A detection reagent for Mycobacterium tuberculosis

Abstract

The present invention relates to the field of immunoassay technology, and in particular to a detection reagent for Mycobacterium tuberculosis, wherein the detection system comprises an antigen repair agent, an immune nanomagnetic bead capture agent, a fluorescent dye, a counterstain and an eluent, and the antigen repair agent is a periodic acid solution. The Mycobacterium tuberculosis detection reagent provided by the present invention greatly reduces the detection limit of combined mycobacteria in clinical samples, and at the same time greatly improves the sensitivity and accuracy of Mycobacterium tuberculosis detection.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a detection reagent for Mycobacterium tuberculosis and a preparation method thereof. Background Art

[0002] Tuberculosis (TB) is a zoonotic infectious disease caused by Mycobacterium tuberculosis (MTB). According to WHO statistics in 2021, there were 9.87 million new cases of tuberculosis and about 1.28 million deaths worldwide. TB remains one of the world's deadliest infectious killers. The incidence of tuberculosis in my country ranks second in the world, second only to India. Early rapid diagnosis and prevention of tuberculosis are key links in my country's control of tuberculosis. Laboratory etiological examination, that is, through the detection of Mycobacterium tuberculosis or its nucleic acid, can provide the most important laboratory basis for clinical diagnosis.

[0003] MTB is a slender, slightly curved bacillus, with a size of 1 to 0.4 μm. Common clinical detection methods for MTB include Ziehl-Neelsen acid-fast staining (ZN test), culture method, and auramine O fluorescence staining.

[0004] However, for patients with mild tuberculosis infection in the early stages of clinical illness, the bacterial count is low, and the common ZN detection method, culture method, and auramine O fluorescence staining method are prone to missed diagnosis due to excessively high detection limits. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defect of missed detection caused by too low tuberculosis content in samples of early tuberculosis patients in the prior art, thereby providing a detection reagent for Mycobacterium tuberculosis and a preparation method thereof.

[0006] The invention provides a detection reagent for Mycobacterium tuberculosis, comprising an antigen repair agent, an immune nanomagnetic bead capture agent, a fluorescent dye, a counterstain and an eluent, wherein the antigen repair agent is a periodic acid solution.

[0007] Preferably, the concentration of the periodic acid solution is 5-10 mg / mL.

[0008] Preferably, the concentration of the periodic acid solution is 8 mg / mL.

[0009] Preferably, the mass concentration of the immune nanomagnetic bead capture agent is 5-10 mg / mL.

[0010] Preferably, the mass concentration of the immune nanomagnetic bead capture agent is 10 mg / mL.

[0011] Preferably, the fluorescent dye is composed of: each 100 mL of fluorescent dye contains:

[0012] Auramine o 0.5-0.9g;

[0013] Rhodamine B 0.1-0.3g;

[0014] Phenol 1-2mL;

[0015] Ethanol 17-28.5mL;

[0016] Glycerol 19.5-30.5mL;

[0017] Dimethyl sulfoxide 10-11.5 mL;

[0018] The balance is water.

[0019] Preferably, the fluorescent dye is composed of: each 100 mL of fluorescent dye contains:

[0020] Auramine o 0.6-0.9g;

[0021] Rhodamine B 0.1-0.3g;

[0022] Phenol 2mL;

[0023] Ethanol 23.5 mL;

[0024] Glycerol 19.5 mL;

[0025] Dimethyl sulfoxide 10 mL;

[0026] The balance is water.

[0027] Preferably, the eluent composition is as follows: each 100 mL of eluent contains:

[0028] Hydrochloric acid 0.5-1mL;

[0029] Ethanol 59.5-64ml;

[0030] Water 35-40m1.

[0031] Preferably, the counterstain is a potassium permanganate solution with a concentration of 30-50 mg / mL.

[0032] Preferably, the counterstain is a potassium permanganate solution with a concentration of 30 mg / mL.

[0033] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0034] 1. The detection reagent for Mycobacterium tuberculosis provided by the present invention adopts periodic acid as an antigen repair solution, which plays the role of oxidative repair of specimens. The immune nanomagnetic beads can specifically enrich and purify MTB in the sample, increase the unit concentration of the specimen bacteria, increase the detection rate of specimen positives, and reduce the nonspecific staining in the next step. Auramine O plays the role of marking acid-fast mycobacteria, and rhodamine B is a red fluorescent pigment, which can make the staining effect of acid-fast bacilli better, so that acid-fast bacilli appear golden yellow. Ethanol is an organic cosolvent, which makes the powder dissolve better and maintains the stability of the solution. Glycerol is a humectant, which can increase the overall moisture retention and refractive index of the solution, improve the contrast, and thus make the staining clearer. Phenol is a penetrant, which can increase the penetration and staining effect of the reagent on the specimen cells. The hydrochloric acid in the eluent plays the role of washing and decolorization after staining. Mycobacterium tuberculosis is not decolorized due to its acid-fast characteristics, reducing the nonspecific staining of other bacteria. Potassium permanganate counterstain enhances the color contrast after the specimen is counterstained, which is convenient for clearer bacterial imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 This is a picture of the staining effect of sputum specimens treated with ZN acid-fast staining method;

[0037] Figure 2 This is a staining effect diagram of a sputum sample treated with the detection reagent provided in Example 2 of the present invention; DETAILED DESCRIPTION

[0038] Example 1

[0039] Test kits for Mycobacterium tuberculosis include:

[0040] (1) 100 mL of antigen retrieval agent, which is a periodic acid solution with a mass concentration of 10 mg / mL, and the preparation method thereof is as follows: weigh 1 g of periodic acid and fully dissolve it in 100 mL of distilled water;

[0041] (2) 100 mL of immune nano-magnetic bead capture agent, the concentration of which is 10 mg / mL, and the preparation method thereof is as follows:

[0042] Take 1g amino mesoporous silica-coated Fe3O4 / SiO2 nanoparticles as raw material (purchased from Angxing New Carbon Materials Changzhou Co., Ltd.), add 50mL dimethylformamide (DMF) into a conical flask, and after ultrasonic dispersion for 1h, add 10mL DMF dissolved with 3g succinic anhydride (SA) into the conical flask and stir at room temperature for 12h; after separation on a magnetic stand, wash with anhydrous ethanol 6 times, and vacuum dry at 50°C to obtain carboxylated magnetic microspheres. Add 30 mg of the carboxylated magnetic microspheres in the previous step to a 50 mL conical flask, then add 15 mL of distilled water, ultrasonically disperse for 5 min, then add 30 mg / mL of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and 30 mg / mL of N-hydroxysuccinimide (NHS) 1 mg each to a 50 mL conical flask, activate the carboxyl group by shaking in a water bath at room temperature for 0.5 h, then add 15 ml of a mixed solution of fuchsin and trehalose (the mass concentration of fuchsin and trehalose is 1 mg / mL), shake at room temperature for 24 h, separate on a magnetic stand, repeatedly wash with anhydrous ethanol and distilled water until the washing solution is no longer red, and vacuum dry at 40 ° C to obtain an active nano-magnetic bead stock solution coated with trehalose and fuchsin. Dissolve the active nano-magnetic bead stock solution in distilled water to reach the expected concentration of 10 mg / ml.

[0043] (3) 100 mL of fluorescent dye, which contains: 0.9 g of auramine O, 0.1 g of rhodamine B, 1 mL of phenol, 32.5 mL of distilled water, 28.5 mL of ethanol, 26.5 mL of glycerol, and 11.5 mL of dimethyl sulfoxide, with a total volume of 100 mL. Among them, the reagents (phenol, ethanol, glycerol, and dimethyl sulfoxide) are all of analytical grade (AR); the preparation method thereof is as follows: phenol, glycerol, and ethanol are fully mixed to obtain an organic solvent; a portion of distilled water is mixed with auramine O and rhodamine B to obtain a saturated solvent; and the saturated solvent, the organic solvent, and the remaining water are stirred and fully mixed to obtain the obtained product.

[0044] (4) 100 mL of eluent, comprising: 1 mL of hydrochloric acid, 64 mL of ethanol, and 35 mL of water; the concentration of hydrochloric acid is 1 mol / L, and the concentration of ethanol is anhydrous ethanol (analytical grade AR); the preparation method thereof is: fully mixing hydrochloric acid, ethanol, and water.

[0045] (5) 100 mL of counterstain, which is a potassium permanganate solution with a mass concentration of 50 mg / mL. The preparation method thereof is as follows: weigh 5 g of potassium permanganate and fully dissolve it in 100 mL of distilled water.

[0046] Example 2

[0047] Test kits for Mycobacterium tuberculosis include:

[0048] (1) 100 mL of antigen retrieval agent, which is a periodic acid solution with a mass concentration of 8 mg / mL, and is prepared by weighing 0.8 g of periodic acid and fully dissolving it in 100 mL of distilled water;

[0049] (2) 100 mL of immune nano-magnetic bead capture agent, the concentration of which is 5 mg / mL, and the preparation method thereof is the same as that in Example 1.

[0050] Dissolve the nanomagnetic bead stock solution in distilled water to reach the desired concentration of 5 mg / ml.

[0051] (3) 100 mL of fluorescent dye, which contains: 0.6-0.9 g of auramine O, 0.1-0.3 g of rhodamine B, 2 mL of phenol, 45.0 mL of distilled water, 23.5 mL of ethanol, 19.5 mL of glycerol, and 10 mL of dimethyl sulfoxide, with a total volume of 100 mL. The concentrations of the reagents (phenol, ethanol, glycerol, and dimethyl sulfoxide) are all of analytical grade (AR). The preparation method is as follows: phenol, glycerol, and ethanol are fully mixed to obtain an organic solvent; a portion of distilled water is mixed with auramine O and rhodamine B to obtain a saturated solvent; and the saturated solvent, the organic solvent, and the remaining water are stirred and fully mixed to obtain the dye.

[0052] (4) 100 mL of eluent, comprising: 0.5 mL of hydrochloric acid, 59.5 mL of ethanol, and 40 mL of water; the concentration of hydrochloric acid is 1 mol / L, and the concentration of ethanol is anhydrous ethanol (analytical grade AR); the preparation method thereof is: fully mixing hydrochloric acid, ethanol, and water.

[0053] (5) 100 mL of counterstain, which is a potassium permanganate solution with a mass concentration of 30 mg / mL. The preparation method thereof is as follows: weigh 3 g of potassium permanganate and fully dissolve it in 100 mL of distilled water.

[0054] Example 3

[0055] Test kits for Mycobacterium tuberculosis include:

[0056] (1) 100 mL of antigen retrieval agent, which is a periodic acid solution with a mass concentration of 5 mg / mL, and is prepared by weighing 0.5 g of periodic acid and fully dissolving it in 100 mL of distilled water;

[0057] (2) 100 mL of immune nano-magnetic bead capture agent, the concentration of which is 5 mg / mL, and the preparation method thereof is the same as that in Example 1.

[0058] Dissolve the active nanomagnetic bead stock solution in distilled water to reach the desired concentration of 5 mg / ml.

[0059] (3) 100 mL of fluorescent dye, which contains: 0.5 g of auramine O, 0.2 g of rhodamine B, 2 mL of phenol, 40.5 mL of distilled water, 17 mL of ethanol, 30.5 mL of glycerol, and 10 mL of dimethyl sulfoxide. The reagents (phenol, ethanol, glycerol, and dimethyl sulfoxide) are all of analytical grade (AR). The preparation method is as follows: phenol, glycerol, and ethanol are fully mixed to obtain an organic solvent; a portion of distilled water is mixed with auramine O and rhodamine B to obtain a saturated solvent; and the saturated solvent, the organic solvent, and the remaining water are stirred and fully mixed to obtain the obtained product.

[0060] (4) 100 mL of eluent, comprising: 0.5 mL of hydrochloric acid, 59.5 mL of ethanol, and 40 mL of water; the concentration of hydrochloric acid is 1 mol / L, and the concentration of ethanol is anhydrous ethanol (analytical grade AR); the preparation method thereof is: fully mixing hydrochloric acid, ethanol, and water.

[0061] (5) 100 mL of counterstain, which is a potassium permanganate solution with a mass concentration of 50 mg / mL. The preparation method thereof is as follows: weigh 5 g of potassium permanganate and fully dissolve it in 100 mL of distilled water.

[0062] The following is a representative example of the Mycobacterium tuberculosis detection reagent prepared in Example 2 of the present invention, and tests on indicators such as minimum detection limit, sensitivity, specificity and stability are performed on the Mycobacterium tuberculosis detection reagent provided by the present invention to illustrate the technical effect of the combined mycobacterium detection reagent provided by the present invention.

[0063] Experimental preparation

[0064] (1) Bacterial species and sputum specimen sources

[0065] Mycobacterium tuberculosis ATCC25177 and Escherichia coli CMCC(B)44102 were purchased from Xinyang Laiyao Biotechnology Co., Ltd., Staphylococcus aureus CMCC(B)26003, Lactobacillus rhamnosus ATCC53103 and Lactobacillus plantarum ATCC8014 were all purchased from Beijing Sanyao Technology Development Co., Ltd., and sputum specimens were obtained from 145 patients with suspected pulmonary tuberculosis who were initially diagnosed and admitted to Shandong Second General Hospital from May 2021 to February 2022. There were 85 males and 60 females, aged 26 to 71 years, with an average age of (52.3±5.6) years. The patients had no other infections, no other heart, liver, kidney, lung and other dysfunctions, and all patients voluntarily signed informed consent.

[0066] (2) Instruments and reagents

[0067] NGPro-32 liquid-based purification automatic staining machine was purchased from Jiangsu Nuoge Biotechnology Co., Ltd., the fully automatic microscope scanning system was purchased from Jiangsu Nuoge Biotechnology Co., Ltd., and the LEICADM500 fluorescence microscope was purchased from Hangzhou Tuxian Technology Co., Ltd.; BACTEC MGIT960 Mycobacterium Rapid Culture Analyzer was purchased from BD Company, USA, Thermo Scientific MK3 microplate reader was purchased from Jinan Cunchang Biotechnology Co., Ltd., desktop high-speed centrifuge H / T16MM was purchased from Hunan Hercy Instrument Equipment Co., Ltd., ZN acid-fast staining kit was purchased from Zhuhai BASO Biotechnology Co., Ltd., comprehensive Sutong liquid culture medium was purchased from Shanghai Ruichu Biotechnology Co., Ltd., brain heart infusion broth culture medium and MRS broth culture medium were purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.

[0068] (3) Establishment of immunomagnetic bead fluorescence staining method

[0069] The fully automatic NGPro-32 liquid-based purification automatic staining machine equipment was used for detection. The deep-well plate pre-packaged with the Mycobacterium tuberculosis detection reagent prepared in Example 2 of the present invention was taken out and inverted and mixed several times to resuspend the immune magnetic beads. The aluminum foil sealing film was carefully torn off before use. 1-3.5 ml of specimens were added to the 1st and 6th columns of the deep-well plate, and the deep-well plate was placed on the base of the deep-well plate of the fully automatic purification staining machine. Insert the magnetic rod sleeve into the slot of the magnetic rod sleeve holder of the fully automatic purification instrument, edit the extraction program, select the automated extraction program and run it; after the automated program ends, take out the purified specimens in the 5th and 10th columns for preparation. Open the fully automatic microscope scanning system all-in-one device, enter the main interface of the scanning software, click the complete mode, select the sample tray position where the slide is placed (5 specimens can be placed at the same time), click Refresh Preview Image-Wait for the preview image to be successfully jigsawed, adjust the focus position appropriately according to the actual situation, click Scan Now-Wait for the scan to end (12mm×12mm area, scanning time is less than 240S), after the scan is completed, click Diagnostic Report and Print. The device automatically detects under a high-power microscope in the UV band (excitation wavelength 330nm~380nm, emission wavelength 420nm). It contains an AI algorithm database, adopts a high-performance neural network algorithm, and integrates multiple optimization mechanisms such as SE and FPN. It achieves real-time detection of specimen MTB while ensuring a high recognition rate.

[0070] Index test

[0071] 1. Minimum detection limit experiment

[0072] Equilibrate the strain bottle of Mycobacterium tuberculosis ATCC 25177 strain to room temperature and open it in a biosafety cabinet; use a pipette to draw the matching strain recovery solution and add it to the strain bottle, gently rotate the bottle and blow for 5-10 seconds to fully mix the freeze-dried strain and the liquid and completely dissolve it; inoculate the liquid in the bottle into the comprehensive Sutong liquid culture medium and place it in a 37°C incubator for 28-35 days. Take an appropriate amount of positive Mycobacterium tuberculosis suspension and use an ELISA instrument to detect OD600=1 (bacterial number 3×10 8 CFU / ml) as the initial concentration, and then gradient dilution was performed to make the bacterial solution concentration 3×10 5 , 3×10 4 , 3×10 3 , 3×10 2 , 1.5×10 2 , 1×10 2 , 3×10, and the bacterial solutions of each concentration were respectively entered into the immunomagnetic bead fluorescence staining analysis system using the Mycobacterium tuberculosis detection reagent prepared in Example 2 of the present invention for detection, and the detection limit was analyzed. The results are shown in Table 1.

[0073] Table 1 Detection limit of immunomagnetic bead fluorescence staining method

[0074]

[0075] *“+” indicates a positive result under the microscope of this method.

[0076] As can be seen from Table 1, the lower limit of detection of the mycobacterium detection reagent provided in Example 2 is 30 CFU / ml. When the concentration is lower than this, the result may be judged as negative.

[0077] 2. Sensitivity and specificity tests

[0078] 50 portions of the above-mentioned Mycobacterium tuberculosis ATCC 25177 strain culture fluids of different concentrations were used as pure positive specimens, and were entered into the immunomagnetic bead fluorescence staining analysis system of the Mycobacterium tuberculosis detection reagent prepared by Example 2 of the present invention for detection, and sensitivity tests were performed. Staphylococcus aureus CMCC (B) 26003 strains and Escherichia coli CMCC (B) 44102 strains were balanced to room temperature, and after being resuscitated by a pipette with a matching strain resuscitation solution, they were inoculated into brain heart infusion broth culture medium, and were placed in a 37°C incubator for cultivation for 24-48h. Lactobacillus rhamnosus ATCC53103 strains and Lactobacillus plantarum ATCC8014 strains were balanced to room temperature, and after being resuscitated with corresponding strain resuscitation solution, they were inoculated into MRS broth culture medium, and were placed in a 37°C incubator for cultivation for 24-48h. After diluting and shaking the cultured bacterial suspension, cover the clean and dry blood cell counting plate with a cover glass, and then use a sterile capillary to drop a small drop of bacterial solution from the edge of the cover glass, allowing the bacterial solution to automatically enter the counting chamber along the gap by capillary penetration, so that the counting chamber is filled with bacterial solution, and observe the number of bacteria in the small squares under a microscope. [9] , and then the total number of bacteria in 1 mL of bacterial suspension was calculated. Then the four strains of bacterial suspension were diluted and mixed so that the concentration of each bacterial suspension in the mixed bacterial suspension (unit CFU / ml) was 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 10, 50 pure negative specimens were made from each concentration of the mixed bacterial suspension and entered into the immunomagnetic bead fluorescence staining analysis system for detection and specificity test. The results are shown in Tables 2 and 3.

[0079] Table 2 Sensitivity of immunomagnetic bead fluorescence staining method

[0080]

[0081] Table 3 Specificity of immunomagnetic bead fluorescence staining method (%)

[0082]

[0083] As shown in Table 2, the sensitivity of the Mycobacterium tuberculosis detection reagent provided in Example 2 of the present invention changes with the change of the initial bacterial solution concentration, and the change is obvious when it is close to the detection limit of 30 CFU / ml; when the concentration is lower than this, the sensitivity drops to 56%;

[0084] It can be seen from the specific experimental results of the immunomagnetic bead fluorescent staining method in Table 3 that when the other four strain suspensions were at different concentrations, the detection results of the mixed bacterial solution were all negative after the two-step treatment of magnetic absorption of immunomagnetic beads and fluorescent staining using the detection reagent for Mycobacterium tuberculosis provided in Example 2, indicating that the specificity of the detection method for combining mycobacteria provided in Example 2 was 100%.

[0085] 3. Stability test

[0086] A stability test was performed on the Mycobacterium tuberculosis detection reagent provided in an embodiment of the present invention. The specific experimental steps are: the Mycobacterium tuberculosis detection reagent provided in an embodiment of the present invention is placed at three different temperatures of 0°C, 20°C, and 30°C, respectively, for 30 days, 60 days, 90 days, 180 days, and 365 days, and the treated Mycobacterium tuberculosis detection reagent is used to detect sputum specimens. The results show that the sensitivity, specificity, and accuracy of the treated Mycobacterium tuberculosis are all above 95%.

[0087] The following is a clinical comparative experiment of the Mycobacterium tuberculosis detection reagent prepared in Example 2 of the present invention and the traditional Mycobacterium tuberculosis detection method. The comparison indicators include positive detection rate, sensitivity and accuracy, and staining experiment.

[0088] Experimental preparation

[0089] (1) Bacterial species and specimen sources

[0090] Mycobacterium tuberculosis ATCC25177 and Escherichia coli CMCC(B)44102 were purchased from Xinyang Laiyao Biotechnology Co., Ltd., Staphylococcus aureus CMCC(B)26003, Lactobacillus rhamnosus ATCC53103 and Lactobacillus plantarum ATCC8014 were all purchased from Beijing Sanyao Technology Development Co., Ltd., and sputum specimens were obtained from 145 patients with suspected pulmonary tuberculosis who were initially diagnosed and admitted to Shandong Second General Hospital from May 2021 to February 2022. There were 85 males and 60 females, aged 26 to 71 years, with an average age of (52.3±5.6) years. The patients had no other infections, no other heart, liver, kidney, lung and other dysfunctions, and all patients voluntarily signed informed consent.

[0091] (2) Instruments and reagents

[0092] NGPro-32 liquid-based purification automatic staining machine was purchased from Jiangsu Nuoge Biotechnology Co., Ltd., the fully automatic microscope scanning system was purchased from Jiangsu Nuoge Biotechnology Co., Ltd., and the LEICADM500 fluorescence microscope was purchased from Hangzhou Tuxian Technology Co., Ltd.; BACTEC MGIT960 Mycobacterium Rapid Culture Analyzer was purchased from BD Company, USA, Thermo Scientific MK3 microplate reader was purchased from Jinan Cunchang Biotechnology Co., Ltd., desktop high-speed centrifuge H / T16MM was purchased from Hunan Hercy Instrument Equipment Co., Ltd., ZN acid-fast staining kit was purchased from Zhuhai BASO Biotechnology Co., Ltd., comprehensive Sutong liquid culture medium was purchased from Shanghai Ruichu Biotechnology Co., Ltd., brain heart infusion broth culture medium and MRS broth culture medium were purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.

[0093] (3) Establishment of immunomagnetic bead fluorescence staining method

[0094] The fully automatic NGPro-32 liquid-based purification automatic staining machine equipment was used for detection. The deep-well plate pre-packaged with the Mycobacterium tuberculosis detection reagent prepared in Example 2 of the present invention was taken out and inverted and mixed several times to resuspend the immune magnetic beads. The aluminum foil sealing film was carefully torn off before use. 1-3.5 ml of specimens were added to the 1st and 6th columns of the deep-well plate, and the deep-well plate was placed on the base of the deep-well plate of the fully automatic purification staining machine. Insert the magnetic rod sleeve into the slot of the magnetic rod sleeve holder of the fully automatic purification instrument, edit the extraction program, select the automated extraction program and run it; after the automated program ends, take out the purified specimens in the 5th and 10th columns for preparation. Open the fully automatic microscope scanning system all-in-one device, enter the main interface of the scanning software, click the complete mode, select the sample tray position where the slide is placed (5 specimens can be placed at the same time), click Refresh Preview Image-Wait for the preview image to be successfully jigsawed, adjust the focus position appropriately according to the actual situation, click Scan Now-Wait for the scan to end (12mm×12mm area, scanning time is less than 240S), after the scan is completed, click Diagnostic Report and Print. The device automatically detects under a high-power microscope in the UV band (excitation wavelength 330nm~380nm, emission wavelength 420nm). It contains an AI algorithm database, adopts a high-performance neural network algorithm, and integrates multiple optimization mechanisms such as SE and FPN. It achieves real-time detection of specimen MTB while ensuring a high recognition rate.

[0095] (4) Clinical specimen collection

[0096] Collect the patient's general information, such as clinical symptoms, physical signs, X-ray, tuberculosis blood routine, blood biochemistry and other test results. Collect deep sputum specimens from each patient, with a qualified specimen volume of ≥1ml, and put the sputum specimens into a sterile plastic specimen box for sputum smear microscopy and culture.

[0097] 1. Positive detection rate, sensitivity and accuracy experiments

[0098] (1) Zn-Acid Fast Staining

[0099] A sputum specimen was directly smeared from each patient and stained according to the instructions of the kit; finally, it was observed under an optical microscope with an oil immersion lens; if the result was positive, Mycobacterium tuberculosis would appear red, and other bacteria and cells would appear blue.

[0100] (2) Immunomagnetic Fluorescence Staining

[0101] The sputum specimens of clinical patients (without special treatment) were added to the pre-packaged deep-well plate sample nest (pre-loaded with 4% sodium hydroxide digestion solution) combined with the mycobacterium detection reagent prepared in Example 2 of the present invention by a pipette; after purification and staining, the preparation was entered into a fully automatic fluorescent biological microscope for detection, and automatically detected under a high-power microscope in the UV band, and a result report was issued. If the result was positive, it could be seen that Mycobacterium tuberculosis was enriched and purified, and after fluorescent staining, the bacteria appeared golden yellow against a blue background.

[0102] (3) BACTEC 960 system culture method

[0103] Pipette the patient's sputum specimen into a 15ml centrifuge tube, add an equal volume of 4% sodium hydroxide solution, tighten the lid and vortex for 15-20s, and let it stand at room temperature for 15-20min. Add sterile phosphate buffer to 15ml, mix thoroughly and centrifuge at 3000g for 30min, discard the supernatant and add 1ml sterile phosphate buffer to resuspend the precipitate, take 0.5ml of the resuspended liquid and inoculate it in MGIT960 liquid rapid tuberculosis culture medium, and place it in the MGIT 960 rapid culture system for culture. According to the fluorescence intensity in the measurement tube, regularly judge the growth of Mycobacterium tuberculosis in the tube.

[0104] (5) Observation indicators

[0105] To analyze the feasibility of immunomagnetic bead fluorescence staining for the detection of Mycobacterium tuberculosis and to evaluate its diagnostic value in clinical tuberculosis detection; the diagnostic criteria were based on the results of the culture method as the gold standard, sensitivity = true positive number / total positive number; specificity = true negative number / total negative number; accuracy = (true positive number + true negative number) / (total positive number + total negative number).

[0106] (6) Statistical methods

[0107] The data of the test results were processed and analyzed using SPSS 22.0 statistical software. The measurement data were expressed as mean ± standard deviation (x ± s) and t-test was performed; the enumeration data were expressed as number of cases (n) and rate (%); the inter-group comparison was performed using the χ2 test, and if P < 0.05, the difference was considered to be significant and statistically significant.

[0108] Positive detection rate comparison test results

[0109] The comparison of the detection results of Mycobacterium tuberculosis by the three clinical detection methods is shown in Table 4. Among the 145 specimens, the Mycobacterium tuberculosis detection reagent provided in Example 2 was used, and the Mycobacterium tuberculosis was enriched and purified by immunomagnetic beads and then automatically stained. The positive rate detected by the fully automatic fluorescence scanner was 73.1%, which was significantly higher than the positive rate of the traditional ZN acid-fast staining method of 40.7%, and the difference was statistically significant (*χ2=31.0599, P<0.05); the positive rate of the immunomagnetic bead fluorescent staining method was lower than the positive rate of the culture method of 78.6%, but the difference was not significant and not statistically significant (**χ2=1.2052, P>0.05).

[0110] Table 4 Comparison of the detection results of three detection methods for Mycobacterium tuberculosis infection in TB patients (n)

[0111]

[0112] (* 2 =31.0599, P<0.05, **χ 2 =1.2052, P>0.05)

[0113] Comparative experimental results of sensitivity, specificity and accuracy

[0114] The comparison of the detection results of the two clinical staining methods and the culture method is shown in Tables 5 and 6. The sensitivity, specificity and accuracy of the combined mycobacterium detection reagent provided in Example 2 are 92.98%, 100% and 94.48%, which are significantly higher than the sensitivity, specificity and accuracy of the ZN acid-fast staining method (47.37%, 83.87% and 55.17%), and the difference is statistically significant (χ2=56.6647, P<0.05; χ2=5.4386, P<0.05; χ2=59.4792, P<0.05).

[0115] Table 5 Comparison of the test results of Example 2 and ZN acid-fast staining method with the results of the culture method (n)

[0116]

[0117] Table 6 Comparison of detection value of Example 2 and ZN acid-fast staining method (%)

[0118]

[0119] 2. Staining test

[0120] The sputum specimens were stained using the ZN method and the detection reagent provided in Example 2 of the present invention, respectively. The staining results of the ZN method are shown in Figure 1, and the staining results of the detection reagent provided in Example 2 of the present invention are shown in Figure 2. Figure 2 shown.

[0121] from Figure 1 It can be seen that acid-fast mycobacteria are red and slightly curved; other bacteria are blue. The overall background difference is not large, and it is not easy to identify acid-fast mycobacteria. For patients with early mild infection, due to the low content of mycobacteria, false negatives are easy to occur, and the probability of missed diagnosis is high.

[0122] from Figure 2 It can be seen that the detection reagent provided in Example 2 of the present invention can effectively enhance and improve the sensitivity, specificity and accuracy of the detection results by performing antigen repair, nanomagnetic bead enrichment and purification, and multiple fluorescent staining on Mycobacterium tuberculosis in sputum specimens. In the staining results, Mycobacterium tuberculosis appears golden yellow, with a slightly curved rod-shaped, spiral-shaped and cross-rod-shaped structure. The contrast between the stained Mycobacterium tuberculosis and the blue background is large, which is easy to identify with the naked eye, and can effectively improve the recognition efficiency and recognition accuracy. In addition, the staining results can clearly observe Mycobacterium tuberculosis without an oil immersion lens, and the result judgment is simpler and more efficient.

[0123] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A detection reagent for Mycobacterium tuberculosis, characterized in that: The detection reagent comprises an antigen repair agent, an immune nano-magnetic bead grabber, a fluorescent dye, a counterstain and an eluent, wherein the antigen repair agent is a periodic acid solution; the preparation method of the immune nano-magnetic bead grabber is as follows: Fe3O4 / SiO2 nanoparticles are used as raw materials, dissolved in ethanol, ultrasonically dispersed at room temperature, activated by amino modification, and treated with a solution containing fuchsin and trehalose to obtain active nano-magnetic beads coated with trehalose and fuchsin; the mass concentration of the nano-magnetic beads is 5-10 mg / mL; the fluorescent dye is composed of: each 100 mL of the fluorescent dye contains: Auramine o 0.5-0.9g; Rhodamine B 0.1-0.3g; Phenol 1-2mL; Ethanol 17-28.5mL; Glycerol 19.5-30.5mL; Dimethyl sulfoxide 10-11.5 mL; The balance is water.

2. The detection reagent according to claim 1, characterized in that The concentration of the periodic acid solution is 5-10 mg / mL.

3. The detection reagent according to claim 2, characterized in that The concentration of the periodic acid solution is 8 mg / mL.

4. The detection reagent according to claim 1, characterized in that The mass concentration of the immune nanomagnetic bead capture agent is 10 mg / mL.

5. The detection reagent according to claim 1, characterized in that The fluorescent dye is composed of: each 100 mL of fluorescent dye contains: Auramine o 0.6-0.9g; Rhodamine B 0.1-0.3g; Phenol 2mL; Ethanol 23.5mL; Glycerol 19.5 mL; Dimethyl sulfoxide 10 mL; The balance is water.

6. The detection reagent according to claim 1, characterized in that The eluent composition is as follows: each 100 mL of the eluent contains: Hydrochloric acid 0.5-1mL; Ethanol 59.5-64mL; 35-40mL of water.

7. The detection reagent according to claim 1, characterized in that The counterstain is a potassium permanganate solution with a concentration of 30-50 mg / mL.

8. The detection reagent according to claim 1, characterized in that The counterstain is a potassium permanganate solution with a concentration of 30 mg / mL.

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