阅读说明：本技术 一种基于CRISPR/Cas12a蛋白对基因的表面增强拉曼光谱检测方法 (Surface-enhanced Raman spectroscopy detection method for gene based on CRISPR/Cas12a protein ) 是由 徐抒平 苏艾玲 梁重阳 徐蔚青 于 2021-09-06 设计创作，主要内容包括：本发明公开了一种基于CRISPR/Cas12a蛋白对基因的表面增强拉曼光谱检测方法。本发明利用目标病毒核酸与Cas12a、crRNA形成三元复合体,开启切割体系中的底物(即：ssDNA)。ssDNA是探针1和探针2上DNA-(1)和DNA-(2)的桥连互补序列。探针1和探针2是否被桥连可引起SERS信号的变化,即可知待检样品中是否含有目标核酸。利用特异性的crRNA可以实现对病毒核酸的准确快速检测。本发明将CRISPR@Cas12a与SERS技术结合最大的优势是避免繁琐的修饰制备过程,SERS探针可以长期稳定存在。该方法信号放大关键步骤发生在ssDNA的溶液相,而非纳米组装体的界面上,因此具有更稳定和更高反式切割效率,可保障该检测方法的灵敏度和重复性,对疾病早期监测诊断与防控具有重要意义。(The invention discloses a surface-enhanced Raman spectroscopy detection method for a gene based on CRISPR/Cas12a protein. The invention utilizes target virus nucleic acid, Cas12a and crRNA to form a ternary complex, and opens a substrate (namely ssDNA) in a cutting system. ssDNA is DNA on Probe 1 and Probe 2 1 And DNA 2 The bridged complementary sequence of (a). Whether the probe 1 and the probe 2 are bridged can cause the change of the SERS signal, namely, whether the sample to be detected contains the target nucleic acid or not can be known. The accurate and rapid detection of the virus nucleic acid can be realized by utilizing the specific crRNA. The method has the greatest advantage of combining the CRISPR @ Cas12a and the SERS technology, avoids a complicated modification preparation process, and ensures that the SERS probe can stably exist for a long time. The key step of signal amplification in this method occurs in the solution phase of ssDNA, rather than at the interface of the nano-assembly, becauseThe method has more stable and higher trans-cutting efficiency, can ensure the sensitivity and repeatability of the detection method, and has important significance for early monitoring, diagnosis, prevention and control of diseases.)

1. A surface enhanced Raman spectroscopy detection method for genes based on CRISPR/Cas12a protein is characterized in that: the formed CRISPR/Cas12a and crRNA and DNA ternary complex can cause effective cleavage of substrate ssDNA, causing signal change of SERS probe, comprising the following steps:

step (1), respectively modifying DNA on the surfaces of the metal nanoparticles₁And DNA₂Modifying Raman reporter molecules on the surface to prepare a probe 1 and a probe 2; mixing the probe 1 and the probe 2 in equal proportion to prepare a solution 3;

step (2), mixing 15 microliters of CRISPR/Cas12a reaction solution with 1 microliter of crRNA and 4 microliters of DNA solution to be detected for 30 minutes at 37 ℃ to form solution 4; simultaneously mixing 15 microliter CRISPR/Cas12a reaction solution with 1 microliter crRNA and 4 microliter water at 37 ℃ for 30 minutes to form solution 5;

step (3), adding a bridging substrate (ssDNA)6 into the solution 4 and the solution 5 respectively, and reacting at 37 ℃ for 30 minutes to obtain a solution 7 and a solution 8;

step (4), adding the solution 7 and the solution 8 into the solution 3 respectively, and reacting for 2-5 minutes at 50 ℃ to obtain a solution 9 and a solution 10;

detecting the Raman signal intensity of the solution 9 and the solution 10 by using a Raman spectrometer; if the signals are close, the result is proved to be negative; if the two detection signals are obviously different, the result is proved to be positive.

2. The surface-enhanced Raman spectroscopy detection method for genes based on CRISPR/Cas12a protein of claim 1, wherein the metal nanoparticle probe is a nanoparticle with SERS enhancement capability.

3. The CRISPR/Cas12a protein-based gene pair table of claim 1The surface-enhanced Raman spectroscopy detection method is characterized in that the DNA₁And DNA₂One end has a sulfhydryl group and the remaining sequence is complementary to the ssDNA sequence for hybridization.

4. The CRISPR/Cas12a protein-based surface-enhanced Raman spectroscopy detection method for genes as claimed in claim 1, wherein the Raman reporter molecule is a molecule with high Raman activity and can be grafted with the surface of the metal nanoparticles.

5. The CRISPR/Cas12a protein-based surface-enhanced Raman spectroscopy detection method for genes as claimed in claim 1, wherein the crRNA is a 42-44nt RNA sequence and can specifically recognize viral genes.

6. The method for detecting the surface-enhanced Raman spectroscopy of the gene based on the CRISPR/Cas12a protein as claimed in claim 1, wherein the solution 9 is a colloidal solution in a dispersion state under a positive result; in the negative result, the colloidal solution is in an aggregated state.

7. The CRISPR/Cas12a protein-based gene surface-enhanced Raman spectroscopy detection method as claimed in any one of claims 1-4, wherein the solution 3 is a homogeneous and dispersed colloidal solution.

Technical Field

The invention belongs to the technical field of gene detection, and particularly relates to a spectral detection method for a virus gene based on CRISPR/Cas12a protein and Surface Enhanced Raman Scattering (SERS).

Background

The rapid diagnosis of virus types can effectively reduce the large-area spread and infection of viral infectious diseases, and provide important scientific basis for prevention and control policies of the virus types in time. The traditional virus nucleic acid detection method generally has the problems of harsh detection conditions, high cost, long detection time consumption, sensitivity to be improved and the like, so that a rapid, high-sensitivity and specific detection technology for virus nucleic acid is urgently needed to be developed.

In recent years, Surface-enhanced Raman Scattering (SERS) biosensing technology has been widely used for biological detection and analysis due to its ultra-high sensitivity and "fingerprint" property of SERS spectrum. The SERS spectrum is an analysis method capable of obtaining abundant structural information of substances on a molecular scale, and is widely applied to the fields of biology, medicine, environmental detection, explosive detection and the like due to the advantages of rapidness, simplicity, no damage and high sensitivity in analysis. The SERS technology has the following advantages in the application of virus gene detection: (1) the sensitivity of the SERS detection technology is very high, and the level of single molecule detection is achieved; (2) SERS detection has small demand on a sample to be detected, and is a trace detection technology; (3) the Raman scattering of water is very weak, and the in-situ identification and identification can be carried out even if the concentration of an analyte in an aqueous solution is very small, so that a complex sample pretreatment process is not needed; (4) compared with fluorescence technology, SERS is not easy to photobleach. The SERS technology is used in nucleic acid detection, and is mostly reported to be based on the change of the aggregation morphology of the metal nanoparticles caused by nucleic acid hybridization, thereby causing the enhancement or the reduction of the SERS signal. For example, Harpster et al have constructed a virus DNA SERS sensor using gold nanoparticle colloid as the SERS substrate, which uses gold nanoparticle colloid as the SERS substrate and methylene blue as the raman Reporter molecule, and after West Nile Virus (WNV) RNA is added, the sensor is simultaneously hybridized with a methylene blue modified Probe molecule (Reporter Probe) and a thiol modified Capture Probe (Capture Probe), and the structure is fixed to the gold nanoparticles through Au — S bonds to generate SERS signals. Ngo et al have constructed a magnetic nanoparticle colloid substrate SERS sensing strategy based on a "sandwich" structure for the ultra-sensitive detection of dengue virus. The magnetic nanoparticles are connected with the prepared SERS (Raman molecule is contained in the gold-silver nucleocapsid) probe with the nano gold-silver nucleocapsid structure through target DNA, then the SERS probe is gathered by the magnetic nanoparticles, and the target nucleic acid is detected through an SERS signal.

The CRISPR/Cas system is an adaptive immune defense mechanism of bacteria and archaea against foreign substances. The CRISPR/Cas system has attracted extensive attention as a gene editing tool in the fields of food industry, human health, and the like. Recently, more and more studies have shown that certain Cas proteins can activate the function of non-specific cleavage of nearby single-stranded dna (ssdna) (called trans-cleavage) after specific cleavage (called cis-cleavage) of the target double-stranded deoxyribonucleic acid sequence (dsDNA). Among the numerous CRISPR proteins, Cas12a (also known as Cpf1) is a ribonucleic acid (RNA) -dependent endonuclease that can trans-cleave nearby non-target single-stranded DNA. The Cas12a protein can specifically recognize and cleave T-rich target dsDNA with the aid of designed crRNA, and Cas12a is a protein that recognizes target DNA guided by a single 42-44nt crRNA without needing trans-acting CRISPR RNA (tracrRNA). Figure 1 shows a schematic representation of Cas12a cleavage principle: cas12a recognizes T-rich target DNA under the guidance of crRNA, and cleaves to form a sticky end. Currently, there are many applications for the detection of viral nucleic acid using CRISPR/Cas12 a. For example, the invention patent of application publication No. CN110453011A introduces a method for rapidly and accurately detecting African swine fever virus based on CRISPR/Cas12a and application thereof; and the invention patent of application patent No. CN201910364284.5 introduces a specific HPV nucleic acid detection method based on CRISPR/Cas12 a; and the invention patent of application publication No. CN113046450A introduces a method for visually detecting phytoplasma based on CRISPR/Cas12a point cutting. However, the signal output of the current detection of viral nucleic acid by using CRISPR/Cas12a mainly depends on fluorescence and colorimetric methods. The CRISPR/Cas12a is combined with SERS technology for gene detection, and three reports have been found so far: (1) hongki Kim et al, which fully utilizes the capture of DNA in complex systems by magnetogold-complexed nanoparticles followed by simple magnetic separation and enhancement of Raman signal molecules (ACS Nano 2020,14, 17241-. (2) Jin Ha Choi et al utilizes CRISPR/Cas12a in combination with SERS technology for the detection of viral nucleic acids. The article mainly develops a nucleic acid amplification-free biosensor based on CRISPR @ Cas12a and an ultrasensitive detection system assisted by SERS. The shearing of ssDNA immobilized between a Graphene Oxide (GO)/triangular gold SERS active substrate and a Raman reporter functionalized gold nanoparticle by the CRISPR @ Cas12a is activated by the presence of viral DNA, so as to cause the change of SERS signals (ACS Nano 2021,15, 13475-13485). (3) Jianghua Liu et al reported a multifunctional strategy based on CRISPR @ Cas12 a-mediated liposome SERS signal amplification and macroscopic colorimetric detection (anal. chem.2021,93, 10167-.

The CRISPR/Cas12a and the SERS technology are combined for rapid and accurate detection of genes. Compared with the reported method, the method has the greatest advantages of combining the CRISPR @ Cas12a with the SERS technology, and the method avoids a fussy modification preparation process, so that the SERS probe can stably exist for a long time. The method takes place in the solution phase of ssDNA in the critical step of signal amplification (i.e., trans-cleavage step), rather than the interface of the nano-assembly reported in the past, so that the method has more stable and higher trans-cleavage efficiency, and can ensure the sensitivity and repeatability of the detection method.

Disclosure of Invention

The invention discloses a method for rapidly and accurately detecting dsDNA based on combination of CRISPR/Cas12a and SERS technology and application thereof.

The invention uses target virus nucleic acid, Cas12a and crRNA to form a ternary complex, and the RuvC structural domain of Cas12a in the complex performs nuclease activity to open a substrate (namely ssDNA) in a cutting system. ssDNA is DNA on Probe 1 and Probe 2₁And DNA₂The bridging complementary sequence of (a) allows for efficient bridging of probe 1 and probe 2. When ssDNA is cleaved, probe 1 and probe 2 are in a dispersed state. And probe 1 and probeWhether the needle 2 is bridged or not can cause the change of the SERS signal, and whether the sample to be detected contains the target nucleic acid or not can be known. The accurate and rapid detection of the virus nucleic acid can be realized by utilizing the specific crRNA.

The technical scheme of the invention is as follows:

detailed procedure for preparation of solution 3

(1) Preparation of gold nanoparticles (AuNPs)

Gold nanoparticles preparation reference (j.am. chem. soc.2009,131,17042) method. The method specifically comprises the following steps: to a clean three-necked flask was added 100mL of 0.01% HAuCl₄The aqueous solution is heated and stirred until slightly boiling. 1mL of 1% aqueous sodium citrate solution was added thereto, and the resulting mixture was subjected to controlled slight boiling under reflux. The solution color was observed to range from light yellow to black to wine red. Cooling to 90 deg.C, and maintaining the temperature for 40min to cure the particles. After which heating and stirring were stopped. And (5) obtaining the gold nano particle solution after the solution is recovered to the room temperature. The solution was stored at 4 ℃ until use.

(2) Preparation of Probe 1 and Probe 2

The probe 1 and the probe 2 are prepared by modifying DNA sequences (DNA) containing sulfydryl on the surface of gold nano particles₁And DNA₂) A reference method was prepared (Nature Protocols,2006,247) in conjunction with raman reporter. The method specifically comprises the following steps:

(2-1) to 10. mu.L of 5. mu.M DNA₁To the solution were added pH 5.2, 500mM sodium acetate buffer solution 1.5. mu.L and 1. mu.L 10mM TCEP, and the mixture was incubated at room temperature for 1 hour to activate the DNA₁A mercapto group of (a).

(2-2) SH-DNA treated with TCEP of (2-1)₁Added to 5mL of the synthesized gold nanoparticle solution and stored at room temperature for 24 hours in the dark.

(2-3) to the (2-2) solution was added 50. mu.L of a tris-acetate buffer solution at pH 8.2 at 500mM to give a final concentration of 5mM of the tris-acetate buffer solution.

(2-4) adding 5M NaCl after the solution is uniformly mixed and aging. The process was repeated five times with at least 1h interval for 10. mu.L each addition to avoid aggregation of the nanoparticles due to too rapid addition of NaCl and was allowed to react at room temperature for at least 24 hours.

(2-5) the reacted solution was centrifuged (2-4) at 6400rpm, and washed twice with a buffer (150mM sodium chloride and 25mM Tris-acetic acid, pH 8.2).

(2-6) dispersing the nanoparticles after (2-5) centrifugation in 2.5mL of water for subsequent experiments.

(2-7) taking SH-DNA successfully prepared in (2-6)₁@ AuNPs 1mL, then 5. mu.L of 1mM Raman reporter (e.g., 4-mercaptobenzonitrile, abbreviated as: MBN) was added, and the reaction was allowed to stand overnight.

(2-8) centrifuging the solution in (2-7) twice to remove unbound Raman reporter molecules, redispersing into 1mL of buffer solution, and storing in a refrigerator at 4 ℃ for subsequent use.

Preparation method of Probe 2 DNA was used for removing the DNA chain₂Otherwise, the procedure was the same as that for preparing probe 1.

(2-9) the successfully prepared probe 1 was mixed with the probe 2 to prepare a solution 3, which was stored in a refrigerator at 4 ℃ for subsequent use.

Specific step (3) CRISPR/Cas12a @ crRNA @ DNA ternary Complex formation for preparation of solution 4

(3-1) to a 20. mu.L system, 1. mu.L Cas12a protein, 1. mu.L corresponding crRNA, 4. mu.L target DNA were added.

(3-2) to the (3-1) solution was added a buffer (20mM tris-HCl pH 7.5100 mM KCl 5mM MgCl)²) The reaction was carried out at 37 ℃ for 30 min.

(3-3) Add 4. mu.L of the bridged substrate ssDNA to the reacted (3-2) solution, and react at 37 ℃ for 30 min.

And (3-4) carrying out water bath on the solution after the reaction of the (3-3) is finished at 85 ℃, and removing protein for 10min to stop the reaction of the system.

Detection of viral nucleic acids using SERS

(4-1) adding 5 mu L of the solution 4 after the reaction in (3-4) into 40 mu L of the solution 3 prepared in (2-9) in a water bath at 50 ℃ for 2min, and then carrying out the following SERS detection.

And (4-2) ultrasonically dispersing the solution after the reaction in the step (4-1) in an ultrasonic machine for 5 min. Then 10. mu.L of the solution was dropped onto a piece of cleaned glass or quartz. Testing by using a confocal Raman spectrometer. The method is realized by pulling on a probe 1 and a probe 2And (3) detecting the signal intensity of the Raman reporter molecule to indicate whether the substrate ssDNA exists in the detection system. During the test, the excitation light source is 633nm, the excitation light and the Raman scattered light are collected through a 50X eyepiece, and the range of the collected Raman spectrum is 200cm^-1To 2400cm^-1The integration time was 10 s.

(4-3) control group sample test except that the target DNA in the specific step (3-1) of preparing solution 4 is changed to H₂The reaction of the remaining solution 5 was exactly the same as in the above step (3) except that O was used as a control, and then 5. mu.L of this solution was added to 40. mu.L (2-9) of the prepared solution 3 in a water bath at 50 ℃ for 2min, followed by SERS detection.

(5) Discrimination of detection result

When no target DNA is present in the system, the ternary complex of Cas12a @ crRNA @ DNA cannot be formed, i.e., the trans-cleavage function cannot be turned on. Therefore, the substrate ssDNA in the system is completely reserved, the effective aggregation structure of the probe 1 and the probe 2 can be promoted, and the corresponding SERS signal is stronger. On the contrary, when the target DNA exists, a ternary complex of Cas12a @ crRNA @ DNA can be formed, and then the shearing function of the nearby substrate ssDNA is activated, so that the substrate ssDNA in the system is greatly degraded and consumed, an effective aggregation structure of the probe 1 and the probe 2 cannot be formed, and the obtained SERS signal is weak.

(5-1) the Raman data obtained in (4-2) and (4-3) were processed and plotted.

And (5-2) if the SERS signal intensity of the (4-2) group is equivalent to that of the (4-3) group, the result is negative.

And (5-3) if the SERS signal intensity of the (4-2) group is weaker than that of the (4-3) group, the result is positive.

Compared with the prior art, the invention has the following effects:

1) the invention can efficiently and accurately detect the virus gene sequence, has the advantage of simple operation, and has important significance for early monitoring, diagnosis, prevention and control of diseases.

2) The method has small measurement interference, which is mainly because the trans-shearing is generated on the unmodified free ssDNA in the solution phase, the shearing is not influenced by the interface, and the success rate is high; and the required sample amount is small, the signal is strong, the sensitivity is high, and the high-sensitivity detection of the low-concentration virus nucleic acid can be realized.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.

Fig. 1 is a schematic diagram of CRISPR/Cas12a binding to target DNA.

FIG. 2 shows Probe 1(AuNPs @ DNA)₁@ MBN) and Probe 2(AuNPs @ DNA)₂@ MBN) and the addition of substrate ssDNA to cause nanoparticle coupling.

Fig. 3 is a flow chart of detection experiment combining CRISPR/Cas12a with SERS technology.

The names of the parts are as follows: the method comprises the following steps of preparing a nanoparticle probe 1, a nanoparticle probe 2, a solution 3 prepared by mixing the nanoparticle probe 1 and the nanoparticle probe 2, Cas12a protein, corresponding crRNA 5, a substrate ssDNA6, virus DNA 7 to be detected, a solution 4 formed by the Cas12a protein, the crRNA, the substrate ssDNA and the virus DNA to be detected, adding the solution 4 into the solution 3 for SERS detection 8, and obtaining a positive SERS result with target DNA (depicted in a figure 9) and a negative SERS result without the target DNA (depicted in a figure 10).

Fig. 4 is a uv result of the gold nanoparticles prepared in example 1 and example 2.

FIG. 5 is a transmission electron micrograph of the gold nanoparticles prepared in example 1 and example 2.

FIG. 6 shows Probe 1(AuNPs @ DNA) in examples 1 and 2₁@ MBN) and Probe 2(AuNPs @ DNA)₂@ MBN).

FIG. 7 shows the comparative results of the detection of Raman signals in example 1 and example 2.

FIG. 8 shows the result of positive detection of HPV16 DNA by Raman signal in example 1.

FIG. 9 shows the result of negative detection of HPV18 DNA Raman signal in example 2.

Detailed Description

Example 1CRISPR/Cas12a in combination with SERS technology for detection of HPV16 nucleic acids

In this embodiment, the metal nanoparticle probes are gold nanoparticle probes.

In this embodiment, the raman signal molecule is MBN.

In this example, the DNA₁Has the sequence of SH-AAAAAAAAAACACACTCACACACAC.

In this example, the DNA₂Has the sequence of SH-AAAAAAAAAACCTCACCACCAACAC.

In this example, the substrate ssDNA has the sequence GTGTGAGTGTGTGTGGTGTTGGTGGTGAGG.

In this example, the target nucleic acid is HPV16 DNA with the specific sequence of CTATGGATTACAAACAAACACAATTGTGTTTAATTGGTTGCAAACCACCTATAGGGGAACACTGGGGCAAAGGATCCCCATGTACCAATGTTGCAGTAAATCCAGGTGATTGTCCACCATTAGAGTTAATAAACACAGTTATTCAGGATGGTGATATGGTTGATACTGGCTTTGGTGCTATGGACTTTACTACATTACAGGCTAACAAAAGTGAAGTTCCACTGGATATTTGTACATCTA.

In this embodiment, the crRNA is the corresponding crRNA of HPV16₁₆The specific sequence is UAAUUUCUACUAAGUGUAGAUCUACAUUACAGGCUAACAAA.

(1) Preparation of gold nanoparticles (AuNPs)

A method for preparing reference sodium citrate hydrothermally reducing chloroauric acid by gold nanoparticles. The method specifically comprises the following steps: to a clean three-necked flask was added 100mL of 0.01% HAuCl₄The aqueous solution is heated and stirred until slightly boiling. 1mL of 1% aqueous sodium citrate solution was added thereto, and the resulting mixture was subjected to controlled slight boiling under reflux. The solution color was observed to range from light yellow to black to wine red. Cooling to 90 deg.C, and maintaining the temperature for 40min to cure the particles. After which heating and stirring were stopped. And (5) obtaining the gold nano particle solution after the solution is recovered to the room temperature. The solution was stored at 4 ℃ until use.

(2) Preparation of Probe 1(AuNPs @ DNA)₁@ MBN) and Probe 2(AuNPs @ DNA)₂@MBN)

The probe 1 and the probe 2 are prepared by modifying DNA sequences (DNA) containing sulfydryl on the surface of gold nano particles₁And DNA₂) Realized by Raman reporter molecules, the method specifically comprises the following steps:

(2-1) to 10. mu.L of 5. mu.M DNA₁Adding 500mM acetic acid with pH 5.2 into the solutionSodium buffer solution 1.5 u L and 1L 10mM TCEP, at room temperature 1 hours incubation activation of DNA₁A mercapto group of (a).

(2-2) SH-DNA treated with TCEP₁Added to 5mL of the synthesized gold nanoparticle solution and stored at room temperature for 24 hours in the dark.

(2-3) 50. mu.L of 500mM tris-acetate buffer solution at pH 8.2 was added to give a final concentration of 5mM tris-acetate buffer solution.

And (2-4) adding 5M NaCl after the solutions in the (2-3) are uniformly mixed, and aging. The process was repeated five times with at least 1h interval for 10. mu.L each addition to avoid aggregation of the nanoparticles due to too rapid addition of NaCl and was allowed to react at room temperature for at least 24 hours.

(2-5) the solution in (2-4) was centrifuged at 6400rpm and washed twice with buffer (150mM sodium chloride and 25mM Tris-acetic acid, pH 8.2).

(2-6) dispersing the nanoparticles obtained in (2-5) after centrifugal washing in 2.5mL of water for subsequent experiments.

Next, a raman reporter molecule (e.g. MBN) is grafted on the surface of the DNA-modified gold nanoparticles by the following method:

(2-7) obtaining successfully prepared SH-DNA₁@ AuNPs 1mL, then 5. mu.L of 1mM MBN Raman probe molecule was added and the reaction was allowed to stand overnight.

(2-8) centrifugation twice to remove unbound Raman probe molecules and re-dispersing into 1mL of buffer solution, and storing in a refrigerator at 4 ℃ for subsequent use.

Probe 2 (SH-DNA)₂@ AuNPs @ MBN) is otherwise the same as described above except that the DNA strand is different. The successfully prepared probe 1 was mixed with probe 2 to achieve the preparation of solution 3, which was stored in a refrigerator at 4 ℃ for subsequent use.

(3) Recognition and cleavage of HPV16 DNA by CRISPR/Cas12a

(3-1) reaction solution 4 and solution 5 were prepared, and 1. mu.L of Cas12a protein, 1. mu.L of crRNA (of HPV 16) were added to a 20. mu.L system₁₆) Wherein 4. mu.L of target DNA (HPV 16 DNA) was added to solution 4 and 4. mu.L of water was added to solution 5 as a control.

(3-2) adding a buffer to the solution (3-1)Rinses (20mM tris-HCl pH 7.5100 mM KCl 5mM MgCl₂) The reaction was carried out at 37 ℃ for 30 min.

(3-3) Add 4. mu.L of the bridged substrate ssDNA to the reacted (3-2) solution, and react at 37 ℃ for 30 min.

And (3-4) carrying out water bath on the solution after the reaction of the (3-3) is finished at 85 ℃, and removing protein for 10min to stop the reaction of the system.

(3-5) adding 5 mu L of each of the solution 4 and the solution 5 after the reaction into the solution 3(40 mu L) prepared in the same volume in a water bath at 50 ℃ for 2min, and then carrying out the following SERS detection.

(3-6) carrying out ultrasonic treatment on the solution after the reaction in the step (3-5) for 5min to improve the dispersibility of the particles in the solution, then dripping 10 mu L of the solution on a cleaned glass sheet, testing by using a confocal Raman spectrometer, wherein an excitation light source is 633nm and 7.1mW, the excitation light and Raman scattering light are collected through a 50X eyepiece, and the range of the collected Raman spectrum is 200cm^-1To 2400cm^-1The integration time was 10 s.

(4) Discrimination of detection result

When the Raman data obtained in (3-6) was processed and plotted, it was found that the detection intensity was lower than 2500 counts and lower than that of the control group. The test result is a positive result.

Example 2 detection of HPV18 nucleic acids in combination with the SERS technique of CRISPR/Cas12a

In this embodiment, the metal nanoparticle probes are gold nanoparticle probes.

In this embodiment, the raman signal molecule is MBN.

In this example, the DNA₁Has the sequence of SH-AAAAAAAAAACACACTCACACACAC.

In this example, the DNA₂Has the sequence of SH-AAAAAAAAAACCTCACCACCAACAC.

In this example, the substrate ssDNA has the sequence GTGTGAGTGTGTGTGGTGTTGGTGGTGAGG.

In this example, the target nucleic acid is a HPV18 DNA fragment, with the specific sequence of GGGAACACTGGGCTAAAGGCACTGCTTGTAAATCGCGTCCTTTATCACAGGGCGATTGCCCCCCTTTAGAACTTAAAAACACAGTTTTGGAAGATGGTGATATGGTAGATACTGGATATGGTGCCATGGACTTTAGTACATTGCAAGATACTAAATGTGAGGTACCATTGGATATTTGTCAGTCTATTTGTAAATATCCTGATTATTTACAAATGTCTGCAGATCCTTATGGGGATTCCA.

In this embodiment, the crRNA is HPV18 crRNA₁₈The specific sequence is UAAUUUCUACUAAGUGUAGAUGUACAUUGCAAGAUACUAAA.

(1) Step (2) preparation of gold nanoparticles (AuNPs) and preparation of Probe 1(AuNPs @ DNA)₁@ MBN) and Probe 2(AuNPs @ DNA)₂@ MBN) is the same as example 1.

(3) Recognition and cleavage of HPV18 DNA by CRISPR/Cas12a

(3-1) reaction solution 4 and solution 5 were prepared, and 1. mu.L of Cas12a protein, 1. mu.L of crRNA (HPV 18 crRNA) were added to a 20. mu.L system₁₈) mu.L of target DNA (HPV 18 DNA) was added to solution 4 and 4. mu.L of water was added to solution 5 as a control.

(3-2) to the (3-1) solution was added a buffer (20mM tris-HCl pH 7.5100 mM KCl 5mM MgCl)₂) The reaction was carried out at 37 ℃ for 30 min.

(3-3) Add 4. mu.L of the bridged substrate ssDNA to the reacted (3-2) solution, and react at 37 ℃ for 30 min.

And (3-4) carrying out water bath on the solution after the reaction of the (3-3) is finished at 85 ℃, and removing protein for 10min to stop the reaction of the system.

(3-5) adding 5 mu L of each of the solution 4 and the solution 5 after the reaction into the solution 3(40 mu L) prepared in the same volume in a water bath at 50 ℃ for 2min, and then carrying out the following SERS detection.

(3-6) performing ultrasonic treatment on the solution after the reaction in the step (3-5) to improve the dispersibility of the particles in the solution, then dripping 10 mu L of the solution onto a clean glass sheet, testing by using a confocal Raman spectrometer, wherein an excitation light source is 633nm and 7.1mW, the excitation light and Raman scattering light are collected through a 50X eyepiece, and the range of the collected Raman spectrum is 200cm^-1To 2400cm^-1The integration time was 10 s.

(4) Discrimination of detection result

And (4) processing the Raman data obtained in (3-6) to prepare a graph. The signal intensity is higher than 10000 and is equivalent to that of a control group. Cas12a @ crRNA can not be formed in a determinable system₁₈@HPAnd (3) a ternary complex of V18 DNA, so that a substrate ssDNA in the system exists completely, and the probe 1 and the probe 2 form an aggregation form to bring a strong SERS signal. Therefore, the result of the detection can be judged to be negative.