Kit for detecting bacteria and bacterial detection method based on CRISPRCas-SDA-AuNPs

Abstract

The present invention belongs to the technical field of bacterial detection, and specifically relates to a kit for detecting bacteria and a bacterial detection method based on CRISPR / Cas-SDA-AuNPs. The present invention introduces the CRISPR-Cas system, specifically identifies bacteria through CRISPR / Cas12a and SDA to amplify the detection signal, and proposes a bacterial detection method based on the photothermal effect of CRISPR / Cas12a-SDA and DNA-AuNPs, which can achieve simple, rapid and highly sensitive detection of bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of bacteria detection, and in particular relates to a kit for detecting bacteria and a bacteria detection method based on CRISPR / Cas-SDA-AuNPs. Background Art

[0002] Bacteria are important pathogens that cause many diseases. Rapid and accurate bacterial detection can achieve early diagnosis of pathogens of emerging infectious diseases; it can guide rational clinical drug use and reduce the increasingly serious problem of drug resistance caused by unknown pathogens or empirical drug use. However, current traditional bacterial detection technologies such as bacterial culture methods are very time-consuming, and polymerase chain reaction (PCR) or enzyme-linked immunosorbent assay (ELISA) rely on expensive instruments and skilled operators, which cannot meet the detection needs of countries and regions with backward medical conditions, and also limit their widespread application in instant detection.

[0003] Point-of-care detection methods based on nanomaterials have attracted interest in rapid, sensitive and specific bacterial detection. Gold nanoparticles (AuNPs) are one of the nanomaterials with the longest history of synthesis and the widest application field. They have size-dependent optical properties and can exhibit a range of color properties. Dispersed AuNPs exhibit typical plasmon absorption in the visible light range. When aggregation occurs, its absorption peak red-shifts and the color changes from red to blue. Due to this characteristic, AuNPs-based bacterial colorimetric detection methods have been widely used in food safety, disease diagnosis and other detection fields. This method can intuitively judge the color change by the naked eye and realize qualitative or semi-quantitative detection of the target. However, in order to achieve its quantitative measurement, it is often necessary to use large instruments such as spectrophotometers, which is not suitable for rapid on-site detection.

[0004] In addition, existing high-sensitivity nucleic acid detection technologies usually use nucleic acid amplification technology to amplify signals to further improve the sensitivity of detection. Strand displacement amplification (SDA) is an enzymatic DNA in vitro isothermal amplification method based on the continuous cutting and polymerization / displacement process catalyzed by restriction endonucleases and DNA polymerases. It has the advantages of high efficiency, high specificity, and low equipment requirements. Although it has obvious advantages in nucleic acid analysis, SDA cannot be directly used for the detection of bacterial genomes and often requires specific binding with aptamers. Limited by the screening of highly specific aptamers, the current aptamer-based SDA bacterial detection method often has the problem of low specificity. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a kit for detecting bacteria and a bacterial detection method based on CRISPR / Cas-SDA-AuNPs.

[0006] In a first aspect of the present invention, a kit for detecting bacteria is provided, comprising an SDA system, a CRISPR / Cas system and DNA-AuNPs;

[0007] The SDA system includes Template, Primer, Bst3.0 DNA polymerase, Nt.BbvCI nicking endonuclease, and dNTPs;

[0008] The CRISPR / Cas system includes Cas12a and crRNA, and the Cas12a and crRNA can recognize the bacterial genome and non-specifically cut the Template after identifying the target bacterial genome;

[0009] The DNA on the DNA-AuNPs is complementary to the two ends of the ssDNA chain generated by the Template and Primer in the SDA system after chain displacement isothermal amplification under the action of Bst3.0 DNA polymerase and Nt.BbvCI endonuclease.

[0010] Preferably, the DNA-AuNPs are obtained by mixing and incubating SH-DNA1 and SH-DNA2 with AuNPs;

[0011] The sequence of SH-DNA1 is 5′- TTG TCG TTG CGT GCT GA-SH-3′;

[0012] The sequence of SH-DNA2 is 5′- SH-CCC AGG TTC TCT -3′;

[0013] The template sequence was 5′- CCC AGG TTC TCT TTG TCG TTG CGT GCT GAG GTT AGC AAAGTA GCG TG-3′;

[0014] The primer sequence is 5′- CAC GCT ACT TTG CTA A-3′;

[0015] The crRNA sequence was 5′- UAA UUU CUA CUA AGU GUA GAU GGG CCU AGA UAA AGA AGAAC-3′.

[0016] Preferably, the DNA-AuNPs are SH-DNA1 and SH-DNA2, which are mixed with AuNPs and incubated with shaking at 36-38° C. for 10-50 minutes, and then citric acid / hydrochloric acid buffer is added and incubated with shaking for 8-12 hours.

[0017] Preferably, the incubation time in the citric acid / hydrochloric acid buffer is 10 hours.

[0018] Preferably, the optimal ratio of DNA to AuNPs is 200:1.

[0019] A second aspect of the present invention provides a bacterial detection method based on CRISPR / Cas-SDA-AuNPs, the method being a non-disease diagnosis or treatment application, and using a kit for detecting bacteria as described in any one of claims 1 to 5;

[0020] The method comprises the following steps:

[0021] (1) Using the CRISPR / Cas system to perform an enzyme digestion reaction with the test sample to obtain an enzyme digestion reaction solution;

[0022] (2) using the SDA system to amplify the enzyme digestion reaction solution obtained in step (1) to obtain an SDA amplification product;

[0023] (3) Mix the SDA amplification product with DNA-AuNPs for reaction.

[0024] Preferably, the enzyme digestion reaction solution obtained in the enzyme digestion reaction in step (1) is first incubated at an elevated temperature to inactivate the Cas enzyme activity, and after inactivation, a hybridization reaction is performed to restore the hybrid double strands opened during the inactivation process to hybrid double strands, and then amplification is performed using the SDA system in step (2).

[0025] Preferably, the SDA amplification product obtained in step (2) is inactivated by incubation at elevated temperature.

[0026] Preferably, after the reaction in step (3), photothermal detection is performed and when Vibrio vulnificus is present, the temperature rises after irradiation with a 660 nm laser.

[0027] Preferably, after the reaction in step (3), the bacteria are Vibrio vulnificus through UV-vis detection, and the concentration of the bacteria is proportional to the absorbance value of the reaction solution obtained in step (3) at 530 nm.

[0028] The beneficial effects of the present invention are as follows: the present invention introduces the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR-Associated Protein), specifically recognizes bacteria through CRISPR / Cas12a and SDA to amplify the detection signal, and proposes a bacterial detection method based on CRISPR / Cas12a-SDA and DNA-AuNPs photothermal effect, which can realize simple, rapid and highly sensitive detection of bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0030] Figure 1 The schematic diagram of the detection principle of the DNA-AuNPs photothermal system based on SDA (A) and the schematic diagram of the bacterial photothermal detection principle based on the photothermal effect of CRISPR / Cas12a-SDA and DNA-AuNPs (B);

[0031] Figure 2 Color change (A) and UV-visible absorption spectra of AuNPs and DNA-AuNPs after adding 1 M NaCl (B), optimization of incubation time in citric acid / hydrochloric acid buffer (C), and optimization of the ratio of DNA to AuNPs (D);

[0032] Figure 3 The 9% gel electrophoresis pattern (A), solution color change (B), UV-vis (C), and photothermal effect (D) verification results of SDA feasibility;

[0033] Figure 4 Schematic diagram of fluorescence verification of CRISPR cutting system (A), fluorescence value results (B);

[0034] Figure 5 UV-vis (A) and temperature change results (B) of the feasibility of the CRISPR / Cas12a-SDA bacterial detection solution;

[0035] Figure 6 The results are validated for the detection performance of Vibrio vulnificus. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0037] The present invention constructs a DNA-AuNPs photothermal system based on SDA by designing the template and primer of SDA and the DNA sequence in DNA-AuNPs. The detection principle diagram is shown in FIG. Figure 1 As shown in A. In this system, Template and Primer undergo strand displacement isothermal amplification under the action of Bst3.0 DNA polymerase and Nt.BbvCI endonuclease to produce a large amount of ssDNA. The two ends of these ssDNA chains are complementary to DNA1 and DNA2, respectively, inducing hybridization and aggregation of DNA1-AuNPs and DNA2-AuNPs. The higher the Template concentration, the higher the Linker concentration of the SDA amplification product, which in turn leads to a greater degree of aggregation of DNA-AuNPs and a greater temperature change after laser irradiation; and vice versa.

[0038] The CRISPR / Cas system (Clustered Regularly Interspaced Short PalindromicRepeat and CRISPR-Associated Protein) is a prokaryotic immune defense system used to resist the invasion of foreign genetic material, such as bacteriophage viruses. At the same time, it provides bacteria with acquired immunity (similar to the secondary immunity of mammals), and when bacteria are invaded by viruses, they will produce corresponding "memory". When the virus invades for the second time, the CRISPR system can identify exogenous DNA, cut them off, silence the expression of exogenous genes, and resist viral interference. It is precisely because of this precise targeting function that the CRISPR / Cas system has been developed into an efficient gene editing tool. CRISPR / Cas can perform precise editing of genes at a fixed point.

[0039] The present invention innovatively introduces the CRISPR / Cas system, which can be used for the detection of bacterial genomes. The detection principle is as follows: Figure 1As shown in B. When the system contains Vibrio vulnificus DNA, the trans-cleavage activity of Cas12a / crRNA is activated, and ssDNA (i.e., Template) is non-specifically cut. The SDA system lacking Template cannot amplify the Linker chain that causes AuNPs aggregation, so AuNPs do not aggregate. When there is no Vibrio vulnificus amplified DNA, the Template is not cut by Cas12a / crRNA. After SDA, a large number of Linkers are amplified, inducing DNA-AuNPs aggregation, and its photothermal conversion efficiency is significantly improved. After 660 nm laser irradiation, the temperature rise value is obvious. Finally, the result is determined by the change of temperature signal, thereby establishing a detection method based on CRISPR / Cas-SDA-AuNPs, and the method is applied to the quantitative detection of Vibrio vulnificus and other bacteria.

[0040] The sources of materials and equipment used in the following examples are as follows:

[0041] 1. Materials and Reagents

[0042] Chloroauric acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Trisodium citrate and 4-mercaptophenylboronic acid were purchased from Sigma-Aldrich (USA). Ammonium bicarbonate was purchased from Aladdin Chemical Co., Ltd. Bst3.0 DNA polymerase and Nt.BbvCI nicking endonuclease were purchased from New England Biolabs (NEB). dNTPs and DNA Ladder (20 bp) were purchased from Beijing Bio-Rad Biotechnology Co., Ltd. Ultra GelRed dye was purchased from Beijing Novozyme Biotech Co., Ltd. DNA sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. All experimental water was ultrapure water with a conductivity of 18.2 MΩ cm.

[0043] Table 1 Main DNA sequences

[0044] name Sequence (5'—3') SH-DNA1 TTG TCG TTG CGT GCT GA-SH SH-DNA2 SH-CCC AGG TTC TCT Template CCC AGG TTC TCT TTG TCG TTG CGT GCT GAG GTT AGC AAA GTA GCG TG Primer CAC GCT ACT TTG CTA A crRNA UAA UUU CUA CUA AGU GUA GAU GGG CCU AGA UAA AGA AGA AC Probe FAM-TTATT-BHQ1

[0045] Note: (1) FAM is a fluorescent group, and BHQ1 is a fluorescence quenching group;

[0046] (2) -SH is a thiol modification.

[0047] 2. Instruments and equipment

[0048] Multifunctional microplate reader (SpectraMax iD3, Meigu Molecular Instrument Co., Ltd., Shanghai), high-speed refrigerated centrifuge (5417 R model, Eppendorf, Germany), heat-collecting constant temperature heating magnetic stirrer (DF-101S model, Shanghai Lichen Bangxi Instrument Technology Co., Ltd.), infrared photothermal detection platform (MW-GX-660 / 2000 MW, Changchun Leishi Optoelectronics Technology Co., Ltd.), ultraviolet-visible spectrometer (Lingxi V-3000, Shanghai Lingxi Instrument Co., Ltd.), portable thermometer (Zhenli GL637, Fuzhou Zhenli Optoelectronics Technology Co., Ltd.), JEM-2100F high-resolution transmission electron microscope (JEM-2100F, Tokyo, Japan), etc.

[0049] Example 1 Preparation of DNA-AuNPs:

[0050] (1) Add 100 mL HAuCl4 (0.01% w / w) to a 250 mL three-necked round-bottom flask washed with aqua regia, heat to 110°C in an oil bath, and quickly add 10 mL sodium citrate (38.8 mM) while stirring vigorously with a magnetic stirrer. Continue heating for 15 min. The solution changes from light yellow to purple to black and finally to wine red. Stop heating and continue stirring until the solution cools to room temperature, about 2 hours, and store in a refrigerator at 4°C for later use.

[0051] (2) According to the extinction coefficient ε of AuNPs is 2.7×10 8 M cm -1 , and Lambert-Beer law A=εbc (where c is the concentration of the solution being measured; b is the width of the cuvette 1 cm), the absorbance at 520 nm was measured by a spectrophotometer and the concentration of the prepared AuNPs was optimized to 10 nM.

[0052] (3) 8 μL of 100 μM SH-DNA1 and SH-DNA2 were mixed with 400 μL of 10 nM AuNPs and incubated at 37°C for 30 min. Then, citric acid / hydrochloric acid buffer (final concentration of 10 nM) was added and incubated for different periods of time. The obtained solution was centrifuged at 12,000 rpm for 10 min at 4°C, washed twice with ultrapure water to remove unbound DNA, and resuspended in 400 μL of 1 M NaCl containing 0.05% Tween-20 and stored at 4°C.

[0053] The successful preparation of DNA-AuNPs was investigated in a stable dispersed state in a high concentration salt solution. Figure 2As shown in AB, naked AuNPs aggregated immediately after adding 1 M NaCl, and the solution color quickly changed from red to blue and quickly precipitated to the bottom of the tube. Its absorbance at 520 nm also dropped significantly. However, DNA-AuNPs remained red after adding the same concentration of NaCl, and the absorbance did not change. The results show that DNA-functionalized AuNPs were successfully synthesized.

[0054] In the process of synthesizing DNA-AuNPs by acidification, the incubation time of citric acid / hydrochloric acid buffer and the ratio of DNA to AuNPs are important factors affecting the stability of DNA-AuNPs. Figure 2 As shown in CD, according to the absorbance at 520 nm, the optimal incubation time with citric acid / hydrochloric acid buffer was 10 h, and the optimal ratio of DNA to AuNPs was 200:1.

[0055] Embodiment 2:

[0056] (1) CRISPR / Cas12a trans-cleavage

[0057] First, incubate Template (1 μM) and Primer (1 μM) in a 95°C metal bath for 5 minutes, then slowly cool to 37°C and keep for 1 hour to form a double-stranded hybrid. Incubate Cas12a and crRNA at a concentration of 200 nM at 37°C for 30 minutes, then add the RPA amplified gene of Vibrio vulnificus, the double-stranded hybrid of Template and Primer (25 nM), and 1×2.1NEBuffer, and add ultrapure water to the system to 10 μL. Mix the mixed system and incubate it in a 37°C metal bath for 60 minutes for enzyme digestion.

[0058] (2) Strand displacement isothermal amplification (SDA)

[0059] The SDA system consists of Template, Primer, Bst3.0 DNA polymerase, Nt.BbvCI nicking endonuclease, dNTPs and rCutSmart Buffer. Since the inactivation process of Cas enzyme activity will open the hybrid double strands of Template and Primer, for the subsequent SDA process, the hybridization reaction is performed again, that is, the temperature is adjusted back to 95°C for incubation for 5 minutes, and then slowly cooled to 37°C and maintained for 1 hour to form a hybrid double strand. Then, 0.05 U Bst3.0 DNA polymerase, 0.05 U Nt.BbvCI nicking endonuclease, 250 μM dNTPs, 1×rCutSmart Buffer and 20 mM phosphate buffer (PB) were added to the above system, amplified at 37°C for 100 minutes, and finally incubated at 80°C for 20 minutes to inactivate the enzyme activity.

[0060] (3) Photothermal detection

[0061] Take 10 μL of SDA amplification product, add 40 μL of DNA-AuNPs and react for 10 minutes, then perform 1UV-vis and photothermal detection (1 W cm -2 , 2 min) and use a thermometer to detect temperature changes.

[0062] The SDA process can amplify a large number of ssDNA chains, thereby inducing the aggregation of DNA-AuNPs. When the Template chain is missing in the SDA system, the SDA process cannot proceed. The experimental group is the one with both Template and Primer, and the negative control group is the one without Template (i.e., only Primer). Figure 3 As shown in the electrophoresis results of A, in the experimental group (lane 1), the Template and Primer formed a double-stranded hybrid after annealing, and a linker band was generated after amplification reaction. In the control group (lane 2), when there was no Template, ssDNA could not be amplified. Figure 3 As shown in BD, the color of DNA-AuNPs in the experimental group changed from red to blue-purple, the absorbance value red-shifted, and the temperature rose by about 6°C after laser irradiation. However, DNA-AuNPs in the negative control group remained in a dispersed red state, and the temperature rise after laser irradiation was significantly lower than that of the experimental group. The results proved the feasibility of the SDA-induced DNA-AuNPs aggregation scheme.

[0063] Schematic diagram of CRISPR / Cas12a cutting process Figure 4 As shown in A, the complete cutting system includes: Cas12a, crRNA, a probe (Probe) labeled with a fluorescent group (FAM) and a fluorescent quencher group (BHQ) at the same time, and a target (Vibrio vulnificus DNA). Only when the system is complete (that is, Vibrio vulnificus DNA exists), the trans-cutting activity of Cas12a / crRNA is activated, and the exogenous ssDNA (fluorescent probe) is non-specifically cut. Since the Probe is modified with fluorescent groups and fluorescent quencher groups at both ends, when the probe is intact, the fluorescence of the fluorescent group is quenched by the quencher group; and when the probe is cut, the fluorescent groups and quenchers modified at both ends emit a strong fluorescent signal due to the distance. The trans-cutting activity of Cas12a / crRNA can be judged by visual observation with ultraviolet light or quantitative detection of the system fluorescence with an enzyme reader. In the experiment, the experimental group was in the presence of Vibrio vulnificus DNA, and the control group was in the absence of target DNA. As shown in Figure 4As shown in B, the test tubes in the experimental group produced green fluorescence under ultraviolet light, and the fluorescence value at 522 nm was strong, indicating that the Cas12a / crRNA trans-cleavage activity was activated and the probe was non-specifically cleaved. No fluorescence was produced in the control group, indicating that non-specific cleavage did not occur when the target DNA was not present. The above results prove the successful establishment of the CRISPR / Cas12a cleavage system.

[0064] Feasibility verification of Vibrio vulnificus detection Figure 5 As shown. In the experimental group, the target gene was amplified by RPA in the presence of Vibrio vulnificus. After the target gene and crRNA were combined, the Cas12 enzyme cleavage process was induced, and the exogenous ssDNA was non-specifically cut (i.e., after the long-chain Template and the short-chain Primer were hybridized, the non-hybridized single-stranded fragments on the Template). Due to the lack of Template, the efficiency of Linker generation after SDA amplification was significantly reduced, so the DNA-AuNPs remained dispersed and the solution was red ( Figure 5 A), the temperature rises by about 5°C after laser irradiation ( Figure 5 B). The control group did not have the target gene of Vibrio vulnificus, so the Template and Primer in the system remained intact. After SDA, a large amount of ssDNA was amplified, inducing the aggregation of DNA-AuNPs. After laser irradiation, the temperature rose by about 6.7°C ( Figure 5 B). The experimental results show the feasibility of the bacterial detection strategy based on CRISPR / Cas12a-SDA.

[0065] Vibrio vulnificus detection performance Figure 6 As shown in Figure 2, the logarithm of the concentration of the target gene amplified by RPA of Vibrio vulnificus is proportional to the absorbance value at 530 nm. The higher the bacterial concentration, the more obvious the dispersion of DNA-AuNPs. 0 cfu / mL-10 2 There was a good linear relationship between the cfu / mL.

[0066] The above embodiments provide applications for detecting Vibrio vulnificus. Those skilled in the art can design and replace the crRNA in the embodiments for detecting other bacteria. The specific crRNA design process is as follows: a 35 bp dsDNA containing a PAM sequence (TTTN) is randomly designed as the target nucleic acid, and the scaffold sequence of Cas12a (UAA UUU CUA CUAAGU GUA GAU) is added to the 20 bases after the PAM sequence of the target nucleic acid.

[0067] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A bacterial detection method based on CRISPR / Cas-SDA-AuNPs, the method being used for non-disease diagnosis or treatment, characterized in that: The kits used include the SDA system, CRISPR / Cas system, and DNA-AuNPs; The SDA system includes Template, Primer, Bst3.0 DNA polymerase, Nt.BbvCI nicking endonuclease, and dNTPs; The CRISPR / Cas system includes Cas12a and crRNA, and the Cas12a and crRNA can recognize the bacterial genome and non-specifically cut the Template after identifying the target bacterial genome; The DNA on the DNA-AuNPs is complementary to the two ends of the ssDNA chain generated by the Template and Primer in the SDA system after chain displacement isothermal amplification under the action of Bst3.0 DNA polymerase and Nt.BbvCI endonuclease; The method comprises the following steps: (1) using the CRISPR / Cas system to perform an enzyme digestion reaction with the test sample to obtain an enzyme digestion reaction solution; (2) using the SDA system to amplify the enzyme digestion reaction solution obtained in step (1) to obtain an SDA amplification product; (3) mixing the SDA amplification product with DNA-AuNPs for reaction; The DNA-AuNPs are obtained by mixing SH-DNA1 and SH-DNA2 with AuNPs for incubation; The sequence of SH-DNA1 is 5′-TTG TCG TTG CGT GCT GA-SH-3′; The sequence of SH-DNA2 is 5′-SH-CCC AGG TTC TCT-3′; The template sequence was 5′-CCC AGG TTC TCT TTG TCG TTG CGT GCT GAG GTT AGC AAA GTAGCG TG-3′; The primer sequence is 5′-CAC GCT ACT TTG CTA A-3′; The crRNA sequence was 5′-UAA UUU CUA CUA AGU GUA GAU GGG CCU AGA UAA AGA AGA AC-3′; The bacteria is Vibrio vulnificus.

2. The bacterial detection method based on CRISPR / Cas-SDA-AuNPs according to claim 1, characterized in that: The DNA-AuNPs are SH-DNA1 and SH-DNA2 mixed with AuNPs and incubated at 36-38° C. for 10-50 minutes with shaking, and then a citric acid / hydrochloric acid buffer is added and incubated with shaking for 8-12 hours.

3. The bacterial detection method based on CRISPR / Cas-SDA-AuNPs according to claim 2, characterized in that: The incubation time in the citric acid / hydrochloric acid buffer was 10 hours.

4. The bacterial detection method based on CRISPR / Cas-SDA-AuNPs according to claim 1, characterized in that: The ratio of DNA to AuNPs was 200:

1.

5. The bacterial detection method based on CRISPR / Cas-SDA-AuNPs according to claim 1, characterized in that: The enzyme digestion reaction solution obtained in step (1) is first incubated at elevated temperature to inactivate the Cas enzyme activity, and then a hybridization reaction is performed after inactivation to restore the hybrid double strands opened during the inactivation process to hybrid double strands, and then amplified using the SDA system in step (2).

6. The bacterial detection method based on CRISPR / Cas-SDA-AuNPs according to claim 1, characterized in that: The SDA amplification product obtained in step (2) is inactivated by incubation at elevated temperature.

7. The bacterial detection method based on CRISPR / Cas-SDA-AuNPs according to claim 1, characterized in that: After the reaction in step (3), through photothermal detection, when Vibrio vulnificus is present, the temperature rise after 660nm laser irradiation is less than that of the control group without Vibrio vulnificus.

8. The bacterial detection method based on CRISPR / Cas-SDA-AuNPs according to claim 1, characterized in that: After the reaction in step (3), the bacterial concentration is proportional to the absorbance value of the reaction solution obtained in step (3) at 530 nm through UV-vis detection.

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