CRISPR-Cas12a-LFIA detection method for bovine-derived cryptosporidium in combination with RPA

Abstract

The invention provides a bovine-derived cryptosporidium CRISPR-Cas12a-LFIA (CRISPR-Cas12a-LFIA) detection method combined with RPA (recombinase polymerase amplification), which is characterized in that primers used in the CRISPR-Cas12a-LFIA detection method comprise an RPA primer group, the RPA primer group comprises an upstream primer and a downstream primer, the sequence of the upstream primer is SEQ ID NO.1, the sequence of the downstream primer is SEQ ID NO.2, and the sequence of the downstream primer is SEQ ID NO.2; the sequence of the downstream primer is SEQ ID NO. 2. And the sequence of the sgRNA used in the CRISPR / Cas12a system is as shown in SEQ ID NO. 3. According to the CRISPR-Cas12a-LFIA detection method for the bovine-derived cryptosporidium in combination with the RPA, provided by the invention, the detection efficiency of the bovine-derived cryptosporidium is greatly improved, and convenience is brought to animal husbandry quarantine operation.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular biology, and particularly relates to a detection method of bovine Cryptosporidium. BACKGROUND

[0002] Cryptosporidium is a parasitic pathogen of human and animals, which is widely spread in the world. The main clinical symptom is to cause severe diarrhea in human and animals, and even death in serious cases. The harm of Cryptosporidium parvum (C. parvum) is the most serious. Cryptosporidium mainly parasitizes in the small intestinal mucosa cells of the host, but not in the blood, and the oocyst is extremely small, only 6-10 μm in size. Therefore, the detection of Cryptosporidium is particularly difficult, and can only rely on the traditional microscopic examination of oocysts in feces or nested PCR amplification for diagnosis. The conventional PCR detection method adopts a variable temperature amplification program, and needs to use a PCR amplifier for variable temperature amplification, which is complex and high in cost. In the early stage of infection, the number of Cryptosporidium oocysts in feces is extremely small, and the current detection method often depends on complex instruments and professional operators, which greatly limits the rapid and accurate detection of Cryptosporidium in the field application of the disease.

[0003] Research has found that there are 45 kinds of Cryptosporidium, and the hosts include human, cattle, turkey and many other mammals and oviparous animals. There are four kinds of Cryptosporidium infecting cattle, including C. parvum, C. bovis, C. andersoni and C. ryanae. The existing detection method can only detect C. parvum and C. andersoni, which is inconvenient for livestock quarantine personnel.

[0004] Therefore, it is urgent to provide a simple, fast, sensitive and specific rapid diagnosis method of Cryptosporidium, to protect the development of animal husbandry and have great public health significance in the early diagnosis of Cryptosporidium disease. LFIA is a labeling technique for applying tracer markers to antigen-antibody immune reactions, and the negative or positive can be judged by observing the test strip with the naked eye, which is simple and intuitive. The lateral flow immunochromatographic test strip is composed of five parts of sample pad, conjugate pad, nitrocellulose membrane, absorbent paper and polyvinyl chloride bottom plate. The tracer marker-antibody (antigen) complex is laid on the conjugate pad, and the antibody for capturing the signal is laid on the chromatography membrane. The tracer markers include radionuclides and their compounds, enzymes, colloidal gold, latex microspheres, carbon nanoparticles and organic fluorescent molecules. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides a bovine Cryptosporidium CRISPR-Cas12a-LFIA (lateral flow immunoassay) detection method combined with RPA, which is a visual detection method and is simple to operate and more efficient.

[0006] The technical solution for achieving the above-mentioned object of the present application is:

[0007] A bovine Cryptosporidium CRISPR-Cas12a-LFIA detection method combined with RPA, the primer used in the CRISPR-Cas12a-LFIA detection method comprises an RPA primer set, the RPA primer set comprises an upstream primer and a downstream primer, the sequence of the upstream primer is SEQ ID NO. 1, and the sequence of the downstream primer is SEQ ID NO. 2.

[0008] The sgRNA (guide RNA) sequence used in the CRISPR / Cas12a system is SEQ ID NO. 3.

[0009] The RPA-CRISPR / Cas12a visual detection method is a nucleic acid detection method combining the trans-cleavage activity of CRISPR / Cas12a with the RPA amplification technology and realizing the visualization of results through a lateral flow chromatographic test strip. The principle is that specific sgRNA guides CRISPR / Cas12a to specifically recognize the target DNA sequence amplified by RPA, activates the trans-cleavage activity of CRISPR / Cas12a, cuts the ssDNA reporter probe in the reaction system, thereby realizes the release of the signal, and realizes the visual reaction result through the lateral flow chromatographic test paper, which is more intuitive than electrophoresis and fluorescence reading, and does not need complex operation and expensive instruments.

[0010] The bovine Cryptosporidium CRISPR-Cas12a-LFIA detection method combined with RPA comprises the following steps:

[0011] S1: extracting DNA of a sample to be detected;

[0012] S2: performing RPA amplification on the DNA to be detected by using the SEQ ID NO. 1 and the SEQ ID NO. 2;

[0013] S3: cutting the DNA obtained in S2 by using a CRISPR / Cas12a reaction system, the sgRNA (guide RNA) sequence used in the CRISPR / Cas12a system is SEQ ID NO. 3, and the CRISPR / Cas12a reaction condition is 34 DEG C to 42 DEG C for 10 min to 40 min;

[0014] S4: Result determination: insert the test strip into the reaction mixture obtained in S3, with the sample pad end facing down, and place it at room temperature for 10 min to determine the result. If the C line of the test strip develops color and the T line does not, it is determined to be negative. If the C line develops color and the T line is visible to the naked eye, it is determined to be positive. If the C line does not develop color and the T line is visible to the naked eye, it is determined to be positive. If neither the C line nor the T line develops color, it is determined to be invalid.

[0015] Further, in step S1, the sample to be detected is cow dung.

[0016] In step S2, the RPA amplification reaction system used includes: 1 μL of each of the upstream and downstream primers, 14.7 μL of A buffer (a finished product in a commercially available kit), 1.2-1.3 μL of B buffer, 2-3 μL of template DNA, and sterilized deionized water to make up to 25 μL; more preferably: 1 μL of each of the upstream and downstream primers, 14.7 μL of A buffer, 1.3 μL of B buffer, and 2 μL of template DNA.

[0017] Further preferably, in step S2, the concentrations of the upstream primer and the downstream primer are both 10 μmol / L.

[0018] In the above reaction tube, the reaction is carried out at 37℃ for 20-30 min.

[0019] In step S3, the CRISPR / Cas12a reaction system includes: 2 μL of 1xHOLMES Buffer; 0.6 μL of LbCas12a; 0.5-1.0 μL of sgRNA; 1 μL of Target DNA (the RPA amplification product is used as the reaction Target DNA); and 0.1-1.0 μL of ssDNA reporter, supplemented with sterilized deionized water to make up to 20 μL.

[0020] More preferably, in step S3, the concentrations of the sgRNA and the ssDNA reporter are both 10 μmol / L, the addition amount of the sgRNA is 1.0 μL, the addition amount of the ssDNA reporter is 0.5-0.6 μL, and the concentration of the LbCas12a is 300 nM.

[0021] In another preferred technical solution of the present application, in step S3, the CRISPR / Cas12a reaction conditions are 37℃ for 40 min.

[0022] In the test strip, the antibody 1 specifically recognizing the target is labeled with a label and dispersedly fixed on a binding pad; the antibody 2 specifically recognizing the target and the "anti-antibody" (commonly known as secondary antibody) specifically recognizing IgG are fixed on a cellulose membrane to form a "test line" (referred to as T line) and a "quality control line" (referred to as C line) respectively.

[0023] In the step S4, two bands are used in the detection test strip, the lower quality control line is referred to as C line, coated with avidin SA (streptavidin), and the upper test line is referred to as T line, coated with goat anti-mouse secondary antibody, and the anti-FITC / FAM monoclonal antibody is labeled on the colloidal gold.

[0024] The present application has the following beneficial effects:

[0025] The method can detect four kinds of cryptosporidium which mainly infect cattle, and is more efficient than the existing detection method; the present application limits sampling from cattle manure, and excludes other hosts; and the method brings convenience to livestock quarantine operation.

[0026] RPA amplification can automatically complete isothermal amplification in a rapid and specific manner without the aid of DNA annealing and other thermal cycle operations. The amplification relies on three enzymes: a recombination enzyme combined with a primer to form a DNA protein complex, which locates and matches homologous sequences through dsDNA, and then undergoes strand displacement under the action of a strand displacement enzyme, and then binds with single-stranded DNA binding protein (SSB) to prevent being displaced again, and then initiates DNA synthesis. The three enzymes remain active after being mixed, and the amplification reaction can occur at 34℃-42℃, and the optimal reaction temperature is about 37℃. The constant temperature can realize exponential amplification of nucleic acids, and the reaction time can be as low as 5-10 min. The CRISPR-Cas12a-LFIA detection method for bovine cryptosporidium combined with RPA greatly improves the detection efficiency of bovine cryptosporidium. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 In the specificity experiment results in the test example 1 in the present application, 1-7 respectively represent Haemonchus contortus, Caenorhabditis elegans, coccidia, trematode, tapeworm, coenurus cerebralis, and cryptosporidium, and NC is a negative control;

[0028] Figure 2 In the sensitivity detection results in the test example 2 in the present application, the first eight test strips represent the positive plasmid concentration of 1×10 6 copies / μL-1×10 -1 copies / μL after RPA amplification, and NC is a negative control;

[0029] Figure 3For the test example 3 in the present application, 1-3 represent the standard positive plasmid with the concentration of 1.0x10 6 copies / μL, 1.0x10 5 copies / μL, 1.0x10 4 copies / μL, respectively, under the same conditions, and NC is the negative control.

[0030] Figure 4 For the test example 4 in the present application, 1-36 represent the detection results of 36 calf diarrhea fecal samples, wherein sample No. 26 and No. 27 are negative, and the rest are positive.

[0031] Figure 5 For the comparative example 1 in the present application, 1-6 represent the standard positive plasmid with the concentration of 1.0x10 6 copies / μL-10 copies / μL, respectively, after nested PCR amplification of Cryptosporidium, and NC is the negative control.

[0032] Figure 6 For the example 1 in the present application, 1-6 represent the amplification results of 6 pairs of different RPA primers, wherein M is 1000 bp DNA Marker.

[0033] Figure 7 For the example 3 in the present application, 50nM, 100nM, 250nM, 300nM, and 500nM represent the final concentration of ssDNA reporter, respectively, and NC is the negative control.

[0034] Figure 8 For the example 4 in the present application, 50nM, 100nM, 150nM, 200nM, and 300nM represent the concentration of LbCas12a protein, respectively, and NC is the negative control.

[0035] Figure 9 For the example 5 in the present application, 10min, 20min, 30min, 40min, 50min, and 60min represent the reaction time, respectively, and NC is the negative control. DETAILED DESCRIPTION

[0036] The following examples are used to illustrate the present application, but not to limit the scope of the present application.

[0037] The technical means employed in the specification, unless otherwise specified, are those known in the art. The raw materials used are commercially available.

[0038] Design and screening of RPA primers in Example 1

[0039] By comparing the 18S rRNA sequences of four bovine Cryptosporidium, specific gene sequences for diagnosis were found, and RPA primers and sgRNA were designed to establish a bovine Cryptosporidium CRISPR-Cas12a-LFIA detection method combined with RPA.

[0040] 1. Primer and sgRNA design

[0041] According to the 18S rRNA sequences of Microsporidium parvum, Cryptosporidium bovis, Cryptosporidium andersoni and Cryptosporidium ryanae, specific gene sequences were screened and primers and sgRNA were designed. The design principles are as follows:

[0042] (1) The length of RPA primer is preferably 30-35 bp, which affects the activity of recombinase, and the length of more than 45 nt may cause secondary structure.

[0043] (2) Primer sequence: The 5' end should avoid continuous 5 or more CGs, the length of single nucleotide repeat sequence is within 5 nucleotides, and C is preferably selected to promote fragment recombination; the 3' end preferably uses GC to improve amplification performance: the GC% content value is preferably 30%-70%; secondary structure or hairpin structure is avoided as much as possible to reduce primer dimer; the Tm value of the primer is preferably 55-65℃.

[0044] (3) RPA can amplify DNA products up to 1.5 kb, and the effect of 100-200 bp amplicon is best.

[0045] (4) There should be a PAM region (TTTN, N is any base) between the primer and the sequence

[0046] RPA primer screening: The best primer pair was screened by RPA amplification reaction of 6 pairs of primers, and the specific screening results are as follows:

[0047] According to a large number of literatures consulted and research comparison, the 18S rRNA gene sequence of bovine Cryptosporidium was finally selected as the target gene, and 6 pairs of primers were designed, as shown in Table 1.

[0048] Table 1 Sequence list of 6 pairs of RPA primers to be selected

[0049]

[0050]

[0051] The bovine Cryptosporidium genomic DNA was used as a template to amplify the sequence by RPA amplification method. The amplified product was subjected to 2% agarose gel electrophoresis to read the result.

[0052] 6The amplification results of the candidate primers are shown in Figure 6 The results show that the No. 1 primer has the brightest target band after RPA amplification, and the size is about 172 bp, and there is no non-specific structure.

[0053] Through the above exploration, the specific primer sequence for rapid detection of bovine Cryptosporidium is designed as follows:

[0054] The upstream primer (primer F) is:

[0055] 5'-ATACAGGGAGGTAGTGACAAGAAATAACAA-3', SEQ ID NO. 1; and the downstream primer (primer R) is:

[0056] 5'-GCTTTTTAACTGCAACAACTTTAATATACGC-3', SEQ ID NO. 2.

[0057] Example 2: Design and synthesis of sgRNA

[0058] In the present application, 20 nt sequences after PAM sites (TTTN, N is any base) in four bovine Cryptosporidium 18S rRNA specific sequences are selected to design sgRNA. The selected sequences are analyzed by BLAST comparison, and the results show that the sequences only exist in the bovine Cryptosporidium genome. The design process is as follows:

[0059] The sgRNA of the LbCas12a protein used in the present application includes a full-length repeated sequence (DR region) of 20 nt, and a guide sequence region (spacer region) of 20 nt, wherein the DR region sequence is fixed, and the sequence is 5'-AAUUUCUACUAAGUGUAGAU-3', SEQ ID NO. 4, and the spacer region sequence is a 20 nt sequence complementary to the specific target sequence, 5'-GAGGGCAAGUCUGGUGCCAG-3', SEQ ID NO. 5 (target sequence). The complete sgRNA sequence after design is:

[0060] 5'-AAUUUCUACUAAGUGUAGAUGAGGGCAAGUCUGGUGCCAG-3', SEQ ID NO. 3.

[0061] The designed RPA primer and sgRNA are synthesized by Shengong Biotechnology (Shanghai) Co., Ltd.

[0062] Example 3: Establishing a bovine-derived Cryptosporidium parvum CRISPR-Cas12a-LFIA detection method combined with RPA

[0063] The detection principle of the method is as follows:

[0064] RPA amplification mainly relies on three enzymes: a recombination enzyme combined with ssDNA and a primer to form a DNA protein complex, which locates and matches the homologous sequence of dsDNA, undergoes strand displacement under the action of strand displacement enzyme, then binds with single-stranded DNA binding protein (SSB) to prevent being displaced again, and then initiates DNA synthesis. The product after RPA amplification is used as a template in the CRISPR-Cas12a reaction.

[0065] Transcleavage activity of CRISPR / Cas12a: both double-stranded and single-stranded DNA targets can activate the transcleavage activity of Cas12a, i.e. when Cas12a enzyme, sgRNA and target DNA form a ternary complex, the transcleavage activity of Cas12a enzyme against ssDNA molecules will be activated, which will cut any sequence of ssDNA molecules in the system, releasing signals.

[0066] In the CRISPR / Cas12a reaction system of the method, Cas12a enzyme, sgRNA and RPA amplification product form a ternary complex, which activates the transcleavage activity of Cas12a enzyme, cuts any ssDNA molecule and releases signals, and the test strip develops color.

[0067] The specific steps are as follows:

[0068] S1: Extraction of template DNA: the sample is bovine feces, and the DNA of the sample to be tested is extracted;

[0069] S2: RPA amplification reaction, using SEQ ID NO. 1 and SEQ ID NO. 2 designed in Example 1 to perform RPA amplification on the DNA to be tested;

[0070] Prepare the reaction system (TwistAmp TM Liquid Basic kit) in a 200 μL PCR tube: Abuffer 14.7 μL, primer F (10 μM) 1 μL, primer R (10 μM) 1 μL, template DNA 2 μL, starting liquid B buffer 1.3 μL, and ddH2O to 25 μL, and place the above reaction tube in a 37°C incubator for 25 min to obtain the RPA amplification product;

[0071] S3: The DNA obtained in S2 was cut by using a CRISPR / Cas12a reaction system, and the sgRNA (guide RNA) sequence used in the CRISPR / Cas12a system was SEQ ID NO. 3, and the concentration was 10 μmol / L;

[0072] RPA combined with CRISPR / Cas12a amplification reaction: the reaction system was prepared in a 200 μL PCR tube (TwistAmp TM Liquid Basic kit): 1xHOLMES Buffer 2 μL, LbCas12a (200 nM) 0.6 μL, sgRNA (10 μM) 1.0 μL, CRISPR-LIFA ssDNA reporter of TOLOBIO company 0.1-1 μL (concentration of 10 μmol / L), RPA amplification product 1 μL, and ddH2O was added to 20 μL, and the above reaction tube was placed at 37°C for 10 min;

[0073] S4: result determination: after the above reaction, 30 μL of ddH2O was added to dilute the amplification product, and the CRISPR single system detection test strip (FAM / FITC) of TOLOBIO company was incubated for 10 min, and then the color development of the test strip was observed. If the detection line is colored, it is positive for Cryptosporidium; the test strip used has two bands, the lower control line is called C line, which is coated with avidin SA (streptavidin), and the upper detection line is called T line, which is coated with goat anti-mouse secondary antibody, and the anti-FITC / FAM monoclonal antibody is labeled with colloidal gold.

[0074] The specific operation is as follows:

[0075] The test strip was inserted into the above reaction tube, the sample pad end was downward, and the result was judged at room temperature for 5 min. If the test strip C line is colored and the T line is not colored, it is negative, indicating that the nucleic acid probe has not been cut by Cas enzyme; if the C line is colored and the T line is visible to the naked eye, it indicates that part of the nucleic acid probe has been cut by Cas enzyme, and it is judged as positive; if the C line is not colored and the T line is visible to the naked eye, it indicates that the nucleic acid probe has been almost completely cut by Cas enzyme, and it is judged as positive; if the C line and the T line are not colored, it indicates that the operation is wrong or the test strip has been deteriorated and is invalid.

[0076] CRISPR-LFA ssDNA reporter addition amount screening: choose the cow dung of known bovine Cryptosporidium, set 6 samples, one of which is NC;

[0077] The final concentration of the CRISPR-LFA ssDNA reporter in step S3 was set to 50nM, 100nM, 250nM, 300nM, and 500nM, respectively, and the ssDNA reporter was added in an amount of 0.1 μL, 0.2 μL, 0.5 μL, 0.6 μL, and 1.0 μL, respectively. The results are shown in FIG. 3. Figure 7 As shown in FIG. 3, when the final concentration of the ssDNA reporter was 300nM, the test strip detection line was the most obvious, so 0.6 μL was selected as the optimal amount of ssDNA added in this experiment. Figure 7

[0078] Example 4 Screening of LbCas12a Protein Concentration

[0079] In step S3, the LbCas12a protein concentration was set to 50nM, 100nM, 150nM, 200nM, and 300nM, respectively, and the LbCas12a protein and ssDNA reporter were added in an amount of 0.6 μL. The remaining operations were equivalent to those in Example 3.

[0080] The results are shown in FIG. 4. As shown in FIG. 4, when the LbCas12a protein concentration was 300nM, the T line was the most obvious, and the C line was not visible, indicating that the probe was completely cut at this time, so the optimal concentration of the LbCas12a protein was 300nM. Figure 8

[0081] Example 5 Optimization of Reaction Time

[0082] According to the RPA-CRISPR / Cas12a procedure, the reaction time in step S3 was set to 10min, 20min, 30min, 40min, 50min, and 60min, respectively. The LbCas12a protein concentration was 300nM. The remaining operations were equivalent to those in Example 4.

[0083] The results are shown in FIG. 5. As shown in FIG. 5, when the reaction time was 40min, the test strip showed the clearest red band, so 40min was selected as the optimal reaction time. Figure 9 Figure 9

[0084] Example 6 CRISPR-Cas12a-LFIA Detection Method for Cattle-derived Cryptosporidium parvum in Combination with RPA

[0085] The method comprises the following steps:

[0086] S1: Extracting DNA from the sample to be tested, wherein the sample to be tested is cattle feces.

[0087] ​​​​S2: The DNA to be tested is amplified by RPA using SEQ ID NO. 1 and SEQ ID NO. 2 designed in Example 1; a reaction system is prepared in a 200 μL PCR tube: Abuffer

[0088] 14.7 μL, primer F (10 μM) 1 μL, primer R (10 μM) 1 μL, template 2 μL, Bbuffer 1.3 μL, and ddH2O is added to 25 μL, and the above reaction tube is placed at 37°C for 25 min to obtain the RPA amplification product;

[0089] S3: The DNA obtained in S2 is cut by using a CRISPR / Cas12a reaction system, and the sgRNA sequence used in the CRISPR / Cas12a system is SEQ ID NO. 3, and the concentration is 10 μmo1 / L;

[0090] A reaction system is prepared in a 200 μL PCR tube: 1xHOLMES Buffer 2 μL, LbCas12a (300 nM) 0.6 μL, sgRNA (10 μmo1 / L) 1.0 μL, CRISPR-LFA ssDNA reporter of TOLOBIO company 0.6 μL, concentration is 10 μmo1 / L, RPA amplification product 1 μL, and ddH2O is added to 20 μL, and the above reaction tube is placed at 37°C for 40 min;

[0091] S4: Result determination: insert the test strip into the reaction mixture obtained in S3, with the sample pad end facing down, and place it at room temperature for 10 min to determine the result, if the C line of the test strip develops color and the T line does not develop color, it is judged as negative; if the C line develops color and the T line is visible to the naked eye, it is judged as positive; if the C line does not develop color and the T line is visible to the naked eye, it is judged as positive; if neither the C line nor the T line develops color, it is judged as invalid.

[0092] Specificity test of test example 1

[0093] The method of Example 6 of the application is used to simultaneously detect Cryptosporidium, Trichuris trichiura, Caenorhabditis elegans, coccidia, trematode, tapeworm, and Coenurus cerebralis, and the results are shown in Table 2. Figure 1

[0094] The results show that only Cryptosporidium is positive, and the other related pathogens are negative. The application designs a pair of primers according to the specific 18S rRNA gene sequence of bovine-derived Cryptosporidium, and performs three times of signal method and specific recognition through three steps of RPA technology, CRISPR system, and test strip detection, which has high specificity.

[0095] Sensitivity test of test example 2

[0096] ​The RPA primer positive standard plasmid was prepared, and the ten-fold dilution concentrations were 1 x 10 6 ~ 1 x 10 -1 copies / μL, and the plasmid at each gradient concentration was used as a template to perform a sensitivity test, and the reaction conditions and reaction system were the same as in Example 6. The detection results are shown in Figure 2 .

[0097] The detection results show that the detection method of the application has high sensitivity, and the minimum detection limit of Cryptosporidium parvum is 1 x 10 -1 copies / μL.

[0098] Test Example 3 Reproducibility Test

[0099] In order to evaluate the stability and inter-group reproducibility of the method, three concentrations of standard positive plasmid with a concentration of 1.0 x 10 4 ~ 1.0 x 10 6 copies / μL were used to perform three repetitions under the same conditions to determine the inter-group reproducibility.

[0100] The results are shown in Figure 3 , and the test strips in the inter-group reproducibility test were all positive, indicating that the method has good inter-group reproducibility and stability.

[0101] Test Example 4 Field Sample Detection

[0102] Using the method of Example 6, 36 fecal samples of diarrhea calves were detected, and 34 positive samples of Cryptosporidium and 2 negative samples were detected, and the detection results are shown in Figure 4 .

[0103] At the same time, the nested PCR method was used to recheck the 36 samples, and the detection results of the RPA-CRISPR / Cas12a-LFIA method were consistent with the detection results of the nested PCR, and the consistency rate was 100%.

[0104] Comparative Example 1 Sensitivity of Industry Standard

[0105] According to the method for detecting Cryptosporidium by nested PCR in the People's Republic of China (GB / T 35942-2018), the size of the primer amplification fragment is 830 bp.

[0106] The sequence of the first round of amplification primer is: P1-1: 5'-TTCTAGAGCTAATACATGCG-3';

[0107] P1-2: 5'-CCCATTTCCTTCGAAACAGGA-3'.

[0108] The second round of amplification primer sequence: P2-1: 5'-GGAAGGGTTGTATTTATTAGA TAAAG-3'

[0109] P2-2: 5'-CTCATAAGGTGCTGAAGGAGTA-3'

[0110] Using the prepared Cryptosporidium positive plasmid, the concentration of 1×10 6～ 10 copies / μL was diluted by ten times, and the sensitivity test was carried out using the plasmid at each gradient concentration as a template according to the Cryptosporidium national standard detection method (GB / T 35942-2018), and the results are shown in Table 6. Figure 5 The results show that the detection limit is 1.0×10 2 copies / μL, and compared with the results of Example 6, it can be known that the method has higher sensitivity.

[0111] Although the above describes the present application through examples, those skilled in the art should understand that improvements and modifications made to the present application without departing from the spirit and essence of the present application shall all belong to the protection scope of the present application.

Claims

1. A CRISPR-Cas12a-LFIA detection method for bovine Cryptosporidium in conjunction with RPA, characterized in that, The primers used in the CRISPR-Cas12a-LFIA detection method include the RPA primer set, which includes an upstream primer and a downstream primer. The upstream primer sequence is SEQ ID NO.1, and the downstream primer sequence is SEQ ID NO.

2. The sgRNA sequence used in the CRISPR / Cas12a system is SEQ ID NO.

3.

2. The method for detecting bovine Cryptosporidium CRISPR-Cas12a-LFIA in conjunction with RPA according to claim 1, characterized in that, Includes the following steps: S1: Extract DNA from the sample to be tested; S2: RPA amplification of the DNA to be tested using SEQ ID NO.1 and SEQ ID NO.2 as described in claim 1; S3: The DNA obtained in S2 was cut using the CRISPR / Cas12a reaction system. The sgRNA sequence used in the CRISPR / Cas12a system is SEQ ID NO.

3. The CRISPR / Cas12a reaction conditions were 34℃~42℃ for 10~40 min. S4: Result Interpretation: Insert the test strip into the reaction mixture obtained in S3, with the sample pad end facing down, and place at room temperature for 10 minutes to interpret the results. If the C line of the test strip is colored and the T line is not colored, it is considered negative; if the C line is colored and the T line is visible to the naked eye, it is considered positive; if the C line is not colored and the T line is visible to the naked eye, it is considered positive; if neither the C line nor the T line is colored, it is considered invalid.

3. The method for detecting bovine Cryptosporidium CRISPR-Cas12a-LFIA in conjunction with RPA according to claim 2, characterized in that, In step S1, the sample to be tested is cow dung.

4. The method for detecting bovine Cryptosporidium CRISPR-Cas12a-LFIA in conjunction with RPA according to claim 2, characterized in that, In step S2, the RPA amplification reaction system used includes: 1 μL each of upstream and downstream primers, 14.7 μL of Abuffer, 1.3 μL of Bbuffer, 2 μL of template DNA, and sterile deionized water to make up to 25 μL.

5. The method for detecting bovine Cryptosporidium CRISPR-Cas12a-LFIA in conjunction with RPA according to claim 4, characterized in that, In step S2, the concentrations of both the upstream and downstream primers are 10 μmol / L.

6. The method for detecting bovine Cryptosporidium CRISPR-Cas12a-LFIA in conjunction with RPA according to claim 2, characterized in that, In step S3, the CRISPR / Cas12a reaction system includes: 2 μL of 1×HOLMES Buffer; 0.6 μL of LbCas12a; 0.5–1.0 μL of sgRNA; 1 μL of Target DNA; 0.1–1.0 μL of ssDNA reporter, and is brought up to 20 μL with sterile deionized water.

7. The method for detecting bovine Cryptosporidium CRISPR-Cas12a-LFIA in conjunction with RPA according to claim 6, characterized in that, In step S3, the concentrations of both sgRNA and ssDNA reporter are 10 μmol / L, the amount of sgRNA added is 1.0 μL, and the amount of ssDNA reporter added is 0.5–0.6 μL.

8. The method for detecting bovine Cryptosporidium CRISPR-Cas12a-LFIA in conjunction with RPA according to claim 6, characterized in that, In step S3, the concentration of LbCas12 is 300 nM.

9. The method for detecting bovine Cryptosporidium CRISPR-Cas12a-LFIA in conjunction with RPA according to claim 6, characterized in that, In step S3, the CRISPR / Cas12a reaction conditions are 37°C for 40 min.

10. The method for detecting bovine Cryptosporidium CRISPR-Cas12a-LFIA in conjunction with RPA according to any one of claims 1 to 9, characterized in that, The test strip used in step S4 has two bands. The lower control line is called the C line, which is coated with avidin SA. The upper test line is called the T line, which is coated with goat anti-mouse secondary antibody and colloidal gold labeled with anti-FITC / FAM monoclonal antibody.

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