CrRNA based on thf modification and application thereof in detection of helicobacter pylori drug-resistant mutation

Abstract

The application discloses crRNA based on THF modification and application thereof in detection of drug-resistant mutation of Helicobacter pylori, and relates to the technical field of biology.A THF molecule is introduced into a position corresponding to a to-be-detected mutation site in a crRNA sequence, so that the discrimination ability of the system is changed.The introduction of the THF molecule causes a vacancy in space, the vacancy can optimize the interaction between the crRNA and a mutant target containing a mismatched base by adjusting the spatial conformation between the crRNA and the mutant target, so that the difference between the mutant target and the wild-type target in activating the Cas12a trans-cleavage activity is significantly amplified, which makes the discrimination based on a fluorescence signal extremely intuitive and reliable, and the detection performance of the CRISPR system on a known single-base mutation can be systematically improved, and the pain point of insufficient discrimination ability of a traditional method is successfully solved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to THF-modified crRNA and its application in the detection of drug-resistant mutations in Helicobacter pylori. Background Technology

[0002] Helicobacter pylori (Hp) infection is a major pathogenic factor in chronic gastritis, peptic ulcers, and gastric cancer. Clinically, antibiotic therapy (such as clarithromycin and levofloxacin) is commonly used to eradicate Hp. However, Hp is prone to gene mutations under antibiotic pressure (such as 23S rRNA gene point mutations), leading to drug resistance, which is a major cause of treatment failure. Therefore, rapid and accurate detection of Hp and its drug resistance before treatment is crucial for guiding clinical medication. Currently, the main methods for detecting Hp drug resistance in clinical practice include microbial susceptibility testing and quantitative real-time PCR (qPCR). Microbial susceptibility testing involves isolating and culturing Hp from gastric mucosal biopsy samples, followed by drug susceptibility testing; this is the traditional "gold standard" for detecting Hp drug resistance. Quantitative real-time PCR (qPCR) directly detects whether clinical samples contain specific gene mutations that lead to drug resistance. Existing methods for detecting Hp resistance have many shortcomings. For example, the drug sensitivity culture method has the following disadvantages: (1) Difficult to culture and low positive rate: Hp has strict requirements for growth conditions, and the success rate of culture is greatly affected by sample transportation and storage conditions, resulting in false negative results; (2) Long time consumption: It usually takes 7-10 days from sample collection to obtaining drug sensitivity results, which seriously delays the treatment time; (3) Invasive: It relies on gastroscopy to obtain tissue samples and is not suitable for large-scale screening or patient follow-up. The qPCR method has the following disadvantages: (1) Limited sensitivity: It has limited ability to detect trace amounts of mutant genes in samples, and it is easy to miss when the proportion of mutant bacteria is low; (2) Complex operation and expensive equipment: It requires a professional laboratory environment, precision instruments and well-trained technicians, which makes it difficult to apply in primary hospitals or point-of-care testing (POCT) scenarios; (3) Limited multiple detection capability: The process of detecting multiple mutation sites at the same time is complicated and costly. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide THF-modified crRNA and its application in the detection of drug resistance mutations in Helicobacter pylori.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a method for modifying crRNA, wherein the method replaces the bases in the crRNA sequence corresponding to the mutation sites of the target gene with tetrahydrofuran.

[0005] Existing technologies mostly use CRISPR-Cas12a fluorescence detection to detect drug-resistant mutations in ice sheet microorganisms. However, when a single-base mutation occurs in the target DNA, the fluorescence signal intensity generated when using traditional crRNA to detect the mutated DNA is often not significantly different from the fluorescence intensity generated by the unmutated DNA. This makes it impossible to effectively distinguish between wild-type (WT, sensitive) and single-base mutant (Mutation, drug-resistant) target DNA.

[0006] This invention also provides crRNA prepared using the method described above. The inventors of this application have discovered that introducing a THF molecule at the position corresponding to the mutation site to be tested in the crRNA sequence can alter the system's discrimination capability. The introduction of THF creates a spatial gap, which may optimize the interaction between the crRNA and the mutated target containing mismatched bases by adjusting the spatial conformation, thereby significantly amplifying the difference between mutant and wild-type targets in activating Cas12a trans-cleavage activity. This makes fluorescence signal-based discrimination extremely intuitive and reliable, systematically improving the detection performance of the CRISPR system for known single-base mutations and successfully solving the pain point of insufficient discrimination capability in traditional methods.

[0007] The present invention also provides a CRISPR-Cas detection system, wherein the detection system includes the aforementioned crRNA.

[0008] In a preferred embodiment of the detection system described in this invention, the Cas protein in the CRISPR / Cas system is Cas12a.

[0009] The present invention also provides a kit comprising the described crRNA or the described detection system.

[0010] As a preferred embodiment of the kit described in this invention, it also includes 1×Cas12a buffer, LbCas12a protein, and fluorescent probe.

[0011] The present invention also provides the application of the crRNA, the CRISPR-Cas12a detection system, or the kit described herein in the preparation of nucleic acid detection products or products for detecting drug resistance mutations in pathogenic microorganisms.

[0012] The present invention also provides the use of the crRNA, the CRISPR-Cas12a detection system, or the kit in the preparation of products for detecting drug-resistant mutations in Helicobacter pylori or in the preparation of drugs for inhibiting Helicobacter pylori.

[0013] The present invention also provides a method for detecting drug resistance mutations in the Helicobacter pylori 23S rRNA gene, comprising designing a crRNA targeting the Helicobacter pylori 23S rRNA and replacing the bases in the crRNA sequence corresponding to the mutation sites of the Helicobacter pylori 23S rRNA gene with tetrahydrofuran.

[0014] The beneficial effects of the present invention: The present invention provides a THF-modified method with the following advantages: (1) Ultra-high sensitivity and specificity: Utilizing the enzymatic amplification effect of CRISPR-Cas12a and the enhanced recognition ability of THF for mismatches, it can detect single-base mutations with a sensitivity far higher than that of traditional qPCR, effectively avoiding false negatives. (2) Fast and convenient: The entire detection process can be completed within 1-2 hours, which is much faster than the drug sensitivity culture that requires 7-10 days. (3) Simple operation and easy to promote: The reaction is carried out at a constant temperature, without the need for a complex thermal cycler, and the results can be directly interpreted by fluorescence intensity, which is very suitable for rapid detection in primary hospitals or on-site. (4) Low cost: The reagent cost is low, far lower than the expensive reagents and instruments required for qPCR. (5) High throughput and multiplex detection potential: By designing different THF-modified crRNAs, multiple drug resistance mutation sites can be detected simultaneously in the same reaction. (6) Non-invasive application potential: It can detect DNA in fecal samples, avoiding endoscopic examination, and is convenient for screening and follow-up. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the fluorescence detection principle of CRISPR-Cas12a.

[0016] Figure 2 Figures A and D show schematic diagrams of CRISPR-cas12a detection. Figures A and D show fluorescence curves, with the vertical axis representing relative fluorescence intensity (RFU) and the horizontal axis representing the number of cycles. Figures C and F show fluorescence intensity bars when the number of cycles is 15.

[0017] Figure 3 The systematic screening of crRNA guide regions was conducted to optimize detection performance. A represents the process of altering the crRNA sequence to cause it to slide against the complementary region of the target DNA (TS), thereby placing the fixed mutation site at different relative positions on the crRNA. B represents the real-time fluorescence curve. C represents the fluorescence intensity bar chart after 30 cycles.

[0018] Figure 4 The effect of the insertion position of THF in the crRNA sequence on its detection efficacy against mutant Hp (Mutation-10) is shown in Figure A, where A is the real-time fluorescence curve and B is the fluorescence intensity bar chart after 30 cycles.

[0019] Figure 5 A shows the fluorescence curves and fluorescence intensity bars at 15 cycles for detecting wild-type (WT) and mutant (Mutation) Helicobacter pylori (HP) using unmodified conventional crRNA; B shows the fluorescence curves and fluorescence intensity bars at 15 cycles for detecting wild-type (WT) and mutant (Mutation) Helicobacter pylori (HP) using THF-modified crRNA at site 10. Detailed Implementation

[0020] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0021] The principle of fluorescence detection of CRISPR-Cas12a ( Figure 1 First, the Cas12a enzyme binds to crRNA, forming a ribonucleoprotein complex. When the guide region of the crRNA specifically binds to the target DNA (TS), it triggers a conformational change in the Cas12a protein, thereby activating its trans-cleavage activity. The activated Cas12a enzyme non-specifically cleaves any single-stranded DNA molecule (ssDNA) in the reaction system. If a single-stranded DNA reporter probe (ssDNA-FQ) with a fluorescent and quenching group is present in the system, its cleavage will cause the fluorescent group (F) and the quenching group (Q) to separate. The quenching effect then disappears, resulting in a detectable fluorescent signal.

[0022] This invention provides a CRISPR-Cas12a system comprising THF-modified crRNA, such as... Figure 2 As shown, introducing a THF molecule at the position corresponding to the mutation site in the crRNA sequence alters the system's discrimination capability. The introduction of THF creates a spatial gap, which may optimize the interaction between the crRNA and the Cas12a protein by adjusting the spatial conformation between the crRNA and the mutant target containing mismatched bases. This significantly amplifies the difference between mutant and wild-type targets in activating Cas12a trans-cleavage activity, making fluorescence-based discrimination extremely intuitive and reliable.

[0023] The RNA sequences involved in the embodiments of the present invention are shown in Table 1, and the DNA sequences are shown in Table 2.

[0024] Table 1 Table 2 Example 1 This embodiment provides a THF-modified crRNA for detecting drug resistance mutations in the 23S rRNA gene of Helicobacter pylori (Hp). The screening process for this crRNA is as follows: 1.1. Determine the optimal relative position of the mutation site on the crRNA sequence: A series of crRNAs were designed ( Figure 3 A) To ensure that the fixed mutation site, when complementary to the target sequence, is located at different positions (e.g., positions 8, 9, 10, 11, 12, and 13) in the portion of the crRNA binding to the target sequence, a series of crRNAs were synthesized and purified by a professional company: mutation-8-crRNA, mutation-9-crRNA, mutation-10-crRNA, mutation-11-crRNA, mutation-12-crRNA, and mutation-13-crRNA (sequences shown in Table 1). These were then prepared as a 100 μM stock solution. Simultaneously, 200 nM of wild-type DNA (HP-TS-WT) was prepared. The detection reaction was performed in a 20 μL system containing 1×Cas12a buffer, 200 nM LbCas12a protein, 200 nM crRNA, 200 nM DNA template, and 500 nM FAM-BHQ1-labeled reporter probe. The reaction was incubated at 37°C for 60 minutes, and the fluorescence signal was monitored in real time. By analyzing real-time fluorescence curves and endpoint fluorescence intensity. Figure 3 According to BC, the detection system exhibits the highest initial activity and identification potential when the mutation site is located at the 10th relative position of the crRNA guide sequence.

[0025] 2.2. Further study was conducted on the crRNA conformation with the mutation site located at position 10: THF molecules were introduced into the crRNA with this specific conformation at positions 8, 9, and 10, respectively, to obtain 8-THF-crRNA, 9-THF-crRNA, and 10-THF-crRNA (sequences shown in Table 1). The ability of unmodified crRNA to distinguish mutant *Hp* was tested, using unmodified crRNA as a control. The above-mentioned THF-modified crRNAs and the unmodified control were incubated with the reaction system (containing 1×Cas12a buffer, 200 nM bCas12a protein, 200 nM mutant HP target DNA template, and 500 nM FAM-BHQ1 reporter probe) at 37°C for 60 minutes, and the fluorescence signal was monitored in real time. The results are as follows: Figure 4 As shown, only when THF is introduced at site 10 (i.e., the location corresponding to the mutation site) can the trans-cleavage activity of Cas12a be strongly activated, resulting in a significantly enhanced fluorescence signal; while when THF is inserted at sites 8 or 9, the system produces almost no effective signal. These results indicate that only when THF modification is precisely placed in the crRNA guide region at a specific location corresponding to the mutation site can a highly efficient signal amplification effect be achieved.

[0026] Example 2 In this embodiment, the 10-THF-crRNA and unmodified crRNA obtained in Example 1 were used to detect and verify the DNA of clinical samples that had been sequenced and validated. The wild-type (sensitive) (HP-TS-WT) and mutant (drug-resistant) (HP-TS-Mutation) Helicobacter pylori genomic DNA templates used were obtained by accurately identifying the 23S rRNA genotype of clinical strains provided by the Seventh Affiliated Hospital of Sun Yat-sen University using the Sanger sequencing method.

[0027] The specific experimental steps are as follows: Equal amounts (200 nM) of wild-type or mutant DNA templates were mixed with 20 μL of reaction system containing 1×Cas12a buffer, 200 nM LbCas12a protein, 200 nM 10-THF-crRNA (or unmodified crRNA control), and 500 nM FAM-BHQ1 reporter probe. The reaction system was incubated at 37°C for 60 minutes, and the fluorescence signal of the FAM channel was collected in real time using a real-time fluorescence quantitative PCR instrument.

[0028] The results are as follows Figure 5As shown in Figure A, when using unmodified crRNA, the fluorescence signal curves triggered by wild-type and mutant samples highly overlap and have similar intensities, making effective differentiation impossible. This indicates an inherent deficiency in the identification of single-base mutations using traditional CRISPR detection methods. In contrast, when using the 10-THF-crRNA prepared in this invention, a significant difference in fluorescence signal was observed between the two, with the fluorescence intensity of the mutant sample being greatly enhanced. Figure 5 As shown in B, this result makes interpretation clear at a glance, and can clearly and accurately distinguish between sequencing-confirmed mutant and wild-type clinical samples, proving its effectiveness and reliability in real clinical testing scenarios.

[0029] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for modifying crRNA, characterized in that, The method replaces the bases in the crRNA sequence corresponding to the mutation sites of the target gene with tetrahydrofuran.

2. crRNA prepared by the method described in claim 1.

3. A CRISPR-Cas detection system, characterized in that, The detection system includes the crRNA described in claim 2.

4. The detection system according to claim 3, characterized in that, The Cas protein in the CRISPR / Cas system is Cas12a.

5. The detection system according to claim 3, characterized in that, The reaction temperature of the CRISPR-Cas detection system is 37 °C.

6. A reagent kit, characterized in that, The kit comprises the crRNA as described in claim 2 or the detection system as described in any one of claims 3-4.

7. The reagent kit according to claim 6, characterized in that, It also includes 1×Cas12a buffer, LbCas12a protein, and fluorescent probe.

8. The use of the crRNA of claim 2, the CRISPR-Cas12a detection system of any one of claims 3-5, or the kit of any one of claims 6-7 in the preparation of nucleic acid detection products or products for detecting drug resistance mutations of pathogenic microorganisms.

9. The use of the crRNA of claim 2, the CRISPR-Cas12a detection system of any one of claims 3-5, or the kit of any one of claims 6-7 in the preparation of products for detecting drug-resistant mutations in Helicobacter pylori or in the preparation of drugs for inhibiting Helicobacter pylori.

10. A method for detecting drug resistance mutations in the 23S rRNA gene of Helicobacter pylori, characterized in that, This includes designing crRNA targeting Helicobacter pylori 23S rRNA and replacing the bases in the crRNA sequence corresponding to the mutation sites in the Helicobacter pylori 23S rRNA gene with tetrahydrofuran.

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