A method for detecting mycobacterium paratuberculosis based on CRISPR-cas12a technology

Abstract

The application belongs to the field of nucleic acid detection, and discloses a method for detecting Mycobacterium avium paratuberculosis based on CRISPR-Cas12a technology. In view of the problems of time-consuming, high cost, low sensitivity and specificity of the existing MAP detection method, and the problems of poor adaptability and insufficient detection of low bacterial load of the existing CRISPR technology, the method combines recombinase polymerase amplification (RPA) and nested polymerase chain reaction (PCR), takes the high-conserved genomic sequence of MAP as a target, and fuses the high sensitivity of nucleic acid amplification with the high specificity and signal amplification capacity of CRISPR-Cas12a. The minimum detection limit reaches 10 −20 M, which is much higher than the effect of traditional PCR or CRISPR alone, and realizes ultra-sensitive, high-specificity, rapid and low-cost detection, and is suitable for early infection or low bacterial load sample detection of paratuberculosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nucleic acid detection, and particularly relates to a method for detecting Mycobacterium paratuberculosis (MAP) based on CRISPR-Cas12a technology. Mycobacterium avium subsp. paratuberculosis , MAP. BACKGROUND

[0002] Paratuberculosis is a chronic infectious disease caused by Mycobacterium paratuberculosis (MAP) in various animals, characterized by progressive weight loss, intractable diarrhea, granulomatous enteritis and mesenteric lymph node hyperplastic inflammation in diseased animals, and even death, which is an infectious disease affecting the economy of animal husbandry and public health safety. At present, the detection methods of MAP include bacterial culture, serological test, PCR and qPCR, etc., but these methods have the disadvantages of long detection time, high cost, low sensitivity and specificity, etc.

[0003] The CRISPR-Cas12a technology has become a core tool for ultra-sensitive detection due to its unique trans-cleavage activity. When Cas12a recognizes the target DNA sequence under the guidance of crRNA, it activates the trans-cleavage activity of non-specific cleavage of single-stranded nucleic acids. However, the combination of the high specificity of CRISPR-Cas12a with the amplification technology of different sample loads, while solving the problem of sample impurity interference and on-site detection adaptability, is the bottleneck of existing technology research. Existing CRISPR technology has made significant progress in nucleic acid detection. Chinese patent CN114457073B discloses a method for detecting MAP based on CRISPR technology, which uses gRNA, V-type Cas protein (such as Cas12) and single-stranded nucleic acid detector for detection. This method improves detection efficiency and specificity by optimizing gRNA sequence, but this method mainly relies on the characteristics of V-type Cas protein and only uses single nucleic acid amplification method (PCR), which is not optimized for low bacterial load samples, and the detection effect of clinical samples is not verified, and the detection range is not presented. Chinese patent CN110541022B discloses a method for detecting Mycobacterium tuberculosis complex based on CRISPR technology, which uses gRNA, V-type Cas protein (such as Cas12a) and single-stranded nucleic acid detector for detection. This method optimizes the reaction conditions to improve the detection efficiency and specificity of Mycobacterium tuberculosis complex by combining recombinase polymerase amplification (RPA) with the trans-cleavage activity of Cas12a, but this method focuses on Mycobacterium tuberculosis complex, does not design specific target (spacer) sequences and matching crRNA for the highly conserved and specific genomic sequence of MAP, and only uses single nucleic acid amplification method (RPA), which cannot accurately identify MAP-specific sequences, cannot adapt to different bacterial load differences in different infection stages, and has insufficient detection capability for low bacterial load samples. Chinese patent CN118895375A discloses a CRISPR-Cas12 detection method for Mycobacterium tuberculosis rpoB gene mutation and application, which uses nested PCR amplification system and CRISPR-Cas12 system to detect drug-resistant gene mutation through specific sgRNA sequence and fluorescent probe. This method improves the sensitivity and specificity of detection by optimizing sgRNA sequence and CRISPR-Cas12 system, and is suitable for detecting rifampicin resistance of Mycobacterium tuberculosis, but this method mainly detects drug-resistant genes of Mycobacterium tuberculosis and does not involve MAP detection, and is not optimized for low bacterial load samples.

[0004] Therefore, developing a method for efficiently detecting MAP and improving detection efficiency and specificity has become a difficult problem that needs to be continuously optimized in the development of current CRISPR technology. SUMMARY

[0005] The first object of the present application is to overcome the shortcomings of the prior art and provide a method for detecting Mycobacterium avium paratuberculosis (MAP) based on CRISPR-Cas12a technology. The method combines recombinase polymerase amplification (RPA) and nested polymerase chain reaction (PCR) to target the highly conserved genomic sequence of MAP, combining the high sensitivity of nucleic acid amplification with the high specificity and signal amplification ability of CRISPR-Cas12a, achieving ultra-sensitive, high-specificity, rapid and low-cost nucleic acid detection, with a detection limit of aM (10 −20 M), far exceeding the ability of traditional PCR or CRISPR alone, suitable for early infection or low bacterial load sample detection of paratuberculosis.

[0006] To achieve the above object, the specific technical solutions adopted by the present application are as follows:

[0007] 1. Sample pretreatment and total DNA extraction

[0008] MAP culture sample: collect the bacterial body by centrifuging the culture bacterial liquid at 8000 r / min for 10 min, use bacterial genomic DNA extraction kit (TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0), lyse the bacterial body with lysis buffer and protease K (56℃ incubation for 30 min), wash with purification column (500μL wash buffer repeated washing 1 time), then elute DNA with 100μL Elution Buffer (room temperature for 2 min, then centrifuge at 12000 r / min for 1 min).

[0009] Tissue sample: use universal genomic DNA extraction kit (TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0), complete tissue homogenization, lysis and DNA purification according to the kit instructions.

[0010] Fecal sample: feces (omega BIO-TEK E.Z.N.A. ®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℃.

[0011] 2. Stepwise amplification of the target DNA fragment

[0012] 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):

[0013] 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.

[0014] Low bacterial load samples (bacterial load <1×10) −10μg / μL): Amplified by nested PCR, the reaction system (total volume 25 μL) contains Takara Premix Taq™ 12.5 μL, ddH2O 9.5 μL, 10 μM upstream primer 1 μL, DNA template 1 μL; one round of amplification uses outer primer pair (7132F1 SEQ ID NO. 3 / 7132R1 SEQ ID NO. 4), the reaction conditions are 94°C pre-denaturation for 2 min, 25 cycles (94°C 30 s→60°C 30 s→72°C 30 s), 72°C final extension for 3 min; the second round of amplification uses inner primer pair (7132F1 SEQ ID NO. 5 / 7132R1 SEQ ID NO. 6), the reaction conditions are 94°C pre-denaturation for 2 min, 25 cycles (94°C 30 s→55°C 30 s→72°C 30 s), 72°C final extension for 3 min, and the target fragment is obtained by amplification.

[0015] 3. Amplification product purification

[0016] Tris-saturated phenol-chloroform-isoamyl alcohol mixture (volume ratio 25:24:1) was added to the RPA or nested PCR amplification product in an equal volume of the above reaction system, mixed well, and centrifuged at 12000 r / min for 5 min. The supernatant was stored at low temperature after taking.

[0017] 4. CRISPR-Cas12a reaction system construction

[0018] The CRISPR-Cas12a reaction system includes Cas12a protein, target guide RNA, detection probe and buffer. The target guide RNA includes a region combined with the Cas12a protein and a guide sequence hybridized with the target nucleic acid sequence. The detection probe includes a test strip probe or a fluorescent probe. The reaction system guides the Cas12a protein to specifically bind to the target nucleic acid sequence through the target guide RNA, activates the transcleavage activity of the Cas12a protein, cuts the detection probe, and produces a detectable signal.

[0019] According to the detection scene requirements, the test strip probe or fluorescent probe reaction system is selected, and the transcleavage activity of LbCas12a is used to realize target signal amplification:

[0020] Table 1 Test strip probe reaction system (total volume 50 μL)

[0021]

[0022] 10×HOLMES Buffer:

[0023] 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, 1 mg / mL BSA, pH 7.9;

[0024] 10 μM MAP-specific crRNA: SEQ ID NO. 7;

[0025] 10 μM test strip probe: SEQ ID NO. 8.

[0026] Table 2 Fluorescent probe reaction system (total volume 30 μL)

[0027]

[0028] 10x Cleavage Buffer:

[0029] 100 mM Tris-HCl, 50 mM MgCl2, 100 mM NaCl, pH 7.5;

[0030] 4 μM fluorescent probe: SEQ ID NO. 9.

[0031] 5. Result interpretation

[0032] According to the selection of the reaction system used, the appropriate interpretation method is selected to realize qualitative or semi-quantitative detection:

[0033] Test strip system: the test strip probe reaction system is reacted at 37°C for 30 min, the CRISPER test strip is inserted into the reaction product, and the results are observed after 3 min. If the detection line (T) and the quality control line (C) both appear red bands, it is determined that the MAP is positive; only the quality control line develops color, which is determined to be negative. Figure 1 ).

[0034] Fluorescent system: ① ultraviolet lamp interpretation: if green fluorescence is observed, it is determined to be positive; Figure 2 ② qPCR instrument interpretation: monitor for 30 min at 30 s / time, and increase to determine that the probe is effectively cut, and the sample is positive. Figure 3 ).

[0035] The minimum detection limit of the above detection method is 2.5x10 −20 M, which is suitable for early infection or low bacterial load sample detection.

[0036] The second object of the present application is to provide a kit for detecting Mycobacterium paratuberculosis, comprising:

[0037] Reagents for extracting total DNA;

[0038] Reagents for RPA or nested PCR amplification reaction system;

[0039] Reagents for CRISPR-Cas12a reaction system, including reagents for test strip probe reaction system or fluorescent probe detection system;

[0040] Specific target guide RNA (SEQ ID NO. 7);

[0041] RPA primers: RPA-7132F1 (SEQ ID NO. 1) and RPA-7132R1 (SEQ ID NO. 2);

[0042] Nested PCR primers: 7132F1 (SEQ ID NO. 3), 7132R1 (SEQ ID NO. 4), 7132F2 (SEQ ID NO. 5), and 7132R2 (SEQ ID NO. 6).

[0043] Tris-saturated phenol-chloroform-isoamyl alcohol mixture for processing the amplification product.

[0044] A third object of the present application is to provide a primer combination for detecting Mycobacterium paratuberculosis, comprising:

[0045] RPA primers: RPA-7132F1 (SEQ ID NO. 1) and RPA-7132R1 (SEQ ID NO. 2);

[0046] Nested PCR primers: 7132F1 (SEQ ID NO. 3), 7132R1 (SEQ ID NO. 4), 7132F2 (SEQ ID NO. 5), and 7132R2 (SEQ ID NO. 6).

[0047] Target guide RNA of CRISPR-Cas12a (SEQ ID NO. 7).

[0048] A fourth object of the present application is to provide a detection system based on CRISPR-Cas12a technology, characterized in that it comprises:

[0049] A sample processing device for extracting total DNA from a sample to be tested;

[0050] An amplification device for amplifying a target gene fragment using RPA or nested PCR method;

[0051] A detection device for detecting the amplification product after processing by adding it to the CRISPR-Cas12a reaction system.

[0052] A fifth object of the present application is to provide the use of a method for detecting Mycobacterium paratuberculosis based on CRISPR-Cas12a technology in detecting Mycobacterium paratuberculosis nucleic acid in environmental or food samples to evaluate the hygiene of the environment or food.

[0053] The method for detecting paratuberculosis mycobacterium has the advantages of high sensitivity, high specificity, rapidness and low cost. -20 M, far beyond the effect of traditional PCR or CRISPR alone, suitable for early infection or low sample detection of paratuberculosis. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 : CRISPR test strip detection result figure, the left side is a positive sample (detection line C, quality control line T color development), the right side is a negative sample (only quality control line T color development);

[0055] Figure 2 : Fluorescent probe ultraviolet light observation result figure, from left to right tube 1~6, 8 are positive (green fluorescence), tube 7 is negative (no fluorescence), tube 9 is blank control (no fluorescence), tube 10 is positive control (strong fluorescence);

[0056] Figure 3 : Fluorescent probe qPCR instrument detection result figure, curve rising is positive (target exists), curve is stable negative (no target);

[0057] Figure 4 : The position of RPA primer, nested PCR primer and crRNA in MAP target sequence (SEQ ID NO. 10);

[0058] Figure 5 : RPA-CRISPR-Cas12a minimum detection limit observation result;

[0059] Figure 6 : RPA-CRISPR-Cas12a minimum detection limit detection result;

[0060] Figure 7 : 50 sample fluorescent probe reaction system ultraviolet light observation result. DETAILED DESCRIPTION

[0061] Example 1 Detection of high bacterial load sample (MAP culture bacteria) (RPA+test strip system)

[0062] 1. Material preparation

[0063] Sample: MAP K-10 standard strain (ATCC 19698, purchased from Shanghai Bai Feng Biological Technology Co., Ltd.) culture bacteria (bacterial load ≥1×10 −10 μg / μL);

[0064] Target sequence: The MAP highly conserved genomic target sequence detected in this example is SEQ ID NO. 10, and the positional relationship between the primers and the crRNA on the target sequence is as follows Figure 4 ;

[0065] 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 probe, 10xHOLMES Buffer, CRISPR test strip;

[0066] Instruments: constant temperature water bath, centrifuge, vortex.

[0067] 2. Operation steps

[0068] (1) Nucleic acid extraction

[0069] Take 1 mL of MAP K-10 culture bacteria, centrifuge at 8000 r / min for 10 min, discard the supernatant, and use TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver. 3.0 to extract total DNA from the precipitate:

[0070] Add 200 μL of lysis buffer and 20 μL of proteinase K, and incubate at 56°C for 30 min;

[0071] Mix 200 μL of ethanol, transfer to the purification column, and centrifuge at 12000 r / min for 1 min to discard the filtrate;

[0072] Add 500 μL of washing buffer and centrifuge for 1 min, repeat 1 time;

[0073] Add 100 μL of Elution Buffer to elute DNA, and store at -20°C.

[0074] (2) RPA amplification

[0075] System preparation: Add 10 μM RPA-7132F1 (SEQ ID NO. 1) 2.4 μL, 10 μM RPA-7132R1 (SEQ ID NO. 2) 2.4 μL, no-primer rehydration buffer 29.5 μL, the above DNA template 5 μL, and enzyme-free water 8.2 μL in a sterile PCR tube, mix to 47.5 μL of rehydration solution;

[0076] Reaction initiation: transfer the rehydration solution to the TwistAmp® Basic reaction microsphere tube, beat to dissolve the microspheres; add 2.5 μL of 280 mM magnesium acetate, mix and centrifuge briefly (5000 r / min, 10 s);

[0077] Amplification reaction: 39°C constant temperature water bath for 30 min to obtain the RPA amplification product (the primer amplification region corresponds Figure 4 to the middle RPA primer coverage region).

[0078] (3) Purification of amplification product

[0079] 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 and store at -20°C.

[0080] (4) CRISPR-Cas12a reaction

[0081] System preparation: add 10x HOLMES Buffer 4 μL, 10 μM LbCas12a 1 μL, 10 μM crRNA (SEQ ID NO. 7) 1 μL, purified RPA product 1 μL, 10 μM test strip probe (SEQ ID NO. 8) 1 μL, and enzyme-free water 42 μL in a sterile centrifuge tube, total volume 50 μL (the crRNA target region corresponds Figure 4 to the middle crRNA target region, containing PAM sequence 5'-TTTC-3').

[0082] Reaction: 37°C for 30 min.

[0083] (5) Result interpretation

[0084] Insert the CRISPR test strip sample end into the reaction solution (the liquid surface does not exceed the quality control line), and place it at room temperature for 3 min; observe that the detection line (C) and the quality control line (T) both appear red bands (such as Figure 1 on the left side), which is determined as MAP positive.

[0085] 3. Results and conclusions

[0086] The high-bacteria-load MAP culture sample is positive by this method, which is consistent with the actual situation, proving the effectiveness of this method for detecting high-bacteria-load samples.

[0087] Example 2 Detection of low-bacteria-load samples (latent infection of sheep feces or tissues) (nested PCR + fluorescence system)

[0088] 1. Material preparation

[0089] Sample: Clinically collected feces of latently infected sheep (bacterial load <1 x 10 −10 μg / μL);

[0090] Reagents: omega BIO-TEK E.Z.N.A. Stool DNA Kit, Takara Premix Taq™ Ex Taq™ 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 Kingsway), 10x Cleavage Buffer (containing 100 mM Tris-HCl, 50 mM MgCl2, 100 mM NaCl, pH 7.5), nuclease-free water;

[0091] Target sequence: The high-conserved genomic target sequence of MAP detected in this example is SEQ ID NO. 10. The positional relationship of the nested PCR primers and the crRNA on the target sequence is shown in Figure 4 ;

[0092] Instruments: constant temperature water bath, centrifuge, ultraviolet lamp, qPCR instrument.

[0093] 2. Operation steps

[0094] (1) Nucleic acid extraction

[0095] According to the instructions of omega BIO-TEK E.Z.N.A. ® Stool DNA Kit:

[0096] Take 1 g of feces and add 3 mL of sterile water to make a paste. Filter through a 60 mesh screen and centrifuge at 8000 r / min for 10 min, discard the supernatant;

[0097] Take 200 mg of the precipitate and add 200 mg of Glass Beads X to ice bath for 3 min; add 540 μL of SLX-Mlus Buffer and vortex for 10 min;

[0098] Add 60 μL of DS Buffer and 20 μL of proteinase K, shake for 1 min; after incubation at 70°C for 10 min, shake for 30 s, and after incubation at 95°C for 5 min, shake for 30 s;

[0099] Add 200 μL of SP2 buffer and shake for 30 s; ice bath for 5 min, centrifuge at 15000 r / min for 5 min;

[0100] Take 400 μL supernatant and add 200 μL HTR Reagent, shake for 10 s; room temperature for 2 min, 12000 r / min centrifugation for 2 min;

[0101] Take 250 μL supernatant and add 250 μL BL buffer and 250 μL anhydrous ethanol, shake for 10 s; transfer to HiBind purification column, 12000 r / min centrifugation for 1 min, discard the filtrate;

[0102] Add 500 μL VHB Buffer and centrifuge for 1 min, add 700 μL DNA Wash Buffer and centrifuge for 1 min, repeat DNA Wash Buffer washing 1 time;

[0103] 12000 r / min centrifugation for 2 min to dry the column; add 100 μL 65℃ Elution Buffer, room temperature for 2 min, 12000 r / min centrifugation for 1 min to elute DNA.

[0104] (2) Nested PCR amplification

[0105] One round of amplification (outer primer): 25 μL system contains Takara Premix Taq™ 12.5 μL, ddH2O 9.5 μL, 10 μM 7132F1 (SEQ ID NO. 3) 1 μL, 10 μM 7132R1 (SEQ ID NO. 4) 1 μL, 1 μL of the above DNA; Reaction conditions: 94℃ 2min, 25 cycles (94℃ 30s, 60℃ 30s, 72℃ 30s), 72℃ 3min (outer primer coverage region corresponds to Figure 4 Middle nested PCR outer primer region, covering RPA primer region);

[0106] Two rounds of amplification (inner primer): take 1 μL of the first round of product as template, system is the same as the first round, replace 10 μM 7132F2 (SEQ ID NO. 5) and 10 μM 7132R2 (SEQ ID NO. 6); Reaction conditions: 94℃ 2min, 25 cycles (94℃ 30s, 55℃ 30s, 72℃ 30s), 72℃ 3min;

[0107] Verification: 1% agarose gel electrophoresis can see the expected band (inner primer coverage region corresponds to Figure 4 Middle nested PCR inner primer region, covering crRNA target region).

[0108] (3) Purification of amplification products

[0109] 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 take the upper aqueous phase (about 20 μL) for standby.

[0110] (4) CRISPR-Cas12a reaction

[0111] System preparation: 30 μL system contains 10x Cleavage Buffer 3 μL, 1 μM LbCas12a 1 μL, 1 μM crRNA 1 μL, 4 μM fluorescent probe (SEQ ID NO. 9) 3 μL, purified PCR product 1 μL, nuclease-free water 21 μL;

[0112] Reaction: 37°C constant temperature water bath for 30 min.

[0113] (5) Result interpretation

[0114] Ultraviolet lamp observation: place the reaction tube under the 485 nm ultraviolet lamp, and obvious green fluorescence can be seen (such as Figure 2 ), which is preliminarily determined as positive;

[0115] qPCR instrument detection: place the reaction tube into the qPCR instrument, set the excitation wavelength to 485 nm and the emission wavelength to 520 nm, and monitor for 30 min at 30 s / time; the fluorescence signal shows an obvious upward trend (such as Figure 3 ), which further confirms the positive result.

[0116] 3. Results and conclusions

[0117] The low-bacterial-load latent sheep fecal sample was detected as positive by the method, which is consistent with the culture identification result, proving the ultra-sensitive detection ability of the method for low-bacterial-load samples.

[0118] Example 3 Method verification

[0119] 1. Sensitivity verification

[0120] Sample: Gradient dilution of standard target DNA by isothermal amplification to 1x10 −5 ~1x10 −12 μg / μL

[0121] Detection method: nested PCR + fluorescent system of Example 2;

[0122] Results: When the DNA concentration is ≥1x10 −10 μg / μL, fluorescence can be seen under the ultraviolet lamp (such as Figure 5 , tube 5 from left to right), and the qPCR signal rises (such as Figure 6 positive curve); the conversion to molar concentration is about 2.5x10-15 M, which proved that the minimum detection limit of the method was 2.5 x 10 -15 M.

[0123] Sample: The standard DNA ladder of nested PCR was diluted to 1 x 10 −9 ~1 x 10 −16 μg / μL

[0124] When the DNA concentration was ≥1 x 10 −15 μg / μL, fluorescence could be observed under the ultraviolet lamp, and the qPCR signal rose; converted to molar concentration, it was about 2.5 x 10 -20 M, which proved that the minimum detection limit of the method was 2.5 x 10 -20 M.

[0125] 2. Accuracy and specificity verification

[0126] Sample: 50 clinical sheep fecal samples (22 MAP culture positive and 28 negative were known);

[0127] Detection method: RPA or nested PCR combined with double reporting system according to the bacterial load;

[0128] Results: The detection results of 50 samples were completely consistent with the culture identification (22 positive and 28 negative), the ultraviolet lamp observation results were as shown in Figure 7 , the positive coincidence rate was 100%, the negative coincidence rate was 100%, which proved that the method had high accuracy.

Claims

1. A method for detecting Mycobacterium paratuberculosis based on CRISPR-Cas12a technology, applied to the detection of Mycobacterium paratuberculosis nucleic acid in environmental or food samples to assess the sanitary condition of the environment or food, characterized in that, The method is for non-disease diagnosis purposes and includes the following steps: S1. Provide a sample to be tested, which may be an environmental sample or a food sample; S2. Extract total DNA from the sample to be tested; S3. The target nucleic acid sequence is amplified using a nested polymerase chain reaction (PCR) system. The nested PCR system includes: an upstream primer with the nucleotide sequence shown in SEQ ID NO.3; a downstream primer with the nucleotide sequence shown in SEQ ID NO.4; an inner upstream primer with the nucleotide sequence shown in SEQ ID NO.5; and an inner downstream primer with the nucleotide sequence shown in SEQ ID NO.

6. 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 is a fluorescent probe; the guide sequence of the target guide RNA is shown in SEQ ID NO.7; 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 to assess the hygiene status of the environment or food; The CRISPR-Cas12a reaction system utilizes 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.

2. The application according to claim 1, characterized in that, The CRISPR-Cas12a reaction system also includes: The fluorescent probe detection system includes Cas12a 10×Cleavage buffer, LbCas12a nuclease, target guide RNA, fluorescent probe, DNA sample, and nuclease-free water.

3. The application according to claim 2, characterized in that, The limit of detection for the method for detecting Mycobacterium paratuberculosis is 2.5 × 10⁻⁶. −20 M is suitable for testing samples with low bacterial load.

4. A kit for implementing the application of claim 1, characterized in that, include: Reagents used to extract total DNA from environmental or food samples; Reagents for nested polymerase chain reaction systems; The reagents in the CRISPR-Cas12a reaction system are also the reagents in the fluorescent probe detection system; A specific target guide RNA, the nucleotide sequence of which is shown in SEQ ID NO.7; The primers for the nested polymerase chain reaction system are: 7132F1, whose nucleotide sequence is shown in SEQ ID NO.3; 7132R1, whose nucleotide sequence is shown in SEQ ID NO.4; 7132F2, whose nucleotide sequence is shown in SEQ ID NO.5; and 7132R2, whose nucleotide sequence is shown in SEQ ID NO.

6.

5. The reagent kit according to claim 4, characterized in that, It also includes a Tris-saturated phenol-chloroform-isoamyl alcohol mixture for processing amplification products.

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