum collecting pipe is that can accommodate sputum sample and protection
The container of agent, the container can be closed and can carried with；Protective agent is pre-placed in sputum collecting pipe, protective agent includes nuclease
Inhibitor, pH value maintain agent and dehydrating agent.
2. the sputum collecting pipe according to claim 1 for detection of nucleic acids, it is characterised in that the sputum collecting pipe is
One kind in 50ml swivelling covers conical centrifuge tube, 50ml swivelling covers round bottom centrifuge tube and the flat centrifuge tube of 50ml swivelling covers.
3. the sputum collecting pipe according to claim 1 for detection of nucleic acids, it is characterised in that nucleic acid inhibitor is salt
It is sour guanidine, guanidinium isothiocyanate, lauryl sodium sulfate, NaTDC, 4-ASA sodium, naphthalene -1,5- sodium disulfonates, three different
One kind or more in naphthalene sulfonate.
4. the sputum collecting pipe according to claim 1 for detection of nucleic acids, it is characterised in that the matter of nucleic acid inhibitor
Measure as 0.5-4.0g.
5. the sputum collecting pipe according to claim 1 for detection of nucleic acids, it is characterised in that it is three hydroxyls that pH value, which maintains agent,
Aminomethane, 3- (N- morpholines) propane sulfonic acid, double (2- (methylol) amino-three (methylol) methane, piperazine -1,4- two
One or more in ethyl sulfonic acid, N-2- hydroxyethyl piperazine-N-2- ethyl sulfonic acids.
6. the sputum collecting pipe according to claim 1 for detection of nucleic acids, it is characterised in that pH value maintains the quality of agent
For 1.5-4.0mg.
7. the sputum collecting pipe according to claim 1 for detection of nucleic acids, it is characterised in that dehydrating agent is poly- second two
Alcohol, n-butanol, sec-butyl alcohol, the one or more of the tert-butyl alcohol.
8. the sputum collecting pipe according to claim 1 for detection of nucleic acids, it is characterised in that the quality of dehydrating agent is
0.5-2.5g or volume are 1.0-3.0ml.
9. the sputum collecting pipe according to claim 1 for detection of nucleic acids, it is characterised in that the sputum collecting pipe
50ml swivelling cover conical centrifuge tubes, built-in protective agent include：1.0g guanidine hydrochlorides, 2.5mg Tris and 1.0g polyethylene glycol.
A kind of 10. sputum store method for detection of nucleic acids, it is characterised in that：
(1) the sputum sample collected, the volume of sputum sample is 1-5ml；
(2) sputum sample is placed directly within to the sputum collecting pipe for detection of nucleic acids as described in claim 1-9 any one
In, sputum sample is mixed with the protective agent in sputum collecting pipe, the storage temperature of collecting pipe is less than or equal to 25 DEG C.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711190683.1A CN107904152B (en) | 2017-11-24 | 2017-11-24 | Sputum collecting pipe for nucleic acid detection and sputum preservation method |
| PCT/CN2018/077930 WO2019100620A1 (en) | 2017-11-24 | 2018-03-02 | Method for storing sputum and method for rapidly extracting nucleic acid from sputum |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711190683.1A CN107904152B (en) | 2017-11-24 | 2017-11-24 | Sputum collecting pipe for nucleic acid detection and sputum preservation method |
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| CN107904152B CN107904152B (en) | 2020-04-14 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN111218444A (en) * | 2020-04-24 | 2020-06-02 | 广州安必平医药科技股份有限公司 | Sputum preserving fluid |
| 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 |
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| 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 |
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