CRISPR-cas12b system-mediated single-tube one-step detection method for shewanella algae

Abstract

This invention discloses a CRISPR-Cas12b system-mediated one-step single-tube detection method for *Shewanella algae*, specifically comprising the following steps: (1) mixing the DNA template, MCDA primers, CRISPR-Cas12b / gRNA complex, fluorescent probe, Bst strand displacement DNA polymerase, and 2× DNA polymerase buffer of the sample to be tested to obtain a reaction system; (2) performing MCDA-mediated isothermal amplification and CRISPR-Cas12b-mediated trans-cutting reaction on the reaction system obtained in step (1) at a constant temperature to obtain a mixed MCTOS product; (3) detecting the fluorescence signal or using a lateral chromatography test strip to detect the mixed MCTOS product after the reaction is completed. The detection method of this invention has the advantages of high sensitivity, strong specificity, and simple operation, and is suitable for large-scale application in the detection of *Shewanella algae*.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, and in particular to a single-tube one-step detection method for Shewanella algae mediated by the CRISPR-Cas12b system. Background Technology

[0002] Shewanella algae ( Shewanella algae Shewanella algae is a Gram-negative bacillus widely distributed in seawater, estuaries, sediments, and seafood. It is halophilic, growing at salinities of 20-50 ppt. This bacterium is an opportunistic pathogen that can cause various infections in humans and aquatic animals. It has a wide host range, infecting not only farmed aquatic animals (such as the tongue sole) but also frequently causing opportunistic infections in humans, especially those with weakened immune systems, liver disease, malignant tumors, or those who have recently undergone invasive procedures (such as ERCP or PTCD). Shewanella algae can cause sepsis, skin and soft tissue infections, abdominal and hepatobiliary system infections, respiratory tract infections, ear infections, eye infections, bone infections, meningitis, and endocarditis, among other diseases, and has been listed as an important zoonotic pathogen.

[0003] Currently, the detection of *Shewanella algae* mainly relies on traditional enrichment culture and biochemical identification methods. This method takes approximately 3 to 7 days, produces H2S but does not ferment sugars, is protease-positive, and is negative for amylase / lipase / β-galactosidase. Traditional isolation and identification methods are time-consuming and labor-intensive, and the interpretation of biochemical results depends on human subjective judgment, leading to poor repeatability and a high risk of misidentification. Furthermore, some automated identification systems (such as API 20E and VITEK) may misidentify *Shewanella algae* as *Shewanella putrefactive* because their databases do not include *Shewanella algae*. Shewanella putrefaciens In molecular biology detection, 16S rRNA gene or... gyrB Genes were used as targets for PCR detection. However, the 16S rRNA gene was... Shewanella Highly conserved within the genus, with interspecific similarity often >99%, especially in *Shewanella algae*, *Shewanella putrefactive*, and *Shewanella hafni*. Shewanella hafniensis The ability to distinguish closely related species is limited, which can easily lead to false negatives or inaccurate species-level identification. gyrB While gene sequencing offers higher resolution than 16S rRNA, strains with less than 10% sequence difference in the gyrB gene cannot be classified as the same species without supporting DNA-DNA hybridization data. Therefore, current methods based on 16S rRNA or... gyrB Gene detection methods still have limitations in identifying algal Shewanella. Research on rapid and accurate molecular diagnostic techniques for algal Shewanella is still lacking, and there is a shortage of diagnostically specific target genes for algal Shewanella.

[0004] In recent years, the CRISPR-Cas system has been successfully applied to rapid gene detection, showing great potential as a next-generation molecular diagnostic method. The principle behind CRISPR / Cas systems for pathogen detection is based on the side-branch cleavage activity of Cas proteins (such as Cas12a, Cas12b, and Cas13a). Cas12b, as a member of the CRISPR-Cas nuclease family, possesses strong single-stranded nucleic acid cleavage activity. When crRNA binds to and is activated by target DNA, it can non-specifically cleave non-target ssDNA. Combining Cas12b with isothermal amplification techniques (such as MCDA, RPA, and LAMP) can develop DETECTR-type detection technologies, enabling rapid and sensitive nucleic acid detection. Compared to PCR technology, this type of detection method has similar or higher sensitivity and does not require expensive equipment, making it suitable for rapid on-site detection.

[0005] Therefore, finding a rapid, highly sensitive, and highly specific single-tube one-step detection method for Shewanella algae has become a research hotspot. Summary of the Invention

[0006] To address the aforementioned deficiencies in existing technologies, this invention proposes a CRISPR-Cas12b system-mediated one-step single-tube detection method for Shewanella algae, thereby resolving the problems mentioned in the background section.

[0007] This invention aims to establish a detection method for screening highly conserved and specific target genes. Based on a pan-genome analysis of 296 whole genomes of *Shewanella algae* and other species in the genus, the invention seeks to identify conserved gene targets specific to *Shewanella algae*. Furthermore, it combines multiple crossdisplacement amplification (MCDA) technology with the CRISPR-Cas12b system to establish an MCTOS detection method (Multiple crossdisplacement amplification-CRISPR-Cas12b-based testing in one step) for the identified conserved gene targets of *Shewanella algae*. To achieve the above objectives, the present invention provides the following technical solution: This invention provides a CRISPR-Cas12b system-mediated one-step single-tube detection method for Shewanella algae, comprising the following steps: (1) Mix the DNA template, MCDA primer, CRISPR-Cas12b / gRNA complex, fluorescent probe, Bst strand displacement DNA polymerase, and 2× DNA polymerase buffer of the sample to be tested to obtain the reaction system; (2) At a constant temperature, the reaction system obtained in step (1) was subjected to MCDA-mediated isothermal amplification and CRISPR-Cas12b-mediated trans cleavage reaction to obtain MCTOS mixed products. (3) After the reaction is completed, the fluorescence signal value generated by the reverse cleavage reaction is detected by a fluorescence instrument or the MCTOS mixed product is detected by a lateral chromatography test strip; The MCDA primers described in step (1) include cross primers, amplification primers, and substitution primers. The gRNA in the CRISPR-Cas12b / gRNA complex is complementary to the target sequence. The cross primers include CP1 and CP2, the amplification primers include C1, C2, D1, D2, R1 and R2, and the substitution primers include F1 and F2; The sequence of CP1 is shown in SEQ ID NO.1, the sequence of CP2 is shown in SEQ ID NO.2, the sequence of C1 is shown in SEQ ID NO.3, the sequence of C2 is shown in SEQ ID NO.4, the sequence of D1 is shown in SEQ ID NO.5, the sequence of D2 is shown in SEQ ID NO.6, the sequence of R1 is shown in SEQ ID NO.7, the sequence of R2 is shown in SEQ ID NO.8, the sequence of F1 is shown in SEQ ID NO.9, and the sequence of F2 is shown in SEQ ID NO.10. The sequence of the gRNA in the CRISPR-Cas12b / gRNA complex is shown in SEQ ID NO. 11.

[0008] Preferably, the sample to be tested in this invention is a clinical sample. The clinical sample includes, but is not limited to, blood, wound secretions, eye secretions, and sputum.

[0009] Preferably, the preparation method of the CRISPR-Cas12b / gRNA complex in this invention is as follows: gRNA and Cas12b are mixed in 1×Reaction Buffer and pre-incubated at 37°C for 10 min to form the CRISPR-Cas12b / gRNA complex. The prepared CRISPR-Cas12b / gRNA complex should be used immediately or stored at 4°C for no more than 12 h.

[0010] Preferably, the constant temperature in step (2) is 59-62℃.

[0011] Preferably, the constant temperature in step (2) is 61°C.

[0012] Preferably, the MCDA amplification product obtained by isothermal amplification in step (2) contains a PAM site, wherein the PAM site is TTTC. The PAM site is a basic condition for the CRISPR-Cas12b system to perform its cleavage function. If the target sequence does not have a PAM site, even if the target sequence perfectly matches the gRNA sequence, the CRISPR-Cas12b protein will not cleave the sequence, nor will it cleave the surrounding single-stranded DNA.

[0013] Preferably, the fluorescent probe is a single-stranded DNA reporter molecule (ssDNA); when fluorescence detection is used, the single-stranded DNA reporter molecule is a fluorescent reporter molecule with the sequence shown in SEQ ID NO.12; when lateral chromatography test strip is used for detection, the single-stranded DNA reporter molecule is a lateral chromatography reporter molecule shown in SEQ ID NO.13.

[0014] Preferably, the 5′ end of the fluorescent reporter molecule is modified with a fluorescent reporter group, and the 3′ end is modified with a fluorescent quencher group; the 5′ end of the lateral chromatography reporter molecule is modified with a fluorescent reporter group, and the 3′ end is modified with a biotin group.

[0015] Preferably, the fluorescent reporter group includes any one of FAM, HEX, JOE, TET, ROX or TAM; the fluorescent quencher group includes any one of BHQ1, BHQ2 or MGB.

[0016] Preferably, the fluorescent reporter group of the present invention is FAM, and the fluorescent quencher group is BHQ1.

[0017] Preferably, the target gene that isothermally amplified in step (2) is the SA gene of Shewanella algae, and the sequence of the SA gene is shown in SEQ ID NO.14.

[0018] Preferably, the final concentration of the MCDA primers is: 1.08 μM for each cross primer, 0.56 μM for each amplification primer, and 0.24 μM for each substitution primer.

[0019] The present invention also provides a detection kit for Shewanella algae, comprising the above-mentioned MCDA primers, CRISPR-Cas12b / gRNA complex, fluorescent probe, Bst strand displacement DNA polymerase, and 2× DNA polymerase buffer.

[0020] This invention also relates to the application of the detection kit in the preparation of Shewanella algae detection products.

[0021] Compared with the prior art, the beneficial effects of the present invention are: the existing technology often uses the conserved region of 16S rRNA or gyrBThe gene is a target gene for the detection of *Shewanella algae*. However, the applicant discovered a conserved region of 16S rRNA and gyrB Genes are highly homologous among closely related species in the genus *Shewanella*, making them lack specificity as target genes. This invention, for the first time, selects the SA gene as the target gene for detecting algal *Shewanella*, and establishes a novel MCTOS detection method by combining multiple crossover isothermal amplification (MCDA) technology with the CRISPR-Cas12b system. This detection method has a sensitivity of 10 copies / μL and a specificity of 100%. MCTOS detection results can be directly visualized using a real-time fluorescence instrument or lateral chromatography test strips. Attached Figure Description

[0022] Figure 1 This is a sequence alignment diagram of the conserved region of the SA target gene in the seaweed Shewanella. Figure 2 The sequence and position of the primers and gRNA used in this invention are shown; the left and right arrows indicate the forward and complementary sequences used, respectively. Figure 3 Results of primer screening for MCDA reaction in Shewanella algae; Figure 4 Flowchart for the MCTOS method of detecting Shewanella algae; Figure 5 The diagram shows the principle of using a lateral chromatography biosensor for the visualization detection of MCTOS products of Shewanella algae; (a) is the design diagram of the lateral chromatography biosensor (LFB); (b) is the principle diagram of the visualization detection of MCTOS amplification products of Shewanella algae using LFB; in (c), "+" indicates a positive result for MCTOS detection of Shewanella algae, with red bands in the CL and TL regions; "-" indicates a negative result for MCTOS detection of Shewanella algae, with only a red band in the CL region. Figure 6 The reaction conditions for target gene detection were optimized; (a) shows the background correction fluorescence at different reaction temperatures; (b) shows the background correction fluorescence with different primer systems; and the error bars represent the mean ± standard error (SEM) of three replicate experiments. Figure 7 The sensitivity of the MCTOS detection method for *Shewanella algae* is shown in (a) for real-time fluorescence PCR analysis with different template amounts; (b) for lateral chromatography strip analysis with different template amounts, where 1-7 represent different template amounts of plasmid, respectively 1×10⁻⁶. 5 copies / μL, 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 copies / μL, 1×10 0Copy / μL and blank control (1μL ultrapure water); Figure 8 The specificity of the MCTOS detection method for *Shewanella algae* is shown in (a), where (a) is the real-time fluorescence result of the MCTOS detection method for *Shewanella algae*; (b) is the result of the lateral chromatography test strip for the MCTOS detection method for *Shewanella algae*, and 1-16 represent *Shewanella algae*, 1 μL ultrapure water, *Escherichia coli*, *Enterococcus faecalis*, *Pseudomonas aeruginosa*, *Acinetobacter baumannii*, *Klebsiella pneumoniae*, *Streptococcus pneumoniae*, *Staphylococcus aureus*, *Salmonella enterica*, *Burkholderia melioides*, *Enterobacter cloacae*, *Candida albicans*, *Stenotrophomonas maltophilia*, *Haemophilus influenzae*, and *Moraxella catarrhalis*, respectively. Figure 9 The results show the clinical application of the MCTOS detection method for *Shewanella algae*. Rows 1-4 show the real-time fluorescence results of the MCTOS detection method; rows 5-8 show the lateral chromatography biosensor results. In the figure, 1A (5A) to 2Y (5Y) represent clinically isolated *Shewanella algae*, 3A (7A) to 3T (7T) represent positive samples isolated from clinical patients and sea turtles, and 3U (7U) to 4V (8V) represent negative samples isolated from clinical *Shewanella algae*. P indicates positive, and N indicates negative. Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical content of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0024] The reagents involved in this invention were sourced from the following sources: the deoxyribonucleic acid isothermal amplification kit was purchased from Beijing Haitai Zhengyuan Biotechnology Co., Ltd., the DNA extraction kit was purchased from Tiangen Biotech Co., Ltd., Cas12b was purchased from Hainan Fenwei Biotechnology Co., Ltd. (manufacturer is Meg Biotechnology Co., Ltd., i.e., AapCas12b protein), and the lateral chromatography test strips were purchased from EZassay Ltd.

[0025] The main instruments used in this invention were: a MA-6000 real-time quantitative PCR instrument purchased from Yarui Biotechnology Co., Ltd.; and an LA-320C constant-temperature real-time turbidimeter (Eiken Chemical Co., Ltd., Japan) purchased from Eiken Corporation, Japan.

[0026] Example 1: Screening of target genes in *Shewanella algae* 1.1 This invention conducts an in-depth study of the genomes of 193 *Shewanella algae* strains with <200 contigs in the NCBI database and the genomes of 103 other species within the genus (strain information is shown in Table 1). Prokka was used to annotate the genomes of the 193 *Shewanella algae* strains and the 103 other species within the genus, and the annotation results were used for Roary analysis to resolve pan-genome and gene presence / deletion. Based on this, core genes unique to *Shewanella algae* were screened, i.e., genes present in all *Shewanella algae* strains but absent in the genomes of the other 103 species within the genus. To verify the specificity of the screened genes, the unique core gene sequences of *Shewanella algae* were compared and analyzed using the NCBI BLAST online tool.

[0027] Table 1. Genomic information of 193 *Shiva* strains and 103 other species within the genus from this study.

[0028] 1.2 Results: NCBI BLAST comparison revealed that early LAMP detection methods targeting the conserved region of the 16S rRNA gene lacked target specificity. Shewanella carassii , Shewanella amazonensis and Shewanella The coverage and similarity of new species such as *S. sp.* were 100%, making accurate identification of *Shewanella algae* impossible (Table 2). A total of 47,741 genes were identified in the pan-genome of 193 *Shewanella algae* strains. Among them, 557 core genes were present in all *Shewanella algae* genomes. After comparison with the genomes of 103 other species within the genus, 43 of these core genes were found to be unique to *Shewanella algae*, representing *Shewanella algae*-specific core genes. Among these specific core genes, a 1302 bp gene showed good conservation among the 193 *Shewanella algae* strains. This gene is located at positions 201534–202835 in the genome of the type strain CECT5071, encoding a protein family of type II and type III secretion systems. A highly conserved 284 bp region within this gene was selected as a target. Figure 1 The gene was named the SA target gene, and its specific sequence is shown in SEQ ID NO.14. It was used for the synthesis of primers for subsequent detection of Shewanella algae.

[0029] Table 2. Comparison results of conserved regions of 16S rRNA gene used in existing LAMP detection methods.

[0030] The SA target gene sequence (SEQ ID NO.14) is shown below:

[0031] Example 2: Primer, gRNA, and reporter molecule design 2.1 The SA target gene is present in *Shewanella algae*, exhibiting good conservation and specificity, which can distinguish *Shewanella algae* from other closely related pathogens. A plasmid was constructed targeting the SA gene sequence of *Shewanella algae* as a standard template and serially diluted to 1×10⁻⁶. 5 copies / μL, 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 copies / μL, 1×10 0 Copy / μL, aliquot and store at -20℃ for later use. Serially diluted plasmid templates were used to establish the MCTOS amplification system and explore optimal reaction conditions.

[0032] 2.2 To verify the feasibility, sensitivity, specificity, and reliability of the MCTOS technology, based on the principle of MCDA amplification, four candidate MCDA primer sets for the SA target gene of *Shewanella algae* were designed using Primer3 software (version 0.4.0, http: / / bioinfo.ut.ee / primer3-0.4.0 / ). Specific primer information is shown in Table 3 (primer set 1) and Table 4 (primer sets 2, 3, and 4). A schematic diagram of the primer design is shown below. Figure 2 Each MCDA primer set targets 10 different regions of the target sequence, including two substitution primers (F1 and F2), two cross primers (CP1 and CP2), and six amplification primers (C1, D1, R1, C2, D2, and R2). Secondary structure and primer dimer analysis were performed using OligoAnalyzer software (version 3.1, Integrated DNA Technologies, Coralville, IA), and the obtained specific primers were sequence aligned in the NCBI database (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to rule out non-specific matches between primers and sequences from other species.

[0033] 2.3 Standard MCDA reaction system: 12.5 μL of 2× DNA polymerase buffer, 8 U of Bst chain displacement DNA polymerase, 1.6 μM each of cross-primers CP1 and CP2, 0.8 μM each of amplification primers C1, C2, D1, D2, R1, and R2, 0.4 μM each of displacement primers F1 and F2, 1 μL of plasmid as template (final template concentration 0.4 copies / μL), and ultrapure water adjusted to a 25 μL amplification system. The primer stock solution concentration is 100 μM. That is, under standard MCDA reaction system conditions, 1×10⁻⁶ plasmid targeting the gene of *Shewanella algae* is added. 1 The template and the designed corresponding MCDA primers were copied, and the reaction was carried out at a constant temperature of 63℃ for 60 minutes. The primers were screened using a real-time turbidimeter (LA-320C), and different dynamic curves were obtained, indicating that the MCDA reaction targeting the target was successfully established. An equal volume of ultrapure water was used as a blank control, and the results are shown below. Figure 3 .

[0034] 2.4 Primer set 1 was found to have the highest amplification efficiency, and was subsequently selected to establish the MCTOS system. Furthermore, gRNA was designed based on the MCTOS principle; for details, see [link to relevant documentation]. Figure 2 Fluorescent reporter molecules and biosensor-specific lateral chromatography reporter molecules were designed for reporting detection results. All primers and reporter molecules were synthesized and purified by Sangon Biotech Ltd. gRNA was synthesized and purified by Genscript Biotech Ltd. Specific MCDA primer, gRNA, and reporter molecule sequence information for primer set 1 is shown in Table 3.

[0035] Table 3. Sequence information of primer set 1, gRNAs, and reporter molecules used in this invention.

[0036] Table 4. Sequence information of primer set 2, primer set 3, and primer set 4

[0037] Example 3 MCTOS Detection 3.1 The specific testing includes the following steps: (1) Add 12.5 μL of 2× DNA polymerase buffer, 8 U of Bst chain replacement DNA polymerase, 1.6 μM each of cross primers CP1 and CP2, 0.8 μM each of amplification primers C1, C2, D1, D2, R1 and R2, 0.4 μM each of replacement primers F1 and F2, 4.0 μL of CRISPR-Cas12b / gRNA complex, fluorescent probe, 1 μL of DNA template of the sample to be tested, add water to 25 μL, mix well to obtain the reaction system; the fluorescent probe is 1.6 μM fluorescent reporter molecule or 0.04 μM lateral chromatography reporter molecule; the preparation method of the CRISPR-Cas12b / gRNA complex includes the following steps: mix 150 nM gRNA and 100 nM Cas12b in 1×Reaction Buffer, pre-incubate at 37 °C for 10 min to form CRISPR-Cas12b / gRNA complex, and then add it to the above reaction system; (2) At a constant temperature, the reaction system obtained in step (1) was subjected to MCDA-mediated isothermal amplification and CRISPR-Cas12b-mediated trans cleavage reaction to obtain MCTOS mixed products. (3) After the reaction is complete, if the fluorescent probe is a 1.6 μM fluorescent reporter molecule, the fluorescence signal is detected by a real-time quantitative PCR instrument; if the fluorescent probe is a 0.04 μM lateral chromatography reporter molecule, the MCTOS mixed product is detected by a lateral chromatography test strip.

[0038] 3.2 MCTOS Detection Method Detection Principle The MCTOS detection process is as follows: Figure 4 As shown, the process, including rapid preparation of Shewanella algae DNA template in 15 minutes (step 1) and MCTOS reaction in 45 minutes, with results detected by real-time fluorescence analysis, can be completed within 60 minutes. Specifically, lateral chromatography strips can be used to visualize MCTOS detection results in 5 minutes, completing the process within 65 minutes (step 2). Figure 4As shown in step 2, the MCDA amplification system contains 10 primers, including two cross primers (CP1 and CP2), two substitution primers (F1 and F2), and six amplification primers (C1, C2, D1, D2, R1, and R2), which can recognize 10 different regions of the target sequence and perform exponential amplification within 45 minutes at a constant temperature. The PAM sequence of the MCDA amplification product and the target sequence can be recognized by the corresponding CRISPR-Cas12b / gRNA system. The CRISPR-Cas12b-gRNA complex binds to the MCDA product complementary to the guide strand, thereby activating the cis-cleavage activity of the Cas12b enzyme in the CRISPR-Cas12b / gRNA system to cleave the target, and activating its trans-cleavage activity to cleave any ss-DNA. Therefore, this method can simultaneously perform MCDA reaction amplification and CRISPR-Cas12b system cleavage amplification signal for the SA target gene in the Shewanella algae DNA template, realizing single-tube one-step detection of the SA target gene.

[0039] 3.3 Detection Principle of Lateral Chromatography Test Strips like Figure 5 As shown in (a), the lateral chromatography test strip comprises a sample pad, a gold-labeled pad, a nitrocellulose membrane, and an absorbent pad. Two capture reagents, streptoavidin and goat anti-mouse secondary antibody, are coated onto the nitrocellulose membrane. Within the reaction region of the nitrocellulose membrane, there are a control line (CL) for binding to streptoavidin and a test line (TL) for binding to goat anti-mouse secondary antibody. A gold nanoparticle-conjugated FAM monoclonal antibody is coated onto the gold-labeled pad. Figure 5 As shown in (b), the detection principle of the lateral chromatography test strip is as follows: Take 3 μL of the MCTOS mixture and dilute it with 47 μL of ultrapure water. Insert the lateral chromatography test strip with the sample pad facing down and place it at room temperature. Read the results after 5-10 minutes (step 1). The liquid level should not exceed the max line. The MCTOS mixture moves from bottom to top through capillary action (from the sample pad towards the absorbent pad), thereby rehydrating the FAM monoclonal antibody conjugated to the gold nanoparticles in the gold-labeled pad. When the MCTOS mixture reaches the gold-labeled pad, the FAM-labeled end of the ssDNA reacts with the FAM monoclonal antibody conjugated to the gold nanoparticles (step 2). As the product continues to move, the ssDNA with the biotin-labeled end binds to streptavidin in the control line region, fixing the ssDNA in the control line region (step 3). If the ssDNA is cleaved by activated CRISPR-Cas12b, the biotin-labeled end and the FAM-labeled end of the ssDNA separate. Therefore, the FAM / gold nanoparticle-conjugated FAM monoclonal antibody complex is captured by goat anti-mouse secondary antibody in the detection line region (step 3). As the product accumulates in the detection line region, a colorimetric reaction occurs through the gold nanoparticles at the other end, thereby enabling visual detection of the product. Figure 5 As shown in (c), the interpretation of the lateral chromatography test strip results is as follows: Positive result: When a red band appears in the TL region, and a red band is present or absent in the CL region (no CL band when ssDNA is completely cleaved by CRISPR-Cas 12b; a weak CL band appears when ssDNA is not completely cleaved by CRISPR-Cas 12b), the detection of the SA target is positive. Negative result: A red band appears only in the CL region, indicating a negative result and no positive product. When no red band appears on the lateral chromatography test strip, the lateral chromatography test strip is invalid.

[0040] Example 4: Optimization of reaction conditions for MCTOS detection method 4.1 Optimization of reaction temperature Under the standard MCTOS reaction system conditions described in Example 2, section 2.3, a target plasmid template and the MCDA primers listed in Table 3 were added, with a template concentration of 1 × 10⁻⁶. 4 Copy the template and replace it with an equal volume of ultrapure water as a blank control (NC). The reaction was carried out under isothermal conditions (59-62℃), and the results were detected using a real-time quantitative PCR instrument. Different fluorescence curves were obtained at different temperatures, as shown in the figure. Figure 6 (a) Considering amplification efficiency, 61°C is recommended as the optimal reaction temperature, i.e., the fluorescence signal peaks early (17 minutes), and the highest fluorescence signal is second only to 59°C. In the subsequent validation of this invention, 61°C was chosen as the isothermal condition for the MCTOS reaction.

[0041] 4.2 Primer volume optimization In the standard MCTOS reaction system conditions described in Example 2.3, the total primer volumes were set as follows: 1.0 μL (0.18 μL each of cross primers, 0.09 μL each of amplification primers, and 0.05 μL each of substitution primers), 1.5 μL (0.27 μL each of cross primers, 0.14 μL each of amplification primers, and 0.06 μL each of substitution primers), 2.0 μL (0.36 μL each of cross primers, 0.18 μL each of amplification primers, and 0.1 μL each of substitution primers), 2.2 μL (0.4 μL each of cross primers, 0.2 μL each of amplification primers, and 0.1 μL each of substitution primers), and 2.4 μL (0.44 μL each of cross primers, 0.22 μL each of amplification primers, and 0.1 μL each of substitution primers). The target plasmid template and the MCDA primers described in Table 3 were added, with a template concentration of 1 × 10⁻⁶. 4 Copy the template and replace it with an equal volume of ultrapure water as a blank control (NC). Amplification was performed at the optimal reaction temperature of 61℃, and the results were detected using a real-time quantitative PCR instrument. Fluorescence curves for different primer volumes were obtained (see [link]). Figure 6(b) Considering both amplification efficiency and signal intensity, a primer volume of 1.5 μL is recommended as the optimal amount, exhibiting an early fluorescence signal peak time (approximately 17 minutes), a large curve rise slope, and stable fluorescence values ​​during the plateau phase. Therefore, 1.5 μL was selected as the optimal primer volume for MCDA amplification in subsequent verifications of this invention.

[0042] Example 5: Sensitivity Test of MCTOS Detection Method 5.1 To further verify the sensitivity of MCTOS technology in detecting Shewanella algae, MCTOS amplification was analyzed using real-time quantitative PCR. Serially diluted SA plasmid templates (1×10⁻⁶) were used. 5 copies / μL, 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 copies / μL, 1×10 0 MCTOS amplification reactions were performed using 1 μL of ultrapure water (NC, blank control) and 1 μL of template (copy / μL). Each template concentration was tested at least three times. Results were interpreted using a real-time quantitative PCR instrument. The results showed... Figure 7 (a) of the results: The lowest detection limit of MCTOS technology is 1 × 10⁻⁶. 1 The mass concentration is approximately 4.40 × 10⁻⁶ copies / μL. -11 μg / μL. A positive amplification curve was observed. When the amount of genomic template in the reaction system was reduced to 1×10 μg / μL. 1 If the following copy does not produce a positive amplification curve, it indicates a negative result.

[0043] 5.2 Using serially diluted SA plasmid templates (1×10⁻⁶) 5 copies / μL, 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 copies / μL, 1×10 0 After performing MCTOS amplification with 1 μL of ultrapure water (NC, blank control) and 1 μL of water (copy / μL), the results were detected by lateral chromatography strips. Figure 7 (b) of the table: The lowest detection limit of MCTOS is 1 × 10⁻⁶. 1 Copy / μL, a red line appears in the TL region on the lateral chromatography strip ( Figure 7 (b)). When the amount of genomic template in the reaction system is reduced to 1×10 1When the number of copies is below a certain threshold, the lateral chromatography strip only shows a red line in the CL region, indicating a negative result. The sensitivity of the lateral chromatography strip and the real-time quantitative PCR instrument for detecting Shewanella algae using MCTOS was consistent, with a LoD of 10 copies / μL.

[0044] Example 6: Specificity Test of MCTOS Detection Method 6.1 Nucleic acid of common bacterial pathogens (Escherichia coli (Escherichia coli (E. ... Escherichia coli ), Enterococcus faecalis ( Enterococcus faecalis ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa Acinetobacter baumannii ( Acinetobacter baumannii ), Klebsiella pneumoniae ( Klebsiella pneumoniae Streptococcus pneumoniae () Streptococcus pneumoniae Staphylococcus aureus ( Staphylococcus aureus ), enteric Salmonella ( Salmonella enterica Burkholderia melioides ( ), Burkholderia pseudomallei Enterobacter cloacae () Enterobacter cloacae ), Candida albicans ( Candida albicans Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia Haemophilus influenzae ( ) Haemophilus influenzae The specificity of the MCTOS technique was evaluated using *Moraxella catarrhalis* as a template, with 1 μL of ultrapure water used as a blank control (NC) when testing with lateral chromatography strips. The nucleic acids of the aforementioned bacterial pathogens were isolated by the inventors from standard strains purchased from commercial institutions.

[0045] 6.2 The results showed that the positive control *Shewanella algae* was positive, while the other templates were negative, indicating no cross-reaction. The MCTOS detection method can accurately identify *Shewanella algae*. (See [link to MCTOS]) Figure 8 .

[0046] Example 7: Feasibility Test of Clinical Application of MCTOS Detection Method 7.1 Clinical samples consisted of 50 strains of Shewanella algae obtained through isolation and culture, 7 swabs of wound secretions and deep sputum from clinical patients with positive isolation and culture, 13 swabs of wounds from rescued sea turtles with positive isolation and culture, and 27 sputum and wound swabs with negative isolation and culture. MCTOS amplification reaction was performed for detection.

[0047] 7.2 The specificity of the above-mentioned *Shewanella algae* and samples detected by MCTOS was 100% (27 / 27), the positive detection rate of real-time fluorescence was 98.6% (69 / 70), and the positive detection rate of lateral chromatography strips was 95.7% (67 / 70). (See [link to MCTOS]). Figure 9The MCTOS technique established in this invention showed high consistency with the isolation and culture methods, with Kappa values ​​ranging from 0.926 to 0.975 (Table 5). These preliminary results indicate that the MCTOS technique has high sensitivity and specificity for the diagnosis of Shewanella algae infection.

[0048] Table 5 Comparison of MCTOS detection methods and isolation / culture methods for detecting clinical Shewanella algae

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A detection kit for Shewanella algae, characterized in that, Includes MCDA primers, CRISPR-Cas12b / gRNA complex, fluorescent probe, Bst strand displacement DNA polymerase, and 2× DNA polymerase buffer; The MCDA primers include cross primers, amplification primers, and substitution primers, and the gRNA in the CRISPR-Cas12b / gRNA complex is complementary to the target sequence. The cross primers include CP1 and CP2, the amplification primers include C1, C2, D1, D2, R1 and R2, and the substitution primers include F1 and F2; The sequence of CP1 is shown in SEQ ID NO.1, the sequence of CP2 is shown in SEQ ID NO.2, the sequence of C1 is shown in SEQ ID NO.3, the sequence of C2 is shown in SEQ ID NO.4, the sequence of D1 is shown in SEQ ID NO.5, the sequence of D2 is shown in SEQ ID NO.6, the sequence of R1 is shown in SEQ ID NO.7, the sequence of R2 is shown in SEQ ID NO.8, the sequence of F1 is shown in SEQ ID NO.9, and the sequence of F2 is shown in SEQ ID NO.

10. The sequence of the gRNA in the CRISPR-Cas12b / gRNA complex is shown in SEQ ID NO.

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2. A method of using the Shewanella algae detection kit of claim 1 for non-diagnostic purposes, characterized in that, Includes the following steps: (1) Mix the DNA template, MCDA primer, CRISPR-Cas12b / gRNA complex, fluorescent probe, Bst strand displacement DNA polymerase, and 2× DNA polymerase buffer of the sample to be tested to obtain the reaction system; (2) At a constant temperature, the reaction system obtained in step (1) was subjected to MCDA-mediated isothermal amplification and CRISPR-Cas12b-mediated trans cleavage reaction to obtain MCTOS mixed products. (3) After the reaction is complete, detect the fluorescence signal or use a lateral chromatography test strip to detect the MCTOS mixed product.

3. The method of use according to claim 2, characterized in that, The constant temperature mentioned in step (2) is 59-62℃.

4. The method of use according to claim 3, characterized in that, The constant temperature mentioned in step (2) is 61°C.

5. The method of use according to claim 2, characterized in that, The MCDA amplification product obtained by isothermal amplification in step (2) contains a PAM site, which is TTTC.

6. The method of use according to claim 2, characterized in that, The fluorescent probe is a single-stranded DNA reporter molecule; when fluorescence detection is used, the single-stranded DNA reporter molecule is the fluorescent reporter molecule with the sequence shown in SEQ ID NO.12; when lateral chromatography test strip is used for detection, the single-stranded DNA reporter molecule is the lateral chromatography reporter molecule shown in SEQ ID NO.

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7. The method of use according to claim 6, characterized in that, The fluorescent reporter molecule is modified with a fluorescent reporter group at its 5′ end and a fluorescent quencher group at its 3′ end; the lateral chromatography reporter molecule is modified with a fluorescent reporter group at its 5′ end and a biotin group at its 3′ end.

8. The method of use according to claim 2, characterized in that, The target gene that is amplified isothermally in step (2) is the SA gene of Shewanella algae, and the sequence of the SA gene is shown in SEQ ID NO.

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9. The method of use according to claim 2, characterized in that, The final concentrations of the MCDA primers are: 1.08 μM for each cross primer, 0.56 μM for each amplification primer, and 0.24 μM for each substitution primer.

10. The application of the Shewanella algae detection kit according to claim 1 in the preparation of Shewanella algae detection products.

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