A detection system for mycoplasma pneumoniae

Abstract

The application discloses a detection system of mycoplasma pneumoniae. According to the P1 gene of mycoplasma pneumoniae, RPA primers and crRNA required by a CRISPR system with LbCas12a as a core enzyme are designed and screened, an RPA system and a CRISPR system are established, the influence of each component of the CRISPR-LbCas12a system on the RPA system reaction and the influence of each component of the RPA system on each component of the CRISPR-LbCas12a system are tested, components are optimized according to the test results, and an RPA-LbCas12a integrated system capable of detecting a conserved fragment on the P1 gene of mycoplasma pneumoniae is established. The RPA-LbCas12a integrated system established in the application has high sensitivity, high detection efficiency, reduces nucleic acid pollution, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a detection system for Mycoplasma pneumoniae. Background Technology

[0002] Mycoplasma pneumoniae, a Gram-negative bacterium, can survive both intracellularly and extracellularly. It primarily infects children and the elderly, and without effective treatment, it can lead to extrapulmonary complications. Therefore, rapid and sensitive detection systems for Mycoplasma pneumoniae have always been a focus of attention. Currently, rapid detection methods for Mycoplasma pneumoniae mainly include PCR and colloidal gold-based detection kits. The former offers fast amplification, high sensitivity, and high detection efficiency, but requires temperature-controlled equipment and is cumbersome to operate, limiting its use to laboratory settings. The latter is more common, simple to operate, and can be used outside the laboratory for self-testing, but it has a high detection threshold and low sensitivity. Previous infection-related IgG can influence the results, leading to false negatives and false positives, making it suitable only for simple initial screening of infected patients.

[0003] Therefore, establishing an integrated system for detecting Mycoplasma pneumoniae that is easy to detect and free from nucleic acid contamination is of great importance to this field. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a detection system for Mycoplasma pneumoniae.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides a Mycoplasma pneumoniae detection system, the detection system comprising RPA primers, the RPA primers comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in any one of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, and the nucleotide sequence of the reverse primer being shown in any one of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7.

[0007] Furthermore, the forward and reverse primers of the RPA primers are shown in SEQ ID NO:6 and SEQ ID NO:3, respectively.

[0008] Furthermore, the detection system also includes crRNA, the nucleotide sequence of which is shown in SEQ ID NO:8 or SEQ ID NO:9.

[0009] Furthermore, the nucleotide sequence of the crRNA is shown in SEQ ID NO:8.

[0010] Furthermore, the detection system also includes an ssDNA probe, the nucleotide sequence of which is shown in any one of SEQ ID NO:10-14.

[0011] Furthermore, the nucleotide sequence of the ssDNA probe is shown in SEQ ID NO:12 or SEQ ID NO:14.

[0012] Furthermore, the ssDNA probe is linked to a fluorescent group and / or a quencher group.

[0013] Furthermore, the fluorescent group is selected from FAM.

[0014] Furthermore, the quenching group is selected from BHQ1.

[0015] Furthermore, the detection system also includes RPA-related reagents and CRISPR-related reagents.

[0016] Furthermore, the RPA-related reagents include reaction buffer, E-mix, MgOAc, and dNTPs.

[0017] Furthermore, the reaction buffer includes a Reaction Buffer and a Core Reaction Buffer.

[0018] Furthermore, the dNTPs include dATP, dGTP, dCTP, and dTTP.

[0019] Furthermore, the CRISPR-related reagents include LbCas12a and restriction endonuclease buffer.

[0020] Furthermore, the restriction endonuclease buffer is selected from NEBuffer.

[0021] Furthermore, the NEBuffer is selected from NEBuffer r2.1.

[0022] Furthermore, the detection system also includes DEPC-treated water.

[0023] Furthermore, the ratio of LbCas12a to crRNA was 1:(1-2).

[0024] Furthermore, the final concentration of LbCas12a was 50-250 nM.

[0025] Furthermore, the final concentration of Reaction Buffer, NEBuffer r2.1, and Core Reaction Buffer is 1×.

[0026] Furthermore, the final concentration of MgOAc was 14-18 mM.

[0027] Furthermore, the final concentrations of dATP, dGTP, dCTP, and dTTP were 0.64 mM.

[0028] Furthermore, the final concentration of the primers was 480 nM.

[0029] Furthermore, the final concentration of the ssDNA probe is 250 nM.

[0030] A second aspect of the present invention provides a Mycoplasma pneumoniae detection product, the product comprising the detection system described in the first aspect of the present invention.

[0031] Furthermore, the products include reagent kits, test strips, and chips.

[0032] The third aspect of the present invention provides the application of the detection system described in the first aspect of the present invention in the detection of Mycoplasma pneumoniae or in the preparation of detection products for Mycoplasma pneumoniae.

[0033] Furthermore, the detection of Mycoplasma pneumoniae is for non-diagnostic purposes.

[0034] A fourth aspect of the present invention provides a method for detecting Mycoplasma pneumoniae, the method comprising using the detection system described in the first aspect of the present invention or the product described in the second aspect of the present invention for detection.

[0035] Furthermore, the temperature range for testing was 37℃-42℃.

[0036] Furthermore, the number of templates detected was over 700 copies.

[0037] Furthermore, the method described is not for diagnostic purposes.

[0038] Advantages and beneficial effects of the present invention:

[0039] This application designs and screens RPA primers and crRNAs required for a CRISPR system with LbCas12a as the core enzyme, based on the Mycoplasma pneumoniae P1 gene. An RPA and CRISPR system are then established. The effects of each component of the CRISPR-LbCas12a system on the RPA system reaction and the effects of each component of the RPA system on each component of the CRISPR-LbCas12a system are tested. Based on the test results, the components are optimized to establish an integrated RPA-LbCas12a system capable of detecting conserved fragments on the Mycoplasma pneumoniae P1 gene. The integrated RPA-LbCas12a system established in this application has high sensitivity, high detection efficiency, and reduces nucleic acid contamination, showing broad application prospects. Attached Figure Description

[0040] Figure 1This is an image showing the nucleic acid electrophoresis results of RPA amplification of Mp-P1 template at room temperature for 30 minutes using different primers;

[0041] Figure 2 This is a diagram of the CRISPR-LbCas12a system constructed using different crRNAs at room temperature for detecting target fragments.

[0042] Figure 3 The fluorescence signal curves are for negative results of the two-step RPA-CRISPR amplification detection method at 37℃ (A, left figure) or room temperature (B, right figure).

[0043] Figure 4 The images show CRISPR test strips with negative results from the two-step RPA-CRISPR amplification detection method at 37℃ (A, left) or room temperature (B, right).

[0044] Figure 5 The graphs show the sensitivity test results of the two-step RPA-CRISPR amplification detection method at room temperature. The fluorescence signal at the reaction endpoint was observed by a chemiluminescent gel imaging analyzer (A), and the changes in fluorescence signal during the reaction process were monitored by a multi-functional microplate reader (B). (The negative group used an equal volume of DEPC-treated water instead of template.)

[0045] Figure 6 This is a CRISPR test strip image used for sensitivity testing of the two-step RPA-CRISPR amplification detection method at room temperature.

[0046] Figure 7 This is a fluorescence signal image obtained from the sensitivity test of the two-step RPA-CRISPR amplification detection method at 37℃.

[0047] Figure 8 The images show the application of primers, templates, probes, and crRNA designed in the Mycoplasma pneumoniae RPA-LbCas12a two-step method to the AIOD-CRISPR system at 37℃ using a multifunctional microplate reader (A), the detection of nucleic acid amplification using agarose gel electrophoresis of the reaction solution (B), and a chemiluminescent gel imaging analyzer (C).

[0048] Figure 9 The image shows the results of preparing a 1% CRISPR gel using primers, probes, crRNA, and Mp-P1 template in the RPA-LbCas12a two-step method. The total system was reacted at 37°C for 1 hour, and then CRISPR test strips were diluted with DEPC-treated water at different ratios.

[0049] Figure 10 This is a nucleic acid electrophoresis result image after RPA reaction for 30 min at room temperature (400 nM-4 μM LbCas12a and 1×NE Buffer were added to the RPA system before the reaction).TM r2.1, 4 μM ssDNA-TA-BHQ, negative control group used an equal volume of DEPC-treated water instead of template);

[0050] Figure 11 This is the result of nucleic acid electrophoresis after RPA reaction for 30 min at room temperature (400 nM-4 μM of Mp-P1-crRNA1 was added to the RPA system before the reaction, and the negative control group used an equal volume of DEPC-treated water instead of template).

[0051] Figure 12 This is the nucleic acid electrophoresis result after RPA reaction at room temperature for 30 min (400 nM-4 μM ssDNA-TA-BHQ and 0.5× NE Buffer were added to the RPA system before the reaction). TM r2.1 and 1×NEBuffer TM r2.1, the negative control group used an equal volume of DEPC-treated water instead of template);

[0052] Figure 13 The images show the results of nucleic acid electrophoresis after adding ssDNA probes of different concentrations and sequences to a 20 μL RPA system and reacting at room temperature for 30 min (the positive control group used an equal volume of DEPC-treated water instead of template in the RPA system).

[0053] Figure 14 It uses 1×NEBuffer TM r2.1, 0.5×NEBuffer TM The changes in fluorescence signal were observed using a multifunctional microplate reader (A) or an image was obtained using a chemiluminescent gel imaging analyzer (B) for the detection of the CRISPR system of r2.1.

[0054] Figure 15 It is to completely replace NEBuffer with DEPC for water treatment. TM Imaging results of the CRISPR system reaction in r2.1;

[0055] Figure 16 The fluorescence curves show the effects of each RPA component on the CRISPR system using ssDNA-TA5C-BHQ(A) or ssDNA-5C-BHQ(B).

[0056] Figure 17 The effect of each RPA component on the CRISPR system using ssDNA-TA5C-BHQ(A) or ssDNA-5C-BHQ(B) is shown in the blue light image of the system at the reaction endpoint using a chemiluminescent gel imaging analyzer.

[0057] Figure 18The fluorescence signal of the integrated CRISPR gel electrophoresis system obtained by adding ssDNA-TA-BHQ(A), ssDNA-TA5C-BHQ(B) and ssDNA-5C-BHQ(C) changes over time at 42°C.

[0058] Figure 19 The image shows the blue light imaging results of the CRISPR gel-gel integrated system obtained by adding ssDNA-TA-BHQ(A), ssDNA-TA5C-BHQ(B) and ssDNA-5C-BHQ(C) after reacting at 42℃ for 1 hour.

[0059] Figure 20 The image shows a CRISPR gel-based integrated system obtained using 250 nM ssDNA-TA5C-BHQ(A) and ssDNA-5C-BHQ(B). The probe was added to the RPA reaction solution before the reaction started, and the image was obtained by blue light imaging after reacting at 42°C for 1 hour.

[0060] Figure 21 The integrated CRISPR gel electrophoresis system was prepared using 250 nM (A), 50 nM (B), and 10 nM (C) LbCas12a to detect the Mp-P1 template. The reaction was carried out at 42°C for 1 hour, and the integrated system image analysis at the reaction endpoint was performed using blue light.

[0061] Figure 22 The fluorescence curves of Mp-P1 template at 42℃ over time are monitored using an integrated CRISPR gel assay system with LbCas12a and Mp-P1-crRNA1 in a ratio of 1:1 (A) and 1:2 (B).

[0062] Figure 23 The images show the blue light imaging results after reacting at 42°C for 1 hour using an integrated CRISPR gel assay system with LbCas12a and Mp-P1-crRNA1 in a ratio of 1:1 (A) and 1:2 (B) to detect Mp-P1 template.

[0063] Figure 24 The image shows the blue light imaging results after 1 hour of reaction at room temperature using an integrated CRISPR gel electrophoresis system with concentrations of 250 nM (A) and 50 nM (B) to detect the Mp-P1 template.

[0064] Figure 25 This is a partial nucleic acid electrophoresis result of RPA after reacting at room temperature for 1 hour in an integrated CRISPR gel electrophoresis system.

[0065] Figure 26The RPA-LbCas12a integrated system was tested by changing the concentration and ratio of LbCas12a and Mp-P1-crRNA1 at 39℃, and the image was taken under blue light after 1 hour of reaction.

[0066] Figure 27 These are blue light images of the system after reacting at room temperature for 1 h (A) and 2 h (B) with different ratios of LbCas12a and Mp-P1-crRNA1.

[0067] Figure 28 The image shows the blue light imaging results after RPA-LbCas12a solution was stored at 4°C in the dark for 72 hours, the template was added, and the reaction was carried out at room temperature for 1 hour (the negative control group used an equal volume of DEPC-treated water instead of the template).

[0068] Figure 29 This is a blue light image of the sensitivity test of the RPA-LbCas12a integrated system at room temperature. Detailed Implementation

[0069] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0070] This invention provides a Mycoplasma pneumoniae detection system, the detection system comprising RPA primers, the RPA primers comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in any one of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, and the nucleotide sequence of the reverse primer being shown in any one of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7.

[0071] In some embodiments, the primers disclosed in this application are not limited to the exact sequences shown. As those skilled in the art will recognize, the sequences can be modified if necessary without significantly affecting the primers' ability to function. For example, the primers of this application may also be sequences having at least 90% identity with any of SEQ ID NO:2-7, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, such as 100% sequence identity. Those skilled in the art will recognize that these provided sequence identity ranges are merely for guidance purposes; primers outside these ranges may also be used.

[0072] The detection system also includes crRNA, the nucleotide sequence of which is shown in SEQ ID NO:8 or SEQ ID NO:9.

[0073] The detection system also includes an ssDNA probe, the nucleotide sequence of which is shown in any one of SEQ ID NO:10-14.

[0074] In some implementations, ssDNA (and / or crRNA) contains one or more modifications, such as a base modification, a backbone modification, a glycosyl modification, etc., to provide new or enhanced functions to the nucleic acid (e.g., improved stability).

[0075] In some embodiments, the ssDNA probe is linked to a fluorescent group and / or a quencher group.

[0076] In some embodiments, the reporter group includes FAM, FITC, HEX, VIC, TET, JOE, ROX, CY3, CY5, or CY7.

[0077] In a specific embodiment, the fluorescent group is selected from FAM.

[0078] In some embodiments, the quenching group includes BHQ1, BHQ2, BHQ3, or DABCYL.

[0079] In a specific embodiment, the quenching group is selected from BHQ1.

[0080] This invention provides a Mycoplasma pneumoniae detection product, which includes the above-mentioned detection system.

[0081] The products include reagent kits, test strips, and chips.

[0082] In some embodiments, a kit refers to any delivery system for delivering materials, including kits for research and clinical applications. Suitable containers in a kit typically include at least one vial, test tube, long-necked flask, PET bottle, syringe, or other container in which one component can be placed, and preferably, can be suitably aliquoted. When more than one component is present in the kit, the kit will also typically include a second, third, or other additional container in which the additional components are placed separately. However, different combinations of components may be contained in a single vial. The kits of this application will also typically include a container for containing the reactants, sealed for commercial sale. Such a container may include injection-molded or blow-molded plastic containers in which the desired vials can be held.

[0083] In some implementations, the chip can be understood as any three-dimensional shape. The material used as the chip substrate can possess desired properties, including optical properties such as flatness, transparency, well-defined optical absorption spectrum, minimal autofluorescence, and high reflectivity; and chemical properties such as surface reactivity that allows for covalent bonding. Non-limiting examples of suitable substrates include inorganic materials such as silicon, glass and ceramics (e.g., low-temperature cofired ceramics (LTCC)), polymer substrates, composite materials, and paper. Polymers may include elastomers such as polydimethylsiloxane (PDMS; dimethicone), polyesters (e.g., thermoset polyester, TPE); thermoplastic polymers such as polystyrene (PS), polycarbonate (PC), polymethyl methacrylate (PMMA) and polyethylene glycol diacrylate (PEGDA), perfluorinated compounds / polymers (e.g., perfluoroalkoxy (Teflon PFA), fluorinated ethylene propylene (Teflon FEP) and polyfluoropolyether diol methacrylate (PFPE-DMA)) and polyurethane (PU); and thermosets, as well as polyimides and acrylics. Paper, flexible cellulose-based materials, composite materials such as amorphous materials, cyclic olefin polymers (COP), and polymers based on cyclic olefin monomers and ethylene, such as cyclic olefin copolymers (COC).

[0084] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0085] Example

[0086] 1. Experimental materials

[0087] Experimental equipment and apparatus: pipettes, conical flasks, shakers, STRIP TUBES (Axygen), BSA224S-CW electronic balance (Sartorius), CU-420 electric thermostatic water bath, nucleic acid electrophoresis apparatus and electrophoresis tank (Takara), NanoPhotometer N50, MicroStar 17 mini benchtop high-speed refrigerated centrifuge (VWR), ChemiDoc TM MPImaging System (Bio-Rad), Tecan Multifunctional Microplate Reader, QuantStudio TM 15. Quantitative Real-Time PCR System (Thermo Fisher Scientific), Chemiluminescent Gel Imaging Analyzer (Bio-Rad)

[0088] Experimental reagents: ultrapure water, DEPC-treated water (Beyotime, R0021), TwistAmp@Basic (Merray, TABAS03KIT), LbCas12a (new England biolabs, M0653T, including NEBuffer r2.1), Cas12 / 13 dedicated nucleic acid detection test strips (Leshan, TEC-LS05). Liquid Basic Kit (Merck, TALQBAS01), 100mM dNTP Set (ThermoFisher Scientific, 10297018), diethyl pyrocarbonate (Merck, V900882), 1×TAE buffer, 10×DNA loading buffer, 100bp-2000bp DNA marker, gel red nucleic acid gel strain, MLtraPure TM Invitrogen TM (16500100), Reaction Buffer, Core Reaction Buffer, E-mix, and MgOAc all originate from Liquid Basic Kit (Merray, TALQBAS01).

[0089] 2. Experimental Methods

[0090] 2.1 Preparations before the experiment

[0091] The nucleic acid sequence of the M129 Mycoplasma pneumoniae P1 gene can be found in the NCBI database. Conserved regions were obtained by BLAST alignment of the P1 gene. The target fragment was carried using the pUC-57 plasmid (see Table 1 for details). Primers were provided by Yizhi Biotechnology, designed to be 28-32 nt in length with a CG content of 40%-60%, and primer specificity was verified using NCBI's Primer-BLAST. 6-FAM and BHQ1 were modified at the 5' and 3' ends of the ssDNA, respectively, to serve as probes for the CRISPR reaction. A classic 14 nt TATA-rich ssDNA probe was designed. The crRNA specifically recognizing the conserved sequence on the P1 gene was designed using the CAS-Designer online software. All target fragments, primers, probes, and crRNA were synthesized by General Biotechnology and shipped as finished products in dry powder form. The following preparations are required before starting the experiment:

[0092] (1) The obtained dry powder plasmids, primers, probes, and crRNA were centrifuged at 3000g for 3 min. Then, in a windless environment, the tube cap was carefully opened and a certain amount of DEPC-treated water was added. After mixing by shaking, the mixture was centrifuged at 3000g for 3 min. After aliquoting, the mixture was stored at -20℃ for later use.

[0093] (2) The concentration of nucleic acid (ng / μL) after mixing and centrifugation was detected using NanoPhotometer N50, and the concentration of plasmid (copy / μL) was converted using SnapGene.

[0094] (3) Clean the surface of the clean bench with alcohol and RNase / DNase detergent in sequence, wipe it dry, turn on the exhaust fan and blow for 3 minutes before starting the experiment.

[0095] 2.2 Construction of a two-step RPA-LbCas12a system for detecting Mycoplasma pneumoniae

[0096] The two-step method requires establishing an RPA system and a CRISPR system for sequential reaction. 29.5 μL Mix rehydration buffer, 2.4 μL of 10 μM forward primer (F), 2.4 μL of 10 μM reverse primer (R), and 11.2 μL of DEPC-treated water. Add this mixture to a PCR tube containing the lyophilized RPA enzyme mixture and mix thoroughly by pipetting until the solution is clear. Add 2 μL of template to the tube, followed by 2.5 μL of 280 mM MgOAc. Vortex to construct a total RPA system of 50 μL. Amplify the reaction at room temperature for 30 min or at 37°C for 20 min. Timing begins from the last addition of MgOAc. For the negative control, use an equal volume of DEPC-treated water instead of template.

[0097] To prepare a 20 μL CRISPR system, add 5 μL of 1 μM LbCas12a, 1 μL of 10 μM crRNA, 9.5 μL of LEPC-treated water, and 2 μL of 10×NEBuffer in sequence. TM r2.1, 0.5μL 10μM ssDNA-TA-BHQ or ssDNA-TA-biotin, 2μL RPA amplification reaction solution, react at 37℃ for 10min or at room temperature for 20min. Results are detected using a multi-functional microplate reader (Tecan) or CRISPR test strips, depending on the type of probe used. Before using CRISPR test strips, DEPC-treated water must be added to mix the CRISPR system to a volume of at least 50μL.

[0098] 2.3 RPA primer screening and crRNA screening

[0099] The forward / reverse primers for amplifying conserved fragments on the P1 gene are shown in Table 2. The corresponding templates were amplified using the 50 μL RPA reaction system above, and the reaction was carried out at room temperature for 30 min. The results were observed by agarose gel electrophoresis.

[0100] Table 3 shows the crRNAs designed for P1 gene detection and for testing mutations. After selecting suitable forward / reverse primers, they were used to amplify the template. RPA amplification reaction solution was added to the 20 μL CRISPR system above to compare and select suitable crRNAs. Fluorescence signal changes under different crRNAs were observed using a Tecan microplate reader. Imaging results under different crRNAs at the reaction endpoint were observed using a chemiluminescent gel imaging analyzer. The program was set to Dylight488 blotting, 532 / 28, Blue Epi.

[0101] 2.4 Agarose gel electrophoresis was used to detect the RPA reaction effect and the amplification effect in the integrated system.

[0102] Weigh 2g of agarose using an electronic balance and add it to a conical flask containing 100ml of 1×TAE buffer, being careful not to add it to the flask walls. Microwave for 3 minutes to obtain a 2% agarose solution. After slightly cooling to approximately 60℃, add 5μL of [unspecified ingredient]. Mix the nucleic acid gel staining agent thoroughly and pour it into the gel mold. Insert the gel comb at the appropriate position and remove air bubbles with a pipette. After complete solidification, remove the gel comb and place the 2% agarose gel into the electrophoresis tank, ensuring the gel is completely submerged in 1×TAE buffer (Tris-acetic acid-EDTA). Store for later use. Mix the RPA amplification reaction solution or RPA-LbCas12a integrated system reaction solution and 10×DNA Loading Buffer at a volume ratio of 9:1 and add the mixture to the wells on the gel. Simultaneously, add 10 μL of a 100bp-2000bp DNA marker to the outermost well. Set the voltage to 135V and react for 30 min. After the reaction, image the gel using a chemiluminescent gel imaging analyzer with the program set to GelRed gel, 590 / 110, UV Trans.

[0103] 2.5 Multifunctional microplate reader for detecting fluorescence signals

[0104] To test fluorescence signal changes in the reaction using a multi-functional microplate reader (Tecan), the FAM-BHQ1 probe and a 96-well plate are required. Set the detection program for FAM fluorescence detection to detect the signal once per minute. Before the fluorescence detection program, allow 5 seconds of shaking to mix. Add at least 50 μL of reaction mixture to each well on the 96-well plate used for the test sample group, positive control group, or negative control group to ensure complete coverage of the bottom of the well. When detecting fluorescence signals at room temperature, no temperature setting is required. Add the corresponding RPA amplification reaction solution, template, or DEPC-treated water to the wells containing pre-added CRISPR reaction mixture (excluding RPA amplification product) and start shaking to detect the fluorescence signal. When detecting fluorescence signals at 37°C, a preheating program is required. Preheat the CRISPR reaction mixture (excluding RPA amplification reaction solution) to 37°C and maintain this temperature. After reaching 37°C, add the RPA amplification reaction solution to the corresponding wells, click start, and the pre-set program will mix and detect fluorescence signal changes in the reaction system at 37°C.

[0105] 2.6 Real-time fluorescence PCR system for detecting fluorescence signals

[0106] QuantStudio TM The Thermo Fisher Scientific 15-voltaic quantitative PCR system heats up rapidly, completing the reaction in under 1 minute, so preheating is unnecessary. The reaction system should use the FAM-BHQ1 probe. PCR tubes containing the RPA-LbCas12a conjugate reaction system (excluding the template) should be placed directly into the QuantStudio. TM15. A real-time PCR system was used. After adding the corresponding template, detection began immediately. A pre-heating program (Hold Stage) was pre-set, with the temperature rising to 42℃ within 25 seconds. An amplification program (PCR Stage) was then set, detecting the FAM fluorescence signal once per minute, maintaining the temperature at 42℃, for 60 cycles. This method was primarily used in testing the integrated CRISPR gel electrophoresis system, so the total volume was set to 30 μL as used in gel electrophoresis.

[0107] 2.7 Test on the effects of various components on the reaction environment of RPA and LbCas12a in the integrated system

[0108] A 20 μL RPA system consisted of 10 μL 2×Reaction Buffer, 2 μL 10×E-mix, 1 μL 20×CoreReaction Buffer, 1 μL 280 mM MgOAc, 0.16 μL dATP, 0.16 μL dGTP, 0.16 μL dCTP, 0.16 μL dTTP, 0.96 μL primer F, 0.96 μL primer R, 0.5 μL template, and 2.94 μL DEPC-treated water. The positive control group consisted of a standard 20 μL RPA system, with the sample groups containing different concentrations of the ssDNA probe, LbCas12a, crRNA, and NE Buffer used in the RPA-LbCas12a two-step method. TM r2.1. After reacting at room temperature for 30 min, the amplification results were tested by agarose gel electrophoresis.

[0109] The concentrations of each component in a 50 μL CRISPR system were the same as in the RPA-LbCas12a two-step method. An RPA reaction solution incubated at room temperature for 30 min was added as a positive control, and an RPA reaction solution containing DEPC-treated water (as a template) incubated at room temperature for 30 min was added as a negative control. Sample groups were treated with 1×Reaction Buffer, 1×E-mix, 1×Core Reaction Buffer, and 14 mM MgOAc, respectively, and reacted at room temperature. Fluorescence signal changes were observed using a Tecan microplate reader.

[0110] 2.8 Construction of an integrated CRISPR gel electrophoresis system for detecting Mycoplasma pneumoniae

[0111] First, prepare the CRISPR gel: Add 100 μL of ultrapure water to the DURAN reagent bottle, then add 100 μL of DEPC stock solution in a fume hood. Tighten the cap and invert the bottle several times, then place it in the fume hood overnight to inactivate the RNase. After overnight, autoclave the bottle to remove DEPC. Weigh an appropriate amount of agarose into the reagent bottle, heat it, and prepare a 2% agarose solution. Store the solution in a 50°C water bath for later use. Add 0.5 μL of 1 μM LbCas12a, 1 μL of 1 μM crRNA, and 1 μL of 10×NEBuffer to the PCR tube. TM Add 0.5 μL of ssDNA probe and 2 μL of DEPC-treated water, mix well, and let stand for 10 min. Add 5 μL of 2% agarose solution to the tube to prepare a CRISPR gel containing all CRISPR components and 1% agarose, and store at 4℃ for later use. The CRISPR gels used in this experiment were stored at 4℃ for 12-24 hours after preparation, and used after preparing the corresponding RPA system. The conditions described here are those after component optimization.

[0112] A 20 μL RPA system was prepared with a volume ratio of 2:1 to the CRISPR gel. The concentrations of each component in the RPA system were consistent with those used in the 20 μL RPA system for the influence of each component in the integrated system. First, all components except the template were mixed and added to the CRISPR gel, followed by 0.5 μL of template. The reaction was carried out at 42°C or room temperature using QuantStudio. TM 15. A real-time PCR system (Thermo Fisher Scientific) measures fluorescence once per minute, or a chemiluminescent gel imaging analyzer (Bio-Rad) is used to observe the results. The program is set to Dylight 488, 532 / 28, Blue Epi.

[0113] 2.9 Constructing an integrated system for Mycoplasma pneumoniae amplification and detection with optimized components

[0114] Based on the results of component influence testing and literature review, a 20 μL integrated system was constructed. The optimized system contained 1×Reaction Buffer, 1×E-mix, 1×Core Reaction Buffer, 14 mM MgOAc, 50 nM LbCas12a, 50 nM crRNA, and 0.5×NEBuffer. TMThe reagents included 480 nM primer F, 480 nM primer R, 0.64 mM dATP, 0.64 mM dGTP, 0.64 mM dCTP, 0.64 mM dTTP, and 0.5 μL of 10 μM ssDNA probe. The total volume after adding all reagents was 19.5 μL. Then, 0.5 μL of template was added, and the amplification and detection reaction was performed at 39°C or room temperature (around 25-27°C). The concentration of some components was adjusted or substituted depending on their impact on the test. The test results were visualized using a chemiluminescent gel imaging analyzer (Bio-Rad), with the program set to Dylight 488 blotting, 532 / 28, and BlueEpi.

[0115] Table 1. Nucleic acid amplification templates for Mycoplasma pneumoniae

[0116]

[0117] Table 2 Primers designed in this study (* indicates the primers used after screening)

[0118]

[0119] Table 3. crRNAs designed in this experiment

[0120]

[0121] 3. Experimental Results

[0122] 3.1 RPA primer selection for amplifying conserved fragments of the Mycoplasma pneumoniae P1 gene

[0123] Add 3×10 to a 50 μL RPA system 5 Copy the Mp-P1 template and react at room temperature for 30 min. The gel electrophoresis results are as follows: Figure 1 As shown, the results indicate that the F3 and R1 primer set yielded the best amplification efficiency and produced fewer heterogeneous bands in the electrophoresis results compared to other primer sets. Therefore, the F3 and R1 primer set will be used in subsequent RPA-LbCas12a two-step method and integrated system construction experiments.

[0124] 3.2 crRNA screening used in the RPA-LbCas12a two-step method

[0125] Amplification was performed using the F3 and R1 primer sets at room temperature for 30 min, using a 50 μL RPA system with 3 × 10⁻⁶ primers added. 5The Mp-P1 template was copied. Two 20 μL CRISPR-LbCas12a systems were prepared, one containing P1-crRNA1 and the other containing P1-crRNA2. A TATA-rich FAM-biotin probe, ssDNA-TA-biotin, was used (see Table 4). These were added to the RPA amplification reaction solution in the same PCR tube and reacted at room temperature for 20 min. After the reaction, 30 μL of DEPC-treated water was added to a total volume of 50 μL. After mixing, the mixture was added to CRISPR test paper, and the reading was taken after 10 min. The results are shown below. Figure 2 As shown, the LbCas12a-P1-crRNA1 complex can recognize the target and cleave the probe normally, while the LbCas12a-P1-crRNA2 complex cannot. Based on the screening results, P1-crRNA1 will be used as the crRNA for the RPA-LbCas12a two-step method and the construction of an integrated amplification and detection system.

[0126] Table 4. ssDNA probes used in the classic RPA-LbCas12a two-step method.

[0127]

[0128] 3.3 Negative Control Experiment

[0129] In a 50 μL RPA system, an equal volume of DEPC-treated water was used to replace the template, with everything else remaining the same, creating a water-negative group. Based on the role of the RPA initiator MgOAc, an equal volume of DEPC-treated water was used to replace MgOAc, with everything else remaining the same, creating a -MgOAc-negative group. An equal volume of DEPC-treated water was used to replace the crRNA in the CRISPR system, with everything else remaining the same, creating a -crRNA-negative group. 3 × 10⁻⁶ DEPC-treated water was added to the normal RPA system. 5 The template was positive. Amplification reaction conditions were 37℃ for 20 min. A 20 μL CRISPR system was used to detect the target fragment at 37℃. According to literature, classic high-TA content ssDNA-TA-BHQ or ssDNA-TA-biotin probes of equal concentration (250 nM) were used. Results were observed using CRISPR test strips after 10 min of CRISPR reaction at 37℃, or using a multi-functional microplate reader (Tecan) to observe changes in fluorescence signal in the CRISPR reaction system after 10 min at 37℃. A negative control experiment was performed using the same RPA-LbCas12a amplification and detection two-step system, but the reaction temperature was changed to room temperature. Fluorescence signal changes within 1 hour were detected using a multi-functional microplate reader, and results were detected using CRISPR test strips after 20 min of reaction. Results are as follows: Figure 3 and Figure 4As shown, regardless of whether the reaction was carried out at 37℃ or room temperature, only the fluorescence signal increased in the positive group, and the detection line showed color development; the results of the other groups were negative, demonstrating the reliability of the detection results of the RPA and CRISPR systems. Furthermore, the CRISPR reaction at 37℃ reached its fluorescence signal peak within 10 minutes, while the CRISPR reaction at room temperature reached its peak at approximately 30 minutes. Subsequent two-step experiments used either 10 minutes at 37℃ or 30 minutes at room temperature to determine the reaction endpoint time.

[0130] 3.4 RPA-LbCas12a Two-Step Sensitivity Test

[0131] The Mp-P1 template was gradient diluted to 1.5 × 10⁻⁶. 2 copy / μL, 1.5×10 1 copy / μL, 1.5×10 0 copy / μL, 1.5×10 -1 2 μL of the sample was added to each of the 50 μL RPA systems in different PCR tubes, and the reaction was carried out at room temperature for 30 min. After the RPA reaction was completed, the RPA amplification reaction solutions with different template concentrations were added to a 50 μL CRISPR-LbCas12a reaction system with the same concentration and volume ratio. The changes in FAM fluorescence signal of the reaction system were detected using a 250 nM TATA-rich ssDNA-TA-BHQ probe and a multi-functional microplate reader (Tecan). The CRISPR reaction solution was then collected in PCR tubes, and the FAM fluorescence signal at the reaction endpoint was observed under blue light using a chemiluminescent gel imaging analyzer (Bio-Rad). The results are shown below. Figure 5 As shown, the lowest detection sensitivity of the RPA-LbCas12a two-step method can reach 3 copies. Instead, a 250 nM ssDNA-TA-biotin probe was used, with a CRISPR reaction volume of 20 μL. The sensitivity of the RPA-LbCas12a two-step method was tested. After reacting for 20 min, DEPC-treated water was added to dilute to 50 μL, and the results were detected using CRISPR test strips. Figure 6 As shown, bands were still observed even when the amount of template added was 3 copies, further demonstrating the reliability of the sensitivity test results. The reaction temperature was changed to 37℃, the RPA system was reacted for 20 min, and the CRISPR system for 10 min. The fluorescence signal at the reaction endpoint was observed under blue light using a chemiluminescent gel imaging analyzer (Bio-Rad). The results are as follows. Figure 7As shown, the highest fluorescence signal observed using the RPA-LbCas12a two-step method at 37℃ was observed when 3 copies of the template were added, which is the same sensitivity as the detection at room temperature. In summary, for the Mycoplasma pneumoniae RPA-LbCas12a two-step detection system constructed in this experiment, lowering the temperature from 37℃ to room temperature does not affect the negative control or the detection sensitivity. The reaction time was determined to be 20 min RPA + 10 min CRISPR at 37℃, or 30 min RPA + 20 min CRISPR at room temperature.

[0132] 3.5 Preliminary Experiment on the Construction of an Integrated System for RPA-CRISPR Amplification and Detection of Mycoplasma pneumoniae

[0133] The RNA template and reverse transcriptase were modified to a plasmid template carrying a conserved fragment of the P1 gene used in this experiment, and the primers, probes, and crRNA of the RPA-LbCas12a two-step method described above were used. The AIOD-CRISPR integrated system applied to Mycoplasma pneumoniae included 4 μM ssDNA-TA-BHQ, 1.26 μM LbCas12a-crRNA complex, 320 nM each of F3 and R1, 1×Reaction Buffer, 1×E-mix, 14 mM MgOAc, 1.2 mM dNTPs, and 1×Core Reaction Buffer. After mixing, 3×10⁻⁶ ppm of the solution was added. 5 The Mp-P1 template was copied and reacted at 37℃. Fluorescence signal changes during the reaction were monitored using a Tecan multi-mode microplate reader. The reaction solution was then collected in PCR tubes, and the fluorescence signal at the reaction endpoint was collected using a chemiluminescent gel imaging analyzer. Finally, the nucleic acid amplification results of this system were detected by agarose gel electrophoresis. The results are as follows: Figure 8 As shown, it was impossible to distinguish between negative and positive results, and the background signal was too high. The fluorescence signal of the negative result showed an abnormal increase, and the agarose gel electrophoresis did not detect the expected target fragment bands.

[0134] A 10 μL CRISPR gel containing 1% agarose, 100 nM LbCas12a-crRNA complex, 500 nM ssDNA-TA-biotin, and 1×NE Buffer was prepared for the gel electrophoresis test. TM r2.1. The gel contains 20 μL of RPA reaction system, to which 3 × 10⁻⁶ ppm was added. 5 The copied Mp-P1 template was reacted at 42°C for 1 hour, and the reaction results were detected using CRISPR test paper. Figure 9As shown, no positive bands could be observed, and due to the increased solution viscosity caused by the RPA system, the CRISPR test paper could not be completely wetted when placed directly into it. After the reaction, it was necessary to dilute it at least 10 times with DEPC-treated water before the CRISPR test paper could be used for detection.

[0135] In preliminary experiments, the effectiveness of two integrated systems already used for detecting RNA viruses in the detection of Mycoplasma pneumoniae was tested. Despite significant differences in the concentrations of some components between the two systems, neither showed any fluorescence signal in positive samples, and both exhibited an abnormal increase in fluorescence signal in negative samples. This indicates that there is a concentration-independent, but highly decisive, factor influencing the integrated reaction between the two systems. This component will be verified in the following steps. Furthermore, the concentration of RPA in the integrated system is significantly higher than that in the CRISPR test strip detection system previously used in the RPA-CRISPR two-step method. This increased solution viscosity necessitates dilution with DEPC-treated water after the reaction, which may interfere with the results. Therefore, subsequent experiments will only use fluorescent probes.

[0136] 3.6 Results of the influence of each component of the CRISPR-LbCas12a system on the RPA system

[0137] First, multiple identical 20 μL RPA amplification reaction systems were prepared, containing 1×Reaction Buffer, 1×E-mix, 1×Core Reaction Buffer, 14 mM MgOAc, 0.8 mM dATP, 0.8 mM dGTP, 0.8 mM dCTP, 0.8 mM dTTP, 480 nM F3, and 480 nM R1. A negative control group was set up using a 20 μL RPA amplification reaction system without additional components and with an equal volume of DEPC-treated water instead of template. The remaining groups each received 7 × 10⁻⁶ ppm of the buffer. 4 Copy the Mp-P1 template and add different concentrations of LbCas12a, ssDNA-TA-BHQ, and NEBuffer respectively. TM r2.1, Mp-P1-crRNA1. Since previous studies have mentioned that LbCas12a-crRNA recognizes and cis-cleaves target nucleic acids in the early stages of amplification, preventing the reaction from proceeding, it was hypothesized that LbCas12a alone might also affect the RPA amplification reaction. Therefore, the following tests were conducted with the addition of 4 μM LbCas12a, 400 nM LbCas12a, and 1×NEBuffer to a 20 μL RPA amplification reaction system. TM The results were obtained by nucleic acid electrophoresis after reacting 2.1, 4 μM ssDNA-TA-BHQ at room temperature for 30 min. Figure 10 As shown, only 1×NEBuffer TM Both the r2.1 group and the 400nM LbCas12a group were able to amplify the target fragment, but the amplification process in both groups was affected to some extent. This demonstrates that LbCas12a itself affects the RPA process, and even significantly inhibits the RPA reaction at high concentrations. In addition, the 4μM ssDNA-TA-BHQ probe also completely inhibited the amplification reaction.

[0138] Next, the effect of crRNA on the RPA amplification reaction system was tested, and an attempt was made to reduce the inhibitory effect of CRISPR components on nucleic acid amplification by lowering their concentrations. The results are as follows: Figure 11 , 12 As shown, single Mp-P1-crRNA1, regardless of concentration, has virtually no effect on the RPA amplification process, 0.5×NEBuffer TM The amplification band ratio of group r2.1 was 1×NEBuffer TM The amplified bands in group r2.1 were clearer, indicating that changing the concentration to 0.5× reduced its impact on the RPA reaction. Surprisingly, ssDNA-TA-BHQ completely inhibited the RPA reaction regardless of its concentration, demonstrating that this probe plays a decisive role in inhibiting the integration process.

[0139] In summary, ssDNA probes have the greatest impact on the RPA process. Meanwhile, due to LbCas12a and NEBuffer... TM r2.1 also had some impact on RPA, and this impact decreased as the concentration decreased. The following will test NEBuffer at low LbCas12a concentrations. TM r2.1 The effect of final concentrations from 1×, 0.5× to complete replacement of DEPC-treated water on the CRISPR reaction process.

[0140] 3.7 Optimize the components of the CRISPR system to reduce their impact on the RPA system.

[0141] The new FAM-BHQ probe was used to construct an integrated system for Mycoplasma pneumoniae amplification and detection, as detailed in Table 5. The previous 20 μL CRISPR-LbCas12a system was still used, with only the probe and its concentration changed. A 20 μL RPA system without additional components was set up as a positive control group. All systems were supplemented with 7 × 10⁻⁶ CRISPR-LbCas12a. 4 Copy the Mp-P1 template. For example... Figure 13As shown, the effects of ssDNA-TA-BHQ, ssDNA-TA5C-BHQ, ssDNA-5C-BHQ, and ssDNA-5T-BHQ on the RPA process were compared through nucleic acid electrophoresis results. Even with the addition of a low concentration of ssDNA-TA-BHQ, the target band amplified by the RPA process remained blurry. Although the nucleic acid electrophoresis bands of the RPA system with the ssDNA-5T-BHQ probe were clearer than before, they were still not as comparable to the positive control group as the bands amplified by the RPA system with the ssDNA-TA5C-BHQ or ssDNA-5C-BHQ probes. This demonstrates that in the probe design of the integrated RPA-LbCas12a amplification and detection system, the 5C design is superior to the classic TA-rich design. ssDNA-TA5C-BHQ and ssDNA-5C-BHQ have a relatively small impact on the RPA process and can be used subsequently.

[0142] Table 5 shows the optimized ssDNA probe design for constructing the RPA-LbCas12a amplification and detection integrated system.

[0143]

[0144] The previous text mentioned reducing NEBuffer TM The r2.1 concentration is beneficial for the RPA process; here, we use 1×NEBuffer. TM r2.1, 0.5×NEBuffer TM The 20 μL CRISPR-LbCas12a system of r2.1 resulted in an LbCas12a-Mp-P1-crRNA1 complex with a final concentration of 250 nM and an ssDNA-TA-BHQ concentration of 250 nM. The addition of 3 × 10⁻⁶ CRISPR-LbCas12a was detected using these two CRISPR-LbCas12a reaction systems. 2 The Mp-P1 template was reacted at room temperature for 30 min, and a 20 μL RPA amplification reaction solution was prepared. For the negative control group, an equal volume of DEPC-treated water was used instead of the template in the RPA system, and the CRISPR system used 1×NEBuffer. TM r2.1. Fluorescence signal changes during the reaction were monitored using a multi-functional microplate reader (Tecan), and the system at the reaction endpoint was imaged using blue light using a chemiluminescent gel imaging analyzer (Bio-Rad). Results are as follows: Figure 14 As shown, all indicate that NEBuffer TM Decreasing the r2.1 concentration does not affect the CRISPR reaction, proving that 0.5×NEBuffer TM r2.1 can be applied to the construction of integrated systems. Furthermore, a configuration was developed that completely replaces NEBuffer with DEPC-treated water. TMThe CRISPR reaction system of r2.1 was imaged at the reaction endpoint using a chemiluminescent gel imaging analyzer (Bio-Rad). Results are as follows: Figure 15 As shown, DEPC is used to treat water, completely replacing NEBuffer. TM Group r2.1 showed no fluorescence signal. In summary, subsequent experiments will use lower LbCas12a concentrations and 0.5×NEBuffer. TM r2.1 Test the effects of each component of RPA on the CRISPR-LbCas12a system.

[0145] 3.8 Effects of each component of the RPA system on the reaction of the CRISPR-LbCas12a system

[0146] Similar to the previous two-step RPA-LbCas12a system, a 20 μL CRISPR-LbCas12a reaction system was finally constructed, using a FAM-BHQ1 ssDNA probe concentration of 250 nM, an LbCas12a concentration of 100 nM, an Mp-P1-crRNA1 concentration of 100 nM, and NE buffer. TM For r2.1, the optimized concentration of 0.5× was used. The optimized ssDNA probes ssDNA-TA5C-BHQ and ssDNA-5C-BHQ were used. Additional components of 14 mM MgOAc, 1× Reaction Buffer, 1× E-mix, and 1× Core Reaction Buffer were added to each system to set up the sample group. The system without additional components was considered the positive or negative control group. 700 copies of Mp-P1 template or an equal volume of DEPC-treated water were added to a 20 μL RPA system, mixed, and reacted at room temperature for 30 min. After the reaction, the reaction solution containing the template was added to the positive control group and the sample group, and the reaction solution using DEPC-treated water instead of the template was added to the negative control group. CRISPR reactions were performed at room temperature. Fluorescence signal changes during the reaction were monitored using a multi-functional microplate reader (Tecan). The results are shown below. Figure 16 As shown. During RPA amplification, the components had virtually no impact on the CRISPR detection results; only 1×E-mix and 1×Reaction Buffer slightly prolonged the time it took for the CRISPR reaction to reach its endpoint. The background fluorescence signals of ssDNA-5C-BHQ and ssDNA-TA5C-BHQ used for CRISPR detection were significantly lower than those of the classic TA-rich ssDNA-TA-BHQ probe. Furthermore, the background fluorescence signal of ssDNA-5C-BHQ was even lower than that of ssDNA-TA5C-BHQ, reaching approximately ten times the level of the black well with no additives. In addition, Figure 16(A) The curve in the positive control group showed a jump, indicating that ssDNA-TA5C-BHQ may be less stable in the CRISPR reaction system compared to ssDNA-5C-BHQ. Blue light imaging of the CRISPR system at the reaction endpoint was performed using a chemiluminescent gel imaging analyzer (Bio-Rad). Figure 17 As shown, the imaging results also indicate that the components of RPA have no effect on the CRISPR reaction endpoint, consistent with the above results, and further confirm that 0.5×NEBuffer TM The r2.1 conditions can be used in CRISPR systems without affecting the reaction.

[0147] 3.9 Construction of an integrated CRISPR gel electrophoresis system for detecting conserved fragments of the Mycoplasma pneumoniae P1 gene

[0148] Prepare 10 μL of CRISPR gel containing 1% agarose at the bottom of a PCR tube, add 20 μL of RPA to the CRISPR gel, and use 1×NE Buffer. TM r2.1 was used as the final concentration for the CRISPR system. Then, 7 × 10⁻⁶ ppm was added on top. 4 Copy the Mp-P1 template or add DEPC-treated water, and start the reaction at 42°C. First, add 500 nM of ssDNA-TA-BHQ, ssDNA-TA5C-BHQ, and ssDNA-5C-BHQ to the CRISPR gel, and set up a negative control group with each probe added (i.e., using an equal volume of DEPC-treated water instead of the Mp-P1 template as the negative control group for the corresponding probe, assuming consistent probe usage). Figure 18 The reaction effects of the CRISPR gel-gel integrated system under the action of these three probes (water curve) were compared. QuantStudio was used to analyze the reaction curves. TM 15. A real-time PCR system (Thermo Fisher Scientific) monitors changes in fluorescence signals during the reaction process, such as... Figure 18As shown, the background fluorescence signal of the ssDNA-5C-BHQ probe group added to the CRISPR gel was relatively low, and the reaction reached its endpoint within 1 hour, proving that it could normally recognize the Mp-P1 template. The addition of the ssDNA-TA-BHQ group and the ssDNA-TA5C-BHQ probe group failed to recognize the Mp-P1 template. Furthermore, the negative groups with DEPC-treated water instead of the Mp-P1 template all showed abnormal increases in fluorescence signal and a significant increase in background signal, further demonstrating that the ssDNA-TA-BHQ and ssDNA-TA5C-BHQ probes are unstable in the RPA-LbCas12a integrated system and cannot be trans-cleaved by the LbCas12a-Mp-P1-crRNA1 complex. Based on the previous test results regarding the effects of ssDNA-TA-BHQ, ssDNA-TA5C-BHQ, ssDNA-5C-BHQ, and ssDNA-5T-BHQ on the RPA system, it is demonstrated that probe design is crucial in constructing the RPA-LbCas12a integrated system. The 5C sequence design is superior to the classic TA-rich sequence design, and the final designed length should not be too long. A chemiluminescent gel imaging analyzer (Bio-Rad) was used to image the CRISPR gel-based integrated system at the reaction endpoint. The imaging results are as follows... Figure 19 As shown, only the CRISPR gel electrophoresis system with 500 nM ssDNA-5C-BHQ added could detect a fluorescence signal sufficient to distinguish it from the negative results of the corresponding system after reacting at 42°C for 1 h. This is consistent with QuantStudio's findings. TM The results obtained from the 15-digit real-time PCR system (Thermo Fisher Scientific) are consistent, further proving the accuracy of the above results.

[0149] The CRISPR gel-based integrated system was further optimized below. Since previous results showed that ssDNA-TA5C-BHQ had little impact on the RPA amplification process and was only slightly unstable in the two-step RPA-LbCas12a system, we first tested whether changing the probe position would allow ssDNA-TA5C-BHQ to function normally. 20 μL of RPA system and 10 μL of CRISPR gel were prepared, but the probe was transferred from the CRISPR gel to the RPA reaction system, and its concentration was changed to 250 nM. The concentrations of primers and Mp-P1 template remained unchanged. After reacting at 42℃ for 1 h, the entire system was imaged using a chemiluminescent gel imaging analyzer (Bio-Rad). The results are as follows. Figure 20As shown, only the CRISPR gel electrophoresis system with the ssDNA-5C-BHQ probe could recognize the target fragment and distinguish negative results. This indicates that the probe's position does not affect the reaction results of this integrated system, suggesting that the factor causing ssDNA probe instability is mainly determined by the RPA reaction system. In conclusion, given the excellent performance of the ssDNA-5C-BHQ probe in the integrated system, subsequent experiments will use this probe as a component of either the CRISPR detection system or the RPA-LbCas12a amplification detection integrated system.

[0150] The effect of LbCas12a concentration on the detection of conserved fragments of the Mycoplasma pneumoniae P1 gene using a CRISPR gel-based integrated system was tested. The concentrations of LbCas12a in the CRISPR gel were varied to 10 nM, 50 nM, and 250 nM, and the concentrations of Mp-P1-crRNA1 added to the CRISPR gel were simultaneously changed to 10 nM, 50 nM, and 250 nM, respectively. 7 × 10⁻⁶ LbCas12a was added to a PCR tube containing 20 μL of LbCas12a (excluding the template) and 10 μL of 1% CRISPR gel. 4 A copy number of Mp-P1 template or an equal volume of DEPC-treated water, with 500 nM ssDNA-5C-BHQ contained in a CRISPR gel before the reaction. The negative control group, in addition to using an equal volume of DEPC-treated water instead of template, also used the same concentrations of LbCas12a and Mp-P1-crRNA1 as the corresponding sample group. The reaction was carried out at 42°C for 1 hour, and the system at the reaction endpoint was imaged under blue light using a chemiluminescent gel imaging analyzer (Bio-Rad). Figure 21 As shown, 250 nM LbCas12a, 50 nM LbCas12a, and 10 nM LbCas12a can all normally trans-cleave the ssDNA-5C-BHQ probe in the CRISPR gel assay system. However, at a concentration of 10 nM LbCas12a, the fluorescence signal detected by this integrated system drops sharply, and the signal is mainly concentrated in the CRISPR gel. This proves that the amount of LbCas12a added to the CRISPR gel should not be less than 50 nM to ensure the normal progress of the reaction.

[0151] Using 50 nM LbCas12a, 500 nM ssDNA-5C-BHQ, and 1×NEBuffer TM CRISPR gels of r2.1 were used to set up sample groups by changing the concentration of Mp-P1-crRNA1 to 50 nM and 100 nM, and 7 × 10⁻⁶ m³ / h was added. 4Copy the Mp-P1 template. The CRISPR gel of the negative control group contained the same concentration of Mp-P1-crRNA1 as the corresponding sample group, but used an equal volume of DEPC-treated water. After the above system was prepared, it was reacted at 42°C and processed using QuantStudio. TM 15. A real-time PCR system (Thermo Fisher Scientific) was used to monitor changes in fluorescence signals during the reaction process. For example... Figure 22 As shown, the negative control group with LbCas12a:Mp-P1-crRNA1 = 1:1 showed an excessively high initiation signal, but the fluorescence signal changes indicated a more complete reaction at LbCas12a:Mp-P1-crRNA1 = 1:2. After the reaction, the corresponding reaction solutions were transferred to PCR tubes and imaged using a chemiluminescent gel imaging analyzer (Bio-Rad). The results are shown below. Figure 23 As shown, it is clear that the relative fluorescence signal of the LbCas12a:Mp-P1-crRNA1 = 1:2 group is stronger, further proving that adding crRNA at twice the concentration of LbCas12a when preparing CRISPR gel is beneficial to the detection reaction of the integrated CRISPR gel method.

[0152] To test whether the optimized CRISPR gel system could detect the Mp-P1 template at room temperature, the optimized reaction conditions were used for 1 hour at room temperature. Only two groups were tested: LbCas12a concentrations of 50 nM and 250 nM, with the Mp-P1-crRNA1 concentration being twice the corresponding LbCas12a concentration. The negative control group used the same LbCas12a and Mp-P1-crRNA1 concentrations as the sample groups. Results obtained using a chemiluminescent gel imaging analyzer (Bio-Rad) are shown below. Figure 24 As shown, no obvious fluorescence signal was observed that differed from the negative control group. Finally, the RPA reaction solution from the CRISPR gel electrophoresis system was collected for nucleic acid electrophoresis, and the results are as follows. Figure 25 As shown, clear amplification bands can be observed, proving that the RPA process in this integrated system proceeds normally at room temperature. However, the CRISPR monitoring response imaging results, which should have occurred normally, did not appear, and... Figure 24 The imaging results showed that the ssDNA probe signal was entirely concentrated in the CRISPR gel.

[0153] In summary, this experiment constructed an integrated CRISPR gel electrophoresis system capable of detecting conserved fragments of the Mycoplasma pneumoniae P1 gene at 42℃ and optimized the components for the primers and crRNA designed in this experiment. However, since this system prevents reactions that could originally be performed at room temperature, the optimized integrated reaction conditions will be used to construct and test an integrated RPA-LbCas12a amplification and detection system without agarose gel to detect the template.

[0154] 3.10 Constructing an integrated RPA-LbCas12a system capable of detecting conserved fragments of the Mycoplasma pneumoniae P1 gene.

[0155] The total system volume was 20 μL, containing 1×Reaction Buffer, 1×E-mix, 1×Core Reaction Buffer, 14 mM MgOAc, 50 nM LbCas12a, 50 nM Mp-P1-crRNA1, and 0.5×NE Buffer. TM The following were added: 480 nM F3, 480 nM R1, 0.64 mM dATP, 0.64 mM dGTP, 0.64 mM dCTP, 0.64 mM dTTP, and 0.5 μL 10 μM ssDNA-5C-BHQ. After the above system was constructed, 0.5 μL of DEPC-treated water was added to set up a negative control group, and 7 × 10⁻⁶ ppm of ssDNA-5C-BHQ was added. 4 Two sample groups were set up using the Mp-P1 template to demonstrate the feasibility of the system. Simultaneously, the concentrations of Mp-P1-crRNA1 were varied from 100 nM to 10 nM, with the concentrations of LbCas12a and Mp-P1-crRNA1 also varied to 10 nM, adding two sample groups to test the effects of LbCas12a concentration and the ratio of LbCas12a to Mp-P1-crRNA1 on the system. After reacting at 39°C for 1 h, the RPA-LbCas12a integrated system at the reaction endpoint was imaged using a chemiluminescent gel imaging analyzer (Bio-Rad). The results are as follows. Figure 26 As shown, all sample groups emitted fluorescence distinct from the negative control group, proving the successful construction of the RPA-LbCas12a system. However, the fluorescence emitted by the system with 10 nM LbCas12a and Mp-P1-crRNA1 added decreased sharply, consistent with the previous CRISPR gel electrophoresis results. Therefore, 10 nM LbCas12a will no longer be used in subsequent tests; the integrated system in this experiment must use at least 50 nM LbCas12a. Furthermore, it is evident that the fluorescence of the integrated system at 39°C with a 1:2 ratio of LbCas12a to Mp-P1-crRNA1 is slightly stronger than that at a 1:1 ratio. Further testing will be conducted below to examine the effect of the LbCas12a to Mp-P1-crRNA1 ratio on the RPA-LbCas12a integrated system reaction at room temperature.

[0156] Next, we tested whether the RPA-LbCas12a integrated system could detect Mp-P1 templates at room temperature. We constructed the aforementioned 20 μL RPA-LbCas12a integrated system containing 50 nM LbCas12a. Two sample groups contained either 50 nM or 100 nM Mp-P1-crRNA1, and 7 × 10⁻⁶ μL of the solution was added. 4 The reaction was initiated after copying the Mp-P1 template. A negative control group was established where DEPC-treated water was added instead of the template. Chemiluminescence gel imaging was performed at 1 h and 2 h of reaction at room temperature using a Bio-Rad analyzer. The results are as follows: Figure 27 As shown, clear fluorescence was observed after 1 hour of reaction at room temperature, proving that the RPA-LbCas12a integrated system can reach the reaction endpoint within 1 hour at room temperature. The RPA and CRISPR components used to construct this system did not exhibit the strong inhibitory effect on the reaction that was seen with ssDNA-TA-BHQ previously, further demonstrating that the inability of the CRISPR gel-based integrated system to react at room temperature is independent of the component composition, but solely determined by the different diffusion rates of DNA fragments by the CRISPR gel at room temperature and 42°C. Simultaneously, the fluorescence was significantly stronger when Mp-P1-crRNA1 was added at twice the amount of LbCas12a compared to the normal 1:1 ratio, indicating that this condition is more suitable for the RPA-LbCas12a integrated system. It is worth noting that the RPA-LbCas12a integrated system constructed in this experiment for detecting the conserved fragment of the Mycoplasma pneumoniae P1 gene has a certain preservation capacity. Even after preparing the RPA-LbCas12a integrated system without template, and storing it directly at 4°C in the dark for 72 hours without storing it as a dry powder, followed by adding the template and reacting at room temperature for 1 hour for imaging testing, it still showed a clear fluorescence distinct from the negative control group. Figure 28 As shown.

[0157] Finally, the sensitivity of the RPA-LbCas12a integrated system for detecting Mp-P1 template at room temperature was tested using 50 nM MbCas12a and 100 nM Mp-P1-crRNA1. The Mp-P1 template was serially diluted with DEPC-treated water, and 7000 copies, 700 copies, 70 copies, and 7 copies of Mp-P1 template were added to the prepared RPA-LbCas12a integrated system, respectively. The negative control group used an equal volume of DEPC-treated water instead of the added template. After reacting at room temperature for 1 h, imaging was performed using a chemiluminescent gel imaging analyzer (Bio-Rad). The results are shown below. Figure 29As shown, a weaker fluorescence, distinct from the negative control group, could still be observed when 700 copies of Mp-P1 template were added, while no fluorescence was observed at all in the 70 copy and 7 copy groups. This indicates that the minimum detection template amount of the RPA-LbCas12a integrated system for detecting Mycoplasma pneumoniae constructed in this experiment is 700 copies at room temperature.

[0158] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An RPA-CRISPR Mycoplasma pneumoniae detection system, characterized in that, The detection system includes RPA primers, and the forward and reverse primers of the RPA primers are shown in SEQ ID NO:6 and SEQ ID NO:3, respectively. The detection system also includes crRNA, the nucleotide sequence of which is shown in SEQ ID NO:8; The detection system also includes an ssDNA probe, the nucleotide sequence of which is shown in SEQ ID NO:

14.

2. The detection system according to claim 1, characterized in that, The ssDNA probe is linked to a fluorescent group and / or a quencher group.

3. The detection system according to claim 2, characterized in that, The fluorescent group is selected from FAM.

4. The detection system according to claim 2, characterized in that, The quenching group is selected from BHQ1.

5. The detection system according to claim 3 or 4, characterized in that, The detection system also includes RPA-related reagents and CRISPR-related reagents.

6. The detection system according to claim 5, characterized in that, The RPA-related reagents include reaction buffer, E-mix, MgOAc, and dNTPs.

7. The detection system according to claim 6, characterized in that, The reaction buffer includes a Reaction Buffer and a Core Reaction Buffer.

8. The detection system according to claim 6, characterized in that, The dNTPs include dATP, dGTP, dCTP, and dTTP.

9. The detection system according to claim 5, characterized in that, The CRISPR-related reagents include LbCas12a and restriction endonuclease buffer.

10. The detection system according to claim 9, characterized in that, The restriction endonuclease buffer is selected from NEBuffer.

11. The detection system according to claim 10, characterized in that, The NEBuffer is selected from NEBuffer r2.

1.

12. The detection system according to claim 5, characterized in that, The detection system also includes DEPC-treated water.

13. The detection system according to claim 9, characterized in that, The ratio of LbCas12a to crRNA is 1:1-2.

14. The detection system according to claim 9, characterized in that, The final concentration of LbCas12a is 50-250 nM.

15. The detection system according to claim 7 or 11, characterized in that, The final concentration of Reaction Buffer, NEBufferr2.1, and Core Reaction Buffer is 1×.

16. The detection system according to claim 6, characterized in that, The final concentration of MgOAc was 14-18 mM.

17. The detection system according to claim 8, characterized in that, The final concentrations of dATP, dGTP, dCTP, and dTTP were 0.64 mM.

18. The detection system according to claim 1, characterized in that, The final concentration of the primers was 480 nM.

19. The detection system according to claim 1, characterized in that, The final concentration of the ssDNA probe was 250 nM.

20. A Mycoplasma pneumoniae detection product, characterized in that, The product includes the testing system described in any one of claims 1-19.

21. The product according to claim 20, characterized in that, The product is a reagent kit, test strip, or chip.

22. The use of the detection system according to any one of claims 1-19 in the detection of Mycoplasma pneumoniae for non-diagnostic purposes or in the preparation of detection products for Mycoplasma pneumoniae.

23. A method for detecting Mycoplasma pneumoniae for non-diagnostic purposes, characterized in that, The method includes testing using the testing system according to any one of claims 1-19 or the product according to any one of claims 20-21.

24. The method according to claim 23, characterized in that, The temperature ranged from 37℃ to 42℃.

25. The method according to claim 23, characterized in that, The number of templates tested is over 700.

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