Mycobacterium paratuberculosis nucleic acid extraction pretreatment splitting module and application thereof

Abstract

The invention belongs to the technical field of biology. In particular to a pretreatment splitting module for nucleic acid extraction of mycobacterium paratuberculosis and application thereof. Specifically, the invention relates to a low-cost and efficient MAP nucleic acid extraction pretreatment splitting module and a method for efficiently and conveniently pretreating an MAP sample by using the splitting module, after the sample is treated by using the method, the MAP can fully release nucleic acid, and the concentration of the extracted nucleic acid is increased, so that the accuracy is higher when the MAP is detected by qPCR (quantitative polymerase chain reaction); the effect of the device is not obviously different from that of a commercialized mechanical cracking module in actual excrement sample detection. More importantly, the splitting decomposition module and the splitting decomposition method are extremely low in economic cost, suitable for large-scale MAP detection in a pasture, and beneficial to reduction of economic loss of livestock breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. In particular, it relates to a pretreatment lysis module for nucleic acid extraction from Mycobacterium paratuberculosis and its application. Background Technology

[0002] Paratuberculosis, also known as John's disease, is a disease caused by Mycobacterium paratuberculosis (Mycobacterium paratuberculosis). Mycobacterium avium subsp. paratuberculosis Paratuberculosis (MAP) is a chronic intestinal granulomatous disease that primarily affects ruminants. Affected animals typically exhibit symptoms such as enteritis, diarrhea, and emaciation. Autopsy of the affected area reveals typical "brain-like changes" in the intestines. Paratuberculosis leads to decreased productivity in affected animals, including but not limited to reduced milk production and reproductive performance. Furthermore, the disease has a long incubation period and subtle subclinical symptoms, easily causing large-scale outbreaks and resulting in nearly $4 billion in economic losses to the global livestock industry annually. Because MAP is an intracellular bacterium, infected animals are difficult to cure, and vaccines are ineffective and may interfere with antibody detection. Therefore, timely diagnosis and early culling of affected animals are the primary means of paratuberculosis control.

[0003] Currently, the main methods for detecting paratuberculosis (MAP) include isolation and culture, PCR, and ELISA. Because MAP culture is highly specific, isolation and culture is recognized by the World Organisation for Animal Health (WOAH) as the gold standard for MAP diagnosis. However, the isolation and culture process for MAP is complex and extremely time-consuming, with a culture cycle that can last for several months, potentially leading to missed opportunities for culling infected animals. ELISA offers advantages such as standardization and a short cycle time; however, current ELISA kits primarily detect MAP antibodies, and their sensitivity varies significantly depending on antibody levels, resulting in low sensitivity for diagnosing subclinical cases. In the past, PCR methods suffered from insufficient sensitivity, but the advent of qPCR has provided a sensitive and rapid detection method for MAP. Compared to isolation and culture, qPCR significantly reduces the detection time and better meets the practical diagnostic needs of paratuberculosis.

[0004] Feces are a non-invasive and easy-to-collect sample, making them the primary sample for detecting MAP pathogens. However, they are quite difficult to process, mainly because ruminant feces contain a rich variety of microorganisms, as well as undigested particulate matter, plant matter, humic acid, bile, and proteins. Many of these substances can interact with the components involved in DNA amplification during qPCR, inhibiting the qPCR reaction and potentially leading to false negative results.

[0005] Furthermore, MAPs have thick lipid cell walls, making it difficult for conventional nucleic acid extraction methods to fully lyse the bacterial cells and release nucleic acids. Therefore, developing a reasonable sample pretreatment method to ensure the complete lysis and release of nucleic acids from MAPs is a prerequisite for accurate detection of MAPs using qPCR. While many bacteria can typically be extracted using only chemical lysis pretreatment, MAPs require both chemical and mechanical lysis pretreatment methods for DNA extraction.

[0006] Automated magnetic bead extraction (MBE) is a widely used nucleic acid extraction method in recent years. It primarily relies on chemical lysis, supplemented by heating and mechanical motion to achieve thorough mixing and reaction, enabling convenient, flexible, and efficient nucleic acid extraction. It has been applied in pathogen detection in various laboratories and testing platforms. However, MBE's detection performance is not ideal when dealing with metastatic MAP (MAP). Compared with culture results, nucleic acids obtained using MBE alone exhibit low sensitivity in detection. This widely used and considered highly effective nucleic acid extraction method cannot overcome the detection challenge of MBE.

[0007] Feedback from ranch testing revealed that the inventors learned that the MagMAX™ CORE mechanical lysis module from Thermo Fisher Scientific could significantly improve nucleic acid extraction from MAP fecal samples. This mechanical lysis module, based on bead milling, does not rely on expensive MP instruments or ultrasonic equipment; the grinding step can be completed using a common, inexpensive vortex mixer adapted to 2mL EP tubes. It is suitable for use in ranch molecular laboratories or environments with limited experimental conditions. However, the product's formulation is unknown and it is expensive (approximately 3000 RMB / 100 reactions). There is still a need to develop a more convenient and economical MAP nucleic acid extraction pretreatment lysis module, as well as an effective and simple-to-operate MAP nucleic acid extraction method. Summary of the Invention

[0008] To address the problems in existing technologies, the inventors have developed a low-cost and efficient MAP nucleic acid extraction pretreatment lysis module, along with a method for efficiently and conveniently pretreating MAP samples using this lysis module. After sample treatment using this method, MAP can fully release nucleic acids, increasing the concentration of extracted nucleic acids and resulting in higher accuracy in qPCR detection of MAP. Furthermore, in actual fecal sample testing, the effect is not significantly different from commercially available mechanical lysis modules. More importantly, this lysis module and method are extremely cost-effective (less than 2 yuan per reaction), significantly reducing the cost of large-scale detection. All raw materials and reagents used can be stored at room temperature, making it suitable for use in pasture molecular laboratories or environments with limited experimental conditions, facilitating timely detection or diagnosis of Mycobacterium paratuberculosis or paratuberculosis disease.

[0009] Based on this, one object of the present invention is to provide a MAP nucleic acid extraction pretreatment lysis module. Another object of the present invention is to provide the application of the pretreatment lysis module described herein in the detection of MAP or the diagnosis of paratuberculosis. Yet another object of the present invention is to provide a MAP nucleic acid extraction pretreatment method.

[0010] In this regard, the present invention includes, but is not limited to, the following: In one aspect, the present invention provides a grinding buffer for pretreatment of MAP nucleic acid extraction, the grinding buffer being prepared from buffer, EDTA-2Na, NaCl and SDS (sodium dodecyl sulfonate).

[0011] In one aspect, the grinding fluid of the present invention has a pH between 7.50 and 9.00, preferably between 7.66 and 8.62; And / or, the concentration of EDTA-2Na in the grinding slurry is 0-40 mM; And / or, the NaCl concentration in the grinding slurry is 75 mM or higher; And / or, the SDS mass percentage in the polishing slurry is between 0.5% and 1%; And / or, the buffer solution is a Tris-HCl buffer solution, preferably with a Tris-HCl concentration of 0-40 mM.

[0012] In one aspect, the pH of the polishing slurry of the present invention is between 7.50 and 9.00. In another aspect, the pH of the polishing slurry of the present invention is between 7.66 and 8.62. In yet another aspect, the pH of the polishing slurry of the present invention is 7.66, 7.87, 8.05, 8.23, 8.40, or 8.62. Preferably, the pH of the polishing slurry of the present invention is 8.40.

[0013] In one aspect, the buffer solution described in this invention is a Tris-HCl buffer solution.

[0014] In one aspect, the Tris-HCl concentration in the polishing slurry of the present invention is between 0 and 40 mM. In another aspect, the Tris-HCl concentration in the polishing slurry of the present invention is 2.5 mM, 5 mM, 10 mM, 20 mM, 30 mM, or 40 mM. Preferably, the Tris-HCl concentration of the present invention is 2.5 mM.

[0015] In one aspect, the concentration of EDTA-2Na in the polishing slurry of the present invention is 0-40 mM. In another aspect, the concentration of EDTA-2Na in the polishing slurry of the present invention is 10 mM, 20 mM, 30 mM, or 40 mM. Preferably, the concentration of EDTA-2Na in the polishing slurry of the present invention is 10 mM.

[0016] In one aspect, the NaCl concentration in the polishing slurry of the present invention is 75 mM or higher. In another aspect, the NaCl concentration in the polishing slurry of the present invention is 75 mM, 150 mM, 300 mM, 600 mM, or 1200 mM or higher. Preferably, the NaCl concentration in the polishing slurry of the present invention is 1200 mM or higher.

[0017] In one aspect, the NaCl concentration in the grinding slurry of the present invention is 1200 mM.

[0018] In one aspect, the SDS mass percentage in the polishing slurry of the present invention is between 0.5% and 1%. In another aspect, the SDS mass percentage in the polishing slurry of the present invention is 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%. Preferably, the SDS mass percentage in the polishing slurry of the present invention is 1%.

[0019] In one aspect, the grinding slurry formulation of the present invention comprises 0-40 mM Tris-HCl, 0-40 mM EDTA-2Na, more than 75 mM NaCl, SDS with a mass percentage between 0.5% and 1%, and a pH between 7.50 and 9.00.

[0020] In one aspect, the grinding slurry formulation of the present invention comprises 0-40 mM Tris-HCl, 0-40 mM EDTA-2Na, more than 1200 mM NaCl, SDS with a mass percentage between 0.5% and 1%, and a pH between 7.66 and 8.62.

[0021] In one aspect, the grinding slurry formulation of the present invention comprises 2.5 mM Tris-HCl, 10 mM EDTA-2Na, more than 1200 mM NaCl, 1% SDS by mass, and a pH of 8.40.

[0022] In one aspect, the grinding slurry formulation of the present invention comprises 2.5 mM Tris-HCl, 10 mM EDTA-2Na, 1200 mM NaCl, 1% SDS by mass, and a pH of 8.40.

[0023] In one aspect, the present invention provides a MAP nucleic acid extraction pretreatment lysis module, the pretreatment lysis module comprising a chemical lysis part and a mechanical lysis part, the chemical lysis part comprising the grinding fluid described in the present invention, and the mechanical lysis part comprising a grinding tube and grinding beads, preferably, the grinding beads being zirconium oxide beads or silica beads.

[0024] In one aspect, the grinding tube of the present invention has a volume of 1 or 2 mL; And / or, the mass of the grinding beads is 0.1-2 g. Preferably, the mass of the grinding beads is 0.1-1 g, more preferably, the mass of the grinding beads is 0.5-0.9 g. The mass of the grinding beads is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 g.

[0025] In one aspect, the grinding tube of the present invention has a volume of 2 mL.

[0026] In one aspect, the grinding beads of the present invention are zirconia beads or silicon dioxide beads.

[0027] In one aspect, the grinding beads described in this invention are zirconia beads.

[0028] In one aspect, the grinding beads / zirconia beads of the present invention have a particle size of 0.1 mm.

[0029] In one aspect, the mass of the grinding bead / zirconia bead according to the present invention is 0.1-2 g. Preferably, the mass of the grinding bead / zirconia bead according to the present invention is 0.5-0.9 g. More preferably, the mass of the grinding bead / zirconia bead according to the present invention is 0.7-0.9 g. Most preferably, the mass of the grinding bead / zirconia bead according to the present invention is 0.9 g.

[0030] In another aspect, the present invention provides a kit comprising the grinding fluid or pretreatment lysis module described in the present invention.

[0031] In another aspect, the present invention provides the use of the grinding fluid, pretreatment lysis module or kit described herein in the preparation of kits for detecting MAP or diagnosing paratuberculosis.

[0032] In another aspect, the present invention provides a pretreatment method for MAP nucleic acid extraction, the method comprising: mixing the sample to be tested with the pretreatment lysis module described in the present invention and then grinding it.

[0033] In one aspect, during the grinding process described in this invention, the volume ratio of the grinding tube to the sample and grinding fluid is 1:0.3.

[0034] In one aspect, the sample to be tested in this invention is selected from fecal samples and milk samples. Preferably, the sample to be tested is a fecal sample.

[0035] In one aspect, the present invention involves grinding in an MP instrument, an ultrasonic instrument, or a vortex mixer, preferably in a vortex mixer, and particularly, in a vortex mixer at 2000-3000 rpm / min for more than 5 minutes.

[0036] In one aspect, the present invention involves grinding in a vortex mixer (e.g., a vortex mixer, Jingqi, VM-02U (HYQ-3111)).

[0037] In one aspect, in the pretreatment method of the present invention, the grinding is performed at a speed of 2000-3000 rpm / min for 5-15 minutes.

[0038] In one aspect, in the pretreatment method of the present invention, the grinding is performed at a speed of 3000 rpm / min for 5-15 minutes.

[0039] In one aspect, in the pretreatment method of the present invention, the grinding is performed at a speed of 3000 rpm / min for 15 minutes.

[0040] In one aspect, in the pretreatment method of the present invention, the sample to be tested is mixed with the grinding beads and the grinding liquid described in the present invention in a grinding tube, and then ground at a speed of 2000-3000 rpm / min for 5-15 minutes.

[0041] In one aspect, in the pretreatment method of the present invention, the grinding tube has a volume of 2 mL, the total volume of the sample and the grinding liquid is about 600 μL, and the mass of the grinding beads is 0.1-2 g.

[0042] In one aspect, in the pretreatment method of the present invention, the sample to be tested is mixed with the grinding beads and the grinding solution described in the present invention in a grinding tube, and then ground at a speed of 2000-3000 rpm / min for more than 5 minutes, wherein the volume of the grinding tube is 1-2 mL, the total volume of the sample and the grinding solution is about 300-600 μL, and the mass of the grinding beads is 0.1-2 g.

[0043] In one aspect, in the pretreatment method of the present invention, the sample to be tested is mixed with the grinding beads and the grinding liquid described in the present invention in a grinding tube, and then ground at 3000 rpm / min for 15 minutes, wherein the volume of the grinding tube is 2 mL, the total volume of the sample and the grinding liquid is about 600 μL, and the mass of the grinding beads is 0.9 g.

[0044] In another aspect, the present invention provides a method for detecting MAP in a sample to be tested, the method comprising: (1) Pre-treatment of the test sample: The test sample will be ground using the pre-treatment pyrolysis module of the present invention; (2) Nucleic acid extraction: Extract MAP nucleic acid from the pretreated test sample obtained in step (1); (3) qPCR reaction: The nucleic acid extracted in step (2) is subjected to qPCR reaction to detect MAP.

[0045] In one aspect, in step (1), the sample to be tested is ground using the pretreatment method of the present invention.

[0046] In one aspect, in step (2), nucleic acids are extracted using an automated magnetic bead nucleic acid extraction kit (e.g., Virus DNA / RNA Extraction Kit 2.0 (Prepackaged) purchased from Nanjing Novizan Biotechnology Co., Ltd.) on an automated extractor (e.g., Fully Automated Nucleic Acid Extractor-VNP-32P purchased from Nanjing Novizan Biotechnology Co., Ltd.).

[0047] In one aspect, in step (3), the detection targets of the qPCR reaction are F57, 251 and / or hsp X. In this invention, MAP targets F57, 251 and / or hsp The primer and probe design and synthesis method for X can be found in the following literature: Liu Tian et al., Establishment of triple TaqMan qPCR detection method for Mycobacterium paratuberculosis, Journal of Animal Husbandry and Veterinary Medicine, 2025.

[0048] In one aspect, in step (3), the qPCR reaction is a triple qPCR reaction, and the primers and probes are shown in Table 1.

[0049] In one aspect, in step (3), the triple qPCR reaction program is as follows: 37 ℃ for 2 min; 95 ℃ for 20 s; 95 ℃ for 10 s and 60 ℃ for 30 s for 40 cycles, collecting fluorescence signals at 60 ℃, and automatically setting the threshold during detection.

[0050] In one aspect, in step (3), each system of the triple qPCR reaction contains 12.5 μL of fluorescent PCR enzyme reaction solution; 0.4 μL of each upstream and downstream primer (10 μmol / L); 0.2 μL of each probe (10 μmol / L); 4.5 μL of ddH2O; 5 μL of template or sample; totaling 25 μL.

[0051] The beneficial technical effects of the present invention include at least the following: (1) The MAP nucleic acid extraction pretreatment grinding solution or lysis module of the present invention can efficiently and conveniently lyse MAP, ensuring that the extracted MAP nucleic acid concentration is suitable for qPCR detection and improving detection accuracy.

[0052] (2) The preparation and storage costs of the MAP nucleic acid extraction pretreatment grinding solution or lysis module of the present invention are extremely low, making it suitable for large-scale MAP detection in ranches. In addition, the MAP nucleic acid extraction pretreatment method of the present invention does not require expensive instruments during operation and is more suitable for use in ranch molecular laboratories or environments with limited experimental conditions, which is conducive to rapid detection of MAP or rapid diagnosis of paratuberculosis, thereby helping to reduce economic losses in livestock farming. Detailed Implementation

[0053] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0054] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0055] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions were performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0056] 1. Sample Source MAP-positive fecal samples used to establish the qPCR pretreatment method were obtained through routine testing and are stored at the WOAH Swine Streptococcal Disease Reference Laboratory of Nanjing Agricultural University. Thirty-two fecal samples used to compare the nucleic acid extraction performance of commercially available pretreatment kits and the self-made pretreatment method were provided by large-scale farms.

[0057] 2. Main Instruments and Reagents The QuantStudio 6 Pro real-time quantitative PCR instrument was purchased from Thermo Fisher Scientific China Co., Ltd. The fluorescent PCR enzyme reaction solution was purchased from Qingdao Lijian Biotechnology Co., Ltd. The Virus DNA / RNA Extraction Kit 2.0 (Prepackaged) and the fully automated nucleic acid extractor - VNP-32P were both purchased from Nanjing Novizan Biotechnology Co., Ltd. Zirconia beads were purchased from Pingxiang Jinrui New Materials Co., Ltd. The MagMAX™ CORE MechanicalLysis Module (the commercial pretreatment kit used in Table 8) was purchased from Thermo Fisher Scientific. The MAP detection qPCR kit (commercial qPCR kit) was purchased from Bioway (Tianjin) Biotechnology Co., Ltd. The crude DNA extraction grinding buffer (the commercially available grinding buffer used in Table 7) was purchased from Hangzhou Fuwalk Biotechnology Co., Ltd.

[0058] Example 1. Establishment of qPCR pretreatment method 1) Nucleic acid extraction efficiency evaluation method The efficiency of nucleic acid extraction was evaluated using a triple qPCR method for MAP detection previously established in our laboratory. This method targets three conserved single-copy non-insertion sequence targets of MAP: F57, 251, and... hsp The detection of X showed that the amplification efficiencies (E) of all three targets were between 97% and 100%, and R... 2 All values ​​are greater than 0.999, indicating good linearity and quantitative accuracy. Multiple detections can be performed in a single reaction. Specific primers and probes are shown in Table 1.

[0059] Table 1 Primer and probe sequences

[0060] GB-F, GB-P, and GB-R are primers disclosed in the national standard GB / T27637—2011. For details, please refer to "Liu Zhongyong, Chen Ru, Yang Guohai, et al. Real-time fluorescent PCR detection method for Mycobacterium paratuberculosis: GB / T27637—2011 [S]. General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, Standardization Administration of China, 2011".

[0061] The triple qPCR system and reaction program are as follows: 37 ℃ for 2 min; 95 ℃ for 20 s; 95 ℃ for 10 s and 60 ℃ for 30 s, repeated 40 times. Fluorescence signals are collected at 60 ℃, and the detection threshold is automatically set. Each system contains 12.5 μL of fluorescent PCR enzyme reaction solution; 0.4 μL each of each forward and reverse primer (10 μmol / L); 0.2 μL each of each probe (10 μmol / L); 4.5 μL of ddH2O; 5 μL of template or sample; totaling 25 μL.

[0062] The results of triple qPCR are calculated as follows: Each reaction uses 10 μC containing three target sequences. 5 copies·μL -1The standard plasmid was used as a positive control. Based on the detection results of the positive control and the previously constructed standard curve (y... F57 = -3.3495x + 41.459; y hspX = -3.3956x + 42.044; y 251 = -3.373x + 41.913) to correct the CT results of the three targets, and calculate the nucleic acid concentration corresponding to the CT value using a standard curve. The mean nucleic acid concentration of the three targets detected in each fecal sample (in copies / μL) was calculated, and the pretreatment lysis module corresponding to the highest mean nucleic acid concentration in each reaction was selected as the optimal pretreatment lysis module. The method for obtaining the positive standard plasmid and standard curve can be found in the following literature: Liu Tian et al., 2025, Establishment of a triple TaqMan qPCR detection method for Mycobacterium paratuberculosis, Journal of Animal Husbandry and Veterinary Medicine. The specific correction method is as follows: 10 5 The mass of copies / μL concentration serves as a positive control. The positive control detection CT results (A) and 10 in the standard curve are shown. 5 The difference between the CT values ​​(B) corresponding to the copies / μL concentration and the difference (C=BA) is used as the correction value. The results of all sample tests are added to the difference (C) to obtain the corrected CT result. The CT result is then substituted into the standard curve to calculate the corrected nucleic acid concentration.

[0063] In this application, samples are processed and nucleic acids are extracted using a commercially available pretreatment kit or a self-made pretreatment grinding module. The nucleic acid extraction efficiency is then evaluated using a commercially available qPCR kit based on the results of the aforementioned triplet PCR. The qPCR detection steps are performed according to the instructions of the commercially available kit. The same sample treated with different pretreatment methods is detected in a single qPCR reaction, with three replicates for each treatment. The nucleic acid extraction efficiency is evaluated by comparing the detected CT values.

[0064] 2) Preparation of homemade grinding fluid 12.1135 g of Tris was dissolved in 500 mL of ultrapure water to prepare a 200 mM Tris solution. 4.5 mL of 37% concentrated hydrochloric acid (12 mol / mL) was added to 500 mL of deionized water to obtain 100 mM hydrochloric acid. 25 mL of the 200 mM Tris solution was taken, and 27.5 mL of the 100 mM hydrochloric acid was added. The solution was then brought to a final volume of 100 mL with ultrapure water to obtain a 50 mM Tris-HCl (23℃; pH = 8.05) buffer. The 50 mM Tris-HCl buffer was diluted to 10 mM with ultrapure water. EDTA·2Na, Triton X-100, NaCl, and SDS (sodium dodecyl sulfate) were added to bring the final concentrations to 20 mM, 0.6% (w / w), 300 mM, and 2.00% (w / w), respectively. The solution was then mixed on a shaker at 37℃ until completely dissolved to obtain the homemade homogenate.

[0065] 3) Comparison of grinding beads and grinding fluid Three grinding buffers (PBS, commercial lysis buffer (purchased from Qingdao Lijian Biotechnology Co., Ltd., requires storage at 4℃), and homemade grinding buffer) and three grinding methods (no grinding beads, 1 mm steel beads, and 0.1 mm zirconia beads) were combined in pairs to detect actual MAP-positive fecal samples collected from pastures according to the following scheme: (1) Take a 2 mL screw-type grinding tube, weigh 1 g of grinding beads into the tube, and add 400 μL of grinding liquid into the grinding tube; (2) Homogenize the fecal sample, take 0.3-0.4 g of fecal sample into a 2 mL EP tube, and add 1 mL of sterile water or sterile PBS; (3) Vortex for 3 min, or use other methods to thoroughly mix the sample; (4) Centrifuge at 500 g for 2 min, and take 200 μL of supernatant into a grinding tube; (5) Fix the grinding tube to a vortex mixer that is compatible with microcentrifuge tubes and oscillate at the highest power for 15 minutes; (6) Centrifuge at 15,000 g for 3 min, take 300 μL of supernatant as a sample, and extract nucleic acid using an automated magnetic bead nucleic acid extraction kit on an automated extractor; (7) Use the aforementioned self-made triple qPCR to detect the nucleic acid in the samples, according to the aforementioned 10 5 The positive plasmid results and the constructed standard curve are used to calculate the nucleic acid concentration corresponding to the CT value. Based on the nucleic acid concentration, the optimal combination of the corresponding grinding solution and grinding beads is selected.

[0066] The test results showed that the highest nucleic acid concentration (28004.80 copies / μL) was obtained when the samples were treated with a combination of 0.1 mm zirconia beads and homemade grinding solution. Compared with pretreatment with PBS or commercial lysis buffer alone, the extracted nucleic acid concentration was increased by at least 3.24-100 times. Therefore, 0.1 mm steel beads and homemade grinding solution are the best grinding combination. The specific test results are shown in Table 2.

[0067] Table 2 Nucleic acid detection results of different grinding methods

[0068] Note: a indicates that one of the three replicate detection channels in the PCR instrument is negative; b indicates that two of the three replicate detection channels in the PCR instrument are negative; NA indicates that all three channels are negative. The unit is copies per microliter (copies / μL).

[0069] Table 2 shows that the self-made grinding buffer of this invention, combined with 0.1 mm zirconia beads, significantly increases the concentration of extracted nucleic acids. In contrast, when using PBS and commercially available lysis buffers for bacterial extraction, even with the addition of zirconia beads or steel beads, the extracted nucleic acid concentration remains low. In the detection of bacteria in fecal samples, conventional pretreatment methods only require PBS or conventional lysis buffer. However, as shown in Table 2, this method is clearly unsuitable as a pretreatment method for extracting MAP nucleic acids.

[0070] Example 2. Optimization of grinding slurry formulation and grinding scheme (1) Optimization of grinding fluid formulation The concentrations of the self-made grinding fluid components in Example 1 were optimized sequentially. After optimizing a component, the optimal concentration of the optimized component was used for subsequent component optimizations. All samples were actual positive fecal samples from the ranch collected for MAP testing. The specific optimization order was as follows: SDS (0.50, 1.00, 1.50, 2.00, 2.50, 3.00%); Triton X 100 (0, 0.08, 0.15, 0.3, 0.6, 1.2%); EDTA·2Na (0, 10, 20, 40, 80, 160 mM); NaCl (0, 75, 150, 300, 600, 1200 mM); Tris-HCl (0, 2.5, 5, 10, 20, 40 mM). The pH was adjusted by changing the ratio of Tris to HCl. The volume ratios of 200 mM Tris to 100 mM hydrochloric acid were 2:1, 10:7, 10:9, 10:11, 10:13, and 2:3, respectively, which corresponded to pH values ​​of 8.62, 8.40, 8.23, 8.05, 7.87, and 7.66.

[0071] The results showed that the optimal grinding slurry formulation was 2.5 mM Tris-HCl with a pH of 8.40, containing 1.0% SDS, 10 mM EDTA·2Na, and 1200 mM NaCl. Specific optimization results are shown in Tables 3 and 4. Furthermore, Table 3 also shows that the concentration of NaCl in the self-made grinding slurry has a positive correlation with the grinding effect; that is, the higher the NaCl concentration, the better the grinding effect.

[0072] Table 3 Results of Optimization of Grinding Fluid Composition

[0073] Note: 'a' represents the nucleic acid concentration corresponding to the indicator (unit: copies per microliter (copies / μL)) expressed as a logarithm (base 10).

[0074] Table 4 Results of pH optimization of grinding fluid

[0075] Note: 'a' represents the nucleic acid concentration (in copies per microliter (copies / μL)) of the index as a logarithm (base 10). (2) Optimization of grinding scheme Using 0.1 mm steel balls and the above-described optimal homemade grinding fluid formulation, actual positive fecal samples were tested according to the procedure in Example 1, but the amount of grinding balls added was varied. The initial optimization used 0.8, 0.9, 1.0, 1.1, 1.2, and 1.3 g of grinding balls, respectively, and the secondary optimization used 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 g of grinding balls, respectively.

[0076] In addition, the grinding time and speed were optimized using the best grinding conditions. The three-level speeds (3000, 2500, 2000 rpm / min) and the three-level grinding times (15, 10, 5 min) were combined in pairs, and the method steps of Example 1 were used to detect actual positive fecal samples.

[0077] The results show that the optimal grinding method involves adding 0.9 g of grinding beads to each grinding tube and vortexing at 3000 rpm / min for 15 min. Specific optimization results are shown in Tables 5 and 6.

[0078] Table 5 Results of Optimization of Grinding Bead Addition Amount

[0079] Note: 'a' represents the nucleic acid concentration (in copies per microliter (copies / μL)) of the corresponding indicator as a logarithm (base 10).

[0080] Table 6 Optimization results of grinding speed and grinding time

[0081] Note: The unit for nucleic acid concentration is copies per microliter (copies / μL).

[0082] The effectiveness of the optimized grinding buffer was evaluated using the optimal grinding protocol (adding 0.9 g grinding beads and vortexing at 3000 rpm / min for 15 min). The effects of the unoptimized grinding buffer (the formulation in Example 1), the optimized grinding buffer (the optimal formulation in Example 2), and the commercially available crude DNA extraction grinding buffer (Hangzhou Fuwalk Biotechnology Co., Ltd.) on sample treatment were compared. The evaluation was conducted twice: first, the effects of the unoptimized and optimized grinding buffers were compared, and then the effects of the optimized grinding buffer and the commercially available crude DNA extraction grinding buffer were compared, with three replicates treated each time. The results showed that the optimized grinding buffer yielded a higher nucleic acid extraction rate compared to the unoptimized grinding buffer; the nucleic acid concentration extracted after treatment with the optimized grinding buffer increased by 58% compared to the unoptimized grinding buffer. Furthermore, the optimized self-made grinding buffer was very similar to the commercially available grinding buffer, but the cost of the optimized self-made grinding buffer was significantly lower, specifically less than 2 yuan / 100 mL, while the cost of the commercially available grinding buffer was 14.2 yuan / 100 mL. Specific test results are shown in Table 7.

[0083] Table 7 Comparison of grinding fluid effects before and after optimization

[0084] Note: "A" is the self-made homogenate before optimization; "B" is the self-made homogenate after optimization; "C" is the commercial homogenate. 'a' represents the nucleic acid concentration (in copies per microliter, base 10) corresponding to this indicator.

[0085] Example 3. Clinical Sample Testing In this embodiment, a total of 32 clinical samples were tested, all of which were fecal samples from cattle suspected of having paratuberculosis, collected in June 2025. Each fecal sample was processed separately using a self-made pretreatment lysis module and processing protocol (including an optimized self-made grinding buffer and an optimized grinding protocol) and a commercial pretreatment kit (Thermo Fisher Scientific's MagMAX™ CORE mechanical lysis module). The concentration of nucleic acids extracted by the two lysis modules or methods was detected using a commercial MAP qPCR kit to determine the presence of Mycobacterium paratuberculosis.

[0086] The results of nucleic acid testing on 32 individual fecal samples showed that the concordance rate for Mycobacterium paratuberculosis detection between the two pretreatment modules was 100% (32 / 32). Specific results are shown in Table 8. To assess whether there was a significant difference in CT values ​​for positive samples detected by the two methods, a two-tailed paired samples t-test was performed. The test results showed no significant difference in CT values ​​for positive samples detected by the two methods (…). p = 0.90836).

[0087] Table 8 Comparison of pretreatment effects between self-made and commercial products

[0088] Note: "+" indicates a positive test; "-" indicates a negative test; the numbers in the table are the CT values ​​of the test.

Claims

1. A grinding solution for pretreatment of MAP nucleic acid extraction, characterized in that, The grinding solution is prepared from buffer solution, EDTA-2Na, NaCl and SDS.

2. The grinding fluid according to claim 1, characterized in that, The pH of the grinding fluid is between 7.50 and 9.00, preferably between 7.66 and 8.62; And / or, the concentration of EDTA-2Na in the grinding slurry is 0-40 mM; And / or, the NaCl concentration in the grinding slurry is 75 mM or higher; And / or, the percentage of SDS in the polishing slurry is between 0.5% and 1%; And / or, the buffer solution is a Tris-HCl buffer solution, preferably with a Tris-HCl concentration of 0-40 mM.

3. A pretreatment lysis module for MAP nucleic acid extraction, the pretreatment lysis module comprising a chemical lysis section and a mechanical lysis section, characterized in that, The chemical pyrolysis section includes the polishing fluid according to claim 1 or 2, and the mechanical pyrolysis section includes a polishing tube and polishing beads, preferably, the polishing beads are zirconia beads or silica beads.

4. The pretreatment pyrolysis module according to claim 3, characterized in that, The grinding tube has a volume of 1 or 2 mL; And / or, the mass of the grinding beads is 0.1-2 g.

5. A reagent kit, characterized in that, The kit contains the grinding slurry according to claim 1 or 2 or the pretreatment lysis module according to claim 3 or 4.

6. The use of the grinding fluid according to claim 1 or 2, the pretreatment lysis module according to claim 3 or 4, or the kit according to claim 5 in the preparation of a kit for detecting MAP or diagnosing paratuberculosis.

7. A pretreatment method for MAP nucleic acid extraction, characterized in that, The method includes: mixing the sample to be tested with the pretreatment pyrolysis module according to claim 3 or 4 and then grinding it.

8. The pretreatment method according to claim 7, characterized in that, The samples to be tested were selected from fecal samples and milk samples.

9. The pretreatment method according to claim 7, characterized in that, Grinding is performed in an MP instrument, an ultrasonic instrument, or a vortex mixer, preferably in a vortex mixer, and in particular, in a vortex mixer at a speed of 2000-3000 rpm / min for more than 5 minutes.

10. A method for detecting MAP in a sample, characterized in that, The method includes: (1) Pretreatment of the sample to be tested: The sample to be tested will be ground using the pretreatment pyrolysis module according to claim 3 or 4; (2) Nucleic acid extraction: Extract MAP nucleic acid from the pretreated test sample obtained in step (1); (3) qPCR reaction: The nucleic acid extracted in step (2) is subjected to qPCR reaction to detect MAP.

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