Method for one-step closed amplification of mycobacterium tuberculosis DNA by tongue swab

Abstract

This invention provides a one-step, fully enclosed method for amplifying Mycobacterium tuberculosis DNA using a tongue swab, belonging to the field of Mycobacterium tuberculosis nucleic acid detection technology. The method includes the following core steps: non-invasive sampling using a tongue swab; breaking off the swab tip into a sampling tube pre-filled with lysis buffer; connecting the sampling tube to a PCR tube containing a built-in lyophilized bulb to form a fully enclosed device; squeezing the sampling tube to mix the swab with the lysis buffer to release the Mycobacterium tuberculosis DNA; inverting the device to allow the mixture to drip into the PCR tube to dissolve the lyophilized bulb; finally, inserting the PCR tube into a detection instrument, and obtaining a positive or negative result directly within 30 minutes. This invention achieves non-invasive and convenient sampling, fully enclosed safety protection, simplified one-step operation, and rapid diagnosis, without relying on a biosafety level 2 laboratory or requiring specialized technical training for operators, effectively solving the problems of difficult sampling, slow detection, and high risk in grassroots tuberculosis screening.

Description

Technical Field

[0001] This invention belongs to the field of Mycobacterium tuberculosis nucleic acid detection technology, and more specifically, it relates to a one-step fully enclosed method for amplifying Mycobacterium tuberculosis DNA using a tongue swab. Background Technology

[0002] Tuberculosis is a serious infectious disease that threatens human health worldwide. Early detection, early diagnosis, and early treatment are key to controlling the spread of tuberculosis and reducing the disease burden. However, current mainstream clinical methods for detecting Mycobacterium tuberculosis have significant limitations and cannot meet the screening needs of primary healthcare institutions and special populations.

[0003] The existing detection technologies mainly have the following problems: Traditional sputum specimen testing methods are inefficient: sputum smear microscopy is a commonly used preliminary screening method in clinical practice, but its positive detection rate is only 30% to 40%, which easily leads to missed diagnoses due to the low detection rate; although sputum culture is the gold standard for diagnosis, the culture cycle is as long as 2 to 8 weeks, which seriously delays the early treatment of patients and cannot meet the needs of rapid screening.

[0004] Current molecular detection technologies have significant shortcomings: Mainstream technologies (such as those from brands like Zhishan, Youstar, Zhiwei, and Kunpeng) still primarily rely on sputum sampling—requiring subjects to actively cough up sputum, making it difficult for the elderly, frail individuals, children, and those who cannot expectorate to provide adequate sputum samples. For some individuals without sputum, invasive methods such as bronchoscopy are required, leading to poor patient compliance and posing a risk of trauma. Furthermore, existing molecular detection procedures are mostly open-access, easily generating aerosols containing Mycobacterium tuberculosis during sample processing, posing a high biosafety risk. Therefore, these procedures must be conducted in biosafety level 2 laboratories, limiting their widespread application in primary healthcare institutions (such as community health service centers and township hospitals).

[0005] A few attempts at swab testing have failed to overcome core bottlenecks: Currently, only a few companies are trying to use swabs to replace sputum samples for testing, but they have not yet successfully achieved "fully enclosed one-step" testing. The testing process still needs to be broken down into three independent steps: "sample extraction, nucleic acid amplification, and result interpretation." A single test takes more than 1.5 hours, the operation process is complex and the technical threshold is high, requiring operators to receive specialized training, which makes it difficult to meet the urgent needs of primary healthcare institutions for rapid and convenient screening technology.

[0006] In summary, existing tuberculosis detection technologies generally suffer from problems such as "limited sampling methods (invasive or dependent on specific specimens), high requirements for the testing environment (requiring a level 2 biosafety laboratory), and lengthy and complex procedures." There is an urgent need for a technical solution that can achieve non-invasive sampling, fully enclosed and safe testing, and rapid one-step operation to address the core pain points of tuberculosis screening at the grassroots level. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a one-step fully enclosed method for amplifying Mycobacterium tuberculosis DNA using a tongue swab, thereby resolving the technical issues of limited sampling methods and poor applicability to different populations.

[0008] A one-step, fully blocked method for amplifying Mycobacterium tuberculosis DNA using a tongue swab includes the following steps: 1) Samples were taken using a tongue swab; 2) Break off the tongue swab head after sampling and place it inside the sampling tube; 3) Take the PCR tube with the built-in lyophilized bulb and detachably connect the PCR tube to the sampling tube; 4) Squeeze the bottom of the sampling tube to mix the tongue swab inside the sampling tube with the lysis solution; 5) Invert the connected sampling tube and PCR tube, and squeeze the sampling tube again to allow the mixed lysis buffer inside the tube to drip into the PCR tube to dissolve the lyophilized bulbs inside the PCR tube. 6) Insert the PCR tube after dissolving the lyophilized bulbs into the detector, let it stand for the preset time, and then interpret the results.

[0009] Preferably, the tongue swab described in step 1) is suitable for people of all ages and special populations, has no contraindications for sampling, and can be used for sampling at any time.

[0010] Preferably, the lyophilized bulbs in the PCR tube in step 3) contain reagents that enable sample processing, nucleic acid purification, and isothermal amplification.

[0011] Preferably, in step 3), the sampling tube is connected to the PCR tube to form a fully enclosed structure, which can prevent the environment, medical staff and screening subjects from being contaminated and reduce the risk of cross-infection. In step 4), squeezing the sampling tube mixes the tongue swab with the lysis buffer, which is used to dissolve the Mycobacterium tuberculosis attached to the tongue swab and release the Mycobacterium tuberculosis DNA.

[0012] Preferably, in step 6), the detector outputs the detection result of Mycobacterium tuberculosis DNA by displaying a positive "+" or a negative "-". The preset time in step 6) is 30 minutes, that is, the result is interpreted within 30 minutes (including 30 minutes) after the PCR tube is inserted into the detector. After the tongue swab head is broken off into the sampling tube in step 2), the sampling tube is in a sealed state and no separate pretreatment operation is required for the tongue swab head.

[0013] Preferably, the entire testing process does not rely on a biosafety level 2 laboratory, and operators do not need to receive specialized technical training; novices can quickly master the operating procedures.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention uses a tongue swab as a sampling carrier. The swab is made of medical-grade, non-irritating material and has no contraindications for sampling in any population—whether it is an infant, a frail elderly person, a patient with difficulty swallowing, or a patient who does not expectorate, all can tolerate the sampling procedure. Moreover, sampling does not require the subject to actively cough up phlegm, and can be carried out at any time in any scenario. This completely solves the problem of traditional testing techniques relying on sputum samples and requiring invasive sampling for those without sputum, and greatly improves subject compliance and sampling success rate.

[0015] This invention features a fully enclosed system from the moment of sampling: after the swab head breaks off into the sampling tube, the tube is immediately sealed; subsequently, it connects with a PCR tube to form an integrated closed device. The entire detection process (sample processing, nucleic acid purification, and isothermal amplification) is completed within this enclosed space, eliminating the need for any separate operations (such as sample processing, nucleic acid extraction and purification, and reagent preparation and addition in traditional techniques). This design minimizes the leakage of Mycobacterium tuberculosis aerosol, effectively protecting the testing environment, medical personnel, and test subjects from contamination, completely eliminating the risk of cross-infection, and allowing testing to be conducted without relying on a biosafety level 2 laboratory.

[0016] This invention integrates multiple steps, such as "sample processing (lysis to release DNA), nucleic acid purification, isothermal amplification, and fluorescence signal capture," into a fully enclosed device and detector. Operators only need to complete the simple operation of "sampling - breaking the swab - connecting the device - squeezing and mixing - inserting into the detector," without needing to master complex molecular biology experimental skills. Novices can complete the operation independently after a short period of guidance. Furthermore, no additional sample extraction equipment or purification reagents are required, which greatly reduces the technical operation threshold and equipment investment costs, making it fully suitable for the operational capabilities of primary healthcare institutions.

[0017] The entire detection process of this invention (from sampling to obtaining results) takes only 30 minutes. Compared with traditional sputum culture (2-8 weeks) and existing molecular detection (more than 1.5 hours), the detection efficiency is improved by 3-168 times, enabling "instant screening and instant interpretation", which saves critical time for early diagnosis and treatment of tuberculosis. At the same time, the instrument directly displays the results in the form of "positive (+)" or "negative (-)", without the need for operators to perform complex signal analysis or data interpretation, avoiding misdiagnosis or missed diagnosis due to interpretation errors.

[0018] This invention requires no biosafety level 2 laboratory, nor expensive professional equipment or specialized training. It only requires a basic testing instrument and disposable consumables to carry out testing. The equipment is small in size and low in cost, and can be widely deployed in community health service centers, township health centers, and grassroots network sites of disease control centers. It effectively solves the problem of "difficulty in popularizing" existing technologies at the grassroots level, and provides a feasible technical solution for the large-scale promotion of early diagnosis and treatment of tuberculosis. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the present invention. Detailed Implementation

[0020] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0021] Please see Figure 1 This invention provides a one-step fully enclosed method for amplifying Mycobacterium tuberculosis DNA using a tongue swab. It details the material selection, operating procedures, result interpretation, and implementation of the core technological advantages of this invention in conjunction with actual testing scenarios, ensuring that those skilled in the art can replicate the testing process of this invention based on this embodiment.

[0022] Preparations before implementation: Material and equipment specifications The implementation of this invention relies on specialized testing consumables and a universal testing instrument. All materials are designed around the core requirements of non-invasive sampling, fully enclosed testing, and rapid testing. Specific specifications are as follows: Tongue swab: Made of medical-grade nylon flocked swab, with a polypropylene swab handle featuring a breakable groove in the middle (the groove can withstand a weight of ≤5N, ensuring it can be broken manually without producing debris); swab head length 8-15mm, flocking density ≥180 strands / mm². 2 It can efficiently adsorb mucosal cells on the tongue and any Mycobacterium tuberculosis that may be present. It is non-irritating and suitable for special populations such as infants, the elderly, and those with difficulty swallowing. There are no contraindications for sampling.

[0023] Sampling tubes: Made of transparent polypropylene, with a capacity of 1.5-2 mL, pre-filled with 0.3-0.5 mL of lysis buffer (containing Tris-HCl buffer, EDTA, proteinase K, and surfactant; pH 8.0-8.5; rapidly dissolves the cell wall of Mycobacterium tuberculosis, releasing its DNA, while inhibiting nuclease activity to prevent DNA degradation). The tubes have two threads, an inner and an outer one. The inner thread (with a cross-shaped stop to prevent large particles from passing through) matches the tube body, while the outer thread (1-2 mm inner diameter, allowing only liquid passage) matches the inner thread of PCR tube C. All threaded connections have nitrile rubber sealing rings to ensure a liquid-tight seal after connection to the PCR tube.

[0024] PCR tubes: Made of high-temperature resistant (≥95℃) transparent polypropylene, with a tube capacity of 0.1-0.5mL. Each tube contains a pre-packaged lyophilized bulb (3-5mm in diameter, composed of nucleic acid purification reagent, isothermal amplification reagent, and fluorescent probe). The nucleic acid purification reagent contains silica beads that specifically bind to DNA; the isothermal amplification reagent contains working enzymes (Bst DNA polymerase, etc.), amplification raw materials (dNTPs, Mg2+, etc.), and amplification primers (designed for Mycobacterium tuberculosis-specific genes such as IS6110, IS1081, rpoB, 6S rRNA, etc.); the fluorescent probe contains FAM fluorescent groups that can bind to amplification products to generate fluorescent signals or generate other detectable FAM channel fluorescent signals. The PCR tube opening has an internal thread that matches the external thread of the sampling tube, and a nitrile rubber sealing ring is installed at the tube opening thread to ensure no liquid leakage when inserted into the detector.

[0025] The detector uses a real-time fluorescence isothermal amplification detector with a FAM channel (excitation wavelength 485nm, emission wavelength 520nm). It has a constant temperature control function, and the display screen can intuitively show "+" (positive) or "-" (negative) results. It can automatically stop heating after 30 minutes. The interpretation time is 30 minutes ("+" is displayed when the positive threshold is reached, and "-" is displayed when the positive threshold is not reached after 30 minutes). The instrument size is ≤30cm×20cm×15cm and the weight is ≤3kg. It is suitable for desktop use in primary healthcare institutions and does not require a professional laboratory environment.

[0026] Detailed operating procedures (full-process closed-loop control): The operation process of this invention strictly follows the logic of non-invasive sampling - sealing treatment - one-step amplification - rapid interpretation, consisting of 6 core steps. The entire process does not require opening the sealed structure, eliminating the risk of aerosol leakage. Details are as follows: Step 1: Non-invasive tongue swab sampling: The operator wears disposable gloves, takes out the sterile tongue swab (unopened), tears off the packaging paper, and holds the swab stick away from the swab head; Have the examinee tilt their head back slightly and open their mouth wide. The operator gently touches the middle and back 1 / 3 of the examinee's tongue with the swab head (avoiding contact with teeth and gums to reduce interference from impurities) and wipes in a clockwise direction for 5-8 circles. The wiping pressure should be such that the examinee does not feel pain. After sampling, remove the swab from the mouth, keeping the swab tip uncontaminated (avoid contact with clothing, tabletops, etc.), and prepare for subsequent procedures; Advantages of this procedure: Unlike traditional sputum sample collection, it does not require the subject to cough up sputum, nor does it require invasive procedures such as bronchoscopy. Infants and young children can have their heads held down by their guardians to complete the sampling, while the elderly or bedridden patients can complete the sampling while lying on their side, enabling sampling anytime, anywhere, and without contraindications.

[0027] Step 2: Seal the swab head into the sampling tube: Take out the sampling tube pre-filled with lysis solution, unscrew the inner interface of the sampling tube (the transparent tube cap of the sampling tube is threaded to match the outer interface of the sampling tube, keeping it closed), and align the tongue swab rod after sampling with the opening of the sampling tube, so that the swab head faces the inside of the tube; Pinch the easily broken groove of the swab stick with your thumb and forefinger, and forcefully break the swab stick towards the sampling tube, ensuring that the swab head falls completely into the sampling tube (after the swab stick breaks, discard the remaining stick into the medical waste bag); Immediately tighten the inner interface of the sampling tube to ensure a seal between the inner interface and the tube opening (tighten until there is no looseness). At this time, the swab head is immersed in the lysis solution, and the sampling tube is in a completely sealed state. No separate pretreatment of the swab head is required (such as centrifugation, filtration, etc.). Key function: After this step is completed, the sample enters a closed system, and all subsequent operations are carried out in a closed state, which avoids contamination caused by the swab head coming into contact with the air and also avoids the risk of operators coming into contact with the sample.

[0028] Step 3: Completely seal the connection between the sampling tube and the PCR tube: Take out the PCR tube pre-loaded with lyophilized bulbs, and make sure the PCR tube cap is tightened (to prevent the lyophilized bulbs from getting wet). Unscrew the PCR tube cap and place it on a sterile operating mat (with the inside of the cap facing up to avoid contamination). Align the outer interface of the sampling tube with the opening (uncapped end) of the PCR tube. Using the engagement of the internal and external threads, rotate the sampling tube clockwise until the sampling tube and the PCR tube are fully connected (keep the two tubes coaxial during rotation to avoid damaging the threads). After the connection is completed, check whether the nitrile rubber sealing ring at the connection of the two tubes fits tightly and ensures that there are no gaps (you can gently squeeze the sampling tube body and observe that there is no liquid leakage at the connection, which means that the seal is qualified). Structural features: This is a fully enclosed device with the sampling tube and PCR tube connected top to bottom. The entire enclosed system contains the sample (swab head), lysis buffer, and lyophilized bulbs. Subsequent reactions are all carried out within this enclosed system, without the need to transfer liquids.

[0029] Step 4: Mix the swab with the lysis buffer to release the DNA. The operator holds the sealed sampling tube and PCR tube (PCR tube on top, sampling tube on the bottom) and gently squeezes the sampling tube with the palm of their hand (the squeezing force should be enough to slightly deform the tube, and avoid excessive squeezing that could cause the tube to break). During the squeezing process, the lysis solution in the sampling tube will fully contact the swab head. The liquid flow generated by squeezing allows the lysis solution to penetrate into the flocking gaps of the swab head, dissolving the Mycobacterium tuberculosis cell wall adsorbed by the swab head and releasing the Mycobacterium tuberculosis DNA. Squeeze and gently shake the sampling tube continuously (shaking frequency 1-2 times / second, for 10-15 seconds) to ensure that the swab head is fully mixed with the lysis buffer and that the DNA is completely released into the lysis buffer; Reaction principle: Proteinase K in the lysis buffer degrades bacterial proteins, surfactants disrupt cell membranes, and Tris-HCl buffer maintains pH stability. Together, they achieve bacterial lysis and DNA release, preparing for subsequent purification and amplification.

[0030] Step 5: Transfer the lysis buffer and dissolve the lyophilized pellets (achieving one-step integration): Keep your hands steady and invert the closed sampling tube and PCR tube (PCR tube down, sampling tube up). At this time, the mixture (containing DNA lysis buffer) in the sampling tube will flow to the connection between the two tubes due to gravity. Gently squeeze the inverted sampling tube again with your palm to allow the mixture inside the tube to drip into the PCR tube through the channel at the connection between the two tubes (the channel diameter is 1-2 mm, allowing only liquid to pass through, and avoiding the swab head from entering the PCR tube); Continue squeezing until most (≥90%) of the mixture in the sampling tube enters the PCR tube. After stopping squeezing, keep the tube inverted for 10-20 seconds to ensure that any remaining liquid flows into the PCR tube to the control line (≥50%). L); After the mixture enters the PCR tube, it will come into contact with the lyophilized bulbs inside the tube. The lyophilized bulbs will dissolve rapidly in the liquid environment (dissolution time ≤30 seconds), releasing nucleic acid purification reagents, isothermal amplification reagents, and fluorescent probes. Core integration: This step completes sample processing (DNA release), nucleic acid purification (silicon bead binding to DNA), and isothermal amplification reagent activation in one go, eliminating the need for adding reagents in steps as in traditional molecular detection, achieving a one-step operation and greatly simplifying the process.

[0031] Step 6: Analyze the results using the detector (results available in 30 minutes): Unscrew the connection between the PCR tube and the sampling tube (rotate the sampling tube counterclockwise to separate them), remove the PCR tube, and confirm that the PCR tube is undamaged and there is no liquid leakage; Turn on the power of the detector and wait for the instrument to complete its self-test (self-test time ≤ 2 minutes, the display screen will show green or "Ready" after the self-test is passed). Open the sample compartment cover of the detector, place the PCR tube into the matching slot inside the sample compartment (ensure that the bottom of the PCR tube is in contact with the bottom of the slot and is not tilted), and close the sample compartment cover. The instrument automatically starts the detection program and enters the isothermal amplification stage (maintaining 37℃±1℃~68℃±1℃, adapting to isothermal amplification reactions with different principles and temperature conditions), while simultaneously starting the fluorescence signal acquisition function (acquiring fluorescence signals at regular intervals and automatically determining whether the positive threshold has been reached). After the detection program runs for 30 minutes, the instrument automatically stops the reaction and outputs the results in real time based on the fluorescence signal intensity: if the fluorescence signal reaches the preset positive threshold, the display shows "+" (indicating that Mycobacterium tuberculosis DNA has been detected, which is positive); if the fluorescence signal does not reach the threshold, the display shows "-" (indicating that Mycobacterium tuberculosis DNA has not been detected, which is negative). After interpreting the results, remove the PCR tube, place it in a medical waste container, turn off the power to the detector, and complete the entire testing process. Operational advantages: No professional molecular biology knowledge is required from the operator. The operation only requires following the simple steps of "place tubes - start - wait for results". Even a novice can complete the operation independently after 5 minutes of verbal guidance. Moreover, it does not rely on a biosafety level 2 laboratory and can be carried out in places such as primary health centers and community service centers.

[0032] Result interpretation criteria and validity verification: Result interpretation criteria: Positive result (+): The detector display clearly shows a red "+" symbol, accompanied by a beeping prompt (optional function), indicating that Mycobacterium tuberculosis-specific DNA was detected in the test sample, suggesting that the subject may be infected with Mycobacterium tuberculosis and further clinical diagnosis is required; Negative result (-): The detector display clearly shows a blue "-" symbol without a beep, indicating that no Mycobacterium tuberculosis-specific DNA was detected in the sample, suggesting that the subject is not currently infected or the infection level is below the detection limit; Invalid results: If the instrument displays "ERROR" or no symbol after 30 minutes, check whether the PCR tube is placed correctly and whether the instrument is powered on properly. After ruling out operation or instrument failure, replace with new testing consumables and repeat the test.

[0033] Through sampling tests on 100 individuals from different groups (including 20 infants, 20 elderly people, and 10 patients with dysphagia), all subjects experienced no significant discomfort, the sampling success rate was 100%, and there were no cases of refusal or failure due to the sampling method. Compared with traditional sputum sample sampling (success rate of approximately 75%), compliance was significantly improved. The testing process was simulated inside a biosafety cabinet, and an aerosol sampler was used to collect microorganisms in the operating environment. The test results showed no leakage of Mycobacterium tuberculosis aerosols. Compared with traditional open extraction (aerosol leakage rate of about 15%), the biosafety risk was completely eliminated. The results of 50 known positive samples were interpreted within 30 minutes, which is 3-168 times more efficient than traditional sputum culture (2-8 weeks) and mainstream molecular detection (1.5-2 hours). Ten primary healthcare workers with no experience in molecular testing were given a 10-minute training session and were able to independently complete the testing process with 100% accuracy in both operation and result interpretation, proving that no professional technical training is required to master the technique.

[0034] All consumables (tongue swabs, sampling tubes, PCR tubes) are for single use only and must not be reused. Before use, check that the packaging is intact (no damage, no leaks). If the packaging is damaged, do not use. During the sampling process, if the subject has a large amount of secretions or food residue in their mouth, the subject should rinse their mouth with water before sampling to avoid impurities interfering with the test results. When connecting the sampling tube to the PCR tube, ensure that the threads are fully engaged to avoid liquid leakage due to loose connection; when inverting and squeezing, avoid contact between the PCR tube opening and other objects to prevent contamination. Before using the detector, ensure that the power supply voltage is stable (220V±10V). Do not open the sample compartment cover during the detection process to avoid temperature fluctuations affecting the amplification reaction. Medical waste after testing (used tongue swabs, PCR tubes, sampling tubes, etc.) must be classified and disposed of in accordance with the "Regulations on the Management of Medical Waste" and must not be discarded at will.

[0035] This specific implementation method is entirely based on the core technical solution in the disclosure document, without adding any additional technical content. By clarifying material specifications, detailing operation steps, and verifying technical advantages, it fully presents the realization process of "non-invasive sampling + fully enclosed one-step detection". It can effectively solve the pain points of "difficult sampling, slow detection, and high risk" in tuberculosis screening at the grassroots level, and meet the needs of grassroots medical institutions for rapid screening.

[0036] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A one-step fully blocked amplification method for Mycobacterium tuberculosis DNA using a tongue swab, characterized in that, Includes the following steps: 1) Samples were taken using a tongue swab; 2) Break off the tongue swab head after sampling and place it inside the sampling tube; 3) Take the PCR tube with the built-in lyophilized bulb and detachably connect the PCR tube to the sampling tube; 4) Squeeze the bottom of the sampling tube to mix the tongue swab inside the sampling tube with the lysis solution; 5) Invert the connected sampling tube and PCR tube, and squeeze the sampling tube again to allow the mixed lysis buffer inside the tube to drip into the PCR tube to dissolve the lyophilized bulbs inside the PCR tube. 6) Insert the PCR tube after dissolving the lyophilized bulbs into the detector, let it stand for the preset time, and then interpret the results.

2. The method according to claim 1, characterized in that, The tongue swab described in step 1) is suitable for people of all ages and special populations, has no contraindications for sampling, and can be used for sampling at any time.

3. The method according to claim 1 or 2, characterized in that, The lyophilized bulbs in the PCR tube described in step 3) contain reagents that enable sample processing, nucleic acid purification, and isothermal amplification.

4. The method according to claim 1, characterized in that, The sampling tube described in step 3) is connected to the PCR tube to form a fully enclosed structure, which can prevent the environment, medical staff and screening subjects from being contaminated and reduce the risk of cross-infection.

5. The method according to claim 1, characterized in that, In step 4), the sampling tube is squeezed to mix the tongue swab with the lysis buffer, which is used to dissolve the Mycobacterium tuberculosis attached to the tongue swab and release the Mycobacterium tuberculosis DNA.

6. The method according to claim 1, characterized in that, The detector described in step 6) outputs the detection results of Mycobacterium tuberculosis DNA by displaying a positive "+" or a negative "-".

7. The method according to claim 1 or 6, characterized in that, The preset time mentioned in step 6) is 30 minutes, that is, the result interpretation is completed 30 minutes after the PCR tube is inserted into the detector.

8. The method according to claim 1, characterized in that, After the tongue swab head is broken off and placed inside the sampling tube in step 2), the sampling tube is in a sealed state, and no separate pretreatment operation is required for the tongue swab head.

9. The method according to claim 1, characterized in that, The entire testing process does not rely on a biosafety level 2 laboratory, and operators do not need to receive specialized technical training; novices can quickly master the operating procedures.

Images