A method for liquefying sputum and a special liquefying reagent therefor

Abstract

The application discloses a sputum liquefaction method and a special liquefaction reagent thereof, and belongs to the technical field of biotechnology. The sputum liquefaction method comprises the following steps: mixing the sputum liquefaction reagent with a sputum sample, and then performing high-temperature treatment or high-temperature and ultrasonic combined treatment. The sputum can be completely liquefied and nucleic acid can be released from pathogens in one step, the nucleic acid of the pathogens can be protected from degradation at the same time, the operation is simple and convenient, the time consumption is short, the liquefaction efficiency of the sputum sample can be greatly improved, and the positive detection rate can be obviously improved. The sputum liquefaction reagent has simple composition and low component concentration, and the production cost can be greatly reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology methods, and particularly relates to a sputum liquefaction method and a special liquefaction reagent thereof. BACKGROUND

[0002] Nucleic acid detection is one of the important methods for pathogen detection. Current pathogen nucleic acid detection is divided into two categories: one is to take DNA as the detection target, and the other is to take RNA as the detection target. The copy number of DNA in pathogens is generally low, while the copy number of RNA in pathogens is high, especially ribosomal RNA, which can have hundreds or even tens of thousands of RNA target copies in a pathogen. Therefore, the pathogen detection method taking the RNA of the pathogen as the target can improve the detection rate of the pathogen. However, due to the characteristics of RNA being easily degraded, once the pathogen dies, the RNA will be quickly degraded, so that the pathogen cannot be detected.

[0003] Sputum pathogen detection is an important way for respiratory disease detection. Sputum is the secretion of the human respiratory tract, which is pushed from the lungs to the upper respiratory tract through the movement of bronchial epithelial cilia, and finally, it is coughed out from the trachea and discharged outside the body through the normal cough reflex of the human body. Normal human sputum is very small, only a small amount of mucus is secreted to keep the respiratory tract moist. When a person inhales irritating gases, dust, pathogenic bacteria, viruses and other harmful microorganisms, inflammation of the upper respiratory tract may occur; or lung diseases such as bronchiectasis, lung abscess, lung cancer, etc. occur, and the respiratory tract sputum will increase, and the sputum contains various pathogenic microorganisms, various inflammatory cells, exfoliated necrotic mucosal epithelial cells and tumor cells, etc. Detecting the nucleic acid of the pathogen in the sputum sample to identify the type of pathogen is one of the most accurate and fastest respiratory pathogen diagnosis methods. With the rapid development of real-time fluorescent nucleic acid isothermal amplification detection technology (Simultaneous Amplification and Testing, SAT) in the field of pathogen detection, this technology has the characteristics of strong specificity, high sensitivity, simple operation, rapid detection, high detection rate, etc. This technology has gradually occupied an important position in the field of clinical diagnosis.

[0004] However, sputum samples contain mucin and other various proteins, various enzymes, exfoliated cells, microorganisms and other inhaled impurities, etc., with high viscosity, high protein, complex composition and other characteristics. It is difficult to directly detect nucleic acid using sputum samples, so it is necessary to liquefy the sputum sample to reduce the viscosity and safely release the nucleic acid in the pathogen without being degraded, and reduce the interference of various nucleic acid detection inhibitors. The common sputum liquefaction methods at present are sodium hydroxide method (such as CN106867998A, CN104988237A), DTT (dithiothreitol) method (such as He Hui et al. "Comparison of different liquefaction treatment and nucleic acid co-extraction methods on the extraction effect of viral nucleic acid in sputum samples", International Journal of Epidemiology and Infectious Diseases, April 2016, Vol. 43, No. 2), protease method (such as CN104232621A), combination method of protease method and DTT (such as CN108048450A), and sputum liquefaction reagent composed of guanidine and cysteine derivatives (such as CN108949748A). Among them, the sodium hydroxide method is the most commonly used sputum liquefaction method. This method is relatively simple, which can liquefy sputum by using a sodium hydroxide solution with a certain concentration as the main component. However, when this method is used for sputum sample liquefaction, most of the pathogens in the sputum will die quickly, resulting in RNA degradation, which ultimately leads to a low detection rate of pathogen RNA. Moreover, the liquefied sample obtained by this method is difficult to be directly used for subsequent pathogen nucleic acid detection, and often needs to be enriched with pathogen precipitate after liquefaction, followed by nucleic acid extraction, which is relatively complex. In addition, many sputum samples of pathogens with low tolerance to sodium hydroxide solution are not suitable for this liquefaction method. The principle of protease method is to digest sputum mucin with protease. This method requires high-cost protease, and the enzyme reaction takes a long time (usually 37-65°C for 1-5 hours), which is easy to liquefy unevenly and has low efficiency. The DTT method uses DTT containing sulfhydryl (-SH) to break the main component of sputum mucin, which leads to viscosity. This method requires high-concentration DTT, takes a long time, and often needs centrifugation. Moreover, due to the poor stability of DTT, it needs to be stored at low temperature. The sputum liquefied by this method is often not uniform, and it is easy to change from solution state to gel state. In addition, DTT has certain toxicity, which is not suitable for high-concentration use in clinical sputum liquefaction.The combination of protease method and DTT method can reduce the transition of sputum solution state to gel state to some extent after liquefaction, but this method still needs to use high-cost protease and high-concentration DTT, and the composition is complex. Since the sputum liquefaction using protease needs to be incubated at room temperature or above for a long time, and DTT has poor stability and needs to be stored at low temperature, the protease liquefaction operation and DTT liquefaction operation must be carried out in steps, which also leads to complicated operation, long time consumption, low liquefaction efficiency, and may also need to be centrifuged, which is not suitable for sputum samples for clinical liquefaction and direct nucleic acid detection. Although the sputum liquefaction reagent composed of guanidine and cysteine derivatives can protect the nucleic acid in the sample at room temperature for a long time, it is not suitable for effective protection of the nucleic acid released by the pathogen under high-temperature treatment conditions, which may cause low positive detection rate. SUMMARY

[0005] In view of one or more of the problems in the prior art, one aspect of the present application provides a sputum liquefaction method, which comprises the following steps:

[0006] 1) mixing sputum liquefaction reagent with sputum sample to obtain a mixed solution;

[0007] 2) subjecting the mixed solution to high-temperature treatment at 90-100°C for 10-30 min;

[0008] The sputum liquefaction reagent comprises 3-5M guanidine salt, 0.1-0.5% detergent, 0.1-3% thiol reducing agent, 10-100mM Tris-HCl, 0.1-0.5mM EDTA, pH≤7.5, preferably pH≤6.9.

[0009] The guanidine salt is selected from one or more of guanidine hydrochloride, guanidine isothiocyanate, guanidine sulfate, and guanidine carbonate.

[0010] The detergent is selected from one or more of sodium dodecyl sulfate and lithium dodecyl sulfate.

[0011] The thiol reducing agent is selected from one or more of cysteine, N-acetyl cysteine, and DTT.

[0012] The thiol reducing agent is a combination of N-acetyl cysteine and DTT, wherein the concentration of N-acetyl cysteine is 0.3-3%, and the concentration of DTT is 0.1-0.3%.

[0013] The method further comprises the following steps:

[0014] 3) simultaneously or subsequently to the high-temperature treatment of the mixed solution, ultrasonic treatment is also performed, with an ultrasonic power of 200-400W for 10-20 min.

[0015] In the method, the volume ratio of the sputum liquefaction reagent to the sputum sample in step 1) is (1-3):1.

[0016] The present application also provides a sputum liquefaction reagent for use in the above method, comprising: 3-5 M guanidine salt, 0.1-0.5% detergent, 0.1-3% thiol reducing agent, 10-100 mM Tris-HCl, 0.1-0.5 mM EDTA, pH≤7.5, preferably pH≤6.9. The guanidine salt is selected from one or more of guanidine hydrochloride, guanidine isothiocyanate, guanidine sulfate, guanidine carbonate; the detergent is selected from one or more of sodium dodecyl sulfate, lithium dodecyl sulfate; the thiol reducing agent is selected from one or more of cysteine, N-acetyl cysteine, DTT.

[0017] The present application also provides a pathogen nucleic acid detection kit comprising the above sputum liquefaction reagent.

[0018] The sputum liquefaction method provided in the above technical solution combines chemical and physical treatment, and one-step operation can effectively liquefy the sputum sample into a solution state while inactivating and lysing the pathogen, so that the nucleic acid in the pathogen is released into the solution, and the nucleic acid (especially RNA) released into the solution is protected from degradation. Therefore, the method is simple and convenient to operate, time-consuming is short, and the obtained sputum liquefaction solution can be directly used as a sample to be tested for pathogen nucleic acid detection, without the need for nucleic acid enrichment or extraction operation, thereby providing a powerful tool for automatic detection of pathogens in sputum samples. In the method, the sputum liquefaction reagent used in the chemical treatment can only contain low concentrations of effective components such as guanidine salt, detergent and thiol reducing agent. Compared with the prior art, although the composition of the sputum liquefaction reagent is very simple, it can cooperate with the physical treatment (heating treatment or heating + ultrasonic combined treatment) operation in the method to effectively break the disulfide bond of mucin, the main component that causes the viscosity of sputum, through the thiol reducing agent in the sputum liquefaction reagent, thereby effectively reducing the viscosity of sputum, and cooperating with the physical treatment operation to completely liquefy the sputum sample (without the sputum changing from a solution state to a gel state after liquefaction), while inactivating the pathogen to reduce the biological safety hazard, and breaking the pathogen to release the nucleic acid into the sputum liquefaction solution, and effectively protecting the nucleic acid from degradation through the synergistic effect of the guanidine salt and the detergent in the sputum liquefaction reagent during and after the sputum liquefaction treatment, thereby significantly improving the positive detection rate.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] 1) The sputum liquefaction method provided by the application is a method of combining chemical and physical treatment, which can achieve complete liquefaction of sputum and release of nucleic acid from pathogens in one step, and at the same time, long-term protection of pathogen nucleic acid from degradation, so the method is simple and convenient to operate, short in time consumption, and greatly improves the liquefaction efficiency of sputum samples, and can be used for liquefaction of sputum of various viscosities;

[0021] 2) The method of the application combines chemical and physical treatment to liquefy the sputum sample, and only needs a short time (for example, about 15 minutes) to obtain a well-liquefied sputum liquefaction solution, greatly shortening the time required for liquefaction;

[0022] 3) The sputum liquefaction solution obtained by the method of the application can be directly used as a sample for RNA isothermal amplification nucleic acid detection for pathogen nucleic acid detection, without complex operation steps such as bacterial enrichment;

[0023] 4) The sputum liquefaction reagent provided by the application can only contain low concentrations of guanidine salt, detergent and thiol reducing agent, etc. Therefore, the sputum liquefaction reagent has simple components, and does not use sodium hydroxide which can easily cause nucleic acid degradation and high-cost protease, so the cost of the reagent can be significantly reduced. When the thiol reducing agent used is DTT, its concentration can be only about 0.1-0.3% (about 6-18mM), which is significantly lower than the DTT content in the sputum liquefaction reagent in the prior art (for example, the DTT content disclosed in CN108048450A is 45-55mM), so the cost of the reagent can be significantly reduced and the toxicity of DTT and the interference caused by the unstable nature of DTT can be reduced. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The nucleic acid detection amplification curve of the sample A1 to be tested in Example 1;

[0025] Figure 2 The nucleic acid detection amplification curve of the sample A2 to be tested in Example 1;

[0026] Figure 3 The nucleic acid detection amplification curve of the sample B1 to be tested in Example 1;

[0027] Figure 4 The nucleic acid detection amplification curve of the sample C1 to be tested in Example 2;

[0028] Figure 5 The nucleic acid detection amplification curve of the sample C1 to be tested in Example 2;

[0029] Figure 6 The nucleic acid detection amplification curve of the sample D1 to be tested in Example 2;

[0030] Figure 7This is the nucleic acid detection amplification curve of sample E11 in Example 3;

[0031] Figure 8 This is the nucleic acid detection amplification curve of sample E21 in Example 3;

[0032] Figure 9 This is the nucleic acid detection amplification curve of sample F11 in Example 3;

[0033] Figure 10 This is the nucleic acid detection amplification curve of sample F21 in Example 3;

[0034] Figure 11 The nucleic acid detection amplification curve of sample G11 in Example 3;

[0035] Figure 12 This is the nucleic acid detection amplification curve of sample G21 in Example 3. Detailed Implementation

[0036] To address the shortcomings of existing sputum liquefaction methods, this invention provides a sputum liquefaction method that combines chemical and physical processing operations, along with a dedicated sputum liquefaction reagent. This method achieves complete liquefaction of sputum and release of nucleic acids from pathogens in a single step, while simultaneously providing long-term protection against degradation of pathogen nucleic acids.

[0037] Chemical treatment uses a sputum liquefaction reagent containing guanidine salts, detergents, and thiol reducing agents. It also contains 10–100 mM Tris-HCl and 0.1–0.5 mM EDTA, with a pH ≤ 7.5, optionally pH ≤ 6.9. (The following is a partial list of ingredients and their components.)

[0038] The role of guanidine salt in this invention is as a nuclease inhibitor, which can protect the nucleic acids released by pathogens from being degraded in large quantities. It can be selected from one or more of guanidine hydrochloride, guanidine isothiocyanate, guanidine sulfate, and guanidine carbonate. The final concentration of guanidine salt in the sputum liquefaction reagent is 3-5M, and can be selected as 3-4M, 4-5M, etc.

[0039] The detergent in this invention works synergistically with the guanidine salt in the sputum liquefaction reagent to effectively protect the nucleic acids released by lysed pathogens under the high-temperature treatment conditions or the combined high-temperature and ultrasonic treatment conditions of this invention. It can be selected from one or more of sodium dodecyl sulfate and lithium dodecyl sulfate. The final concentration of the detergent in the sputum liquefaction reagent is 0.1-0.5% (mass-volume concentration, for example, 0.1% means 0.1g / 100ml, the same below), and can be selected as 0.1-0.2%, 0.1-0.3%, 0.1-0.4%, 0.2-0.3%, 0.2-0.4%, 0.3-0.4%, 0.3-0.5%, or 0.4-0.5%, etc.

[0040] The role of the mercapto reducing agent in the present application is to break the disulfide bond of the main component of sputum, mucin, which causes viscosity, thereby effectively reducing the viscosity of sputum, and cooperating with the physical treatment operation of the present application to completely liquefy the sputum sample (without the sputum changing from a solution state to a gel state after liquefaction). The mercapto reducing agent can be selected from one or more of cysteine, N-acetyl cysteine, and DTT; the final concentration of the mercapto reducing agent in the sputum liquefaction reagent is 1-3% (mass / volume concentration). When a combination of multiple mercapto reducing agents containing DTT is used, for example, a combination of N-acetyl cysteine and DTT, wherein the final concentration of N-acetyl cysteine is 0.3-3%, which can be selected from 0.3-0.5%, 0.3-1.0%, 0.5-1.0%, 0.5-3%, 1.0-3%, etc., and the final concentration of DTT can be only 0.1-0.3%, which can be selected from 0.1-0.2%, 0.2-0.3%, etc.

[0041] Tris-HCl in the sputum liquefaction reagent serves as a pH buffer medium, and EDTA is used to stabilize the pH value of the system and remove metal ions as nuclease activity cofactors.

[0042] The physical treatment includes high temperature treatment or high temperature + ultrasonic combined treatment: the high temperature treatment refers to treatment at 90-100°C for 10-30 min, which can be selected from 90°C for 10-20 min, 90°C for 20-30 min, 95°C for 10-20 min, 95°C for 20-30 min, 100°C for 10-20 min, 100°C for 20-30 min, etc. The role in the present application is to completely liquefy the sputum sample (without the sputum changing from a solution state to a gel state after liquefaction), while inactivating the pathogen to reduce biological safety hazards, and breaking the pathogen to release nucleic acids into the sputum liquefaction solution; the ultrasonic treatment refers to ultrasonic treatment at a power of 200-400W for 10-20 min after or at the same time as the high temperature treatment, which can be selected from 200W for 10-15 min, 200W for 15-20 min, 300W for 10-15 min, 300W for 15-20 min, 400W for 10-20 min, 400W for 15-20 min, 400W for 10-15 min, etc. The role in the present application is to completely liquefy sputum with high viscosity in cooperation with the high temperature treatment, and to lyse the pathogen in the sputum to release more nucleic acids into the sputum liquefaction solution.

[0043] The following will be described in detail in conjunction with specific embodiments.

[0044] In the following text, only some exemplary embodiments are briefly described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the present invention. Therefore, the accompanying drawings and the description are considered to be exemplary in nature rather than restrictive.

[0045] In the following embodiments, to verify the effectiveness of the method provided by the present invention, a magnetic bead extraction reagent (Shanghai Rendu Biotechnology Co., Ltd., Shanghai Medical Device Preparation No. 20190131) and a Mycobacterium tuberculosis nucleic acid detection kit TB-SAT (National Medical Device Approval No. 20153401875) are used for illustration. The sputum samples used in the embodiments are collected according to the clinical standard sputum collection procedure. Referring to the "China Tuberculosis Control Program - Standardized Operation and Quality Assurance Manual for Sputum Smear Microscopy", specimens that are yellow, gray, rust-colored, bloody, purulent, thick, and in a clumpy form are the acceptance criteria by visual inspection.

[0046] The acquisition routes of various biological materials described in the embodiments are only provided as a way to obtain through experiments for the purpose of specific disclosure, and should not be a limitation on the source of biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the prompts in the embodiments.

[0047] Example 1: Sputum liquefaction method

[0048] The sputum liquefaction method provided in this example includes the following steps:

[0049] 1) Prepare 20 sputum samples from Mycobacterium tuberculosis-positive patients (collected from Shanghai Pulmonary Hospital, informed consent has been signed), mix them evenly and divide them into two equal parts: Samples A and B are placed in test tubes respectively;

[0050] 2) Add an equal volume of sputum liquefaction reagent (containing: 5M guanidine hydrochloride, 0.5% sodium dodecyl sulfate (SDS), 0.5% N-acetylcysteine, 0.1% DTT, 20 mM Tris-HCl, 0.3 mM EDTA) to Sample A and mix well, for example, by shaking or using a mixer. Then place it in a boiling water bath at 95°C for 10 minutes, and take 400 μl of the sample for Mycobacterium tuberculosis nucleic acid detection (test sample A1); put the remaining sample into an ultrasonic disruptor and ultrasonicate at 300 W for 15 minutes, and then take 400 μl of the sample for Mycobacterium tuberculosis nucleic acid detection (test sample A2). The Mycobacterium tuberculosis nucleic acid detection is carried out according to the instructions of the magnetic bead extraction reagent (Shanghai Medical Device Preparation No. 20190131) and the Mycobacterium tuberculosis nucleic acid detection kit TB-SAT (National Medical Device Approval No. 20153401875) respectively. The same applies to the following examples; the detection results of test sample A1 and test sample A2 are respectively asFigure 1 and Figure 2 As shown;

[0051] 3) Add an equal volume of 4% sodium hydroxide solution to sample B, mix well, incubate at room temperature for 20 min, then take 1 mL of sample and centrifuge and wash. Perform Mycobacterium tuberculosis nucleic acid detection on the washed precipitate according to the kit instructions (sample B1). The detection results for sample B1 are as follows: Figure 3 As shown;

[0052] according to Figures 1-3 The results show that by first mixing the sputum sample with the sputum liquefaction reagent provided by this invention, and then performing a high-temperature treatment step, a positive result for Mycobacterium tuberculosis in the sputum sample (A1) can be detected (see [reference]). Figure 1 Furthermore, the sample preparation process before detection took only about 10 minutes, which is relatively short. The amplification curve showed a high relative fluorescence intensity, indicating that the pathogen nucleic acid in the sample was released in significant quantities and well protected. Further ultrasonic treatment resulted in even greater release of Mycobacterium tuberculosis nucleic acid from sample A2, and its amplification curve showed a higher relative fluorescence intensity. Figure 1 The test results were higher (see Figure 2 Therefore, the positive detection sensitivity is correspondingly higher. However, the existing technology, which uses 4% sodium hydroxide solution to digest sputum to obtain the test sample (B1), yields a negative result (see...). Figure 3 Therefore, the test results are unreliable and time-consuming, and complex operations such as centrifugation, nucleic acid precipitation and enrichment, and nucleic acid extraction are required.

[0053] Example 2: Sputum liquefaction method

[0054] The sputum liquefaction method provided in this embodiment includes the following steps:

[0055] 1) Prepare 20 sputum samples from patients who are positive for Mycobacterium tuberculosis (collected from Shanghai Pulmonary Hospital, with informed consent signed), mix them evenly (the viscosity of the sputum samples in this example is higher than that of the sputum samples in Example 1), and divide them into two equal portions: Sample C and Sample D are placed in test tubes respectively.

[0056] 2) To sample C, add an equal volume of sputum liquefaction reagent (containing: 3M guanidine hydrochloride, 0.2% sodium dodecyl sulfate (SDS), 1.0% N-acetyl cysteine, 0.2% DTT, 20mM Tris-HCl, 0.3mM EDTA) and mix well, for example by shaking or mixing on a mixer, then boil at 95°C for 10 min, take 400 μl sample for Mycobacterium tuberculosis nucleic acid detection (test sample C1); the remaining sample is placed in an ultrasonic disrupter and ultrasonicated at 300W for 15 min, then take 400 μl sample for Mycobacterium tuberculosis nucleic acid detection (test sample C2); the detection results of test sample C1 and test sample C2 are shown in Figure 4 and Figure 5 respectively;

[0057] 3) To sample D, add an equal volume of 4% sodium hydroxide solution and mix well, incubate at room temperature for 20 min, then centrifuge 1 mL sample, the precipitate after washing is used for Mycobacterium tuberculosis nucleic acid detection according to the kit instructions (test sample D1); the detection result of test sample D1 is shown in Figure 6 ;

[0058] According to the results shown in Figures 4-6 , it can be seen that when using the sputum liquefaction method provided by the present application to prepare test samples for Mycobacterium tuberculosis nucleic acid detection, for some sputum samples (for example, sputum samples with high viscosity or samples with low pathogen content), the test sample C1 still cannot release a detectable amount of pathogen nucleic acid (see Figure 4 , the detection result is negative), and ultrasonic treatment is needed to release a detectable amount of nucleic acid from the pathogen in the sputum sample (test sample C2) (see Figure 5 , the detection result is positive), so for such sputum samples, chemical (sputum liquefaction reagent) and physical treatment (ultrasonic treatment after or at the same time as high-temperature treatment, and preferably simultaneous high-temperature treatment and ultrasonic treatment, which can effectively save the time of the treatment process) need to be combined to liquefy the sputum, in order to improve the positive detection rate, the three (liquefaction reagent + high temperature + ultrasonic) synergistically achieve complete liquefaction of the sputum and release of nucleic acid from the pathogen, and at the same time long-term protection of pathogen nucleic acid from degradation. However, the detection result of the test sample (D1) obtained by using 4% sodium hydroxide solution to digest the sputum sample with high viscosity in the prior art is negative (see Figure 6 ), so the detection result is unreliable, and it takes a long time and needs complex operations such as centrifugation, Mycobacterium tuberculosis nucleic acid precipitation enrichment and nucleic acid extraction.

[0059] Example 3: Sputum liquefaction method

[0060] The sputum liquefaction method provided by the embodiment comprises the following steps:

[0061] 1) Prepare 20 sputum samples from patients positive for Mycobacterium tuberculosis (collected from Shanghai Pulmonary Hospital, with signed informed consent), evenly mix the samples, and divide the mixed sample into three parts: sample E, sample F and sample G, and place them in test tubes;

[0062] 2) Add an equal volume of sputum liquefaction reagent 1 (containing 4M guanidine hydrochloride, 0.3% sodium dodecyl sulfate (SDS), 0.5% N-acetyl cysteine, 0.1% DTT, 20mM Tris-HCl, 0.3mM EDTA) to sample E, mix well, and then divide the mixed sample into two parts (E1 and E2), wherein the E1 sample is placed in a 95°C boiling water bath for 25 min, and 400 μl of the sample is taken for Mycobacterium tuberculosis nucleic acid detection (test sample E11) ; the E2 sample is placed in an ultrasonic cell disruptor, boiled at 95°C for 25 min, and simultaneously ultrasonically treated at 300W for 10 min, and 400 μl of the sample is taken for Mycobacterium tuberculosis nucleic acid detection (test sample E21), and the detection results of test samples E11 and E21 are shown in Figure 7 and 8

[0063] 3) Add an equal volume of liquefaction reagent 2 (containing 4M guanidine hydrochloride, 0.5% N-acetyl cysteine, 0.1% DTT, 20mM Tris-HCl, 0.3mM EDTA) to sample F, mix well, and then divide the mixed sample into two parts (F1 and F2), wherein the F1 sample is placed in a 95°C boiling water bath for 25 min, and 400 μl of the sample is taken for Mycobacterium tuberculosis nucleic acid detection (test sample F11) ; the F2 sample is placed in an ultrasonic cell disruptor, boiled at 95°C for 25 min, and simultaneously ultrasonically treated at 300W for 10 min, and 400 μl of the sample is taken for Mycobacterium tuberculosis nucleic acid detection (test sample F21), and the detection results of test samples F11 and F21 are shown in Figure 9 and 10

[0064] 4) Add an equal volume of sputum liquefaction reagent 3 (containing 0.3% sodium dodecyl sulfate (SDS), 0.5% N-acetyl cysteine, 0.1% DTT, 20mM Tris-HCl, 0.3mM EDTA) to sample G, mix well, for example, by shaking or mixing on a sample mixer, and then divide the mixed sample into two parts (G1 and G2), wherein the G1 sample is placed in a 95°C boiling water bath for 25 min, and 400 μl of the sample is taken for Mycobacterium tuberculosis nucleic acid detection (test sample G11) ; the G2 sample is placed in an ultrasonic cell disruptor, boiled at 95°C for 25 min, and simultaneously ultrasonically treated at 300W for 10 min, and 400 μl of the sample is taken for Mycobacterium tuberculosis nucleic acid detection (test sample G21), and the detection results of test samples G11 and G21 are shown in​​Figure 11 and 12 as shown in the formula (I) ;

[0065] According to Figure 7 and Figure 8 It can be seen from the results that, first, the sputum liquefaction reagent 1 provided by the present application is mixed with sputum, and then high-temperature treatment is performed (test sample E11), and the combination of the two can fully liquefy the sputum and release the nucleic acid of the pathogen in the sputum, and the sputum liquefaction reagent used at the same time can effectively protect the released nucleic acid from being degraded, and the amplification curve of the nucleic acid detection has a relatively high relative fluorescence intensity (see Figure 7 ). After the sputum liquefaction reagent 1 provided by the present application is mixed with sputum, synchronous operation of high-temperature treatment and ultrasonic treatment is performed (test sample E21), which will further promote the liquefaction of the sputum and the release of the nucleic acid in the pathogen, and effectively protect the released nucleic acid from being degraded, and the amplification curve of the nucleic acid detection has a higher relative fluorescence intensity (see Figure 8 ), thus having a higher positive detection sensitivity. It can be seen that the sputum liquefaction method provided by the present application can help to detect lower concentrations of pathogens in sputum, thereby effectively improving the positive detection rate.

[0066] According to Figure 9 and Figure 10 It can be seen from the results that, first, the sputum liquefaction reagent 2 (which does not contain a detergent relative to the sputum liquefaction reagent 1 provided by the present application) is mixed with sputum, and then high-temperature treatment is performed (test sample F11), which can also liquefy the sputum and release the nucleic acid in the pathogen, but cannot effectively protect the released nucleic acid from being degraded, thus the relative fluorescence intensity of the amplification curve of the nucleic acid detection result is low (see Figure 9 ), and the corresponding positive detection sensitivity is also low. In addition, although the scheme of first mixing the sputum liquefaction reagent 2 with sputum and then synchronously performing high-temperature treatment and ultrasonic treatment (test sample F21) can further promote the release of the nucleic acid in the pathogen, the sputum liquefaction reagent 2 used cannot effectively protect the released nucleic acid, which will cause a large amount of degradation of the nucleic acid, thus causing the fluorescence intensity of the amplification curve of the detection result to be significantly lower than that of Figure 7 and Figure 8 (see Figure 10 ), and the corresponding positive detection sensitivity is also significantly lower.

[0067] According to Figure 11 and Figure 12 It can be seen from the results that, first, the sputum liquefaction reagent 3 (which does not contain guanidine salt relative to the sputum liquefaction reagent 1 provided by the present application) is mixed with sputum, and then high-temperature treatment is performed, and the nucleic acid detection result of the obtained test sample (G11) is negative (see Figure 11), it can be seen that the sputum liquefaction reagent 3 cannot protect the nucleic acid released by the pathogen, and the released nucleic acid is substantially completely degraded. In addition, the nucleic acid detection result of the sample to be tested (G21) obtained by the scheme of mixing the sputum liquefaction reagent 3 with the sputum first and then simultaneously performing high-temperature treatment and ultrasonic treatment is positive, but the relative fluorescence intensity of the amplification curve of the detection result is very low (see Figure 12 ), it can be seen that the sputum liquefaction reagent 3 cannot protect the nucleic acid released by the pathogen, and the released nucleic acid is substantially completely degraded. In addition, the nucleic acid detection result of the sample to be tested (G21) obtained by the scheme of mixing the sputum liquefaction reagent 3 with the sputum first and then simultaneously performing high-temperature treatment and ultrasonic treatment is positive, but the relative fluorescence intensity of the amplification curve of the detection result is very low (see

[0068] From the above results of the examples, it can be seen that in the sputum liquefaction reagent provided by the present application, although only a low concentration of guanidine salt, detergent and sulfhydryl reducing agent, etc. are included, and the effective components are very simple, but in the case of cooperating with high-temperature treatment or cooperating with the combined treatment of high-temperature and ultrasonic, one-step operation is enough to make the sputum completely liquefied, and promote the nucleic acid of the pathogen (such as Mycobacterium tuberculosis) in the sputum sample to be released into the sputum liquefaction solution in large quantities, and the time consumption is short and the operation is simple. At the same time, the low concentration of guanidine salt and detergent in the sputum liquefaction reagent synergistically act, which can effectively protect the nucleic acid released into the sputum liquefaction solution from being degraded or being degraded little, so that the positive detection rate can be significantly improved, and it is especially suitable for the liquefaction of the sputum sample with low concentration of Mycobacterium tuberculosis or high viscosity sputum sample.

[0069] Finally, it should be noted that: the above only describes the preferred embodiments of the present application and is not used to limit the present application, although the present application has been described in detail with reference to the foregoing embodiments, and for those skilled in the art, the technical solutions recorded in the foregoing embodiments can be modified, or some technical features can be replaced equivalently. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for sputum liquefaction, comprising the following steps: 1) mixing a sputum liquefaction reagent with a sputum sample to obtain a mixture; 2) subjecting the mixture to high temperature treatment at 90-100℃ for 10-30 min; wherein the sputum liquefaction reagent comprises 3-5 M guanidine salt, 0.1-0.5% detergent, 0.1-3% thiol reducing agent, 10-100 mM Tris-HCl, 0.1-0.5 mM EDTA, and pH≤7.5; wherein the detergent is selected from one or more of sodium dodecyl sulfate and lithium dodecyl sulfate, and the thiol reducing agent is selected from one or more of cysteine, N-acetyl cysteine and DTT.

2. The method of claim 1, wherein, The guanidine salt is selected from one or more of guanidine hydrochloride, guanidine isothiocyanate, guanidine sulfate and guanidine carbonate.

3. The method of claim 1, wherein, The thiol reducing agent is a combination of N-acetyl cysteine and DTT, wherein the concentration of N-acetyl cysteine is 0.3-3% and the concentration of DTT is 0.1-0.3%.

4. The method according to any one of claims 1-3, characterized in that, The method further comprises the following step: 3) simultaneously or subsequently to the high temperature treatment, subjecting the mixture to ultrasonic treatment at a power of 200-400 W for 10-20 min.

5. The method according to any one of claims 1-3, characterized in that, In step 1), the volume ratio of the sputum liquefaction reagent to the sputum sample is (1-3):

1. 6.A sputum liquefaction reagent for use in the method of any one of claims 1-5, comprising: 3-5 M guanidine salt, 0.1-0.5% detergent, 0.1-3% thiol reducing agent, 10-100 mM Tris-HCl, 0.1-0.5 mM EDTA, and pH≤7.5; wherein the detergent is selected from one or more of sodium dodecyl sulfate and lithium dodecyl sulfate, and the thiol reducing agent is selected from one or more of cysteine, N-acetyl cysteine and DTT.

7. The sputum liquefaction reagent according to claim 6, wherein, The guanidine salt is selected from one or more of guanidine hydrochloride, guanidine isothiocyanate, guanidine sulfate and guanidine carbonate. 8.A pathogen nucleic acid detection kit comprising the sputum liquefaction reagent of claim 6 or 7.

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