Mycobacterium tuberculosis detection method and kit

Abstract

The invention belongs to the technical field of biology, and provides a mycobacterium tuberculosis detection method and a kit, the detection method comprises the following steps: adding a to-be-detected sample containing mycobacterium tuberculosis into a lysis working solution, carrying out a heat preservation reaction, centrifuging, and taking a supernatant, the cracking working solution contains a biotinylated metal complexing agent and biotinylated nanoparticles, and guanidine isothiocyanate and hydrophobic alkyl groups are connected to the biotinylated nanoparticles; adding a magnetic bead suspension into the supernatant, stirring at room temperature for reaction, standing, taking an upper solution, adding the upper solution into a purification column, purifying and washing to obtain a detection sample, and the surface of a magnetic bead contains streptavidin; and adding a detection sample into an amplification reagent for fluorescent PCR amplification, and analyzing a PCR amplification product by using a fluorescence melting curve to finish detection. The method can effectively reduce impurities contained in the detection sample and improve the detection accuracy.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a method and kit for detecting Mycobacterium tuberculosis. Background Technology

[0002] Fluorescence melting curve detection, as an emerging molecular diagnostic technology, has shown significant advantages in the identification of Mycobacterium tuberculosis species and the detection of drug resistance. Based on real-time fluorescence PCR, this technology involves slowly heating the product after amplification and monitoring changes in fluorescence signal in real time. Utilizing the differences in melting temperature (Tm value) of different DNA sequences, it can simultaneously identify Mycobacterium tuberculosis complexes and rapidly and accurately identify gene mutations related to resistance to key first-line drugs such as rifampin and isoniazid within a single closed reaction tube. Its core advantages lie in its high sensitivity and specificity, enabling the detection of low-bacterial-weight samples that are easily missed by traditional smear methods; its high efficiency and speed, reducing the traditional culture and drug susceptibility testing process, which can take weeks, to 2-4 hours, providing a valuable window for early clinical diagnosis and precision medicine; and its ability to simultaneously detect multiple targets, achieving multiple interpretations with a single test, effectively improving diagnostic efficiency.

[0003] While fluorescence melting curve detection technology for Mycobacterium tuberculosis offers advantages such as speed and sensitivity, its performance is highly dependent on the quality of the nucleic acid template. In practical applications, it is often limited by interference from complex impurities in clinical samples. These impurities mainly originate from two sources: first, the bacteria themselves, with their unique cell walls containing mycolic acid, arabinogalactan, and peptidoglycan, which release small molecular fragments after lysis; second, impurities in sputum samples, including polysaccharides derived from mucus, hemoglobin exudated from the lungs, and the complex mucus matrix of the sputum itself. The small molecular fragments generated by the lysis of cell wall components such as mycolic acid, arabinogalactan, and peptidoglycan not only form gel-like substances in solution, reducing DNA extraction efficiency, but also inhibit the activity of key enzymes such as proteinase K, leading to incomplete DNA release and reduced DNA yield. Furthermore, these impurities can compete for binding sites or clog purification columns, further hindering effective DNA recovery. Meanwhile, polysaccharides in sputum samples can inhibit the activity of DNA polymerase, while hemoglobin can disrupt the redox microenvironment required for enzyme reactions. Furthermore, viscous substances not only physically encapsulate bacteria, leading to a sharp drop in genomic DNA extraction efficiency, but also non-specifically adsorb primers, enzymes, and other reaction components. This interaction directly results in a significant decrease in sensitivity, leading to false negatives. Low genomic DNA extraction efficiency and low or even complete inhibition of target DNA amplification significantly increase the risk of detection failure in low-load samples. It also causes abnormal melting curve morphology and Tm value shifts, affecting the accuracy of result interpretation and potentially misclassifying drug-resistant mutations as sensitive ones. Summary of the Invention

[0004] The technical problem to be solved by this invention is to propose a method for detecting Mycobacterium tuberculosis, which aims to solve the problem that the detection sample contains too many impurities during the detection of Mycobacterium tuberculosis by fluorescence melting curve, resulting in a decrease in the accuracy of result interpretation.

[0005] To address the aforementioned technical problems, this invention proposes a method for detecting Mycobacterium tuberculosis, the method comprising the following steps: S1. Add the sample containing Mycobacterium tuberculosis to the lysis working solution, keep it warm, centrifuge, and take the supernatant. The lysis working solution contains a biotinylated metal complexing agent and biotinylated nanoparticles, and the biotinylated nanoparticles are connected to guanidine isothiocyanate and hydrophobic alkyl groups. S2. Add the magnetic bead suspension to the supernatant, stir and react at room temperature, let stand, take the supernatant and add it to the purification column, purify and wash to obtain the test sample, in which the magnetic bead surface contains streptavidin; S3. Add the test sample to the amplification reagent for fluorescent PCR amplification, and then use the fluorescence melting curve to analyze the PCR amplification product to complete the detection.

[0006] In some embodiments, the biotinylated metal complexing agent in step S1 includes at least one of N1-ethylenediaminetetraacetic acid-biotin, N1-diethylenetriaminetetraacetic acid-biotin, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin; the biotinylated nanoparticles include at least one of biotinylated silica nanoparticles, biotin-cholesterol-polycaprolactone-polylactic acid nanoparticles, and biotin-palmitoyl-chitosan nanoparticles; and the hydrophobic alkyl group is derived from at least one of sodium dodecyl sulfate, n-dodecyl-β-D-maltopyranoside, and hexadecyltrimethylammonium bromide.

[0007] In some embodiments, step S1 includes: S1.1 Prepare biotinylated metal complexing agents and biotinylated nanoparticles with guanidine isothiocyanate and hydrophobic alkyl groups on their surfaces, respectively. S1.2 Add the biotinylated metal complexing agent and biotinylated nanoparticles connected with guanidine isothiocyanate and hydrophobic alkyl groups to the lysis working solution, stir, then add the sample to be tested containing Mycobacterium tuberculosis, incubate at 45~50℃ for 15~20 min, centrifuge at 10000~12000*g for 8~10 min, filter, and obtain the supernatant. The lysis working solution is a 100mM Tris-HCl buffer solution with pH 8.0, and the concentration of the biotinylated metal complexing agent in the lysis working solution is 4~6mM and the concentration of the biotinylated nanoparticles is 0.5~1.5μg / mL.

[0008] In some embodiments, the method for preparing the biotinylated metal complex in step S1.1 includes: S1.1.1 The metal complexing agent, N-(3-dimethylhydropropyl)-N'-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide are sequentially added to dimethyl sulfoxide at 0-4°C. After stirring and dispersing, the mixture is heated to room temperature and reacted for 2-4 hours. Then, biotinylate and an organic base are added, and the reaction is carried out at 30-35°C for 6-12 hours. The mixture is then added to diethyl ether at 0-4°C, the precipitate is collected by filtration and dried to obtain the biotinylated metal complexing agent, wherein the metal complexing agent includes ethylenediaminetetraacetic acid, ... At least one of diethylenetriaminetetraacetic acid and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, the organic base being N,N-diisopropylethylamine and / or triethylamine, and the molar ratio of the metal complexing agent N-(3-dimethylhydropropyl)-N'-ethylcarbodiimide hydrochloride: N-hydroxysuccinimide: biotinylate: organic base being 1:(1.02~1.03):(1.02~1.03):(1.05~1.07):(2~3).

[0009] In some embodiments, the method for preparing biotinylated nanoparticles in step S1.1 includes: S1.1.2 Tetraethyl orthosilicate, anhydrous ethanol, pure water, and concentrated ammonia were mixed and stirred at room temperature for 6-8 hours. After centrifugation, washing with anhydrous ethanol, and drying, nanoparticles were obtained. These nanoparticles were then dispersed in anhydrous ethanol, and 3-aminopropyltriethoxysilane was added. The mixture was heated to 70°C and refluxed for 10-12 hours. After centrifugation, washing with anhydrous ethanol, and drying, ammoniated nanoparticles were obtained. Finally, these nanoparticles were dispersed in dimethyl sulfoxide, and biotin-N-hydroxysuccinimide ester was added. The mixture was stirred at room temperature in the dark for 20-24 hours, followed by centrifugation, filtration, and subsequent... Biotinylated nanoparticles were obtained by washing and drying with N,N-dimethylformamide and pure water, wherein the volume ratio of tetraethyl orthosilicate: anhydrous ethanol: pure water: concentrated ammonia was 1:(30~50):(8~12):(1.2~2), the mass ratio of silica: anhydrous ethanol:3-aminopropyltriethoxysilane was 1:(10~20):(0.8~1.5), and the mass ratio of aminated silica:dimethyl sulfoxide:biotin-N-hydroxysuccinimide ester was 1:(10~15):(0.3~0.8). S1.1.3. Biotinylated nanoparticles were dispersed in a borate buffer solution at pH 8.5, and lauric acid-N-hydroxysuccinimide and benzyl isothiocyanate EDTA were added sequentially. The mixture was stirred at room temperature for 10-12 h, and the precipitate was collected by centrifugation. The precipitate was washed sequentially with ethanol and pure water and dried to obtain biotinylated nanoparticles with guanidine isothiocyanate and hydrophobic alkyl groups. The mass ratio of biotinylated nanoparticles to lauric acid-N-hydroxysuccinimide to benzyl isothiocyanate EDTA was 1:(0.2-0.5):(0.5-1.0).

[0010] In some embodiments, the method for preparing biotinylated nanoparticles in step S1.1 further includes: S1.1.2. Cholesterol, polycaprolactone, polylactic acid, and stannous octoate were dissolved in dichloromethane and reacted at 120-130°C for 6-8 hours under inert gas protection. After filtration, washing, and vacuum drying, a copolymer was obtained. Then, poloxamer was added to a mixed solvent of dimethylformamide and acetone as the oil phase, and added to an aqueous solution of polyvinyl alcohol under stirring. The mixture was stirred at room temperature for 8-12 hours, centrifuged, and washed with deionized water to obtain nanoparticles. These nanoparticles were dispersed in a phosphate buffer solution at pH 7.4, and a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide was added. The mixture was activated at room temperature for 0.5-1 hour, followed by the addition of biotin-hydrazide. The mixture was reacted at room temperature for 12-16 hours in the dark. After the reaction, the nanoparticles were centrifuged and then processed sequentially with a phosphate buffer solution containing 0.05% polysorbate-20 and pure water. Washing and drying yielded biotin-cholesterol-polycaprolactone-polylactic acid nanoparticles, wherein the mass ratio of cholesterol, polycaprolactone, polylactic acid, stannous octoate, and dichloromethane was 1:(3~5):(5~10):(0.03~0.08):(15~25), the mass ratio of copolymer, poloxamer, and mixed solvent was 1:(0.5~2):(15~25), the mass ratio of dimethylformamide to acetone in the mixed solvent was 3:7, the concentration of polyvinyl alcohol in the polyvinyl alcohol aqueous solution was 1~3wt%, the mass ratio of polyvinyl alcohol aqueous solution to oil phase was (8~12):1, and the mass ratio of nanoparticles, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and biotin-hydrazide was 1:(0.15~0.3):(0.03~0.08):(0.05~0.15).

[0011] In some embodiments, the method for preparing biotinylated nanoparticles in step S1.1 further includes: S1.1.2. Chitosan was dissolved in a 1% (w / w) aqueous acetic acid solution to prepare a 0.5-2% (w / w) chitosan solution. Then, N-hydroxysuccinimide-activated palmitate was added at a palmitoyl to chitosan unit molar ratio of 1:(0.2-0.5). The mixture was stirred at 50-60°C for 12-16 hours. After the reaction, the mixture was transferred to a dialysis bag and purified by dialysis with an ethanol aqueous solution containing 0.1% SDS and then by lyophilization with pure water. The purified solution was then obtained by freeze-drying to obtain a palmitoyl-chitosan polymer. This polymer was then dispersed in a phosphate buffer solution at pH 7.4, and 1-ethyl-3-(3-dimethylaminopropyl) was added. Carbodiimide hydrochloride and N-hydroxysuccinimide were reacted at room temperature for 0.5-1 h. Biotin-hydrazide was added, and the mixture was reacted at room temperature for 12-16 h under light-protected conditions. The precipitate was collected by centrifugation and washed and dried sequentially with phosphate buffer containing 0.05% polysorbate-20 and pure water to obtain biotin-palmitoyl-chitosan nanoparticles. The mass ratio of palmitoyl-chitosan polymer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and biotin-hydrazide was 1:(0.15-0.3):(0.03-0.08):(0.05-0.15).

[0012] In some embodiments, step S2 includes: S2.1 Dissolve ferric nitrate nonahydrate and ferrous chloride hexahydrate in ultrapure water. Under nitrogen atmosphere, add ammonia dropwise, heat to 80°C and stir for 1-1.5 h. Then add sodium citrate solution and continue stirring for 2-2.5 h to obtain carboxyl-modified magnetic beads. The mass ratio of ferric nitrate nonahydrate: ferrous chloride hexahydrate: ammonia: sodium citrate: ultrapure water is 1:(0.55-0.62):(3.2-4.5):(1.4-1.6):(70-80). S2.2 Disperse carboxyl-modified magnetic beads in 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.0, add N-(3-dimethylhydropropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, activate at room temperature for 30 min, separate by magnetic field, discard the supernatant to obtain activated magnetic beads, wherein the mass ratio of carboxyl-modified magnetic beads:N-(3-dimethylhydropropyl)-N'-ethylcarbodiimide hydrochloride:N-hydroxysuccinimide is 1:(0.1~0.3):(0.05~0.15); S2.3. Disperse the activated magnetic beads in Tris buffer at pH 7.5 containing 1.5~2.5 mg / mL streptavidin and react at room temperature for 4~6 h. Separate them with a magnetic field to obtain magnetic beads containing streptavidin. Then add Tris buffer at pH 8.0 containing 100 mM ethanolamine and 1% bovine serum albumin and react for 1~2 h to obtain blocked magnetic beads. Disperse them in Tris buffer at pH 7.5 containing 0.1% polysorbate-20 and 0.1% bovine serum albumin to obtain a magnetic bead suspension. S2.4 Add magnetic bead suspension to the supernatant and stir at room temperature for 1-2 hours. Separate by magnetic field, take the supernatant and add it to the purification column for purification and washing to obtain the test sample.

[0013] In some embodiments, the amplification reagent in step S3 comprises: The sequences of the upper primer IS6110-F, the lower primer IS6110-R, and the probe IS6110-Probe used for the identification of Mycobacterium tuberculosis complexes are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3. The sequences of the upper primer rpoB-F, lower primer rpoB-R, probe rpoB-Probe1, probe rpoB-Probe2, probe rpoB-Probe3, and probe rpoB-Probe4 for detecting rifampicin resistance gene mutations are shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9. The sequences of the upper primer KatG-F, lower primer KatG-R, and probe KatG-Probe for detecting isoniazid resistance gene mutations are shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12; the sequences of the upper primer inhA-F, lower primer inhA-R, and probe inhA-Probe are shown in SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15; and the sequences of the upper primer ahpC-F, lower primer ahpC-R, probe ahpC-Probe1, and probe ahpC-Probe2 are shown in SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.19. The sequences of the upper primer gyrA-F, lower primer gyrA-R, and probe gyrA-Probe for gene mutation detection of quinolone drugs are shown in SEQ ID NO.20, SEQ ID NO.21, and SEQ ID NO.22. The sequences of the upper primer embB306-F, lower primer embB306-R, and probe embB306-Probe for detecting ethambutol resistance gene mutations are shown in SEQ ID NO.23, SEQ ID NO.24, and SEQ ID NO.25; the sequences of the upper primer embB406-F, lower primer embB406-R, and probe embB406-Probe are shown in SEQ ID NO.26, SEQ ID NO.27, and SEQ ID NO.28; and the sequences of the upper primer embB497-F, lower primer embB497-R, and probe embB497-Probe are shown in SEQ ID NO.29, SEQ ID NO.30, and SEQ ID NO.31.

[0014] In addition, a kit is disclosed, which contains a lysis reagent tube, a suspension reagent tube, and an amplification reagent tube. The lysis reagent tube, the suspension reagent tube, and the amplification reagent tube respectively contain the lysis working solution, the magnetic bead suspension, and the amplification reagent in the above-described method for detecting Mycobacterium tuberculosis.

[0015] The beneficial effects of this invention are: Biotinylated nanoparticles linked with guanidine isothiocyanate and hydrophobic alkyl groups, thanks to their extremely small size and huge specific surface area, can effectively penetrate the viscous network structure of mucus through Brownian motion, reaching the cell wall of Mycobacterium tuberculosis and thus locally constructing an ultra-high concentration lysis microenvironment. In this synergistic lysis process, while guanidine isothiocyanate strongly denatures proteins to disrupt the cell wall structure, its mechanism of action does not break the stable phosphodiester backbone structure of DNA; simultaneously, the hydrophobic alkyl groups are mainly responsible for solubilizing and disintegrating lipid components in the cell membrane and cell wall. Both work synergistically and efficiently open the cell, while preserving the integrity of the DNA molecule due to the specificity of their target sites. This improves lysis efficiency while ensuring the template quality for subsequent fluorescent PCR detection. During this process, biotinylated nanoparticles can actively adsorb small molecule fragments of disintegrated mycolic acid, arabinogalactan, and peptidoglycan, effectively preventing them from re-aggregating or non-specifically adsorbing DNA. The subsequently added biotinylated metal complexing agent can efficiently chelate metal ions such as calcium ions in the solution, thereby disrupting the gel structure of mucopolysaccharides and eliminating the potential inhibition of PCR enzymes by metal ions. At the same time, the hydrophobic alkyl groups in the system can effectively disintegrate and emulsify the lipid components in the mucus, and work synergistically with the biotinylated nanoparticles. Through hydrophobic interactions and adsorption, the system captures and solidifies the hemoglobin exudated from the lungs, the disintegrated mucopolysaccharide fragments, and the viscous mucus matrix fragments of the mucus itself, uniformly assigning them a biotinylated tag. In the subsequent purification stage, magnetic beads coated with streptavidin utilize the high affinity interaction between biotin and streptavidin to efficiently and specifically capture and remove labeled inhibitor complexes in solution, including metal ion-metal complexes, lipid-hydrophobic alkyl group fragments, and small molecule fragments captured by nanoparticles. Finally, fine purification using a purification column removes impurities from the sample, significantly improving PCR amplification efficiency and enhancing the accuracy of the entire fluorescence melting curve detection method. Attached Figure Description

[0016] Figure 1 This is a schematic flowchart of a method for detecting Mycobacterium tuberculosis in one embodiment of the present invention; Figure 2 Image of the lyophilized powder product of the amplification system. Detailed Implementation

[0017] In the description of this application, it should be noted that, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0018] Please refer to Figure 1This invention provides a method for detecting Mycobacterium tuberculosis, the method comprising the following steps: S1. Add the sample containing Mycobacterium tuberculosis to the lysis working solution, keep it warm, centrifuge, and take the supernatant. The lysis working solution contains a biotinylated metal complexing agent and biotinylated nanoparticles, and the biotinylated nanoparticles are connected to guanidine isothiocyanate and hydrophobic alkyl groups. In step S1, the biotinylated metal complexing agent includes at least one of N1-ethylenediaminetetraacetic acid-biotin, N1-diethylenetriaminetetraacetic acid-biotin, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin. The biotinylated nanoparticles include at least one of biotinylated silica nanoparticles, biotin-cholesterol-polycaprolactone-polylactic acid nanoparticles, and biotin-palmitoyl-chitosan nanoparticles. The hydrophobic alkyl group is derived from at least one of sodium dodecyl sulfate, n-dodecyl-β-D-maltopyranoside, and hexadecyltrimethylammonium bromide.

[0019] Biotinylated nanoparticles linked with guanidine isothiocyanate and hydrophobic alkyl groups, thanks to their extremely small size and huge specific surface area, can effectively penetrate the viscous network structure of mucus through Brownian motion, reaching the cell wall of Mycobacterium tuberculosis and thus locally constructing an ultra-high concentration lysis microenvironment. In this synergistic lysis process, while guanidine isothiocyanate strongly denatures proteins to disrupt the cell wall structure, its mechanism of action does not break the stable phosphodiester backbone structure of DNA; simultaneously, the hydrophobic alkyl groups are mainly responsible for solubilizing and disintegrating lipid components in the cell membrane and cell wall. Both work synergistically and efficiently open the cell, while preserving the integrity of the DNA molecule due to the specificity of their target sites. This improves lysis efficiency while ensuring the template quality for subsequent fluorescent PCR detection. During this process, biotinylated nanoparticles can actively adsorb small molecule fragments of disintegrated mycolic acid, arabinogalactan, and peptidoglycan, effectively preventing them from re-aggregating or non-specifically adsorbing DNA. The subsequently added biotinylated metal complexing agent can efficiently chelate metal ions such as calcium ions in the solution, thereby disrupting the gel structure of mucopolysaccharides and eliminating the potential inhibition of PCR enzymes by metal ions. At the same time, the hydrophobic alkyl groups in the system can effectively disintegrate and emulsify the lipid components in the mucus, and work synergistically with the biotinylated nanoparticles. Through hydrophobic interactions and adsorption, the system captures and solidifies the hemoglobin exudated from the lungs, the disintegrated mucopolysaccharide fragments, and the viscous mucus matrix fragments of the mucus itself, uniformly assigning them a biotinylated tag.

[0020] Step S1 includes: S1.1 Prepare biotinylated metal complexing agents and biotinylated nanoparticles with guanidine isothiocyanate and hydrophobic alkyl groups on their surfaces, respectively. S1.1.1 Preparation of biotinylated metal complexes: A metal complexing agent, N-(3-dimethylhydropropyl)-N'-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide were sequentially added to dimethyl sulfoxide at 0–4 °C. After stirring and dispersing, the mixture was heated to room temperature and reacted for another 2–4 hours. Then, biotinylate and an organic base were added, and the reaction was carried out at 30–35 °C for 6–12 hours. The mixture was then added to diethyl ether at 0–4 °C, and the precipitate was collected by filtration and dried to obtain the biotinylated metal complexing agent, wherein the metal complexing agent included ethylenediaminetetraacetic acid and diethylidene. At least one of triaminetetraacetic acid and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, the organic base being N,N-diisopropylethylamine and / or triethylamine, and the metal complexing agent being N-(3-dimethylhydropropyl)-N'-ethylcarbodiimide hydrochloride: N-hydroxysuccinimide: biotinylate: the molar ratio of the organic base being 1:(1.02~1.03):(1.02~1.03):(1.05~1.07):(2~3).

[0021] Using a low-temperature initiation reaction of 0-4℃ effectively reduced the hydrolysis side reaction of the activator and ensured the efficient activation of the carboxyl group of the metal complexing agent. Subsequently, the temperature was raised to room temperature and the reaction time was extended to promote the formation of stable intermediates. After adding biotinylate hydrazine, the reaction time was extended under mild heating conditions to ensure the full formation of amide bonds. The addition of organic base neutralized the hydrochloric acid byproduct generated by the reaction and maintained the pH stability of the system. Finally, the product was efficiently separated by low-temperature diethyl ether precipitation. The strict molar ratio not only avoided side reactions but also ensured the complete reaction, ultimately yielding the target product with high yield and high biotin labeling.

[0022] S1.1.2 Preparation of biotinylated nanoparticles: Preparation of biotin-containing silica nanoparticles: Tetraethyl orthosilicate, anhydrous ethanol, pure water, and concentrated ammonia were mixed and stirred at room temperature for 6-8 hours. After centrifugation, washing with anhydrous ethanol, and drying, nanoparticles were obtained. These nanoparticles were then dispersed in anhydrous ethanol, and 3-aminopropyltriethoxysilane was added. The mixture was heated to 70°C and refluxed for 10-12 hours. After centrifugation, washing with anhydrous ethanol, and drying, ammoniated nanoparticles were obtained. Finally, these nanoparticles were dispersed in dimethyl sulfoxide, and biotin-N-hydroxysuccinimide ester was added. The mixture was stirred at room temperature in the dark for 20-24 hours, followed by centrifugation and filtration. Biotinylated nanoparticles were obtained by sequentially washing with N,N-dimethylformamide and pure water and then drying. The volume ratio of tetraethyl orthosilicate: anhydrous ethanol: pure water: concentrated ammonia was 1:(30~50):(8~12):(1.2~2), the mass ratio of silica: anhydrous ethanol:3-aminopropyltriethoxysilane was 1:(10~20):(0.8~1.5), and the mass ratio of aminated silica:dimethyl sulfoxide:biotin-N-hydroxysuccinimide ester was 1:(10~15):(0.3~0.8).

[0023] In the silica nucleus synthesis stage, tetraethyl orthosilicate was catalyzed in an alcohol-water system with an appropriate amount of concentrated ammonia to ensure an efficient and controllable hydrolysis-condensation reaction, generating nanoparticles with good monodispersity and uniform particle size. Subsequently, amino functional groups were firmly introduced onto the particle surface by reflux treatment with 3-aminopropyltriethoxysilane at 70°C, providing efficient reaction sites for subsequent biotin labeling. Finally, under light-protected conditions, it was reacted with biotin-N-hydroxysuccinimide ester (biotin-NHS ester) at room temperature. The precise feed ratio and long-term mild reaction ensured that biotin was covalently coupled to the surface of the aminated particles with high density and stability. Unreacted substances were effectively removed by step washing with N,N-dimethylformamide (DMF) and water, finally obtaining functionalized nanoparticles with uniform biotin modification and good dispersibility.

[0024] Preparation of Biotin-Cholesterol-Polycaprolactone-Polylactic Acid Nanoparticles: Cholesterol, polycaprolactone, polylactic acid, and stannous octoate were dissolved in dichloromethane and reacted at 120-130°C for 6-8 hours under inert gas protection. The mixture was filtered, washed, and vacuum dried to obtain a copolymer. Then, poloxamer was added to a mixed solvent of dimethylformamide and acetone as the oil phase, and the mixture was added to an aqueous solution of polyvinyl alcohol under stirring. The mixture was stirred at room temperature for 8-12 hours, centrifuged, and washed with deionized water to obtain nanoparticles. These nanoparticles were dispersed in a phosphate buffer solution at pH 7.4, and a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide was added. The mixture was activated at room temperature for 0.5-1 hour, followed by the addition of biotin-hydrazide. The reaction was carried out at room temperature for 12-16 hours in the dark. After the reaction, the nanoparticles were centrifuged and then processed sequentially with a phosphate buffer solution containing 0.05% polysorbate-20. The nanoparticles were washed and dried with acid salt buffer and pure water to obtain biotin-cholesterol-polycaprolactone-polylactic acid nanoparticles. The mass ratio of cholesterol, polycaprolactone, polylactic acid, stannous octoate and dichloromethane was 1:(3~5):(5~10):(0.03~0.08):(15~25). The mass ratio of copolymer, poloxamer and mixed solvent was 1:(0.5~2):(15~25). The mass ratio of dimethylformamide and acetone in the mixed solvent was 3:7. The concentration of polyvinyl alcohol in the polyvinyl alcohol aqueous solution was 1~3wt%. The mass ratio of polyvinyl alcohol aqueous solution and oil phase was (8~12):1. The mass ratio of nanoparticles, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and biotin-hydrazide was 1:(0.15~0.3):(0.03~0.08):(0.05~0.15).

[0025] In the copolymer synthesis stage, cholesterol, polycaprolactone, and polylactic acid react under trace amounts of stannous octoate catalysis and an inert high-temperature environment, ensuring the efficient synthesis of block copolymers and the controllable self-assembly of molecular chains, endowing the nanoparticle core with good biocompatibility and hydrophobic drug loading capacity. Secondly, by optimizing the mass ratio and composition of the oil phase and polyvinyl alcohol solution, nanoparticles with uniform particle size and high stability are formed using an emulsification-solvent evaporation method, and the introduction of poloxamer further enhances its hydrophilicity and cycle stability. Finally, in the biotinylation modification, the carboxyl group is mildly activated at physiological pH using a system of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide (EDC / NHS), and reacts with biotin-hydrazide in the dark. By precisely controlling the ratio of coupling reagents and reaction time, high-density, directional fixation of biotin is achieved, and step washing effectively removes unreacted substances.

[0026] Preparation of biotin-palmitoyl-chitosan nanoparticles: Chitosan was dissolved in a 1% (w / w) aqueous acetic acid solution to prepare a 0.5-2% (w / w) chitosan solution. N-hydroxysuccinimide-activated palmitate was then added at a palmitoyl to chitosan unit molar ratio of 1:(0.2-0.5). The mixture was stirred at 50-60°C for 12-16 h. After the reaction, the mixture was transferred to a dialysis bag and purified by dialysis with an ethanol aqueous solution containing 0.1% SDS and then by lyophilization to obtain the palmitoyl-chitosan polymer. This polymer was then dispersed in a phosphate buffer solution at pH 7.4, and 1-ethyl-3-(3-diethyl-3- ... 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were reacted at room temperature for 0.5-1 h. Biotin-hydrazide was added, and the mixture was reacted at room temperature for 12-16 h under light-protected conditions. The precipitate was collected by centrifugation and washed and dried sequentially with phosphate buffer containing 0.05% polysorbate-20 and pure water to obtain biotin-palmitoyl-chitosan nanoparticles. The mass ratio of palmitoyl-chitosan polymer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and biotin-hydrazide was 1:(0.15-0.3):(0.03-0.08):(0.05-0.15).

[0027] In the hydrophobic modification stage, by controlling the molar ratio of palmitoyl groups to chitosan units and using N-hydroxysuccinimide-activated palmitate to react for a long time at 50-60℃, uniform grafting of hydrophobic chains was ensured, effectively enhancing the amphiphilicity of the polymer and laying the foundation for its self-assembly in the aqueous phase to form structurally stable nanoparticles. Subsequently, unreacted reagents and byproducts were efficiently removed by dialysis purification, ensuring the purity of the intermediate product, palmitoyl-chitosan polymer. Finally, in the biotinylation modification, the carboxyl groups of chitosan were gently activated under physiological pH conditions using an EDC / NHS system and fully reacted with biotin-hydrazide under light-protected conditions. By precisely controlling the mass ratio of each component, high-density, directional coupling of biotin molecules was achieved, and physically adsorbed impurities were effectively removed by stepwise washing with a buffer containing polysorbate-20 and pure water.

[0028] S1.1.3 Preparation of biotinylated nanoparticles with guanidine isothiocyanate and hydrophobic alkyl groups attached to their surface: Biotinylated nanoparticles were dispersed in a borate buffer solution at pH 8.5, and lauric acid-N-hydroxysuccinimide and benzyl isothiocyanate EDTA were added sequentially. The mixture was stirred at room temperature for 10-12 h, and the precipitate was collected by centrifugation. The precipitate was washed sequentially with ethanol and pure water and dried to obtain biotinylated nanoparticles with guanidine isothiocyanate and hydrophobic alkyl groups. The mass ratio of biotinylated nanoparticles to lauric acid-N-hydroxysuccinimide to benzyl isothiocyanate EDTA was 1:(0.2-0.5):(0.5-1.0).

[0029] Using a borate buffer solution at pH 8.5 as the reaction medium ensured both the high reactivity of lauric acid-N-hydroxysuccinimide and benzyl isothiocyanate EDTA and the dispersion stability of the nanoparticles. By precisely setting the mass ratio and allowing a long reaction at room temperature, hydrophobic alkyl chains and guanidine isothiocyanate groups were simultaneously introduced. The guanidine isothiocyanate groups attached to the surface of the alkyl chains can target the protein components of the Mycobacterium tuberculosis cell wall, efficiently cleaving the cell wall structure through strong hydrogen bond disruption and protein denaturation while strictly maintaining the integrity of DNA phosphodiester bonds. The hydrophobic alkyl chains, on the other hand, synergistically enhance cell wall permeability by penetrating and interfering with the ordered structure of lipid layers such as mycolic acid in the cell wall. These two components form a spatiotemporal synergistic effect on the nanoparticle carrier, effectively overcoming the mucus barrier to reach the bacterial surface and constructing a locally high-concentration lysis microenvironment around the bacterial cells through a directional lysis mechanism. This completely disintegrates the cell wall while perfectly preserving the integrity of nucleic acid molecules, providing a high-quality template for subsequent PCR detection and significantly improving the sensitivity and reliability of Mycobacterium tuberculosis detection.

[0030] S1.2 Add the biotinylated metal complexing agent and biotinylated nanoparticles to the lysis working solution, stir, then add the sample to be tested containing Mycobacterium tuberculosis, incubate at 45~50℃ for 15~20 min, centrifuge at 10000~12000*g for 8~10 min, filter, and obtain the supernatant. The lysis working solution is a 100mM Tris-HCl buffer solution with a pH of 8.0. The concentration of the biotinylated metal complexing agent in the lysis working solution is 4~6mM, and the concentration of the biotinylated nanoparticles is 0.5~1.5μg / mL.

[0031] In a Tris-HCl buffer at pH 8.0, 4–6 mM of biotinylated metal complexing agent efficiently chelates metal ions in the solution, not only disrupting the polysaccharide gel structure of the mucus and reducing sample viscosity, but also eliminating metal ions that inhibit PCR enzymes. Simultaneously, 0.5–1.5 μg / mL of biotinylated nanoparticles, with their surface guanidine isothiocyanate and hydrophobic alkyl groups, synergistically act on the cell wall of Mycobacterium tuberculosis under mild heating conditions of 45–50 °C, efficiently denaturing proteins and disintegrating lipid components, thereby achieving targeted lysis while preserving DNA integrity. After the reaction, high-speed centrifugation and filtration effectively separate the lysis products from biotin-labeled impurity complexes (such as cell wall fragments adsorbed by nanoparticles and complexed metal ions).

[0032] S2. Add the magnetic bead suspension to the supernatant, stir and react at room temperature, let stand, take the supernatant and add it to the purification column, purify and wash to obtain the test sample, in which the magnetic bead surface contains streptavidin.

[0033] Magnetic beads with streptavidin on their surface efficiently and specifically capture and remove labeled inhibitor complexes in solution, including metal ion-metal complexes, lipid-hydrophobic alkyl group fragments, and small molecule fragments captured by nanoparticles, through the high affinity interaction between biotin and streptavidin. Finally, the samples are finely purified by a purification column to remove impurities from the test samples.

[0034] Step S2 includes: S2.1 Dissolve ferric nitrate nonahydrate and ferrous chloride hexahydrate in ultrapure water. Under nitrogen atmosphere, add ammonia dropwise, heat to 80°C and stir for 1-1.5 h. Then add sodium citrate solution and continue stirring for 2-2.5 h to obtain carboxyl-modified magnetic beads. The mass ratio of ferric nitrate nonahydrate: ferrous chloride hexahydrate: ammonia: sodium citrate: ultrapure water is 1:(0.55-0.62):(3.2-4.5):(1.4-1.6):(70-80). Under nitrogen protection, ferric nitrate nonahydrate and ferrous chloride hexahydrate react in an alkaline ammonia environment at 80℃, ensuring the efficient generation and excellent magnetic responsiveness of magnetic Fe3O4 nanocrystal nuclei. Subsequently, sufficient sodium citrate is added for surface ligand exchange and stabilization modification, forming a robust hydrophilic modification layer on the surface of the magnetic beads through its carboxyl groups. This not only significantly improves the colloidal stability and dispersibility of the magnetic beads, but also provides abundant active sites for their subsequent immobilization with streptavidin. Finally, the reaction is carried out in an optimized ultrapure water system, effectively controlling the uniformity and monodispersity of the nanoparticle size, thereby obtaining magnetic beads with high magnetic saturation strength, good biocompatibility, and surface carboxyl functionalization.

[0035] S2.2 Disperse carboxyl-modified magnetic beads in 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.0, add N-(3-dimethylhydropropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, activate at room temperature for 30 min, separate by magnetic field, discard the supernatant to obtain activated magnetic beads, wherein the mass ratio of carboxyl-modified magnetic beads:N-(3-dimethylhydropropyl)-N'-ethylcarbodiimide hydrochloride:N-hydroxysuccinimide is 1:(0.1~0.3):(0.05~0.15).

[0036] In a 2-(N-morpholino)ethanesulfonic acid (MES) buffer system at pH 6.0, by optimizing the mass ratio of carboxyl-modified magnetic beads to EDC and NHS, and activating them at room temperature for 30 minutes, the carboxyl groups on the surface of the magnetic beads were efficiently and directionally converted into active NHS ester intermediates. This significantly improved the efficiency and specificity of subsequent covalent coupling with streptavidin. Furthermore, the short activation time and precise reagent dosage effectively reduced the hydrolysis or side reactions of the activating groups, maintaining the reactivity of the intermediates. Simultaneously, after activation, unreacted EDC / NHS and byproducts were rapidly removed by magnetic field separation, avoiding excessive cross-linking or aggregation, ultimately yielding functionalized magnetic beads with high activation and good stability.

[0037] S2.3. Disperse the activated magnetic beads in Tris buffer at pH 7.5 containing 1.5~2.5 mg / mL streptavidin and react at room temperature for 4~6 h. Separate them with a magnetic field to obtain magnetic beads containing streptavidin. Then add Tris buffer at pH 8.0 containing 100 mM ethanolamine and 1% bovine serum albumin and react for 1~2 h to obtain blocked magnetic beads. Disperse them in Tris buffer at pH 7.5 containing 0.1% polysorbate-20 and 0.1% bovine serum albumin to obtain a magnetic bead suspension.

[0038] Reacting in Tris buffer at pH 7.5 for 4–6 hours ensured the formation of efficient and stable amide bonds between the NHS esters on the surface of the activated magnetic beads and the primary amino groups of streptavidin, achieving high-density, directional immobilization of streptavidin. Subsequently, double blocking with ethanolamine and bovine serum albumin in a buffer system at pH 8.0 effectively quenched residual activation sites and blocked non-specific binding sites, significantly reducing background interference in subsequent bioseparation processes. Finally, the magnetic beads were dispersed in a stabilizing buffer containing polysorbate-20 and bovine serum albumin, further enhancing the colloidal stability of the magnetic beads and preventing their aggregation. Ultimately, streptavidin magnetic beads with high bioactivity, low non-specific adsorption, and good storage stability were obtained, laying a reliable foundation for the efficient targeted capture and separation application based on the biotin-streptavidin system.

[0039] S2.4 Add magnetic bead suspension to the supernatant and stir at room temperature for 1-2 hours. Separate by magnetic field, take the supernatant and add it to the purification column for purification and washing to obtain the test sample.

[0040] The supernatant was reacted with the magnetic bead suspension at room temperature for 1-2 hours to ensure that residual biotin-labeled impurity complexes were efficiently and specifically captured by the magnetic beads through high-affinity biotin-streptavidin interactions. Magnetic field separation rapidly and thoroughly separated these interfering substances from the solution, significantly reducing background interference. The collected supernatant was then finely purified using a purification column to further remove residual salts, unbound biotinylated molecules, and other trace contaminants, ultimately obtaining a high-purity, high-integrity nucleic acid sample. This provided a high-quality template for subsequent fluorescent PCR detection, thereby improving the overall sensitivity, specificity, and reliability of the detection.

[0041] S3. Add the test sample to the amplification reagent for fluorescent PCR amplification, and then use the fluorescence melting curve to analyze the PCR amplification product to complete the detection.

[0042] The amplification reagent in step S3 includes: The sequences of the upper primer IS6110-F, the lower primer IS6110-R, and the probe IS6110-Probe used for the identification of Mycobacterium tuberculosis complexes are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3. The sequences of the upper primer rpoB-F, lower primer rpoB-R, probe rpoB-Probe1, probe rpoB-Probe2, probe rpoB-Probe3, and probe rpoB-Probe4 for detecting rifampicin resistance gene mutations are shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9. The sequences of the upper primer KatG-F, lower primer KatG-R, and probe KatG-Probe for detecting isoniazid resistance gene mutations are shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12; the sequences of the upper primer inhA-F, lower primer inhA-R, and probe inhA-Probe are shown in SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15; and the sequences of the upper primer ahpC-F, lower primer ahpC-R, probe ahpC-Probe1, and probe ahpC-Probe2 are shown in SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.19. The sequences of the upper primer gyrA-F, lower primer gyrA-R, and probe gyrA-Probe for gene mutation detection of quinolone drugs are shown in SEQ ID NO.20, SEQ ID NO.21, and SEQ ID NO.22. The sequences of the upper primer embB306-F, lower primer embB306-R, and probe embB306-Probe for detecting ethambutol resistance gene mutations are shown in SEQ ID NO.23, SEQ ID NO.24, and SEQ ID NO.25; the sequences of the upper primer embB406-F, lower primer embB406-R, and probe embB406-Probe are shown in SEQ ID NO.26, SEQ ID NO.27, and SEQ ID NO.28; and the sequences of the upper primer embB497-F, lower primer embB497-R, and probe embB497-Probe are shown in SEQ ID NO.29, SEQ ID NO.30, and SEQ ID NO.31.

[0043] A probe labeled with a fluorescent group and a quencher group at both ends is added to the PCR system. During PCR, a single-stranded oligonucleotide sequence complementary to the probe sequence is amplified. After amplification, a melting curve analysis process is added, and the change in fluorescence value is monitored in real time. By calculating the negative derivative of fluorescence value with temperature, the melting peak curve of the probe-probe hybridization product can be obtained, and the Tm value can be derived, thereby inferring the mutation information of the sequence. If the target sequence and the probe are perfectly matched, the Tm value of the probe-probe hybridization with the sequence is the highest; if the probe and the sequence are not perfectly matched, such as due to point mutation, insertion, or deletion, the Tm value of the probe-probe hybridization with the sequence is lower than the Tm value of the probe-probe hybridization with a perfectly matched sequence. The degree of decrease in Tm value is related to the type of point mutation, the number of inserted or deleted bases, and the location of the mutation site.

[0044] The above detection methods can simultaneously identify Mycobacterium tuberculosis complexes and detect resistance mutations to rifampin, isoniazid, quinolones, and ethambutol in the same detection procedure.

[0045] In addition, a kit is provided, which contains a lysis reagent tube, a suspension reagent tube, and an amplification reagent tube, wherein the lysis reagent tube, the suspension reagent tube, and the amplification reagent tube respectively contain the lysis working solution, the magnetic bead suspension, and the amplification reagent in the above-described method for detecting Mycobacterium tuberculosis.

[0046] It should be noted that the samples used in this application are all animal experimental samples obtained by injecting known mutation types of Mycobacterium tuberculosis, such as oral mucus from experimental monkeys.

[0047] For example, the present invention illustrates a detection method that simultaneously performs the identification of Mycobacterium tuberculosis complex and the detection of resistance mutations to rifampin, isoniazid, quinolones, and ethambutol in the same detection procedure: 1. Mycobacterium tuberculosis nucleic acid primer and probe set: A set of primers and probes was obtained through screening, containing 9 pairs of primers and 13 probes for 7 genes (IS6110, rpoB, katG, inhApromoter, aphCpromoter, gyrA, embB). The Mycobacterium tuberculosis complex and its drug resistance genes for four drugs can be identified simultaneously by fluorescent PCR, which increases the number of drugs that can be detected. Identification of Mycobacterium tuberculosis using the IS6110 gene; detection of rifampicin resistance gene mutations using mutations in the 27 amino acid codon regions (81 bp, rifampicin resistance-determining region) of the rpoB gene from 426 to 452; detection of isoniazid resistance gene mutations using mutations in the ahpC promoter region (-44 to -30 and -15 to -4), the inhA promoter region (-17 to -8), and the katG315 codon; detection of fluoroquinolone resistance gene mutations using mutations in codons 88 to 94 of the gyrA gene; and detection of ethambutol resistance gene mutations using mutations in codons 306, 406, and 497 of the embB gene.

[0048] The nucleic acid primer and probe set for Mycobacterium tuberculosis is shown in Table 1: Table 1 Primer and probe sets 2. Composition and components of amplification reagent tubes: 2.1 The composition and components of the reagent tubes are shown in Table 2: Table 2. Composition of reagent tubes 2.2 Amplification reagent formulation: The 10×Buffer formula is shown in Table 3: Table 3 10×Buffer Recipe The amplification reagent formulations are shown in Table 4: Table 4 Amplification reagent formulation 2.3 Freeze-drying: Eight amplification reagents were sequentially aliquoted into PCR tubes and lyophilized according to the lyophilization process curves shown in Table 5. The lyophilized tubes exhibited the following shapes: Figure 2 As shown, remove the PCR tubes from the container under conditions of humidity below 20% and cap them. Place the PCR reagent tubes into the inner packaging bag, vacuum seal, and store at 2–8°C.

[0049] Table 5 Freeze-drying process 2.4 The reagent tube testing procedure is shown in Table 6: Table 6. Reagent Kit Testing Procedure 2.5 Positive quality control process: 2.5.1 Preparation of positive and negative control samples: The positive control is a plasmid containing the target gene, which includes IS6110, rpoB, katG, inhA promoter, ahmC promoter, gyrA, and embB, all of which are sensitive genes. The positive control is prepared using TE buffer at a concentration of 0.01 ng / µL.

[0050] The negative control was TE solution.

[0051] 2.5.2 Lyophilized positive control samples: After preparation, 64µL of the positive control solution was dispensed into 1.5mL centrifuge tubes, and lyophilized powder was prepared according to the lyophilization process curve of the amplification reagent.

[0052] 3. Combined detection kit for Mycobacterium tuberculosis and drug resistance genes (identification + rifampin + isoniazid + quinolones + ethambutol): Resistance to rifampin and isoniazid was determined using national reference standards from the China National Institutes for Food and Drug Control. Resistance to quinolones and ethambutol was determined using externally synthesized plasmid reference standards containing target gene fragments. The gene synthesis company was General Biotechnology (Anhui) Co., Ltd. The resistance sites of the involved resistance genes (rpoB, katG, inhA, ahpC, gyrA, embB) were all referenced to the World Health Organization's "Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance" and other literature. The national reference material for the detection reagent of rifampicin resistance gene of Mycobacterium tuberculosis was purchased from the National Institutes for Food and Drug Control, product batch number: 230033-202304; National Reference Material for Detection of Isoniazid Resistance Gene in Mycobacterium Tuberculosis (purchased from China National Institutes for Food and Drug Control, batch number: 230034-202303); The Mycobacterium tuberculosis quinolone resistance gene detection strain H37Ra was donated by the Shanghai Key Laboratory of Tuberculosis (Lung) (Shanghai Pulmonary Hospital), with the number ATCC25277.

[0053] The test results are explained below: 3.1 Interpretation of positive control: If all 8 wells of the PCR reaction tube have corresponding reference values ​​or melting peaks and all meet the reference value range specified in Table 7, the positive control can be judged as qualified; otherwise, the positive control is unqualified and the experimental results are deemed invalid. Table 7. Detection range of positive controls in each channel. The Tm values ​​were common values ​​obtained from the fully automated medical PCR analysis system (SLAN-96S; Shanghai Hongshi Medical Technology Co., Ltd.) as reference values. The Tm values ​​for this experiment were based on those obtained automatically by the instrument.

[0054] 3.2 Interpretation of Negative Controls: The negative control contains template-free tris(hydroxymethyl)aminomethane, which monitors whether contamination occurred during sample addition or aerosol contamination from the air. If any channel of the negative control shows a reference value or melting peak, the negative control is considered unqualified, indicating possible nucleic acid contamination in the operating environment. Simultaneous testing of samples may produce false positive results, rendering the experimental results invalid. The results of the drug resistance assessment of the samples are shown in Table 8-11 below. The final results of the drug resistance assessment of rifampin, isoniazid, fluoroquinolones and ethambutol are obtained by combining the results of Table 8-11.

[0055] Table 8 Interpretation of Rifampicin Resistance Table 9 Interpretation of Isoniazid Resistance Table 10 Interpretation of Quinolone Drug Resistance Table 11 Interpretation of Ethambutol Resistance 3.3 Kit Detection Data: The compositions of rifampin, isoniazid, quinolone drugs, and ethambutol-sensitive and resistant reference materials are shown in Table 12. Table 12 Composition of Sensitive and Resistant Reference Materials Continued from Table 12 Continued from Table 12 Continued from Table 12 Table 13 shows the test data for rifampin, isoniazid, quinolones, and ethambutol sensitive and resistant reference standards: Table 13 Detection Data Continued from Table 13 Continued from Table 13 The test results for rifampin, isoniazid, quinolones, and ethambutol sensitivity and resistance reference standards are shown in Table 14: Table 14 Test Results Continued from Table 14 Continued from Table 14 As shown in Tables 13-14, based on the detection data and results of rifampin, isoniazid, quinolones, and ethambutol sensitive and resistant reference standards, this method can simultaneously complete the identification of Mycobacterium tuberculosis complex and the accurate detection of resistance mutations in rifampin, isoniazid, quinolones, and ethambutol in the same detection procedure.

[0056] The above embodiments are preferred embodiments of this application, but the implementation of this application is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of this application shall be considered equivalent substitutions and shall be included within the protection scope of this application.

Claims

1. A method for detecting Mycobacterium tuberculosis, characterized in that, The detection method includes the following steps: S1. Add the sample containing Mycobacterium tuberculosis to the lysis working solution, keep it warm, centrifuge, and take the supernatant. The lysis working solution contains a biotinylated metal complexing agent and biotinylated nanoparticles, and the biotinylated nanoparticles are connected to guanidine isothiocyanate and hydrophobic alkyl groups. S2. Add the magnetic bead suspension to the supernatant, stir and react at room temperature, let stand, take the supernatant and add it to the purification column, purify and wash to obtain the test sample, in which the magnetic bead surface contains streptavidin; S3. Add the test sample to the amplification reagent for fluorescent PCR amplification, and then use the fluorescence melting curve to analyze the PCR amplification product to complete the detection.

2. The method for detecting Mycobacterium tuberculosis according to claim 1, characterized in that, In step S1, the biotinylated metal complexing agent includes at least one of N1-ethylenediaminetetraacetic acid-biotin, N1-diethylenetriaminetetraacetic acid-biotin, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin. The biotinylated nanoparticles include at least one of biotinylated silica nanoparticles, biotin-cholesterol-polycaprolactone-polylactic acid nanoparticles, and biotin-palmitoyl-chitosan nanoparticles. The hydrophobic alkyl group is derived from at least one of sodium dodecyl sulfate, n-dodecyl-β-D-maltopyranoside, and hexadecyltrimethylammonium bromide.

3. A method for detecting Mycobacterium tuberculosis according to claim 1 or 2, characterized in that, Step S1 includes: S1.1 Prepare biotinylated metal complexing agents and biotinylated nanoparticles with guanidine isothiocyanate and hydrophobic alkyl groups on their surfaces, respectively. S1.2 Add the biotinylated metal complexing agent and biotinylated nanoparticles with guanidine isothiocyanate and hydrophobic alkyl groups on their surface to the lysis working solution, stir, then add the sample to be tested containing Mycobacterium tuberculosis, incubate at 45~50℃ for 15~20 min, centrifuge at 10000~12000*g for 8~10 min, filter, and obtain the supernatant. The lysis working solution is a 100mM Tris-HCl buffer solution with pH 8.0, and the concentration of the biotinylated metal complexing agent in the lysis working solution is 4~6mM and the concentration of the biotinylated nanoparticles is 0.5~1.5μg / mL.

4. The method for detecting Mycobacterium tuberculosis according to claim 3, characterized in that, The preparation method of the biotinylated metal complex in step S1.1 includes: S1.1.1 The metal complexing agent, N-(3-dimethylhydropropyl)-N'-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide are sequentially added to dimethyl sulfoxide at 0-4°C. After stirring and dispersing, the mixture is heated to room temperature and reacted for 2-4 hours. Then, biotinylate and an organic base are added, and the reaction is carried out at 30-35°C for 6-12 hours. The mixture is then added to diethyl ether at 0-4°C, the precipitate is collected by filtration and dried to obtain the biotinylated metal complexing agent, wherein the metal complexing agent includes ethylenediaminetetraacetic acid, ... At least one of diethylenetriaminetetraacetic acid and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, the organic base being N,N-diisopropylethylamine and / or triethylamine, and the molar ratio of the metal complexing agent N-(3-dimethylhydropropyl)-N'-ethylcarbodiimide hydrochloride: N-hydroxysuccinimide: biotinylate: organic base being 1:(1.02~1.03):(1.02~1.03):(1.05~1.07):(2~3).

5. The method for detecting Mycobacterium tuberculosis according to claim 3, characterized in that, The preparation method of biotinylated nanoparticles with guanidine isothiocyanate and hydrophobic alkyl groups attached to their surface in step S1.1 includes: S1.1.2 Tetraethyl orthosilicate, anhydrous ethanol, pure water, and concentrated ammonia were mixed and stirred at room temperature for 6-8 hours. After centrifugation, washing with anhydrous ethanol, and drying, nanoparticles were obtained. These nanoparticles were then dispersed in anhydrous ethanol, and 3-aminopropyltriethoxysilane was added. The mixture was heated to 70°C and refluxed for 10-12 hours. After centrifugation, washing with anhydrous ethanol, and drying, ammoniated nanoparticles were obtained. Finally, these nanoparticles were dispersed in dimethyl sulfoxide, and biotin-N-hydroxysuccinimide ester was added. The mixture was stirred at room temperature in the dark for 20-24 hours, followed by centrifugation, filtration, and subsequent... Biotinylated nanoparticles were obtained by washing and drying with N,N-dimethylformamide and pure water, wherein the volume ratio of tetraethyl orthosilicate: anhydrous ethanol: pure water: concentrated ammonia was 1:(30~50):(8~12):(1.2~2), the mass ratio of silica: anhydrous ethanol:3-aminopropyltriethoxysilane was 1:(10~20):(0.8~1.5), and the mass ratio of aminated silica:dimethyl sulfoxide:biotin-N-hydroxysuccinimide ester was 1:(10~15):(0.3~0.8). S1.1.

3. Biotinylated nanoparticles were dispersed in a borate buffer solution at pH 8.5, and lauric acid-N-hydroxysuccinimide and benzyl isothiocyanate EDTA were added sequentially. The mixture was stirred at room temperature for 10-12 h, and the precipitate was collected by centrifugation. The precipitate was washed sequentially with ethanol and pure water and dried to obtain biotinylated nanoparticles with guanidine isothiocyanate and hydrophobic alkyl groups. The mass ratio of biotinylated nanoparticles to lauric acid-N-hydroxysuccinimide to benzyl isothiocyanate EDTA was 1:(0.2-0.5):(0.5-1.0).

6. The method for detecting Mycobacterium tuberculosis according to claim 3, characterized in that, The preparation method of biotinylated nanoparticles in step S1.1 further includes: S1.1.

2. Cholesterol, polycaprolactone, polylactic acid, and stannous octoate were dissolved in dichloromethane and reacted at 120-130°C for 6-8 hours under inert gas protection. After filtration, washing, and vacuum drying, a copolymer was obtained. Then, poloxamer was added to a mixed solvent of dimethylformamide and acetone as the oil phase, and added to an aqueous solution of polyvinyl alcohol under stirring. The mixture was stirred at room temperature for 8-12 hours, centrifuged, and washed with deionized water to obtain nanoparticles. These nanoparticles were dispersed in a phosphate buffer solution at pH 7.4, and a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide was added. The mixture was activated at room temperature for 0.5-1 hour, followed by the addition of biotin-hydrazide. The mixture was reacted at room temperature for 12-16 hours in the dark. After the reaction, the nanoparticles were centrifuged and then processed sequentially with a phosphate buffer solution containing 0.05% polysorbate-20 and pure water. Washing and drying yielded biotin-cholesterol-polycaprolactone-polylactic acid nanoparticles, wherein the mass ratio of cholesterol, polycaprolactone, polylactic acid, stannous octoate, and dichloromethane was 1:(3~5):(5~10):(0.03~0.08):(15~25), the mass ratio of copolymer, poloxamer, and mixed solvent was 1:(0.5~2):(15~25), the mass ratio of dimethylformamide to acetone in the mixed solvent was 3:7, the concentration of polyvinyl alcohol in the polyvinyl alcohol aqueous solution was 1~3wt%, the mass ratio of polyvinyl alcohol aqueous solution to oil phase was (8~12):1, and the mass ratio of nanoparticles, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and biotin-hydrazide was 1:(0.15~0.3):(0.03~0.08):(0.05~0.15).

7. The method for detecting Mycobacterium tuberculosis according to claim 3, characterized in that, The preparation method of biotinylated nanoparticles in step S1.1 further includes: S1.1.

2. Chitosan was dissolved in a 1% (w / w) aqueous acetic acid solution to prepare a 0.5-2% (w / w) chitosan solution. Then, N-hydroxysuccinimide-activated palmitate was added at a palmitoyl to chitosan unit molar ratio of 1:(0.2-0.5). The mixture was stirred at 50-60°C for 12-16 hours. After the reaction, the mixture was transferred to a dialysis bag and purified by dialysis with an ethanol aqueous solution containing 0.1% SDS and then by lyophilization with pure water. The purified solution was then obtained by freeze-drying to obtain a palmitoyl-chitosan polymer. This polymer was then dispersed in a phosphate buffer solution at pH 7.4, and 1-ethyl-3-(3-dimethylaminopropyl) was added. Carbodiimide hydrochloride and N-hydroxysuccinimide were reacted at room temperature for 0.5-1 h. Biotin-hydrazide was added, and the mixture was reacted at room temperature for 12-16 h under light-protected conditions. The precipitate was collected by centrifugation and washed and dried sequentially with phosphate buffer containing 0.05% polysorbate-20 and pure water to obtain biotin-palmitoyl-chitosan nanoparticles. The mass ratio of palmitoyl-chitosan polymer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and biotin-hydrazide was 1:(0.15-0.3):(0.03-0.08):(0.05-0.15).

8. The method for detecting Mycobacterium tuberculosis according to claim 1, characterized in that, Step S2 includes: S2.1 Dissolve ferric nitrate nonahydrate and ferrous chloride hexahydrate in ultrapure water. Under nitrogen atmosphere, add ammonia dropwise, heat to 80°C and stir for 1-1.5 h. Then add sodium citrate solution and continue stirring for 2-2.5 h to obtain carboxyl-modified magnetic beads. The mass ratio of ferric nitrate nonahydrate: ferrous chloride hexahydrate: ammonia: sodium citrate: ultrapure water is 1:(0.55-0.62):(3.2-4.5):(1.4-1.6):(70-80). S2.2 Disperse carboxyl-modified magnetic beads in 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.0, add N-(3-dimethylhydropropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, activate at room temperature for 30 min, separate by magnetic field, discard the supernatant to obtain activated magnetic beads, wherein the mass ratio of carboxyl-modified magnetic beads:N-(3-dimethylhydropropyl)-N'-ethylcarbodiimide hydrochloride:N-hydroxysuccinimide is 1:(0.1~0.3):(0.05~0.15); S2.

3. Disperse the activated magnetic beads in Tris buffer at pH 7.5 containing 1.5~2.5 mg / mL streptavidin and react at room temperature for 4~6 h. Separate them with a magnetic field to obtain magnetic beads containing streptavidin. Then add Tris buffer at pH 8.0 containing 100 mM ethanolamine and 1% bovine serum albumin and react for 1~2 h to obtain blocked magnetic beads. Disperse them in Tris buffer at pH 7.5 containing 0.1% polysorbate-20 and 0.1% bovine serum albumin to obtain a magnetic bead suspension. S2.4 Add magnetic bead suspension to the supernatant and stir at room temperature for 1-2 hours. Separate by magnetic field, take the supernatant and add it to the purification column for purification and washing to obtain the test sample.

9. The method for detecting Mycobacterium tuberculosis according to claim 1, characterized in that, The amplification reagent in step S3 includes: The sequences of the upper primer IS6110-F, the lower primer IS6110-R, and the probe IS6110-Probe used for the identification of Mycobacterium tuberculosis complexes are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.

3. The sequences of the upper primer rpoB-F, lower primer rpoB-R, probe rpoB-Probe1, probe rpoB-Probe2, probe rpoB-Probe3, and probe rpoB-Probe4 for detecting rifampicin resistance gene mutations are shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.

9. The sequences of the upper primer KatG-F, lower primer KatG-R, and probe KatG-Probe for detecting isoniazid resistance gene mutations are shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12; the sequences of the upper primer inhA-F, lower primer inhA-R, and probe inhA-Probe are shown in SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15; and the sequences of the upper primer ahpC-F, lower primer ahpC-R, probe ahpC-Probe1, and probe ahpC-Probe2 are shown in SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.

19. The sequences of the upper primer gyrA-F, lower primer gyrA-R, and probe gyrA-Probe for detecting gene mutations in quinolone drugs are shown in SEQ ID NO.20, SEQ ID NO.21, and SEQ ID NO.

22. The sequences of the upper primer embB306-F, lower primer embB306-R, and probe embB306-Probe for detecting ethambutol resistance gene mutations are shown in SEQ ID NO.23, SEQ ID NO.24, and SEQ ID NO.25; the sequences of the upper primer embB406-F, lower primer embB406-R, and probe embB406-Probe are shown in SEQ ID NO.26, SEQ ID NO.27, and SEQ ID NO.28; and the sequences of the upper primer embB497-F, lower primer embB497-R, and probe embB497-Probe are shown in SEQ ID NO.29, SEQ ID NO.30, and SEQ ID NO.

31.

10. A reagent kit, characterized in that, The kit contains lysis reagent tubes, suspension reagent tubes, and amplification reagent tubes, each containing a lysis working solution, a magnetic bead suspension, and an amplification reagent as described in any one of claims 1-9 for the detection of Mycobacterium tuberculosis.

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