A kit and method for detecting pachysolen tannophilus based on rpa-crispr / cas12a

Abstract

The application discloses a kit and a detection method for detecting Leucotrichia bicuspidata based on RPA-CRISPR / Cas12a. 16S rRNA The application takes Leucotrichia bicuspidata as a specific target, designs and optimizes a RPA amplification primer pair and a specific crRNA recognition sequence, realizes high-sensitivity and high-specificity detection of a target nucleic acid under the condition of not depending on a variable temperature instrument by integrating the isothermal amplification technology of RPA and the precise target recognition and trans-cleavage activity of the CRISPR / Cas12a system, and the detection result can be observed by naked eyes under blue light. ‑3 The application has the characteristics of simple operation, low cost and strong practicability, and can realize sensitive and visual detection of the target gene under the condition that the DNA template concentration is not lower than 2.9*10 The method is not only suitable for laboratory environment, but also can be used for instant and rapid detection on site, such as a crab breeding pond, thereby providing an effective technical means for early diagnosis and prevention and control of diseases caused by Leucotrichia bicuspidata, and having important practical application value.

Description

Technical Field

[0001] This invention belongs to the field of aquatic animal pathogen detection technology, and mainly relates to a kit and detection method for detecting *Saccharomyces cerevisiae* based on RPA-CRISPR / Cas12a. Background Technology

[0003] Two-pointed malt yeast ( Metschnikowia Bicuspidata Yeasts (Saccharomyces cerevisiae) are a type of pathogenic yeast that harms aquatic animals. They belong to the class Fungi, order Ascomycetes, and genus Saccharomyces, and are considered opportunistic pathogens. Their vegetative cells are mostly oval, cylindrical, or spherical, but may also be elongated. Reproduction mainly occurs through polygonal budding, with cells existing singly or in chains. Aquatic animal pathogens are generally transmitted horizontally, vertically, or both. According to transmission experiments by Jiang et al., *Saccharomyces cerevisiae* was not detected in the reproductive cells, fertilized eggs, and hatched larvae of the Chinese mitten crab, indicating that *Saccharomyces cerevisiae* is mainly transmitted horizontally (JIANG H, BAO J, CAO G, et al. Experimental transmission of the yeast, Metschnikowia bicuspidata , in the Chinese mittencrab, Eriocheir sinensis [J]. Journal of Fungi,2022,8(2): 210-221.).

[0004] Currently, the detection methods for *Saccharomyces cerevisiae* mainly rely on pathogen detection and molecular biological detection. Molecular biological detection technology is a nucleic acid amplification technique based on the amplification of specific nucleic acid target sequences within an organism. In recent years, nucleic acid-based detection technologies for *Saccharomyces cerevisiae* have been extensively researched and developed. Sequencing and polymerase chain reaction (PCR) and their derivative technologies are currently the most commonly used molecular biological methods for detecting various pathogens. Xing et al. established a real-time quantitative PCR (qPCR) detection method targeting the mitochondrial cytochrome c oxidase subunit VIA (COX 6A) of *Saccharomyces cerevisiae* (XING Y, CHEN Y, FENG C, et al. Establishment and Application of Real-Time Fluorescence Quantitative PCR Detection Technology for...). Metschnikowia bicuspidata Disease in Eriocheir sinensis[J], Fungi (Basel), 2023, 9(8):791-802.); Bao et al. developed a nested PCR based on the cell wall protein (HYR) gene regulated by hyphae (BAO J, CHEN Y, XING Y, et al. Development of a Nested PCR Assay for Specific Detection of Metschnikowia Bicuspidata Infecting Eriocheir Sinensis [J]. Frontiers in Cellular and Infection Microbiology, 2022, 12: 930585-930594), the above detection methods all achieve high sensitivity, strong specificity, and good repeatability, and can rapidly and accurately detect *Saccharomyces cerevisiae*. However, temperature-switching methods such as PCR are highly dependent on temperature-switching equipment, which cannot meet the detection needs of aquaculture sites and limits the application of these methods.

[0005] Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique. The entire reaction can achieve exponential amplification within 30 minutes at a constant temperature of 35-41℃, and it has been widely used in pathogen detection. CRISPR sequences were first reported in 1987 by Japanese scientists in their research on the genome of *E. coli*, and were subsequently identified and formally named in various prokaryotes by Janson et al. in 2002 (JANSEN R, EMBDEN J. D, GAASTRA W, et al. Identification of genes that are associated with DNA repeats in prokaryotes. *Molecular microbiology*, 2002, 43(6): 1565-1575.). Related Cas proteins are further divided into many types, such as Cas9, Cas12a, Cas12b, Cas13a, and Cas13b. In CRISPR detection technology, the most common one is Cas12a, also known as Cpf1. It was originally used for gene editing. Due to its target recognition and cutting characteristics, it can amplify the detection signal through cascade reactions and is now widely used in the field of detection.

[0006] However, to date, there have been no reports of combining recombinase polymerase amplification technology and CRISPR / Cas12a technology for the visualization detection of Saccharomyces cerevisiae. Summary of the Invention

[0007] To enable timely, sensitive, specific, and visual detection of *D. mitralovichthys*, the pathogen causing "milk disease" in crabs and other aquatic animals, this invention aims to provide a kit and detection method for *D. mitralovichthys* based on RPA-CRISPR / Cas12a by combining RPA isothermal amplification technology with a CRISPR / Cas12a system. A rapid and visual detection system suitable for *D. mitralovichthys* has been established. The kit mainly includes specific primer pairs (SEQ ID NO.1, SEQ ID NO.2) for detecting *D. mitralovichthys* based on RPA-CRISPR / Cas12a, Cas12a protein, crRNA (SEQ ID NO.7), and a fluorescent reporter probe (nucleotide sequence shown in SEQ ID NO.10). The detection limit of this method for *D. mitralovichthys* is as low as 2.9 × 10⁻⁶. -3 The designed RPA primers and crRNA, at a mol / L concentration, specifically react with *Saccharomyces cerevisiae*, exhibiting no cross-reactivity with other yeasts, bacteria, or viruses, demonstrating excellent specificity. Furthermore, this detection can be performed at room temperature (35-45℃), requiring no complex instruments, and can be completed within 60 minutes. The results can be directly visualized under blue light, meeting the need for rapid detection. It is particularly suitable for immediate, pond-side detection in aquaculture, providing a reliable and portable technical means for the early diagnosis and control of aquatic animal diseases caused by *Saccharomyces cerevisiae*.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a kit for detecting *Saccharomyces cerevisiae* based on RPA-CRISPR / Cas12a, the kit comprising RPA amplification reaction reagents and CRISPR / Cas12a detection reagents; the RPA amplification reaction reagents mainly include specific primer pairs for amplifying *Saccharomyces cerevisiae*; the CRISPR / Cas12a detection reagents mainly include crRNA, Cas12a protein and fluorescent reporter probe.

[0010] Based on the above technical solution, the nucleotide sequence of the primer pair is shown in SEQ ID NO.1-6, and preferably, the nucleotide sequence of the primer pair is shown in SEQ ID NO.1-2.

[0011] Based on the above technical solution, the nucleotide sequence of the crRNA is shown in SEQ ID NO.7-9, and preferably, the nucleotide sequence of the crRNA is shown in SEQ ID NO.7.

[0012] Based on the above technical solution, the structure of the fluorescent reporter probe is further shown below: 5'-FAM-TTATTATT-BHQ1-3'.

[0013] Based on the above technical solution, the CRISPR / Cas12a detection reagent further includes a CRISPR buffer, which comprises 10×NE Buffer. TM r2.1.

[0014] Based on the above technical solution, the RPA amplification reaction reagent further includes RPA amplification reaction dry powder.

[0015] Based on the above technical solution, the RPA amplification reaction reagent further includes buffer (buffer A and buffer B) and nuclease-free water.

[0016] Based on the above technical solution, the kit further includes *Saccharomyces cerevisiae* genomic DNA.

[0017] Secondly, the present invention provides a method for detecting *Saccharomyces cerevisiae* using the above-mentioned kit, comprising the following steps: (1) Extraction of genomic DNA from the sample: Take liver, pancreas or blood lymph samples from the animal to be tested and extract genomic DNA from the sample; (2) RPA amplification: The genomic DNA of the sample to be tested extracted in step (1) was amplified by recombinase polymerase (RPA) using specific primers for amplifying *Saccharomyces cerevisiae* to obtain RPA amplification products; (3) CRISPR / Cas12a detection: Take 0.5~5 μL of the RPA amplification product obtained in step (2) and add it to 20~50 μL of CRISPR / Cas12a reaction system. The composition of the CRISPR / Cas12a reaction system is as follows: 1~3 μL crRNA, 1~3 μL Cas12a protein, 3~5 μL fluorescent reporter probe, 1~3 μL CRISPR buffer, and the remainder is nuclease-free water. Detect the fluorescence signal every 20~40 s at 43~47℃ for a total of 50~150 times. Interpret the results according to the fluorescence intensity-time curve or observe the fluorescence color of the reaction system under blue light after the reaction for naked-eye visualization. Positive samples can be observed to have obvious green fluorescence, while negative samples have no fluorescence.

[0018] Based on the above technical solution, further, the hepatopancreas or hemolymph sample mentioned in step (1) is derived from a diseased Chinese mitten crab.

[0019] Based on the above technical solution, further, the total volume of the RPA amplification reaction system in step (2) is 50 μL, including: 1 tube of reaction dry powder, 2 μL each of upstream and downstream primers, 25 μL of buffer A, 2.5 μL of buffer B, 5.0 μL of genomic DNA, and nuclease-free water to make up to 50 μL.

[0020] Based on the above technical solution, further, the concentrations of the upstream and downstream primers in step (2) are both 1~100 μmol / L, preferably 10 μmol / L.

[0021] Based on the above technical solution, further, step (2) RPA amplification reaction is carried out under constant temperature conditions, with a temperature range of 35-41℃ and a reaction time of 5-50 min; preferably, the reaction is carried out at a constant temperature of 39℃ for 30 min.

[0022] Based on the above technical solution, further, in step (2), in order to ensure the reliability of the experiment, negative control and positive control are set up at the same time: an equal volume of nuclease-free water is used to replace the DNA template as a blank control; and the known genomic DNA of *Saccharomyces cerevisiae* is used as a positive control. Both are amplified using the same amplification process as the sample to be tested.

[0023] Based on the above technical solution, further, the final concentration of crRNA in step (3) is 0.05~1 μmol / L, the final concentration of Cas12a protein is 0.2~4 μmol / L, and the final concentration of fluorescent reporter probe is 0.1~1 μmol / L.

[0024] Based on the above technical solution, further, the final molar concentration ratio of crRNA and Cas12a protein in the system in step (3) is 1:4.

[0025] Based on the above technical solution, further, in step (3), the Cas12a protein and crRNA are incubated at 35~38℃ for 5~30 min in advance to promote the effective formation of the ribonucleoprotein complex.

[0026] Based on the above technical solution, further, in step (3), when observing the fluorescence color of the reaction system under blue light to determine the result, if the reaction solution of the test sample tube and the blank control and negative control tubes does not show visible green fluorescence, it is determined to be negative; if the test sample tube shows obvious green fluorescence, while the control tube does not show fluorescence, it is determined to be positive.

[0027] All detection steps in this method are performed under a single isothermal condition, eliminating the need for thermal cycling or complex temperature gradient control, thus completely freeing it from dependence on large, sophisticated instruments such as real-time quantitative PCR instruments. This characteristic makes it particularly suitable for resource-constrained field environments or grassroots laboratories, enabling rapid, naked-eye visualization screening of pathogens in the field, and demonstrating broad application prospects in areas such as point-of-care diagnostics.

[0028] Thirdly, the present invention provides the application of the above-described kit or method in detecting whether crabs are infected with Diffused Gram-Thomsonia.

[0029] This invention establishes a novel, rapid, sensitive, specific, and visualized method for detecting *Saccharomyces cerevisiae* based on recombinase polymerase amplification (RPA) technology and a CRISPR / Cas12a system. The method employs a two-cascade detection strategy: First, RPA amplification primers are designed, and isothermal RPA amplification technology is used to efficiently and rapidly amplify the target nucleic acid fragment, generating a large amount of double-stranded DNA product within a short time at a constant temperature. Subsequently, the RPA amplification product is transferred to a CRISPR / Cas12a reaction system for specific recognition and signal transduction. In this step, a synthesized crRNA guides the Cas12a protein to precisely recognize the target sequence, activating its trans-cleavage activity. Because a double-labeled fluorescent reporter probe (Reporter) is pre-added to the system, with a fluorophore (FAM) at its 5' end and a quencher (BHQ1) at its 3' end, the activated Cas12a protein non-specifically cleaves the reporter probe, causing the fluorophore and quencher to separate, thereby releasing a detectable fluorescent signal. After the fluorescence signal collection is completed, the sample is placed under blue light to observe whether there is fluorescence. The appearance of green fluorescence indicates that the sample is positive and has been successfully detected; no fluorescence indicates that the sample is negative and there is no detection target, thus achieving specific and visual detection of Saccharomyces cerevisiae nucleic acid.

[0030] Compared with the prior art, the beneficial results of the present invention are as follows: 1. The RPA-based invention provided by this invention This document describes specific primers, kits, and detection methods for CRISPR / Cas12a detection in *Saccharomyces cerevisiae*. After RPA amplification of the *Saccharomyces cerevisiae* gene target, the Cas12a protein is activated under the guidance of a designed specific crRNA, achieving its non-specific trans-cleavage activity. The signal is output by cleaving a fluorescently labeled reporter probe, and the result is visible to the naked eye. The synergistic effect of RPA and the CRISPR / Cas12a system constructs a dual cascade reaction system of "nucleic acid amplification" and "enzyme-catalyzed signal amplification," significantly surpassing the detection sensitivity of traditional qPCR technology that relies on a single amplification mechanism. Since the entire RPA and CRISPR / Cas12a reaction can be completed under isothermal conditions, eliminating the need for complex thermal cycling processes and providing visualized results, it completely eliminates the reliance on large, precision instruments such as real-time quantitative PCR instruments. This significantly simplifies the hardware requirements and technical barriers of the detection operation, making it more suitable for rapid on-site diagnosis and high-throughput screening scenarios.

[0031] 2. The detection method of the present invention has high sensitivity; it can detect 2.9 × 10⁻⁶. -3 amol / L of *Saccharomyces cerevisiae* can achieve precise detection of trace amounts.

[0032] 3. The detection method of this invention has high specificity, accurately detecting only *Saccharomyces cerevisiae*, and is different from other yeasts (…). Candida oleophila, Candida lusitaniae ), bacterial pathogens ( Pseudomonas putida、Vibrio alginolyticus ), and viral pathogens (Grass carp reovirus virus) 、 Cyprinid herpes virus3 、 There is no cross-reactivity with White spot syndrome virus, which is of great significance for the prevention and control of diseases in aquatic animals. Attached Figure Description

[0033] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0034] Figure 1 This is a diagram showing the results of RPA primer screening in Example 1, where 1—negative, 2—positive control in the kit, 3—primer 1 + strain DNA, 4—primer 1 + hepatopancreatic DNA, 5—primer 2 + strain DNA, 6—primer 2 + hepatopancreatic DNA, 7—primer 3 + strain DNA, and 8—primer 3 + hepatopancreatic DNA.

[0035] Figure 2This is a diagram showing the results of crRNA screening in Example 1. In the diagram, A: fluorescence amplification effect diagram, B: visual fluorescence result diagram, 1—negative, 2—crRNA 1+ strain DNA, 3—crRNA 1+ hepatopancreatic DNA, 4—crRNA 2+ strain DNA, 5—crRNA 2+ hepatopancreatic DNA, 6—crRNA 3+ strain DNA, 7—crRNA 3+ hepatopancreatic DNA.

[0036] Figure 3 The graph shows the optimization results of RPA reaction parameters in Example 2, where A: RPA amplification temperature optimization, B: RPA amplification time optimization, and C: RPA primer concentration optimization.

[0037] Figure 4 This is a graph showing the sensitivity results of RPA-CRISPR / Cas12a detection of *Saccharomyces cerevisiae* in Example 3. In the graph, A: fluorescence amplification effect, B: amplification efficiency, and C: visible fluorescence results under blue light. In the graph, 1—2.9×10⁻⁶ 1 amol / L, 2—2.9×10 -1 amol / L, 3—2.9×10 -2 amol / L, 4—2.9×10 -3 amol / L, 5—2.9×10 -5 amol / L, 6—negative.

[0038] Figure 5 The images shown are from Example 4, illustrating the specific detection results of RPA-CRISPR / Cas12a on Saccharomyces cerevisiae. A: Fluorescence amplification effect of different pathogens; B: Fluorescence amplification efficiency of different pathogens; C: Visual fluorescence results of different pathogen detection systems under blue light.

[0039] Figure 6 These are the results of RPA-CRISPR / Cas12a detection of actual samples in Example 5. Among them, A: Fluorescence amplification effect of diseased crabs in summer, B: Visual fluorescence result of diseased crabs in summer, C: Fluorescence amplification effect of diseased crabs in winter, and D: Visual fluorescence result of diseased crabs in winter.

[0040] Figure 7 This is a diagram showing the qPCR primer screening results in Example 6, where 1—negative, 2—primer 1+ bacterial DNA, 3—primer 1+ hepatopancreatic DNA, 4—negative, 5—primer 2+ bacterial DNA, and 6—primer 2+ hepatopancreatic DNA.

[0041] Figure 8 This is a graph showing the qPCR sensitivity detection results from Example 6. In the graph, A: amplification curve, B: standard curve, and C: melting curve. 1—2.9×10⁻⁶1 amol / L, 2—2.9×10 -1 amol / L, 3—2.9×10 -2 amol / L, 4—2.9×10 -3 amol / L, 5—2.9×10 -5 amol / L.

[0042] Figure 9 The graphs shown are the results of qPCR detection of actual samples in Example 6. Among them, A: Amplification curve of diseased crabs in summer, B: Melting curve of diseased crabs in summer, C: Amplification curve of diseased crabs in winter, and D: Melting curve of diseased crabs in winter. Detailed Implementation

[0043] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0044] Example 1: RPA primers and CRISPR / Cas12a crRNA design Materials and Methods 1. Materials Positive samples of *Eriocheir mitrichoides* were collected from Panjin, Liaoning Province, and were subsequently isolated and purified in the laboratory; *Eriocheir sinensis* infected with *Eriocheir mitrichoides* were collected from Panjin, Liaoning Province.

[0045] 2. Samples and reagents Oligonucleotides were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; 2×Taq Master Mix was purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). SYBR qPCR Master Mix was purchased from Novizan Biotechnology Co., Ltd. (Nanjing, China). Marine animal tissue genomic DNA extraction kit was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). DNA Good View™ nucleic acid dye was purchased from Beijing Saibaisheng Gene Technology Co., Ltd. (Beijing, China). Cas12a protein was purified in our laboratory using 10×NEBuffer. TM r2.1 was purchased from New England Biolabs, Inc. (USA). RAA ® Nucleic acid amplification reagent (basic type) was purchased from Lingyu Biotechnology Co., Ltd. (Zhejiang, China). DNA extraction phenol reagent was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China).

[0046] 3. DNA extraction Hemolymph and hepatopancreas of Chinese mitten crab were collected, and DNA was extracted from the hemolymph and hepatopancreas according to the instructions of the DNA extraction kit.

[0047] 4. RPA primer design RPA amplification reaction conditions are one of the important factors affecting the subsequent detection efficiency of the CRISPR / Cas12a system. RPA primers must be designed according to the crRNA location, with each primer pair covering the crRNA binding site to amplify DNA fragments containing the crRNA binding site. This invention is based on *Saccharomyces cerevisiae*. 16S rRNA Genes were used to design RPA primer sequences using BLAST comparative analysis (details are shown in Table 1).

[0048] Table 1. Three pairs of RPA primers designed in this embodiment

[0049] The optimal primers for RPA amplification in *Saccharomyces cerevisiae* were selected and designed as follows: In this embodiment, the RPA amplification reaction used a 50 μL system, comprising the following components: 1 tube of reaction powder, 2 μL of forward and reverse primers (added accordingly, 10 μmol / L), 25 μL of buffer A, 2.5 μL of buffer B, 5 μL of genomic DNA, and nuclease-free water to a final volume of 50 μL. The reaction powder, buffer A, and buffer B were all obtained from a commercial kit (purchased from Zhejiang Lingyu Biotechnology Co., Ltd., China); the genomic DNA was extracted from *Saccharomyces cerevisiae* (strain DNA) at a concentration of 370.5 ng / μL; and the genomic DNA was extracted from the hepatopancreatic tissue of a diseased *Eriocheir sinensis* (hepatopancreatic DNA) at a concentration of 41.7 ng / μL. The specific operating procedure is as follows: First, according to the reaction quantity, prepare a premix containing water, buffer A, upstream primer (10 μmol / L), and downstream primer (10 μmol / L) according to the reaction system. Mix well and add it to the detection unit tube containing the reaction powder. Then, add the DNA sample to be tested to the detection unit tube. Next, add 2.5 μL of buffer B to the cap of the detection unit tube, cap it, and gently shake it upside down 5-6 times to mix thoroughly. After centrifugation at low speed for 10 s, place the detection unit tube in a 39℃ constant temperature metal bath (or constant temperature water bath) and incubate for 30 min. After the reaction is complete, add 50 μL of DNA extraction phenol reagent to the detection unit tube, mix thoroughly, centrifuge at 12000 rpm for 5 min, and take the supernatant for electrophoresis. Select a 1.5% concentration agarose gel for electrophoresis at 100V for 45 min. Select the optimal primers based on the electrophoresis results. The agarose gel electrophoresis results are shown below. Figure 1As shown, the negative control was very dark or showed no band, while the positive control was very bright; the amplified fragment of primer 3 was in the wrong position, and the band of primer 1 was clearer and brighter than that of primer 2. Therefore, subsequent experiments will design crRNA based on primer 1 for secondary screening.

[0050] 5. Design of CRISPR / Cas12a crRNA Three crRNAs were designed based on the selected optimal RPA primer pairs. The specific sequences of the three crRNAs are shown in Table 2.

[0051] Table 2. Three crRNAs designed in this embodiment

[0052] The fluorescent reporter probe is shown below (nucleotide sequence as shown in SEQ ID NO.10): 5'-FAM-TTATTATT-BHQ1-3'.

[0053] In this embodiment, RPA amplification was performed as described above to obtain 1 μL of RPA amplification product, which was then added to a 20 μL CRISPR / Cas12a reaction system for reaction. The system composition is as follows: 10×NEBuffer TM The reaction mixture consisted of 2 μL r2.1, 2 μL Cas12a protein (4 μmol / L), 2 μL crRNA (1 μmol / L), 4 μL fluorescent reporter probe (1 μmol / L), 1 μL RPA amplification product, and nuclease-free water to a final volume of 20 μL. The specific procedure was as follows: First, according to the reaction quantity, prepare the reaction system containing water and 10×NE Buffer. TM The premixed solution of r2.1 and 1 μmol / L fluorescent reporter probe was mixed thoroughly and added to the detection unit tube; then 1 μL of the obtained RPA amplification product was added to the detection unit tube; finally, 4 μL of the Cas12a protein and crRNA incubation complex was added; to promote the effective formation of the ribonucleoprotein complex, the Cas12a protein and crRNA were incubated at 37℃ for 10 min in advance to form a complex with a final concentration ratio of 1:4 in the system. The fluorescence signal was then detected every 30 s at 45℃ for a total of 99 detections. The results are as follows. Figure 2 As shown in the results, crRNA 1 had the best effect, so crRNA 1 was selected for subsequent experiments.

[0054] Example 2: RPA Reaction System Optimization and Establishment of RPA-CRISPR / Cas12a System This embodiment optimizes the temperature, time, and primer concentration of RPA amplification to maximize detection efficiency and reduce detection costs.

[0055] 1. RPA amplification temperature optimization The reaction system and method of Example 1 were used, with a reaction time of 30 min and reaction temperatures of 35℃, 37℃, 39℃, and 41℃, respectively. The target bands of the amplified products at different temperatures were compared by agarose gel electrophoresis to screen for the optimal reaction temperature.

[0056] The results are as follows Figure 3 As shown in Figure A, the difference in band brightness was not significant within the temperature range of 35℃ to 41℃. Therefore, the temperature of 39℃ recommended in the instructions of the commercial kit was selected for subsequent experiments.

[0057] 2. RPA amplification time optimization The reaction system and reaction method of Example 1 were used, the reaction temperature was 39°C, and the reaction time was set to 5 min, 10 min, 20 min and 30 min respectively.

[0058] The results are as follows Figure 3 As shown in Figure B, the band is brightest when the RPA amplification time is 30 min, therefore the optimal RPA amplification time is 30 min.

[0059] 3. Optimization of primer concentrations used for RPA amplification The reaction system and method of Example 1 were used, the reaction temperature was 39℃, and the reaction time was 30 min. The primer concentrations were set to 1 μmol / L, 10 μmol / L, and 100 μmol / L, respectively.

[0060] The results are as follows Figure 3 As shown in C, the optimal banding effect is achieved when the RPA primer concentration is 10 μmol / L. Therefore, the optimal primer concentration for RPA amplification is 10 μmol / L.

[0061] 4. Establishment of the RPA-CRISPR / Cas12a system After determining the optimal reaction conditions for RPA, the RPA-CRISPR / Cas12a system was constructed in conjunction with the aforementioned CRISPR system for subsequent experiments. The optimal system was as follows: 1 tube of reaction powder, 2 μL each of forward and reverse primers (primer 1, 10 μmol / L), 25 μL of buffer A, 2.5 μL of buffer B, 5.0 μL of genomic DNA, and nuclease-free water to a final volume of 50 μL. The reaction was carried out at 39°C for 30 min.

[0062] CRISPR / Cas12a detection system: 10×NEBuffer TMr2.1 2 μL, 4 μmol / L Cas12a protein 2 μL, 1 μmol / L crRNA 2 μL, 1 μmol / L fluorescent reporter probe 4 μL, RPA amplification product 1 μL, nuclease-free water to make up to 20 μL; fluorescence signal was detected every 30 s at 45℃ for a total of 99 detections.

[0063] Example 3 Sensitivity analysis of *Saccharomyces cerevisiae* based on RPA-CRISPR / Cas12a method The extracted *Saccharomyces cerevisiae* genomic DNA (original concentration 2900 μmol / L) was diluted to obtain concentrations of 2.9 × 10⁻⁶. 1 amol / L, 2.9×10 -1 amol / L, 2.9×10 -2 amol / L, 2.9×10 -3 amol / L, 2.9×10 - 5 Genomic DNA template at amol / L. Sensitivity experiments were performed using the optimal RPA-CRISPR / Cas12a reaction system obtained in Example 2.

[0064] The results are as follows Figure 4 As shown, the lowest detectable concentration was ultimately 2.9 × 10⁻⁶. -3 amol / L.

[0065] Example 4: Specificity analysis of *Saccharomyces cerevisiae* based on RPA-CRISPR / Cas12a method Using the optimal RPA-CRISPR / Cas12a reaction system and detection method obtained in Example 2, *Saccharomyces cerevisiae* was compared with other yeasts. Candida oleophila , Candida lusitaniae ), bacterial pathogens ( Pseudomonas putida , Vibrio alginolyticus ) and viral pathogens (Grass carp reovirus vitus) 、 Cyprinid herpesvirus 3 、 White spot syndrome virus (BSD) was detected at concentrations of 2.9 amol / L and 2.9 × 10⁻⁶ mcg / L, respectively. -3 amol / L Metschnikowia bicuspidata Nucleic acid is reacted, and the result is as follows: Figure 5 As shown, the detection method of the present invention is effective for low concentrations of... Metschnikowia bicuspidata It can be effectively amplified, while the other 7 high concentrations did not amplify, indicating that the detection method of the present invention has good specificity.

[0066] Example 5: Detection of actual samples of *Saccharomyces cerevisiae* based on RPA-CRISPR / Cas12a method This embodiment uses the optimal RPA-CRISPR / Cas12a reaction system and detection method obtained in Example 2 to detect actual samples of *Eriocheir mitralovichthys* (Chinese mitral crabs infected with *Eriocheir mitralovichthys*): Hepatopancreatic DNA and hemolymph DNA were extracted from *Eriocheir mitralovichthys*-infected Chinese mitral crabs (the specific operation procedure is the same as step 3 DNA extraction in Example 1). The concentration and purity were then determined using an ultra-micro spectrophotometer, with the absorbance ratio A260 / A280 used as the evaluation index. The concentrations of all extracted hepatopancreatic tissue DNA were in the range of 20-1000 ng / μL, and the concentrations of hemolymph DNA were in the range of 10-200 ng / μL, with A260 / A280 values ​​between 1.8 and 2.0. Hepatopancreatic tissue DNA samples with a concentration of approximately 100 ng / μL (30 samples each in summer and winter) and hemolymph DNA samples with a concentration of approximately 50 ng / μL (30 samples each in summer and winter) were selected for subsequent detection. The results are presented for each group with 6 samples as an example, as follows: Figure 6 As shown in AD, DNA from the hepatopancreas and hemolymph of crabs infected with *Dictyophora indicum* in both summer and winter can be successfully detected.

[0067] Example 6: Detection of *Saccharomyces cerevisiae* by qPCR The results of qPCR assay were consistent with those of RPA-CRISPR / Cas12a sensitivity detection and actual sample detection. 1. qPCR primer screening Using Primer Premier 5.0 with mitochondrial yeast 16S rRNA Two primer pairs were designed for gene sequencing (see Table 3), and primer selection was performed by PCR amplification. The PCR reaction system consisted of 10 μL 2× Taq Master Mix; 1 μL each of forward and reverse primers (10 μmol / L); 2 μL DNA template; and nuclease-free water to a final volume of 20 μL. The DNA templates used were *Saccharomyces cerevisiae* genomic DNA (strain DNA, concentration 370.5 ng / μL) and hepatopancreatic tissue DNA from diseased *Eriocheir sinensis* (hepatopancreatic DNA, concentration 41.7 ng / μL). The PCR reaction program was 95 ℃ for 3 min, 95 ℃ for 15 s, 60 ℃ for 30 s, 72 ℃ for 30 s, for 40 cycles; followed by a final extension at 72 ℃ for 5 min. The amplified products were interpreted by 1.5% agarose gel electrophoresis. The results are shown below. Figure 7 As shown, the band of primer 1 is more regular and has no extraneous bands than that of primer 2. Therefore, primer 1 was selected for the subsequent qPCR experiments.

[0068] Table 3. Two sets of qPCR primer pairs designed in this embodiment.

[0069] 2. Establishment of qPCR program The qPCR reaction system consisted of 10 μL of SYBR qPCR Master Mix, 0.4 μL each of forward and reverse primers (primer 1, 10 μmol / L), and 1 μL of DNA template, with the volume brought to 20 μL using nuclease-free water. The reaction program was as follows: 95 ℃ pre-denaturation for 30 s, 95 ℃ denaturation for 15 s, 60 ℃ annealing for 30 s, and 72 ℃ extension for 30 s, for 40 cycles. The melting curve was photographed at 60 ℃, and the remaining operations were performed according to the specifications of the real-time quantitative PCR instrument (CFX Opus 96, Shanghai Bio-Rad Laboratories Co., Ltd.).

[0070] 3. qPCR sensitivity detection The same target concentration gradient as in the sensitivity detection in Example 3 was set (Yeastra mitochondrial genomic DNA concentration was 2.9 × 10⁻⁶). 1 amol / L, 2.9×10 -1 amol / L, 2.9×10 -2 amol / L, 2.9×10 -3 amol / L, 2.9×10 - 5 (amol / L) qPCR detection was performed according to the established reaction system and procedure, with three replicates for each concentration gradient. The minimum detectable concentration of this method was determined by observing the standard curve. A standard curve was established with Ct value on the ordinate and target concentration on the abscissa to evaluate its linearity and assess the sensitivity of the method. The results are as follows: Figure 8 As shown, this demonstrates that the established qPCR method is effective for... Metschnikowia bicuspidata The DNA template amplification quality was good, with a clear template concentration gradient, reasonable amplification Ct values, and only one peak appearing in the melting curve, verifying the homogeneity of the amplification products and the absence of non-specific products. The calculated minimum detection concentration for the qPCR method was 0.029 amol / L.

[0071] 4. qPCR detection of actual samples Using samples identical to the actual samples in Example 5, qPCR detection was performed according to the established reaction system and procedure described above. The results are as follows: Figure 9 As shown, the amplification curve is smooth and the melting curve has a single peak, proving that the hepatopancreatic DNA and hemolymph DNA of crabs infected with *Dictamnus cusiae* in summer and winter can be successfully detected, which is consistent with the method of this invention.

[0072] 5. Comparison of detection results between qPCR and RPA-CRISPR / Cas12a methods The detection results of qPCR and the detection results of the detection method of the present invention are shown in Table 4. The detection rate and accuracy of the two methods are consistent, indicating the feasibility and reliability of the RPA-CRISPR / Cas12a method.

[0073] Table 4 Comparison of detection results between qPCR and RPA-CRISPR / Cas12a methods

[0074] The specific embodiments of the present invention have been described in detail above. It should be noted that, for those skilled in the art, some modifications or improvements can be made to the present invention without departing from the principle of the present invention and under the limitations of the embodiments. Such modifications or improvements should also be considered within the protection scope of the technical content disclosed in the present invention.

Claims

1. A kit for detecting *Saccharomyces cerevisiae* based on RPA-CRISPR / Cas12a, characterized in that, The kit includes RPA amplification reaction reagents and CRISPR / Cas12a detection reagents; the RPA amplification reaction reagents mainly include specific primer pairs for amplifying *Saccharomyces cerevisiae*; the CRISPR / Cas12a detection reagents mainly include crRNA, Cas12a protein, and fluorescent reporter probes. The nucleotide sequences of the primer pairs are shown in SEQ ID NO.1-6, and preferably, the nucleotide sequences of the primer pairs are shown in SEQ ID NO.1-2; The nucleotide sequence of the crRNA is shown in SEQ ID NO.7-9, preferably, the nucleotide sequence of the crRNA is shown in SEQ ID NO.

7.

2. The reagent kit according to claim 1, characterized in that, The structure of the fluorescent reporter probe is shown below: 5'-FAM-TTATTATT-BHQ1-3'; the CRISPR / Cas12a detection reagent also includes CRISPR buffer, which contains 10×NE Buffer. TM r2.1; The RPA amplification reaction reagents mentioned above include RPA amplification reaction dry powder.

3. The reagent kit according to claim 1, characterized in that, The RPA amplification reaction reagent also includes buffer and nuclease-free water; the kit also includes *Saccharomyces cerevisiae* genomic DNA.

4. A method for detecting *Saccharomyces cerevisiae* using the kit according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Extraction of genomic DNA from the sample: Take liver, pancreas or blood lymph samples from the animal to be tested and extract genomic DNA from the sample; (2) RPA amplification: The genomic DNA of the sample to be tested extracted in step (1) was amplified by recombinase polymerase (RPA) using specific primers for amplifying *Saccharomyces cerevisiae* to obtain RPA amplification products; (3) CRISPR / Cas12a detection: Take 0.5~5 μL of the RPA amplification product obtained in step (2) and add it to 20~50 μL of CRISPR / Cas12a reaction system. The composition of the CRISPR / Cas12a reaction system is as follows: 1~3 μL crRNA, 1~3 μL Cas12a protein, 3~5 μL fluorescent reporter probe, 1~3 μL CRISPR buffer, and the remainder is nuclease-free water. Detect the fluorescence signal every 20~40 s at 43~47℃ for a total of 50~150 times. Interpret the results according to the fluorescence intensity-time curve or observe the fluorescence color of the reaction system under blue light after the reaction for naked-eye visualization. Positive samples can be observed to have obvious green fluorescence, while negative samples have no fluorescence.

5. The method according to claim 4, characterized in that, The hepatopancreas or hemolymph sample mentioned in step (1) was obtained from a diseased Chinese mitten crab.

6. The method according to claim 4, characterized in that, Step (2) The total volume of the RPA amplification reaction system is 50 μL, including: 1 tube of reaction powder, 2 μL each of upstream and downstream primers, 25 μL of buffer A, 2.5 μL of buffer B, 5.0 μL of genomic DNA, and nuclease-free water to make up to 50 μL; The concentrations of the upstream and downstream primers are both 1~100 μmol / L, preferably 10 μmol / L.

7. The method according to claim 4, characterized in that, Step (2) The RPA amplification reaction is carried out under isothermal conditions, with a temperature of 35-41℃ and a reaction time of 5-50 min; preferably, the reaction is carried out at 39℃ for 30 min.

8. The method according to claim 4, characterized in that, The final concentration of crRNA in step (3) is 0.05~1 μmol / L, the final concentration of Cas12a protein is 0.2~4 μmol / L, and the final concentration of fluorescent reporter probe is 0.1~1 μmol / L.

9. The method according to claim 4, characterized in that, In step (3), the final molar ratio of crRNA and Cas12a protein in the system is 1:4; the Cas12a protein and crRNA are incubated at 35~38℃ for 5~30 min in advance to promote the effective formation of the ribonucleoprotein complex.

10. The use of the kit according to any one of claims 1-3 or the method according to any one of claims 4-9 in detecting whether crabs are infected with *Dictyophora inerme*.

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