Dual amplification nucleic acid detection method based on crispr-cas12a

Abstract

This invention proposes a CRISPR-Cas12a bioassay platform based on AuNPs-labeled magnetic probes for HIV-related nucleic acid detection. Nucleic acid amplification strategies are commonly used to improve sensitivity, but they suffer from drawbacks such as long processing time, complex operation, high risk of contamination, and mismatch-related inaccuracies. This invention proposes a method that eliminates the need for traditional nucleic acid amplification. It achieves sensitive detection of target nucleic acids by combining the self-amplification effect of CRISPR-Cas12a with a dual amplification method utilizing the strong signal detectable by gold nanoparticles in inductively coupled plasma mass spectrometry. The method of this invention achieves a low detection limit of 1.05 amol for HIV-related nucleic acids and has been successfully validated in spiked recovery experiments using fetal bovine serum.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, and in particular to a dual-signal amplification method that combines CRISPR-Cas12a trans cleavage and gold nanoparticle labeling. Background Technology

[0002] In quantitative detection methods for nucleic acids, nucleic acid amplification strategies are frequently used to amplify target sequences to improve detection performance. These methods can effectively amplify target nucleic acids by several orders of magnitude. However, nucleic acid amplification methods also have some drawbacks: complex operation, long processing time, risk of contamination due to multi-step operations, and uncertainties related to mismatches; nucleic acid amplification methods can produce non-specific amplification byproducts that increase background signal; and performing this experiment requires sophisticated instruments and highly skilled technicians. Therefore, researchers believe that developing non-nucleic acid amplification signal amplification methods for quantitative nucleic acid detection is an effective alternative.

[0003] Clustered regularly interspaced short palindromic repeats (CRISPR) have attracted widespread attention as an emerging gene editing tool. Since the indiscriminate cleavage of side-stranded single-stranded DNA (ssDNA) by the Cas12a protein in 2018 (i.e., trans-cleavage), diagnostic technologies based on the CRISPR-Cas system have been extensively studied. The trans-cleavage ability of the Cas12a protein is key to establishing related biosensing technologies. The encoding crRNA binds to Cas12a first, and when the target sequence successfully hybridizes and complements the coding portion of the crRNA, the CRISPR-Cas12a system is activated to trans-cleave side-stranded ssDNA. This cleavage activity of side-stranded ssDNA can reach up to 1250 times per second, exhibiting a considerable self-amplification effect.

[0004] Meanwhile, biosensing methods based on stable metal isotope labeling have also been widely reported. These methods mainly include two types: metal ion labeling and metal nanoparticle labeling. Compared with metal ions, metal nanoparticles contain a large number of metal atoms, and after digestion, they can be detected by ICP-MS with a stronger elemental signal, which can effectively improve the sensitivity of the method. Summary of the Invention

[0005] The purpose of this invention is to propose a dual-signal amplification method that combines CRISPR-Cas12a trans-cleavage and gold nanoparticle labeling. This method avoids the risks associated with traditional nucleic acid amplification methods, simplifies the procedure, and reduces high blank background signal caused by nonspecific amplification products.

[0006] The CRISPR-Cas12a-based double amplification method of the present invention is used for inductively coupled plasma mass spectrometry (ICP-MS) detection of HIV-DNA, comprising the following steps:

[0007] 1. Preparation of AuNPs;

[0008] 2. Preparation of AuNPs-labeled magnetic probes;

[0009] 3. Trans-cleavage reaction of CRISPR-Cas12a;

[0010] 4. Perform magnetic separation on the product from step 3, collect the supernatant, and send it to ICP-MS for detection;

[0011] 5. Calculate the target DNA content based on the measured results.

[0012] The dual-signal amplification nucleic acid detection method of this invention utilizes the characteristic of the target nucleic acid to activate the CRISPR-Cas12a trans-cleavage activity, causing the AuNPs-labeled magnetic oligonucleotide probes to be cleaved. The released AuNPs are then magnetically separated, collected, digested, and detected. This process converts the signal of the target nucleic acid into... 197 The signal detected by Au in ICP-MS. The method of this invention enables sensitive detection of low concentrations of HIV-DNA in a short time. This not only saves detection time and cost but also avoids the generation of non-specific amplification products associated with nucleic acid amplification steps. Attached Figure Description

[0013] Figure 1 In 10 -16 ~10 -13 HIV-DNA and within the range of mol 197 Linear relationship of Au's ICP-MS signal.

[0014] Figure 2 In 10 -12 ~10 -10 HIV-DNA and within the range of mol 197 Linear relationship of Au's ICP-MS signal.

[0015] Figure 3 Results of spiked recovery experiment of HIV-DNA in fetal bovine serum.

[0016] Figure 4 Results of HIV-DNA selectivity detection using a CRISPR-Cas12a double amplification method. Detailed Implementation

[0017] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0018] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. The implementation conditions used in the examples may be further adjusted according to the specific manufacturer's conditions, and the implementation conditions not specified are generally those in routine experiments.

[0019] Example 1: HIV-DNA detection.

[0020] (1) Synthesis of AuNPs

[0021] The entire reaction was carried out in a three-necked round-bottom flask. First, 100 mL of 0.01% HAuCl4 solution was added, boiled, and refluxed. After boiling, 1 mL of freshly prepared 3% sodium citrate solution was quickly added, and heating was continued until the solution turned wine-red. After cooling to room temperature, it was stored in a refrigerator at 4°C for later use.

[0022] (2) Preparation of AuNPs-labeled magnetic probes

[0023] The first step is the ligation of AuNPs to reporter DNA. We used the classic salt aging method to ligate AuNPs and reporter DNA: First, the reporter DNA was treated in 10 mM TCEP solution for 1 hour. Then, 120 μL of the treated reporter DNA (10 μmol / L) was added to 1 mL of AuNP solution, followed by 10 μL of 0.1% Tween-20 solution and 10 μL of freshly prepared 0.5 mol / L sodium citrate solution (pH = 7.0). Subsequently, 200 μL of 3 mol / L NaCl solution was added in eight portions, bringing the final NaCl concentration in the mixture to 0.5 mol / L. Each addition was spaced 20 minutes apart and sonicated for 30 seconds to prevent AuNPs from agglomerating under high salinity. After the NaCl addition was complete, the mixture was allowed to react at room temperature for at least 6 hours to ensure sufficient reporter DNA was immobilized on the AuNP surface. The solution was then centrifuged at 16,500 rpm for 12 minutes and washed three times with 20 mmol / L PB buffer (pH = 7.4, main components NaH₂PO₄ and Na₂HPO₄) to remove excess unreacted reporter DNA from the mixture, yielding AuNPs-reporter. This was then dispersed in 1 mL of 10 mmol / L Tris-HCl buffer (containing 100 mmol / L NaCl) for later use.

[0024] The second step involves immobilizing the AuNPs-reporter on the surface of SA-MB. First, 20 μL of a 10 mg / mL SA-MB dispersion was washed three times with binding buffer (10 mmol / L Tris-HCl, 0.1% PEG 4000, 0.1% Tween 20, 50 mmol / L NaCl) and diluted to 40 μL. Then, 1 mL of the AuNPs-reporter in Tris-HCl solution was added. After reacting overnight at room temperature, the solution was magnetically separated, and the supernatant was collected. The supernatant was then washed three times with a 40 mmol / L Hepes solution (containing 100 mmol / L NaCl). Finally, the supernatant was dispersed in 40 μL of a 40 mmol / L Hepes solution for later use. The final product is the AuNPs-labeled magnetic probe solution.

[0025] (3) Trans-cleavage reaction of CRISPR-Cas12a

[0026] The total volume of the reaction solution was 100 μL, containing 1× TOLO buffer 3 and 1.5 mmol / L Mg2+. 2 + 0.1 μmol / L crRNA, 30 nmol / L Cas12a, and amounts of substance from 10 -16 ~10 -10 1 mol of HIV-DNA and 2.25 μmol / L of AuNPs-labeled magnetic probe solution prepared in step (2) were finally diluted to 100 μL with DEPC-treated water.

[0027] Reaction conditions: Reaction at 37℃ for 40 minutes.

[0028] (4) Elimination of AuNPs

[0029] The mixture after the above reaction was magnetically separated using a magnet, the supernatant was collected, and then digested with 50 μL of aqua regia (HCl:HNO3 = 3:1) for 20 minutes.

[0030] (5) ICP-MS detection

[0031] The solution after the above reaction was diluted to 4 mL with ultrapure water, and then detected by ICP-MS. 197 Au isotopic signals.

[0032] (6) Test Results

[0033] like Figure 1 and Figure 2 As shown, in a 100 μL reaction system, HIV-DNA was measured at 10... -16~10 -13 mol and 10 -12 ~10 -10 The two mol ranges were compared with those measured by ICP-MS. 197 Au isotope signals exhibit a good linear relationship. Their linear regression equations are I = 8268.2lgn + 150067 and I = 65035lgn + 872714, with correlation coefficients of 0.996 and 0.987, respectively, where I represents... 197 The ICP-MS intensity of Au was used, where n represents the amount of HIV-DNA. The detection limit of this method was calculated to be 1.05 amol.

[0034] Example 2: Spiked recovery of HIV-DNA from fetal bovine serum.

[0035] (1) Prepare fetal bovine serum samples containing different amounts of HIV-DNA.

[0036] First, fetal bovine serum purchased from Shanghai Sangon Biotech Co., Ltd. was diluted 10 times with DEPC-treated water, and then different concentrations of HIV-DNA were added to prepare a series of test samples.

[0037] (2) Synthesis of AuNPs

[0038] The synthesis process is the same as in Example 1.

[0039] (3) Preparation of AuNPs-labeled magnetic probes

[0040] The preparation process is the same as in Example 1.

[0041] (4) Trans-cleavage reaction of CRISPR-Cas12a

[0042] The total volume of the reaction solution was 100 μL, containing 1× TOLO buffer 3 and 1.5 mmol / L Mg2+. 2 + The sample consisted of 0.1 μmol / L crRNA, 30 nmol / L Cas12a, different amounts of HIV-DNA-spiked fetal bovine serum samples, and 2.25 μmol / L AuNPs-labeled magnetic probe solution prepared in step (3). Finally, the solution was diluted to 100 μL with DEPC-treated water.

[0043] Reaction conditions: Reaction at 37℃ for 40 minutes.

[0044] (5) ICP-MS detection

[0045] The solution after the above reaction was diluted to 4 mL with ultrapure water, and then detected by ICP-MS. 197Au isotopic signals.

[0046] (6) Test Results

[0047] like Figure 3 As shown, when using the method of the present invention to detect HIV-DNA-spiked fetal bovine serum samples, the recovery rate of HIV-DNA was between 91.1% and 110%, while the relative standard deviation was between 1.56% and 4.43%, proving that the method of the present invention can be applied to the nucleic acid detection of complex biological samples.

[0048] Example 3: Selectivity experiment for detecting HIV-DNA using a CRISPR-Cas12a-based double amplification method.

[0049] (1) The selected viral nucleic acids are:

[0050] HAV, HBV, HCV, and HPV-16.

[0051] (2) Synthesis of AuNPs

[0052] The synthesis process is the same as in Example 1.

[0053] (3) Preparation of AuNPs-labeled magnetic probes

[0054] The preparation process is the same as in Example 1.

[0055] (4) Trans-cleavage reaction of CRISPR-Cas12a

[0056] The total volume of the reaction solution was 100 μL, containing 1× TOLO buffer 3 and 1.5 mmol / L Mg2+. 2 + 0.1 μmol / L crRNA, 30 nmol / L Cas12a, 10 nmol / L HIV-DNA or 100 nmol / L HAV, HBV, HCV, HPV-16 and 2.25 μmol / L AuNPs-labeled magnetic probe solution prepared in step (3), and finally diluted to 100 μL with DEPC-treated water.

[0057] Reaction conditions: Reaction at 37℃ for 40 minutes.

[0058] (5) ICP-MS detection

[0059] The solution after the above reaction was diluted to 4 mL with ultrapure water, and then detected by ICP-MS. 197 Au isotopic signals.

[0060] (6) Test Results

[0061] like Figure 4 As shown, except for HIV-DNA, other viral nucleic acid sequences were not clearly detected in ICP-MS. 197 The isotopic signals of Au demonstrate the high specificity of this method. sequence list <110> Sichuan University <120> CRISPR-Cas12a-based double amplification nucleic acid detection method <141> 2022-02-11 <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 42 <212> RNA <213> crRNA(2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 1 uaauuuucua cuaaguguag auauguggaa aaucucuagc ag 42 <210> 2 <211> 75 <212> DNA <213> reporter DNA(2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 2 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60 tttttttttt ttttt 75 <210> 3 <211> twenty one <212> DNA <213> HIV‑DNA(2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 3 actgctagag attttccaca t 21 <210> 4 <211> twenty three <212> DNA <213> HAV(2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 4 ggacttgata cctcaccgcc 20 <210> 5 <211> 30 <212> DNA <213> HBV(2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 5 ataccacatc atccatataa ctgaaagcca 30 <210> 6 <211> 31 <212> DNA <213> HCV(2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 6 atctccaggc attgagcggg tttatccagg a 31 <210> 7 <211> 39 <212> DNA <213> HPV‐16(2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 7 aatatgtcat tatgtgctgc catatctact tcagaaact 39

Claims

1. A double-amplification nucleic acid detection method based on CRISPR-Cas12a, characterized by, The double amplification is double signal amplification, and the double signal amplification comprises gold nanoparticle labeling and CRISPR-Cas12a trans cleavage, and specifically comprises the following steps: Preparation of gold nanoparticle solution: after boiling the HAuCl4 solution, 3% sodium citrate solution is added, heating is continued until the solution turns into wine red, and then the heating is stopped, and the gold nanoparticle solution, namely AuNPs solution, is obtained after cooling; Preparation of gold nanoparticle-labeled magnetic probe solution: after treating the reporter DNA in the TCEP solution, the AuNPs solution is added, and 0.1% Tween-20 solution and 0.5 mol / L sodium citrate solution are added to obtain a mixed solution; NaCl solution is added in the mixed solution in batches and ultrasonic is performed, and then the solution is reacted at room temperature for more than 6 hours, and after centrifugation and PB flushing, AuNPs-reporter is obtained, and the precipitate is dispersed in Tris-HCl buffer to obtain AuNPs-reporter Tris-HCl solution; the SA-MB dispersion liquid is diluted by 1 times, and then added into the AuNPs-reporter Tris-HCl solution to react overnight, magnetic separation is performed, and the supernatant is collected, and the magnetic precipitate is washed and dispersed by Hepes solution to obtain the gold nanoparticle-labeled magnetic probe solution; the main structure of the gold nanoparticle-labeled magnetic probe is a reporter DNA containing 75 thymine, the 5' end of which is connected with SA-MB magnetic nanoparticles to facilitate magnetic separation, and the 3' end is modified with AuNPs as a reporter signal; Trans -cleavage reaction of CRISPR-Cas12a: the reaction system contains 1xTOLO buffer 3 buffer, Mg 2+ , crRNA, Cas12a, gold nanoparticle labeled magnetic probe solution, target nucleic acid and DEPC water; ICP-MS detection: The products of the trans-cleavage reaction were magnetically separated, and the supernatant was collected, digested with aqua regia, and diluted before being sent to ICP-MS for detection 197 Au detection, and the target nucleic acid content was calculated according to the measured results; The above method is for non-disease diagnosis.

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