Rapid detection method for mycoplasma pneumoniae nucleic acid

Abstract

The invention relates to the technical field of biological detection, and discloses a mycoplasma pneumoniae nucleic acid rapid detection method, which comprises the following steps: mixing a sample to be detected with a multifunctional pretreatment buffer solution containing tris (hydroxymethyl) aminomethane, potassium acetate, polyethylene glycol, saponin, spermine tetrahydrochloride and tris (2-carboxyethyl) phosphine hydrochloride, and carrying out cracking treatment; a cracking mixed solution is obtained; redissolving the enzymatic isothermal amplification basic reagent, the specific primer and the probe to obtain a redissolving amplification system; sucking the supernatant of the cracking mixed solution, adding the supernatant into a redissolving amplification system, and adding a magnesium acetate solution to obtain a reaction mixed solution to be detected; and carrying out isothermal amplification and signal acquisition to obtain a detection conclusion. The multifunctional pretreatment buffer solution is used for cracking the to-be-detected sample, sample liquefaction, cracking and nucleic acid release are completed in a single system, the nucleic acid extraction and purification step of centrifugal column adsorption or magnetic bead elution is avoided, and the operation process is simplified.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a rapid nucleic acid detection method for Mycoplasma pneumoniae. Background Technology

[0002] Mycoplasma pneumoniae is a common pathogen causing respiratory infections, easily leading to pneumonia and other diseases in children and adolescents. Clinical diagnosis relies on accurate etiological detection to guide medication. Currently, nucleic acid detection technology, due to its high sensitivity and specificity, has become the main method for confirming Mycoplasma pneumoniae infection. Among them, polymerase chain reaction (PCR) and real-time quantitative PCR are widely used detection methods in clinical laboratories, capable of effectively detecting low viral loads of pathogen nucleic acid.

[0003] However, traditional nucleic acid testing procedures are quite cumbersome, typically requiring separate nucleic acid extraction and purification steps. Conventional methods often employ centrifugation columns or magnetic beads to separate nucleic acids from the sample matrix, involving multiple steps such as lysis, binding, washing, and elution, increasing the time required for testing and reliance on the professional skills of the operators. Furthermore, clinically collected respiratory samples, especially mucopurulent or high-viscosity sputum, contain large amounts of mucin and cell debris, which can easily encapsulate pathogens, leading to incomplete lysis. Conventional chemical lysis buffers often fail to completely liquefy high-viscosity samples, while physical-mechanical homogenization methods pose a risk of generating aerosols that contaminate the laboratory environment. Simultaneously, endogenous inhibitors carried in complex sample matrices, if not completely removed during purification, can inhibit subsequent enzymatic amplification reactions, resulting in reduced detection sensitivity or false negative results.

[0004] Furthermore, existing sample pretreatment reagents are often incompatible with downstream amplification reaction systems in terms of ionic environment and chemical composition. Lysis products typically require buffer replacement or significant dilution before being added to the amplification system, limiting the continuity of the detection process. While isothermal amplification technology possesses the potential for rapid detection due to its elimination of thermal cycling equipment, the lack of a pretreatment scheme highly compatible with its reaction system and capable of liquefaction and lysis makes it difficult to meet the timeliness and convenience requirements of point-of-care testing. Therefore, developing a nucleic acid detection method that can effectively handle high-viscosity samples, balances lysis and amplification compatibility, and simplifies the operational process is a problem that needs to be addressed in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a rapid nucleic acid detection method for Mycoplasma pneumoniae, which solves the problems of cumbersome sample pretreatment steps, difficulty in effectively processing high-viscosity sputum samples, and susceptibility of amplification reactions to interference from inhibitors in existing technologies.

[0006] To address the above problems, the present invention provides the following technical solution: This invention provides a rapid nucleic acid detection method for Mycoplasma pneumoniae, employing the following technical solution: A rapid nucleic acid detection method for Mycoplasma pneumoniae includes the following steps: The sample to be tested was mixed with a multifunctional pretreatment buffer and then lysed to obtain a lysate mixture. The enzyme-catalyzed isothermal amplification reagents, Mycoplasma pneumoniae-specific primers, and Mycoplasma pneumoniae-specific probes were mixed with reconstitution buffer and reconstituted to obtain the reconstitution amplification system. The supernatant of the lysis mixture was added to the reconstitution amplification system, and magnesium acetate solution was added to start the reaction to obtain the reaction mixture to be tested. The test reaction mixture was subjected to isothermal amplification and signal acquisition, and the detection conclusion was obtained based on the real-time fluorescence curve; The solvent of the multifunctional pretreatment buffer is pure water, and it contains the following components: tris(hydroxymethyl)aminomethane, potassium acetate, polyethylene glycol, saponins, spermine tetrahydrochloride, and tris(2-carboxyethyl)phosphine hydrochloride.

[0007] By adopting the above technical solution, a rapid detection process for sample input and result output is achieved by combining a multifunctional pretreatment buffer with enzymatic isothermal amplification technology. Specifically, the components in the multifunctional pretreatment buffer exert a synergistic effect, resulting in the following beneficial effects: It enables extraction-free one-step lysis, as the multifunctional pretreatment buffer can directly complete sample liquefaction, pathogen lysis, and nucleic acid release during the lysis step, eliminating the need for purification steps such as centrifugal column adsorption or magnetic bead elution, significantly shortening the operation time. It efficiently processes high-viscosity samples; the tris(2-carboxyethyl)phosphonic acid hydrochloride in the system acts as an effective reducing agent, effectively breaking the disulfide bonds of mucin in sputum, achieving rapid liquefaction of high-viscosity mucopurulent sputum, and solving the problem of difficult sample loading using conventional methods. Meanwhile, compared to dithiothreitol, the tris(2-carboxyethyl)phosphonic acid hydrochloride is more stable and odorless over a wide pH range. It exhibits mild lysis and resistance to inhibition; the saponin, as a nonionic surfactant, specifically binds to cholesterol on the cell membrane, effectively disrupting the membrane structure of Mycoplasma pneumoniae and releasing nucleic acids. Furthermore, compared to strong denaturing ionic detergents, the saponin has a lower inhibitory effect on subsequent enzymatic reactions. It enhances and stabilizes the reaction system; the spermine tetrahydrochloride, as a polyamine compound, neutralizes the negative charge of the DNA backbone, stabilizes the nucleic acid structure, prevents trace nucleic acid degradation, and counteracts interference from some polymerase inhibitors in the sample. The polyethylene glycol, as a molecular crowding reagent, simulates the intracellular environment, increasing the effective collision probability between the enzyme and substrate, significantly improving the reaction rate and sensitivity of subsequent isothermal amplification. It ensures buffer system compatibility; the tris(hydroxymethyl)aminomethane and potassium acetate construct a basic ionic environment highly compatible with downstream enzymatic isothermal amplification reactions, allowing the supernatant after lysis to be used directly for amplification reactions without dilution or buffer replacement.

[0008] Further, the concentration ranges of each component in the final system of the multifunctional pretreatment buffer are as follows: the concentration of tris(hydroxymethyl)aminomethane is 10 mM to 50 mM; the concentration of potassium acetate is 10 mM to 50 mM; the mass-volume concentration of polyethylene glycol is 2.0% to 15.0%; the mass-volume concentration of saponin is 0.05% to 2.0%; the concentration of spermine tetrahydrochloride is 0.5 mM to 10.0 mM; the concentration of tris(2-carboxyethyl)phosphine hydrochloride is 1.0 mM to 20.0 mM; and the pH of the multifunctional pretreatment buffer is 7.2 to 7.8.

[0009] By employing the above technical solution and limiting the concentrations of each component within the aforementioned range, a better balance between lysis efficiency and enzyme compatibility can be achieved. If the concentration of tris(2-carboxyethyl)phosphonic acid hydrochloride or the saponin is too high, it may inhibit recombinant enzyme activity; if the concentration is too low, liquefaction of sputum or release of nucleic acids cannot be completely achieved. Maintaining the pH value between 7.2 and 7.8 ensures the reducing activity of the tris(2-carboxyethyl)phosphonic acid hydrochloride while remaining within the optimal pH range for the amplification enzyme, further guaranteeing the stability of the reaction system.

[0010] Furthermore, in the step of obtaining the reconstitution amplification system, the basic reagents for enzyme-catalyzed isothermal amplification include single-stranded binding proteins, recombinases, and polymerases.

[0011] By adopting the above technical solution, the amplification system utilizes recombinase polymerase amplification or enzymatic recombination isothermal amplification mechanisms. The recombinase forms a protein-DNA complex with primers, scans double-stranded DNA at room temperature, and performs homologous substitution. The single-stranded binding protein stabilizes the substituted single strand, and the polymerase initiates strand extension. This mechanism does not require thermal cycling denaturation and has higher tolerance to impurities in the sample than polymerase chain reaction (PCR) technology, making it suitable for use in conjunction with the extraction-free pretreatment method of this invention.

[0012] Furthermore, the preparation method of the multifunctional pretreatment buffer includes: firstly dissolving the tris(hydroxymethyl)aminomethane, the potassium acetate, and the polyethylene glycol in water to form a clear solution, then sequentially adding the saponin, the spermine tetrahydrochloride, and the tris(2-carboxyethyl)phosphonic acid hydrochloride, adjusting the pH value and making up the volume, and then filtering the solution using a filter membrane with a pore size of 0.10 μm to 0.45 μm.

[0013] By adopting the above technical solution, the above feeding sequence prevents the precipitation or separation of high-concentration polymers and other salt components; using a 0.10μm to 0.45μm filter membrane not only removes insoluble particles, but also plays a sterilization role, preventing the deterioration of the multifunctional buffer solution or the introduction of non-specific nucleic acid interference caused by exogenous microbial contamination.

[0014] Furthermore, the purity of the saponin used in the multifunctional pretreatment buffer is 90% to 98%.

[0015] By adopting the above technical solution, the use of high-purity saponins can effectively reduce the quenching of fluorescence signals or background interference by impurities in the raw materials, while avoiding the nuclease residues that may exist in low-purity raw materials, thereby ensuring the signal-to-noise ratio and accuracy of the detection results.

[0016] Furthermore, in the step of obtaining the lysis mixture, the volume ratio of the sample to be tested to the multifunctional pretreatment buffer is 1:1 to 1:5; the lysis treatment conditions are: incubation at 15°C to 30°C for 2 to 5 minutes.

[0017] By employing the above technical solution, the sample processing ratio and mild lysis conditions are defined. A ratio of 1:1 to 1:5 ensures the effective concentration of the buffer components while avoiding excessive sample dilution that could lead to decreased sensitivity. Lysis can be completed within 2 to 5 minutes at 15°C to 30°C, meaning that this method requires no heating equipment, is easy to operate, and meets the requirements of point-of-care testing for room temperature operation and rapid pretreatment.

[0018] Furthermore, in the step of obtaining the reaction mixture to be tested, the specific operation of aspirating the supernatant of the pyrolysis mixture is as follows: aspirate the supernatant from 2 mm to 5 mm below the surface of the pyrolysis mixture.

[0019] By adopting the above technical solution, the specified sampling position can effectively avoid cell debris sediment or high-density mucus residue at the bottom, while avoiding foam or oil that may exist on the liquid surface, ensuring that the liquid added to the amplification system is homogeneous, clear and contains nucleic acid, and preventing impurities from interfering with optical path detection or quenching fluorescence.

[0020] Furthermore, in the step of obtaining the reconstitution amplification system, the Mycoplasma pneumoniae-specific primers and the Mycoplasma pneumoniae-specific probes are primers designed for the Mycoplasma pneumoniae P1 gene and probes with fluorescent groups; in the step of obtaining the reaction mixture to be tested, the concentration of the magnesium acetate solution is 260mM to 300mM.

[0021] By employing the above-mentioned technical solution, a target gene for the Mycoplasma pneumoniae P1 adhesion protein was designed. Due to the high species specificity and multiple copy characteristics of the P1 gene, the specificity and sensitivity of the detection can be guaranteed. Using a high-concentration magnesium acetate solution as a reaction initiator can rapidly provide a high concentration of magnesium ions, precisely control the onset time of the enzymatic reaction, and prevent premature non-specific amplification.

[0022] Furthermore, in the step of obtaining the detection conclusion, the temperature of the isothermal amplification is 37°C to 42°C, and the continuous running time of the isothermal amplification is 15 min to 25 min.

[0023] By adopting the above technical solution, the reaction temperature is close to the human body temperature, the energy consumption is low, and the accuracy requirements of the temperature control equipment are low; the amplification and result interpretation can be completed in only 15 to 25 minutes. Compared with the traditional polymerase chain reaction technology, the detection efficiency is significantly improved, making it suitable for rapid screening in emergency and outpatient departments.

[0024] Furthermore, the sample to be tested is selected from throat swab wash, mucopurulent sputum, or high-viscosity sputum.

[0025] By adopting the above technical solution, the sample types applicable to this method have been clarified. In particular, for mucopurulent sputum or high-viscosity sputum that are difficult to handle in clinical practice, this method can directly detect them through chemical liquefaction, avoiding the risk of aerosol contamination caused by physical mechanical homogenization, and expanding the clinical applicability of the method.

[0026] This invention provides a rapid nucleic acid detection method for Mycoplasma pneumoniae. It has the following beneficial effects: 1. This invention uses a multifunctional pretreatment buffer containing tris(hydroxymethyl)aminomethane, potassium acetate, polyethylene glycol, saponins, spermine tetrahydrochloride, and tris(2-carboxyethyl)phosphine hydrochloride to lyse the test sample. The sample liquefaction, lysis, and nucleic acid release are completed in a single system, avoiding the nucleic acid extraction and purification steps of centrifugal column adsorption or magnetic bead elution, and simplifying the operation process of Mycoplasma pneumoniae nucleic acid detection.

[0027] 2. This invention utilizes tris(2-carboxyethyl)phosphonic acid hydrochloride to reduce and break the disulfide bonds of mucin to liquefy mucopurulent sputum or high-viscosity sputum, utilizes saponins to disrupt cell membranes to release nucleic acids and reduce the inhibition of subsequent enzymatic reactions, and utilizes spermine tetrahydrochloride to stabilize the nucleic acid structure, thereby achieving effective processing of complex samples and reducing the interference of inhibitors on detection results.

[0028] 3. This invention utilizes the molecular crowding effect generated by polyethylene glycol and combines it with tris(hydroxymethyl)aminomethane and potassium acetate to construct a suitable ionic environment, so that the supernatant of the lysis mixture can be directly added to the reconstitution amplification system without dilution or replacement of the buffer. Combined with enzyme-catalyzed isothermal amplification technology, rapid amplification and detection of nucleic acids were completed at 37°C to 42°C. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the preparation examples, examples, comparative examples, and test examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Preparation Examples 1-3: Preparation Example 1: This preparation example provides a multifunctional pretreatment buffer for Mycoplasma pneumoniae nucleic acid detection, including the following steps: Add 80 mL of autoclaved deionized water to a clean glass container. Place the container on a magnetic stirrer, set the stirring speed to 300 rpm, and maintain the ambient temperature at 20°C. Add 121.1 mg of tris(hydroxymethyl)aminomethane to the water to prepare a buffer system with a final concentration of 10 mM. Add 98.1 mg of potassium acetate (KOAc) to achieve a final concentration of 10 mM, and then add 2.0 g of polyethylene glycol (PEG). Continue stirring for 30 minutes until the solution is clear and transparent, with no visible particles.

[0031] Subsequently, 0.05 g of 90% pure saponin was added to the above clear solution to achieve a mass-volume concentration of 0.05% (w / v); 17.4 mg of arginine tetrahydrochloride was added to achieve a final concentration of 0.5 mM. Finally, add 0.029 g of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP·HCl) to achieve a final concentration of 1.0 mM. After all solids have completely dissolved, adjust the pH of the solution dropwise with 1 M hydrochloric acid solution (HCl) until the pH meter reading stabilizes at 7.2. Finally, bring the volume to 100 mL with deionized water and filter the solution using a 0.10 μm polyethersulfone membrane to obtain the final buffer solution. Aliquot the buffer solution and store it at 2 °C.

[0032] Preparation Example 2: This preparation example provides a multifunctional pretreatment buffer for Mycoplasma pneumoniae nucleic acid detection, including the following steps: Add 400 mL of autoclaved deionized water to a clean glass container. Place the container on a magnetic stirrer, set the stirring speed to 500 rpm, and maintain the ambient temperature at 25°C. Add 1.51 g of tris(hydroxymethyl)aminomethane to the water to prepare a buffer system with a final concentration of 25 mM. Add 1.23 g of potassium acetate (KOAc) to achieve a final concentration of 25 mM, and then add 40.0 g of polyethylene glycol (PEG). Continue stirring for 45 minutes until the solution is completely homogeneous and viscous with no residual bubbles.

[0033] Subsequently, 5.0 g of 95% pure saponin was added to the above solution to bring its mass-volume concentration to 1.0% (w / v); 348.2 mg of arginine tetrahydrochloride was added to bring its final concentration to 2.0 mM. Finally, add 0.72 g of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP·HCl) to achieve a final concentration of 5.0 mM. After all solids have completely dissolved, fine-tune the pH of the solution using 2 M sodium hydroxide solution (NaOH) or hydrochloric acid solution until the pH meter reading stabilizes at 7.5. Finally, bring the volume to 500 mL with deionized water and filter the solution using a 0.22 μm mixed cellulose ester membrane to obtain the final buffer solution. Aliquot the buffer solution and store it at 4 °C.

[0034] Preparation Example 3: This preparation example provides a multifunctional pretreatment buffer for Mycoplasma pneumoniae nucleic acid detection, including the following steps: Add 800 mL of autoclaved deionized water to a clean glass container. Place the container on a magnetic stirrer, set the stirring speed to 800 rpm, and maintain the ambient temperature at 30°C. Add 6.06 g of tris(hydroxymethyl)aminomethane to the water to prepare a buffer system with a final concentration of 50 mM. Add 4.91 g of potassium acetate (KOAc) to achieve a final concentration of 50 mM, and then add 150.0 g of polyethylene glycol (PEG). Continue stirring for 60 minutes to ensure the polymer is fully hydrated and dissolved at a high concentration.

[0035] Subsequently, 20.0 g of saponin with a purity of 98% was added to the above solution to bring its mass-volume concentration to 2.0% (w / v); 3.48 g of arginine tetrahydrochloride was added to bring its final concentration to 10.0 mM. Finally, 5.73 g of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP·HCl) was added to achieve a final concentration of 20.0 mM. After all solids were completely dissolved, the pH of the solution was adjusted dropwise with 5 M sodium hydroxide solution (NaOH) until the pH meter reading stabilized at 7.8. The solution was then brought to a final volume of 1000 mL with deionized water and filtered through a 0.45 μm polyvinylidene fluoride membrane to obtain the final buffer solution, which was then aliquoted and stored at 8°C.

[0036] Examples 1-3: Example 1: This embodiment provides a rapid nucleic acid detection method for Mycoplasma pneumoniae, using the multifunctional pretreatment buffer prepared in Preparation Example 1, including the following steps: S01. Sample pretreatment: Take the pharyngeal swab wash fluid collected clinically as the sample to be tested. Take 50 μL of the sample to be tested and place it in a 0.5 mL sterile centrifuge tube. Add 50 μL of the multifunctional pretreatment buffer prepared in Example 1 (sample to buffer volume ratio is 1:1). Tighten the cap and manually invert the tube 5 times. Then place the centrifuge tube in a constant temperature metal bath at 15°C and incubate for 2 minutes. The liquefaction of the sample and the release of nucleic acid are completed by utilizing the osmotic pressure difference and enzyme inhibitor shielding mechanism in the system to obtain the lysis mixture. S02. Construction of the amplification system: Take a basic lyophilized reagent tube of enzyme isothermal amplification (ERA) powder (containing single-stranded binding protein, recombinase and polymerase), add 29.5 μL of reconstitution buffer, 1.5 μL of 10 μM Mycoplasma pneumoniae P1 gene-specific upstream primer, 1.5 μL of 10 μM specific downstream primer, 0.4 μL of 10 μM specific probe with FAM fluorescent group, and 12.7 μL of deionized water, and mix thoroughly to reconstitute. S03, Sample addition and reaction start-up: Directly aspirate 2.0 μL of the supernatant 2 mm below the surface of the lysis mixture in step S01 and add it to the amplification reagent tube after reconstitution in step S02. Finally, add 2.4 μL of 260 mM magnesium acetate solution to start the reaction. Immediately cap the tube and shake it downwards to centrifuge the liquid to the bottom of the tube. S04. Amplification and Detection: Place the reaction tube in the fluorescence detector, set the isothermal amplification temperature to 37℃, set the fluorescence signal acquisition channel to the FAM channel (excitation wavelength 495nm, emission wavelength 520nm), acquire the fluorescence signal once every 30 seconds, and run continuously for 15 minutes. Judge the detection result based on the peak time of the real-time fluorescence curve.

[0037] Example 2: This embodiment provides a rapid nucleic acid detection method for Mycoplasma pneumoniae, using the multifunctional pretreatment buffer prepared in Preparation Example 2, including the following steps: S01. Sample pretreatment: Take the clinically collected mucopurulent sputum as the test sample. Take 100 μL of the test sample and place it in a 1.5 mL sterile centrifuge tube. Add 300 μL of the multifunctional pretreatment buffer prepared in Example 2 (sample to buffer volume ratio is 1:3). Tighten the cap and vortex at low speed for 3 seconds. Then place the centrifuge tube in a room temperature environment of 25°C for 3 minutes. The liquefaction of the sample and the release of nucleic acid are completed by utilizing the osmotic pressure difference and enzyme inhibitor shielding mechanism in the system to obtain the lysis mixture. S02. Construction of the amplification system: Take a basic lyophilized reagent tube of enzyme isothermal amplification (ERA) powder (containing single-stranded binding protein, recombinase and polymerase), add 29.5 μL of reconstitution buffer, 2.1 μL of 10 μM Mycoplasma pneumoniae P1 gene-specific upstream primer, 2.1 μL of 10 μM specific downstream primer, 0.6 μL of 10 μM specific probe with HEX fluorescent group, and 8.2 μL of deionized water, and mix thoroughly to reconstitute. S03, Sample addition and reaction start-up: Directly aspirate 5.0 μL of the supernatant 3 mm below the surface of the lysis mixture in step S01 and add it to the amplification reagent tube after reconstitution in step S02. Finally, add 2.5 μL of 280 mM magnesium acetate solution to start the reaction. Immediately cap the tube and shake it downwards to centrifuge the liquid to the bottom of the tube. S04. Amplification and Detection: Place the reaction tube in the fluorescence detector, set the isothermal amplification temperature to 39℃, set the fluorescence signal acquisition channel to the HEX channel (excitation wavelength 535nm, emission wavelength 556nm), acquire the fluorescence signal every 45 seconds, and run continuously for 20 minutes. The detection result is judged based on the peak time of the real-time fluorescence curve.

[0038] Example 3: This embodiment provides a rapid nucleic acid detection method for Mycoplasma pneumoniae, using the multifunctional pretreatment buffer prepared in Preparation Example 3, including the following steps: S01. Sample pretreatment: High-viscosity sputum collected clinically was used as the test sample. 200 μL of the test sample was placed in a 2.0 mL sterile centrifuge tube, and 1000 μL of the multifunctional pretreatment buffer prepared in Example 3 was added (sample to buffer volume ratio of 1:5). The tube was capped tightly and vortexed at high speed for 5 seconds. Then the centrifuge tube was placed in a constant temperature metal bath at 30°C and incubated for 5 minutes. The liquefaction of the sample and the release of nucleic acids were completed by utilizing the osmotic pressure difference and enzyme inhibitor shielding mechanism in the system to obtain the lysis mixture. S02. Construction of the amplification system: Take a basic lyophilized reagent tube of enzyme isothermal amplification (ERA) powder (containing single-stranded binding protein, recombinase and polymerase), add 29.5 μL of reconstitution buffer, 3.0 μL of 10 μM Mycoplasma pneumoniae P1 gene-specific upstream primer, 3.0 μL of 10 μM specific downstream primer, 1.0 μL of 10 μM specific probe with Cy5 fluorescent group, and 2.9 μL of deionized water, and mix thoroughly to reconstitute. S03, Sample addition and reaction start-up: Directly aspirate 8.0 μL of the supernatant 5 mm below the surface of the lysis mixture in step S01 and add it to the amplification reagent tube after reconstitution in step S02. Finally, add 2.6 μL of 300 mM magnesium acetate solution to start the reaction. Immediately cap the tube and shake it downwards to centrifuge the liquid to the bottom of the tube. S04. Amplification and Detection: Place the reaction tube in the fluorescence detector, set the isothermal amplification temperature to 42℃, set the fluorescence signal acquisition channel to Cy5 channel (excitation wavelength 650nm, emission wavelength 670nm), acquire the fluorescence signal once every 60 seconds, and run continuously for 25 minutes. Judge the detection result based on the peak time of the real-time fluorescence curve.

[0039] Comparative Examples 1-6: Comparative Example 1: Compared with Example 2, the difference is that no saponins were added to the pretreatment buffer during the preparation process, and the volume was made up with an equal volume of deionized water. All other parameters and steps are the same.

[0040] Comparative Example 2: Compared with Example 3, the difference is that the pretreatment buffer used did not contain tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) during preparation, and the volume was made up with an equal volume of deionized water. All other parameters and steps were the same.

[0041] Comparative Example 3: Compared with Example 2, the difference is that the pretreatment buffer used did not contain succinamine tetrahydrochloride during preparation, and the volume was made up with an equal volume of deionized water. All other parameters and steps were the same.

[0042] Comparative Example 4: Compared with Example 2, the difference is that polyethylene glycol (PEG) was not added to the pretreatment buffer during the preparation process, and the mass was made up with an equal mass of deionized water. All other parameters and steps are the same.

[0043] Comparative Example 5: Compared with Example 2, the difference is that in the preparation of the pretreatment buffer, tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) was replaced with an equimolar concentration of dithiothreitol (DTT), while the other parameters and steps were the same.

[0044] Comparative Example 6: Compared with Example 2, the difference is that in step S01 sample pretreatment, a multifunctional pretreatment buffer is not used. Instead, 100 μL of the sample to be tested is mixed with 100 μL of deionized water and then boiled in boiling water at 100°C for 10 minutes for thermal lysis. After cooling, the supernatant is taken for subsequent amplification. All other parameters and steps are the same.

[0045] Test Examples 1-4: Test Example 1: Sensitivity and Linearity Analysis Experimental description: This test was used to verify the detection limit and linear response performance of the detection method provided in the embodiments under different concentrations of samples. The Mycoplasma pneumoniae standard strain ATCC 29342 was selected as a reference. After incubation and counting, a series of simulated positive samples with varying concentrations were prepared to examine the nucleic acid release efficiency and sensitivity of the multifunctional pretreatment buffer described in this invention under low copy number conditions.

[0046] Experimental steps: The revived standard strains were cultured to the logarithmic growth phase, and their concentrations were double-measured before being serially diluted 10-fold with sterile physiological saline to prepare a concentration of 1.0 × 10⁻⁶. 5 copies / mL up to 2.5 × 10 1 A series of simulated samples (copies / mL) were prepared, with a negative control included. For each concentration sample, 100 μL of the sample was mixed with 300 μL of the buffer solution obtained in Preparation Example 2, vortexed, and incubated at 25°C. The supernatant was then added to the amplification system containing the HEX probe, and the reaction was performed in parallel 20 times. Fluorescence signals were acquired every 45 seconds. If an S-shaped curve appeared within 20 minutes and the fluorescence value was significantly higher than the baseline, it was considered positive, and the peak time was recorded.

[0047] Experimental data: The detection results for each concentration gradient are shown in Table 1 below.

[0048] Table 1. Sensitivity test data for gradient concentration samples Note: "--" indicates that no fluorescence peak signal or typical amplification curve was detected within the specified detection time (20 min).

[0049] Experimental conclusion: Table 1 shows that this method works at 1.0 × 10⁻⁶. 2 100% detection was achieved at a concentration of 2.5 × 10⁻⁶ copies / mL. 1 Even at the limit concentration of copies / mL, the detection rate still reaches 95%, and the average peak time is controlled within 13 minutes. This high sensitivity is attributed to the saponin component in the buffer, which specifically binds to Mycoplasma pneumoniae membrane cholesterol, enabling rapid chemical pore breaking and nucleic acid release under mild conditions, avoiding damage to trace amounts of nucleic acid template caused by high-temperature lysis.

[0050] The peak time distribution shows a linear increase in time with decreasing concentration and a controllable coefficient of variation. This is attributed to the synergistic effect of tris(2-carboxyethyl)phosphonic acid hydrochloride and spermine: TCEP effectively reduces mucin disulfide bonds and removes enzyme inhibitors, eliminating matrix interference; spermine stabilizes primer binding by neutralizing the charge of the nucleic acid backbone, and, combined with the molecular crowding effect of polyethylene glycol, significantly increases the collision probability and reaction rate between polymerase and substrate under low template concentration conditions.

[0051] Test Example 2: Comparative Analysis of the Influence of Key Components on Detection Performance Experimental description: This test examines the contribution of each key component in the buffer solution to the detection performance and compares the differences between room-temperature chemical lysis and traditional physical thermal lysis. A concentration of approximately 1.0 × 10⁻⁶ was selected. 4 Using clinical high-viscosity sputum (copies / mL) as test samples, the mechanisms of action of saponins, TCEP, spermine, and PEG were analyzed by comparing the reaction kinetic parameters of Example 2 with those of Comparative Examples 1 to 6.

[0052] Experimental steps: High-viscosity sputum was thoroughly mixed and then divided into 7 experimental groups. The experimental groups used the full-component buffer from Example 2, mixed at a 1:3 ratio, incubated at room temperature, and amplified. The control group was treated similarly using buffers from Comparative Examples 1 to 5, with or without specific components replaced. Comparative Example 6 did not use buffer; the sample was mixed with water, boiled at 100°C for 10 minutes, and the supernatant was collected for amplification. All groups were tested in parallel 5 times, and the peak time and plateau fluorescence values ​​were recorded.

[0053] Experimental data: The test performance data for each group are summarized in Table 2 below.

[0054] Table 2. Detection performance of high-viscosity sputum samples Note: "--" indicates that no fluorescence peak signal was detected within the specified detection time, and it is judged as not detected.

[0055] Experimental conclusion: Experimental results confirmed the necessity of the full-component buffer. Example 2 showed the shortest peak time and the strongest fluorescence signal, indicating that the components reached optimal equilibrium. The group without added saponins was undetectable, demonstrating that for the robust cell membrane structure of Mycoplasma pneumoniae, the specific binding action of saponins is necessary to disrupt the membrane structure and release nucleic acids. The group without added TCEP showed a severely delayed reaction, indicating that for high-viscosity sputum, TCEP is essential to break the disulfide bonds of mucin to liquefy and inactivate enzyme inhibitors encapsulated in the mucus.

[0056] The absence of cofactors also affected reaction efficiency. The peak time was prolonged in groups without spermine and PEG, confirming that spermine's compressive effect on nucleic acid conformation and the molecular crowding environment created by PEG effectively lower the reaction energy barrier. Furthermore, the room-temperature chemical lysis method of this invention is superior to the boiling water thermal lysis method in both detection speed and signal intensity. It is not only simpler to operate but also avoids the risk of long-fragment DNA breakage caused by high temperatures, making it more suitable for rapid on-site detection.

[0057] Test Example 3: Clinical Sample Conformity Test Experimental description: This test, based on clinical applications, uses magnetic bead-based nucleic acid extraction combined with real-time fluorescence PCR as the gold standard to evaluate the diagnostic efficacy of the method of this invention for complex samples such as pharyngeal swabs and sputum. The focus is on clinical sensitivity, specificity, and overall concordance rate.

[0058] Experimental steps: One hundred and eight clinical samples were collected (including 58 pharyngeal swabs and 50 sputum samples). After resuscitation and mixing, the samples were divided into two groups. The experimental group underwent direct amplification after lysis with buffer solution following the procedure in Example 2, running for 20 minutes. The control group underwent nucleic acid extraction using magnetic beads and followed by fluorescent PCR detection, running for approximately 110 minutes. Based on the PCR results, true positive and false positive data were statistically analyzed, and evaluation indicators were calculated.

[0059] Experimental data: The comparative statistical results of the two methods on 108 clinical samples are shown in Table 3 below.

[0060] Table 3. Contingency Table of Clinical Detection Results by Real-Time Fluorescent PCR Indicator Calculation: Clinical sensitivity = 94.67%; clinical specificity = 93.94%; overall concordance rate = 94.44%; Kappa coefficient = 0.88.

[0061] Experimental conclusion: Statistical analysis shows that the method of this invention has a high degree of consistency with the gold standard PCR method (Kappa=0.88). The high sensitivity of 94.67% is mainly attributed to the combination of saponins and spermines in the buffer. The former ensures the complete disintegration of the host cell and pathogen membrane structures, while the latter prevents trace nucleic acids from being degraded or adsorbed in the crude lysis buffer, thereby achieving effective detection of low-load samples under extraction-free conditions.

[0062] In terms of specificity, this method produced only two false positives, thanks to the highly efficient reduction properties of TCEP. Particularly in processing sputum samples, TCEP rapidly disrupts the mucin network structure, inactivating or dissociating endogenous polymerase inhibitors, thus overcoming the shortcomings of traditional extraction-free methods that are prone to false positives due to impurities. In summary, this invention, by replacing physical extraction with multi-component chemical lysis, shortens the entire detection process to less than 25 minutes while maintaining high accuracy, demonstrating significant clinical application value.

Claims

1. A method for rapid detection of Mycoplasma pneumoniae nucleic acid, characterized by, The method comprises the following steps: mixing a sample to be tested with a multifunctional pretreatment buffer and performing lysis treatment to obtain a lysis mixture; mixing an enzyme isothermal amplification base reagent, Mycoplasma pneumoniae specific primers and Mycoplasma pneumoniae specific probes with a reconstitution buffer and performing reconstitution to obtain a reconstitution amplification system; adding supernatant of the lysis mixture to the reconstitution amplification system and adding a magnesium acetate solution to start the reaction to obtain a reaction mixture to be tested; performing constant temperature amplification and signal collection on the reaction mixture to be tested and obtaining a detection conclusion according to a real-time fluorescence curve; the solvent of the multifunctional pretreatment buffer is pure water, and the multifunctional pretreatment buffer comprises the following components: tris, potassium acetate, polyethylene glycol, saponin, spermine tetrahydrochloride and tris (2-carboxyethyl) phosphine hydrochloride.

2. The method for rapid detection of Mycoplasma pneumoniae nucleic acid according to claim 1, characterized in that, The concentration ranges of the components in the multifunctional pretreatment buffer in the final system are as follows: the concentration of the tris is 10 mM to 50 mM; the concentration of the potassium acetate is 10 mM to 50 mM; the mass / volume concentration of the polyethylene glycol is 2.0% to 15.0%; the mass / volume concentration of the saponin is 0.05% to 2.0%; the concentration of the spermine tetrahydrochloride is 0.5 mM to 10.0 mM; the concentration of the tris (2-carboxyethyl) phosphine hydrochloride is 1.0 mM to 20.0 mM; the pH value of the multifunctional pretreatment buffer is 7.2 to 7.

8.

3. The method for rapid detection of Mycoplasma pneumoniae nucleic acid according to claim 1, characterized in that, In the step of obtaining the reconstitution amplification system, the enzyme isothermal amplification base reagent comprises single-strand binding protein, recombinase and polymerase.

4. The method for rapid detection of Mycoplasma pneumoniae nucleic acid according to claim 1, characterized in that, The preparation method of the multifunctional pretreatment buffer comprises the following steps: dissolving the tris, the potassium acetate and the polyethylene glycol in water to form a clear solution, then sequentially adding the saponin, the spermine tetrahydrochloride and the tris (2-carboxyethyl) phosphine hydrochloride, adjusting the pH value and constant volume, and performing filtration treatment using a filter membrane with a pore size of 0.10 µm to 0.45 µm.

5. The method for rapid detection of Mycoplasma pneumoniae nucleic acid according to claim 1, characterized in that, The raw material purity of the saponin used in the multifunctional pretreatment buffer is 90% to 98%.

6. The method for rapid detection of Mycoplasma pneumoniae nucleic acid according to claim 1, characterized in that, The preparation method of the multifunctional pretreatment buffer comprises: dissolving the tris, the potassium acetate and the polyethylene glycol in water to form a clear solution, then sequentially adding the saponin, the spermine tetrahydrochloride and the tris (2-carboxyethyl) phosphine hydrochloride, adjusting the pH value and constant volume, and performing filtration treatment using a filter membrane with a pore size of 0.10 µm to 0.45 µm.

7. The method for rapid detection of Mycoplasma pneumoniae nucleic acid according to claim 1, characterized in that, In the step of obtaining the reaction mixture to be tested, the specific operation of aspirating the supernatant of the lysis mixture is as follows: aspirating the supernatant at a position 2 mm to 5 mm below the liquid level of the lysis mixture.

8. The method for rapid detection of Mycoplasma pneumoniae nucleic acid according to claim 1, characterized in that, In the step of obtaining the reconstitution amplification system, the Mycoplasma pneumoniae specific primers and the Mycoplasma pneumoniae specific probes are primers and probes with fluorescent groups designed for the P1 gene of Mycoplasma pneumoniae; In the step of obtaining the reaction mixture to be tested, the concentration of the magnesium acetate solution is 260 mM to 300 mM.

9. The method for rapid detection of Mycoplasma pneumoniae nucleic acid according to claim 1, characterized in that, In the step of obtaining the detection conclusion, the temperature of the isothermal amplification is 37-42 DEG C, and the continuous operation time of the isothermal amplification is 15-25 min.

10. The method for rapid detection of Mycoplasma pneumoniae nucleic acid according to claim 1, characterized in that, The sample to be detected is selected from a throat swab eluate, mucous purulent sputum or high viscosity sputum.

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