Method, kit and system for detecting intestinal microsporidia based on CRISPR-Cas12a (clustered regularly interspaced short palindromic repeats / CRISPR-Cas12a) technology

Abstract

The invention provides a method, a kit and a system for detecting intestinal microsporidia based on a CRISPR-Cas12a technology, belongs to the field of nucleic acid detection, and aims to solve the problems that an existing intestinal microsporidia 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 DNA template quantity and the like. Recombinase polymerase amplification (RPA) and nested polymerase chain reaction (PCR) are combined, a conserved genome sequence of the enterosporidia is taken as a target, high sensitivity of nucleic acid amplification and high specificity and signal amplification capability of CRISPR-Cas12a are fused, the lowest detection limit reaches 1021M, the lowest detection limit is far better than the single use effect of traditional PCR or CRISPR-Cas12a, ultra-sensitive, high-specificity, rapid and low-cost detection is realized, and the kit is suitable for clinical application. The kit is suitable for detecting enterosporidium pichia early infection or samples with low DNA template quantity.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection, and more particularly to a method for detecting Enterospora bifidum based on CRISPR-Cas12a technology (CRISPR-Cas12a). Enterocytozoon bieneusi The method. Background Technology

[0002] Bispora bengaliformis ( Enterocytozoon bieneusi *Pichia pastoris* is an obligate intestinal parasitic protozoan that can be transmitted via the fecal-oral route through contaminated food or water. Symptoms include chronic emaciation, diarrhea, and even death in infected animals, severely impacting livestock economics and public health. Currently, detection methods for *Pichia pastoris* include electron or optical microscopy, serological tests, nested polymerase chain reaction (PCR), and qPCR. However, these methods have drawbacks such as time-consuming testing, high equipment requirements, high cost, and low sensitivity or 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 target guide RNA, it activates its 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 amounts of DNA template, while simultaneously addressing issues of sample impurity interference and on-site detection adaptability, remains a bottleneck in current research. Existing CRISPR technology has made significant progress in the field of nucleic acid detection. Currently, Chinese patent CN117286144A discloses a method for detecting microsporidia based on CRISPR technology, utilizing gRNA, type V Cas protein (such as Cas12), and a single-stranded nucleic acid detector. However, the amplification method and CRISPR system in this invention only showed detection effectiveness against *Microsporidia helenii*, *Microsporidia enterica*, and *Microsporidia rabbitis*, and failed to successfully amplify and detect *Microsporidia bidentata*. Chinese patent CN119265356A discloses a method for detecting *P. pylori* based on CRISPR technology. This method utilizes gRNA, type V Cas protein (such as Cas12a), and a single-stranded nucleic acid detector. By leveraging the trans-cleavage activity of Cas12a and combining it with recombinase polymerase amplification (RPA) technology to optimize reaction conditions, the method aims to improve the detection efficiency and specificity of *P. pylori*. The sensitivity of this method is 1 copy / μL, but it has not shown detection performance with lower DNA template amounts, and its effectiveness in clinical samples has not been verified. Therefore, developing an efficient method for detecting *P. pylori* and improving its detection efficiency and specificity remains a challenging problem that requires continuous optimization in the development of CRISPR technology. Summary of the Invention

[0004] The first object of the present application is to overcome the shortcomings of the prior art and provide a method, kit and system for detecting Enterocytozoon bieneusi based on CRISPR-Cas12a technology. The method for detecting Enterocytozoon bieneusi combines recombinase polymerase amplification (RPA) and nested polymerase chain reaction (PCR), takes the conserved sequence of Enterocytozoon bieneusi genome (GeneBank: KY972259.1) as the target, and combines the high sensitivity of nucleic acid amplification with the high specificity and signal amplification ability of CRISPR-Cas12a, so as to realize the ultra-sensitive, high-specific, rapid and low-cost nucleic acid detection, and the detection lower limit is pushed to aM (10 −21 M), far exceeding the ability of traditional PCR or CRISPR alone, and is suitable for detecting Enterocytozoon bieneusi early infection or low DNA template amount samples.

[0005] To achieve the above object, the specific technical solutions adopted by the present application are as follows: 1. Sample pretreatment and total DNA extraction The stool is adjusted with sterile water, filtered through a 60-mesh screen, and the precipitate is taken after centrifugation at 8000 r / min for 10 min. The total DNA of the sample is extracted according to the operation of omegaBIO-TEK E.Z.N.A. ® Stool DNA Kit. Take 200 mg of the precipitate and place it in 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 shake for 10 min to homogenize; add 60 μL of DS Buffer and 20 μL of proteinase K, shake for 1 min; place the centrifuge tube in a 70°C incubator for 10 min, then shake for 30 s, and incubate at 95°C for 5 min, shake for 30 s; add 200 μL of SP2 buffer, shake for 30 s, ice bath for 5 min, and centrifuge at 15000 r / min for 5 min; transfer 400 µL of supernatant to a 1.5 mL centrifuge tube, add 200 μL of HTR Reagent solution, and shake immediately for 10 s; room temperature for 2 min, 12000 r / min, centrifuge for 2 min; transfer 250 µL of supernatant to a new 1.5 mL centrifuge tube, add 250 μL of BL buffer and 250 μL of anhydrous ethanol, shake for 10 s to mix thoroughly; transfer the 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°C.

[0006] 2. Ladder amplification of target DNA fragments According to the amount of sample DNA template, select the appropriate amplification method to ensure efficient enrichment of the target fragment (containing the specific target sequence of N. binlatiens): High DNA template sample (template amount ≥1×10 −12 μg / μL): RPA amplification was used, the reaction system (total volume 50 μL) contained 10 μM RPA-ITSF1 (SEQ ID NO: 1) 2.4 μL, 10 μM RPA-ITSR1 (SEQ ID NO: 2) 2.4 μL, no primer rehydration buffer 29.5 μL, DNA template and water 13.2 μL, mixed and centrifuged briefly, then the rehydration solution was transferred to the reaction microspheres, and the whole microspheres were resuspended by blowing; 2.5 μL of 280 mM magnesium acetate was added and mixed thoroughly, and then centrifuged quickly again and placed at 39°C for 30 min, and the target fragment was amplified.

[0007] Low DNA template sample (template amount <1×10 −12 μg / μL): Nested PCR amplification was used, the reaction system (total volume 25 μL) contained Takara Premix Taq™ 12.5 μL, ddH2O 9.5 μL, 10 μM upper and lower primers 1 μL each, DNA template 1 μL; one round of amplification used outer primer pair (ITSF1 SEQ ID NO: 3 / ITSR1 SEQ ID NO: 4), the reaction condition was 94°C pre-denaturation for 5 min, 30 cycles (94°C 30 s→57°C 30 s→72°C 40 s), 72°C final extension for 10 min; the second round of amplification used inner primer pair (ITSF2 SEQ ID NO: 5 / ITSR2 SEQ ID NO: 6), the reaction condition was 94°C pre-denaturation for 5 min, 30 cycles (94°C 30 s→55°C 30 s→72°C 40 s), 72°C final extension for 10 min, and the target fragment was amplified.

[0008] 3. Purification of amplification products Tris-saturated phenol-chloroform-isoamyl alcohol mixture (volume ratio 25:24:1) was added to the RPA or nested PCR amplification products, mixed well, and centrifuged at 12000 r / min for 5 min, and the supernatant was stored at low temperature.

[0009] 4. Construction of CRISPR-Cas12a reaction system The CRISPR-Cas12a reaction system comprises a Cas12a protein, a target guide RNA (crRNA), a detection probe, and a buffer. The crRNA comprises a region combined with the Cas12a protein and a guide sequence hybridized with the target nucleic acid sequence. The detection probe comprises 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 crRNA, activates the transcleavage activity of the Cas12a protein, cuts the detection probe, and generates a detectable signal.

[0010] According to the detection scene requirements, the test strip probe or fluorescent probe reaction system is selected, and the transcleavage activity of LbCas12a is utilized to realize target signal amplification: Table 1 Test strip probe reaction system (total volume 50 μL)

[0011] 10×HOLMES Buffer: 500mM NaCl, 100mM Tris-HCl, 100mM MgCl2, 1mg / mL BSA, pH 7.9; 10μM Entamoeba bieneusi specific crRNA: SEQ ID NO: 7; 10μM test strip probe: SEQ ID NO: 8.

[0012] Table 2 Fluorescent probe reaction system (total volume 30 μL)

[0013] 10×Cleavage Buffer: 100mM Tris-HCl, 50mM MgCl2, 100mM NaCl, pH 7.5; 4μM fluorescent probe: SEQ ID NO: 9.

[0014] A second object of the present application is to provide a kit for detecting Entamoeba bieneusi, comprising: reagents for extracting total DNA; reagents for RPA or nested PCR amplification reaction system; reagents for CRISPR-Cas12a reaction system, including reagents for test strip probe reaction system or fluorescent probe detection system; specific target guide RNA (SEQ ID NO: 7); RPA primers: RPA-ITSF1 (SEQ ID NO: 1) and RPA-ITSR1 (SEQ ID NO: 2); Nested PCR primers: ITSF1 (SEQ ID NO: 3), ITRR1 (SEQ ID NO: 4), IITSF2 (SEQ ID NO: 5) and IITSR2 (SEQ ID NO: 6).

[0015] Tris-saturated phenol-chloroform-isoamyl alcohol mixture for processing the amplification product.

[0016] A third object of the present application is to provide a primer combination for detecting Enterocytozoon bieneusi, comprising: RPA primers: RPA-ITSF1 (SEQ ID NO: 1) and RPA-ITSR1 (SEQ ID NO: 2); Nested PCR primers: ITSF1 (SEQ ID NO: 3), ITRR1 (SEQ ID NO: 4), IITSF2 (SEQ ID NO: 5) and IITSR2 (SEQ ID NO: 6); Target guide RNA of CRISPR-Cas12a (SEQ ID NO: 7).

[0017] A fourth object of the present application is to provide a detection system based on CRISPR-Cas12a technology, comprising: A sample processing device for extracting total DNA from a sample to be tested; An amplification device for amplifying the target gene fragment using RPA or nested PCR method; A detection device for adding the amplification product after processing to the CRISPR-Cas12a reaction system for detection.

[0018] A fifth object of the present application is to provide an application of a method for detecting Enterocytozoon bieneusi based on CRISPR-Cas12a technology, for preparing a product for detecting Enterocytozoon bieneusi in environmental samples / food samples; it can also be used for detecting Enterocytozoon bieneusi nucleic acid in animal feces, environmental or food samples to evaluate the infection or health status of animals, environment or food.

[0019] A sixth object of the present application is to provide a nucleic acid combination for detecting Enterocytozoon bieneusi based on CRISPR-Cas12a technology, comprising: RPA amplification primer pair, the gene sequence of which is shown in SEQ ID NO: 1 and SEQ ID NO: 2; Nested PCR primer set, the gene sequence of which is shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; crRNA, the gene sequence of which is shown in SEQ ID NO: 7; The detection probe is a test strip probe or a fluorescent probe; the gene sequence of the test strip probe is shown in SEQ ID NO:8, and the gene sequence of the fluorescent probe is shown in SEQ ID NO:9.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides a highly sensitive, specific, rapid, and low-cost method for detecting *Microsporidium pichii*, combining recombinase polymerase amplification (RPA) and nested polymerase chain reaction (PCR). Targeting conserved sequences in the *Microsporidium pichii* genome, it integrates the high sensitivity of nucleic acid amplification with the high specificity and signal amplification capabilities of CRISPR-Cas12a, achieving highly sensitive, specific, rapid, and low-cost nucleic acid detection, pushing the detection limit to aM (0.7 × 10⁻⁶). −21 M), far exceeding the capabilities of traditional PCR or CRISPR alone, is suitable for detecting early infection of Enterospora bifidum or samples with low DNA template content. It can achieve early infection diagnosis and accurate detection of samples with low pathogen load, solving the industry problem of difficult detection of latent and asymptomatic infections.

[0021] 2. The method for detecting *Microsporidium pichii* provided by this invention uses RPA technology for high template load samples, achieving rapid (30 minutes) and isothermal (39°C) detection without the need for complex thermal cyclers, making it suitable for on-site screening. For low template load samples, nested PCR technology is used, which greatly improves sensitivity and specificity through two rounds of amplification, ensuring the detection rate of extremely small sample volumes. It can also be quickly interpreted under ultraviolet light. This method can cover both high and low template load sample types, making it more widely applicable.

[0022] 3. The method for detecting *Microsporidium pichii* provided by this invention uses crRNA to precisely guide the Cas12a protein to target and bind to the specific gene sequence of *Microsporidium pichii*, effectively avoiding cross-reactions with other microorganisms and ensuring high reliability of the detection results.

[0023] 4、The method of the present application provides a "amplification-identification-re-amplification" reaction system for Enterocytozoon bieneusi, which first processes and extracts DNA from the sample to obtain a DNA template; according to the initial DNA template amount, a suitable nucleic acid amplification method is selected, a recombinase polymerase amplification (RPA) is used for rapid enrichment of the target DNA at room temperature for high template amount samples, and a nested polymerase chain reaction (nested PCR) is used for two rounds of amplification to improve the sensitivity and specificity for low template amount samples; then in the CRISPR-Cas12a stage, a complex is formed with a guide RNA (crRNA), which specifically recognizes the conserved target sequence of Enterocytozoon bieneusi in the amplification product, activates the trans-cleavage activity of Cas12a, and non-specifically cleaves the single-stranded DNA probe; when a fluorescent probe is used, the fluorescent reporter group and the quencher group are separated to generate a fluorescent signal after cleavage; when a test strip probe is used, the detection line changes color after cleavage; finally, the results are interpreted according to the physical signals (fluorescence or color bands) generated by the cleavage of the probe; the appearance of the signal indicates that the target sequence exists in the sample; through multi-stage amplification, the system breaks through the sensitivity limit of traditional methods and realizes ultra-sensitive and high-specificity detection. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The CRISPR test strip detection result, 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); Figure 2 The fluorescent probe ultraviolet lamp observation result, from left to right tube 1-4 is positive (fluorescent signal), tube 5-8 is negative (no fluorescent signal); Figure 3 The fluorescent probe qPCR instrument detection result, the curve rises is positive (target exists), the curve is stable is negative (no target); Figure 4 The position of the RPA primer, the nested PCR primer and the crRNA in the Enterocytozoon bieneusi target sequence (SEQ ID NO: 10); Figure 5 The RPA-CRISPR-Cas12a minimum detection limit eye observation result; Figure 6 The RPA-CRISPR-Cas12a minimum detection limit machine detection result; Figure 7 The fluorescent probe reaction system ultraviolet light observation result of 50 samples, from left to right tube 6 is positive control, tube 7 is negative control, tube 8 is blank on the left side of the fourth row; from left to right tube 6 is positive control, tube 7 is negative control, tube 8 is blank on the right side of the third row; Figure 8 The nested PCR-CRISPR-Cas12a minimum detection limit eye observation result; Figure 9 The minimum detection limit of the nested PCR-CRISPR-Cas12a was determined by machine detection. DETAILED DESCRIPTION

[0025] Example 1: Detection of high template amount samples (RPA + test strip system) 1. Material preparation Sample: clinically collected feces of animals with latent infection (template amount ≥ 1 x 10 −12 μg / μL); Reagent: omega BIO-TEK E.Z.N.A. 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 Kingsray), 10x Cleavage Buffer (containing 100mM Tris-HCl, 50mM MgCl2, 100mM NaCl, pH 7.5), nuclease-free water; Target sequence: The conserved genomic target sequence of the Enterocytozoon bieneusi detected in this example is SEQ ID NO: 10, and the positional relationship of the nested PCR primers and the crRNA on the target sequence is shown in Figure 4 ; Instrument: constant temperature water bath, centrifuge.

[0026] 2. Operation steps (1) Nucleic acid extraction According to the operation manual of omega BIO-TEK E.Z.N.A. ® Stool DNA Kit: Take 1 g of feces and add 3 mL of sterile water to make a paste, filter through a 60 mesh sieve, and centrifuge at 8000 r / min for 10 min to discard the supernatant; 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; 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; Add 200 μL of SP2 buffer and shake for 30 s; ice bath for 5 min, centrifuge at 15000 r / min for 5 min; 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.

[0027] (2) RPA amplification System preparation: Add 2.4 μL of 10 μM RPA-ITSF1 (SEQ ID NO:1), 2.4 μL of 10 μM RPA-ITSR1 (SEQ ID NO:2), 29.5 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).

[0028] (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℃.

[0029] (4) CRISPR-Cas12a reaction System preparation: add 10x HOLMES Buffer 4μL, 10μM LbCas12a 1μL, 10μM crRNA (SEQ ID NO. 7) 1μL, purified RPA product 1μL, 10μM test strip probe (SEQ ID NO. 8) 1μL, nuclease-free water 42μL in a sterile centrifuge tube, total volume 50μL, (crRNA target region corresponds to Figure 4 the PAM sequence 5'-TTTC-3' in the middle crRNA target region); Reaction: 37℃ for 30min.

[0030] (5) Result interpretation Insert the sample end of the CRISPR test strip into the reaction solution (the liquid level does not exceed the quality control line), observe after standing at room temperature for 3min. If the detection line (C) and the quality control line (T) both appear red bands, it is determined to be positive for N. binlatiensis; if only the quality control line (T) develops color, it is determined to be negative. In this example, it was observed that the detection line (C) and the quality control line (T) both appeared red bands (Fig. 2, left side), so the sample was determined to be positive for N. binlatiensis. Figure 1

[0031] 3. Result conclusion This high DNA template amount (template amount ≥1x10 −12 μg / μL) sample was detected as positive by this method, which is consistent with the actual situation, proving the effectiveness of this method for detecting high DNA template amount samples.

[0032] Example 2 Detection of low template amount samples (feces of animals with latent infection) (nested PCR + fluorescence system or test strip system) 1. Material preparation Sample: clinically collected feces of animals with latent infection (bacterial load <1x10 −12 μg / μL); Reagent: 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 Kingsray), 10xCleavage Buffer (containing 100mM Tris-HCl, 50mM MgCl2, 100mM NaCl, pH7.5), nuclease-free water; ​Target sequence: The conserved genomic target sequence of the detected Enterozytom bietschlii in this embodiment is SEQ ID NO: 10, and the positional relationship between the nested PCR primer and the crRNA on the target sequence is shown in the following table: Figure 4 ; Instrument: constant temperature water bath, centrifuge, ultraviolet lamp, qPCR instrument.

[0033] 2. Operation steps (1) Nucleic acid extraction According to the operation instruction of omega BIO-TEK E.Z.N.A. ® Stool DNA Kit: 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 to discard the supernatant; 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; 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; Add 200 μL of SP2 buffer and shake for 30 s; ice bath for 5 min, centrifuge at 15000 r / min for 5 min; Take 400 μL of supernatant and add 200 μL of HTR Reagent, shake for 10 s; room temperature for 2 min, centrifuge at 12000 r / min for 2 min; Take 250 μL of supernatant and add 250 μL of BL buffer and 250 μL of anhydrous ethanol, shake for 10 s; transfer to HiBind purification column, centrifuge at 12000 r / min for 1 min to discard the filtrate; Add 500 μL of VHB Buffer and centrifuge for 1 min, add 700 μL of DNA Wash Buffer and centrifuge for 1 min, repeat the DNA Wash Buffer washing for 1 time; Centrifuge at 12000 r / min for 2 min to dry the column; add 100 μL of 65°C Elution Buffer, room temperature for 2 min, centrifuge at 12000 r / min for 1 min to elute DNA.

[0034] (2) Nested PCR amplification One round of amplification (outer primer): 25 μL system contains Takara Premix Taq™ 12.5 μL, ddH2O 9.5 μL, 10 μM ITSF1 (SEQ ID NO: 3) 1 μL, 10 μM ITSR1 (SEQ ID NO: 4) 1 μL, 1 μL of the above DNA; reaction conditions: 94°C for 5 min, 30 cycles (94°C for 30 s, 57°C for 30 s, 72°C for 40 s), 72°C for 10 min (outer primer coverage area corresponds to Figure 4 Middle nested PCR outer primer region, covering RPA primer region); Second round 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 ITSF2 (SEQ ID NO: 5) and 10 μM ITSR2 (SEQ ID NO: 6); reaction conditions: 94°C for 5 min, 30 cycles (94°C for 30 s, 55°C for 30 s, 72°C for 40 s), 72°C for 10 min; Verification: 1% agarose gel electrophoresis can see the expected band (inner primer coverage area corresponds to Figure 4 Middle nested PCR inner primer region, covering crRNA target region).

[0035] (3) Purification of amplification products Take 25 μL of the second round of 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.

[0036] (4) CRISPR-Cas12a reaction Fluorescent probe reaction system preparation: 30 μL system contains 10×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; Test strip reaction system configuration: add 10×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, enzyme-free water 42 μL in a sterile centrifuge tube, total volume 50 μL, (crRNA target region corresponds to Figure 4 Middle crRNA target region, containing PAM sequence 5'-TTTC-3') Reaction: 37°C constant temperature water bath for 30 min.

[0037] (5) Result interpretation UV lamp observation: Place the reaction tube under a 485nm UV lamp, and observe obvious fluorescence (such as Figure 2 ), which is preliminarily determined as positive; qPCR instrument detection: Place the reaction tube into the qPCR instrument, set the excitation wavelength to 485nm and the emission wavelength to 520nm, and monitor for 30min at 30s / time; the fluorescence signal shows an obvious upward trend (such as Figure 3 ), which further confirms the positive result.

[0038] Test strip interpretation: Insert the CRISPR test strip sample end into the reaction solution (the liquid level does not exceed the quality control line), and place it at room temperature for 3min; observe that the detection line (C) and the quality control line (T) both appear red bands, which is determined as positive for B. intestinalis.

[0039] 3. Results and conclusions The low DNA template amount (template amount <1×10 −12 μg / μL) of the latent infected animal fecal sample was detected as positive by the method, which is consistent with the identification result of sequencing analysis, proving the ultra-sensitive detection ability of the method for low DNA template amount samples.

[0040] Example 3 Method validation 1. Sensitivity verification (1) High template amount sample detection sensitivity verification Sample: Gradient dilution of isothermal amplification standard target DNA to 1×10 −4 to 1×10 −15 μg / μL; Detection method: Use the RPA amplification method of Example 1, and cooperate with the fluorescence probe reaction system for detection; Results: When the DNA concentration is ≥1×10 −12 μg / μL, fluorescence can be observed under a UV lamp (such as Figure 5 , tube 9 from left to right), and the qPCR signal rises (such as Figure 6 positive curve); converted to molar concentration, it is about 1.18×10 -17 M, proving that the minimum detection limit of the method is 1.18×10 -17 M (( Figure 5 , Figure 6 ).

[0041] (2) Low template amount sample detection sensitivity verification Sample: Gradient dilution of nested PCR standard target DNA to 1×10 −10 to 1×10 −18 μg / μL; Detection method: Use the nested PCR amplification method of Example 2, and cooperate with the fluorescence probe reaction system for detection; Result: When DNA concentration ≥ 1 × 10 −16 At μg / μL, fluorescence is visible under ultraviolet light (e.g., Figure 8 From left to right, tube 7), the qPCR signal increases (e.g., Figure 9 (Positive curve); converted to a molar concentration of approximately 0.7 × 10⁻⁶. -21 M, proving that the limit of detection for this method is 0.7 × 10⁻⁶. -21 M ( Figure 8 , Figure 9 ).

[0042] 2. Accuracy and Specificity Validation Samples: 50 clinical animal fecal samples (21 of which were positive for Enteromicrosporidia picarii, and 29 were negative). Detection method: Select RPA or nested PCR based on the amount of DNA template, combined with a dual reporter system (test strip system and fluorescence system). Results: The test results of 50 samples were completely consistent with the sequencing analysis results (21 positive and 29 negative), and 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 *Bietrionis pilosa* based on CRISPR-Cas12a technology, characterized in that, Includes the following steps: (1) Extract total DNA from the sample to be tested; (2) Using the total DNA obtained in step (1) as a template, RPA or nested PCR is performed according to the total DNA template amount to enrich the target DNA fragment containing the target sequence specific to Bishopricia pygmys. (3) Purify the amplification product obtained in step (2); (4) The purified amplification product was mixed with the CRISPR-Cas12a reaction system and reacted. The CRISPR-Cas12a reaction system comprises Cas12a protein, crRNA targeting a specific target sequence of *Bishopus picus*, a detection probe, and a buffer; the gene sequence of the crRNA is SEQ ID NO.7; The crRNA guides the Cas12a protein to specifically bind to the target sequence of *Microsporidium bitum*, activating the trans-cleavage activity of the Cas12a protein, cleaving the detection probe, and generating a detectable signal. (5) By detecting the detectable signal generated after the detection probe is cut, it is determined whether the sample to be tested contains Enterospora bifidum.

2. The method according to claim 1, characterized in that, In step (2): When the amount of DNA template is ≥1×10 -12 At μg / μL, RPA was used for amplification, and the gene sequences of the primers used were SEQ ID NO.1 and SEQ ID NO.2; When the amount of DNA template is <1×10 -12 At a concentration of μg / μL, nested PCR was used for amplification. The gene sequences of the primers used in the first round of amplification were SEQ ID NO.3 and SEQ ID NO.4, and the gene sequences of the primers used in the second round of amplification were SEQ ID NO.5 and SEQ ID NO.

6.

3. The method according to claim 1 or 2, characterized in that, The specific target sequence of *Pichia pastoris* is shown in SEQ ID NO.

10.

4. The method according to claim 1, characterized in that, The sample to be tested in step (1) is a fecal sample; The detection probe mentioned in step (4) is a test strip probe or a fluorescent probe; The gene sequence of the test strip probe is SEQ ID NO.8, and the gene sequence of the fluorescent probe is SEQ ID NO.

9.

5. A kit for use in the method of claim 4, characterized in that, Include: Reagents used to extract total DNA from samples; Primer pairs for RPA amplification, the gene sequences of which are SEQ ID NO.1 and SEQ ID NO.2; and / or primer sets for nested PCR amplification, the gene sequences of which are SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6; The CRISPR-Cas12a reaction system reagents include Cas12a protein, crRNA with the sequence SEQ ID NO.7, detection probe and buffer; Tris-saturated phenol-chloroform-isoamyl alcohol mixture used for purifying amplified products.

6. The reagent kit according to claim 5, characterized in that, The detection probe is a test strip probe with the sequence SEQ ID NO.8 or a fluorescent probe with the sequence SEQ ID NO.

9.

7. The reagent kit according to claim 5, characterized in that, In the Tris-saturated phenol-chloroform-isoamyl alcohol mixture, the volume ratio of Tris-saturated phenol:chloroform:isoamyl alcohol is 25:24:

1.

8. A nucleic acid combination for use in the method of claim 4, characterized in that, Include: Primer pairs used for RPA amplification: SEQ ID NO.1 and SEQ ID NO.2; Primer set for nested PCR amplification: ITSF1: has the nucleotide sequence shown in SEQ ID NO.3; ITSR1: Has the nucleotide sequence shown in SEQ ID NO.4; ITSF2: has the nucleotide sequence shown in SEQ ID NO.5; ITSR2: has the nucleotide sequence shown in SEQ ID NO.6; crRNA used in the CRISPR-Cas12a reaction system: SEQ ID NO:7; The detection probe is a test strip probe or a fluorescent probe; the gene sequence of the test strip probe is shown in SEQ ID NO:8, and the gene sequence of the fluorescent probe is shown in SEQ ID NO:

9.

9. A system for detecting *Bieterina pentaphyllum* 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 selectively amplify target gene fragments using RPA or nested PCR methods based on the amount of DNA template. The detection device is used to purify the amplification product and add it to a CRISPR-Cas12a reaction system containing Cas12a protein, crRNA and detection probe for detection and result interpretation.

10. The application of the method for detecting *Bishoprus pentodesmus* based on CRISPR-Cas12a technology as described in claim 1, characterized in that... This is used to detect Bispora picaridinia nucleic acid in animal feces to assess the Bispora picaridinia infection status in animals.

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