CN107904152B

CN107904152B - Sputum collecting pipe for nucleic acid detection and sputum preservation method

Info

Publication number: CN107904152B
Application number: CN201711190683.1A
Authority: CN
Inventors: 陈燃; 曹文静; 宣金聪
Original assignee: Zhejiang Jfk Biological Technology Co ltd
Current assignee: Zhejiang Jfk Biological Technology Co ltd
Priority date: 2017-11-24
Filing date: 2017-11-24
Publication date: 2020-04-14
Anticipated expiration: 2037-11-24
Also published as: CN107904152A

Abstract

The invention provides a sputum collection pipe for nucleic acid detection and a sputum preservation method. Aiming at the problem that nucleic acid in a sputum sample is easy to degrade, the invention designs the collecting pipe containing the nucleic acid protective agent, so that a receiver needing sputum nucleic acid detection can conveniently collect sputum at any time by using the collecting pipe carried with the receiver when the sputum exists, meanwhile, the nucleic acid in the sputum sample can be protected from being degraded at room temperature, and the receiver can be given enough time (72 hours) to send the sample to (or express) hospitals or other inspection institutions for nucleic acid detection.

Description

Sputum collecting pipe for nucleic acid detection and sputum preservation method

Technical Field

The method belongs to the technical field of sample preservation for medical detection, and relates to a sputum preservation method and a collection tube for nucleic acid detection.

Background

Sputum is the secretion of the respiratory tract of a human body, is pushed from the lung to the upper respiratory tract by the movement of epithelial cilia through bronchial cilia movement, and is finally expectorated from the trachea and discharged out of the body through the normal cough reflex of the human body, and the normal human body has little sputum and only a small amount of mucus secreted by keeping the respiratory tract moist. When a person inhales harmful microorganisms such as irritant gases, dust, pathogenic bacteria, viruses and the like, inflammation of the upper respiratory tract can occur; or diseases of lung, such as bronchiectasis, lung abscess, lung cancer, etc., increase in respiratory tract secretion, increase in phlegm amount, change in phlegm property, and change from sticky phlegm to cheese phlegm, blood phlegm, yellow purulent phlegm, etc. According to the mechanism of producing and discharging the sputum, the sputum contains various pathogenic microorganisms, various inflammatory cells, desquamated necrotic mucosal epithelial cells, tumor cells and the like.

At present, sputum samples are mainly used for sputum cytology detection and respiratory tract pathogen detection in clinic. Among them, identification of the type of pathogen by detecting the nucleic acid of the pathogen in sputum samples is currently the most accurate and rapid diagnostic method for respiratory pathogens. Among the pathogens that can cause acute respiratory infections, viruses predominate, and the second is bacteria, mycoplasma, molds, protozoa, and the like. In primary acute upper respiratory infections, viral infections are as high as over 90%. Common respiratory pathogens are: mycobacterium tuberculosis, respiratory syncytial virus, adenovirus, EB virus, influenza virus, mycoplasma pneumoniae, rubella virus, streptococcus, staphylococcus, chlamydia pneumoniae, parvovirus B19, cytomegalovirus, coronavirus and the like. The traditional pathogen diagnosis method is separation culture or serological detection, and the separation culture takes long time and is complex to operate and not suitable for the diagnosis of clinical emergency; serological tests are difficult to diagnose early in the disease. With the rapid development of fluorescent quantitative PCR and multiplex PCR technologies in the field of pathogen detection, the method has the characteristics of strong specificity, high sensitivity, simple and convenient operation, rapid detection, high detection rate and the like, and the technology gradually occupies an important position in the field of clinical diagnosis. In addition, nucleic acid is extracted from sputum samples of high risk group of lung cancer and lung cancer patients, and the nucleic acid can be used for early stage lung cancer prediction and gene mutation detection and treatment monitoring of lung cancer patients. In conclusion, the detection of nucleic acid in sputum samples is of great clinical significance. However, in practice, the nucleic acids (especially RNA) in sputum samples are extremely unstable and are usually degraded under greenhouse conditions for several hours. External physical factors causing nucleic acid degradation such as temperature, humidity, ultraviolet rays, etc.; chemical factors such as strong acid or strong base environment, hydrolysis reaction, etc.; biological factors such as enzymolysis, microbial infection and the like. Among them, nuclease released by cell rupture in sputum and nuclease introduced by contamination during operation are the main causes of nucleic acid degradation. In addition, even a patient in the onset stage of disease, a general examinee cannot expectorate a sufficient amount of sputum at any time, and most patients cannot collect and store the sputum in time after the morning hours when the amount of sputum is large.

Chinese patent application CN104263721A discloses a sputum paper for protecting nucleic acid (DNA and RNA) in sputum and a method for extracting nucleic acid, which mainly has the following disadvantages: (1) the sputum paper disclosed by the patent is a square or round paper sheet, a letter seal type or a box type, is easy to break and damage, cannot resist external force extrusion, is easy to overflow (particularly when the sputum is more), and is inconvenient to carry and transport after the sputum is collected; (2) the patent states that the sputum paper is soaked with a nucleic acid protective solution with main components of DTT (dithiothreitol), EDTA (ethylene diamine tetraacetic acid and disodium salt thereof) and Tris-HCl (trihydroxyaminomethane-hydrochloric acid buffer solution), the content of the soaked protective solution is low, the sputum sample cannot be ensured to be fully contacted with the nucleic acid protective solution, and the protective effect on the nucleic acid in the sputum is general.

In addition, the related patents for protecting nucleic acid or cells disclosed in the prior art, such as "nucleic acid protection solution for long-term storage and transportation of tissue samples under normal temperature conditions" disclosed in chinese patent application CN105145545A, "a ribonucleic acid protective agent, kit, application and storage method" disclosed in chinese patent application 106065400a, and "a cell preservation solution" disclosed in chinese patent application CN 104041484 a, can all be used for protecting nucleic acid of tissues or cells. However, these patents relate to techniques that have poor utility for sputum samples, mainly for the following reasons: (1) the sputum sample has the characteristics of high viscosity, high protein, difficulty in liquefaction, easiness in agglomeration and the like, and after the sputum is added into the conventional liquid protective agent, the sputum is easy to be layered with the protective agent, and the contact surface of the protective agent and the sputum sample is limited; (2) some patents relate to complex protective agent components, such as preservatives, buffers, chelating agents, enzyme inhibitors, fixing agents and the like, and the protective agent components are more and high in cost, so that the protective agent components are not suitable for being widely used clinically.

Disclosure of Invention

In order to solve the problems that nucleic acid in a sputum sample is very easy to degrade and sputum cannot be collected at any time in clinical practice, the invention provides a method which is convenient to carry and can collect the sputum at any time and can protect the nucleic acid in the sputum sample from degradation.

The technical scheme of the invention is as follows:

a sputum storage method capable of protecting nucleic acid and a sputum collection tube which is convenient to carry and can be collected at any time. The sputum sample collection device mainly comprises a collection tube and a protective agent, wherein the collection tube can contain a sputum sample and the protective agent, can be closed and can be carried about; the protective agent is placed in the sputum collection tube in advance and comprises a nuclease inhibitor, a pH value maintaining agent and a dehydrating agent, so that the integrity of nucleic acid in a sample is protected.

(1) The used collecting tube is 50ml rotary cover sharp-bottom centrifuge tube, 50ml rotary cover round-bottom centrifuge tube, 50ml rotary cover flat-bottom centrifuge tube (can be vertical), is not limited to the above 3 kinds, and other can contain protective agent and sputum can be sealed and portable's container all can.

(2) The nuclease inhibitor is one or more of guanidine hydrochloride, guanidine isothiocyanate, sodium dodecyl sulfate, sodium deoxycholate, 4-aminosalicylate, naphthalene-1, 5-disulfonate, and sodium triisopropylnaphthalenesulfonate. The mass of the nuclease inhibitor is 0.5-4.0 g.

(3) The pH value maintaining agent is one or a combination of more of Tris (hydroxymethyl) aminomethane (Tris), 3- (N-morpholine) propanesulfonic acid (MOPS), Bis (2- (hydroxymethyl) amino-Tris (hydroxymethyl) methane (Bis Tris), piperazine-1, 4-diethylsulfonic acid (PIPES) and N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid (HEPES). the mass of the pH value maintaining agent is 1.5-4.0 mg.

(3) The dehydrating agent is one or a combination of polyethylene glycol (PEG), n-butyl alcohol, sec-butyl alcohol and tert-butyl alcohol. The dehydrating agent has a mass of 0.5-2.5g or a volume of 1.0-3.0 ml.

Preferably, the nuclease inhibitor and collection tube is: 1.0g guanidine hydrochloride, 2.5mg Tris and 1.0g polyethylene glycol were placed in a 50ml spinner-cap conical centrifuge tube.

The invention also provides a sputum preservation method for nucleic acid detection, which comprises the following steps

(1) Collecting the sputum sample, wherein the volume of the sputum sample is 1-5 ml; the sputum sample is not limited, and the collected sputum sample can be sputum with various characters such as sticky sputum, blood sputum, purulent sputum, thick sputum, yellow/gray/rust sputum and the like.

(2) Placing the sputum sample directly into the sputum collection tube for nucleic acid detection according to any one of claims 1-9, and mixing the sputum sample with the protective agent in the sputum collection tube, wherein the storage temperature of the collection tube is less than or equal to 25 ℃.

The principle of the invention is as follows: the nuclease inhibitor is used for inhibiting the activity of nuclease in a system and preventing nucleic acid in the sputum sample from being degraded; the pH value maintaining agent is used for keeping the sputum sample neutral or alkalescent and providing a relatively stable pH value environment for nucleic acid in the sputum sample; the dehydrating agent has the effects of reducing moisture in the sputum sample and preventing nucleic acid in the sputum sample from being hydrolyzed, and the dehydrating agent only absorbs the moisture and does not dissolve the nucleic acid. The collection pipe with the cover and capable of being sealed is convenient to carry, can be convenient for an examinee to collect sputum at any time when a expectoration reaction is suddenly caused, and effectively protects the integrity of nucleic acid in the sputum.

In order to verify the effectiveness of the method, the method is verified by comparison by using a fluorescence quantitative PCR method and an electrophoresis method respectively, and the experimental steps and results are as follows:

(1) respectively weighing 1.0g of guanidine hydrochloride, 2.5mg of Tris and 1.0g of polyethylene glycol, placing the weighed materials into a 50ml rotary cover pointed-bottom centrifuge tube, fully and uniformly mixing the materials to prepare a sputum collecting tube, and preparing 4 collecting tubes containing protective agents in total, wherein the serial numbers are 1-4; additionally preparing 4 collecting pipes without protective agent, with the number of 5-8;

(2) clinically confirmed lung cancer patient sputum samples provided by the respiratory medicine department of the first hospital affiliated to the Zhejiang university medical college. Telomerase contained in the lung cancer cell endows the lung cancer cell with the capability of unlimited proliferation, and the telomerase is synthesized by translation of hTERT mRNA of the lung cancer cell, so that the hTERT mRNA is a characteristic marker of the lung cancer cell. In the process of sputum production and expectoration, exfoliated lung cancer cells are mixed in the sputum to be expectorated. Therefore, htert mrna could be detected in sputum of lung cancer patients. Fully and uniformly mixing sputum samples of 10 cases of lung cancer patients to prepare 16ml of lung cancer positive mixed sputum samples, and respectively taking 2ml of sputum samples to a No. 1-8 collection tube;

(3) the collection tubes were placed at room temperature (20 ℃) for the following times:

collection pipe numbering	1、3、5、7	2、4、6、8
			Time of standing	5 hours	48 hours

(4) Detecting mRNA in the sputum sample by a fluorescence quantitative PCR method: the telomerase reverse transcriptase subunit (hTERT) mRNA detection kit developed by Zhejiang and Jinkang biotechnology limited company (national institutes of health 20173404247) is adopted, and the kit can detect the hTERT mRNA in a sputum sample. When the PCR amplification result is that the Ct value is less than 33, the detection result is positive, namely the hTERT mRNA is contained in the sample; and when the PCR amplification result is that the Ct value is more than or equal to 33 or No Ct, the detection result is negative, namely No hTERT mRNA exists in the sample. The samples in the collection tubes No. 1, No. 2, No. 5 and No. 6 obtained in the step (3) are detected according to the operation steps of the product instruction of the kit, and the experimental results are shown in the following table after the steps of sputum dissolution, hybridization, washing, elution, enzyme digestion, PCR amplification and the like:

the experimental results show that: by using the method, the lung cancer positive sputum is respectively preserved for 5 hours and 48 hours at room temperature (20 ℃), hTERT mRNA in a sample can be detected, and the integrity is proved to be good; but not applicable to the method, after the lung cancer positive sputum is placed for 5 hours and 48 hours at room temperature (20 ℃),

no hTERT mRNA was detected, indicating that it had been degraded. Therefore, the method can effectively protect mRNA in the sputum sample from being degraded.

(5) The nucleic acid in the sputum sample is detected by electrophoresis, which comprises adding 100 mul guanidine hydrochloride solution (2.0g/ml) into the collection tube No. 3 and No. 4 obtained in the above (3), adding 600 mul guanidine hydrochloride solution (2.0g/ml) into the collection tube No. 7 and No. 8 obtained in the above (3), adding 100 mul β -mercaptoethanol and 30 mul 20% NP-40 into the tubes No. 3, No. 4, No. 7 and No. 8, respectively, keeping the temperature for 5min at 60 ℃, shaking vigorously, centrifuging for 10min at 12000rpm, taking the supernatant, separating the nucleic acid column, centrifuging for 1min at 12000rpm, taking 700 mul washing solution (10mM MES pH6.0, 35% ethanol) for 2 times, placing the column on a clean 1.5ml centrifuge tube, adding 30 mul TE solution into the middle position of the column membrane, keeping the temperature for 3min at 60 ℃, centrifuging for 1min at 12000rpm, collecting the effluent, taking 15 mul for gel electrophoresis, and obtaining the electrophoresis result shown in figure 1.

The experimental results show that: the method is used for respectively preserving lung cancer positive sputum for 5 hours and 48 hours at room temperature (20 ℃), and clear nucleic acid strips can be seen after electrophoresis of corresponding samples (No. 3 and No. 4 respectively), so that the integrity of nucleic acid in a sputum sample is proved to be good; the lung cancer positive sputum samples (No. 7 and No. 8 respectively) which are placed for 5 hours and 48 hours at room temperature (20 ℃) without the method are proved to be degraded after electrophoresis without nucleic acid bands. Therefore, the method can effectively protect nucleic acid in the sputum sample from being degraded.

The invention has the beneficial effects that: the invention provides a method capable of collecting sputum at any time and protecting nucleic acid in the sputum from degradation. The method places the nucleic acid protective agent in a collecting pipe which is convenient to carry, can collect sputum at any time when a detected person has sputum, and the protective agent can well prevent nucleic acid from being degraded. The protective agent is powdery solid and can better permeate into the sputum sample so as to better contact with the sputum sample. In addition, the protective agent has relatively simple components and low cost, is very suitable for being widely used in clinic, is convenient for a receiver needing sputum nucleic acid detection to collect sputum at any time by using a carrying collecting pipe when the receiver has the sputum, can protect the nucleic acid in the sputum sample from being degraded at room temperature, and provides sufficient time for the receiver to send the sample to (or express) hospitals or other inspection institutions.

Drawings

FIG. 1 is an electropherogram of nucleic acid in sputum sample detected by electrophoresis in the invention, which is No. 3, No. 7, No. 4, No. 8 and Marker from left to right;

FIG. 2 is an electropherogram of nucleic acid in the sputum sample detected by the electrophoresis method in example 4, which is shown as No. 5, No. 6, No. 7, No. 8 and Marker from left to right.

Detailed Description

The sputum sample used in the examples was collected by a clinical standard sputum sampling procedure in the respiratory medicine department of the first hospital affiliated to the medical college of Zhejiang university, approved by the department of Biotechnology Limited of Fukang Zhejiang, and by referring to the handbook on Chinese tuberculosis prevention and treatment planning, sputum smear microscopy standardized operation and quality assurance, the specimen in the form of a lump was accepted by visual observation. The lung cancer positive sputum is collected from clinically confirmed lung cancer patients. Telomerase contained in the lung cancer cell endows the lung cancer cell with the capability of unlimited proliferation, and the telomerase is synthesized by translation of hTERT mRNA of the lung cancer cell, so that the hTERT mRNA is a characteristic marker of the lung cancer cell. In the process of sputum production and expectoration, exfoliated lung cancer cells are mixed in the sputum to be expectorated. hTERT mRNA can therefore be detected in sputum from lung cancer patients. The nucleic acid detection adopts a telomerase reverse transcriptase subunit (hTERT) mRNA detection kit (national institutes of health 20173404247) developed by Zhejiang and Jinkang biotechnology limited, and the kit can detect the hTERT mRNA in a sputum sample. When the PCR amplification result is that the Ct value is less than 33, the detection result is positive, namely the hTERT mRNA is contained in the sample; and when the PCR amplification result is that the Ct value is more than or equal to 33 or the NoCt value indicates that the detection result is negative, namely that the hTERT mRNA does not exist in the sample.

Example 1: the sputum sample can be preserved for 72 hours in guanidine hydrochloride-Tris-polyethylene glycol protective agent

Step 1: respectively weighing 1.0g of guanidine hydrochloride, 2.5mg of Tris and 1.0g of polyethylene glycol, placing the weighed materials in a 50ml rotating cover pointed-bottom centrifuge tube, fully and uniformly mixing the materials to prepare a sputum protective agent and a collecting tube, and preparing 4 collecting tubes containing the protective agent in total, wherein the serial numbers are 1-4; additionally preparing 4 collecting pipes without protective agent, with the number of 5-8; taking a plurality of lung cancer positive sputum, fully and uniformly mixing to prepare a 16ml lung cancer positive sputum sample, and respectively taking 2ml sputum samples to each collection pipe;

step 2: the collection tubes were placed at room temperature (20 ℃) for the following times:

collection pipe numbering	1、5	2、6	3、7	4、8
					Time of standing	24 hours	48 hours	72 hours	96 hours

And step 3: and (3) detecting the sputum sample obtained in the step (2) by using a telomerase reverse transcriptase subunit (hTERT) mRNA detection kit (national mechanical Standard 20173404247) according to the operation steps of a product specification, and performing the steps of sputum dissolution, hybridization, washing, elution, enzyme digestion, PCR amplification and the like, wherein the experimental results are shown in the following table. The result shows that the sputum sample preserved by the method can still detect mRNA after being placed at room temperature for 24-72 hours, but the sputum sample directly placed without the method can not detect mRNA after being placed at room temperature for 24 hours, so that the method can protect the integrity of nucleic acid in the sputum sample for 72 hours at room temperature (20 ℃).

Sample numbering

Number 1

Number 2

No. 3

Number 4

Number 5

Number 6

No. 7

Number 8

Ct value

25.97

27.03

30.11

35.76

No Ct

Results of the experiment

Positive for

Negative of

Example 2: the method is suitable for phlegm with sticky, blood, pus, thick, yellow, gray, and rust

Step 1: respectively weighing 1.0g of guanidine hydrochloride, 2.5mg of Tris and 1.0g of polyethylene glycol, placing the weighed materials into a 50ml rotary cover pointed-bottom centrifuge tube, fully and uniformly mixing the materials to prepare a sputum collecting tube, and preparing 7 collecting tubes containing protective agents in total, wherein the serial numbers are 1-7;

step 2: taking one positive lung cancer sample with the characters of sticky sputum, blood sputum, purulent sputum, thick sputum, yellow sputum, gray sputum and rust sputum, respectively, placing the positive lung cancer sample in a No. 1-7 collecting pipe, and placing the collecting pipe at room temperature (20 ℃) for 72 hours;

and step 3: and (3) detecting the sputum sample obtained in the step (2) by using a telomerase reverse transcriptase subunit (hTERT) mRNA detection kit (national mechanical Standard 20173404247) according to the operation steps of a product specification, and performing the steps of sputum dissolution, hybridization, washing, elution, enzyme digestion, PCR amplification and the like, wherein the experimental results are shown in the following table. The result shows that the method can play a better protective role on nucleic acid in sticky sputum, blood sputum, purulent sputum, thick sputum and yellow/gray/rust sputum:

sample numbering

Number 1

Number 2

No. 3

Number 4

Number 5

Number 6

No. 7

Ct value

22.67

25.44

29.05

31.11

25.64

28.92

27.49

Results of the experiment

Positive for

Example 3: the method is suitable for 1-5ml sputum sample

Step 1: respectively weighing 1.0g of guanidine hydrochloride, 2.5mg of Tris and 1.0g of polyethylene glycol, placing the weighed materials in a 50ml rotating cover pointed-bottom centrifuge tube, fully and uniformly mixing the materials to prepare a sputum protective agent and a collecting tube, and preparing 5 collecting tubes containing the protective agent in total, wherein the serial numbers are 1-5;

step 2: taking a plurality of lung cancer positive sputum, fully and uniformly mixing to prepare a 10ml lung cancer positive sputum sample, taking 1ml, 2ml, 3ml, 4ml and 5ml sputum samples into a No. 1-5 collecting pipe respectively, and placing the collecting pipe under the condition of room temperature (20 ℃) for 72 hours;

and step 3: and (3) detecting the sputum sample obtained in the step (2) by using a telomerase reverse transcriptase subunit (hTERT) mRNA detection kit (national mechanical Standard 20173404247) according to the operation steps of a product specification, and performing the steps of sputum dissolution, hybridization, washing, elution, enzyme digestion, PCR amplification and the like, wherein the experimental results are shown in the following table. The result shows that the method can play a good protection role on nucleic acid in 1-5ml of sputum samples:

sample numbering	Number 1	Number 2	No. 3	Number 4	Number 5
						Ct value	29.79	26.74	25.03	21.21	20.65
Results of the experiment	Positive for	Positive for	Positive for	Positive for	Positive for

Example 4: the sputum sample can be preserved for 72 hours in guanidinium isothiocyanate-Bis Tris-n-butanol protective agent

Step 1: respectively weighing 1.2g of guanidinium isothiocyanate, 2.0mg of Bis Tris and 2ml of tert-butyl alcohol, placing in a 50ml rotating cover round bottom centrifuge tube, fully and uniformly mixing to prepare a sputum protective agent and a collecting tube, and preparing 8 collecting tubes containing the protective agent in total, wherein the serial numbers are 1-8;

step 2: taking a plurality of lung cancer positive sputum, fully and uniformly mixing to prepare a 16ml lung cancer positive sputum sample, respectively taking 2ml sputum samples to a No. 1-8 collecting pipe, and placing the collecting pipe for 72 hours at room temperature (20 ℃);

and step 3: mRNA in the sputum sample is detected by using a telomerase reverse transcriptase subunit (hTERT) mRNA detection kit (national mechanical Standard 20173404247), the No. 1-4 sputum sample obtained in the step 2 is detected according to the operation steps of a product specification, and the experimental results are shown in the following table after the steps of sputum dissolution, hybridization, washing, elution, enzyme digestion, PCR amplification and the like. The results show that sputum samples can be preserved in guanidinium isothiocyanate-Bis Tris-tert-butanol protectant for 72 hours:

step 4, detecting nucleic acid in a sputum sample by using an electrophoresis method, adding 600 mu l of guanidine hydrochloride solution (2.0g/ml) into a collection tube No. 5-8 obtained in the step 2, adding 100 mu l of β -mercaptoethanol and 30 mu l of 20% NP-40 into a collection tube No. 3, No. 4, No. 7 and No. 8 respectively, preserving heat at 60 ℃ for 5min, violently shaking and uniformly mixing, centrifuging at 12000rpm for 10min, taking a supernatant to a nucleic acid separation column, centrifuging at 12000rpm for 1min, taking 700 mu l of washing solution (10mM MES pH6.0 and 35% ethanol) to wash for 2 times, placing the column on a clean centrifugal tube 1.5ml, adding 30 mu l of TE solution into the middle position of a column membrane, preserving heat at 60 ℃ for 3min, centrifuging at 12000rpm for 1min, collecting an effluent, taking 15 mu l of effluent to perform gel electrophoresis, and obtaining an electrophoresis result shown in figure 2, wherein after the sputum sample is preserved in 72 hours, the corresponding nucleic acid strips in the sputum sample No. 5-8 are clear.

Example 5: guanidine hydrochloride in the protective agent can play a good role in protecting nucleic acid when the weight of guanidine hydrochloride is 0.5-4.0g

Step 1: respectively weighing 2.5mg Tris and 1.0g polyethylene glycol, placing in a 50ml rotary cover pointed-bottom centrifuge tube, and preparing 8 samples with the number of 1-8; adding 0.5g, 1.0g, 1.5g, 2.0g, 2.5g, 3.0g, 3.5g and 4.0g guanidine hydrochloride into No. 1-8 collection tube, mixing well to obtain sputum protectant and collection tube;

and step 3: and (3) detecting the sputum sample obtained in the step (2) by using a telomerase reverse transcriptase subunit (hTERT) mRNA detection kit (national mechanical Standard 20173404247) according to the operation steps of a product specification, and performing the steps of sputum dissolution, hybridization, washing, elution, enzyme digestion, PCR amplification and the like, wherein the experimental results are shown in the following table. The results show that guanidine hydrochloride in the protective agent can play a good role in protecting nucleic acid at 0.5-4.0 g:

example 6: tris in the protective agent can play a good role in protecting nucleic acid at 1.5-4.0mg

Step 1: respectively weighing 1.0g of guanidine hydrochloride and 1.0g of polyethylene glycol, placing the guanidine hydrochloride and the polyethylene glycol into a 50ml centrifuge tube with a rotary cover and a pointed bottom, preparing 6 pieces in total, and numbering 1-6; adding 1.5mg, 2.0mg, 2.5mg, 3.0mg, 3.5mg and 4.0mg Tris into No. 1-6 collection tube, mixing well to obtain protective agent for sputum and collection tube;

step 2: taking a plurality of lung cancer positive sputum, fully and uniformly mixing to prepare a 12ml lung cancer positive sputum sample, respectively taking 2ml sputum samples to a No. 1-6 collecting pipe, and placing the collecting pipe for 72 hours at room temperature (20 ℃);

and step 3: and (3) detecting the sputum sample obtained in the step (2) by using a telomerase reverse transcriptase subunit (hTERT) mRNA detection kit (national mechanical Standard 20173404247) according to the operation steps of a product specification, and performing the steps of sputum dissolution, hybridization, washing, elution, enzyme digestion, PCR amplification and the like, wherein the experimental results are shown in the following table. The result shows that Tris in the protective agent can play a good role in protecting nucleic acid at 1.5-4.0 mg:

sample numbering

Number 1

Number 2

No. 3

Number 4

Number 5

Number 6

Ct value

21.79

22.35

25.73

22.22

21.75

20.95

Results of the experiment

Positive for

Example 7: the polyethylene glycol in the protective agent can play a good role in protecting nucleic acid at 0.5-2.5g

Step 1: respectively weighing 1.0g of guanidine hydrochloride and 2.5mg of Tris into a 50ml rotating cover pointed-bottom centrifuge tube, and preparing 5 pieces with the number of 1-5; adding 0.5g, 1.0g, 1.5g, 2.0g and 2.5g polyethylene glycol into No. 1-5 collecting tube, and mixing to obtain sputum protectant and collecting tube;

step 2: taking a plurality of lung cancer positive sputum, fully and uniformly mixing to prepare 10ml lung cancer positive sputum samples, respectively taking 2ml sputum samples to a No. 1-5 collecting pipe, and placing the collecting pipe for 72 hours at room temperature (20 ℃);

and step 3: and (3) detecting the sputum sample obtained in the step (2) by using a telomerase reverse transcriptase subunit (hTERT) mRNA detection kit (national mechanical Standard 20173404247) according to the operation steps of a product specification, and performing the steps of sputum dissolution, hybridization, washing, elution, enzyme digestion, PCR amplification and the like, wherein the experimental results are shown in the following table. The result shows that the polyethylene glycol in the protective agent can play a good role in protecting nucleic acid at 0.5-2.5 g:

sample numbering	Number 1	Number 2	No. 3	Number 4	Number 5
						Ct value	20.66	21.47	21.53	20.26	23.67
Results of the experiment	Positive for	Positive for	Positive for	Positive for	Positive for

Claims

1. The sputum collecting tube for nucleic acid detection is characterized in that the sputum collecting tube is a container capable of containing a sputum sample and a protective agent, and the container can be closed and carried about; a protective agent is pre-placed in the sputum collecting tube, and comprises a nuclease inhibitor, a pH value maintaining agent and a dehydrating agent;

the mass of the nuclease inhibitor is 0.5-4.0 g;

the quality of the pH value maintaining agent is 1.5-4.0 mg;

the mass of the dehydrating agent is 0.5-2.5g or the volume is 1.0-3.0 ml;

the nuclease inhibitor is one or more of guanidine hydrochloride and guanidine isothiocyanate;

the pH value maintaining agent is one or more of trihydroxymethyl aminomethane, 3- (N-morpholine) propanesulfonic acid, bis (2- (hydroxymethyl) amino-tris (hydroxymethyl) methane, piperazine-1, 4-diethylsulfonic acid and N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid;

the dehydrating agent is one or more of polyethylene glycol, n-butyl alcohol, sec-butyl alcohol and tert-butyl alcohol.

2. The sputum collection tube for nucleic acid testing of claim 1, wherein the sputum collection tube is one of a 50ml spin-lid conical centrifuge tube, a 50ml spin-lid round-bottom centrifuge tube, and a 50ml spin-lid flat-bottom centrifuge tube.

3. The sputum collection tube for nucleic acid detection of claim 1, wherein the sputum collection tube is a 50ml spin-top, bottom-tip centrifuge tube, and the built-in protectant comprises: 1.0g guanidine hydrochloride, 2.5mg Tris and 1.0g polyethylene glycol.

4. A method for preserving sputum for nucleic acid detection, comprising:

(1) collecting the sputum sample, wherein the volume of the sputum sample is 1-5 ml;

(2) directly placing the sputum sample into the sputum collection tube for nucleic acid detection according to any one of claims 1 to 3, and mixing the sputum sample with the protective agent in the sputum collection tube, wherein the storage temperature of the collection tube is less than or equal to 25 ℃.