Mycobacterium tuberculosis detection system, detection method and application

Abstract

The invention belongs to the technical field of biology, and discloses a detection system and a detection method of mycobacterium tuberculosis and application. In the mycobacterium tuberculosis detection system provided by the invention, magnesium chloride, magnesium sulfate, polyethylene glycol, lysozyme, betaine, lecithin and alkyl trimethyl ammonium bromide are compounded according to the mass ratio of (2-4): (3-5): (40-3000): (0.05-0.2): (650-2500): (50-300): (1-250) to obtain a PCR (Polymerase Chain Reaction) reaction solution, and the PCR reaction solution has excellent splitting capacity on mycobacterium tuberculosis; according to the present invention, in the PCR reaction chamber provided with the magnesium oxide and silver oxide introduced anti-interference layer, the PCR reaction liquid can effectively inhibit the inhibition effect of the mycobacterium tuberculosis cell lysate on the PCR reaction, and can enhance the specific amplification so as to obtain the detection result with characteristics of high reliability and high accuracy; and the kit has a good application prospect in rapid clinical detection of tuberculosis.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a detection system and method for Mycobacterium tuberculosis and applications. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis. Currently, the main methods for detecting TB are microscopy and culture. These methods have drawbacks such as low sensitivity, inability to detect drug resistance, and high sample quality requirements, making them unsuitable for rapid clinical TB testing.

[0003] In vitro nucleic acid amplification technology refers to the technology of amplifying specific DNA or RNA fragments under in vitro conditions. It is widely used in scientific research, medicine, food science and other fields. Among them, polymerase chain reaction (PCR) is one of the most widely studied and used in vitro nucleic acid amplification technologies. It has the advantages of high sensitivity, strong specificity and short detection time, and shows good application potential in the rapid clinical detection of tuberculosis. However, because the cell wall of Mycobacterium tuberculosis is thick and rich in lipids, it is difficult to lyse it with conventional lysis buffers or extraction buffers for DNA extraction. The DNA extraction rate is low, and the extracted samples also contain lipids, polysaccharides and other substances, which will have a significant impact on the test results and lead to false positives and false negatives. Therefore, it needs to be further purified, which makes sample processing complicated and has significant limitations. Summary of the Invention

[0004] The first purpose of the present invention is to solve the problems of low DNA extraction efficiency and complex sample processing in the application of existing PCR technology in tuberculosis detection, and to provide a detection system for Mycobacterium tuberculosis.

[0005] The second object of the present invention is to provide a method for detecting Mycobacterium tuberculosis.

[0006] A third object of the present invention is to provide a use of the above-mentioned detection system for Mycobacterium tuberculosis in detecting Mycobacterium tuberculosis for non-diagnostic purposes.

[0007] Specifically, the detection system for Mycobacterium tuberculosis provided by the present invention includes: a PCR detection device, which is used to provide a reaction space for PCR amplification, the PCR detection device includes a PCR reaction chamber, the inner wall of the PCR reaction chamber is provided with an anti-interference layer, the anti-interference layer includes polyolefin, magnesium oxide and silver oxide in a mass ratio of (100-300):(10-20):1; a PCR reaction solution, which is used to provide biochemical conditions for PCR amplification, the PCR reaction solution includes magnesium chloride, magnesium sulfate, polyethylene glycol, lysozyme, betaine, lecithin and alkyltrimethylammonium bromide in a mass ratio of (2-4):(3-5):(40-3000):(0.05-0.2):(650-2500):(50-300):(1-250).

[0008] Furthermore, the polyolefin is selected from one or more of polyethylene, polypropylene and polybutylene.

[0009] Furthermore, the weight average molecular weight of the polyolefin is 100 kDa to 300 kDa.

[0010] Furthermore, the preparation of the anti-interference layer includes: taking the polyolefin, magnesium oxide and silver oxide, performing melting treatment and cooling molding to form the anti-interference layer.

[0011] Furthermore, the anti-interference layer has a thickness of 1 mm to 5 mm.

[0012] Furthermore, the temperature of the melting treatment is 200° C. to 300° C., and the time is 10s to 60s.

[0013] Furthermore, the cooling molding temperature is 4° C. to 8° C., and the time is 30s to 120s.

[0014] Furthermore, the weight average molecular weight of the polyethylene glycol is 1000Da to 6000Da.

[0015] Furthermore, the alkyltrimethylammonium bromide is selected from one or more of dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide and octadecyltrimethylammonium bromide.

[0016] Furthermore, based on the total volume of the PCR reaction solution, the concentration of the magnesium chloride is 0.5 to 1.8 mM, the concentration of the magnesium sulfate is 0.5 mM to 2 mM, the concentration of the polyethylene glycol is 4 mM to 20 mM, the concentration of the lysozyme is 1.5 μg / mL to 7.5 μg / mL, the concentration of the betaine is 0.1 M to 0.6 M, the concentration of the lecithin is 0.2 wt% to 0.8 wt%, and the concentration of the alkyltrimethylammonium bromide is 0.13 wt% to 0.6 wt%.

[0017] Furthermore, the detection system includes PCR reagents, which include UNG enzyme, Taq enzyme and dNTPs, and the dNTPs include dATP, dUTP, dCTP and dGTP in a molar ratio of (0.9-1.1):(0.9-1.1):(0.9-1.1):(0.9-1.1).

[0018] Furthermore, in the PCR amplification reaction system, the concentration of the UNG enzyme is 0.4 mU / μL to 3.5 mU / μL, the concentration of the Taq enzyme is 0.01 U / μL to 0.2 U / μL, and the concentration of the dNTP is 0.01 mM to 1.5 mM.

[0019] Furthermore, the PCR reaction solution includes a primer-probe composition, which includes a probe, a forward primer and a reverse primer; the nucleotide sequence of the probe is shown in SEQ ID NO: 1, the nucleotide sequence of the forward primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 3.

[0020] Furthermore, in the PCR amplification reaction system, the concentration of the probe is 0.3 μM to 2.8 μM, the concentration of the forward primer is 0.3 μM to 2.8 μM, and the concentration of the reverse primer is 0.3 μM to 2.8 μM.

[0021] Furthermore, the detection device includes: a main body, which includes a first part and a second part that are connected, the first part is structured to form a sample loading chamber, and the second part is structured to form a PCR reaction chamber and an overflow part, and the overflow part is arranged on the side of the second part close to the first part; a puncture part is provided in the PCR reaction chamber; an injector, which is movably arranged in the main body, and the injector is structured to form a mixing chamber and a microfluidic channel that are connected; the mixing chamber is arranged in the PCR reaction chamber, and the mixing chamber can be destroyed by contact with the puncture part; the microfluidic channel passes through the overflow part, and the end of the microfluidic channel away from the mixing chamber is arranged in the sample loading chamber and is connected to the sample loading chamber; a sample loading column, which is inserted in the mixing chamber, and the sample loading column drives the injector to move in a direction close to the PCR reaction chamber, and the sample loading column, injector and qPCR reaction chamber can enclose to form a closed PCR amplification reaction space.

[0022] The Mycobacterium tuberculosis detection method provided by the present invention uses the Mycobacterium tuberculosis detection system to detect a sample to be tested, mixes the sample to be tested with a PCR reaction solution, performs PCR amplification in the PCR reaction chamber, and obtains a detection result.

[0023] Furthermore, the added volume ratio of the sample to be tested and the PCR reaction solution is (0.2-0.6):1.

[0024] Furthermore, the PCR amplification reaction procedure includes pretreatment, pre-denaturation and chain extension performed in sequence; the pretreatment temperature is 45°C to 55°C, and the time is 10s to 20s; the pre-denaturation temperature is 93°C to 97°C, and the time is 1s to 3s; the chain extension includes alternating denaturation and annealing, the denaturation temperature is 93°C to 97°C, the denaturation time is 1s to 5s, the annealing temperature is 58°C to 62°C, the annealing time is 6s to 10s, and the number of alternating cycles of denaturation and annealing is 30 to 40.

[0025] The present invention also provides the use of the above-mentioned Mycobacterium tuberculosis detection system in detecting Mycobacterium tuberculosis for non-diagnostic purposes.

[0026] Beneficial effects:

[0027] In the detection system of Mycobacterium tuberculosis provided by the present invention, a PCR reaction chamber provided with an anti-interference layer is used as a reaction space for PCR amplification, and a PCR reaction solution including magnesium chloride, magnesium sulfate, polyethylene glycol, lysozyme, betaine, lecithin and alkyltrimethylammonium bromide in a specific mass ratio is used to treat the sample to be tested, thereby fully releasing the genomic DNA of Mycobacterium tuberculosis and providing a biochemical environment for PCR amplification. More specifically, the PCR reaction solution can effectively destroy the outer structure of Mycobacterium tuberculosis under simple mixing conditions, fully free and release DNA, and has excellent DNA extraction efficiency. It can also work in conjunction with a PCR reaction chamber with an anti-interference layer to effectively inhibit the PCR inhibition of substances such as mycolic acid, protein and polysaccharide contained in the sample to be tested, enhance specific amplification and reduce non-specific amplification, so as to obtain detection results with high reliability and high accuracy. It effectively solves the problems of low extraction rate of Mycobacterium tuberculosis genomic DNA, high extraction operation requirements, and false positives and false negatives caused by complex interfering substance components in existing tuberculosis detection, so as to achieve rapid and accurate detection of Mycobacterium tuberculosis. It can be well applied to rapid clinical detection of tuberculosis and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic structural diagram of the Mycobacterium tuberculosis detection device provided in Example 1 of the present invention;

[0029] Figure 2 for Figure 1 Cross-sectional view of AA in the figure. DETAILED DESCRIPTION

[0030] Based on a profound understanding of the problems existing in existing molecular detection technologies for Mycobacterium tuberculosis, the inventors of the present invention, after extensive and in-depth research and numerous experiments, have creatively discovered that a PCR reaction solution prepared by compounding magnesium chloride, magnesium sulfate, polyethylene glycol, lysozyme, betaine, lecithin, and alkyltrimethylammonium bromide in specific proportions has excellent lysis ability against Mycobacterium tuberculosis, effectively destroying the integrity of the cell wall and cell membrane of Mycobacterium tuberculosis to release cellular contents, while also further freeing DNA to fully release Mycobacterium tuberculosis genomic DNA. Furthermore, when PCR amplification is performed in a PCR reaction chamber with magnesium oxide and silver oxide introduced into the inner wall, the PCR reaction solution can effectively suppress the inhibitory effect of Mycobacterium tuberculosis cell lysate on the PCR reaction, allowing the processed sample to be directly PCR amplified without the need for additional purification treatment, which can well meet the needs of rapid clinical detection of tuberculosis. Based on this, the technical solution of the present invention was obtained.

[0031] The Mycobacterium tuberculosis detection system provided by the present invention comprises a PCR reaction solution and a PCR detection device.

[0032] In the present invention, the PCR reaction solution is used to provide biochemical conditions for PCR amplification, which specifically includes magnesium chloride, magnesium sulfate, polyethylene glycol, lysozyme, betaine, lecithin and alkyltrimethylammonium bromide in a mass ratio of (2-4):(3-5):(40-3000):(0.05-0.2):(650-2500):(50-300):(1-250), which can specifically be 2:3:40:0.05:650:50:1, 3:3:1000:0.1:1500:200:100, 4:5:3000:0.2:2500:80:1 or any value therebetween.

[0033] In some specific embodiments, the weight average molecular weight of the polyethylene glycol is preferably 1000Da to 6000Da, and specifically can be 1000Da, 2000Da, 3000Da, 4000Da, 5000Da, 6000Da or any value therebetween.

[0034] In some specific embodiments, specific examples of the alkyltrimethylammonium bromide include, but are not limited to, one or more of dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, and octadecyltrimethylammonium bromide.

[0035] In some specific embodiments, based on the total volume of the PCR reaction solution, the concentration of the magnesium chloride is preferably 0.5mM to 1.8mM, specifically 0.5mM, 0.7mM, 0.9mM, 1.3mM, 1.5mM, 1.8mM or any value therebetween; the concentration of the magnesium sulfate is preferably 0.5mM to 2mM, specifically 0.5mM, 0.7mM, 0.8mM, 1.2mM, 1.5mM, 2mM or any value therebetween; the concentration of the polyethylene glycol is preferably 4mM to 20mM, specifically 4mM, 6mM, 8mM, 10mM, 15mM, 18mM, 20mM or any value therebetween; the concentration of the lysozyme is preferably 1.5μg / mL to 7.5μg / mL, specifically 1.5μg / mL, 1 .8μg / mL, 2.3μg / mL, 2.5μg / mL, 3μg / mL, 5μg / mL, 7μg / mL, 7.5μg / mL or any value therebetween; the concentration of the betaine is preferably 0.1M to 0.6M, specifically 0.1M, 0.15M, 0.2M, 0.4M, 0.6M or any value therebetween; the concentration of the lecithin is preferably 0.2wt% to 0.8wt%, specifically 0.2wt%, 0.4wt%, 0.6wt%, 0.8wt% or any value therebetween; the concentration of the alkyltrimethylammonium bromide is preferably 0.13wt% to 0.6wt%, specifically 0.13wt%, 0.15wt%, 0.2wt%, 0.3wt%, 0.5wt%, 0.6wt% or any value therebetween.

[0036] In the present invention, the PCR detection device is used to provide a reaction space for PCR amplification, which includes at least a PCR reaction chamber, and the inner wall of the PCR reaction chamber is provided with an anti-interference layer, and the anti-interference layer includes polyolefin, magnesium oxide and silver oxide in a mass ratio of (100-300):(10-20):1, specifically 100:10:1, 100:13:1, 150:20:1, 300:20:1 or any value therebetween.

[0037] In some specific embodiments, specific examples of the polyolefin include, but are not limited to, one or more of polyethylene, polypropylene, and polybutylene.

[0038] In some specific embodiments, the weight average molecular weight of the polyolefin is preferably 100 kDa to 300 kDa, and specifically can be 100 kDa, 115 kDa, 130 kDa, 150 kDa, 180 kDa, 200 kDa, 250 kDa, 300 kDa or any value therebetween.

[0039] In some specific embodiments, the thickness of the anti-interference layer is preferably 1 mm to 5 mm, and specifically can be 1 mm, 1.5 mm, 2 mm, 3 mm, 4 mm, 5 mm or any value therebetween.

[0040] In the present invention, the method of forming the anti-interference layer inside the PCR reaction chamber can be an integrated molding process or a split manufacturing process. Those skilled in the art can make adaptive choices based on actual needs, and the present invention does not impose any special limitations on it.

[0041] In some specific embodiments, the preparation of the anti-interference layer preferably includes: taking the polyolefin, magnesium oxide and silver oxide for melt treatment and cooling molding to form the anti-interference layer. More specifically, the conditions of the melt treatment include a temperature of preferably 200°C to 300°C, specifically 200°C, 230°C, 250°C, 280°C, 300°C or any value therebetween; a time of preferably 10s to 60s, specifically 10s, 15s, 20s, 35s, 40s, 45s, 60s or any value therebetween. The conditions of the cooling molding include a temperature of preferably 4°C to 8°C, specifically 4°C, 4.5°C, 5°C, 6°C, 7°C, 8°C or any value therebetween; a time of preferably 30s to 120s, such as 30s, 35s, 40s, 50s, 60s, 80s, 100s, 120s or any value therebetween.

[0042] In some specific embodiments, the structure of the detection device preferably includes: a main body, which includes a first part and a second part that are connected, the first part is structured to form a sample loading chamber, and the second part is structured to form a PCR reaction chamber and an overflow part, and the overflow part is arranged on the side of the second part close to the first part; a puncture part is arranged in the PCR reaction chamber; an injector, which is movably arranged in the main body, and the injector is structured to form a mixing chamber and a microfluidic channel that are connected; the mixing chamber is arranged in the PCR reaction chamber, and the mixing chamber can be destroyed by contact with the puncture part; the microfluidic channel passes through the overflow part, and the end of the microfluidic channel away from the mixing chamber is arranged in the sample loading chamber and is connected to the sample loading chamber; a sample loading column, which is inserted in the mixing chamber, and the sample loading column drives the injector to move in a direction close to the PCR reaction chamber, and the sample loading column, injector and qPCR reaction chamber can enclose to form a closed PCR amplification reaction space.

[0043] In the present invention, the Mycobacterium tuberculosis detection system preferably further comprises a PCR reagent, wherein the PCR reagent specifically comprises UNG enzyme, Taq enzyme and dNTP, and the dNTP comprises dATP, dUTP, dCTP and dGTP in a molar ratio of (0.9-1.1):(0.9-1.1):(0.9-1.1):(0.9-1.1).

[0044] In some specific embodiments, in the PCR amplification reaction system, the concentration of the UNG enzyme is preferably 0.4 mU / μL to 3.5 mU / μL, specifically 0.4 mU / μL, 0.8 mU / μL, 1 mU / μL, 1.3 mU / μL, 1.5 mU / μL, 2 mU / μL, 2.5 mU / μL, 3 mU / μL, 3.5 mU / μL or any value therebetween; the concentration of the Taq enzyme is preferably 0.01 U / μL to 0.2 U / The concentration of the dNTPs is preferably 0.01 mM to 1.5 mM, specifically 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.45 mM, 0.6 mM, 0.9 mM, 1 mM, 1.3 mM, 1.5 mM or any value therebetween.

[0045] In the present invention, the PCR reagent preferably also includes a primer-probe combination, which specifically includes a probe, a forward primer and a reverse primer; the nucleotide sequence of the probe is shown in SEQ ID NO: 1, the nucleotide sequence of the forward primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 3.

[0046] In some specific embodiments, in the reaction system of PCR amplification, the concentration of the probe is preferably 0.3 μM to 2.8 μM, specifically 0.3 μM, 0.5 μM, 0.9 μM, 1 μM, 1.5 μM, 1.8 μM, 2 μM, 2.5 μM, 2.8 μM or any value therebetween; the concentration of the forward primer is preferably 0.3 μM to 2.8 μM, specifically 0.3 μM, 0.8 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 2.8 μM or any value therebetween; the concentration of the reverse primer is preferably 0.3 μM to 2.8 μM, specifically 0.3 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 2.8 μM or any value therebetween.

[0047] In the Mycobacterium tuberculosis detection method provided by the present invention, the Mycobacterium tuberculosis detection system described above is used to detect a sample to be tested, specifically comprising: mixing the sample to be tested with a PCR reaction solution, performing PCR amplification in the PCR reaction chamber, and obtaining a detection result.

[0048] In the present invention, simply mixing the PCR reaction solution with the sample to be tested can well achieve the sufficient release of Mycobacterium tuberculosis genomic DNA, and the resulting mixed solution can directly enter the PCR reaction chamber for PCR amplification to achieve the detection of Mycobacterium tuberculosis, which has the advantages of convenience and high efficiency.

[0049] In some specific embodiments, the added volume ratio of the test sample and the PCR reaction solution is preferably (0.2-0.6):1, specifically 0.2:1, 0.3:1, 0.45:1, 0.5:1, 0.6:1 or any value therebetween.

[0050] In some specific embodiments, the reaction procedure of the PCR amplification preferably includes pretreatment, pre-denaturation and chain extension performed in sequence. More specifically, the pretreatment conditions include a temperature of preferably 45°C to 55°C, specifically 45°C, 49°C, 50°C, 52°C, 55°C or any value therebetween; a time of preferably 10s to 20s, specifically 10s, 12s, 13s, 15s, 19s, 20s or any value therebetween. The pre-denaturation conditions include a temperature of preferably 93°C to 97°C, specifically 93°C, 93.5°C, 94°C, 95°C, 96°C, 97°C or any value therebetween; a time of preferably 1s to 3s, specifically 1s, 1.5s, 2s, 3s or any value therebetween. The chain extension specifically includes alternating denaturation and annealing; wherein the denaturation temperature is preferably 93°C to 97°C, specifically 93°C, 94°C, 95°C, 96°C, 97°C or any value therebetween; the denaturation time is preferably 1s to 5s, specifically 1s, 2s, 3s, 4s, 5s or any value therebetween; the annealing temperature is preferably 58°C to 62°C, specifically 58°C, 59°C, 60°C, 61°C, 62°C or any value therebetween; the annealing time is preferably 6s to 10s, specifically 6s, 7s, 8s, 9s, 10s or any value therebetween; and the number of alternating cycles of denaturation and annealing is preferably 30 to 40, specifically 30, 32, 34, 38, 40 or any integer value therebetween.

[0051] The present invention also provides the use of the above-mentioned Mycobacterium tuberculosis detection system in detecting Mycobacterium tuberculosis for non-diagnostic purposes.

[0052] The embodiments of the present invention are described in detail below. The examples of the embodiments are intended to explain the present invention and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0053] The samples and their acquisition methods involved in the following embodiments specifically include:

[0054] (1) Negative respiratory tract pharyngeal swab sample: Insert the swab completely from the mouth into the throat (or nasal cavity), pass the root of the tongue to the posterior pharyngeal wall or the back of the uvula, wipe the posterior pharyngeal wall and the tonsils on both sides with appropriate force, and take care to avoid touching the tongue, oral mucosa and saliva when removing the swab; insert the collected swab into 1 mL of 0.9% saline, rotate it close to the inner wall of the test tube for about 10 times, then squeeze the swab head along the inner wall of the sample preservation liquid tube, remove the swab, tighten the tube cap, and obtain the respiratory tract pharyngeal swab sample to be tested.

[0055] (2) Sputum negative sample: Collect 1 mL of sputum into a sputum bottle, cover it, gently mix the sputum sample, add 1 mL of sodium hydroxide solution and liquefy it for 30 minutes to obtain the sputum sample to be tested.

[0056] The reagents and their sources involved in the following examples specifically include:

[0057] dATP (Shengyi (Xiamen) Technology Co., Ltd., catalog number SJ765); dGTP (Shengyi (Xiamen) Technology Co., Ltd., catalog number SJ00189); dCTP (Shengyi (Xiamen) Technology Co., Ltd., catalog number SJ00188); dUTP (Shengyi (Xiamen) Technology Co., Ltd., catalog number SJ00182); UNG enzyme (Shengyi (Xiamen) Technology Co., Ltd., catalog number SJ277765); Taq enzyme (Anbang (Xiamen) Technology Co., Ltd., catalog number AOPT009); PEG6000 (Aladdin, catalog number P103725); lysozyme (Aladdin, catalog number L105521); betaine (Aladdin, catalog number B106221); lecithin (Aladdin, catalog number L105732).

[0058] Example 1

[0059] This embodiment is used to illustrate a detection device for Mycobacterium tuberculosis. Figure 1 and 2 The detection device includes a main body 1, a sample injector 2 and a sample adding column 3.

[0060] The main body 1 includes a first part 11 and a second part 12 that are detachably connected. The first part 11 is structured to form a sample loading chamber 4, and the cross-sectional area of the sample loading chamber 4 gradually decreases along the direction close to the second part 12 to form a guide portion, and the sample loading column 3 is inserted into the sample loading chamber 4 and is gap-fitted with the sample loading chamber 4. The second part 12 is structured to form a PCR reaction chamber 5 and an overflow chamber 6, and the overflow portion 6 is arranged on the side of the second part 12 close to the first part 11, and the sample loading chamber 4, the PCR reaction chamber 5 and the overflow chamber 6 are connected. The inner wall of the PCR reaction chamber 5 is structured to form a puncture portion 7, and an anti-interference layer 8 is provided on the inner wall of the reaction chamber 4.

[0061] The injector 2 is structured to form a interconnected mixing chamber 21 and microfluidic channel 22. The mixing chamber 21 is installed in the PCR reaction chamber 5. The microfluidic channel 22 extends away from the PCR reaction chamber 5 and passes through the overflow chamber 6. The end of the microfluidic channel 22 away from the mixing chamber 21 is located in the sample loading chamber 4 and is connected to the sample loading chamber 4. The microfluidic channel 22 has an equivalent channel diameter of 1 μm and a length of 25 μm. The total volume of the injector 2 is 50 μL, which means that the volume of solution entering the PCR reaction chamber 5 is 50 μL.

[0062] During the test, the sample to be tested is added to the sample loading chamber 4, and the sample loading column 3 drives the sample to be tested in the sample loading chamber 4 through the microfluidic channel 22 into the mixing chamber 21. The sample loading column 3 moves toward the direction close to the PCR reaction chamber 5 and then contacts the end of the microfluidic channel 22 located in the sample loading chamber 4. The sample loading chamber 4 seals the microfluidic channel 22 and the mixing chamber 21 to accurately control the volume of the sample to be tested that finally enters the PCR reaction chamber 5, and the sample loading column 3 continues to move to drive the injector 2 to move toward the direction close to the PCR reaction chamber 5, so that the mixing chamber 21 contacts the puncture part 7 and is destroyed, and the sample to be tested in the mixing chamber 21 is released into the PCR reaction chamber 5 for PCR amplification.

[0063] In this embodiment, the main body 1, the injector 2, and the sample addition column 3 are all made of polypropylene (weight-average molecular weight of 80,000 Da, the same below) as raw materials, melt-treated at 200°C for 60 seconds, then injected into a mold of corresponding shape, and cooled and formed at 4°C for 120 seconds. The method for forming the anti-interference layer 8 on the inner wall of the reaction chamber 4 includes: taking polypropylene, magnesium oxide, and silver oxide in a mass ratio of 200:4:1 and mixing them uniformly, melting them at 200°C for 60 seconds, injecting them into a mold of corresponding shape, and cooling and shaping them at 4°C for 30 seconds to obtain a film with a thickness of 4 mm. Then, the film is attached to the inner wall of the PCR reaction chamber 5 during the preparation of the second part 12 to prepare the anti-interference layer 8.

[0064] Example 2

[0065] The Mycobacterium tuberculosis detection device provided in this embodiment is basically the same as that in Example 1, except that, in the preparation of the anti-interference layer 8, the added mass ratio of polypropylene, magnesium oxide, and silver oxide is 100:10:1. Other conditions and structures are the same, and a Mycobacterium tuberculosis detection device is obtained.

[0066] Example 3

[0067] The Mycobacterium tuberculosis detection device provided in this embodiment is basically the same as that in Example 1, except that, in the preparation of the anti-interference layer 8, the added mass ratio of polypropylene, magnesium oxide, and silver oxide is 300:20:1. Other conditions and structures are the same, and a Mycobacterium tuberculosis detection device is obtained.

[0068] Comparative Example 1

[0069] The Mycobacterium tuberculosis detection device provided in this comparative example is basically the same as that in Example 1, except that, in the preparation of the anti-interference layer 8, polypropylene of equal mass is used instead of magnesium oxide and silver oxide. Other conditions and structures are the same, thereby obtaining a Mycobacterium tuberculosis detection device.

[0070] Comparative Example 2

[0071] The Mycobacterium tuberculosis detection device provided in this comparative example is basically the same as that in Example 1, except that, in the preparation of the anti-interference layer 8, an equal mass of magnesium oxide is used instead of silver oxide. Other conditions and structures are the same, and a Mycobacterium tuberculosis detection device is obtained.

[0072] Comparative Example 3

[0073] The Mycobacterium tuberculosis detection device provided in this comparative example is basically the same as that in Example 1, except that, in the preparation of the anti-interference layer 8, an equal mass of silver oxide is used instead of magnesium oxide. Other conditions and structures are the same, thereby obtaining a Mycobacterium tuberculosis detection device.

[0074] Example 4

[0075] This embodiment is used to illustrate a detection system and method for Mycobacterium tuberculosis, which specifically includes:

[0076] 1. Assembly of the Mycobacterium tuberculosis detection system

[0077] (1) According to the amount of each PCR reagent including 0.5 μmol of Tris-HCl (pH = 8.3), 0.075 μmol of dNTP (including dATP, dUTP, dCTP and dGTP with a molar ratio of 1:1:1:1), 0.1 nmol of probe, 0.1 nmol of forward primer, 0.1 nmol of reverse primer, 0.1 U of UNG enzyme and 5 U of Taq enzyme, Tris-HCl (pH = 8.3), dNTP, probe, forward primer and reverse primer were mixed and freeze-dried according to the procedure shown in Table 1 to obtain a PCR reagent.

[0078] Table 1.

[0079]

[0080]

[0081] (2) According to the formula shown in Table 2, each raw material was mixed with ddH2O to obtain a PCR reaction solution.

[0082] Table 2.

[0083]

[0084] (3) According to Table 3, the PCR reaction solution was added to the sample injector 2 of each Mycobacterium tuberculosis detection device, and the PCR reagent was added to the PCR reaction chamber 5 (one portion of the PCR reagent was added to each PCR reaction chamber 5) to assemble a Mycobacterium tuberculosis detection system.

[0085] Table 3.

[0086]

[0087]

[0088] 2. Positive detection rate of the Mycobacterium tuberculosis detection system

[0089] (1) According to Table 4, Mycobacterium tuberculosis was inoculated into a negative sputum sample to prepare a positive sample.

[0090] Table 4.

[0091]

[0092] (2) Positive samples, negative respiratory pharyngeal swab samples, and negative sputum samples were added to the Mycobacterium tuberculosis detection device, and PCR amplification was performed according to the procedure shown in Table 5 to obtain the CT value of each positive sample.

[0093] Table 5. qPCR amplification program

[0094]

[0095] Each group of samples was tested 20 times, and the positive detection rate (unit: %) was calculated according to the following formula. The test results are shown in Table 6.

[0096] Detection positive rate = positive samples / total number of samples × 100%

[0097] Among them, positive samples are judged based on the CT value. When the CT value is not greater than 38, it is judged as a positive sample, otherwise it is excluded.

[0098] Table 6.

[0099]

[0100]

[0101] As shown in Table 6, compared with control groups 1 to 7, the detection device provided by Examples 1 to 3 of the present invention, combined with any one of PCR reaction solution 1, PCR reaction solution 2, PCR reaction solution 3 or PCR reaction solution 4, has a good detection ability for Mycobacterium tuberculosis in the sample, and the positive detection rate for positive samples is higher than 95%. No positive results were detected in negative respiratory swab samples and sputum negative samples, and the detection sensitivity, specificity and accuracy were excellent.

[0102] 3. Anti-interference ability of the Mycobacterium tuberculosis detection system

[0103] (1) According to Table 7, each interfering substance was added to the positive sample 3 to prepare an interference test sample.

[0104] Table 7.

[0105] Group Interfering substances Addition concentration Group Interfering substances Addition concentration Interference sample 1 Interferon alpha 10 million U / mL Interference sample 9 Levofloxacin 500mg / mL Interference sample 2 Zanamivir 50mg / mL Interference sample 10 Azithromycin 1g / mL Interference sample 3 Ribavirin 2g / mL Interference sample 11 Ceftriaxone 2g / mL Interference sample 4 Oseltamivir 200mg / mL Interference sample 12 Mometasone 1mg / mL Interference sample 5 Peramivir 1g / mL Interference sample 13 Fluticasone 10 mg / mL Interference sample 6 Histamine hydrochloride 5mg / mL Interference sample 14 Phenylephrine 50mg / mL Interference sample 7 Ritonavir 250mg / mL Interference sample 15 Oxymetazoline 0.5 mg / mL Interference sample 8 Abidor 1g / mL Interference sample 16 Beclomethasone 2mg / mL

[0106] (2) Each interfering sample was added to the Mycobacterium tuberculosis detection device, and PCR amplification was performed according to the procedure shown in Table 5 to obtain the CT value of each positive sample; each group of samples was measured 20 times, and the positive detection rate of each group was calculated according to the method of "2. Positive detection rate of the Mycobacterium tuberculosis detection system", as shown in Table 8.

[0107] Table 8.

[0108]

[0109]

[0110] The test results shown in Table 8 show that compared with control groups 1 to 7, when the PCR reaction solution provided by the present invention is used in combination with a PCR reaction chamber provided with an anti-interference layer to detect interference samples, the interfering substances contained in the interference samples have no effect on the test results, and it has excellent anti-interference ability.

[0111] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A detection system for Mycobacterium tuberculosis, characterized in that: The detection system comprises: A PCR detection device for providing a reaction space for PCR amplification, the PCR detection device comprising a PCR reaction chamber, the inner wall of which is provided with an anti-interference layer, the anti-interference layer comprising polyolefin, magnesium oxide, and silver oxide in a mass ratio of (100-300):(10-20):1; A PCR reaction solution is used to provide biochemical conditions for PCR amplification. The PCR reaction solution includes magnesium chloride, magnesium sulfate, polyethylene glycol, lysozyme, betaine, lecithin and alkyltrimethylammonium bromide in a mass ratio of (2-4):(3-5):(40-3000):(0.05-0.2):(650-2500):(50-300):(1-250).

2. The Mycobacterium tuberculosis detection system according to claim 1, characterized in that: The polyolefin is selected from one or more of polyethylene, polypropylene and polybutylene; Optionally, the weight average molecular weight of the polyolefin is 100 kDa to 300 kDa.

3. The Mycobacterium tuberculosis detection system according to claim 1, characterized in that: The preparation of the anti-interference layer comprises: taking the polyolefin, magnesium oxide and silver oxide, performing melting treatment and cooling molding to form the anti-interference layer; Optionally, the anti-interference layer has a thickness of 1 mm to 5 mm; Optionally, the temperature of the melt treatment is 200° C. to 300° C., and the time is 10s to 60s; Optionally, the cooling molding temperature is 4° C. to 8° C., and the time is 30s to 120s.

4. The Mycobacterium tuberculosis detection system according to claim 1, characterized in that: The weight average molecular weight of the polyethylene glycol is 1000Da to 6000Da; Optionally, the alkyltrimethylammonium bromide is selected from one or more of dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide and octadecyltrimethylammonium bromide; Optionally, based on the total volume of the PCR reaction solution, the concentration of the magnesium chloride is 0.5 mM to 1.8 mM, the concentration of the magnesium sulfate is 0.5 mM to 2 mM, the concentration of the polyethylene glycol is 4 mM to 20 mM, the concentration of the lysozyme is 1.5 μg / mL to 7.5 μg / mL, the concentration of the betaine is 0.1 M to 0.6 M, the concentration of the lecithin is 0.2 wt% to 0.8 wt%, and the concentration of the alkyltrimethylammonium bromide is 0.13 wt% to 0.6 wt%.

5. The Mycobacterium tuberculosis detection system according to claim 1, characterized in that: The detection system includes a PCR reagent, wherein the PCR reagent includes UNG enzyme, Taq enzyme and dNTP, wherein the dNTP includes dATP, dUTP, dCTP and dGTP in a molar ratio of (0.9-1.1):(0.9-1.1):(0.9-1.1); Optionally, in the PCR amplification reaction system, the concentration of the UNG enzyme is 0.4 mU / μL to 3.5 mU / μL, the concentration of the Taq enzyme is 0.01 U / μL to 0.2 U / μL, and the concentration of the dNTP is 0.01 mM to 1.5 mM.

6. The Mycobacterium tuberculosis detection system according to claim 5, characterized in that: The PCR reagent includes a primer-probe combination, which includes a probe, a forward primer, and a reverse primer; the nucleotide sequence of the probe is shown in SEQ ID NO: 1, the nucleotide sequence of the forward primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 3; Optionally, in the PCR amplification reaction system, the concentration of the probe is 0.3 μM to 2.8 μM, the concentration of the forward primer is 0.3 μM to 2.8 μM, and the concentration of the reverse primer is 0.3 μM to 2.8 μM.

7. The Mycobacterium tuberculosis detection system according to claim 1, characterized in that: The detection device comprises: The main body includes a first portion and a second portion that are connected to each other, the first portion being structured to form a sample loading chamber, the second portion being structured to form a PCR reaction chamber and an overflow portion, the overflow portion being arranged on a side of the second portion close to the first portion; a puncture portion being arranged in the PCR reaction chamber; An injector is movably disposed within the main body, the injector being structured to form a mixing chamber and a microfluidic channel that are interconnected; the mixing chamber is disposed within the PCR reaction chamber and can be destroyed by contact with the puncture portion; the microfluidic channel passes through the overflow portion, and an end of the microfluidic channel remote from the mixing chamber is disposed within the sample loading chamber and is in communication with the sample loading chamber; The sample loading column is inserted into the mixing chamber. The sample loading column drives the injector to move in the direction close to the PCR reaction chamber. The sample loading column, the injector and the qPCR reaction chamber can enclose and form a closed PCR amplification reaction space.

8. A method for detecting Mycobacterium tuberculosis, characterized in that: The detection method uses the Mycobacterium tuberculosis detection system according to any one of claims 1 to 7 to detect the sample to be tested, and the detection method includes: mixing the sample to be tested with a PCR reaction solution, performing PCR amplification in the PCR reaction chamber, and obtaining the detection result.

9. The method for detecting Mycobacterium tuberculosis according to claim 8, characterized in that: The added volume ratio of the sample to be tested and the PCR reaction solution is (0.2-0.6):1; Optionally, the reaction procedure of the PCR amplification includes pretreatment, pre-denaturation and chain extension performed in sequence; the temperature of the pretreatment is 45°C to 55°C, and the time is 10s to 20s; the temperature of the pre-denaturation is 93°C to 97°C, and the time is 1s to 3s; the chain extension includes alternating denaturation and annealing, the denaturation temperature is 93°C to 97°C, the denaturation time is 1s to 5s, the annealing temperature is 58°C to 62°C, the annealing time is 6s to 10s, and the number of alternating cycles of denaturation and annealing is 30 to 40.

10. Use of the Mycobacterium tuberculosis detection system according to any one of claims 1 to 7 in detecting Mycobacterium tuberculosis for non-diagnostic purposes.

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