One-tube one-step detection method of burkholderia pseudomallei mediated by crisper-cas12b system

Abstract

This invention discloses a CRISPR-Cas12b system-mediated one-step single-tube detection method for Burkholderia melioides, specifically comprising the following steps: (1) mixing the DNA template, MCDA primers, CRISPR-Cas12b / gRNA complex, fluorescent probe, Bst chain 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 Burkholderia melioides.

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 Burkholderia melioides mediated by the CRISPR-Cas12b system. Background Technology

[0002] Burkholderia melioides ( Burkholderia pseudomallei Melioidosis is a Gram-negative, coccobacillus-shaped bacterium that does not form capsules or spores. It has three or more flagella at one end and is the pathogen of melioidosis. Patients with immunocompromised diseases such as type 2 diabetes, or those frequently exposed to soil and water contaminated with this bacterium, are at higher risk of infection. Currently, there is no effective vaccine for prevention. If left untreated, it can lead to sepsis, with a high mortality rate and a high recurrence rate. It has been classified as a Category B bioterrorism agent and a Class I pathogen by the U.S. Centers for Disease Control and Prevention. The clinical manifestations of melioidosis are diverse, earning it the nickname "a disease that resembles a hundred kinds of diseases." Acute melioidosis has the highest mortality rate and is often associated with underlying diseases such as diabetes, cirrhosis, and chronic renal insufficiency. Subacute melioidosis is more common, with primary manifestations including lung, bone and joint, and urinary tract infections. Chronic melioidosis mainly presents with abscesses in organs such as the neck, periauricular region, parotid gland, testes, liver, and spleen. Its nonspecific clinical manifestations make early detection and diagnosis difficult, easily leading to misdiagnosis and missed opportunities for optimal treatment. Therefore, in order to provide accurate and rapid treatment to clinical patients, it is necessary to conduct environmental monitoring such as soil and water bodies, as well as epidemiological surveys of Burkholderia melioides, and to develop a time-saving, labor-saving, and highly specific detection method that can simultaneously detect and identify Burkholderia melioides.

[0003] Currently, the gold standard for detecting *Burkholderia melioides* relies on traditional enrichment culture and biochemical identification. This method takes approximately 3 to 7 days, including strain culture and subsequent biochemical identification. Traditional isolation and identification methods are time-consuming and labor-intensive, and the interpretation of biochemical results depends on subjective human judgment, leading to poor reproducibility and a high risk of misinterpretation. Therefore, in melioidosis-endemic areas, serological detection methods, such as enzyme-linked immunosorbent assay (ELISA), immunochromatography, latex agglutination, immunofluorescence assay, and indirect hemagglutination techniques, are commonly used as primary screening methods to assist in the diagnosis of melioidosis. However, in practical applications, the indirect hemagglutination test has insufficient sensitivity and specificity, and is prone to false positives due to interference from background antibodies in endemic populations; the indirect fluorescent antibody method requires a fluorescence microscope and specialized technicians, limiting its application in economically underdeveloped areas; while the detection of *Burkholderia melioides* specific antigens has a high positive predictive value, antibody reagents for *Burkholderia melioides* lipopolysaccharide and specific protein components are not yet commercially available, limiting their application in clinical diagnosis. With the rapid development of nucleic acid diagnostic technology, some PCR-based diagnostic techniques (such as conventional PCR and fluorescent PCR) have been used for the rapid detection of Burkholderia melioides. However, these methods rely on expensive equipment, require subsequent electrophoresis, costly probe synthesis, and skilled operators. This limitation restricts their application in some less developed laboratories. Currently, PCR and Real-time PCR methods for diagnosing Burkholderia melioides using these techniques have poor sensitivity and are time-consuming, making them unsuitable for rapid and emergency testing. These drawbacks limit the widespread use of these techniques; therefore, there is an urgent need to develop an easy-to-use, simple, and rapid detection technology for Burkholderia melioides.

[0004] Currently, the CRISPR-Cas system (regularly clustered short palindromic repeats / regularly clustered short palindromic repeats-associated protein system) has been successfully applied to rapid gene detection, showing great potential as a next-generation molecular diagnostic method. Considering the reliability of detection, several CRISPR-Cas nucleases (e.g., Cas9, Cas12a, Cas12b, Cas13a, and Cas13b) have been discovered for gene detection. In particular, several CRISPR-Cas nucleases, including Cas12a, Cas12b, Cas13a, and Cas13b, possess strong single-stranded nucleic acid cleavage activity. CRISPR-Cas nucleases activated by binding to cr-RNA targets can non-specifically cleave non-target ssDNA and ssRNA. Currently, studies are combining Cas12b and Cas13 with isothermal amplification technology RPA (recombinase polymerase amplification) to develop DETECTR and SHERLOCK detection technologies for rapid and sensitive nucleic acid detection.

[0005] To broaden and improve the cost-effectiveness of this detection technology in the fields of biology, medicine, and healthcare, this invention combines MCDA detection technology with the CRISPR-Cas12b system for the detection of Burkholderia melioides. This detection technology is named the MCTOS detection method (Multiple crossdisplacement amplification-CRISPR-Cas12b-based testing in one-step), aiming to establish, validate, and evaluate a rapid, sensitive, and specific MCTOS detection system for Burkholderia melioides. 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 Burkholderia melioides, thereby resolving the problems mentioned in the background section.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This application provides a CRISPR-Cas12b system-mediated one-step single-tube detection method for Burkholderia melioides, including 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; The MCDA primers described in step (1) include substitution primers, cross primers, and amplification primers. The gRNA in the CRISPR-Cas12b / gRNA complex is complementary to the target sequence. The replacement primers include F1 and F2, the cross primers include CP1 and CP2, and the amplification primers include C1, C2, D1, D2, R1, and R2. The sequence of F1 is shown in SEQ ID NO.1, the sequence of F2 is shown in SEQ ID NO.2, the sequence of CP1 is shown in SEQ ID NO.3, the sequence of CP2 is shown in SEQ ID NO.4, the sequence of C1 is shown in SEQ ID NO.5, the sequence of C2 is shown in SEQ ID NO.6, the sequence of D1 is shown in SEQ ID NO.7, the sequence of D2 is shown in SEQ ID NO.8, the sequence of R1 is shown in SEQ ID NO.9, and the sequence of R2 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, abscess drainage fluid, sputum, bronchoalveolar lavage fluid, urine, joint effusion, and wound drainage fluid.

[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 58-63℃.

[0011] Preferably, the constant temperature in step (2) is 60°C.

[0012] Preferably, the MCDA amplification product obtained by isothermal amplification in step (2) contains a PAM site, wherein the PAM site is TTA. 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 LC1 gene of Burkholderia melioides, and the sequence of the LC1 gene is shown in SEQ ID NO.14.

[0018] Preferably, the final concentration of the MCDA primers is: 0.2 μM for each replacement primer, 0.8 μM for each cross primer, and 0.4 μM for each amplification primer.

[0019] This application also provides a detection kit for Burkholderia melioides, comprising the MCDA primers, CRISPR-Cas12b / gRNA complex, fluorescent probe, Bst strand displacement DNA polymerase, and 2× DNA polymerase buffer.

[0020] The application of the detection kit involved in this application is used in the preparation of Burkholderia melioides detection products.

[0021] Compared with existing technologies, the advantages of this invention are as follows: This invention uses the LC1 gene as the target gene for detecting Burkholderia melioides, and for the first time combines multiple crossover isothermal amplification (MCDA) technology with the CRISPR-Cas12b system to establish a novel Burkholderia melioides detection method—the MCTOS method; moreover, this detection method has a sensitivity of 10 copies and a specificity of 100%. The MCTOS detection results can be directly visualized and read using a real-time fluorescence instrument or a lateral chromatography test strip. Attached Figure Description

[0022] Figure 1 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 2 Primers for screening MCDA reactions of Burkholderia melioides; among primer groups a-f, the left side represents a positive reaction, and the right side represents a blank control (1 μL ultrapure water). Figure 3 Flowchart of the MCTOS method for detecting Burkholderia melioides; Figure 4 This diagram illustrates the principle of using a lateral chromatography biosensor for the visualization detection of MCTOS products in Burkholderia melioides. (a) shows the design of the lateral chromatography biosensor (LFB); (b) shows the principle of LFB visualization detection of MCTOS amplification products in Burkholderia melioides. "+" indicates a positive result for MCTOS detection in Burkholderia melioides, with red bands appearing in the CL and TL regions; "-" indicates a negative result for MCTOS detection in Burkholderia melioides, with only a red band appearing in the CL region. Figure 5 The optimal reaction conditions for the detection method of Burkholderia melioides MCTOS were optimized; where a represents the background correction fluorescence at different reaction temperatures; b represents the background correction fluorescence with different primer systems; and the error bar represents the mean ± standard error (SEM) of three replicate experiments. Figure 6 The sensitivity of the MCTOS detection method for Burkholderia melioides is given; where a represents the results analysis of different template amounts using real-time fluorescence PCR; b represents the results analysis of different template amounts using lateral chromatography strips; 1-7 represent different template amounts of LC1 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 0 Copy / μL and blank control (NC, 1μL ultrapure water); Figure 7 The MCTOS detection method for Burkholderia melioides is specific; where a represents the real-time fluorescence result of the MCTOS detection method for Burkholderia melioides; b represents the lateral chromatography test strip result of the MCTOS detection method for Burkholderia melioides; 1-16 represent Burkholderia melioides, blank control (1 μL ultrapure water), Pseudomonas aeruginosa, Acinetobacter baumannii, Burkholderia thamnip, Burkholderia cepacia, Burkholderia gladiolus, Klebsiella pneumoniae, Escherichia coli, Candida albicans, Enterococcus faecalis, Streptococcus pneumoniae, Staphylococcus aureus, Salmonella enterica, Stenotrophomonas maltophilia, and Haemophilus influenzae, respectively. Figure 8This study presents the clinical application of the MCTOS detection method for Burkholderia melioides. Rows 1-3 show the real-time fluorescence results of the MCTOS detection method for Burkholderia melioides; rows 4-6 show the results of the lateral chromatography biosensor detection method for Burkholderia melioides. In the figure, 1A (4A) and 1B (4B) are the LC1 plasmid and blank control (1 μL ultrapure water), respectively. 1C (4C) to 2J (5J) are clinically isolated Burkholderia melioides. 2K (5K) to 2P (5P) are clinically isolated positive samples of Burkholderia melioides, namely, 1 case of abscess drainage fluid, 1 case of deep sputum, 1 case of bronchoalveolar lavage fluid, 1 case of positive blood culture bottle, 1 case of urine, and 1 case of joint effusion. 2Q (5Q) to 3V (6V) are clinically isolated negative samples of Burkholderia melioides. 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: Target gene of Burkholderia melioides was selected. The specific LC1 gene is present in *Burkholderia melioides*. This application selects the specific LC1 gene as the target gene because it has high specificity and high conservation, and can distinguish *Burkholderia melioides* from other closely related pathogens. The specific LC1 gene sequence (SEQ ID NO.14) is shown below:

[0027] Example 2: Primer, gRNA, and reporter molecule design 2.1 A plasmid specific to the LC1 gene sequence of Burkholderia melioides was constructed 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. The serially diluted LC1 plasmid template was used to establish the MCTOS amplification system and explore optimal reaction conditions.

[0028] 2.2 To verify the feasibility, sensitivity, specificity, and reliability of the MCTOS technology, based on the principle of MCDA amplification, six candidate MCDA primer sets targeting the target gene (specific LC1 gene) of Burkholderia melioides were designed using Primer3 software (version 0.4.0, (http: / / bioinfo.ut.ee / primer3-0.4.0 / ). Specific primer information is shown in Table 1 (primer set b) and Table 2 (primer sets a, c, d, e, and f). A schematic diagram of the primer design is shown below. Figure 1 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.

[0029] 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 4000 copies / μL), and ultrapure water adjusted to 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 Burkholderia melioides gene is added. 5 The template and the designed corresponding MCDA primers were copied, and the reaction was carried out at a constant temperature of 65℃ 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. The results are shown below. Figure 2 .

[0030] 2.4 Primer set b 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 1 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 b is shown in Table 1.

[0031] Table 1. Sequence information of primer set b, gRNAs, and reporter molecules used in this invention.

[0032] Table 2. Sequence information of primer sets a, c, d, e, and f.

[0033] 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.

[0034] 3.2 MCTOS Detection Method Detection Principle The MCTOS detection process is as follows: Figure 3 As shown, the process, including rapid preparation of Burkholderia melioides 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 also be used to visualize MCTOS detection results in 5 minutes, completing the process within 65 minutes (step 2). Figure 3As 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 specific LC1 target gene in Burkholderia melioides DNA template, realizing single-tube one-step detection of the LC1 target gene.

[0035] 3.3 Detection Principle of Lateral Chromatography Test Strips like Figure 4 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 4 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 4 As shown, the interpretation of 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 LC1 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 test strip is invalid.

[0036] 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, primers targeting the LC1 plasmid template and the optimal MCDA primers listed in Table 1 were added, with a template size of 1×10⁻⁶. 5 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 (58-63℃), 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 5 In section a, considering amplification efficiency, 60℃ is recommended as the optimal reaction temperature, meaning the fluorescence signal peaks early (14 minutes), and the highest fluorescence signal is second only to 59℃. Subsequent validation in this invention selected 60℃ as the isothermal condition for the MCTOS reaction.

[0037] 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: 1.1 μL (0.8 μM for each cross primer, 0.4 μM for each amplification primer, and 0.2 μM for each substitution primer), 1.5 μL (1.09 μM for each cross primer, 0.55 μM for each amplification primer, and 0.27 μM for each substitution primer), 2.0 μL (1.45 μM for each cross primer, 0.73 μM for each amplification primer, and 0.36 μM for each substitution primer), 2.2 μL (1.6 μM for each cross primer, 0.8 μM for each amplification primer, and 0.4 μM for each substitution primer), and 2.4 μL (1.75 μM for each cross primer, 0.87 μM for each amplification primer, and 0.44 μM for each substitution primer). Primers targeting the LC1 plasmid template and the MCDA primers described in Table 1 were added. The template was 1 × 10⁻⁶. 5 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 60℃, and the results were detected using a real-time quantitative PCR instrument. Fluorescence curves for different primer volumes were obtained (see [link]). Figure 5(b) Considering both amplification efficiency and signal intensity, a primer volume of 1.1 μL is recommended as the optimal amount, exhibiting an early fluorescence signal peak time (approximately 14 minutes), a large curve rise slope, and stable fluorescence values ​​during the plateau phase. Therefore, 1.1 μL was selected as the optimal primer volume for MCDA amplification in subsequent verifications of this invention.

[0038] Example 5: Sensitivity Test of MCTOS Detection Method 5.1 To further verify the sensitivity of MCTOS technology in detecting Burkholderia melioides, MCTOS amplification was analyzed using real-time quantitative PCR. Serially diluted LC1 plasmid template (1×10⁻⁶) was 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 6 (a) The lowest detection limit of MCTOS technology is 1×10⁻⁶. 1 The number of copies, with a mass concentration of approximately 3.60 × 10⁻⁶, was [amount missing]. -11 μg. A positive amplification curve was observed. When the amount of genomic template in the reaction system was reduced to 1×10 μg. 1 If the following copy does not produce a positive amplification curve, it indicates a negative result.

[0039] 5.2 Using serially diluted LC1 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 6 (b) The lowest detection limit of MCTOS is 1×10⁻⁶. 1 Copying, a red line appears in the TL region on the lateral chromatography strip. When the amount of genomic template in the reaction system is reduced to 1×10⁻⁶. 1When the number of copies is below a certain threshold, the lateral chromatography strip shows a red line only in the CL region, indicating a negative result. The sensitivity of the lateral chromatography strip and the real-time quantitative PCR instrument for detecting Burkholderia melioides using MCTOS was consistent, with a LoD of 10 copies.

[0040] Example 6: Specificity Test of MCTOS Detection Method 6.1 Nucleic acids of common bacterial pathogens (Pseudomonas aeruginosa) Pseudomonas aeruginosa Acinetobacter baumannii ( Acinetobacter baumannii Burkholderia tsukia ( ), Burkholderia thailandensis Burkholderia cepacia (), Burkholderia cepacia ), Burkholderia gladioli ( Burkholderia gladioli ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), Escherichia coli ( Escherichia coli Candida albicans ( Candida albicans ), Enterococcus faecalis ( Enterococcus faecalis Streptococcus pneumoniae () Streptococcus pneumoniae Staphylococcus aureus ( Staphylococcus aureus ), enteric Salmonella ( Salmonella enterica Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia Haemophilus influenzae ( ) Haemophilus influenzae To evaluate the specificity of the MCTOS technique, 1 μL of ultrapure water was used as a blank control (NC) when testing with lateral chromatography test strips. The above-mentioned bacterial pathogen nucleic acids were isolated by the inventors from standard strains purchased from commercial institutions.

[0041] 6.2 The results showed that the positive control *Burkholderia melioides* was positive, while the other templates were negative, with no cross-reaction. The MCTOS detection method can accurately identify *Burkholderia melioides*. (See attached results). Figure 7 .

[0042] Example 7: Feasibility Test of Clinical Application of MCTOS Detection Method 7.1 Clinical samples consisted of 30 strains of Burkholderia melioides obtained through isolation and culture, 6 positive samples of abscess drainage, deep sputum, bronchoalveolar lavage fluid, blood culture bottle, urine, and joint effusion, as well as 28 negative samples of sputum, swabs, and wound drainage. MCTOS amplification reaction was performed for detection.

[0043] 7.2 The specificity of the above-mentioned Burkholderia melioides and samples detected by MCTOS was 100% (28 / 28), and the positive detection rates of real-time fluorescence and lateral chromatography test strips were both 97.2% (35 / 36). See the results below. Figure 8The MCTOS technique established in this invention showed high consistency with the isolation and culture methods, with a Kappa value of 0.844 (Table 3). These preliminary results indicate that the MCTOS technique has high sensitivity and specificity for the diagnosis of Burkholderia melioides infection.

[0044] Table 3 Comparison of MCTOS detection method and isolation and culture method for detecting clinical Burkholderia melioides.

[0045] 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 Burkholderia melioides detection kit, 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 substitution primers, cross primers, and amplification primers, and the gRNA in the CRISPR-Cas12b / gRNA complex is complementary to the target sequence. The replacement primers include F1 and F2, the cross primers include CP1 and CP2, and the amplification primers include C1, C2, D1, D2, R1, and R2. The sequence of F1 is shown in SEQ ID NO.1, the sequence of F2 is shown in SEQ ID NO.2, the sequence of CP1 is shown in SEQ ID NO.3, the sequence of CP2 is shown in SEQ ID NO.4, the sequence of C1 is shown in SEQ ID NO.5, the sequence of C2 is shown in SEQ ID NO.6, the sequence of D1 is shown in SEQ ID NO.7, the sequence of D2 is shown in SEQ ID NO.8, the sequence of R1 is shown in SEQ ID NO.9, and the sequence of R2 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.

2. A method of using the Burkholderia melioides detection kit according to claim 1, 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 58-63℃.

4. The method of use according to claim 3, characterized in that, The constant temperature mentioned in step (2) is 60°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 TTA.

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.

13.

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 LC1 gene of Burkholderia melioides, and the sequence of the LC1 gene is shown in SEQ ID NO.

14.

9. The method of use according to claim 2, characterized in that, The final concentrations of the MCDA primers are: 0.2 μM for each replacement primer, 0.8 μM for each cross primer, and 0.4 μM for each amplification primer.

10. The use of the Burkholderia melioides detection kit according to claim 1 in the preparation of Burkholderia melioides detection products.

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