WO2019100620A1 - Method for storing sputum and method for rapidly extracting nucleic acid from sputum - Google Patents

Abstract

A sputum collection tube for nucleic acid detection and a sputum storing method. The sputum collection tube is provided with a nuclease inhibitor, a pH maintainer, and a dehydrating agent. Also provided is a method for rapidly extracting nucleic acid from sputum, comprises the following steps: (1) taking 2-5 ml sputum sample, adding 0.5-2 g nuclease inhibitor and an equal volume of chloroform, oscillating at 50-60ºC and maintaining the temperature for 10-20 min; (2) centrifuging at a rotating speed ≥ 12,000 r/min for 1-5 min, and taking supernatant; and (3a) adding a pre-treatment liquid, oscillating and maintaining the temperature, then centrifugally adding isopropanol, uniformly mixing, centrifuging, and taking liquid supernatant for standby, or (3b) adding isopropanol, centrifuging and then discarding supernatant, re-suspending and merging precipitates in the pre-treatment liquid, water-saturated phenol and chloroform, oscillating, centrifuging and taking liquid supernatant for standby.

Description

Method for preserving sputum and method for rapidly extracting nucleic acid from sputum

The invention belongs to the technical field of sample preservation for medical detection, and relates to a method for storing sputum for nucleic acid detection and a collecting tube. The key of the technology is to protect the nucleic acid in the sputum sample from degradation. The invention also relates to a method for rapidly extracting nucleic acid from sputum, the key of the technology is to simultaneously carry out sputum liquefaction and cell lysis, and quickly complete the nucleic acid extraction process.

痰 is the secretion of the human respiratory tract. It is the movement of the epithelial cilia through the bronchial ciliary movement. It is pushed from the lungs to the upper respiratory tract. Finally, it is coughed out from the trachea through the normal cough reflex of the human body. A small amount of mucus secreted by the respiratory tract. When a person inhales harmful microorganisms such as irritating gases, dust, pathogenic bacteria, viruses, etc., inflammation may occur in the upper respiratory tract; or diseases in the lungs such as bronchiectasis, lung abscess, lung cancer, etc., the respiratory tract will increase secretion, and the amount of sputum will increase. It will increase, and the nature of sputum will change. It can be changed from sputum to cheese sputum, blood sputum, yellow purulent sputum. According to the sputum production and discharge mechanism, the sputum contains a variety of pathogenic microorganisms, various types of inflammatory cells, exfoliative necrotic mucosal epithelial cells, and tumor cells.

Currently, clinically, sputum samples are mainly used for sputum cytology and respiratory pathogen detection. Among them, the identification of the pathogen by detecting the nucleic acid of the pathogen in the sputum sample is the most accurate and fastest diagnostic method for respiratory pathogens. Among the pathogens that can cause acute respiratory infections, viruses are dominant, followed by bacteria, mycoplasma, molds, protozoa, and the like. In the primary acute upper respiratory tract infection, viral infections are as high as 90% or more. Common respiratory pathogens are: Mycobacterium tuberculosis, respiratory syncytial virus, adenovirus, Epstein-Barr virus, influenza virus, Mycoplasma pneumoniae, rubella virus, Streptococcus, Staphylococcus, Chlamydia pneumoniae, Parvovirus B19, Cytomegalovirus, and Coronavirus Wait. The traditional pathogen diagnosis method is separation culture or serological detection. The separation and culture takes a long time and the operation is complicated, and it is not suitable for the diagnosis of clinical emergency; serological detection is difficult to diagnose early in the disease. With the rapid development of real-time PCR and multiplex PCR technology in the field of pathogen detection, it has the characteristics of strong specificity, high sensitivity, easy operation, rapid detection and high detection rate. This technology has gradually occupied an important position in the field of clinical diagnosis. In addition, nucleic acid extraction from sputum samples from high-risk lung cancer patients and lung cancer patients can be used for early lung cancer prediction and gene mutation detection and treatment monitoring for lung cancer patients. In summary, nucleic acid detection in sputum samples is clinically important. However, in practice, the nucleic acids (especially RNA) in the sputum sample are extremely unstable and are usually degraded in greenhouse conditions for several hours. External physical factors that cause nucleic acid degradation such as temperature, humidity, ultraviolet light, etc.; chemical factors such as strong acid or strong alkali environment, hydrolysis reaction, etc.; biological factors such as enzymatic hydrolysis and microbial infestation. Among them, the nuclease released by cell rupture in sputum and the nuclease introduced by pollution during the operation are the main causes of nucleic acid degradation. In addition, the general subject, even in the onset of the disease, is not always able to cough up a sufficient amount of sputum, most patients after the morning sputum volume is more, but can not collect and save sputum in time.

Chinese patent application document CN104263721A discloses a method for extracting sputum and nucleic acid for protecting nucleic acids (DNA and RNA) in sputum, and mainly has the following disadvantages: (1) The crepe paper described in the patent is square or circular paper. , envelope type or box type, such crepe paper is easy to break, can not withstand external force extrusion, sputum is easy to overflow (especially when sputum is more), it is not convenient to carry and transport after sputum collection; (2) its According to the patent, the main components are DTT (dithiothreitol), EDTA (ethylenediaminetetraacetic acid and its disodium salt), and Tris-HCl (trishydroxyaminomethane-hydrochloric acid buffer). The nucleic acid protection solution has a small content of the infiltrating protective liquid, and cannot ensure that the sputum sample is in full contact with the nucleic acid protective solution, and the protection effect on the nucleic acid in the sputum is general.

In addition, the related patents for protecting nucleic acids or cells are disclosed, for example, "Nucleic acid protection liquid for long-term preservation and transportation of tissue samples under normal temperature conditions" disclosed in Chinese Patent Application No. CN105145545A, and disclosed in Chinese Patent Application No. CN106065400A. A ribonucleotide protectant, a kit, an application and a preservation method, and a "cell preservation solution" disclosed in Chinese Patent Application No. CN 104041484 A, the techniques of which are all applicable to the protection of nucleic acids of tissues or cells. However, the techniques involved in these patents have poor practicability for sputum samples. The main reasons are: (1) sputum samples have the characteristics of high viscosity, high protein, not easy to liquefy, easy to agglomerate, etc., adding sputum to conventional liquid protection. After the agent, the sputum is easy to stratify with the protective agent, and the contact surface of the protective agent and the sputum sample is limited; (2) The protective agent components involved in some patents are relatively complex, such as preservatives, buffers, chelating agents, enzyme inhibitors, Fixatives, etc., have many protective agents and high cost, and are not suitable for clinical use.

In addition, regarding the extraction of nucleic acids in sputum, since the sputum sample has the characteristics of high viscosity, high protein, liquefaction, and easy agglomeration, the nucleic acid detection routine of the sputum sample requires at least three steps of pretreatment: sputum liquefaction, Cell lysis and nucleic acid extraction. The conventional pretreatment process mainly has the following deficiencies: (1) The liquefaction process of the sputum alone does not add a strong denaturant because it requires the preservation of cell integrity, so it is easy to cause the RNA in the sample to be catalytically degraded by RNase, resulting in sample nucleic acid. loss. (2) Conventional sputum liquefaction is commonly used in sulfhydryl-based reagents, and the added reagents are 5-10 times larger than the sputum volume, resulting in a larger sample volume, which is highly prone to nucleic acid degradation and loss; (3) operation process More complicated and time-consuming; respectively, sodium hydroxide method, DTT (dithiothreitol) method and protease method. The sodium hydroxide method is the most commonly used sputum liquefaction method. The method is relatively simple. The sputum is liquefied at a temperature of 60-80 ° C (also at room temperature) using a sodium hydroxide solution having a certain concentration as a main component. A strong alkaline environment during nucleic acid detection is liable to cause nucleic acid loss. The DTT (dithiothreitol) method originally used the thiol (-SH)-containing DTT (dithiothreitol) cleavage sputum to cause viscous main component mucin, in PBS buffer (phosphate buffer) Provide physiological buffer, usually add fixed cells such as ethanol. The DTT (dithiothreitol) used in this method is costly, and the method is easy to cause RNA loss, and is not suitable for large-scale treatment of clinical sputum samples. The principle of the protease method is to use protease to digest sputum mucin. The enzymatic hydrolysis reaction of this method takes a long time, the protease cost is high, and the mucin digestion efficiency is low.

The technical solutions disclosed in the above-mentioned Chinese patent application documents CN104263721A and CN106065400A mainly have the following disadvantages: (1) more sputum treatment reagents are added and more sputum treatment steps are involved, which are liable to cause nucleic acid loss; (2) more steps and more It is cumbersome and time-consuming to operate; (3) There are many types of reagents used, and the cost of nucleic acid extraction is high. In addition, there are many invention patents for extracting nucleic acids in tissue or cell samples, but these patented techniques cannot handle sputum samples because they do not contain a sputum liquefaction process.

In order to solve the problem that the nucleic acid in the clinical sputum sample is easily degraded and the sputum cannot be collected at any time, the first aspect of the present invention provides a convenient carrying and sputum collection at any time, and can protect the nucleic acid in the sputum sample. Degraded sputum collection tube.

The technical solution of the first aspect of the invention is:

A sputum collection tube that protects nucleic acids in sputum and is convenient to carry and collect at any time. It mainly comprises a container and a protective agent, and the container can contain a sputum sample and a protective agent, which can be enclosed and can be carried around; a protective agent is pre-positioned in the container, and the protective agent includes a nuclease inhibitor, a pH maintenance agent and a dehydrating agent. The role of protecting the integrity of nucleic acids in a sample.

Preferably, the container is a 50 ml rotating lid bottom centrifuge tube, a 50 ml rotating lid round bottom centrifuge tube or a 50 ml rotating lid flat bottom centrifuge tube (vertical type), not limited to the above three types, and other can accommodate the protective agent and the sputum liquid. It can be sealed and easy to carry.

Preferably, the nuclease inhibitor is guanidine hydrochloride, guanidinium isothiocyanate, sodium lauryl sulfate, sodium deoxycholate, sodium 4-aminosalicylate, sodium naphthalene-1,5-disulfonate, three different One or several combinations of sodium propyl naphthalene sulfonate. The mass of the nuclease inhibitor is from 0.5 to 4.0 g.

Preferably, the pH maintenance agent is Tris, 3-(N-morpholine)propanesulfonic acid (MOPS), bis(2-(hydroxymethyl)amino-tris(hydroxymethyl) One or a combination of methane (Bis Tris), piperazine-1,4-diethanesulfonic acid (PIPES), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES). The pH maintenance agent has a mass of 1.5 to 4.0 mg.

Preferably, the dehydrating agent is one or a combination of polyethylene glycol (PEG), n-butanol, sec-butanol, tert-butanol. The dehydrating agent has a mass of 0.5 to 2.5 g or a volume of 1.0 to 3.0 ml.

Preferably, the nuclease inhibitor and collection tube are: 1.0 g guanidine hydrochloride, 2.5 mg Tris, and 1.0 g polyethylene glycol placed in a 50 ml rotating lid tip centrifuge tube.

The second aspect of the invention also provides a method for preserving sputum for nucleic acid detection, comprising the steps of:

(1) The collected sputum sample, the volume of the sputum sample is 1-5ml; the sputum sample is not limited, the collected sputum sample can be sticky, bloody, purulent, thick sputum, yellow / gray / rust A sputum of various traits such as sputum.

(2) The sputum sample is directly placed in the sputum collection tube for nucleic acid detection described above, and the sputum sample is mixed with the protective agent in the sputum collection tube, and the storage tube is stored at a temperature of less than or equal to 25 °C.

The principles of the first and second aspects of the invention are as follows: the action of the nuclease inhibitor is to inhibit nuclease activity in the system and prevent degradation of the nucleic acid in the sputum sample; the role of the pH maintenance agent is to maintain the sputum sample as medium Sexual or weakly alkaline, providing a relatively stable pH environment for the nucleic acid in the sputum sample; the role of the dehydrating agent is to reduce the moisture in the sputum sample, prevent the nucleic acid in the sputum sample from being hydrolyzed, and the dehydrating agent only sucks away Moisture does not dissolve nucleic acids. The closed collection tube with lid is convenient to carry around, which is convenient for the subject to collect the sputum at any time when there is a sudden cough reaction, and effectively protect the integrity of the nucleic acid in the sputum.

In order to solve the problem that the current clinical extraction of nucleic acid from the sputum sample is complicated and time-consuming, and the nucleic acid is easy to lose, the third aspect of the present invention provides a method for rapidly extracting nucleic acid from sputum, which can not only liquefy sputum quickly, Simplify the sputum nucleic acid extraction process, save operating time, minimize nucleic acid loss, and reduce nucleic acid extraction costs.

A method for rapidly extracting nucleic acid from sputum provided by the third aspect of the invention comprises the following steps:

(1) Take 2-5ml of sputum sample, add 0.5-2g nuclease inhibitor, equal volume of chloroform, shake at 50 ° C ~ 60 ° C for 10-20 min;

(2) Centrifuge for 1-5 min at a speed of ≥ 12000 r/min, and take the supernatant;

(3a) adding the pre-treatment solution, adding isopropyl alcohol by centrifugation after shaking, mixing and centrifuging, and taking the supernatant for use; or

(3b) Add isopropanol, discard the supernatant after centrifugation, and resuspend the pellet in the pretreatment solution, water-saturated phenol and chloroform, shake, centrifuge, and take the supernatant for use.

Preferably, the step (3a) is: adding 1/10-1/5 volume of the pretreatment liquid, shaking at 50 ° C ~ 60 ° C for 8-15 min, and then centrifuging for 8-15 min at a speed of ≥ 12000 r / min, adding 1/3 volume of isopropanol, mix, centrifuge at ≥12000 r/min for 8-15 min, and take the supernatant for use.

Preferably, the step (3b) is: adding 1/2-1.5 volumes of isopropanol, centrifuging at a speed of ≥12000 r/min for 10-15 min, discarding the supernatant, and resuspending the precipitate of the previous step at 300 -500μl pretreatment solution, 300-800μl water-saturated phenol, 80-200μl chloroform, violently oscillate, centrifuge at maximum speed ≥12000 r/min for 1-5min, and take the supernatant for use.

Preferably, the sputum sample of step (1) is provided by the sputum collected in the collection tube according to claims 1-9, and the nuclease inhibitor in step (1) is subjected to the sputum The protective agent in the collection tube is provided.

Preferably, in the step (3b), in the step (1), 20-80 μl of β-mercaptoethanol is further added before the shaking.

Preferably, the pretreatment liquid comprises: 2-(N-morpholine)ethanesulfonic acid, sodium acetate or proteinase K, β-mercaptoethanol, guanidine hydrochloride, Triton.

Preferably, the pretreatment liquid comprises: 1-5 mM 2-(N-morpholine)ethanesulfonic acid, 1-5 mM sodium acetate or 1-20 mg/ml proteinase K, 5-15% β-mercaptoethanol , 3-7M guanidine hydrochloride, 0.01-0.1% Triton.

In the "volume" and "equal volume" as used in the present invention, the corresponding sputum sample volume is referred to.

The principle of the method provided by the third aspect of the present invention is as follows: The inventors have innovatively used a nuclease inhibitor and chloroform simultaneously to pretreat a sputum sample, and the most prominent advantage of combining nuclease inhibitor with chloroform is that it is highly prone to promote 痰. The liquid is liquefied, in addition to removing fat, densifying the protein, allowing the nucleic acid to enter the upper aqueous phase while completely inhibiting the nuclease. This method breaks the tradition of conventional sputum nucleic acid extraction that must be performed in steps, maximizing the operational steps. The pretreatment liquid can further treat the sputum and rupture the cells, releasing the nucleic acids while inhibiting the nucleases (including: DNase and RNase). Among them, the buffer environment provided by 2-(N-morpholine)ethanesulfonic acid is beneficial to maintain the stability of nucleic acid, and Triton X-100 can fully rupture cells, β-mercaptoethanol and guanidine hydrochloride variability proteins and inhibit nuclease. The pretreatment liquid can release and protect the nucleic acid to the greatest extent, and the formulation is simple and cost-effective. The nucleic acid extraction reagent can extract the nucleic acid into the supernatant liquid, thereby achieving the purpose of extracting the nucleic acid of the sputum sample.

The electrophoresis pattern of the nucleic acid in the sputum sample obtained by the sputum preservation method according to the present invention is detected by electrophoresis, and is No. 3, No. 7, No. 4, No. 8, Marker from left to right.

For the electrophoresis method in Example 4, the electrophoresis pattern of the nucleic acid in the sputum sample was detected, and from left to right, the numbers were No. 5, No. 6, No. 7, No. 8, Marker.

For the electrophoresis method in Example 8 in the specific embodiment, the electrophoresis pattern of the nucleic acid in the sputum sample is detected, and the numbers are No. 1, No. 2, No. 3, No. 4, and Marker from left to right.

The electrophoresis pattern of the nucleic acid in the sputum sample is detected by electrophoresis in Example 10 in the specific embodiment, and is No. 1, No. 2, No. 3, No. 4, and Marker from left to right.

For the electrophoresis method in Example 12, the electrophoresis pattern of the nucleic acid in the sputum sample is detected by the electrophoresis method in the specific embodiment, which is No. 1, No. 2, No. 3, No. 4, and Marker from left to right.

According to a first aspect of the present invention, there is provided a sputum collection tube which can protect nucleic acids in sputum and is convenient to carry and can be collected at any time. It mainly comprises a container and a protective agent, and the container can contain a sputum sample and a protective agent, which can be enclosed and can be carried around; a protective agent is pre-positioned in the container, and the protective agent includes a nuclease inhibitor, a pH maintenance agent and a dehydrating agent. The role of protecting the integrity of nucleic acids in a sample.

In some embodiments, the container can be a 50 ml rotating lid tip centrifuge tube, a 50 ml rotating lid round bottom centrifuge tube, and a 50 ml rotating cap flat bottom centrifuge tube (standing). It will be understood by those skilled in the art that the container used in the present invention is not limited to the above three types, and other closable and portable containers capable of containing a protective agent and a sputum can be used.

In some embodiments, the nuclease inhibitor may be guanidine hydrochloride, guanidinium isothiocyanate, sodium lauryl sulfate, sodium deoxycholate, sodium 4-aminosalicylate, naphthalene-1,5-disulfonate. One or a combination of sodium, sodium triisopropylnaphthalenesulfonate. The mass of the nuclease inhibitor is from 0.5 to 4.0 g.

In some embodiments, the pH maintenance agent may be Tris, 3-(N-morpholine)propanesulfonic acid (MOPS), bis(2-(hydroxymethyl)amino-three One of (hydroxymethyl)methane (Bis Tris), piperazine-1,4-diethanesulfonic acid (PIPES), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) Or a combination of several. The pH maintenance agent has a mass of 1.5 to 4.0 mg.

In some embodiments, the dehydrating agent can be one or several combinations of polyethylene glycol (PEG), n-butanol, sec-butanol, tert-butanol. The dehydrating agent has a mass of 0.5 to 2.5 g or a volume of 1.0 to 3.0 ml.

In some embodiments, the nuclease inhibitor and collection tube are: 1.0 g guanidine hydrochloride, 2.5 mg Tris, and 1.0 g polyethylene glycol in a 50 ml rotating lid tip centrifuge tube.

A method for sputum preservation for nucleic acid detection provided by the second aspect of the present invention comprises the following steps:

(1) Collect the sputum sample, the volume of the sputum sample is 1-5ml; the sputum sample is not limited, the collected sputum sample can be sticky, bloody, purulent, thick sputum, yellow / gray / rust Sputum of various traits;

(2) directly placing the sputum sample in the above-mentioned sputum collection tube for nucleic acid detection, and mixing the sputum sample with the protective agent in the sputum collection tube;

(3) The storage tube has a storage temperature of less than or equal to 25 °C.

In order to verify the effectiveness of the method, the method was verified by fluorescence quantitative PCR and electrophoresis respectively. The experimental steps and results are as follows:

(1) Weigh 1.0g of guanidine hydrochloride, 2.5mg of Tris and 1.0g of polyethylene glycol in a 50ml rotating lid tip centrifuge tube, mix well to make a sputum collection tube, and prepare 4 pieces containing protective agent. Collection tube, number 1-4; additionally prepare 4 unprotected collection tubes, number 5-8;

(2) A clinically confirmed sputum sample of lung cancer patients provided by the Department of Respiratory Medicine, the First Affiliated Hospital of Zhejiang University School of Medicine. The telomerase contained in lung cancer cells confers the ability of lung cancer cells to proliferate indefinitely, and telomerase is translated and synthesized by hTERT mRNA of lung cancer cells, so hTERT mRNA is a characteristic marker of lung cancer cells. In the process of sputum production and coughing, the detached lung cancer cells are mixed and coughed out in the sputum. Therefore, hTERT mRNA can be detected in the sputum of lung cancer patients. The sputum samples of 10 lung cancer patients were thoroughly mixed to prepare 16 ml lung cancer positive mixed sputum samples, and 2 ml sputum samples were taken to the 1-8 collection tube;

(3) Place the collection tube at room temperature (20 ° C). The different collection tubes and placement time are shown in Table 1:

Collection tube number	1, 3, 5, 7	2, 4, 6, 8
Placement time	5 hours	48 hours

(4) Detection of mRNA in sputum samples by real-time PCR: "Terminase reverse transcriptase subunit (hTERT) mRNA detection kit developed by Zhejiang Jinfukang Biotechnology Co., Ltd." (National Machinery Note 20173404247) The kit detects hTERT mRNA in sputum samples. When the PCR amplification result is Ct value <33, it indicates that the test result is positive, which means that the sample contains hTERT mRNA; when the PCR amplification result is Ct value ≥33 or No Ct, it indicates that the test result is negative, indicating that there is no sample. hTERT mRNA. The samples in the collection tubes No. 1, No. 2, No. 5, No. 6 obtained in the above (3) are tested according to the product manual of the kit, and are subjected to sputum dissolution, hybridization, washing, elution, enzymatic digestion and PCR amplification and other steps, the experimental results are shown in Table 2:

Sample serial number	number 1	number 2	Number 5	number 6
Ct value	26.93	27.05	No Ct	No Ct
Experimental result	Positive +hTERT mRNA	Positive +hTERT mRNA	negative-hTERT mRNA	negative-hTERT mRNA

The experimental results showed that the lung cancer positive sputum was stored at room temperature (20 °C) for 5 hours and 48 hours, and the hTERT mRNA in the sample was detected enough to prove its integrity; After 5 hours and 48 hours of lung cancer-positive sputum were placed at room temperature (20 ° C), hTERT mRNA was not detected, indicating that it had been degraded. Therefore, the method can effectively protect the mRNA in the sputum sample from degradation.

(5) Detection of nucleic acid in the sputum sample by electrophoresis: 100 μl of a guanidine hydrochloride solution (2.0 g/ml) was added to the collection tubes No. 3 and No. 4 obtained in the above (3), and the No. 7 obtained in the above (3), Add 600 μl of guanidine hydrochloride solution (2.0 g/ml) to the No. 8 collection tube, and add 100 μl β-mercaptoethanol and 30 μl 20% NP-40 to the No. 3, No. 4, No. 7, and No. 8 tubes, respectively; 5 min, vigorously shake and mix, centrifuge at 12000 rpm for 10 min, take the supernatant on the nucleic acid separation column, centrifuge at 12000 rpm for 1 min; take 700 μl of washing solution (10 mM MES pH 6.0, 35% ethanol) for 2 times; place the column in a clean 1.5 ml On the centrifuge tube, add 30 μl of TE solution to the middle of the column membrane, incubate at 60 ° C for 3 min, centrifuge at 12000 rpm for 1 min, collect the effluent, and take 15 μl of the effluent for gel electrophoresis. The electrophoresis results are shown in Figure 1.

The experimental results showed that the lung cancer positive sputum was stored at room temperature (20 °C) for 5 hours and 48 hours, and the corresponding samples (3 and 4 respectively) were electrophoresed to show clear nucleic acid bands, which proved that 痰The nucleic acid in the liquid sample was in good integrity; instead of using this method, the lung cancer positive sputum samples (7 and 8 respectively) were placed at room temperature (20 ° C) for 5 hours and 48 hours, and no nucleic acid strips were observed after electrophoresis. Band, prove that it has been degraded. Therefore, the method can effectively protect the nucleic acid in the sputum sample from degradation.

A beneficial aspect of the first aspect and the second aspect of the present invention is to provide a method of collecting sputum at any time and protecting the nucleic acid in the mash from degradation. The method puts the nucleic acid protectant in a portable carrying tube, and can collect the sputum at any time when the subject has sputum, wherein the protective agent can well prevent the nucleic acid from being degraded. The protective agent according to the present invention is a powdery solid, which can be better penetrated into the sputum sample, so as to be in good contact with the sputum sample. In addition, the protective agent component of the present invention is relatively simple and low in cost, and is very suitable for being widely used in clinical practice. It is convenient for a subject who needs to perform sputum nucleic acid detection to collect sputum at any time by using a portable collecting tube at the time of sputum, and at the same time The nucleic acid in the sputum sample is protected from degradation at room temperature, giving the subject sufficient time to send the sample to (or express) the hospital or other testing facility.

The following is a detailed description of specific examples of the sputum collection tube and the sputum preservation method for nucleic acid detection according to the present invention.

The sputum samples used in the examples were entrusted by Zhejiang Jinfukang Biotechnology Co., Ltd., and the Department of Respiratory Medicine, the First Affiliated Hospital of Zhejiang University Medical College, was collected according to the clinical standard. The reference was made to the Chinese Tuberculosis Control Plan. Operation and Quality Assurance Manual, by visual inspection, yellow, gray, rust, bloody, purulent, thick, showing a specimen of the mass as the receiving standard. Lung cancer-positive sputum was collected from clinically diagnosed lung cancer patients. The telomerase contained in lung cancer cells confers the ability of lung cancer cells to proliferate indefinitely, and telomerase is translated and synthesized by hTERT mRNA of lung cancer cells, so hTERT mRNA is a characteristic marker of lung cancer cells. In the process of sputum production and coughing, the detached lung cancer cells are mixed and coughed out in the sputum. Therefore, hTERT mRNA can be detected in the sputum of lung cancer patients. The nucleic acid test uses the "telomerase reverse transcriptase subunit (hTERT) mRNA detection kit" developed by Zhejiang Jinfukang Biotechnology Co., Ltd. (National Machinery Co., Ltd. 20173404247), which can detect hTERT mRNA in sputum samples. . When the PCR amplification result is Ct value <33, it indicates that the test result is positive, which means that the sample contains hTERT mRNA; when the PCR amplification result is Ct value ≥33 or No Ct, it indicates that the test result is negative, indicating that there is no sample. hTERT mRNA.

Example 1: Sputum sample can be stored in guanidine hydrochloride-Tris-polyethylene glycol protectant for 72 hours

Step 1: Weigh 1.0 g of guanidine hydrochloride, 2.5 mg of Tris and 1.0 g of polyethylene glycol in a 50 ml rotating lid tip centrifuge tube, mix thoroughly to prepare a sputum protection agent and a collection tube, and prepare 4 tubes in total. Collection tube of protective agent, No. 1-4; additionally prepare 4 unprotected collection tubes, No. 5-8; take a few lung cancer positive sputum and mix well to prepare 16ml lung cancer positive sputum samples, take 2ml respectively Liquid sample to each collection tube;

Step 2: Place the collection tube at room temperature (20 ° C). The different collection tubes and placement time are shown in Table 3:

Collection tube number	1,5	2, 6	3,7	4,8
Placement time	24 hours	48 hours	72 hours	96 hours

Step 3: Using the "telomerase reverse transcriptase subunit (hTERT) mRNA detection kit" (National Machinery Co., Ltd. 20173404247), the sputum sample obtained in step 2 is detected by referring to the product manual operation steps, and the sputum solution is dissolved. Hybridization, washing, elution, enzyme digestion and PCR amplification, etc., the experimental results are shown in Table 4. The results showed that the sputum samples preserved by the method could still detect mRNA after being placed at room temperature for 24-72 hours, and the sputum samples directly placed by the method could not be detected for 24 hours at room temperature, so the method can be used. The integrity of the nucleic acid in the sputum sample was protected for up to 72 hours at room temperature (20 ° C).

Sample serial number	number 1	number 2	number 3	No 4	Number 5	number 6	Number 7	number 8
Ct value	25.97	27.03	30.11	35.76	No Ct	No Ct	No Ct	No Ct
Experimental result	Positive	Positive	Positive	negative	negative	negative	negative	negative

Example 2: The method is suitable for sticking, blood stasis, purulent sputum, thick sputum, yellow sputum, gray sputum, rust color 痰

Step 1: Weigh 1.0 g of guanidine hydrochloride, 2.5 mg of Tris and 1.0 g of polyethylene glycol in a 50 ml rotating lid tip centrifuge tube, mix thoroughly to prepare a sputum collection tube, and prepare a total of 7 protective agents. Collection tube, number 1-7;

Step 2: Take lung cancer positive and the traits are phlegm, blood stasis, purulent sputum, thick sputum, yellow sputum, gray sputum, rust sputum, respectively, placed in the collection tube 1-7, the collection tube is placed Leave at room temperature (20 ° C) for 72 hours;

Step 3: Using the "telomerase reverse transcriptase subunit (hTERT) mRNA detection kit" (National Machinery Co., Ltd. 20173404247), the sputum sample obtained in step 2 is detected by referring to the product manual operation steps, and the sputum solution is dissolved. Hybridization, washing, elution, restriction enzyme digestion and PCR amplification, etc., the experimental results are shown in Table 5. The results showed that the method can play a better protective effect on nucleic acids in sputum, blood stasis, purulent sputum, thick sputum, yellow/grey/rust rust:

Sample serial number	number 1	number 2	number 3	No 4	Number 5	number 6	Number 7
Ct value	22.67	25.44	29.05	31.11	25.64	28.92	27.49
Experimental result	Positive	Positive	Positive	Positive	Positive	Positive	Positive

Example 3: This method is suitable for 1-5 ml sputum samples

Step 1: Weigh 1.0g of guanidine hydrochloride, 2.5mg of Tris and 1.0g of polyethylene glycol into a 50ml rotating lid tip centrifuge tube, mix thoroughly to prepare a sputum protection agent and a collection tube. Collection tube of protective agent, number 1-5;

Step 2: Take a few lung cancer-positive sputum and mix well to prepare 10ml lung cancer positive sputum samples, take 1ml, 2ml, 3ml, 4ml, 5ml sputum samples to the 1-5 collection tube, and place the collection tube Leave at room temperature (20 ° C) for 72 hours;

Step 3: Using the telomerase reverse transcriptase subunit (hTERT) mRNA detection kit (National Machinery Co., Ltd. 20173404247), refer to the product manual for the sputum sample obtained in step 2, after sputum dissolution, hybridization The steps of washing, elution, enzymatic digestion and PCR amplification are shown in Table 6. The results show that the method can exert a good protective effect on the nucleic acids in 1-5 ml sputum samples:

Sample serial number	number 1	number 2	number 3	No 4	Number 5
Ct value	29.79	26.74	25.03	21.21	20.65
Experimental result	Positive	Positive	Positive	Positive	Positive

Example 4: A sputum sample can be stored in guanidinium isothiocyanate-Bis Tris-n-butanol protectant for 72 hours.

Step 1: Weigh 1.2 g of guanidinium isothiocyanate, 2.0 mg of Bis Tris and 2 ml of t-butanol in a 50 ml rotating cap round bottom centrifuge tube, mix well to prepare a sputum protection agent and a collection tube. a collection tube containing a protective agent, number 1-8;

Step 2: Take a few lung cancer-positive sputum and mix well to prepare 16ml lung cancer positive sputum samples, take 2ml sputum samples to the 1-8 collection tube, and place the collection tube at room temperature (20 °C). 72 hours;

Step 3: The telomerase reverse transcriptase subunit (hTERT) mRNA detection kit (National Machinery Co., Ltd. 20173404247) is used to detect the mRNA in the sputum sample, and the 1-4 number obtained in step 2 is referred to the product manual. The sputum samples were tested and subjected to steps such as sputum dissolution, hybridization, washing, elution, enzyme digestion and PCR amplification. The experimental results are shown in Table 7. The results showed that the sputum sample was stored in guanidinium isothiocyanate-Bis Tris-tert-butanol protectant for 72 hours:

Sample serial number	number 1	number 2	number 3	No 4
Ct value	23.55	27.34	24.43	23.21
Experimental result	Positive	Positive	Positive	Positive

Step 4: Detection of nucleic acids in the sputum sample by electrophoresis. Add 600 μl of guanidine hydrochloride solution (2.0 g/ml) to the 5-8 collection tube obtained in step 2, and then add 100 μl of β-mercaptoethanol and 30 μl to the tubes No. 3, No. 4, No. 7, and No. 8, respectively. % NP-40; incubated at 60 ° C for 5 min, violently shake and mix, centrifuge at 12000 rpm for 10 min, take the supernatant on the nucleic acid separation column, centrifuge at 12000 rpm for 1 min; take 700 μl of washing solution (10 mM MES pH 6.0, 35% ethanol) for 2 times. Place the column on a clean 1.5ml centrifuge tube, add 30μl TE solution to the middle of the column membrane, incubate at 60°C for 3min, centrifuge at 12000rpm for 1min, collect the effluent, and take 15μl of the effluent for gel electrophoresis. The electrophoresis results are shown in Figure 2. . The results showed that after the sputum sample was stored in guanidinium isothiocyanate-Bis Tris-tert-butanol protective agent for 72 hours, the corresponding sample (No. 5-8) showed clear nucleic acid bands after electrophoresis, which proved sputum. The nucleic acid in the sample is in good integrity.

Example 5: The guanidine hydrochloride in the protective agent can exert good nucleic acid protection at 0.5-4.0 g.

Step 1: Weigh 2.5mg Tris and 1.0g polyethylene glycol separately into a 50ml rotating lid tip centrifuge tube, a total of 8 tubes, number 1-8; add 0.5g to the 1-8 collection tube, 1.0 g, 1.5 g, 2.0 g, 2.5 g, 3.0 g, 3.5 g, 4.0 g of guanidine hydrochloride, thoroughly mixed to prepare a sputum protection agent and a collecting tube;

Step 2: Take a few lung cancer-positive sputum and mix well to prepare 16ml lung cancer positive sputum samples, take 2ml sputum samples to the 1-8 collection tube, and place the collection tube at room temperature (20 °C). 72 hours;

Step 3: Using the "telomerase reverse transcriptase subunit (hTERT) mRNA detection kit" (National Machinery Co., Ltd. 20173404247), the sputum sample obtained in step 2 is detected by referring to the product manual operation steps, and the sputum solution is dissolved. Hybridization, washing, elution, enzyme digestion and PCR amplification, etc., the experimental results are shown in Table 8. The results showed that the guanidine hydrochloride in the protective agent can play a good nucleic acid protection at 0.5-4.0 g:

Sample serial number	number 1	number 2	number 3	No 4	Number 5	number 6	Number 7	number 8
Ct value	22.80	23.09	19.13	25.46	20.16	21.45	26.15	24.36
Experimental result	Positive	Positive	Positive	Positive	Positive	Positive	Positive	Positive

Example 6: Tris in the protective agent can exert good nucleic acid protection at 1.5-4.0 mg.

Step 1: Weigh 1.0 g of guanidine hydrochloride and 1.0 g of polyethylene glycol in a 50 ml rotating lid tip centrifuge tube, and prepare 6 tubes, number 1-6; add 1.5 mg to the 1-6 collection tube respectively. , 2.0mg, 2.5mg, 3.0mg, 3.5mg, 4.0mg Tris, fully mixed to make a sputum protection agent and collection tube;

Step 2: Take a few lung cancer-positive sputum and mix well to prepare 12ml lung cancer positive sputum samples, take 2ml sputum samples to the 1-6 collection tube, and place the collection tube at room temperature (20 °C). 72 hours;

Step 3: Using the "telomerase reverse transcriptase subunit (hTERT) mRNA detection kit" (National Machinery Co., Ltd. 20173404247), the sputum sample obtained in step 2 is detected by referring to the product manual operation steps, and the sputum solution is dissolved. Hybridization, washing, elution, enzyme digestion and PCR amplification, etc., the experimental results are shown in Table 9. The results showed that Tris in the protective agent can exert good nucleic acid protection at 1.5-4.0 mg:

Sample serial number	number 1	number 2	number 3	No 4	Number 5	number 6
Ct value	21.79	22.35	25.73	22.22	21.75	20.95
Experimental result	Positive	Positive	Positive	Positive	Positive	Positive

Example 7: Polyethylene glycol in the protective agent can exert good nucleic acid protection at 0.5-2.5g

Step 1: Weigh 1.0g of guanidine hydrochloride and 2.5mg of Tris into a 50ml rotating lid tip centrifuge tube, and prepare 5 pieces, number 1-5; add 0.5g, 1.0g to the 1-5 collection tube respectively. , 1.5g, 2.0g, 2.5g polyethylene glycol, fully mixed to make a sputum protection agent and collection tube;

Step 2: Take a few lung cancer-positive sputum and mix well to prepare 10ml lung cancer positive sputum samples, take 2ml sputum samples to 1-5 collection tube, and place the collection tube at room temperature (20 °C). 72 hours;

Step 3: Using the "telomerase reverse transcriptase subunit (hTERT) mRNA detection kit" (National Machinery Co., Ltd. 20173404247), the sputum sample obtained in step 2 is detected by referring to the product manual operation steps, and the sputum solution is dissolved. Hybridization, washing, elution, enzyme digestion and PCR amplification, etc., the experimental results are shown in Table 10. The results show that the polyethylene glycol in the protective agent can play a good nucleic acid protection at 0.5-2.5g:

Sample serial number	number 1	number 2	number 3	No 4	Number 5
Ct value	20.66	21.47	21.53	20.26	23.67
Experimental result	Positive	Positive	Positive	Positive	Positive

A third aspect of the invention also provides a method for rapidly extracting nucleic acid from sputum, the method comprising:

(1) Take 2-5ml of sputum sample, add 0.5-2g nuclease inhibitor, equal volume of chloroform, shake at 50 ° C ~ 60 ° C for 10-20 min;

(2) Centrifuge for 1-5 min at a speed of ≥ 12000 r/min, and take the supernatant;

(3a) adding the pre-treatment solution, adding isopropyl alcohol by centrifugation after shaking, mixing and centrifuging, and taking the supernatant for use; or

(3b) Add isopropanol, discard the supernatant after centrifugation, and resuspend the pellet in the pretreatment solution, water-saturated phenol and chloroform, shake, centrifuge, and take the supernatant for use.

In some embodiments, step (3a) is: adding 1/10-1/5 volume of pretreatment liquid, shaking at 50 ° C ~ 60 ° C for 8-15 min, and then centrifuging at a speed of ≥ 12000 r / min 8- 15 min, add 1/3 volume of isopropanol, mix, centrifuge at ≥12000 r/min for 8-15 min, and take the supernatant for use.

In some embodiments, step (3b) is: adding 1/2-1.5 volumes of isopropanol, centrifuging at a speed of ≥12000 r/min for 10-15 min, discarding the supernatant, and weighing the precipitate of the previous step The suspension was suspended in 300-500 μl of the pretreatment solution, 300-800 μl of water-saturated phenol, and 80-200 μl of chloroform, and violently shaken, centrifuged at a maximum speed of ≥12,000 r/min for 1-5 minutes, and the supernatant was taken for use.

In some embodiments, the sputum sample of step (1) is provided by the sputum collected in the aforementioned collection tube, and the nuclease inhibitor in step (1) is collected by the sputum collection tube. Provided in the protective agent.

In some embodiments, in performing step (3b), in step (1), 20-80 μl of β-mercaptoethanol is further added prior to shaking.

In some embodiments, the pretreatment solution comprises: 2-(N-morpholine)ethanesulfonic acid, sodium acetate or proteinase K, beta-mercaptoethanol, guanidine hydrochloride, Triton.

In some embodiments, the pretreatment solution comprises: 1-5 mM 2-(N-morpholine)ethanesulfonic acid, 1-5 mM sodium acetate or 1-20 mg/ml proteinase K, 5-15%巯-mercaptoethanol, 3-7 M guanidine hydrochloride, 0.01-0.1% Triton.

The method combines a nuclease inhibitor and chloroform for both sputum treatment and nucleic acid extraction, and requires only a small amount of reagent to complete sputum liquefaction and inactivate nuclease, thereby minimizing degradation and loss of nucleic acid. The method not only enables simultaneous sputum liquefaction and cell lysis, but also completes nucleic acid extraction rapidly, preferably within 60 minutes. Thereby, the sputum nucleic acid extraction process is simplified, the operation time is saved, the degradation and loss of nucleic acid can be effectively reduced, and the extraction efficiency of sputum nucleic acid is improved. It has been experimentally verified that, compared with the present method, the first step alone uses nuclease inhibitor or chloroform for sputum treatment and nucleic acid extraction, and the nucleic acid extraction efficiency is low. In addition, the traditional DTT (dithiothreitol) method combined with phenol-chloroform extraction method for sputum treatment and nucleic acid extraction, the nucleic acid extraction efficiency is also low.

In summary, the third aspect of the present invention provides a method for rapidly extracting nucleic acid from sputum, which requires only a small amount of reagent to complete sputum liquefaction and inactivate nuclease, thereby minimizing degradation and loss of nucleic acid. The method can not only simultaneously perform sputum liquefaction and cell lysis, but also rapidly complete the nucleic acid extraction process, and also reduce the use of the reagent phenol in the nucleic acid extraction step, thereby simplifying the sputum nucleic acid extraction process, saving operation time and reducing nucleic acid extraction. cost. The method can be applied to clinical or scientific research fields to achieve the goal of rapidly extracting and detecting sputum nucleic acid.

The following examples are illustrated with guanidine hydrochloride as a nuclease inhibitor, but those skilled in the art will appreciate that nuclease inhibitors may also include guanidinium thiocyanate, sodium lauryl sulfate, sodium deoxycholate, 4- One or more of sodium aminosalicylate, sodium naphthalene-1,5-disulfonate, and sodium triisopropylnaphthalenesulfonate.

The following is a description of specific examples of the method for extracting sputum nucleic acids provided by the third aspect of the present invention.

Example 8: Using the method to detect nucleic acids in 1-5 ml sputum samples

Step 1: Take a few lung cancer-positive sputum and mix well to prepare 10ml lung cancer positive sputum samples, and take 1ml, 2ml, 3ml, 5ml sputum samples to the 1-4th centrifuge tube;

Step 2: Add 1 g of guanidine hydrochloride, an equal volume of chloroform, 1/5 volume of pretreatment solution (composition: 1 mM 2-(N-morpholine) ethanesulfonic acid (pH 7.5), 1 mM sodium acetate, 10% β - mercaptoethanol (w/vol), 7M guanidine hydrochloride, 0.05% Triton X-100 (w/vol), fully incubated at 50 ° C ~ 60 ° C for 10 minutes;

Step 3: Centrifuge the sample obtained in the previous step at a speed of ≥12000 r/min for 1 minute, observe the supernatant and the middle layer. If the middle layer has no obvious solid impurities and the supernatant is not sticky, take the supernatant directly. The liquid (containing the sample nucleic acid) is reserved; if the intermediate layer has obvious solid impurities or the supernatant is viscous, continue to extract with an equal volume of chloroform until the intermediate layer is not obvious and the supernatant is not viscous, and the supernatant is taken ( Containing sample nucleic acid)

Step 4: Using the "telomerase reverse transcriptase subunit (hTERT) mRNA detection kit" (National Machinery Co., Ltd. 20173404247), the sample obtained in step 3 is detected by referring to the product manual operation steps, after hybridization, washing, elution , enzyme digestion and PCR amplification steps, the experimental results are shown in Table 11. The results show that the method can extract nucleic acids from 1-5 ml sputum samples:

Sample serial number	number 1	number 2	number 3	No 4
Ct value	20.73	24.44	22.06	24.21
Experimental result	Positive	Positive	Positive	Positive

Step 5: Detection of nucleic acids in the sample by electrophoresis. The supernatant obtained in step 3 was subjected to a nucleic acid separation column, centrifuged at 12000 r/min for 1 min; washed with 700 μl of washing solution (10 mM MES pH 6.0, 35% ethanol); the column was placed on a clean 1.5 ml centrifuge tube. Add 30 μl of TE solution to the middle of the column membrane, incubate at 60 ° C for 3 min, centrifuge at 12000 r/min for 1 min, collect the effluent, and take 15 μl of the effluent for gel electrophoresis. The electrophoresis results are shown in Figure 1. The results showed that clear samples of nucleic acids were observed after electrophoresis of samples No. 1-4, which proved that the corresponding samples contained nucleic acids with good integrity.

Example 9: The method is suitable for sticking/blood sputum/purulent/thick 痰/yellow 痰/grey 痰/rust 痰

Step 1: Take lung cancer positive and the traits are phlegm, blood stasis, purulent sputum, thick sputum, yellow sputum, gray sputum, rust sputum, each in a 1-7th centrifuge tube;

Step 2: Add 1 g of guanidine hydrochloride, an equal volume of chloroform, and 1/5 volume of the pretreatment liquid, and shake well for 10 minutes at 50 ° C to 60 ° C;

Step 3: Centrifuge the sample obtained in the previous step at a speed of ≥12000 r/min for 1 minute, observe the supernatant and the middle layer. If the middle layer has no obvious solid impurities and the supernatant is not sticky, take the supernatant directly. The liquid (containing the sample nucleic acid) is reserved; if the intermediate layer has obvious solid impurities or the supernatant is viscous, continue to extract with an equal volume of chloroform until the intermediate layer is not obvious and the supernatant is not viscous, and the supernatant is taken ( Containing sample nucleic acid)

Step 4: Using the "telomerase reverse transcriptase subunit (hTERT) mRNA detection kit" (National Machinery Note 20173404247), refer to the product manual for the sputum sample obtained in step 3, after hybridization, washing, The steps of elution, restriction enzyme digestion and PCR amplification are shown in Table 12. The results show that the method can extract nucleic acids in sputum, blood sputum, purulent sputum, thick sputum, yellow/grey/rust rust:

Sample serial number	number 1	number 2	number 3	No 4	Number 5	number 6	Number 7
Ct value	23.47	25.64	28.01	31.21	25.44	21.22	23.48
Experimental result	Positive	Positive	Positive	Positive	Positive	Positive	Positive

Example 10: Nucleic acid extraction can be accomplished using 1/10 volume of pretreatment solution

Step 1: Take a few lung cancer-positive sputum and mix well to prepare 8ml lung cancer positive sputum samples, and take 2ml sputum samples to 1-4 centrifuge tubes respectively;

Step 2: Add 1 g of guanidine hydrochloride, an equal volume of chloroform, and then add 1/2, 1/5, 1/10, 1/20 volume of the pretreatment solution to the No. 1-4 tube, and fully shake at 50 ° C. Incubate for 10 minutes at ~60 °C;

Step 3: Centrifuge the sample obtained in the previous step at a speed of ≥12000 r/min for 1 minute, observe the supernatant and the middle layer. If the middle layer has no obvious solid impurities and the supernatant is not sticky, take the supernatant directly. The liquid (containing the sample nucleic acid) is reserved; if the intermediate layer has obvious solid impurities or the supernatant is viscous, continue to extract with an equal volume of chloroform until the intermediate layer is not obvious and the supernatant is not viscous, and the supernatant is taken ( Containing sample nucleic acid)

Step 4: Using the "telomerase reverse transcriptase subunit (hTERT) mRNA detection kit" (National Machinery Note 20173404247), refer to the product manual for the sputum sample obtained in step 3, after hybridization, washing, The steps of elution, restriction enzyme digestion and PCR amplification are shown in Table 13. The results show that the method can complete nucleic acid extraction using 1/10 volume of pretreatment solution:

Sample serial number	number 1	number 2	number 3	No 4
Ct value	19.11	25.43	27.93	No Ct
Experimental result	Positive	Positive	Positive	negative

Step 5: Detection of nucleic acids in the sample by electrophoresis. The supernatant obtained in step 3 was subjected to a nucleic acid separation column, centrifuged at 12000 r/min for 1 min; washed with 700 μl of washing solution (10 mM MES pH 6.0, 35% ethanol); the column was placed on a clean 1.5 ml centrifuge tube. Add 30 μl TE solution to the middle of the column membrane, incubate at 60 °C for 3 min, centrifuge at 12000 r/min for 1 min, collect the effluent, and take 15 μl of the effluent for gel electrophoresis. The electrophoresis results are shown in Figure 2. The results showed that the samples of No. 1-3 showed clear nucleic acid bands after electrophoresis, which proved that the corresponding samples contained nucleic acids with good integrity; No. 4 samples showed no nucleic acid bands after electrophoresis, which proved that the corresponding samples did not contain A nucleic acid of good integrity.

Example 11: Extraction of sputum nucleic acid using a preferred extraction method

Step 1: Take a few lung cancer-positive sputum and mix well to prepare 8ml lung cancer positive sputum samples, and take 2ml sputum samples to 1-4 centrifuge tubes respectively;

Step 2: Add 1 g of guanidine hydrochloride, an equal volume of chloroform, and incubate at 50 ° C ~ 60 ° C for 10-20 min;

Step 3: Centrifuge at ≥12000 r/min for 1-5 min, take the supernatant, add 1/10-1/5 volume of pretreatment solution (containing proteinase K), and incubate for 8-15 min at 50 °C~60 °C. ;

Step 4: Add 1/3 volume of isopropanol, mix and centrifuge for 8-15 min at a speed of ≥12000 r/min, and take the supernatant (containing sample nucleic acid) for use;

Step 5: Using the "telomerase reverse transcriptase subunit (hTERT) mRNA detection kit" (National Machinery Co., Ltd. 20173404247), the sputum sample obtained in step 4 is detected by referring to the product manual operation steps, after hybridization, washing, The steps of elution, restriction enzyme digestion and PCR amplification are shown in Table 14. The results show that extraction of nucleic acids from sputum samples can be achieved by optimal extraction methods:

Sample serial number	number 1	number 2	number 3	No 4
Ct value	21.11	22.13	21.91	22.06
Experimental result	Positive	Positive	Positive	Positive

Example 12: Extraction of nucleic acids using guanidine hydrochloride or chloroform alone and comparison with conventional methods

Step 1: Take a few lung cancer-positive sputum and mix well to prepare 8ml lung cancer positive sputum samples, and take 2ml sputum samples to 1-4 centrifuge tubes respectively;

Step 2: Sample No. 1 was subjected to nucleic acid extraction according to the method of Example 4;

Step 3: Add 1 g of guanidine hydrochloride and 1/5 volume of pretreatment solution to the No. 2 sample tube (composition: 1 mM 2-(N-morpholine) ethanesulfonic acid (pH 7.5), 1 mg/ml proteinase K , 10% β-mercaptoethanol (w/vol), 7M guanidine hydrochloride, 0.05% Triton X-100 (w/vol), fully incubated at 50 ° C for 15 min after shaking; add 1/3 volume difference Propyl alcohol, mix, centrifuge at a speed of ≥ 12000 r / min for 10 min, take the supernatant (containing sample nucleic acid) for use;

Step 4: Add an equal volume of chloroform and 1/5 volume of pretreatment solution to the No. 3 sample tube (composition: 1 mM 2-(N-morpholine) ethanesulfonic acid (pH 7.5), 1 mg/ml proteinase K , 10% β-mercaptoethanol (w/vol), 7M guanidine hydrochloride, 0.05% Triton X-100 (w/vol), fully incubated at 50 ° C for 15 min after shaking; add 1/3 volume difference Propyl alcohol, mix, centrifuge at a speed of ≥ 12000 r / min for 10 min, take the supernatant (containing sample nucleic acid) for use;

Step 5: Add 5 volumes of DTT (dithiothreitol) solution to the No. 4 sample tube (composition: 0.1% DTT + PBS buffer (phosphate buffer), liquefaction and liquefaction for 20 min;

Step 6: Extract the nucleic acid of sample No. 4 by classical phenol-chloroform extraction method: add an equal volume of saturated phenol, shake for 5 min, centrifuge at a speed of ≥12000 r/min for 10 min, take the supernatant and add an equal volume of chloroform. Isoamyl alcohol (24:1), shaking for 5 min, centrifugation at a speed of ≥ 12000 r / min for 10 min, taking the supernatant (containing sample nucleic acid) for use;

Step 7: Detection of nucleic acids in the sample by electrophoresis. The supernatant No. 1-4 obtained in the above step was centrifuged on a nucleic acid separation column, centrifuged at 12000 r/min for 1 min; washed with 700 μl of washing solution (10 mM MES pH 6.0, 35% ethanol); the column was placed in a clean 1.5 On the ml centrifuge tube, add 30 μl TE solution to the middle of the column membrane, incubate at 60 ° C for 3 min, centrifuge at 12000 r/min for 1 min, collect the effluent, and take 15 μl of the effluent for gel electrophoresis. The electrophoresis results are shown in Figure 3. The results showed that the clear sample of nucleic acid was observed after electrophoresis of sample No.1, which proved that the corresponding sample contained nucleic acid with good integrity. The nucleic acid bands of samples 2-4 after electrophoresis were lighter, indicating that the corresponding samples contained less nucleic acid and the nucleic acid extraction efficiency was lower.

Example 13: Extraction of sputum nucleic acid using a preferred extraction method

Step 1: Take a few lung cancer-positive sputum and mix well to prepare 8ml lung cancer positive sputum samples, and take 2ml sputum samples to 1-4 centrifuge tubes respectively;

Step 2: adding 1 g of guanidine hydrochloride, an equal volume of chloroform, 20 μl of β-mercaptoethanol, and shaking at 60 ° C for 10 min;

Step 3: Centrifuge at a speed of ≥12000 r/min for 5 min, take the supernatant, add 1 volume of isopropanol, centrifuge at a speed of ≥12000 r/min for 10 min, discard the supernatant;

Step 4: Resuspend the pellet from the previous step in 350 μl of pretreatment solution (composition: 1 mM 2-(N-morpholine) ethanesulfonic acid (pH 7.5), 1 mg/ml proteinase K, 10% β-mercapto Ethanol (w/vol), 7M guanidine hydrochloride, 0.05% Triton X-100 (w/vol), 500μl water-saturated phenol, 100μl chloroform, violently oscillate, centrifuge at maximum speed ≥12000 r/min for 5min, take Clear liquid (containing sample nucleic acid) for use;

Step 5: Using the "telomerase reverse transcriptase subunit (hTERT) mRNA detection kit" (National Machinery Co., Ltd. 20173404247), the sputum sample obtained in step 4 is detected by referring to the product manual operation steps, after hybridization, washing, The steps of elution, restriction enzyme digestion and PCR amplification are shown in Table 15. The results show that extraction of nucleic acids from sputum samples can be achieved by optimal extraction methods:

Sample serial number	number 1	number 2	number 3	No 4
Ct value	19.14	19.89	20.33	21.07
Experimental result	Positive	Positive	Positive	Positive

Claims (17)

1. A sputum collection tube for nucleic acid detection, comprising: a container capable of containing a sputum sample and a protective agent, the container being closable and portable; a protective agent pre-placed in the container, the protective agent comprising nuclease inhibition Agent, pH maintenance agent and dehydrating agent.
2. The sputum collection tube for nucleic acid detection according to claim 1, wherein the container is one of a 50 ml rotating cap tip centrifuge tube, a 50 ml rotating cap round bottom centrifuge tube, and a 50 ml rotating cap flat bottom centrifuge tube. Kind.
3. The sputum collection tube for nucleic acid detection according to claim 1, wherein the nuclease inhibitor is guanidine hydrochloride, guanidinium isothiocyanate, sodium lauryl sulfate, sodium deoxycholate, 4 One or more of sodium aminosalicylate, sodium naphthalene-1,5-disulfonate, and sodium triisopropylnaphthalenesulfonate.
4. The sputum collection tube for nucleic acid detection according to claim 1, wherein the nuclease inhibitor has a mass of 0.5 to 4.0 g.
5. The sputum collection tube for nucleic acid detection according to claim 1, wherein the pH maintenance agent is tris (hydroxymethyl) aminomethane, 3-(N-morpholine) propane sulfonic acid, and bis (2) One of -(hydroxymethyl)amino-tris(hydroxymethyl)methane, piperazine-1,4-diethanesulfonic acid, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid or A variety.
6. The sputum collection tube for nucleic acid detection according to claim 1, wherein the pH maintenance agent has a mass of 1.5 to 4.0 mg.
7. The sputum collection tube for nucleic acid detection according to claim 1, wherein the dehydrating agent is one or more of polyethylene glycol, n-butanol, sec-butanol, and tert-butanol.
8. The sputum collection tube for nucleic acid detection according to claim 1, wherein the dehydrating agent has a mass of 0.5 to 2.5 g or a volume of 1.0 to 3.0 ml.
9. The sputum collection tube for nucleic acid detection according to claim 1, wherein the sputum collection tube 50 ml rotates the tip-end centrifuge tube, and the built-in protective agent comprises: 1.0 g of guanidine hydrochloride, 2.5 mg of Tris, and 1.0. g polyethylene glycol.
10. A method for preserving sputum for nucleic acid detection, characterized in that:
    (1) collecting a sample of sputum, the volume of the sputum sample is 1-5 ml;
    (2) placing the sputum sample directly in the sputum collection tube for nucleic acid detection according to any one of claims 1-9, mixing the sputum sample with the protective agent in the sputum collection tube;
    (3) The storage tube has a storage temperature of less than or equal to 25 °C.
11. A method for rapidly extracting nucleic acid from sputum, characterized in that it comprises the following steps:
    (1) Take 2-5ml of sputum sample, add 0.5-2g nuclease inhibitor, equal volume of chloroform, shake at 50 ° C ~ 60 ° C for 10-20 min;
    (2) Centrifuge for 1-5 min at a speed of ≥12000 r/min, take the supernatant; and (3a) add the pre-treatment solution, shake and incubate, add isopropyl alcohol by centrifugation, mix and centrifuge, and take the supernatant for use; or (3b) Add isopropanol, centrifuge, discard the supernatant, and resuspend the pellet in the pretreatment solution, water-saturated phenol and chloroform, shake, centrifuge, and take the supernatant for use.
12. The method according to claim 11, wherein the step (3a) is: adding 1/10-1/5 volume of the pretreatment liquid, shaking at 50 ° C to 60 ° C for 8-15 min, and then rotating at ≥ 12000. Centrifuge for 8-15 min at r/min, add 1/3 volume of isopropanol, mix well, centrifuge for 8-15 min at ≤12000 r/min, and take the supernatant for use.
13. The method according to claim 11, wherein the step (3b) is: adding 1/2-1.5 volumes of isopropanol, centrifuging at a rotation speed of ≥12000 r/min for 10-15 minutes, discarding the supernatant, and Resuspend the pellet from the previous step in 300-500 μl pretreatment solution, 300-800 μl water-saturated phenol, 80-200 μl chloroform, shake vigorously, centrifuge at maximum speed ≥12000 r/min for 1-5 min, and take the supernatant for use. .
14. The method according to claim 11, wherein the sputum sample of step (1) is provided by the sputum collected in the collection tube of claims 1-9, and the step (1) The nuclease inhibitor is provided by a protective agent in the sputum collection tube.
15. The method according to claim 11, wherein in the step (3b), in the step (1), 20-80 μl of β-mercaptoethanol is further added before the shaking.
16. The method according to any one of claims 11-15, wherein the pretreatment liquid comprises: 2-(N-morpholine)ethanesulfonic acid, sodium acetate or proteinase K, β-mercaptoethanol, guanidine hydrochloride , Triton.
17. The method according to any one of claims 11-15, wherein the pretreatment liquid comprises: 1-5 mM 2-(N-morpholine)ethanesulfonic acid, 1-5 mM sodium acetate or 1- 20 mg/ml proteinase K, 5-15% β-mercaptoethanol, 3-7 M guanidine hydrochloride, 0.01-0.1% Triton.

Images