CRISPR-cas12a detection primer set for fransisella tularensis and application thereof

Abstract

The application discloses a CRISPR-Cas12a detection primer group for Francisella tularensis and application thereof. The application provides a CRISPR-Cas12a system for detecting Francisella tularensis, and also provides RAA amplification primers for specifically amplifying the target sequence of the Francisella tularensis in the first aspect; and a probe as a fluorescent reporter molecule is also provided. The application obtains a Francisella tularensis specific gene sequence based on whole genome alignment of Francisella tularensis and its close species, further utilizes the specific gene sequence to design RAA amplification primers and crRNA sequences to detect Francisella tularensis, and develops a related kit. The application can specifically detect Francisella tularensis and distinguish it from other close species, and has the characteristics of rapidness, simplicity and accuracy.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a CRISPR-Cas12a detection primer set for *Streptococcus tularensis* and its application, and more specifically to CRISPR-Cas12a detection primers, crRNA, and their uses for *Streptococcus tularensis*. Background Technology

[0002] Tularemia, also known as tularemia, is a zoonotic febrile infectious disease caused by *Francisella tularensis*. Because it initially circulated among wild rodents, especially rabbits, it is also called rabbit fever. *Francisella tularensis* belongs to the genus *Francisella*, which consists of four subspecies. Types A and B are the main subspecies causing the disease in humans. Type A strains are mainly distributed in Canada and the United States and are the most virulent. Type B strains are found almost throughout the Northern Hemisphere, including Europe. Epidemiological surveys show that all current tularemia cases in my country are of the type B subspecies. Tularemia is characterized by fever, lymphadenopathy, and petechial necrosis of the spleen and other internal organs. Clinical manifestations are mostly acute onset, with rapid fever, accompanied by headache, fatigue, loss of appetite, muscle pain, and night sweats. The disease then progresses, leading to delirium, drowsiness, restlessness, and other acute systemic poisoning symptoms. Tularemia, a zoonotic disease with natural focal origin, remains prevalent in many regions. Its aerosol transmission poses significant challenges to testing and identification by prevention and treatment institutions. Traditional methods for identifying Tularemia include routine isolation and culture, enzyme-linked immunosorbent assay (ELISA), microhemagglutination assay, colloidal gold assay, and conventional PCR. Isolation and culture of Tularemia is the gold standard for diagnosis, but the bacterium's slow growth and high nutritional requirements hinder diagnosis. ELISA and latex agglutination assays are widely used, but they exhibit varying degrees of cross-contamination with Brucella, leading to potential misdiagnosis. Quantitative real-time PCR and conventional PCR offer high sensitivity, but require sophisticated and expensive equipment and a suitable experimental environment, making them unsuitable for use in grassroots laboratories and field settings.

[0003] CRISPR-Cas technology is a rapidly developing gene-editing technology in recent years. The CRISPR-Cas system consists of clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas). Originally, the CRISPR-Cas system was an adaptive immune mechanism found in prokaryotes. Currently, after a series of modifications, CRISPR-Cas technology has been widely applied in various life science research fields, including genome editing, gene expression regulation, gene therapy, pathogen detection, high-throughput screening of target genes, and epigenetic modification. Besides being a gene-editing tool, the "accessory cleavage" properties of class II Cas proteins, such as Cas12a, have been developed into nucleic acid detection methods to identify different types of targets, showing great potential in the field of rapid detection.

[0004] Recombinase polymerase amplification (RPA) is a novel isothermal amplification technique widely used for the molecular diagnosis of pathogenic nucleic acids. In China, based on differences in the companies that developed it and the sources of the enzymes used, it is also known as Recombinase Aided Amplification (RAA, Jiangsu Qitian Gene Biotechnology Co., Ltd., Hangzhou Zhongce Biotechnology Co., Ltd.) or Enzymatic Recombinase Amplification (ERA, Suzhou Xianda Gene Technology Co., Ltd.). RAA replaces the thermal cycling required for polymerase chain reaction (PCR) with three core enzymes. Unlike many other isothermal techniques, RAA eliminates the need for a heating or precise temperature control stage; the amplification reaction can be carried out within a temperature range of 25–42°C, and the reaction is usually completed within 5–15 minutes. RAA technology has been gaining prominence since its inception, and has been widely used in the field of nucleic acid detection due to its advantages such as rapid reaction (5-15 min), high specificity (primers), low temperature operation (25-42℃), sample tolerance (particularly lenient requirements on sample type), high sensitivity (single copy), wide applicability, flexible reagent form (liquid reagent or dry powder), and diverse detection methods.

[0005] Combining the CRISPR-Cas12a detection system with RAA technology, the target fragment is amplified at room temperature using RAA technology before the CRISPR-Cas12a detection reaction is performed. The significance of this combination is that the pre-amplification of RAA can compensate for the low sensitivity of CRISPR-Cas12a detection, while the specificity of CRISPR-Cas12a detection ensures the accuracy of the detection results, achieving complementary advantages and improving the detection effect. Summary of the Invention

[0006] The purpose of this invention is to provide a CRISPR detection primer and crRNA for *Tetrandella tularensis*. Using this primer set and crRNA in gene detection based on CRISPR technology can rapidly detect *Tetrandella tularensis* on-site, with the advantages of high specificity, high sensitivity, and ease of use.

[0007] In a first aspect, the present invention provides a CRISPR-Cas12a system for detecting Tula Francisella, comprising Cas12a protein and crRNA, or a complex of the two;

[0008] The crRNA includes an anchoring sequence for binding to the Cas12a protein and a guide sequence for targeting the Tula Francisella target sequence.

[0009] The target sequence of the *Tulafrancsis* strain is derived from the ACX55_1601 gene shown in SEQ ID No. 1 of *Tulafrancsis*.

[0010] In the CRISPR-Cas12a system described above, the target sequence of the *Tulafrancsis* is the DNA encoding the RNA fragment shown in positions 22-43 of SEQ ID No. 18.

[0011] In the CRISPR-Cas12a system described above, the anchoring sequence of the crRNA can be positions 1-21 of SEQ ID No. 18, and the guide sequence can be positions 22-43 of SEQ ID No. 18. Specifically, the sequence of the crRNA is SEQ ID No. 18;

[0012] Alternatively, the Cas12a protein may be the LbCas12a protein.

[0013] Secondly, the present invention provides a kit for detecting *Tulafrancsis*, comprising the following:

[0014] 1) The CRISPR-Cas12a system for detecting *Tulafrancsis* as described in the first aspect;

[0015] 2) RAA amplification primers for specifically amplifying the target sequence of *Tulafrancsis* as described in the first aspect;

[0016] 3) As a probe for fluorescent reporter molecules;

[0017] The probe is labeled with a fluorescent group and a quenching group at its two ends, respectively.

[0018] In the kit described above, the RAA primer pair is a primer pair that can amplify the DNA fragment shown at positions 305-510 of SEQ ID No. 1 using the Tula Francisella genome as a template.

[0019] The RAA amplification primers consist of single-stranded DNA molecules shown in SEQ ID No. 4 and SEQ ID No. 9;

[0020] The sequence of the probe is as shown in SEQ ID No. 24. In a specific embodiment of the present invention, the fluorescent reporter group of the probe is specifically FAM, and the fluorescent quencher group is specifically BHQ1. The probe is: 5`6-FAM-TTTTTTTTTTTT-BHQ1-3'.

[0021] Furthermore, the kit may also contain all or some of the following: recombinases capable of binding single-stranded nucleic acids, single-stranded DNA-binding enzymes, strand displacement DNA polymerases, dNTPs, and magnesium acetate.

[0022] Furthermore, the kit may also contain a positive reference plasmid.

[0023] Furthermore, the positive reference plasmid may be a plasmid containing the DNA fragment shown in positions 305-510 of SEQ ID No. 1, specifically a recombinant plasmid obtained by cloning the DNA fragment shown in positions 305-510 of SEQ ID No. 1 into the pUC57 vector.

[0024] Thirdly, the present invention provides any of the following substances:

[0025] A1) The crRNA described in the first aspect;

[0026] A2) The Cas12a protein and crRNA described in the first aspect, or a complex formed therefrom;

[0027] A3) The RAA amplification primers described in the second aspect;

[0028] The probe described in the second aspect of A4)

[0029] The sequence provided by this invention is the nucleotide sequence shown in SEQ ID No. 1. This sequence is specifically present in the genome of *Tulafrancsis* and is highly conserved among strains, but is not present in the genomes of other closely related species of the *Tulafrancsis* genus.

[0030] Fourthly, the present invention provides any of the following applications:

[0031] B1) The use of the system described in the first aspect, the kit described in the second aspect, or the substance described in the third aspect in the detection or auxiliary detection of Tulafrancsis or its nucleic acid;

[0032] B2) The use of the system described in the first aspect, the kit described in the second aspect, or the substance described in the third aspect in the preparation of products for detecting or assisting in the detection of Tulafrancsis or its nucleic acids;

[0033] B3) The use of the system described in the first aspect, the kit described in the second aspect, or the substance described in the third aspect in detecting or assisting in the detection of whether a sample contains *Tulafrancsis* or its nucleic acid;

[0034] B4) The use of the system described in the first aspect, the kit described in the second aspect, or the substance described in the third aspect in the preparation of products for detecting or assisting in the detection of whether a sample to be tested contains Tulafrancsis or its nucleic acid;

[0035] B5) Use of the system described in the first aspect, the kit described in the second aspect, or the substance described in the third aspect in the diagnosis or auxiliary diagnosis of whether a sample to be tested is infected with *Tulafrancsis*.

[0036] B6) The use of the system described in the first aspect, the kit described in the second aspect, or the substance described in the third aspect in the preparation of products for the diagnosis or auxiliary diagnosis of whether a sample to be tested is infected with *Tulafrancsis*.

[0037] B7) The use of the system described in the first aspect, the kit described in the second aspect, or the substance described in the third aspect in screening or assisting in screening drugs for the prevention and treatment of *Tulafrancsis*.

[0038] B8) Use of the system described in the first aspect, the kit described in the second aspect, or the substance described in the third aspect in the preparation of products for screening or assisting in screening for the prevention and treatment of *Tulafrancsis*.

[0039] B9) Use of the substance described in the third aspect in the preparation of the kit described in the second aspect;

[0040] The application of the DNA molecule shown in SEQ ID No. 1 (B10) as a target gene for detecting *Tula française* in the detection or auxiliary detection of *Tula française* or its nucleic acids;

[0041] The application of the DNA molecule shown in SEQ ID No. 1 (B11) as a target gene for detecting Tula Francisella in the preparation of products for detecting or assisting in the detection of Tula Francisella or its nucleic acids.

[0042] Fifthly, the present invention provides a method for detecting or assisting in the detection of *Tulafrancsis*, comprising the following steps:

[0043] C1) Using the nucleic acid of the sample to be tested as a template, RAA amplification is performed using the RAA amplification primers described in the second aspect to obtain the RAA product;

[0044] C2) Prepare a CRISPR-Cas12a detection system containing the following components: the RAA product, the Cas12a protein described in the first aspect, the crRNA described in the first aspect, the CRISPR reaction buffer, and the probe described in the second aspect; water is used instead of the RAA product as a negative control.

[0045] C3) The CRISPR-Cas12a detection system is reacted, and the reaction products are detected to determine whether the sample to be tested contains Tula Francisella.

[0046] In the method described above, step C1) involves a RAA amplification reaction under the following conditions: 35-41°C for 20-30 minutes. Specifically, in this embodiment of the invention, the RAA amplification reaction temperature is 39°C and the reaction time is 30 minutes.

[0047] Alternatively, in step C2), the CRISPR reaction is performed at 35-39°C for 10-30 minutes. In the specific embodiment of the present invention, the CRISPR reaction temperature is 39°C and the reaction time is 20 minutes.

[0048] In the method described above, the method for detecting the reaction product is to detect the signal using a fluorescence detector, and to determine the detection result based on the detection signal.

[0049] The application of detecting *Tulafrancsis* mentioned above is a non-disease diagnostic and therapeutic application, and the method for detecting *Tulafrancsis* mentioned above is a non-disease diagnostic and therapeutic method; specifically, the purpose of the application and method is to prepare a *Tulafrancsis* drug model or to screen *Tulafrancsis* therapeutic drugs.

[0050] The primers and crRNA provided in this invention relate to in vitro diagnostic reagents used in medical examination and testing instruments and services. They are designed specifically for the *Tetrandella tularensis* gene ACX55_1601 and are detected using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology. In the CRISPR-Cas system, the Cas protein, guided by crRNA (CRISPR-derived RNA), recognizes the target sequence and initiates "collateral cleavage" activity. A fluorescent reporter molecule is added to the system, utilizing the collateral cleavage activity of the Cas enzyme to convert the sequence information into a fluorescent signal. Through the coupling of RAA and the Cas protein, a two-stage amplification process—sequence amplification (completed by RAA) and enzymatic cascade (completed by the Cas enzyme)—is achieved, surpassing the sensitivity of single-stage amplification like Q-PCR. Furthermore, because the RAA amplification method does not require complex temperature changes, it eliminates the dependence on sophisticated instruments such as Q-PCR instruments, making CRISPR-Cas technology a promising candidate for on-site diagnosis of *Tetrandella tularensis*.

[0051] Experiments have demonstrated that the CRISPR detection primers, crRNA, and corresponding detection method provided in this invention have the advantages of short detection time, high amplification efficiency, good sensitivity, and strong specificity. The detection sensitivity for *Streptococcus tularensis* positive reference plasmids can reach 1 copy / reaction. This invention features low cost, convenient operation, short time consumption, high sensitivity, and strong specificity. The entire process is carried out at 37–40℃, effectively eliminating reliance on large laboratory instruments, and has broad application prospects in the field diagnosis of *Streptococcus tularensis*. Attached Figure Description

[0052] Figure 1 This is the result of the Blast gene alignment.

[0053] Figure 2 For screening crRNA.

[0054] Figure 3 For screening RAA primer pairs.

[0055] Figure 4 For screening RAA primer pairs.

[0056] Figure 5 For screening RAA primer pairs.

[0057] Figure 6 Evaluation of plasmid copy sensitivity of CRISPR detection system for Tula Francisella.

[0058] Figure 7 Evaluation of the genome copy sensitivity of the CRISPR detection system for *Tulafrancsis*.

[0059] Figure 8 Evaluation of the specificity of the CRISPR detection system for *Tulafrancsis*. Detailed Implementation

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0061] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0062] The materials and instruments involved in this invention are as follows:

[0063] RAA primers, crRNA, and signal reporter probes were synthesized by Suzhou Genewiz Biotechnology Co., Ltd.; LbCas12a protein was purchased from Nanjing GenScript Biotech Co., Ltd.; and NEBuffer was used. TM r2.1 was purchased from New England Biolabs, and DEPC water was purchased from Beyotime Biotechnology Co., Ltd. The fluorescent RAA kit was purchased from Jiangsu Qitian Gene Biotechnology Co., Ltd., and the standard RAA kit was purchased from Hangzhou Zhongce Biotechnology Co., Ltd. The positive reference plasmid for *Streptococcus tulariensis* ACX55_1601 (pUC57-ACX55_1601) was constructed by Shanghai Sangon Biotech Co., Ltd., and other biochemical reagents were either imported and repackaged or domestically produced analytical grade.

[0064] Metal bath, centrifuge, vortex mixer, RAA-B6100 isothermal oscillator, RAA-F1620 isothermal amplification fluorescence detection system (Jiangsu Qitian Gene Biotechnology Co., Ltd.), etc.

[0065] Example 1: Sequence Design and Establishment of CRISPR Detection Method

[0066] 1. Target sequence screening

[0067] The inventors obtained reference genome sequences of *François tularensis* and other species in the *François* genus from the NCBI database. Using MAUVE software, they screened and identified the *François tularensis*-specific gene ACX55_1601 (gene sequence shown in SEQ ID No. 1). The ACX55_1601 gene sequence was then subjected to Blast sequence alignment, and the alignment results are as follows: Figure 1As shown, the NCBI database contains genomic sequences of 70 strains of *Tetrandella tularensis*. The ACX55-1601 gene is present in the genomes of 69 of these strains and is specific only to *Tetrandella tularensis*. This sequence, as a specific conserved sequence for *Tetrandella tularensis*, can be used for the specific detection of this strain. A plasmid (pUC57-ACX55_1601) containing this gene sequence was synthesized by Shanghai Sangon Biotech Co., Ltd.

[0068] 2. Design of amplification primer pairs, crRNA, and probes

[0069] For the above-mentioned ACX55_1601 gene sequence of *Tulafrancsis* (as shown in SEQ ID No. 1), RAA amplification primer pairs, crRNA and probes were designed using software, and the sequences are shown in Table 1.

[0070] Table 1 lists the candidate RAA amplification primer pairs and crRNAs for this invention.

[0071]

[0072]

[0073] Note: The underlined portion of crRNA is the guide sequence (matching the amplification product sequence of the RAA primer pair), and the preceding portion is the anchoring sequence (used to bind the Cas protein). In the RNA sequences 16-23 shown in the sequence listing, U is represented by T.

[0074] 3. CRISPR detection reaction principle

[0075] CRISPR experimental principle: crRNA binds to LbCas12a to form a complex. This complex can specifically recognize DNA sequences complementary to crRNA. The LbCas12a enzyme is activated and begins to cut single-stranded DNA probes in the reaction system. The probe consists of a fluorescent group and a quenching group linked by several bases. After the probe is cut, it produces fluorescence, indicating that the reaction system contains the target gene, thus playing a role in identification.

[0076] 4. Establishment of CRISPR detection method

[0077] 1) RAA amplification

[0078] Using the nucleic acid of the sample to be tested as a template, RAA amplification primers were used for amplification to obtain RAA amplification products.

[0079] The RAA reaction premix consists of: 25 μL of RAA amplification buffer (Zhongce, S001ZC, provided by a standard RAA kit), 2 μL of 10 μM upstream primer, 2 μL of 10 μM downstream primer, and 17.5 μL of water.

[0080] After preparing the RAA reaction premix, add 46.5 μL of the above reaction solution to each reaction unit of the RAA kit and dissolve it evenly. Add 1 μL of nucleic acid of the sample to be tested, add 2.5 μL of MgAc to the cap, place the tube in the RAA-B6100 instrument, set the reaction temperature to 39℃ and the time to 30 min (1800 s).

[0081] 2) CRISPR cutting

[0082] The RAA amplification product was cleaved by crRNA and LbCas12a protein, and the cleavage reaction product was obtained by using a probe as a fluorescent signal.

[0083] The reaction system for the cleavage reaction is: NEBuffer TM r2.1 2 μL, 1 μL of 26 μM LbCas12a protein diluted 26 times (final concentration in the system is 50 nM), 1 μL of 10 μM specific crRNA diluted 2 times (final concentration in the system is 250 nM), 1 μL of 10 μM probe diluted 4 times (final concentration in the system is 125 nM), 10 μL of DEPC water and 5 μL of RAA amplification product obtained in step 1).

[0084] The cleavage reaction was carried out by incubating at 39°C for 20 minutes to obtain the reaction product.

[0085] 3) Fluorescence detector detection

[0086] The cleavage reaction products were detected using a fluorescence detector, with fluorescence values ​​read every 20 seconds. The cumulative fluorescence value was used as the signal intensity, and the analysis and judgment were based on the following criteria:

[0087] If the fluorescence intensity of the reaction product is less than or equal to twice the fluorescence intensity of the negative control, then the sample to be tested does not contain or does not contain Tulafrancsis or its nucleic acid.

[0088] If the fluorescence intensity of the reaction product is greater than twice that of the negative control, then the sample to be tested contains or is a candidate to contain Tulafrancsis or its nucleic acid.

[0089] The negative control group mentioned above was a negative control set up for each experimental group, using water or other non-Tula Francisella DNA as a template.

[0090] Example 2: Screening of CRISPR-detected reaction crRNA

[0091] crRNAs were designed targeting the ACX55_1601 gene sequence. A crRNA leader sequence was designed downstream of the PAM sequence containing TTTN, and an anchoring sequence was added upstream of the leader sequence, resulting in 13 crRNA sequences (see Table 1).

[0092] Experimental group: The CRISPR reaction system consisted of NEBuffer. TM 2 μL of r2.1 (New England Biolabs, B6002S), 1 μL of 26 μM LbCas12a protein (GenScript, Z03753) diluted 26-fold, 1 μL of 10 μM specific crRNA (any one of SEQ ID NO. 16-23) diluted 2-fold, 1 μL of 10 μM probe (SEQ ID NO: 24) diluted 20-fold, 14 μL of DEPC water, and 1 μL of 40 nM ACX55_1601 plasmid synthesized from the gene were added. The reaction system was incubated at 37 °C for 60 min, and the fluorescence value was detected in real time using a RAA-F1620 instrument. The negative control group used 1 μL of DEPC water instead of the template.

[0093] The ACX55_1601 plasmid synthesized by gene synthesis was obtained by inserting the gene fragment shown in SEQ ID NO.1 between the BamHI and HindIII restriction sites of the pUC57 vector (Shanghai Sangon Biotech Co., Ltd., B522201).

[0094] The results are as follows Figure 2 As shown, ACX55_1601-CrRNA8 (denoted as crRNA8 in the figure) exhibits the best detection response to the ACX55_1601 plasmid, with the experimental group and the negative control group showing the largest ratio of fluorescence curve slope.

[0095] Therefore, this invention selects ACX55_1601-CrRNA8 as the CrRNA for detecting Tula Francisella.

[0096] Example 3: Screening of RAA amplification primers for *Tulafrancsis*

[0097] The synthesized ACX55_1601 plasmid (pUC57-ACX55_1601) stock solution was serially diluted 10-fold to 1000 copies / μL. Each serially diluted solution was used as a template for RAA amplification: 1 μL of plasmid was used to dilute the template for RAA amplification, and 1 μL of H2O was used as a negative control. The RAA amplification reaction was carried out according to the Qitian Gene RAA kit (catalog number: F00000A), and a premixed solution was prepared.

[0098] The RAA premixed solution consists of: 25 μL of buffer (Qitian, F00000A, provided by the fluorescent RAA kit), 2.1 μL of 10 μM upstream primer (SEQ ID NO: 2-6), 2.1 μL of 10 μM downstream primer (SEQ ID NO: 7-14), 0.6 μL of 10 μM probe (SEQ ID NO: 15, which binds to the target gene fragment and emits fluorescence to indicate gene amplification), and 16.7 μL of water.

[0099] After preparing the premix, add 46.5 μL of the premix to each basic reaction unit of the fluorescent RAA kit (included in the kit, containing recombinases that bind single-stranded nucleic acids, single-stranded DNA-binding enzymes, strand displacement DNA polymerases, dNTPs, etc.) and dissolve thoroughly. Then add 1 μL (1×10⁻⁶) of the ACX55_1601 plasmid (pUC57-ACX55_1601). 3 (Copies / μL). Pipette 2.5 μL of MgAc onto the RAA tube cap, place it in the RAA-B6100, press the preprocessing button to perform pre-amplification. After the instrument beeps, remove the reaction tube and place it in the RAA-F1620 instrument. Set the instrument to a reaction temperature of 39℃ and a time of 30 min (1800 s), and monitor the fluorescence in real time.

[0100] The results are as follows Figures 3-5 As shown, the RAA amplification primer pair ACX55-1601-F3 / ACX55-1601-R3 (denoted as F3+R3 in the figure) began to show fluorescence at 6 min, which was significantly better than other primer pairs.

[0101] Therefore, the present invention selected ACX55-1601-F3 / ACX55-1601-R3 as the primer pair for detecting Tula Francisella.

[0102] Example 4: Determination of the sensitivity of the CRISPR detection system for *Tulafrancsis*

[0103] I. Plasmid Sensitivity

[0104] 1. RAA amplification reaction

[0105] The original solution of plasmid ACX55_1601 (pUC57-ACX55_1601) was serially diluted 10-fold, using serial dilution buffer (1×10⁻⁶). 2 copies / μL, 1×10 1 copies / μL, 1×10 0Using copies / μL as a template, and employing the specific RAA primer pair ACX55-1601-F3 / ACX55-1601-R3 obtained in Example 3, the RAA amplification reaction was performed according to the method of the RAA testing kit (catalog number: S001ZC). The recombinase polymerase amplification reaction (RAA) was used to obtain the RAA amplification product. Water was used as a template as a negative control.

[0106] The RAA reaction premix consisted of: 25 μL of buffer (Zhongce, S001ZC, provided by a standard RAA kit), 2 μL of 10 μM upstream primer ACX55-1601-F3 (SEQ ID NO: 4), 2 μL of 10 μM downstream primer ACX55-1601-R3 (SEQ ID NO: 9), and 17.5 μL of water.

[0107] After preparing the RAA reaction premix, add 46.5 μL of the above reaction solution to each reaction unit of the RAA kit and dissolve it evenly. Add 1 μL of ACX55-1601 plasmid of different concentrations, add 2.5 μL of MgAc to the cap, place the tube in the RAA-B6100 instrument, set the reaction temperature to 39℃ and the time to 30 min (1800 s).

[0108] 2. Cas12a cleavage reaction

[0109] The LbCas12a protein (GenScript Biotechnology Co., Ltd., Z03753) guided by the specific ACX55_1601-CrRNA8 (SEQ ID NO: 18) obtained in Example 2 was incubated at 39°C for 20 minutes to cleave the amplification product obtained in step 1, and the reaction product was obtained.

[0110] The reaction system for the cleavage reaction is: NEBuffer TM r2.1 2 μL, 1 μL of 26 μM LbCas12a protein diluted 26 times (final concentration in the system is 50 nM), 1 μL of 10 μM specific crRNA (SEQ ID NO. 18) diluted 2 times (final concentration in the system is 250 nM), 1 μL of 10 μM probe (SEQ ID NO: 24) diluted 4 times (final concentration in the system is 125 nM), 10 μL of DEPC water, and 5 μL of RAA amplification product obtained in step 1.

[0111] 3. Testing

[0112] The reaction product obtained in step 2 was placed in a RAA-F1620 instrument. The instrument was set to a reaction temperature of 39°C and a reaction time of 20 min (1200 s), and fluorescence was detected in real time.

[0113] The results are as follows Figure 6 As shown, 10 0 Copy / tube (referred to as 1 in the diagram), 10 1 Copy / tube (referred to as 10 in the diagram) 1 ), 10 2 Copy / tube (referred to as 10 in the diagram) 2 ) respectively correspond to 1×10 0 1×10 1 1×10 2 RAA amplification was performed using primers ACX55-1601-F3 / ACX55-1601-R3, with the amplification product as the target DNA. The sensitivity of the combination of ACX55_1601-CrRNA8 and LbCas12a can reach 1 copy / reaction.

[0114] II. Genome Sensitivity

[0115] 1. RAA amplification reaction

[0116] The genome of *Tulafrancsis* was serially diluted 10-fold, using a serial dilution buffer (1×10⁻⁶). 3 copies / μL, 1×10 2 copies / μL, 1×10 1 copies / μL, 1×10 0 Using copies / μL) as templates, RAA amplification reaction was performed according to method 1 above to obtain amplification products;

[0117] 2. Cas12a cleavage reaction

[0118] The cleavage reaction was carried out according to method 2 in section 1 above to obtain the reaction product.

[0119] 3. Testing

[0120] The reaction product obtained in step 2 was placed in a RAA-F1620 instrument. The instrument was set to a reaction temperature of 39°C and a reaction time of 20 min (1200 s), and fluorescence was detected in real time.

[0121] The results are as follows Figure 7 As shown, 1 copy (denoted as 1 in the figure), 10 copies (denoted as 10 in the figure), and 100 copies (denoted as 10 in the figure) 2 1000 copies (referred to as 10 in the diagram) 9 ) respectively correspond to 1×10 0 1×10 1 1×10 2 1×10 3Copy / μL; RAA amplification was performed using the ACX55-1601-F3 / ACX55-1601-R3 primer pair, with the amplification product as the target DNA. The sensitivity of the combination of ACX55_1601-CrRNA8 and LbCas12a can reach 1 copy / reaction.

[0122] Example 5: Specificity detection of *Tulafrancsis* using the CRISPR detection system

[0123] The genomes of each tested bacterial species were amplified using primers ACX55-1601-F3 / ACX55-1601-R3. The RAA amplification products were then analyzed for specificity by CRISPR detection using a combination reaction of ACX55_1601-CrRNA8 and Cas12a.

[0124] Specifically as follows:

[0125] 1. RAA amplification reaction

[0126] Genomic DNA was extracted from the following strains: *Francis tularensis* (U112), *Francis mirabilis* (ATCC25015), *Yersinia pestis* (Y. pestis 201, biovar Microtus strain), *Bacillus thuringiensis* (ATCC 10792), *Bacillus subtilis* (ATCC 6051), *Vibrio vulnificus* (ATCC 27562), and *Vibrio parahaemolyticus* (ATCC17802).

[0127] The species *Franciella tularensis* (U112) is described in the following literature, under the name U112: Johnson SL, Daligault HE, Davenport KW, et al. Genome sequencing of 18 *francisella* strains to aid in assay development and testing. *Genome Announc*. 3(2):e00147-15, 2015.

[0128] Yersiniapestis (Y. pestis 201, biovar Microtus strain) is described in the following literature, where it is referred to as biovar Microtus strain 201: Zhang Q, Wang Q, Tian G, et al. Yersiniapestis biovar Microtus strain 201, an avirulent strain to humans, provides protection against bubonic plague in rhesus macaques. Hum Vaccin Immunother. 10(2):368-77, 2014.

[0129] Using the genomic DNA of each bacterial strain and the genomic DNA of the mixed bacteria as templates, RAA amplification reactions were performed according to the method in Example 4-1 above to obtain amplification products.

[0130] The mixed bacterial genomic DNA was a mixture of equal mass of the genomic DNA of the above six non-Tula Francisella strains.

[0131] 2. Cas12a cleavage reaction

[0132] The cleavage reaction was carried out according to the method in Example 4-2 above to obtain the reaction product.

[0133] 3. Testing

[0134] The reaction product obtained in step 2 was placed in a RAA-F1620 instrument. The instrument was set to a reaction temperature of 39°C and a reaction time of 20 min (1200 s), and fluorescence was detected in real time.

[0135] The results are as follows Figure 8 As shown, the CRISPR detection system for *François tularensis* established in this invention exhibits good specificity, detecting only *François tularensis*. It shows no cross-reactivity with engineered bacteria such as *François mirageensis*, *Yersinia pestis*, *Bacillus thuringiensis*, *Bacillus subtilis*, *Vibrio vulnificus*, and *Vibrio parahaemolyticus*, and also shows no cross-reactivity with the mixed genomes of the aforementioned six non-*François tularensis* bacteria.

[0136] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A CRISPR-Cas12a system for detecting *Tulafrancsis*, comprising Cas12a protein and crRNA, or a complex of the two; The crRNA includes an anchoring sequence for binding to the Cas12a protein and a guide sequence for targeting the *Tulafrancsis* target sequence. The target sequence of *Tulafrancsis* is derived from the ACX55_1601 gene of *Tulafrancsis* as shown in SEQ ID No. 1; The target sequence of the *Tulafrancsis* is the DNA encoding the RNA fragment shown in positions 22-43 of SEQ ID No.

18.

2. The CRISPR-Cas12a system according to claim 1, characterized in that: The sequence of the crRNA is SEQ ID No. 18; Alternatively, the Cas12a protein may be the LbCas12a protein.

3. A kit for detecting *Tulafrancsis*, comprising the following: 1) The CRISPR-Cas12a system for detecting Tula Francisella as described in claim 1 or 2; 2) RAA amplification primers for specifically amplifying the target sequence of *Tulafrancsis* as described in claim 1 or 2; 3) As a probe for fluorescent reporter molecules; The probe is labeled with a fluorescent group and a quenching group at its two ends, respectively.

4. The reagent kit according to claim 3, characterized in that: The RAA amplification primers consist of single-stranded DNA molecules shown in SEQ ID No. 4 and SEQ ID No. 9; The nucleotide sequence of the probe is SEQ ID No.

24.

5. Any of the following applications: B1) The use of the system of claim 1 or 2 or the kit of claim 3 or 4 for non-disease diagnostic and therapeutic purposes in the detection or auxiliary detection of Tulafrancsis or its nucleic acid; B2) The use of the system of claim 1 or 2 or the kit of claim 3 or 4 in the preparation of products for detecting or assisting in the detection of Tulafrancsis or its nucleic acids; B3) The use of the system of claim 1 or 2 or the kit of claim 3 or 4 for non-disease diagnostic and therapeutic purposes in detecting or assisting in the detection of whether a sample contains Tulafrancsis or its nucleic acid; B4) The use of the system of claim 1 or 2 or the kit of claim 3 or 4 in the preparation of products for detecting or assisting in the detection of whether a sample to be tested contains Tulafrancsis or its nucleic acid; B5) The use of the system of claim 1 or 2 or the kit of claim 3 or 4 in the preparation of products for the diagnosis or auxiliary diagnosis of whether a sample to be tested is infected with Tulafrancsis.

6. A method for detecting or assisting in the detection of *Tulafrancsis* for non-disease diagnostic and therapeutic purposes, comprising the following steps: C1) Using the nucleic acid of the sample to be tested as a template, perform RAA amplification using the RAA amplification primers described in claim 4 to obtain the RAA product; C2) Prepare a CRISPR-Cas12a detection system containing the following components: the RAA product, the Cas12a protein as described in claim 1 or 2, the crRNA as described in claim 1 or 2, the CRISPR reaction buffer, and the probe as described in claim 4; water is used instead of the RAA product as a negative control. C3) The CRISPR-Cas12a detection system is reacted, and the reaction products are detected to determine whether the sample to be tested contains Tula Francisella.

7. The method according to claim 6, characterized in that: In step C1), the reaction conditions for RAA amplification are: 35-41℃ for 20-30 minutes; Alternatively, in step C3), the reaction conditions are 35-39°C for 10-30 minutes.

8. The method according to claim 6 or 7, characterized in that: The method for detecting the reaction products is to detect the signal using a fluorescence detector, and to determine the detection result based on the detection signal.

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