RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening

Abstract

The invention relates to the technical field of nucleic acid detection, and discloses an RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening, which comprises a sample processing module, an RCA isothermal amplification module, a CRISPR-Cas multiple detection module and a signal output module, and solves the problems that when the existing detection system is used, the detection is often performed through a single mutation detection system, and the detection time is short. The detection method cannot meet the multi-target screening requirement of the drug-resistant tuberculosis, and a multi-channel detection platform has the problems of complex operation, high cost and the like, and is not beneficial to subsequent screening and detection.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid detection technology, and specifically to an RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening. Background Art

[0002] Nucleic acid testing is the gold standard for identifying biological species and an important means of detecting and identifying microorganisms such as viruses and bacteria. CRISPR technology, as a gene editing technology, can accurately identify nucleic acid fragments and can be widely used in nucleic acid testing. During nucleic acid testing, nucleic acid extraction and concentration are usually required before nucleic acid testing, which helps to improve the sensitivity of nucleic acid testing. In recent decades, several isothermal amplification methods, such as recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and rolling circle amplification (RCA), have gradually emerged. Because their reaction conditions are carried out at a constant temperature, they have the potential to develop into powerful alternatives to traditional PCR methods. Among them, RCA has been favored by many researchers for its high amplification efficiency and simple operation. The specific principle of RCA amplification is: using a circular DNA template to recognize and bind to short DNA and RNA primers, amplified under the action of special DNA and RNA polymerases, forming long tandem single-stranded DNA and RNA containing hundreds of tandem repeat sequences (complementary to the circular template). Compared with traditional PCR, isothermal amplification methods such as RCA simplify experimental conditions. For example, patent application number CN202010441655.8 discloses a closed, fully automatic nucleic acid extraction and detection system based on CRISPR technology; It can be seen that there are shortcomings in the above-mentioned patent application: when using existing detection systems, detection is often performed through a single mutation detection system. This detection method cannot meet the needs of multi-target screening of drug-resistant tuberculosis, and the multi-channel detection platform has problems such as complex operation and high cost, which is not conducive to subsequent screening and detection.

[0003] To address the above issues, a RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening was proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide an RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening. By using this device, the problem that existing detection systems often use a single mutation detection system for detection when in use cannot meet the needs of multi-target screening of drug-resistant tuberculosis, and the multi-channel detection platform has problems such as complex operation and high cost, which is not conducive to subsequent screening and detection.

[0005] To achieve the above objectives, the present invention provides the following technical solutions: an RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening, comprising a sample processing module, an RCA isothermal amplification module, a CRISPR-Cas multiple detection module, and a signal output module. The sample processing module is used to lyse the sample and enrich drug resistance-related genes; The RCA isothermal amplification module includes a sample lysis module and a nucleic acid enrichment module, and the RCA isothermal amplification module is used to perform rolling circle amplification on the target gene; The CRISPR-Cas multiple detection module includes a constant temperature reaction module and a multi-channel circular probe module, and the CRISPR-Cas multiple detection module is used to identify mutation sites and generate detection signals; The signal output module includes a fluorescence detection module and a data processing module, and the signal output module is used to collect and analyze detection signals.

[0006] Preferably, the RCA isothermal amplification module comprises a circular probe array targeting the rpoB, katG, and inhA genes of Mycobacterium tuberculosis, and the circular probe sequence is complementary to the drug-resistant mutation hotspot region.

[0007] Using the design steps described above, the rpoB, katG, and inhA genes of Mycobacterium tuberculosis are key hotspots for drug-resistant mutations. By designing circular probe arrays complementary to these regions, precise targeted identification of drug-resistant genes can be achieved, ensuring that the detection covers common clinical drug-resistant mutation sites and improving the comprehensiveness of multi-target screening.

[0008] Preferably, the RCA isothermal amplification module uses the padlock probe connected into a circle as a template and performs rolling circle amplification using Phi29 DNA polymerase under constant temperature conditions to generate a long-chain amplification product with thousands of repeating sequences.

[0009] Using the aforementioned design, the padlock probes can only form a circle when they precisely match the target gene sequence. This serves as a template for constant-temperature rolling circle amplification, leveraging the high extension capacity of Phi29 DNA polymerase to generate long-chain amplification products containing thousands of repeats. This design eliminates the need for variable temperature equipment, simplifying experimental conditions while significantly improving detection sensitivity by exponentially amplifying trace amounts of the target gene.

[0010] Preferably, the CRISPR-Cas multiple detection module uses Cas12a and Cas14a proteins, in combination with multiple sets of guide RNAs, wherein the 5' ends of the multiple sets of guide RNAs are modified with fluorescent groups and the 3' ends are modified with quenching groups.

[0011] Using the above design steps, the Cas12a and Cas14a proteins possess "side-cleavage" activity. When the guide RNA hybridizes with the mutation site in the RCA amplification product, its ability to cleave single-stranded nucleic acids is activated. The fluorophore at the 5' end of the guide RNA and the quencher at the 3' end form a FRET (Frequency Resonance Emission Transition) system. When not cleaved, the fluorescence is quenched; after cleavage, the fluorophore is released, generating a detectable fluorescent signal, enabling visual detection of the mutation site.

[0012] Preferably, the CRISPR-Cas multiple detection module comprises a Cas protein and a specific guide RNA, wherein the guide RNA sequence is complementary to a specific region containing a mutation site in the RCA amplification product, and the Cas protein can activate nuclease activity to cut single-stranded DNA or RNA after the guide RNA hybridizes with the RCA amplification product to form a double-stranded structure.

[0013] Using the design steps described above, the guide RNA sequence is strictly complementary to the region containing the mutation site in the RCA amplification product. Only when the corresponding mutation is present in the target gene can a stable duplex structure be formed, activating the nuclease activity of the Cas protein. This design, through dual specificity (padlock probe recognition + guide RNA hybridization), avoids nonspecific cleavage and reduces the false positive rate.

[0014] Preferably, the signal output module adopts multi-channel fluorescence detection technology, which can simultaneously identify multiple fluorescence signals such as FAM, HEX, ROX, etc.

[0015] By employing the above design steps and utilizing the different emission wavelengths of various fluorophores, such as FAM, HEX, and ROX, combined with multi-channel fluorescence detection technology, multiple fluorescent signals can be simultaneously identified within the same reaction system. For example, by modifying guide RNAs targeting different drug-resistance genes with different fluorophores, multiple mutations can be simultaneously genotyped and quantitatively analyzed, improving detection efficiency.

[0016] Preferably, the multi-channel circular probe module is a series of padlock probes designed for multiple key mutation sites of Mycobacterium tuberculosis resistance-related genes. Both ends of the padlock probes have complementary sequences to the target DNA and can specifically identify specific mutation sites.

[0017] Using the above design steps, the padlock probe's end sequences complement the upstream and downstream sequences of the target DNA mutation site. Only when the target gene is free of mutations can the end sequences precisely hybridize and ligate into a loop; if a mutation is present, the closed loop cannot form, thus blocking subsequent amplification. This design achieves highly specific recognition of single-base mutations through a conformation-dependent ligation reaction.

[0018] Preferably, the sample processing module, RCA isothermal amplification module, CRISPR-Cas multiple detection module and signal output module are all integrated into a microfluidic chip, and the modules are connected through microchannels to achieve automated operations from sample processing to result output.

[0019] Using the aforementioned design steps, sample processing, RCA amplification, CRISPR-Cas detection, and signal output modules are integrated into a microfluidic chip, enabling automated sample flow through microchannels. This design reduces manual handling errors and shortens testing time (from sample to result in just a few hours). Furthermore, the device is portable and energy-efficient, making it suitable for primary care and on-site screening scenarios.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1. This application proposes an RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening. It uses rolling circle amplification technology to exponentially amplify target genes, which can amplify extremely small amounts of mutant genes to detectable levels, greatly improving the sensitivity of detection. It can detect gene mutations as low as a single copy number, which is significantly improved compared to traditional detection methods.

[0021] 2. This application proposes an RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening. The padlock probe design ensures highly specific identification of specific mutation sites. Only when the target gene sequence and the complementary sequences at both ends of the padlock probe are precisely matched can the padlock probes connect to form a loop and undergo subsequent amplification reactions. Furthermore, the specific hybridization of the guide RNA and the RCA amplification product in the CRISPR-Cas system further ensures the specificity of the detection and effectively avoids false-positive results.

[0022] 3. This application proposes an RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening. This system can simultaneously design padlock probes and guide RNAs targeting multiple drug-resistance-related gene mutation sites, enabling the simultaneous detection of multiple drug-resistance gene mutations in the same reaction system. For example, it can simultaneously detect gene mutations associated with resistance to multiple anti-tuberculosis drugs, such as isoniazid, rifampicin, and ethambutol. A single test can provide a comprehensive understanding of a patient's drug resistance status, providing richer and more accurate information for the formulation of clinical treatment plans and greatly improving detection efficiency.

[0023] 4. This application proposes an RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening. The entire detection process, from sample processing to result analysis, can be completed in a relatively short time. Compared with traditional phenotypic drug sensitivity tests and some molecular detection technologies, the detection time is greatly shortened, which can meet the needs of clinical rapid diagnosis and contribute to timely treatment and epidemic prevention and control of patients.

[0024] 5. This application proposes an RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening. This detection system does not require complex instruments and equipment, but only conventional constant temperature equipment, fluorescence detection equipment or colorimetric equipment. It has relatively low professional requirements for operators and is easy to promote and apply in primary medical institutions and on-site testing.

[0025] 6. The present application proposes an RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening. Compared with traditional molecular detection technologies that rely on expensive large instruments and complex diagnostic reagents, the detection system of the present invention requires lower reagent costs and does not require the purchase and maintenance of large equipment, thereby reducing detection costs and facilitating the screening and diagnosis of drug-resistant tuberculosis in resource-limited areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the overall process of the present invention; Figure 2 This is a schematic diagram of the sample processing module flow of the present invention; Figure 3 This is a schematic diagram of the RCA isothermal amplification module process of the present invention; Figure 4 It is a flow chart of the signal output module of the present invention. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0028] In order to further understand the content of the present invention, the present invention is described in detail with reference to the accompanying drawings.

[0029] Combine Figure 1-Figure 4 , an RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening, including a sample processing module, an RCA isothermal amplification module, a CRISPR-Cas multiple detection module, and a signal output module. The sample processing module is used to lyse samples and enrich drug resistance-related genes; The RCA isothermal amplification module includes a sample lysis module and a nucleic acid enrichment module. The RCA isothermal amplification module is used to perform rolling circle amplification of the target gene; The CRISPR-Cas multiplex detection module includes a constant temperature reaction module and a multi-channel circular probe module. The CRISPR-Cas multiplex detection module is used to identify mutation sites and generate detection signals. The signal output module includes a fluorescence detection module and a data processing module, and the signal output module is used to collect and analyze the detection signal.

[0030] The RCA isothermal amplification module contains a circular probe array targeting the rpoB, katG, and inhA genes of Mycobacterium tuberculosis, and the circular probe sequences are complementary to the hotspot regions of drug-resistant mutations.

[0031] The RCA isothermal amplification module uses a padlock probe connected into a ring as a template and performs rolling circle amplification using Phi29 DNA polymerase under constant temperature conditions to generate long-chain amplification products with thousands of repeat sequences.

[0032] The CRISPR-Cas multiplex detection module uses Cas12a and Cas14a proteins, in conjunction with multiple sets of guide RNAs, each of which is modified with a fluorescent group at the 5' end and a quenching group at the 3' end.

[0033] The CRISPR-Cas multiplex detection module contains Cas protein and specific guide RNA. The guide RNA sequence is complementary to the specific region containing the mutation site in the RCA amplification product. After the guide RNA and the RCA amplification product hybridize to form a double-stranded structure, the Cas protein can activate the nuclease activity to cut single-stranded DNA or RNA.

[0034] The signal output module adopts multi-channel fluorescence detection technology, which can simultaneously identify multiple fluorescence signals such as FAM, HEX, ROX, etc.

[0035] The multi-channel circular probe module is a series of padlock probes designed for multiple key mutation sites of Mycobacterium tuberculosis resistance-related genes. Both ends of the padlock probes have complementary sequences to the target DNA and can specifically identify specific mutation sites.

[0036] The sample processing module, RCA isothermal amplification module, CRISPR-Cas multiple detection module and signal output module are all integrated into the microfluidic chip. The modules are connected through microchannels to realize automated operations from sample processing to result output.

[0037] The present invention will be further described below with reference to the embodiments.

[0038] Example 1: Fifty sputum samples from clinically diagnosed tuberculosis patients were collected, including 20 sputum samples from multidrug-resistant tuberculosis patients, 20 sputum samples from sensitive tuberculosis patients, and 10 sputum samples from non-tuberculosis patients as negative controls. 1-2 mL of sputum sample was added to an equal volume of 4% sodium hydroxide solution, the solution was shaken and mixed, and the solution was placed at room temperature for 15-20 minutes for sputum liquefaction.

[0039] First, the liquefied sputum sample was centrifuged at 12000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was washed 2-3 times with PBS buffer.

[0040] Add an appropriate amount of lysis buffer and incubate at 56 degrees Celsius for 30-60 minutes to lyse the cells. Then extract genomic DNA from the lysate and dissolve the DNA with an appropriate amount of TE buffer to serve as a template for subsequent detection.

[0041] In a 20 μl reaction system, add 100 ng of extracted genomic DNA, 100 nM of each padlock probe, ligation buffer, and DNA ligase.

[0042] The reaction system was placed in a constant temperature water bath at 37 degrees Celsius and incubated for 1-2 hours to perform hybridization and ligation reactions between the padlock probe and the target gene.

[0043] Add Phi29 DNA polymerase reaction buffer, 4 mM dNTPs, and 10 U Phi29 DNA polymerase to the ligation reaction system to make the total volume reach 50 μL.

[0044] The reaction system was placed in a constant temperature water bath at 37 degrees Celsius and incubated for 2-3 hours to perform rolling circle amplification reaction.

[0045] In a 20 μL reaction system, add 10 μL RCA amplification product, 50 nM Cas12a protein, 100 nM corresponding guide RNA, Cas12a reaction buffer, and 100 nM single-stranded reporter molecules with fluorescent labels and quenching groups.

[0046] Place the reaction system in a constant temperature water bath at 37 degrees Celsius and incubate for 30-60 minutes to perform the CRISPR-Cas cleavage reaction.

[0047] Finally, for analysis, the system after the CRISPR-Cas cleavage reaction was transferred to a fluorescent quantitative PCR instrument, the detection channel was set to the FAM channel, and the fluorescence signal intensity was read.

[0048] Using the test results of sputum samples from non-tuberculosis patients as a negative control, a fluorescence signal intensity threshold was set. When the fluorescence signal intensity of the sample was higher than the threshold, it was judged as positive, indicating the presence of the corresponding drug-resistant gene mutation; when the fluorescence signal intensity of the sample was lower than the threshold, it was judged as negative.

[0049] The test results of sputum samples from patients with multidrug-resistant tuberculosis and patients with sensitive tuberculosis were statistically analyzed, and the sensitivity, specificity, and accuracy of the detection system were calculated.

[0050] Example 2: Forty blood samples from clinically confirmed tuberculosis patients were collected, including 15 blood samples from multidrug-resistant tuberculosis patients, 15 blood samples from sensitive tuberculosis patients, and 10 blood samples from healthy volunteers as negative controls. 2-3 mL of blood sample was added with an appropriate amount of red blood cell lysis buffer, shaken to mix, and left at room temperature for 5-10 minutes to lyse the red blood cells.

[0051] The lysed sample was centrifuged at 12000 rpm for 10 minutes, the supernatant was removed, and the precipitate was washed 2-3 times with PBS buffer.

[0052] Add an appropriate amount of cell lysis buffer (including proteinase K, etc.) and incubate at 56 degrees Celsius for 30-60 minutes to lyse the cells.

[0053] Extract genomic DNA from the lysate and dissolve the DNA in an appropriate amount of TE buffer to serve as a template for subsequent detection.

[0054] In a 20 μL reaction system, add 100 ng of extracted genomic DNA, 100 nM of each padlock probe, ligation buffer, and DNA ligase.

[0055] The reaction system was placed in a constant temperature water bath at 37 degrees Celsius and incubated for 1-2 hours to perform hybridization and ligation reactions between the padlock probe and the target gene.

[0056] Add Phi29 DNA polymerase reaction buffer, 4 mM dNTPs, and 10 U Phi29 DNA polymerase to the ligation reaction system to make the total volume reach 50 μL.

[0057] The reaction system was placed in a constant temperature water bath at 37 degrees Celsius and incubated for 2-3 hours to perform rolling circle amplification reaction.

[0058] In a 20 μL reaction system, add 10 μL RCA amplification product, 50 nM Cas13a protein, 100 nM corresponding guide RNA, Cas13a reaction buffer, and 100 nM HRP-labeled single-stranded reporter molecule.

[0059] Place the reaction system in a constant temperature water bath at 37 degrees Celsius and incubate for 30-60 minutes to perform the CRISPR-Cas cleavage reaction.

[0060] Finally, for analysis, add an appropriate amount of TMB substrate solution to the system after the CRISPR-Cas cleavage reaction, incubate at room temperature in the dark for 10-15 minutes, and observe the color change of the solution.

[0061] When the solution color turns blue, two moles of sulfuric acid solution is added to terminate the reaction, and then the absorbance value is measured at a wavelength of 450 nm using a microplate reader.

[0062] Using the test results of healthy volunteer blood samples as a negative control, an absorbance threshold is set. When the sample's absorbance value is above the threshold, it is considered positive, indicating the presence of the corresponding drug-resistant gene mutation; when the sample's absorbance value is below the threshold, it is considered negative.

[0063] The test results of blood samples from patients with multidrug-resistant tuberculosis and patients with sensitive tuberculosis were statistically analyzed, and the sensitivity, specificity, and accuracy of the detection system were calculated.

[0064] In summary, as Figure 1-Figure 4 As shown, the present application proposes an RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening, which solves the problem that existing detection systems are often used to detect through a single mutation detection system. This detection method cannot meet the needs of multi-target screening of drug-resistant tuberculosis, and the multi-channel detection platform has problems such as complex operation and high cost, which is not conducive to subsequent screening and detection. The system is not only suitable for the detection of multidrug-resistant tuberculosis and sensitive tuberculosis in sputum samples, but can also effectively identify drug-resistant gene mutations in blood samples. By setting sputum of non-tuberculosis patients and blood of healthy volunteers as negative controls, its stable detection ability in different sample types is further verified, providing a reliable solution for the detection of diverse clinical samples.

[0065] In sputum sample testing, positive results are determined by the intensity of the fluorescent signal, which can accurately distinguish between multidrug-resistant and sensitive tuberculosis patients; blood sample testing is determined by the absorbance value threshold. Both detection methods can accurately identify drug-resistant gene mutations. After statistical analysis, the system has high sensitivity, specificity and accuracy, and can effectively meet the clinical needs for accurate diagnosis of drug-resistant tuberculosis.

[0066] The exponential amplification of target genes by rolling circle amplification technology, combined with the dual specific recognition mechanism of padlock probes and CRISPR-Cas system, enables the precise detection of low-abundance mutant genes in the examples, effectively avoiding false positive results.

[0067] The circular probe array designed for multiple drug-resistance-related genes such as rpoB, katG, and inhA can complete the simultaneous detection of multiple mutations in the same reaction system, which greatly shortens the detection time compared with traditional methods and improves clinical diagnosis efficiency.

[0068] The entire detection process is integrated into the microfluidic chip and automated through microchannel connections. It does not require complex instruments and equipment, and the reagent cost is low. The operation in the embodiment shows its adaptability to primary medical institutions and on-site testing, which is conducive to promotion and application in resource-limited areas.

[0069] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0070] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening, comprising a sample processing module, an RCA isothermal amplification module, a CRISPR-Cas multiple detection module, and a signal output module, characterized in that: The sample processing module is used to lyse the sample and enrich drug resistance-related genes; The RCA isothermal amplification module includes a sample lysis module and a nucleic acid enrichment module, and the RCA isothermal amplification module is used to perform rolling circle amplification on the target gene; The CRISPR-Cas multiple detection module includes a constant temperature reaction module and a multi-channel circular probe module, and the CRISPR-Cas multiple detection module is used to identify mutation sites and generate detection signals; The signal output module includes a fluorescence detection module and a data processing module, and the signal output module is used to collect and analyze detection signals.

2. The RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening according to claim 1, characterized in that: The RCA isothermal amplification module comprises a circular probe array targeting the rpoB, katG, and inhA genes of Mycobacterium tuberculosis, and the circular probe sequence is complementary to the hotspot region of drug-resistant mutation.

3. The RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening according to claim 1, characterized in that: The RCA isothermal amplification module uses the padlock probe connected into a circle as a template and uses Phi29 DNA polymerase to perform rolling circle amplification under constant temperature conditions to generate long-chain amplification products with thousands of repeating sequences.

4. The RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening according to claim 1, characterized in that: The CRISPR-Cas multiplex detection module uses Cas12a and Cas14a proteins in conjunction with multiple sets of guide RNAs, each of which is modified with a fluorescent group at its 5' end and a quenching group at its 3' end.

5. The RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening according to claim 1, characterized in that: The CRISPR-Cas multiple detection module includes Cas protein and specific guide RNA. The guide RNA sequence is complementary to a specific region containing a mutation site in the RCA amplification product. The Cas protein can activate nuclease activity to cut single-stranded DNA or RNA after the guide RNA hybridizes with the RCA amplification product to form a double-stranded structure.

6. The RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening according to claim 1, characterized in that: The signal output module adopts multi-channel fluorescence detection technology and can simultaneously identify multiple fluorescence signals such as FAM, HEX, ROX, etc.

7. The RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening according to claim 1, characterized in that: The multi-channel circular probe module is a series of padlock probes designed for multiple key mutation sites of Mycobacterium tuberculosis resistance-related genes. Both ends of the padlock probes have complementary sequences to the target DNA and can specifically identify specific mutation sites.

8. The RCA-CRISPR multiple mutation detection system for drug-resistant tuberculosis screening according to claim 1, characterized in that: The sample processing module, RCA isothermal amplification module, CRISPR-Cas multiple detection module and signal output module are all integrated into a microfluidic chip. The modules are connected through microchannels to achieve automated operations from sample processing to result output.

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