Method for detecting mycobacterium paratuberculosis based on CRISPR-Cas12a technology

Abstract

The invention belongs to the field of nucleic acid detection, and discloses a method for detecting mycobacterium paratuberculosis based on a CRISPR-Cas12a technology. Aiming at the problems that an existing MAP detection method is time-consuming, high in cost and low in sensitivity and specificity, and an existing CRISPR technology is poor in adaptability, insufficient in detection of low bacterium loading amount and the like, the method is combined with recombinase polymerase amplification (RPA) and nested polymerase chain reaction (PCR), takes an MAP high-conservative genome sequence as a target, and fuses high sensitivity of nucleic acid amplification and high specificity and signal amplification capability of CRISPR-Cas12a (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-CRISPR-CRISPR-CRISPR-CRISPR-CRISPR-Cas12a). The lowest detection limit of the kit reaches 1020M, which is far better than the single use effect of the traditional PCR or CRISPR, and the kit realizes ultra-sensitive, high-specificity, rapid and low-cost detection, and is suitable for paratuberculosis early infection or low-bacterial-loading sample detection.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection, and more particularly to a method for detecting Mycobacterium paratuberculosis based on CRISPR-Cas12a technology. Mycobacterium avium subsp. paratuberculosis The method of MAP. Background Technology

[0002] Paratuberculosis is a chronic infectious disease caused by Mycobacterium paratuberculosis (MAP) in various animals. It is characterized by progressive weight loss, persistent diarrhea, granulomatous enteritis, and mesenteric lymphadenitis, and can even lead to death. It is an infectious disease that impacts both livestock economics and public health. Currently, methods for detecting MAP include bacterial culture, serological tests, PCR, and qPCR, but these methods have drawbacks such as time-consuming detection, high cost, and low sensitivity and specificity.

[0003] CRISPR-Cas12a technology, with its unique trans-cleavage activity, has become a core tool for ultrasensitive detection. When Cas12a recognizes the target DNA sequence under the guidance of crRNA, it activates the non-specific trans-cleavage activity of single-stranded nucleic acids. However, combining the high specificity of CRISPR-Cas12a with amplification techniques adapted to samples with different bacterial loads, while simultaneously solving the problems of sample impurity interference and on-site detection adaptability, is a bottleneck in current technology research. Existing CRISPR technology has made significant progress in the field of nucleic acid detection. Chinese patent CN114457073B discloses a method for detecting MAP based on CRISPR technology, which uses gRNA, type V Cas protein (such as Cas12), and a single-stranded nucleic acid detector for detection. This method improves detection efficiency and specificity by optimizing the gRNA sequence, but it mainly relies on the characteristics of type V Cas protein, uses only a single nucleic acid amplification method (PCR), has not been optimized for low bacterial load samples, has not verified the detection effect on clinical samples, and has not presented the detection range. Chinese patent CN110541022B discloses a method for detecting Mycobacterium tuberculosis complex (MAP) based on CRISPR technology. This method utilizes gRNA, type V Cas protein (such as Cas12a), and a single-stranded nucleic acid detector. It optimizes reaction conditions by combining recombinase polymerase amplification (RPA) with the trans-cleavage activity of Cas12a to improve the detection efficiency and specificity of MAP. However, this method focuses on MAP and does not design specific target (spacer) sequences and matching crRNAs for the highly conserved and specific genomic sequences of MAP. Furthermore, it only uses a single nucleic acid amplification method (RPA), which cannot accurately identify MAP-specific sequences, cannot adapt to differences in bacterial load at different infection stages, and has insufficient detection capability for samples with low bacterial loads. Chinese patent CN118895375A discloses a CRISPR-Cas12 detection method and its application for detecting rpoB gene mutations in Mycobacterium tuberculosis. Utilizing a nested PCR amplification system and a CRISPR-Cas12 system, it detects drug resistance gene mutations through specific sgRNA sequences and fluorescent probes. This method improves the sensitivity and specificity of detection by optimizing the sgRNA sequence and the CRISPR-Cas12 system, making it suitable for detecting rifampicin resistance in Mycobacterium tuberculosis. However, this method primarily targets the detection of drug resistance genes in Mycobacterium tuberculosis and does not involve the detection of MAP (method of metastasis), nor is it optimized for samples with low bacterial loads.

[0004] Therefore, developing an efficient method for detecting MAP and improving detection efficiency and specificity has become a challenging problem that needs continuous optimization in the current development of CRISPR technology. Summary of the Invention

[0005] The first objective of this invention is to overcome the shortcomings of existing technologies and provide a method for detecting Mycobacterium paratuberculosis (MAP) based on CRISPR-Cas12a technology. This method combines recombinase polymerase amplification (RPA) and nested polymerase chain reaction (PCR), targeting highly conserved MAP genomic sequences. It integrates the high sensitivity of nucleic acid amplification with the high specificity and signal amplification capability of CRISPR-Cas12a, achieving ultrasensitive, highly specific, rapid, and low-cost nucleic acid detection, pushing the detection limit to aM (10⁻⁶). −20 M), far exceeding the capabilities of traditional PCR or CRISPR alone, is suitable for detecting early paratuberculosis infection or samples with low bacterial load.

[0006] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows: 1. Sample pretreatment and total DNA extraction MAP cultured bacterial samples: The bacterial culture was centrifuged at 8000 r / min for 10 min to collect the bacterial cells. The bacterial cells were lysed using a bacterial genomic DNA extraction kit (TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0) with lysis buffer and proteinase K (incubated at 56℃ for 30 min). After washing with a purification column (washed once with 500 μL washing buffer), the DNA was eluted with 100 μL Elution Buffer (incubated at room temperature for 2 min, then centrifuged at 12000 r / min for 1 min).

[0007] Tissue samples: Tissue homogenization, lysis, and DNA purification were performed using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver. 5.0, following the kit instructions.

[0008] Fecal sample: feces (omega bio-tek ezna) ®The Stool DNA Kit is prepared by mixing with sterile water, filtering through a 60-mesh sieve, and centrifuging at 8000 rpm for 10 minutes to collect the precipitate. Transfer 200 mg of the precipitate to a 2 mL centrifuge tube, add 200 mg of Glass Beads X, and incubate on ice for 2–5 min. Add 540 μL of SLX-Mlus Buffer to the centrifuge tube and vortex for 10 min to homogenize thoroughly. Add 60 μL of DS Buffer and 20 μL of Proteinase K, and vortex for 1 min. Incubate the centrifuge tube at 70 °C for 10 min, then vortex for 30 s, and incubate at 95 °C for 5 min, then vortex for 30 s. Add 200 μL of SP2 buffer, vortex for 30 s, incubate on ice for 5 min, and centrifuge at 15000 rpm for 5 min. Transfer 400 μL of the supernatant to a 1.5 mL centrifuge tube, add 200 μL of HTR Reagent solution, and vortex for 10 s. Incubate at room temperature for 2 min, then centrifuge at 12000 rpm for 2 min. Transfer 250 μL of the supernatant to a new 1.5 mL centrifuge tube, and add 250 μL of... Mix BL buffer and 250 μL of anhydrous ethanol by vortexing for 10 seconds. Transfer the well-mixed solution to a HiBind DNA purification column, centrifuge at 12000 r / min for 1 min, discard the filtrate (repeat washing with VHB Buffer + DNA Wash Buffer), and finally elute the DNA with Elution Buffer preheated to 65℃.

[0009] 2. Stepwise amplification of the target DNA fragment Select the appropriate amplification method based on the bacterial load of the sample to ensure efficient enrichment of the target fragment (including MAP-specific target sequences): High bacterial load samples (bacterial load ≥ 1 × 10⁻⁶) −10 μg / μL): RPA amplification was performed. The reaction system (total volume 50μL) contained 2.4μL of 10μM RPA-7132F1 (SEQ ID NO.1), 2.4μL of 10μM RPA-7132R1 (SEQ ID NO.2), 29.5μL of primer-free rehydration buffer, 13.2μL of DNA template and water. After mixing and briefly centrifuging, the rehydration solution was transferred to reaction microspheres and resuspended by pipetting. 2.5μL of 280mM magnesium acetate was added and mixed thoroughly. After rapid centrifugation again, the mixture was placed at 39℃ for 30 min to amplify the target fragment.

[0010] Low bacterial load samples (bacterial load <1×10) −10μg / μL: Nested PCR amplification was used. The reaction system (total volume 25μL) contained 12.5μL Takara Premix Taq™, 9.5μL ddH2O, 1μL 10μM upstream primer, and 1μL DNA template. The first round of amplification used the outer primer pair (7132F1 SEQ ID NO.3 / 7132R1 SEQ ID NO.4), and the reaction conditions were: 94℃ pre-denaturation for 2 min, 25 cycles (94℃ 30s→60℃ 30s→72℃ 30s), and final extension at 72℃ for 3 min. The second round of amplification used the inner primer pair (7132F1 SEQ ID NO.5 / 7132R1 SEQ ID NO.6), and the reaction conditions were: 94℃ pre-denaturation for 2 min, 25 cycles (94℃ 30s→55℃ 30s→72℃ 30s), and final extension at 72℃ for 3 min. The target fragment was amplified.

[0011] 3. Purification of amplification products Add an equal volume of Tris-saturated phenol-chloroform-isoamyl alcohol mixture (volume ratio 25:24:1) to the RPA or nested PCR amplification product. Mix well, centrifuge at 12000 r / min for 5 min, collect the supernatant and store at low temperature.

[0012] 4. Construction of the CRISPR-Cas12a reaction system The CRISPR-Cas12a reaction system includes the Cas12a protein, target guide RNA, detection probes, and buffer. The target guide RNA includes a region that binds to the Cas12a protein and a guide sequence that hybridizes to the target nucleic acid sequence. The detection probes include test strip probes or fluorescent probes. This reaction system guides the Cas12a protein to specifically bind to the target nucleic acid sequence via the target guide RNA, activating the trans-cleavage activity of the Cas12a protein, which then cleaves the detection probe, generating a detectable signal.

[0013] Depending on the requirements of the detection scenario, select either a test strip probe or a fluorescent probe reaction system, and utilize the trans-cleavage activity of LbCas12a to amplify the target signal: Table 1. Test strip probe reaction system (total volume 50 μL)

[0014] 10×HOLMES Buffer: 500mM NaCl, 100mM Tris-HCl, 100mM MgCl2, 1mg / mL BSA, pH7.9; 10 μM MAP-specific crRNA: SEQ ID NO.7; 10μM test strip probe: SEQ ID NO.8.

[0015] Table 2. Fluorescent probe reaction system (total volume 30 μL)

[0016] 10×Cleavage Buffer: 100mM Tris-HCl, 50mM MgCl2, 100mM NaCl, pH7.5; 4μM fluorescent probe: SEQ ID NO.9.

[0017] 5. Result Interpretation Choose the appropriate interpretation method based on the reaction system used to achieve qualitative or semi-quantitative detection: Test strip system: The test strip probe reaction system is reacted at 37℃ for 30 min. The CRISPER test strip is inserted into the reaction product, and the results are observed after 3 min. If both the test line (T) and the control line (C) show red bands, it is judged as MAP positive; if only the control line shows color, it is judged as negative. Figure 1 ).

[0018] Fluorescence system: ① UV lamp interpretation: If green fluorescence is observed, it is considered positive. Figure 2 ); ② qPCR instrument interpretation: 30s / time, 30min monitoring; if the reading is increased, the probe is considered to have been effectively cleaved and the sample is positive. Figure 3 ).

[0019] The limit of detection for the above detection method is 2.5 × 10⁻⁶. −20 M is suitable for detecting samples with early infection or low bacterial load.

[0020] A second objective of this invention is to provide a kit for detecting Mycobacterium paratuberculosis, comprising: Reagents used for extracting total DNA; Reagents for RPA or nested PCR amplification reaction systems; Reagents for the CRISPR-Cas12a reaction system, including reagents for the test strip probe reaction system or the fluorescent probe detection system; Specific target guide RNA (SEQ ID NO.7); RPA primers: RPA-7132F1 (SEQ ID NO.1) and RPA-7132R1 (SEQ ID NO.2); Nested PCR primers: 7132F1 (SEQ ID NO.3), 7132R1 (SEQ ID NO.4), 7132F2 (SEQ ID NO.5) and 7132R2 (SEQ ID NO.6).

[0021] Tris-saturated phenol-chloroform-isoamyl alcohol mixture was used to process the amplification products.

[0022] A third objective of this invention is to provide a primer combination for detecting Mycobacterium paratuberculosis, comprising: RPA primers: RPA-7132F1 (SEQ ID NO.1) and RPA-7132R1 (SEQ ID NO.2); Nested PCR primers: 7132F1 (SEQ ID NO.3), 7132R1 (SEQ ID NO.4), 7132F2 (SEQ ID NO.5) and 7132R2 (SEQ ID NO.6); CRISPR-Cas12a target guide RNA (SEQ ID NO.7).

[0023] The fourth objective of this invention is to provide a detection system based on CRISPR-Cas12a technology, characterized in that it comprises: A sample processing device for extracting total DNA from a sample to be tested; Amplification device used to amplify target gene fragments using RPA or nested PCR methods; The detection device is used to process the amplification product and add it to the CRISPR-Cas12a reaction system for detection.

[0024] The fifth objective of this invention is to provide a method for detecting Mycobacterium paratuberculosis based on CRISPR-Cas12a technology for the detection of Mycobacterium paratuberculosis nucleic acid in environmental or food samples, in order to assess the hygiene status of the environment or food.

[0025] The beneficial effects of this invention are that it provides a highly sensitive, highly specific, rapid, and low-cost method for detecting Mycobacterium paratuberculosis. The method achieves a limit of detection of 2.5 × 10⁻⁶. -20 M, far exceeding the effects of traditional PCR or CRISPR alone, is suitable for detecting early paratuberculosis infection or samples with low bacterial load. Attached Figure Description

[0026] Figure 1 Image of CRISPR test strip results: The left side shows a positive sample (both test line C and control line T are colored), and the right side shows a negative sample (only control line T is colored). Figure 2 The results of UV light observation of the fluorescent probe are shown in the figure. From left to right, tubes 1-6 and 8 are positive (green fluorescence), tube 7 is negative (no fluorescence), tube 9 is blank control (no fluorescence), and tube 10 is positive control (strong fluorescence). Figure 3 The graph shows the results of the fluorescent probe qPCR test. An upward curve indicates a positive result (target present), while a flat curve indicates a negative result (no target). Figure 4 The positions of RPA primers, nested PCR primers, and crRNA in the MAP target sequence (SEQ ID NO. 10); Figure 5 Visual results of the lowest detection limit of RPA-CRISPR-Cas12a; Figure 6 Results of RPA-CRISPR-Cas12a minimum detection limit test; Figure 7 Results of visual observation under ultraviolet light on the reaction system of 50 fluorescent probe samples. Detailed Implementation

[0027] Example 1: Detection of high bacterial load samples (MAP culture) (RPA + test strip system) 1. Material Preparation Sample: MAP K-10 standard strain (ATCC 19698, purchased from Shanghai Baifeng Biotechnology Co., Ltd.) cultured bacteria (bacterial load ≥ 1×10⁻⁶). −10 μg / μL); Target sequence: The highly conserved genomic target sequence for MAP detected in this embodiment is SEQ ID NO.10. The positional relationship between the primers and crRNA on this target sequence is as follows: Figure 4 ; Reagents: TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0, TwistAmp® Basic kit, Tris saturated phenol-chloroform-isoamyl alcohol (25:24:1), LbCas12a Nuclease (Novoprotein, purity ≥95%), crRNA, test strip probes, 10×HOLMES Buffer, CRISPR test strips; Instruments: constant temperature water bath, centrifuge, vortex shaker.

[0028] 2. Operating Procedures (1) Nucleic acid extraction Take 1 mL of MAP K-10 cultured bacteria, centrifuge at 8000 r / min for 10 min, discard the supernatant, and extract total DNA from the precipitate using the TaKaRa MiniBESTBacteria Genomic DNA Extraction Kit Ver.3.0. Add 200 μL of lysis buffer and 20 μL of proteinase K, and incubate at 56 °C for 30 min. Add 200 μL of ethanol and mix well. Transfer to a purification column and centrifuge at 12000 r / min for 1 min, then discard the filtrate. Add 500 μL of washing buffer and centrifuge for 1 min. Repeat once. Elute DNA with 100 μL of Elution Buffer and store at -20°C.

[0029] (2) RPA amplification System preparation: Add 2.4 μL of 10 μM RPA-7132F1 (SEQ ID NO.1), 2.4 μL of 10 μM RPA-7132R1 (SEQ ID NO.2), 29.5 μL of primer-free rehydration buffer, 5 μL of the above DNA template, and 8.2 μL of enzyme-free water to a sterile PCR tube, and mix well to obtain a 47.5 μL rehydration solution; Reaction initiation: Transfer the rehydrated solution to a TwistAmp® Basic reaction microsphere tube and pipette until the microspheres dissolve; add 2.5 μL of 280 mM magnesium acetate, mix well, and then briefly centrifuge (5000 r / min, 10 s). Amplification reaction: Incubate at 39℃ for 30 min to obtain RPA amplification products (primer amplification regions corresponding to...). Figure 4 (RPA primer coverage area).

[0030] (3) Purification of amplification products Take 50 μL of RPA product, add 50 μL of Tris-saturated phenol-chloroform-isoamyl alcohol (25:24:1), vortex for 1 min; centrifuge at 12000 r / min for 5 min, and transfer the upper aqueous phase (about 40 μL) to a new tube for later use at -20℃.

[0031] (4) CRISPR-Cas12a reaction System preparation: Add 4 μL of 10×HOLMES Buffer, 1 μL of 10 μM LbCas12a, 1 μL of 10 μM crRNA (SEQ ID NO.7), 1 μL of purified RPA product, 1 μL of 10 μM test strip probe (SEQ ID NO.8), and 42 μL of enzyme-free water to a sterile centrifuge tube, for a total volume of 50 μL. (crRNA targeting region corresponds to...) Figure 4The crRNA targeting region contains the PAM sequence 5′-TTTC-3′. Reaction: React at 37℃ for 30 min.

[0032] (5) Result interpretation Insert the sample end of the CRISPR test strip into the reaction solution (the liquid level should not exceed the control line) and let it stand at room temperature for 3 minutes; observe that both the test line (C) and the control line (T) show red bands (e.g., Figure 1 (Left side), determined to be MAP positive.

[0033] 3. Results and Conclusions The high bacterial load MAP culture sample tested positive by this method, which is consistent with the actual situation, proving the effectiveness of this method in detecting high bacterial load samples.

[0034] Example 2 Detection of samples with low bacterial load (latently infected sheep feces or tissue) (nested PCR + fluorescence system) 1. Material Preparation Samples: Feces from latently infected sheep collected clinically (bacterial load <1×10⁻⁶) −10 μg / μL); Reagents: omega BIO-TEK EZNA® Stool DNA Kit, Takara Premix Taq™ ExTaq™ Version 2.0 plus dye, Tris-saturated phenol-chloroform-isoamyl alcohol (25:24:1), LbCas12a Nuclease, crRNA (SEQ ID NO.7), fluorescent probe (SEQ ID NO.9, synthesized by Nanjing GenScript), 10×Cleavage Buffer (containing 100mM Tris-HCl, 50mM MgCl2, 100mM NaCl, pH 7.5), nuclease-free water; Target sequence: The highly conserved genomic target sequence for MAP detected in this embodiment is SEQ ID NO.10. The positional relationship between the nested PCR primers and crRNA on this target sequence is described in [reference needed]. Figure 4 ; Instruments: constant temperature water bath, centrifuge, ultraviolet lamp, qPCR instrument.

[0035] 2. Operating Procedures (1) Nucleic acid extraction Press omega BIO-TEK EZNA ® Stool DNA Kit Instructions: Take 1g of feces and add 3mL of sterile water to make a paste. Filter the paste through a 60-mesh sieve and centrifuge at 8000r / min for 10min, then discard the supernatant. Take 200 mg of the precipitate and add 200 mg of Glass Beads X to an ice bath for 3 min; add 540 μL of SLX-Mlus Buffer and vortex for 10 min; Add 60 μL DS Buffer and 20 μL proteinase K, and vortex for 1 min; incubate at 70℃ for 10 min, then vortex for 30 s; incubate at 95℃ for 5 min, then vortex for 30 s. Add 200 μL of SP2 buffer, vortex for 30 seconds; incubate on ice for 5 minutes, then centrifuge at 15000 rpm for 5 minutes. Take 400 μL of supernatant and add 200 μL of HTR Reagent, shake for 10 s; let stand at room temperature for 2 min, then centrifuge at 12000 r / min for 2 min; Take 250 μL of supernatant, add 250 μL of BL buffer and 250 μL of anhydrous ethanol, and shake for 10 s; transfer to a HiBind purification column, centrifuge at 12000 r / min for 1 min and discard the filtrate; Add 500 μL VHB Buffer and centrifuge for 1 min, then add 700 μL DNA Wash Buffer and centrifuge for 1 min. Repeat the DNA Wash Buffer wash once. Centrifuge at 12000 rpm for 2 min to dry the column; add 100 μL of 65℃ Elution Buffer, incubate at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min to elute DNA.

[0036] (2) Nested PCR amplification First round of amplification (outer primers): A 25 μL system contained 12.5 μL of Takara Premix Taq™, 9.5 μL of ddH2O, 1 μL of 10 μM 7132F1 (SEQ ID NO.3), 1 μL of 10 μM 7132R1 (SEQ ID NO.4), and 1 μL of the above DNA; Reaction conditions: 94℃ for 2 min, 25 cycles (94℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s), 72℃ for 3 min (for the outer primer coverage area corresponding to...) Figure 4 The nested PCR outer primer region covers the RPA primer region. Second-round amplification (internal primers): Use 1 μL of the first-round product as a template. For the same round, replace the primers with 10 μM 7132F2 (SEQ ID NO.5) and 10 μM 7132R2 (SEQ ID NO.6); Reaction conditions: 94℃ for 2 min, 25 cycles (94℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s), 72℃ for 3 min; Verification: 1% agarose gel electrophoresis showed the expected band (corresponding to the inner primer coverage area). Figure 4 The inner primer region of nested PCR covers the crRNA targeting region.

[0037] (3) Purification of amplification products Take 25 μL of the second round PCR product, add 25 μL of Tris-saturated phenol-chloroform-isoamyl alcohol (25:24:1), shake for 1 min; centrifuge at 12000 r / min for 5 min, and aspirate the upper aqueous phase (about 20 μL) for later use.

[0038] (4) CRISPR-Cas12a reaction System preparation: A 30 μL system contains 3 μL of 10×Cleavage Buffer, 1 μL of 1 μM LbCas12a, 1 μL of 1 μM crRNA, 3 μL of 4 μM fluorescent probe (SEQ ID NO.9), 1 μL of purified PCR product, and 21 μL of nuclease-free water; Reaction: 37℃ constant temperature water bath for 30 min.

[0039] (5) Result interpretation UV observation: When the reaction tube is placed under a 485nm UV lamp, obvious green fluorescence can be seen (e.g., Figure 2 The preliminary assessment is positive. qPCR instrument detection: Place the reaction tube into the qPCR instrument, set the excitation wavelength to 485nm and the emission wavelength to 520nm, monitor for 30 seconds per cycle for 30 minutes; the fluorescence signal should show a clear upward trend (e.g., Figure 3 (This was) further confirmed as positive.

[0040] 3. Results and Conclusions The fecal sample from sheep with latent infection and low bacterial load was positive by this method, which was consistent with the culture identification results, proving the ultrasensitive detection capability of this method for samples with low bacterial load.

[0041] Example 3 Method Validation 1. Sensitivity Verification Sample: Isothermal amplification standard target DNA serially diluted to 1×10 −5 ~1×10 −12 μg / μL Detection method: Nested PCR + fluorescence system of Example 2; Result: When DNA concentration ≥ 1 × 10 −10 At μg / μL, fluorescence is visible under ultraviolet light (e.g., Figure 5 From left to right, tube 5), qPCR signal increases (e.g., Figure 6 (Positive curve); converted to a molar concentration of approximately 2.5 × 10⁻⁶. -15 M, proving that the limit of detection for this method is 2.5 × 10⁻⁶. -15M.

[0042] Sample: Nested PCR standard target DNA serially diluted to 1×10⁻⁶ −9 ~1×10 −16 μg / μL When DNA concentration ≥ 1 × 10 −15 At a concentration of μg / μL, fluorescence is visible under UV light, and the qPCR signal increases; this translates to a molar concentration of approximately 2.5 × 10⁻⁶. -20 M, proving that the limit of detection for this method is 2.5 × 10⁻⁶. -20 M.

[0043] 2. Accuracy and Specificity Validation Samples: 50 clinical sheep fecal samples (22 known to be positive for MAP culture, 28 to be negative); Detection method: Select RPA or nested PCR based on bacterial load, combined with a dual reporter system; Results: The test results of 50 samples were completely consistent with the culture identification (22 positive, 28 negative). The results observed under ultraviolet light were as follows: Figure 7 As shown, the positive concordance rate was 100% and the negative concordance rate was 100%, proving that this method has high accuracy.

Claims

1. A method for detecting Mycobacterium paratuberculosis based on CRISPR-Cas12a technology, characterized in that, Includes the following steps: S1. Provide a sample to be tested, wherein the sample to be tested is feces, tissue, or cultured bacteria sample; S2. Extract total DNA from the sample to be tested; S3. Based on the bacterial load of the sample to be tested, select either the recombinase polymerase amplification (RPA) reaction system or the nested polymerase chain reaction (PCR) amplification reaction system to amplify the target nucleic acid sequence. S4. Prepare the CRISPR-Cas12a reaction system, which includes Cas12a protein, target guide RNA, detection probe and buffer. The target guide RNA includes a region that binds to the Cas12a protein and a guide sequence that hybridizes to the target nucleic acid sequence; the detection probe includes a test strip probe or a fluorescent probe. S5. The amplified target nucleic acid sequence is added to the CRISPR-Cas12a reaction system. The target guide RNA guides the Cas12a protein to specifically bind to the target nucleic acid sequence, activates the trans-cleavage activity of the Cas12a protein, cleaves the detection probe, and generates a detectable signal. S6. Detect the detectable signal and calculate the content of the target nucleic acid sequence.

2. The method for detecting Mycobacterium paratuberculosis based on CRISPR-Cas12a technology according to claim 1, characterized in that, The CRISPR-Cas12a reaction system utilizes a target guide RNA to guide the Cas12a protein to specifically bind to the PAM sequence (5'-TTTV-3') of the target DNA, forming an R-loop structure to activate enzyme activity. This cis-cleaves the double-stranded target DNA to generate sticky ends, simultaneously triggering trans-cleavage activity, and indiscriminately degrading single-stranded nucleic acids in the system, thereby amplifying the target detection signal. The guide sequence of the target guide RNA is shown in SEQ ID NO.

7.

3. The method for detecting Mycobacterium paratuberculosis based on CRISPR-Cas12a technology according to claim 1, characterized in that, The CRISPR-Cas12a reaction system also includes: The test strip probe reaction system includes Cas12a 10×Cleavage buffer, LbCas12a nuclease, target guide RNA, DNA sample, test strip probe, and nuclease-free water; Alternatively, a fluorescent probe detection system may be used, comprising Cas12a 10×Cleavage buffer, LbCas12a nuclease, target guide RNA, fluorescent probe, DNA sample, and nuclease-free water.

4. The method for detecting Mycobacterium paratuberculosis based on CRISPR-Cas12a technology according to claim 1, characterized in that, The recombinase polymerase amplification (RPA) reaction system includes: Forward primer having the nucleotide sequence shown in SEQ ID NO.1; reverse primer having the nucleotide sequence shown in SEQ ID NO.2; primer-free rehydration buffer, template and water, and magnesium acetate; The nested polymerase chain reaction (PCR) amplification system includes Premix Taq; ddH2O; an upstream primer having the nucleotide sequence shown in SEQ ID NO.3; a downstream primer having the nucleotide sequence shown in SEQ ID NO.4; an inner upstream primer having the nucleotide sequence shown in SEQ ID NO.5; an inner downstream primer having the nucleotide sequence shown in SEQ ID NO.6; and a DNA template.

5. The method for detecting Mycobacterium paratuberculosis based on CRISPR-Cas12a technology according to claim 4, characterized in that, The limit of detection for the detection method is 2.5 × 10⁻⁶. −20 M is suitable for detecting samples with early infection or low bacterial load.

6. A kit for detecting Mycobacterium paratuberculosis using the CRISPR-Cas12a technology method as described in claim 1, characterized in that, include: Reagents used for extracting total DNA; Reagents for RPA or nested PCR amplification reaction systems; Reagents for the CRISPR-Cas12a reaction system, including reagents for the test strip probe reaction system or the fluorescent probe detection system; A specific target guide RNA having a nucleotide sequence as shown in SEQ ID NO.7; RPA primers: RPA-7132F1, having the nucleotide sequence shown in SEQ ID NO.1, and RPA-7132R1, having the nucleotide sequence shown in SEQ ID NO.2; Nested PCR primers: 7132F1, having the nucleotide sequence shown in SEQ ID NO.3; 7132R1, having the nucleotide sequence shown in SEQ ID NO.4; 7132F2, having the nucleotide sequence shown in SEQ ID NO.5; and 7132R2, having the nucleotide sequence shown in SEQ ID NO.

6.

7. The kit for detecting Mycobacterium paratuberculosis based on CRISPR-Cas12a technology according to claim 6, characterized in that, It also includes a Tris-saturated phenol-chloroform-isoamyl alcohol mixture for processing amplification products.

8. The use of the method for detecting Mycobacterium paratuberculosis based on CRISPR-Cas12a technology according to claim 4 in detecting Mycobacterium paratuberculosis nucleic acid in environmental or food samples to assess the hygiene status of the environment or food.

9. Primers for an RPA system or nested PCR system used in the method for detecting Mycobacterium paratuberculosis based on CRISPR-Cas12a technology as described in claim 1, characterized in that, RPA primers: RPA-7132F1, having the nucleotide sequence shown in SEQ ID NO.1, and RPA-7132R1, having the nucleotide sequence shown in SEQ ID NO.2; Nested PCR primers: 7132F1, having the nucleotide sequence shown in SEQ ID NO.3; 7132R1, having the nucleotide sequence shown in SEQ ID NO.4; 7132F2, having the nucleotide sequence shown in SEQ ID NO.5; and 7132R2, having the nucleotide sequence shown in SEQ ID NO.

6.

10. A detection system based on CRISPR-Cas12a technology, characterized in that, include: A sample processing device for extracting total DNA from a sample to be tested; Amplification device used to amplify target gene fragments using RPA or nested PCR methods; The detection device is used to process the amplification product and add it to the CRISPR-Cas12a reaction system for detection.

Images