Detection kit based on orthogonal dual-channel label-free CRISPR-Cas and application thereof

Abstract

The invention discloses a detection kit based on orthogonal dual-channel label-free CRISPR-Cas and application of the detection kit, relates to the technical field of nucleic acid detection, and constructs an orthogonal dual-channel response system by utilizing the differentiated cleavage activity of Cas12a targeted DNA and Cas13 targeted RNA to a substrate. A Cas12a pathway is reported by utilizing protoporphyrin IX to be compounded with G-quadruplex G4DNA, and a Cas13a pathway is reported by utilizing DFHBI to be compounded with a Broccoli RNA structure, so that label-free signal transduction is realized. Through accurate screening of fluorophores, channel specificity is ensured, and optical signal crosstalk is eliminated. Mycobacterium tuberculosis (MTB) and respiratory syncytial virus (RSV) are used as model pathogens for verification, and a multiple recombinase polymerase amplification (RPA) technology is combined, so that the detection sensitivity on a synthetic target reaches a single molecule level. The system has high specificity, and even if the concentration of a non-target pathogen is increased by 100 times, no cross reaction exists.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology, specifically relating to a detection kit based on orthogonal dual-channel label-free CRISPR-Cas and its application. Background Technology

[0002] Simultaneous detection of multiple pathogens has significant application value in clinical diagnosis, food safety, and public health surveillance. In practical applications, co-infection of viruses or bacteria not only significantly increases the complexity of disease management but also seriously affects the timeliness of epidemic response. Among existing detection technologies, traditional microbial culture methods, while exhibiting good specificity, suffer from inherent limitations such as long detection cycles and limited sensitivity, making them unsuitable for rapid diagnosis. In contrast, immunoassays (such as lateral flow chromatography strips) offer the advantage of point-of-care testing (POC), but their multiplexing capabilities are insufficient, and their sensitivity for low-abundance targets often falls short of requirements. Currently, nucleic acid detection technologies, represented by qRT-PCR, have become the gold standard for pathogen detection due to their high sensitivity and specificity. However, it is worth noting that although PCR technology can achieve accurate multiplexing, its stringent requirements for specialized equipment (such as thermal cyclers), skilled operators, and lengthy testing procedures (typically requiring 1-2 hours) limit its application primarily to central laboratories, severely restricting its promotion in resource-scarce areas.

[0003] In recent years, the CRISPR-Cas system has emerged as a revolutionary nucleic acid detection tool, attracting significant attention for its rapid, specific, and easy-to-deploy-in-the-field characteristics. However, early CRISPR diagnostic platforms were mainly limited to single-target detection, a technological bottleneck that has spurred researchers to continuously explore innovations. Against this backdrop, orthogonal CRISPR-Cas multiplex detection platforms have emerged. Their core technology lies in integrating Cas enzymes with different properties (such as Cas12 and Cas13 variants) into a single reaction system, coupled with multiplex isothermal pre-amplification technology. Specifically, by designing specific guide RNAs (sgRNAs) for each Cas enzyme and matching them with reporter groups of different fluorescent properties, a unique detection signal is generated when a specific Cas enzyme is activated, thus achieving multiplex detection. Currently, fluorescently labeled probes (such as fluorescent group-quencher pairs) remain the primary signal output method, but their inherent toxicity risks and complex synthesis processes limit further development. In contrast, label-free CRISPR probes exhibit significant advantages in cost control, design simplification, and improved stability, particularly avoiding complex chemical modification processes, opening up new directions for the development of CRISPR diagnostic technology. However, how to utilize these label-free reporter groups to construct an efficient orthogonal CRISPR sensing platform remains an urgent problem to be solved. Summary of the Invention

[0004] Technical problems to be solved:

[0005] This application addresses the problem of constructing an efficient orthogonal CRISPR sensing platform using existing label-free reporter groups. It provides a detection kit based on orthogonal dual-channel label-free CRISPR-Cas and its application. The kit utilizes G4 and Broccoli to establish a label-free orthogonal dual-channel CRISPR-Cas sensing method, enabling simultaneous detection of MTB and RSV pathogen nucleic acids.

[0006] Technical solution:

[0007] To achieve the above objectives, this application provides the following technical solution:

[0008] A detection kit based on orthogonal dual-channel label-free CRISPR-Cas includes:

[0009] Reagents for specific amplification of MTB and RSV genes, the reagents comprising primer pairs of the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 for MTB, SEQ ID NO.7 and SEQ ID NO.8 for RSV, and primer pairs of the sequences shown in SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11;

[0010] The nucleic acid sequences of G4 and Broccoli are specifically shown as G4 in SEQ ID NO.12, Broccoli A in SEQ ID NO.13, and Broccoli B in SEQ ID NO.14.

[0011] The sgRNA-MT and sgRNA-RSV-A are designed to guide CRISPR-Cas12a / Cas13 to perform targeted cleavage. The sgRNA-MT sequence is shown in SEQ ID NO.16, and the sgRNA-RSV-A sequence is shown in SEQ ID NO.17.

[0012] CRISPR-Cas12a / Cas13 protein.

[0013] Further, the MTB is MT-89 as shown in SEQ ID NO.1 and MT-89-C as shown in SEQ ID NO.2.

[0014] Furthermore, the primer pairs of the sequences shown in SEQ ID NO.7 and SEQ ID NO.8 are specifically 30nt FP-89 as shown in SEQ ID NO.7 and 30nt RP-89 as shown in SEQ ID NO.8.

[0015] Furthermore, the primer pairs of the sequences shown in SEQ ID NO.10 and SEQ ID NO.11 are specifically T7-FP as shown in SEQ ID NO.10 and T7-RP as shown in SEQ ID NO.11.

[0016] This application also discloses the application of any of the above-mentioned orthogonal dual-channel label-free CRISPR-Cas detection kits in pathogen detection or auxiliary identification, wherein the detection or auxiliary identification is for non-disease diagnosis purposes, and the specific pathogens are MTB and RSV pathogens.

[0017] Furthermore, the application of the orthogonal dual-channel label-free CRISPR-Cas detection kit, which is not intended for disease diagnosis and treatment, in pathogen detection includes the following steps:

[0018] S1. Multiple amplification of the MTB and RSV samples to be tested yields dsDNA and ssRNA target sequences;

[0019] S2. Construct a system including Cas12a-sgRNA, Cas13-sgRNA, G4 as shown in SEQ ID NO.12, PPIX, and as shown in SEQ ID NO.

[0020] The detection system of Broccoli A shown in NO.13, Broccoli B shown in SEQ ID NO.14, the dsDNA target and ssRNA target sequences described in DFHBI, and the reaction buffer was used for the reaction.

[0021] S3. After the reaction is complete, the above detection system is subjected to fluorescence detection to measure the fluorescence intensity.

[0022] Furthermore, in the detection system described in step S1, the MTB sample is amplified to obtain the dsDNA target sequence, and the RSV sample is amplified and transcribed to obtain the ssRNA target sequence.

[0023] Further, in the detection system described in step S2, the concentrations of sgRNA-MT (as shown in SEQ ID NO.16) and sgRNA-RSV-A (as shown in SEQ ID NO.17) are both 40 nM. In the detection system described in step S2, the concentrations of G4 (as shown in SEQ ID NO.12) are 1 μM, PPIX (as shown in SEQ ID NO.12) are 5 μM, Broccoli A (as shown in SEQ ID NO.13) is 800 nM, Broccoli B (as shown in SEQ ID NO.14) is 800 nM, and DFHBI (as shown in SEQ ID NO.14) is 5 μM.

[0024] Furthermore, in the detection system described in step S2, 200 mM KCl is added to promote the binding of G4 with PPIX and the binding of Broccoli A, Broccoli B and DFHBI.

[0025] Furthermore, the fluorescence detection temperature described in step S3 is 37°C.

[0026] Beneficial effects:

[0027] This application provides a detection kit based on orthogonal dual-channel label-free CRISPR-Cas and its application, which has the following advantages compared with the prior art:

[0028] 1. This invention is based on a label-free Cas12 and Cas3 multiplex nucleic acid detection method, which uses G4 and Broccoli as signal output means, avoiding fluorescent labeling, reducing detection costs, and improving system stability;

[0029] 2. When used in conjunction with RPA amplification technology and T7 transcription, the detection sensitivity can be significantly improved;

[0030] 3. This invention utilizes orthogonal dual-channel label-free CRISPR-Cas sensing technology, which can generate two independent signal channels to detect different pathogens simultaneously, making the detection process simpler. Attached image description:

[0031] Figure 1 This is a schematic diagram illustrating the detection kit based on orthogonal dual-channel label-free CRISPR-Cas for nucleic acid detection of MTB and RSV pathogens in this application.

[0032] Figure 2 This is a PAGE representation of the CRISPR-Cas12a-mediated G4 cleavage in this application.

[0033] Figure 3 This image shows the results of PAGE characterization of CRISPR-Cas13-mediated Broccoli A cleavage in this application.

[0034] Figure 4 This is a schematic diagram of the CRISPR-Cas13-mediated Broccoli cleavage fluorescence signal in this application.

[0035] Figure 5 This is a schematic diagram illustrating the linear relationship between the fluorescence signal and the Cas12a / Cas13 target concentration of 0.1-50 nM in this application;

[0036] Figure 6 This is a PAGE image showing the RPA efficiency analysis using primers with lengths of 20 to 30 nt in this application.

[0037] Figure 7 The image shows the fluorescence signal results of the detection kit based on orthogonal dual-channel label-free CRISPR-Cas in this application for detecting dual targets;

[0038] Figure 8 This figure shows the specificity study results of the detection kit based on orthogonal dual-channel label-free CRISPR-Cas in this application. Detailed Implementation

[0039] To make the technical problems, solutions, and advantages of this invention clearer, a detailed description will be provided below with reference to the accompanying drawings and specific embodiments. The described embodiments are only a part of the embodiments of this invention and not all of them.

[0040] This invention provides a detection method based on an orthogonal dual-channel label-free CRISPR-Cas sensing system for the simultaneous detection of MTB and RSV pathogen nucleic acids. For example... Figure 1 As shown, after amplifying the MTB and RSV pathogen genes, dsDNA and ssRNA target sequences were obtained, respectively. These target sequences can be specifically recognized by Cas12a-sgRNA and Cas13-sgRNA and activate the CRISPR-Cas sensing system, thereby specifically cleaving G4 and Broccoli. Using G4 and Broccoli as fluorescent signal output means, the degradation of G4 and Broccoli leads to a decrease in fluorescence, thereby realizing the detection of MTB and RSV pathogens.

[0041] The technical solution of the present invention will be further illustrated below with reference to several embodiments.

[0042] Example 1:

[0043] To investigate the specific cleavage of G4 by Cas12a-sgRNA, this invention used 8% non-denaturing PAGE to characterize CRISPR-Cas12a-mediated G4 cleavage. The Cas12a (20 nM) / sgRNA-MT (40 nM) complex was pre-assembled in 1×NEBuffer 2.1. After adding 50 nM of double-stranded target MT-89 (as shown in SEQ ID NO. 1), MT-89-C (as shown in SEQ ID NO. 2), and 1 μM of substrate G4 (as shown in SEQ ID NO. 12), the resulting solution was incubated at 39°C for 40 min. 10 μL of the reaction product was quenched with 2 μL of 6×DNA loading buffer; the DNA loading buffer consisted of 60% (v / v) glycerol, 0.05% (w / v) bromophenol blue, and 0.05% (w / v) xylenecyanide containing 60 mM EDTA. Electrophoresis was performed in 1×TBE buffer, which consisted of 89 mM Tris, 89 mM boric acid, and 2 mM EDTA at pH 8.0, and run at a constant voltage of 80 V for 80 minutes. Finally, the gel was stained with 4S Red for imaging. Figure 2 It can be seen that Cas12a-sgRNA only has cleavage activity when the target is present, cleaving the G4 chain in the solution, specifically manifested as a lighter G4 band in lane 1.

[0044] Next, this application investigated the specific cleavage of Broccoli A by Cas13-sgRNA as shown in SEQ ID NO.13. CRISPR-Cas13-mediated Broccoli A cleavage was characterized using 8% non-denaturing PAGE. The Cas13 (20 nM) / sgRNA-RSV-A (40 nM) complex as shown in SEQ ID NO.17 was pre-assembled in 1×NEBuffer 2.1 buffer. After adding RSV-Target (50 nM) as shown in SEQ ID NO.18 and the substrate strand (Broccoli A, 1.5 μM), the resulting solution was incubated at 39 °C for 90 min. 10 μL of the reaction product was quenched with 2 μL of 6×DNA loading buffer. Electrophoresis was performed in 1×TBE buffer at a constant voltage of 80 V for 80 min. Finally, the gel was stained with 4S Red and imaged. Figure 3 It can be seen that Cas13-sgRNA only has cleavage activity when the target is present, cleaving the Broccoli A chain in the solution, specifically manifested as a lighter Broccoli A band in lane 1.

[0045] Example 2:

[0046] Figure 4The fluorescence signal intensities generated by CRISPR-Cas13-mediated cleavage of different substrates (fragmented Broccoli and intact Broccoli) were displayed. The signal output of the detection results was obtained by fluorescence generated by RNA aptamers and DFHBI. Therefore, the substrates were optimized in this application. The Cas13 (20 nM) / sgRNA-RSV-A (40 nM) complex was pre-assembled in 1×NEBuffer 2.1 buffer. RSV-Target (50 nM) and different substrate chains (800 nM Broccoli A, 800 nM Full-length Broccoli as shown in SEQ ID NO.15) were added to this system, and the resulting solutions were incubated at 39 °C for 1 h. After adding 800 nM of substrate chain as shown in SEQ ID NO.14 Broccoli B and without substrate chain, as well as adding 200 mM KCl and 5 μM DFHBI, the fluorescence was measured after incubation at 37 °C for 10 min (λex: 460 nm; λem: 502 nm). like Figure 4 As shown, the cleavage efficiency of CRISPR-Cas13-mediated split-form Broccoli is higher than that of whole Broccoli within the same time frame. Therefore, this application selects split-form Broccoli as the substrate chain.

[0047] Example 3:

[0048] The response of the CRISPR-Cas reaction system to different target concentrations was determined. The Cas12a (20 nM) / sgRNA-MT-89 (40 nM) complex as shown in SEQ ID NO. 16 was pre-assembled in 1×NEBuffer 2.1 buffer. The Cas13 (20 nM) / sgRNA-RSV-A (40 nM) complex was pre-assembled in 1×NEBuffer 2.1 buffer. The two tubes were then mixed together, and different concentrations of the target (0.1, 1, 10, 100 nM) and substrate chains (G4, 1 μM; Broccoli A, 800 nM) were added to this system. The resulting solution was incubated at 39 °C for 1 h, followed by the addition of 800 nM Broccoli B, 200 mM KCl, 5 μM PPIX, and 5 μM DFHBI. After incubation at 37 °C for 10 min, fluorescence was measured (λex(PPIX): 410 nm, λem(PPIX): 635 nm, λex(DFHBI): 460 nm, λem(DFHBI): 502 nm). Figure 5 As shown, the fluorescence signal decreases with increasing target concentration. This result demonstrates that the orthogonal CRISPR-Cas detection method constructed in this application can achieve quantitative analysis of the target sequence.

[0049] Example 4:

[0050] To improve the efficiency of RPA amplification of the MTB gene sequence, this application optimized the primers for RPA amplification. Three different lengths of front primers (e.g., 20nt FP-89 as shown in SEQ ID NO.3, 25nt FP-89 as shown in SEQ ID NO.5, and 30nt FP-89 as shown in SEQ ID NO.7) and back primers (e.g., 20nt RP-89 as shown in SEQ ID NO.4, 25nt RP-89 as shown in SEQ ID NO.6, and 30nt RP-89 as shown in SEQ ID NO.8) were designed. The MTB pseudovirus gene sequence was then amplified using these primers of different lengths, and the amplification was performed using an RPA kit. The following components were added to RPA lyophilized powder tubes: 29.4 μL reconstitution buffer, 4 μL forward and reverse primers (400 nM each), 2 μL DNA amplification template, 12.1 μL ddH2O, for a total volume of 47.5 μL. Then, 2.5 μL of MgOAc (280 mM) was added for amplification. The reaction was carried out at 39 °C for 20 minutes. The resulting samples were annealed from 95 °C to 4 °C in an Applied Biosystems 96-well thermal cycler and characterized by PAGE. Figure 6 As shown, the amplification efficiency was highest and the amount of amplified product was maximized using the front primer (30nt FR) and the back primer (30nt RP). Therefore, this application selected the front primer (30nt FR) and the back primer (30nt RP) for subsequent RPA amplification.

[0051] Example 5:

[0052] This application utilizes the established orthogonal dual-channel label-free CRISPR-Cas sensing technology to analyze MTB and RSV pseudoviruses. Pre-amplification was performed using an RPA kit. The following components were added to the RPA lyophilized powder tubes: 29.4 μL reconstitution buffer, 4 μL MTB forward and reverse primers (400 nM each), 4 μL RSV forward and reverse primers (400 nM each), 2 μL DNA amplification template (MTB, RSV), 10 μL NTP Mix (2 mM), 1 μL T7 RNA Polymerase (1 u / μL), 1 μL DTT (2 mM), and 2.5 μL MgOAc (280 mM). The reaction was carried out at 39 °C for 20 minutes, and the resulting samples were annealed from 95 °C to 4 °C in an Applied Biosystems 96-well thermal cycler.

[0053] In fluorescence detection, this application performed RPA amplification and detection of the MTB and RSV pseudovirus genes. 6 μL of annealed amplification product was added to the CRISPR-Cas12a / Cas13 reaction system. The Cas12a (20 nM) / sgRNA-MT-89 (40 nM) complex was pre-assembled in 1×NEBuffer 2.1. The Cas13 (20 nM) / sgRNA-RSV-A (40 nM) complex was pre-assembled in 1×NEBuffer 2.1 buffer. The two tubes were then combined, and the amplification product and substrate strands (Broccoli A, 800 nM; G4, 1 μM) were added. The resulting solution was incubated at 39 °C for 60 min. Then, 800 nM Broccoli B, 200 mL MMKCl, 5 μM PPIX, and 5 μM DFHBI were added, and the solution was incubated at 37 °C for 10 min. Then, fluorescence was detected (λex: 460nm, λem: 502nm; λex: 410nm; λem: 635nm), and the fluorescence signal was recorded. For example... Figure 7 As shown, Cas12a / Cas13-sgRNA is activated only in the presence of the target, cleaving the G4 and Broccoli A chains in solution, resulting in a decrease in fluorescence signal. This demonstrates that the established RPA-CRISPR-Cas sensing technology can be used for the detection of MTB and RSV pathogen nucleic acids.

[0054] Example 6:

[0055] To achieve specific detection of MTB and RSV pathogens, this application investigated the specificity of the established orthogonal dual-channel label-free CRISPR-Cas detection method. For example... Figure 8As shown, this application selected seven pseudoviruses: 10 copies of MTB, 10 copies of RSV-A, 10 copies of RSV-B, 1000 copies of SARS-CoV-2, 1000 copies of H7N9, 1000 copies of MPXV, and 1000 copies of SARS-CoV. Pre-amplification was then performed using an RPA kit. The RPA amplification reaction was carried out at 39°C for 20 minutes. The resulting samples were annealed from 95°C to 4°C in an Applied Biosystems 96-well thermal cycler. 6 μL of the annealed amplified product was added to a CRISPR-Cas12a / Cas13 reaction system, along with substrate chains (Broccoli A, 800 nM; G4, 1 μM). After reacting at 39°C for 60 minutes, 800 nM Broccoli B, 200 mL MMKCl, 5 μM PPIX, and 5 μM DFHBI were added, and the mixture was incubated at 37°C for 10 minutes. Then, fluorescence was detected (λex(PPIX): 410nm, λem(PPIX): 635nm, λex(DFHBI): 460nm, λem(DFHBI): 502nm), and the fluorescence signals were recorded. The results are as follows: Figure 8 As shown in the results, the orthogonal dual-channel label-free CRISPR-Cas sensing platform established in this application can only detect MTB, RSV-A and RSV-B pseudoviruses as shown in SEQ ID NO.9, confirming that the CRISPR-Cas sensing platform has specificity for pathogen detection and ensuring the accuracy of pathogen nucleic acid detection.

[0056] All sequences used in this invention are shown in Table 1 below:

[0057] Table 1. All nucleic acid sequences involved in this invention.

[0058]

[0059]

[0060] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A detection kit based on orthogonal dual-channel label-free CRISPR-Cas, characterized in that, include: Reagents for specific amplification of MTB and RSV genes, the reagents comprising primer pairs of the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 for MTB, SEQ ID NO.7 and SEQ ID NO.8 for RSV-A, and SEQ ID NO.10 and SEQ ID NO.11 for RSV-A; The nucleic acid sequences of G4 and Broccoli are specifically shown as G4 in SEQ ID NO.12, Broccoli A in SEQ ID NO.13, and Broccoli B in SEQ ID NO.

14. The sgRNA-MT and sgRNA-RSV-A are designed to guide CRISPR-Cas12a / Cas13 to perform targeted cleavage. The sgRNA-MT sequence is shown in SEQ ID NO.16, and the sgRNA-RSV-A sequence is shown in SEQ ID NO.

17. CRISPR-Cas12a / Cas13 protein.

2. The detection kit based on orthogonal dual-channel label-free CRISPR-Cas according to claim 1, characterized in that, The MTB is MT-89 as shown in SEQ ID NO.1 and MT-89-C as shown in SEQ ID NO.

2.

3. The detection kit based on orthogonal dual-channel label-free CRISPR-Cas according to claim 1, characterized in that, The primer pair for the sequences shown in SEQ ID NO.7 and SEQ ID NO.8 is specifically 30ntFP-89 as shown in SEQ ID NO.

7. And 30nt RP-89 as shown in SEQ ID NO.

8.

4. The detection kit based on orthogonal dual-channel label-free CRISPR-Cas according to claim 1, characterized in that, The primer pairs for the sequences shown in SEQ ID NO.10 and SEQ ID NO.11 are specifically T7-FP as shown in SEQ ID NO.10 and T7-RP as shown in SEQ ID NO.

11.

5. The application of the detection kit based on orthogonal dual-channel label-free CRISPR-Cas as described in any one of claims 1-4 in pathogen detection or auxiliary identification, characterized in that, The detection or auxiliary identification is for non-disease diagnosis purposes, and the specific pathogens are MTB and RSV pathogens.

6. The application according to claim 5, characterized in that: The application of the orthogonal dual-channel label-free CRISPR-Cas detection kit, which is not intended for disease diagnosis and treatment, in pathogen detection specifically includes the following steps: S1. Multiple amplification of the MTB and RSV samples to be tested yields dsDNA and ssRNA target sequences; S2. Construct a system including Cas12a-sgRNA, Cas13-sgRNA, G4 as shown in SEQ ID NO.12, PPIX, and as shown in SEQ ID NO. The detection system of Broccoli A shown in NO.13, Broccoli B shown in SEQ ID NO.14, the dsDNA target and ssRNA target sequences described in DFHBI, and the reaction buffer was used for the reaction. S3. After the reaction is complete, the above detection system is subjected to fluorescence detection to measure the fluorescence intensity.

7. The application according to claim 6, characterized in that, In the detection system described in step S1, the dsDNA target sequence is obtained by amplifying the MTB sample, and the ssRNA target sequence is obtained by amplifying and transcribing the RSV sample.

8. The application according to claim 6, characterized in that, In the detection system described in step S2, the concentrations of sgRNA-MT (as shown in SEQ ID NO. 16) and sgRNA-RSV-A (as shown in SEQ ID NO. 17) are both 40 nM, the concentrations of G4 (as shown in SEQ ID NO. 12) are 1 μM, the concentration of PPIX is 5 μM, the concentration of Broccoli A (as shown in SEQ ID NO. 13) is 800 nM, the concentration of Broccoli B (as shown in SEQ ID NO. 14) is 800 nM, and the concentration of DFHBI is 5 μM.

9. The application according to claim 6, characterized in that, In the detection system described in step S2, 200 mM KCl is added to promote the binding of G4 with PPIX and the binding of Broccoli A, Broccoli B and DFHBI.

10. The application according to claim 6, characterized in that, The temperature for fluorescence detection in step S3 is 37°C.

Images