A Tobramycin Detection System and Detection Method Based on CRISPR-Cas12a and Hybridization Chain Reaction

Abstract

The present invention discloses a tobramycin detection system and a detection method based on CRISPR-Cas12a and hybridization chain reaction, including an identification probe TOB-AP-5, hybridization chain reaction amplification elements H1 and H2, LbCas12a protein, crRNA, and F-Q reporter probe. This method uses the identification probe TOB-AP-5 to recognize tobramycin, trigger the hybridization chain reaction, and then activate the nuclease activity of LbCas12a-crRNA to cleave the F-Q reporter probe and output a fluorescence signal. The system designs and optimizes the position of the tobramycin aptamer sequence in the identification probe, improving the detection sensitivity. The system designs part of the activation sequence of CRISPR-Cas12a in the loop region of the hybridization chain reaction element H1, effectively improving the signal-to-noise ratio. The detection linear range of this method is relatively wide, being 0.125 nM - 25 nM and 25 nM - 2500 nM, with high detection sensitivity, the lowest detection limit being 25 pM, and it can detect tobramycin in milk and beef samples, having broad application prospects in the fields of food safety and environmental monitoring.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a tobramycin detection system and method based on CRISPR-Cas12a and hybridization chain reaction. Background Art

[0002] Tobramycin is an aminoglycoside antibiotic widely used in human clinical practice and veterinary medicine to treat bacterial infections. The misuse of tobramycin can cause irreversible side effects on human life and health, including nephrotoxicity, neuromuscular blockade, and hypersensitivity reactions. Because the tobramycin molecule lacks a luminescent or fluorescent group, instrument-based analytical methods such as high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) lack sufficient sensitivity for tobramycin detection, posing significant challenges.

[0003] CRISPR-Cas (clustered regularly interspaced short palindromic repeats and their associated Cas proteins) is a remarkable adaptive immunity mechanism found in many bacteria and archaea. In addition to genome editing, the system has also been developed as a powerful molecular diagnostic tool. Among different CRISPR systems, CRISPR-Cas12a (also known as Cpf1) exhibits efficient nuclease activity, trans-cleaving non-target single-stranded DNA. The combination of the CRISPR-Cas12a system with functional nucleic acids (such as aptamers) has promoted the development of many non-nucleic acid detection methods based on the CRISPR-Cas12a system, which have shown good analytical performance.

[0004] Hybridization chain reaction (HCR) is an isothermal amplification method. This reaction can be performed by continuously opening two hairpin DNAs to amplify long double-stranded DNA without the participation of an enzyme. As a signal amplification method upstream of the CRISPR-Cas12a system, the hybridization chain reaction has the following problems: the recognition sequence and PAM site of the CRISPR-Cas12a system are both present in the hairpin DNA. When the hybridization chain reaction is not activated, the two hairpin DNAs, as hybridization chain reaction elements, may also activate the nuclease cleavage activity of the CRISPR-Cas12a system, generating nonspecific signals, resulting in high detection background, low sensitivity and other problems. Therefore, it is very necessary to optimize the recognition sequence and PAM site of the CRISPR-Cas12a system in the hairpin DNA. Summary of the Invention

[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a tobramycin detection system based on CRISPR-Cas12a and hybridization chain reaction.

[0008] To solve the above technical problems, the present invention provides the following technical solutions: a tobramycin detection system based on CRISPR-Cas12a and hybridization chain reaction, characterized in that it includes: nucleic acid probe TOB-AP-5, signal amplification nucleic acid element H1, signal amplification nucleic acid element H2, LbCas12a protein, crRNA, and FQ reporter probe;

[0009] Among them, the recognition nucleic acid probe TOB-AP-5 is a tobramycin recognition element; the signal amplification nucleic acid elements H1 and H2 are strand displacement isothermal amplification reaction elements; the FQ fluorescent reporter probe is a single-stranded DNA modified with a fluorescent group and a fluorescent quenching group at both ends, which is a trans-cleavage substrate of the LbCas12a protein.

[0010] As a preferred embodiment of the tobramycin detection system of the present invention, the TOB-AP-5 probe sequence is shown in SEQ ID No. 1; the H1 sequence is shown in SEQ ID No. 2; the H2 sequence is shown in SEQ ID No. 3; the crRNA sequence is shown in SEQ ID No. 4; and the FQ reporter probe sequence is shown in SEQ ID No. 5.

[0011] As a preferred embodiment of the tobramycin detection system of the present invention, the recognition probe TOB-AP-5 can specifically recognize tobramycin and induce a subsequent amplification reaction;

[0012] As a preferred embodiment of the tobramycin detection system of the present invention, the signal amplification nucleic acid element H1 comprises a recognition sequence of the CRISPR-Cas12a system, and the 10 nucleotides (5'-TATAGTACAG-3') in the recognition sequence are present in the loop region of H1;

[0013] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for detecting tobramycin based on CRISPR-Cas12a and hybridization chain reaction.

[0014] To solve the above technical problems, the present invention provides the following technical solution: a detection method for tobramycin based on CRISPR-Cas12a and hybridization chain reaction, characterized in that the detection method is:

[0015] The TOB-AP-5 probe was diluted to a concentration of 10 μM in buffer A, heated at 95°C for 5 minutes, and then slowly cooled to room temperature for use;

[0016] H1 and H2 were diluted to 10 μM in buffer A, heated at 95°C for 5 minutes, and then slowly cooled to room temperature for use;

[0017] Take 0.8 μL of the prepared annealed TOB-AP-5, add 2.0 μL of tobramycin standard solution or test solution of different concentrations, 1.0 μL of prepared annealed H1, 1.0 μL of prepared annealed H2, add buffer B to the final volume of 20 μL, and incubate the reaction at 37°C for 2 hours to obtain reaction solution 1;

[0018] Take 2.0 μL of 800 nM LbCas12a protein, 2.0 μL of 1 μM crRNA, 2.0 μL of 5 μM FQ reporter probe, 2 μL of 10× buffer C, and 11 μL of water, mix and incubate at room temperature for 5 minutes, then add 1 μL of the prepared reaction solution 1, react at 37 ° C for 2 hours, inactivate at 65 ° C for 10 minutes, and then measure the fluorescence signal using a fluorescence spectrophotometer;

[0019] As a preferred embodiment of the detection method of the present invention, the formula of the buffer A is: 25mM Tris-HCl, 500mM NaCl, pH=7.4.

[0020] As a preferred embodiment of the detection method of the present invention, the formula of the buffer B is: 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg / mL BSA, pH 7.9.

[0021] As a preferred embodiment of the detection method of the present invention, the formula of the 1× buffer C is: 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 100 μg / mL BSA, pH 7.9.

[0022] As a preferred embodiment of the detection method of the present invention, when measuring the fluorescence signal using a fluorescence spectrophotometer, the excitation wavelength is set to 480 nm and the scanning range is set to 500-600 nm.

[0023] Beneficial effects of the present invention:

[0024] The present invention discloses a tobramycin detection system based on CRISPR-Cas12a and hybridization chain reaction. The recognition probe in the detection system can specifically bind to tobramycin and undergo conformational modification, triggering a hybridization chain reaction to produce a product that can be recognized by the CRISPR-Cas12a system, activating the LbCas12a protein-crRNA complex to cut the fluorescent reporter probe, and analyzing the fluorescence intensity to detect trace amounts of tobramycin. The system designs and optimizes the position of the tobramycin aptamer sequence in the recognition probe, thereby improving the detection sensitivity. The system designs a partial activation sequence of CRISPR-Cas12a in the loop region of the hybridization chain reaction element H1, effectively improving the signal-to-noise ratio. The method has high detection sensitivity, a minimum detection limit of 25pM, and can detect tobramycin in milk and beef samples, and has broad application prospects in the fields of food safety and environmental monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the detection principle diagram of the present invention;

[0026] Figure 2 This is the result of feasibility verification of tobramycin detection in Example 2 of the present invention;

[0027] Figure 3 The effect of the position of the CRISPR-Cas12 activation sequence in the hybrid chain reaction element on the detection results in Example 3 of the present invention;

[0028] Figure 4 Schematic diagram of the structural optimization of the recognition probe in Example 4 of the present invention;

[0029] Figure 5 This is the structural optimization result of the recognition probe in Example 4 of the present invention;

[0030] Figure 6 The fluorescence signal response of different concentrations of tobramycin in Example 5 of the present invention and the drawing of a standard curve;

[0031] Figure 7 The specificity test results in Example 6 of the present invention are as follows; DETAILED DESCRIPTION

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0033] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0034] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0035] Unless otherwise specified, all raw materials used in the examples of the present invention are commercially available, as shown in Table 1.

[0036] Table 1

[0037]

[0038] LbCas12a protein was obtained by prokaryotic induction expression and purification. For details, see: Analytica Chimica Acta, 2023, 1283: 341950

[0039] The oligonucleotide sequences involved in the present invention are shown in Table 2.

[0040] Table 2

[0041]

[0042]

[0043] Note: The underline represents the intramolecular complementary region, the italic represents the tobramycin aptamer sequence, the bold represents the CRISPR-Cas12 activation sequence, 6-FAM is the fluorescent group, and BHQ1 is the fluorescence quenching group.

[0044] Example 1

[0045] The detection principle diagram of the ATP detection system of the present invention is shown in FIG. Figure 1. When there is no tobramycin in the test liquid, the recognition probe and the hybridization chain reaction amplification nucleic acid elements H1 and H2 all exist in a hairpin structure. The amplification reaction is a hybridization chain reaction (HCR). HCR does not require a temperature change program and can be carried out under constant temperature conditions, which simplifies the operation process and reduces dependence on equipment; HCR does not require the participation of enzymes, which reduces the detection cost and operation complexity, improves the biocompatibility of the detection system, and is suitable for the detection of complex samples with high accuracy; HCR signal amplification efficiency is high, and through the cascade hybridization events triggered by the target molecule, a linear double-stranded DNA nanostructure containing a large number of repeating units can be formed to achieve efficient signal amplification; the HCR product sequence is controllable, and different product DNA sequences can be designed as needed, which is very suitable for use with CRISPR technology to construct biosensors. In the present invention, CRISPR-Cas12a activation sequence (green) is designed in the loop region of H1 hairpin structure. When hybridization chain reaction is not triggered, the structure will not activate the nuclease activity of CRISPR-Cas12a system. When there is tobramycin in the liquid to be tested, tobramycin can specifically bind to the aptamer sequence (blue) in the recognition probe, causing the recognition probe to undergo conformational change, thereby triggering hybridization chain reaction. Due to the presence of double-stranded activation sequence (green) in the product of hybridization chain reaction, it can be recognized by LbCas12a-crRNA complex, and stimulate the nuclease hydrolysis activity of LbCas12a protein, cut the reporter probe, and output fluorescent signal. Fluorescence value is measured using a fluorescence spectrophotometer to calculate tobramycin concentration.

[0046] The method for detecting tobramycin using the above detection system comprises the following steps:

[0047] (1) Dilute the TOB-AP-5 probe to a concentration of 10 μM in buffer A (25 mM Tris-HCl, 500 mM NaCl, pH = 7.4), heat at 95°C for 5 minutes, and then slowly cool to room temperature for use;

[0048] (2) H1 and H2 were diluted to 10 μM in buffer A (25 mM Tris-HCl, 500 mM NaCl, pH = 7.4), heated at 95°C for 5 minutes, and then slowly cooled to room temperature for use;

[0049] (3) Take 0.8 μL of the annealed TOB-AP-5, add 2.0 μL of different concentrations of tobramycin standard solution or test solution, 1.0 μL of annealed H1, 1.0 μL of annealed H2, and use buffer B (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg / mL BSA, pH 7.9) to make up to a final volume of 20 μL, and incubate the reaction at 37°C for 2 hours to obtain a reaction solution;

[0050] (4) Take 2.0 μL of LbCas12a protein with a concentration of 800 nM, 2.0 μL of crRNA with a concentration of 1 μM, 2.0 μL of FQ reporter probe with a concentration of 5 μM, 2 μL of 10× buffer C (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 100 μg / mL BSA, pH 7.9) and 11 μL of water, mix and incubate at room temperature for 5 minutes, then add 1 μL of the reaction solution prepared in step (3), react at 37 ° C for 2 hours, inactivate at 65 ° C for 10 minutes, and then use a fluorescence spectrophotometer to set the excitation wavelength to 480 nm, the scanning range to 500-600 nm, and measure the fluorescence intensity at 520 nm.

[0051] Example 2

[0052] Figure 2 The feasibility verification results of the tobramycin detection method in Example 1: In the figure, the red line represents that the detection solution contains tobramycin, recognition probe (TOB-AP-5), hairpin DNA (H1), hairpin DNA (H2), LbCas12a-crRNA and reporter probe (FQ); the orange line represents that the detection solution contains recognition probe (TOB-AP-5), hairpin DNA (H1), hairpin DNA (H2), LbCas12a-crRNA and reporter probe (FQ); the green line represents that the detection solution contains hairpin DNA (H1), hairpin DNA (H2), LbCas12a-crRNA and reporter probe (FQ); the purple line represents that the detection solution contains hairpin DNA (H2), LbCas12a-crRNA and reporter probe (FQ); the black line represents that the detection solution contains hairpin DNA (H1), LbCas12a-crRNA and reporter probe (FQ). The experimental results show that only when tobramycin, LbCas12a-crRNA and all DNA sequences are present at the same time, a strong fluorescent signal can be detected, which is consistent with the detection principle described in Example 1.

[0053] Example 3

[0054] In the present invention, the partial activation sequence of CRISPR-Cas12a is designed in the loop region of the hybrid chain reaction element H1 (SEQ ID No.2), and there is no activation element in H2 (SEQ ID No.3), as shown in Table 2. In the reported design scheme, the partial activation sequence of CRISPR-Cas12a is designed in the neck region of the two hairpin DNAs of the hybrid chain reaction element, and the activity of CRISPR-Cas12a is controlled by the formation of the PAM site, as shown in Microchimica Acta.2024, 191:386; Int J Biol Macromol.2024, 266:130848. In order to compare the effects of the two design strategies on the detection results, two other hybrid chain reaction elements C-H1 (SEQ ID No.12) and C-H1 (SEQ ID No.13) were designed according to the reported strategy, and hybrid chain reaction excitation sequences Op-1 (SEQ ID No.14) and (SEQ ID No.15) Op-2 were designed, and the sequences are shown in Table 2. The excitation sequence Op-1 was used to activate the hybridization chain reaction with H1 and H2 as elements, and the excitation sequence Op-2 was used to activate the hybridization chain reaction with C-H1 and C-H2 as elements. The concentrations of the three activation sequences were set to 1.25 pM, 37.5 pM, and 125 pM, respectively. After the hybridization chain reaction was completed, the CRISPR-Cas12a system was used to identify the reaction products and cut the reporter probe. Figure 3 The effect of the position of the CRISPR-Cas12 activation sequence within the hybrid chain reaction element on the detection results: The two hybrid chain reaction elements H1 and H2 in the present invention significantly outperformed the two hybrid chain reaction elements C-H1 and C-H2 in the reported strategy. Based on these results, the strategy of designing part of the CRISPR-Cas12a activation sequence within the H1 loop region of the hybrid chain reaction element can effectively improve the signal-to-noise ratio of the detection.

[0055] Example 4

[0056] Figure 4This is a schematic diagram of the structural optimization of the recognition probes in the present invention: the red color in the recognition probe represents the hybridization chain reaction activation sequence, the blue color represents the tobramycin aptamer sequence, and the single arrow represents the direction of the nucleic acid sequence (5'-3'). The recognition probe has two important regions: Region 1 is the 3'-terminal complementary region of the hybridization chain reaction activation sequence, which affects the efficiency of the hybridization chain reaction; Region 2 is the 3'-terminal complementary extension region of the tobramycin aptamer sequence, which affects the recognition efficiency of tobramycin. The present invention optimizes Regions 1 and 2 and designs seven different probes: TOB-AP-1 (SEQ ID No. 6), TOB-AP-2 (SEQ ID No. 7), TOB-AP-3 (SEQ ID No. 8), TOB-AP-4 (SEQ ID No. 9), TOB-AP-5 (SEQ ID No. 1), TOB-AP-6 (SEQ ID No. 10), and TOB-AP-7 (SEQ ID No. 11). The sequences are shown in Table 2.

[0057] Figure 5 The structural optimization results of the recognition probes in the present invention are as follows: the bar graph represents the fluorescence intensity in negative (tobramycin concentration of 0 nM) and positive (tobramycin concentration of 1 μM) conditions, and the line graph represents the signal-to-noise ratio (F / F0) of different recognition probes. It can be seen that when the recognition probe is TOB-AP-5, the signal-to-noise ratio is the highest. Therefore, TOB-AP-5 is selected as the optimal recognition probe in the present invention.

[0058] Example 5

[0059] Different concentrations of tobramycin (0 nM, 0.025 nM, 0.125 nM, 0.250 nM, 2.5 nM, 25 nM, 125 nM, 250 nM, 750 nM, 1250 nM, 2500 nM, 5000 nM) were added to the biosensor designed by the present invention, and the fluorescence intensity of the solution at an emission wavelength of 520 nm was measured using a fluorescence spectrophotometer to determine the gradient response of the solution fluorescence intensity to samples with different tobramycin concentrations in the present tobramycin detection invention. The same set of samples was measured three times. The relationship between fluorescence intensity and tobramycin concentration is shown in Figure 6 A, As can be seen, within the range of 0.025nM-5000nM, as the tobramycin concentration increases, the fluorescence intensity increases accordingly. Compared with the blank sample, the fluorescence intensity of all samples is significantly different (p value is 0.01 for tobramycin concentrations of 0.025-0.250nM and p value is 0.001 for tobramycin concentrations of 2.5-5000nM).

[0060] The calculation formula of the detection limit of the present invention is based on the calculation method of the detection limit (LOD) of the Vashist team: the absorbance value corresponding to the minimum detection limit = the average blank absorbance value + the blank value of 3σ (standard deviation), also known as the 3σ principle. According to this principle, the minimum detection limit (LOD) of tobramycin in the present invention is 25pM. Figure 6 B. Based on the above results, the tobramycin detection system designed by the present invention has good detection sensitivity.

[0061] Select Figure 6 The data of tobramycin concentration in A are 0nM, 0.125nM, 0.250nM, 2.50nM, and 25nM. The standard curve is drawn with tobramycin concentration as the horizontal axis and F / F0 value as the vertical axis. Figure 6 C. The results showed that there was a good linear relationship between the fluorescence intensity and the logarithmic concentration of tobramycin, and the linear relationship coefficient R 2 =0.9983, the linear range is 0.125nM to 25.0nM. Figure 6 The data of tobramycin concentration in A are 0nM, 25nM, 125nM, 250nM, 750nM, 1250nM, and 2500nM. The standard curve is drawn with tobramycin concentration as the horizontal axis and F / F0 value as the vertical axis. Figure 6 D. The results showed that there was a good linear relationship between the fluorescence intensity and the concentration of tobramycin, and the linear relationship coefficient R 2 =0.9823, and the linear range is 25nM to 2500nM.

[0062] Example 6

[0063] To evaluate the specificity of the tobramycin detection method designed by the present invention, five antibiotics, including tobramycin (TOB), kanamycin (KANA), ampicillin (AMP), oxytetracycline (OTC), and chloramphenicol (CHL), and their mixture (MIX), were selected under optimal detection conditions. The detection method of Example 1 was applied for simultaneous detection, and the specificity of the detection method was verified by comparing the test results. In the experiment, each antibiotic was set at a concentration of 2.5 μM. The fluorescence intensity of the final sample at 520 nm was measured and compared by a fluorescence spectrophotometer.

[0064] Specificity test results such as Figure 7 As can be seen, there was no significant difference in fluorescence intensity between the non-target antibiotics (KANA, AMP, OTC, and CHL) and the blank sample. Only in the presence of tobramycin (TOB) did the fluorescence intensity increase significantly compared to the blank sample. These results demonstrate that the constructed tobramycin detection method has excellent specificity.

[0065] Example 7

[0066] To evaluate the reliability of the biosensor designed in the present invention for detecting tobramycin in food samples, we added different concentrations of tobramycin to pure milk (brand: Weigang, purchased from a local supermarket) or beef (purchased from a local vegetable market). After treatment using the method reported in the literature (Sensors and Actuators B: Chemical. 2012, 173: 262-267), samples were prepared for testing. These samples were tested using the detection method of Example 1 under optimal detection conditions. The same group of samples was tested three times. The test results were compared with the added amount, and the recovery rate and relative standard deviation were calculated. The results are shown in Table 3. It can be seen that the average recovery rate of the tobramycin biosensor designed in the present invention for detecting food samples was between 95.361% and 99.338%, the detection error was less than 5%, and the relative standard deviation was less than 3.2854%. This shows that the method is reliable and accurate and can be effectively applied to the detection of tobramycin in food samples.

[0067] Table 3

[0068]

[0069] Comparative Example 1

[0070] This comparative example is CN 113640268 B, a tobramycin detection system based on CRISPR-Cas12a: aptamer probe AP, CrRNA, AsCas12a protein, KF polymerase, and a reporter probe modified with a fluorescent group and a quenching group at both ends;

[0071] The sequence of the aptamer probe AP is (5'-3'):

[0072] ATC ATT TGG AGG AAC TGG AGT CAC AAG CTG AGG A TG TGA CTC CAG GCA CTTAGT CAC A;

[0073] The sequence of crRNA is (5'-3'):

[0074] UAA UUU CUA CUC UUG UAG AUG CUU GUG ACU CCA GUU CCU C;

[0075] The sequence of the reporter probe is (5'-3'):

[0076] FAM-TTATT-BHQ1.

[0077] In Comparative Example 1, signal amplification was performed using strand displacement amplification (SDA), a polymerase-dependent DNA amplification method. The product sequence is template-restricted and has low programmability. In contrast, the present invention employs hybridization chain reaction (HCR) for signal amplification. This method does not require enzymes, and the hairpin DNA used in the HCR amplification element is programmable, making it more suitable for use with CRISPR technology to construct biosensors. Compared with Comparative Example 1, the present invention has the following improvements in detection effect: (1) The detection results of the present invention are more accurate. The detection error of the spiked recovery rate of tobramycin in milk or beef samples in Example 7 of the present invention is within 5%, and the relative standard deviation is lower than 3.2854%, while the detection error of the spiked recovery rate of tobramycin in milk or lake water samples in Comparative Example 1 is within 10%; (2) The detection linear range of the present invention is wider, and it can be applied to the detection of tobramycin in a wider concentration range. There are two detection linear regions in Example 5 of the present invention, namely 0.125nM to 25.0nM and 25nM to 2500nM, while there is only a narrower linear detection range in Comparative Example 1, and the detectable upper limit is only 1nM; (3) The amplification reaction in the present invention does not require the participation of enzymes, which reduces the detection cost.

[0078] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, and all of these should be included in the scope of the present invention.

Claims

1. A tobramycin detection system based on CRISPR-Cas12a and hybridization chain reaction, characterized in that: Including nucleic acid probe TOB-AP-5, signal amplification nucleic acid element H1, signal amplification nucleic acid element H2, LbCas12a protein, crRNA, FQ reporter probe; Among them, the nucleic acid probe TOB-AP-5 is a tobramycin recognition element; the signal amplification nucleic acid elements H1 and H2 are strand displacement isothermal amplification reaction elements; the FQ reporter probe is a single-stranded DNA modified with a fluorescent group and a fluorescent quenching group at both ends, which is a trans-cleavage substrate of the LbCas12a protein; The nucleic acid probe TOB-AP-5 sequence is shown in SEQ ID No.1; the H1 sequence is shown in SEQ ID No.2; the H2 sequence is shown in SEQ ID No.3; the crRNA sequence is shown in SEQ ID No.4; and the FQ reporter probe sequence is 6-FAM - TTTATT -BHQ1.

2. A method for detecting tobramycin based on CRISPR-Cas12a and hybridization chain reaction, characterized in that: The detection method is: The nucleic acid probe TOB-AP-5 was diluted to a concentration of 10 μM in buffer A, heated at 95°C for 5 minutes, and then slowly cooled to room temperature for use; The signal amplification nucleic acid elements H1 and H2 were diluted to 10 μM in buffer A, heated at 95°C for 5 minutes, and then slowly cooled to room temperature for use; Take 0.8 μL of the prepared annealed nucleic acid probe TOB-AP-5, add 2.0 μL of tobramycin standard solution or test solution of different concentrations, 1.0 μL of the prepared annealed signal amplification nucleic acid element H1, 1.0 μL of the prepared annealed signal amplification nucleic acid element H2, use buffer B to make up to a final volume of 20 μL, and incubate the reaction at 37°C for 2 hours to obtain reaction solution 1; Take 2.0 μL of 800 nM LbCas12a protein, 2.0 μL of 1 μM crRNA, 2.0 μL of 5 μM FQ reporter probe, 2 μL of 10 × buffer C and 11 μL of water, mix and incubate at room temperature for 5 minutes, then add 1 μL of the prepared reaction solution 1, react at 37°C for 2 hours, inactivate at 65°C for 10 minutes, and then measure the fluorescence signal using a fluorescence spectrophotometer; Wherein, the nucleic acid probe TOB-AP-5 sequence is shown in SEQ ID No.1; the H1 sequence is shown in SEQ ID No.2; the H2 sequence is shown in SEQ ID No.3; the crRNA sequence is shown in SEQ ID No.4; the FQ reporter probe sequence is 6-FAM - TTTATT- BHQ1; The formula of the buffer A is: 25 mM Tris-HCl, 500 mM NaCl, pH = 7.4; The formula of the buffer B is: 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 µg / mL BSA, pH 7.9; The formula of the 10× buffer C is: 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 100 μg / mL BSA, pH 7.

9.

3. The detection method according to claim 2, characterized in that: When measuring fluorescence signals using a fluorescence spectrophotometer, set the excitation wavelength to 480 nm and the scanning range to 500 - 600 nm.

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