Integrated nucleic acid detection method and device based on RPA-CRISPR / Cas12a and lateral flow chromatography and application

Abstract

The invention discloses an integrated device and method for nucleic acid detection. The device comprises a heating base, a disposable reaction bin in which an RPA amplification reagent is pre-stored, a disposable hose which is coated with a CRISPR / Cas12a detection reagent and enzyme-removed water in a segmented manner, and a push-pull test strip. According to the system, constant-temperature amplification, CRISPR detection and chromatography reading are integrated in a closed device by virtue of fluid control of pre-storing reagents and sequentially extruding hoses, so that an integrated detection process of'sample feeding-result discharging 'is realized, the operation is simple and convenient, pollution can be effectively prevented, and the system is suitable for on-site and basic nucleic acid rapid detection.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection, and in particular to an integrated nucleic acid detection method, device and application based on isothermal amplification of nucleic acid, CRISPR-Cas12a system and lateral flow chromatography. Background Technology

[0002] In recent years, molecular biology detection technologies have played a vital role in pathogen diagnosis, genotyping, genetic disease screening, and food safety analysis. Traditional nucleic acid detection methods, such as polymerase chain reaction (PCR), while possessing high sensitivity and specificity, rely on complex instruments, specialized operators, and lengthy testing cycles, making them unsuitable for point-of-care testing (POCT) and home testing needs. Therefore, developing a simple, rapid nucleic acid detection technology that requires no complex equipment has become a current research hotspot.

[0003] CRISPR-Cas, as an emerging gene-editing tool, offers a highly specific and sensitive solution for nucleic acid detection due to its derivative technology, CRISPR-Cas12a, which possesses unique trans-cleavage activity. This activity allows for non-specific cleavage of reporter molecules after recognizing the target nucleic acid. However, traditional CRISPR-Cas12a detection methods typically require fluorescence reading devices or electrochemical detection platforms, limiting their application in resource-constrained scenarios. Lateral flow strips (LFS), as a low-cost, portable, and visual detection tool, have been widely used in immunoassay, but their application in nucleic acid detection still faces challenges such as insufficient sensitivity and complex operation procedures.

[0004] To address the aforementioned issues, this invention proposes a nucleic acid detection method based on an integrated "sample-to-answer" CRISPR-Cas12a system combined with a transverse flow test strip. This method innovatively integrates recombinase polymerase amplification (RPA), the CRISPR-Cas12a detection reaction, and test strip signal reading into a single integrated detection device. This device achieves rapid, highly sensitive, highly specific, and closed-tube contamination-free nucleic acid detection. Through a nucleic acid extraction-free design, raw samples can be directly used for detection, further simplifying the operation process. The device features ease of operation, high portability, and visualized results, making it suitable for various scenarios such as point-of-care testing (POCT) and home testing. This invention not only significantly simplifies the complex process of traditional nucleic acid detection and greatly reduces detection costs by eliminating the need for expensive instruments and equipment, but also provides a new, efficient, reliable, and easily scalable solution for the field of molecular diagnostics. Summary of the Invention

[0005] In view of this, the purpose of this invention is to overcome the aforementioned defects and shortcomings in existing nucleic acid detection technologies and provide a method for detecting nucleic acids based on the RPA-CRISPR-Cas12a system. Combined with a portable nucleic acid detection device, this method achieves highly sensitive, highly specific, and visualized home testing of nucleic acids using a one-pot CRISPR-Cas12a system combined with a transverse flow test strip, enabling integrated "sample-to-answer" testing. This method significantly improves the convenience and practicality of testing through its nucleic acid extraction-free design, efficient combination of isothermal amplification and CRISPR-Cas12a detection, and visualized reading of the test strip signal.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: (1) Place the clinical sample (such as a swab) in a buffer solution and thermally lyse it in a metal bath at 95°C for 10 minutes to release the DNA; (2) Using the DNA from step (1) as a template, perform RPA isothermal amplification in the reaction chamber: The amplification system includes: upstream primers for isothermal amplification, downstream primers for isothermal amplification, ddH2O, isothermal amplification reagent, HPV nucleic acid template (HPV nucleic acid template of the sample to be tested), buffer solution; and ultrapure water (as a negative control). (3) CRISPR / Cas detection: The CRISPR / Cas system was added to the tubing. The reaction system included: crRNA, Cas12a, a single-stranded DNA reporter molecule modified with fluorescent and biotinylated groups, DTT, and 10×NEBuffer 2.1. (4) Start the portable device to perform RPA amplification, keep the temperature at 37℃, and react for 20 min; (5) Squeeze tube 1 to allow the CRISPR / Cas reaction reagent to flow into the reaction chamber, keep the temperature constant at 37℃, and react for 20 min. (6) Squeeze hose 2 to allow DE water to flow into the reaction chamber; (7) By pushing the fixed tube down along the test paper tube, the colloidal gold test paper sample pad is immersed in the reaction solution. Observe the color reaction of the test paper strip after 5 minutes.

[0007] The technical solution provided by this invention is an integrated nucleic acid detection method based on an integrated "sample-to-answer" CRISPR-Cas12a system combined with a transverse flow test strip; The design principle of the crRNA is that the 5' end of the crRNA target sequence should have a 5'-TTN-3' sequence, and the crRNA sequence (including the crRNA backbone sequence) and the crRNA target sequence should not form a stable secondary structure. The Cas12a protein is a Cas12a protein that has both endonuclease activity and accessory nucleic acid cleavage activity, such as LbCas12a, FnCas12a, AsCas12a, ScCas12a, etc. The sequences of the isothermal amplification primers are amplification primers that meet the amplification primer design principles of the present invention and are targeted at the detection target. Furthermore, the detection system also includes isothermal amplification reagents, CRISPR buffer, and single-stranded DNA reporter molecules modified with fluorescent and biotinylate groups; Furthermore, the isothermal amplification reagent consists of: RPA enzyme preparation and RPA reaction buffer; Furthermore, the CRISPR buffer (1×) is composed of: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, pH 7.9; Furthermore, the single-stranded DNA reporter molecule modified with fluorescent and biotin groups is a single-stranded DNA with fluorescent and biotin groups modified at both ends, respectively. The fluorescent groups include, but are not limited to, FAM, VIC, HEX, TET, JOE, Cy5, Cy3, TAMRA, etc. Preferably, the single-stranded DNA reporter molecule modified with fluorescent and biotin groups is: 5'FAM-TTATT-Biotin3'. Beneficial effects

[0008] (1) Isothermal and simple: Compared with traditional PCR technology, the RPA-CRISPR / Cas12a system completes the reaction under isothermal conditions, without the need for complex thermal cycling equipment. It is simple, fast and portable, and suitable for environments with limited resources. (2) Rapid: The entire time of RPA-CRISPR / Cas12a HPV nucleic acid detection (including nucleic acid release, isothermal amplification, CRISPR / Cas12a detection and signal reading) does not exceed one hour, significantly shortening the detection cycle.

[0009] (3) High sensitivity and high specificity: Combining RPA and CRISPR / Cas12a dual signal amplification and specific recognition, the detection sensitivity reaches 10 copies / reaction, which can specifically identify high-risk HPV, while showing no detection signal for low-risk HPV, Candida, yeast, herpes simplex virus, coronavirus, group B streptococcus, Salmonella, Staphylococcus aureus, and human genome. The detection sensitivity and specificity for 14 types of high-risk HPV clinical samples are both 100%.

[0010] (4) Visualization: The results can be interpreted visually through the color reaction of the test strip, without relying on complex instruments; (5) Low cost: No expensive instruments or equipment are required, and the reagent cost is low, which significantly reduces the testing cost and is suitable for large-scale screening and home testing; (6) Integrated design: The isothermal amplification, CRISPR / Cas12a detection and signal reading are integrated into a portable device to achieve one-pot operation, avoid cross-contamination and improve the reliability and convenience of detection; (7) Wide applicability: This method is not only applicable to HPV detection, but can also be extended to other pathogen detection, genotyping and genetic disease screening by adjusting crRNA and primer design. Attached Figure Description

[0011] Figure 1 This section presents the detection principle of RPA-CRISPR / Cas12a, a comparison of results from different lysis methods, and their feasibility. (A shows (a) a schematic diagram of the test strip reaction principle illustrating the RPA-CRISPR / Cas12a detection principle; (b) a schematic diagram of the portable nucleic acid detection device, including a heating base, disposable reaction chamber, tubing, and test strip pusher. B compares different DNA extraction methods. The thermal decomposition method requires only one heating treatment (95℃, 10 minutes) for rapid extraction. The proteinase K method (56℃, 15 minutes) and the column-based detection kit method (60 minutes) require prolonged heating and multiple purification steps. C compares the PCR amplification results of the three extraction methods. D shows the analysis results of the gray value of the T line on the test strip.) Figure 2Sensitivity study of RPA-CRISPR / Cas12a detection. (A shows the analysis results of the T-line grayscale values ​​on the sensitivity test strip for HPV16 detection using the CRISPR / Cas12a lateral flow strip detection method. B shows the test strip results for the sensitivity of HPV16 detection using the CRISPR / Cas12a lateral flow strip detection method. C shows the analysis results of the T-line grayscale values ​​on the sensitivity test strip for HPV18 detection using the CRISPR / Cas12a lateral flow strip detection method. D shows the test strip results for the sensitivity of HPV18 detection using the CRISPR / Cas12a lateral flow strip detection method.) Figure 3 This study examines the specificity of RPA-CRISPR / Cas12a detection. (A shows the analysis results of the T-line grayscale values ​​on the specificity test strip for HPV16 detection using the CRISPR / Cas12a-based lateral flow strip detection method. B shows the specificity test strip results for HPV16 detection using the CRISPR / Cas12a-based lateral flow strip detection method.) Figure 4 This study is for a clinical sample study of RPA-CRISPR / Cas12a detection. (A shows the thermographic analysis results of the T-line grayscale values ​​on the test strip. B shows the visual results of the test strip.) Detailed Implementation

[0012] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0013] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0014] Example 1: Establishment of an HPV detection system based on RPA and CRISPR / Cas and its integrated one-pot detection method The RPA isothermal amplification system included 2.4 μL (10 μmol / L) upstream RPA amplification primers, 2.4 μL (10 μmol / L) downstream RPA amplification primers, 11.2 μL ddH2O, 29.5 μL Rehydration Buffer, 2.5 μL MgoAc (280 mmol / L), and 2 μL HPV viral plasmid template. The RPA amplification program was: isothermal 37℃ for 20 min. The upstream and downstream RPA amplification primers, ddH2O, Rehydration Buffer, and MgoAc were obtained from the RPA kit (Amp-Future). The CRISPR / Cas detection system consists of 10 μM crRNA, 0.5 μL Cas12a, 10 μL 2 μM fluorescent probe, and 1×CRISPR buffer, with ddH2O added to bring the total volume to 100 μL.

[0015] Add the RPA reaction system and target to a disposable reaction chamber, and the CRISPR system to a disposable tubing. Press the device switch to perform RPA amplification. After 20 minutes, the target DNA is amplified in the reaction chamber. Squeeze the end of the tubing to allow the CRISPR system to flow into the reaction chamber, and press the switch again to perform the CRISPR reaction. After the reaction is complete, squeeze the disposable tubing containing DE water to allow it to flow into the reaction chamber. Press the pusher of the test strip until the sample pad of the test strip is immersed in the reaction solution. Observe the color development result after 1 minute.

[0016] Example 2: Specificity study of RPA-CRISPR / Cas12a in detecting high-risk HPV According to the detection reagents and methods in Example 1, the genomes of low-risk HPV (HPV6), Candida, Candida albicans, herpes simplex virus, Group B Streptococcus, Escherichia coli, Staphylococcus aureus, Salmonella, novel coronavirus, influenza A virus, HeLa cells, and human cells were detected. Water was used as a negative control group to analyze the specificity of the detection system provided by the present invention.

[0017] The specific method involves using high-risk HPV16 and HPV18 genomes as positive controls, and low-risk HPV (HPV6), Candida, Candida albicans, herpes simplex virus, Group B Streptococcus, Escherichia coli, Staphylococcus aureus, Salmonella, novel coronavirus, influenza A virus, and HeLa cell genomes as test samples. Amplification reactions and CRISPR / Cas12a detection are performed according to the detection system described in Example 1, and signal readings and analysis are conducted by observing the color development results of the test strips. Specifically, primers and crRNA corresponding to HPV16 are used for HPV16 detection, and primers and crRNA corresponding to HPV18 are used for HPV18 detection. For other microorganisms and human genomes, two independent detections are performed using primers and crRNA corresponding to HPV16 and HPV18, respectively. The experimental results showed that, compared with the negative control group, only the high-risk HPV16 and HPV18 detection groups showed significant signal enhancement (P < 0.0001). However, the detection of low-risk HPV, Candida albicans and other microorganisms, as well as human genomes, showed no significant signal enhancement (no statistical difference). These results indicate that the RPA-CRISPR / Cas12a detection system established in this invention has high specificity for high-risk HPV (HPV16 and HPV18), can effectively distinguish low-risk HPV and other common pathogenic microorganisms and human genomes, and no cross-reaction was observed. This provides reliable technical support for the specific detection of high-risk HPV.

[0018] Example 3: Sensitivity study of RPA-CRISPR / Cas12a in detecting high-risk HPV To determine the sensitivity of the RPA-CRISPR / Cas-based HPV detection system and method, a mixed sample with known HPV viral copy numbers was diluted to 10⁻⁶. 0 copies / μL, 10 1 copies / μL, 10 2 copies / μL, 10 3 copies / μL, 10 4 copies / μL, 10 5 Six concentration gradients were used as templates, and amplification and detection were performed according to the detection system in Example 1.

[0019] The results are shown in the figure, 10 1 ~10 5 After amplification of the template at 1 copy / μl for 20 min, the gray value signal of the C-line was significantly higher than that of the negative control, and the difference was statistically significant (P < 0.0001). These results indicate that the HPV detection system and procedure based on RPA and CRISPR / Cas established in this invention can detect samples containing 10 copies of HPV.

[0020] Example 4: Detection of known clinical samples This application uses the RPA-CRISPR / Cas12a detection system of Example 1 to accurately detect 10 HPV16 positive cervical swab samples, 10 HPV18 positive cervical swab samples, 10 other 12 high-risk HPV types, and 10 HPV negative cervical swab samples (using isothermal amplification primers and crRNA corresponding to the sample typing), and compares the CRISPR / Cas detection results with the TaqMan PCR detection results.

[0021] As shown in the figure, the RPA-CRISPR / Cas12a detection system achieved 100% accuracy in detecting both positive and negative samples, which is completely consistent with the results of TaqMan PCR. These results demonstrate that the RPA-CRISPR / Cas12a detection system established in this invention possesses 100% sensitivity, accuracy, and specificity for detecting 14 high-risk HPV types. It can accurately distinguish between HPV-positive and negative samples, providing highly reliable results and is suitable for precise detection of clinical samples.

[0022] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments and drawings.

Claims

1. A nucleic acid detection method based on an integrated "sample in - result out" CRISPR-Cas12a system combined with a transverse flow test strip, characterized in that, The method uses a detection device, which includes a heating base, a disposable reaction chamber, a disposable tubing, and a push-pull test strip lever; the disposable reaction chamber is pre-coated with a constant-temperature nucleic acid amplification premix, and the disposable tubing is divided into sections coated with CRISPR / Cas12a premix and nuclease-free water.

2. The nucleic acid detection method according to claim 1, characterized in that, The isothermal amplification premix contains: upstream primers for isothermal amplification, downstream primers for isothermal amplification, isothermal amplification reagents, target gene nucleic acid template, buffer solution, and ultrapure water.

3. The nucleic acid detection method according to claim 1 or 2, characterized in that, The CRISPR / Cas12a premix contains: crRNA, Cas12a protein, a single-stranded DNA reporter molecule with fluorescent and biotinylated groups, DTT, and 10×NEBBuffer 2.

1.

4. The nucleic acid detection method according to any one of claims 1-3, characterized in that, The push-pull test strip rod is loaded with a transversely flowing test strip, which includes a sample pad, a gold label pad, a nitrocellulose membrane, and an absorbent pad, and is fixed as a whole on a PVC base plate.

5. The nucleic acid detection method according to any one of claims 1-4, characterized in that, The target gene nucleic acid template is obtained through any of the following methods: (1) Heat the bacterial solution at 90-100℃ for 3-10 minutes; (2) Add proteinase K to the bacterial culture and heat at 56°C for 5-20 minutes; (3) Use a nucleic acid extraction kit for washing and elution.

6. A nucleic acid detection device for implementing the method according to any one of claims 1-5, characterized in that, It includes a heating base, a disposable reaction chamber, two disposable tubing tubes, and a push-pull test strip lever; the heating base is equipped with a constant temperature heating pad, and the push-pull test strip lever is equipped with a transparent observation window at the corresponding position.

7. The nucleic acid detection device according to claim 6, characterized in that, The disposable reaction chamber is pre-filled with a temperature-controlled amplification premix; the disposable tubing is pre-filled with CRISPR / Cas12a premix and nuclease-free water, respectively.

8. A method for nucleic acid detection using the apparatus of claim 6 or 7, characterized in that, Includes the following steps: S1: Add the sample to be tested into the disposable reaction chamber and start the isothermal amplification reaction; S2: Squeeze the disposable tubing to allow the coated CRISPR / Cas12a premixed solution to flow into the reaction chamber for the Cas12a cutting reaction; S3: Continue to squeeze the disposable tubing to allow the coated nuclease-free water to flow into the reaction chamber; S4: Push the test strip lever to immerse the sample pad of the test strip into the reaction mixture. After reacting for 30 seconds to 5 minutes, read the colorimetric results through the transparent observation window.

9. The method according to claim 8, characterized in that, The isothermal amplification reaction is a recombinase polymerase-mediated isothermal amplification reaction, including but not limited to RPA, RAA, and MIRA isothermal amplification reactions.

10. The application of the method according to any one of claims 1-5 or the apparatus according to any one of claims 6-7 in nucleic acid detection in non-disease diagnostic fields.

Images