Methods, compositions, and kits for detecting n-acylhomoserine lactones in a test sample

Abstract

This invention provides a method, composition, and kit for detecting N-acylhomoserine lactone in a sample, relating to the fields of biosensing and molecular diagnostics. The method provided by this invention, based on nucleic acid aptamers, T4 DNA polymerase extension blocking, T7 RNA polymerase in situ transcription, and CRISPR / enAsUCas12a technology, achieves rapid, highly sensitive, and amplification-free detection of the bacterial quorum sensing signal molecule N-acylhomoserine lactone. It has significant application value in food safety, environmental microbial monitoring, clinical diagnosis of drug-resistant bacterial infections, and high-throughput screening of quorum sensing inhibitors (QSI).

Description

Technical Field

[0001] This invention relates to the field of biosensing and molecular diagnostics, and in particular to a method, composition, and kit for detecting N-acylhomoserine lactone in a sample. Background Technology

[0002] Bacterial quorum sensing (QS) is a core mechanism by which Gram-negative bacteria secrete and sense N-acylhomoserine lactones (AHLs) as autoinducers to achieve cell density-dependent gene regulation. This process directly regulates biofilm formation, virulence factor expression, bacterial resistance, and maintenance of chronic infection, and has become a key target for food safety. N-butyryl-L-homoserine lactone (C4-HSL) and other molecules are key signaling molecules in the *Pseudomonas aeruginosa* QS system, and their sensitive detection is crucial for infection early warning and anti-biofilm therapy. Traditional AHL detection methods mainly include thin-layer chromatography and high-performance liquid chromatography-mass spectrometry (HPLC-MS). However, existing methods have limitations such as insufficient sensitivity, cumbersome operation, or the need for specialized instruments, making them unsuitable for rapid on-site detection.

[0003] CRISPR / Cas12a is a type II V CRISPR effector protein characterized by: (1) recognizing the 5'-TTTV-3' (V = A / C / G) PAM sequence; (2) the crRNA consists of a backbone sequence of approximately 21 nt (sfcrRNA) and a spacer sequence of approximately 20 nt (spcrRNA); and (3) after cis-cleaving the target dsDNA, it can continuously perform trans-cleavage on free ssDNA, thereby amplifying the signal. The crRNA splitting strategy involves splitting the complete crRNA into two parts at the junction, and then recombinating them in solution through their complementary pairing regions to form a functional crRNA, thereby activating Cas12a.

[0004] In recent years, the combination of nucleic acid aptamers and CRISPR / enAsuCas12a technology has developed rapidly and has been successfully used for the detection of non-nucleic acid targets such as ATP, uric acid, and proteins. However, these methods generally suffer from problems such as high background signal, high false positive risk, and the need for pre-amplification. Furthermore, there is still no dedicated, accurate, high-signal-intensity, and portable innovative solution for small molecules like AHLs. Therefore, there is an urgent need to develop a novel AHLs detection technology that requires no nucleic acid pre-amplification, has no false positives, and achieves a detection limit in the nM-pM range, to meet the pressing needs of rapid food safety screening, environmental water monitoring, and high-throughput screening of QS-inhibiting drugs. Summary of the Invention

[0005] This invention provides a method, composition, and kit for detecting N-acylhomoserine lactone in a sample.

[0006] In a first aspect, the present invention provides a method for detecting N-acylhomoserine lactone in a sample to be tested, comprising: S1. Configure the reaction system; the reaction system includes a detection unit, a first signal amplification unit, and a second signal amplification unit, wherein the detection unit includes an aptamer sensor with viscous ends, and the aptamer sensor has the structure shown in Formula 1. 5'-n(a)-n(b)-n(c)-3' Equation 1; In Formula 1, n(a) represents the reverse complementary sequence of the polynucleotide fragment shown in SEQ ID NO:4, n(b) represents the positive strand sequence of the T7 promoter, and n(c) is the aptamer sequence that can specifically bind to the target substance N-acylhomoserine lactone that may be present in the sample to be tested. The first signal amplification unit includes a T4 DNA polymerase, a T7 RNA polymerase, and a crRNA backbone, wherein the nucleotide sequence of the crRNA backbone is SEQ ID NO:46; The second signal amplification unit includes a double-stranded DNA substrate, a Cas enzyme, and a fluorescent signal molecule, wherein the nucleotide sequence of the double-stranded DNA substrate is SEQ ID NO:19; S2. The detection unit and the sample to be tested are mixed. After the aptamer sensor binds to the N-acyl homoserine lactone that may exist in the sample to be tested and undergoes a conformational change, the first signal amplification unit is added to the reaction system. The sticky ends in the aptamer sensor that may contain sticky ends are extended and transcribed by T4 DNA polymerase and T7 RNA polymerase to obtain crRNA fragments. S3. After the reaction in S2 is completed, the reaction system is subjected to enzyme inactivation. S4. The reaction system after enzyme inactivation in S3 is mixed with the second signal amplification unit to self-assemble the possible crRNA splitting fragments with the crRNA backbone to obtain crRNA, and activate the cleavage activity of Cas enzyme, thereby cleaving the double-stranded DNA substrate and fluorescent signal molecules. S5. By detecting the fluorescence signal of the reaction system, confirm whether the sample to be tested contains N-acylhomoserine lactone.

[0007] As described above, the samples to be tested are primarily those that may contain Gram-negative bacteria, such as bacterial cultures or cell-free supernatants, environmental samples, food samples, and biological samples. The Gram-negative bacteria are selected from the genus *Pseudomonas*, such as *Pseudomonas aeruginosa* and *Pseudomonas fluorescens*; the genus *Aeromonas*, such as *Aeromonas hydrophila* and *Aeromonas salmonicida*; the genus *Yersinia*, such as *Yersinia enterocolitica*; the genus *Serratia*, such as *Serratia liquefaciens*; *Escherichia*; and *Salmonella*. The environmental and food samples refer to aquatic environments, soil environments, and food samples containing the aforementioned Gram-negative bacteria. The biological samples refer to tissues, feces, etc., derived from animals.

[0008] In the method described above, the N-acylhomoserine lactone includes C4-HSL, C6-HSL, C8-HSL, C12-HSL, etc. Further, the nucleotide sequence of the aptamer specifically binding to C4-HSL is SEQ ID NO:44, the nucleotide sequence of the aptamer specifically binding to C12-HSL is SEQ ID NO:45, and the nucleotide sequences of the aptamers specifically binding to both C4-HSL and C12-HSL are at least one of SEQ ID NO:37-43. Even further, the nucleotide sequence of the aptamer specifically binding to both C4-HSL and C12-HSL is SEQ ID NO:40.

[0009] As described above, the 5' end of the aptamer sensor is modified with a labeling group, and the detection unit further includes magnetic beads modified with an antibody against the labeling group. Further, the labeling group is biotin, and the antibody against the labeling group is streptavidin. Further, the particle size of the magnetic beads is 300 nm-5 μm; even further, the particle size of the magnetic beads is 5 μm.

[0010] As described above, the first signal amplification unit further includes a mixture of dNTPs (including dATP, dTTP, dGTP and dCTP), a mixture of NTPs (ATP, GTP, CTP and UTP), and a reaction buffer required for the catalytic reaction of T4 DNA polymerase and T7 RNA polymerase.

[0011] In the method described above, in step S3, the enzyme inactivation temperature is 55-70°C and the time is 2-10 minutes.

[0012] In the method described above, the Cas enzyme is the Cas12a protein derived from amino acid cocci.

[0013] In a second aspect, the present invention provides a composition comprising a detection unit, a first signal amplification unit, and a second signal amplification unit, wherein the detection unit includes an aptamer sensor containing a sticky end, and the aptamer sensor comprises the structure shown in Formula 1. 5'-n(a)-n(b)-n(c)-3' Equation 1; In Formula 1, n(a) represents the reverse complementary sequence of the polynucleotide fragment shown in SEQ ID NO:4, n(b) represents the reverse complementary sequence of the T7 promoter, and n(c) is the aptamer sequence that can specifically bind to the target substance N-acylhomoserine lactone that may be present in the sample to be tested. The first signal amplification unit includes a T4 DNA polymerase, a T7 RNA polymerase, and a crRNA backbone, wherein the nucleotide sequence of the crRNA backbone is SEQ ID NO:46; The second signal amplification unit includes a double-stranded DNA substrate, a Cas enzyme, and a fluorescent signal molecule, wherein the nucleotide sequence of the double-stranded DNA substrate is SEQ ID NO:19.

[0014] In the composition described above, the aptamer sensor containing the adhesive end is selected from at least one of A1)-A9): A1) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:37; A2) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:38; A3) The nucleotide sequence of the DNA molecule is shown in SEQ ID NO:39; A4) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:40; A5) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:41; A6) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:42; A7) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:43; A8) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:44; A9) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:45.

[0015] In the composition described above, the 5' end of the aptamer sensor is modified with a labeling group, and the detection unit further includes an antibody-modified magnetic bead that resists the labeling group.

[0016] Thirdly, the present invention provides a kit comprising any of the compositions described above.

[0017] The method provided by this invention can achieve rapid, highly sensitive, and amplification-free detection of bacterial quorum sensing signal molecules (such as C4-HSL, C6-HSL, C8-HSL, C12-HSL, etc.), and has important application value in food safety, environmental microbial monitoring, clinical diagnosis of drug-resistant bacterial infections, and high-throughput screening of quorum sensing inhibitors (QSI). Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the detection principle provided by the present invention; Figure 2 Design and screening of spcrRNA and substrates for CRISPR system; where AB is the fluorescence intensity comparison of PAM-D01-PAM-D12; C is the secondary structure of the hairpin switch constructed by spcrRNA-04; Figure 3 CRISPR signals were compared for different hairpin sequences; where A is a comparison of fluorescence intensity of different hairpin sequences; B is the fluorescence intensity of a1; C is the fluorescence intensity of b4; D is the fluorescence intensity of c6; E is the fluorescence intensity of d7; F is the fluorescence intensity of e10; and G is the fluorescence intensity of f11. Figure 4 Comparison of CRISPR signals between the T4 / T7 two-step method and the one-step method; where AB represents the fluorescence intensity of the two-step method and CD represents the fluorescence intensity of the one-step method. Figure 5 Comparison of CRISPR signals for magnetic beads with different particle sizes; where A represents the fluorescence intensity of magnetic beads with different particle sizes; and B represents the fluorescence intensity comparison of two aptamer switches. Figure 6 This study aims to validate the system and investigate false positives at different T4 / T7 enzyme inactivation temperatures. A represents the fluorescence intensity in different reaction systems, and B represents the fluorescence intensity at different inactivation temperatures and times. Figure 7 For system sensitivity verification; Figure 8 For system specificity verification; where A is the fluorescence intensity after the magnetic C4 / C12 hairpin switch reacts with different substrates; B is the fluorescence intensity after the magnetic C4 hairpin switch reacts with different substrates; C is the fluorescence intensity after the magnetic C12 hairpin switch reacts with different substrates.

[0019] Figure 2-8In this context, data are expressed as mean ± standard deviation, and **** indicates p < 0.05. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, embodiments of this invention, and should not be construed as limiting the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. In the description of this invention, it should be understood that the terminology used is for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0022] The streptavidin-modified magnetic beads (SA-Magnetic Beads) used in the following examples were purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.; T4 DNA polymerase (5 U / μL) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; dNTP Mix (10 mM each) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; T7 High Efficiency Transcription Kit (containing a mixture of T7 RNA polymerase and NTPs (100 mM each)) was purchased from Beijing TransGen Biotech; N-(3-Oxopentanoyl)-L-homoserine lactone (CAS No. 148433-21-0, C5-HSL) was purchased from MedChemexpress (MCE); N-Octanoyl-L-homoserine lactone (CAS No. 147852-84-4, C8-HSL) was purchased from MedChemexpress (MCE); N-3-oxo-dodecanoyl-L-homoserine Lactone (CAS No. 168982-69-2, C12-HSL) was purchased from MedChemexpress (MCE); N-[(3S)-tetrahydro-2-oxo-3-furanyl]butyramide (N-Butyryl-L-homoserine lactone, CAS No. 67605-85-0, C4-HSL) was purchased from MedChemexpress (MCE).

[0023] The sequences involved in the following examples are shown in Tables 1-3. The sequences used were synthesized from Qingke Biotechnology Co., Ltd.

[0024] Table 1. Different spcrRNAs and their corresponding candidate substrate sequences in the CRISPR system (5'→3')

[0025] Table 2. Candidate List of Different Card Issuing Sequences

[0026] Table 3. Sequences used in the kit

[0027] The principle of the detection method provided by this invention is as follows: Figure 1 As shown, firstly, a target substance recognition element is constructed. The structure of the target substance recognition element is 5'-n(a)-n(b)-n(c)-3', where n(a) represents the split recognition crRNA (spcrRNA) transcription template sequence of the spacer region crRNA (sfcrRNA) sequence of the target gene (excluding the corresponding crRNA sequence in the scaffold region), n(b) represents the T7 promoter template sequence, and n(c) represents the aptamer sequence that can specifically bind to the target substance N-acylhomoserine lactone (AHL) that may be present in the test sample. The target substance recognition element is formed after annealing. Figure 1The hairpin structure shown includes a sticky end, comprising an exposed T7 promoter b1 and a transcription template strand c1 for spcrRNA. The linker region a1 is complementary to the AHL aptamer sequence, forming a closed hairpin stem structure for controlling the on / off state. When the target substance AHL is absent in the sample, the linker region of this recognition element remains closed, the hairpin structure is intact, and the sticky end is present normally. Subsequently, in the first signal amplification unit, under the action of T4 DNA polymerase and T7 RNA polymerase, the sticky end extends normally, generating a complete double-stranded DNA template, which is then used to transcribe spcrRNA. Next, in the second signal amplification unit, the spcrRNA self-assembles with the pre-added scaffold region sequence of sfcrRNA in solution to form complete crRNA, which guides the Cas protein to recognize the CRISPR substrate DNA, activating its trans-cleavage activity and extensively cleaving the fluorescent probe (5'-FAM-TTATT-BHQ1-3'), generating a strong fluorescent signal. When the target substance AHL is present in the sample, it specifically binds to the aptamer sequence in the target substance recognition element, causing the hairpin structure to open, the linker region to be exposed, and the complementary sticky end structure to disappear. This blocks the elongation reaction of T4 DNA polymerase, significantly reduces spcrRNA transcription, and ultimately decreases the yield of intact crRNA. The trans-cleavage activity of Cas protein is inhibited, reducing the cleavage of the fluorescent probe, and positive samples show a significant decrease in fluorescence signal. By comparing the fluorescence signal difference between the test group and the non-target group, the presence of the target substance AHL in the sample can be determined.

[0028] Example 1: spcrRNA screening The splitting SfcrRNA is responsible for binding to the Cas12a protein, while the spcrRNA region is complementary to the non-template strand of the target DNA, determining sequence specificity. This embodiment utilizes spcrRNA in a splitting crRNA strategy and designs a method to screen for the optimal sequence.

[0029] As shown in Table 1, this embodiment provides 12 different spcrRNA sequences, each 20 nt in length. Candidate spcrRNA-01 to spcrRNA-12 were designed based on principles such as GC content and structure. To systematically investigate the effect of GC content on Cas12a activation efficiency, the 12 candidate sequences cover three GC content gradients: low, medium, and high. The specific groupings are as follows: Low GC approximately 25-35%: spcrRNA-01, 02, 04, 05, 06, 07, 10, and 12; Medium GC approximately 40-55%: spcrRNA-03 and 11; High GC approximately 75-100%: spcrRNA-08 and 09. The secondary structure of the candidate sequences in the complete crRNA conformation was predicted using Mfold software (https: / / www.unafold.org / mfold / applications / rna-folding-form.php), and those with reasonable structures were retained for subsequent experimental verification.

[0030] Simultaneously, the detection system uses related nucleic acid sequences. The aptamer sequences and T7 promoter sequences used in this embodiment are as follows, and each sequence is written in the 5'→3' direction. (1) Aptamer sequences: C4 / C12 dual-target recognition aptamer (C4 / C12 hairpin aptamer): 5'-GCAATGGTACGGTACTTCCTGTGGGTTAGTTTGACTCAAGGCTGGGCTTATACAAAAGTGCACGCTACTTTGCTAA-3'; C4-HSL specific aptamer (C4 hairpin aptamer): 5'-GCAATGGTACGGTACTTCCCGGGGCGGGCTGTCATGCCCATCCTACCGTGACCGCAAAAGTGCACGCTACTTTGCTAA-3'; C12-HSL specific aptamer (C12 hairpin aptamer): 5'-GCAATGGTACGGTACTTCCCGCCGCCGCGTTCTCATCGCGTGATGTAGTGTCCGCAAAAGTGCACGCTACTTTGCTAA-3'. (2) T7 promoter double-stranded sequence: T7 promoter sense strand (T7F): 5'-CCCTATAGTGAGTCGTATTA-3'; T7 promoter antisense strand (T7R): 5'-TAATACGACTCACTATAGGG-3'. The above two sequences form the T7 promoter double-stranded structure in the aptamer switch nucleic acid conformation, driving the transcription of spcrRNA.

[0031] The specific procedure and system for CRISPR detection are as follows: Add 1 μL of different ssDNA substrates (16.6 nM) to the CRISPR detection system. This system contains: 0.4 μL of enAsuCas12a protein (1 mg / mL, the enAsuCas12a protein used was expressed and purified in the laboratory, amino acid sequence is SEQ ID NO:48), 2 μL of corresponding sfcrRNA (1 μM) or enzyme-free water (as a CRISPR reaction blank control, NC), 1 μL of fluorescent reporter molecule (25 μM, FAM-TTATT-BHQ1), and 2 μL of NEB2.1 reaction buffer, and add enzyme-free water to make up the volume to 20 μL. After mixing, immediately place in a microplate reader, at 39℃, excitation wavelength 485 nm, emission wavelength 520 nm, react for 60 minutes, and collect the fluorescence signal intensity once per minute. To eliminate interference from fluorescence background signals, the experimental results are converted into normalized fluorescence intensity (%) to reflect the cleavage efficiency. The calculation formula is as follows: Normalized fluorescence intensity (%) = (F sample F NC ) / (F max F NC ) × 100%. Where, F sample The fluorescence values ​​of each group after 30 minutes of CRISPR reaction, F N C represents the fluorescence value of the negative control group, F max This represents the maximum fluorescence value across all experimental groups. For example... Figure 2 As shown in Figures A and B, ssDNA containing the TTTV sequence and PAM can directly activate the trans-cleavage activity of Cas12a; even when the ssDNA substrates without PAM (D01-D12) are perfectly complementary to spcrRNA, the fluorescence signal is significantly lower. PAM-mediated substrate efficiency is higher when Cas12a protein recognizes and targets ssDNA. The optimal sequence pair was determined to be the combination of PAM-D04 and spcrRNA-04, which exhibited the highest normalized fluorescence intensity.

[0032] The secondary structure of the hairpin switch constructed from spcrRNA-04 was predicted using Mfold, with the folding temperature set to 25°C and Na+. + 100 mM Mg² + Concentration 1 mM. The constructed two-stage structure of the card-issuing switch is as follows: Figure 2 As shown in C, it can pair with the 3' end aptamer bases to form a stem, which provides a structural basis for the subsequent rapid recognition of the AHL open double-stranded structure and serves as an extension transcription switch.

[0033] Example 2: Comparison of background signal and sensitivity between the T4 / T7 two-step method and the one-step method Step 1: Fabrication of the recognition element The nucleic acid strand shown in Table 1, “Magnetic C4 hairpin switch spcrRNA-04”, was synthesized, and 100 μM of aptamer was prepared with sterile water. After unwinding at 95°C for 10 minutes, the temperature was slowly lowered to 25°C at a rate of 1°C per minute to form a stable stem-loop secondary structure. After annealing, the biotin-labeled recognition element was diluted with Buffer I (containing 10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, and 0.01%–0.1% Tween-20, adjusted to pH 7.5), and the prepared recognition element was stored at -4°C. The recognition element was mixed with 5 mM and 10 mM target small molecule C4-HSL standard aqueous solutions at a volume ratio of 2:3 to form the positive control group. A negative control group was also set up, with an equal volume of enzyme-free water added. The mixture was incubated at 37°C for 30–60 minutes. Under these conditions, C4-HSL in the positive group can specifically bind to the recognition element, thereby preventing the formation of the stem-loop structure of the aptamer; while in the negative group, since there is no target, the aptamer retains its stem-loop structure.

[0034] Step 2: Construction of the first signal amplification unit To compare the background signal and sensitivity of the T4 / T7 two-step method with the one-step method, this step is divided into two cases and constructed separately.

[0035] 2.1, T4 / T7 two-step method 2.1.1 T4 DNA polymerase extension Add 0.5 μL of a dNTP mixture (dATP, dGTP, dTTP, and dCTP concentrations of 10 mM each), 0.2 μL of T4 DNA polymerase, and 2 μL of NEBuffer 2.1 to the 3 μL reaction mixture provided in step 1. Then, bring the reaction volume to 20 μL using nuclease-free water. Incubate the mixture at 25°C for 30 minutes. During this time, the sticky ends of the recognition element will extend under the action of T4 DNA polymerase, forming a complete DNA double-stranded structure.

[0036] 2.1.2 T7 RNA polymerase transcription Take 4 μL of the T4 extension product obtained in step 2.1.1 above, and add 0.5 μL of a mixture of NTPs (ATP, UTP, CTP, and GTP, all 100 mM), 0.2 μL of T7 RNA polymerase, and 2 μL of NEBuffer 2.1. Add nuclease-free water to a final volume of 20 μL. Incubate the reaction mixture at 37°C for 30 minutes. During this time, the T7 RNA polymerase will transcribe spcrRNA-04 from the extended double-stranded template. After incubation, heat-inactivate the enzyme at 60°C for 3 minutes.

[0037] 2.2, T4 / T7 one-step method To the 0.6 μL reaction mixture provided in step 1, add 0.2 μL T4 DNA polymerase, 0.5 μL dNTP mixture, 0.2 μL T7 RNA polymerase, 0.5 μL NTP mixture, and 2 μL NEBuffer 2.1. Finally, bring the total volume to 20 μL with enzyme-free water. After reacting at 37°C for 60 minutes, heat at 60°C for 3 minutes to inactivate the enzyme, and store the product at 4°C for later use.

[0038] 3. CRISPR / enAsuCas12a detection 10 μL of the reaction products obtained from the 2.1 T4 / T7 two-step method and the 2.2 T4 / T7 one-step method, along with enzyme-free water (as a CRISPR reaction blank control, NC), were added to the CRISPR detection system, which was the same as the CRISPR detection system in Example 1. This system contained: 0.4 μL of enAsuCas12a protein (1 mg / mL, the enAsuCas12a protein used was expressed and purified in the laboratory), 1 μL of PAM-DO4 (16.6 nM), 1 μL of fluorescent reporter molecule (25 μM, FAM-TTATT-BHQ1), and 2 μL of NEB2.1 reaction buffer, with enzyme-free water added to bring the volume to 20 μL. After mixing, the mixture was immediately placed in a microplate reader and reacted at 39°C with an excitation wavelength of 485 nm and an emission wavelength of 520 nm for 60 minutes, collecting the fluorescence signal intensity every minute. By comparing the fluorescence curves of the positive and negative groups, the inhibitory effect of C4-HSL binding to the aptamer on the CRISPR / enAsuCas12a system cleavage activity was evaluated, thereby achieving the detection of the target molecule. Results are as follows: Figure 4 As shown, Figure 4 The two-step methods in sections A and B, using recognition elements, respectively identify and bind to 5 mM and 10 mM target small molecule C4-HSL standards. Positive test groups produce significant fluorescent signals, showing a significant difference in intensity compared to the negative groups. Figure 4 In the C and D one-step methods, significant signal intensity differences were observed for the 5 mM and 10 mM target small molecule C4-HSL standards and the negative group. The difference in fluorescence signal between the positive group and the target-free negative group was calculated using the following formula: ,in The fluorescence signals represent the target-free negative group, the target-positive group, and the NC blank group, respectively. The optimized T4 / T7 one-step strategy and the two-step strategy showed no significant difference in fluorescence signal intensity between the target-positive group and the target-free negative group, and the background signal of the NC group was extremely low. This indicates that the designed recognition element sequence and the T4 / T7 one-step strategy can achieve highly sensitive AHL small molecule signal generation while reducing the difficulty of human operation.

[0039] Example 3: Optimal Adaptation of Hairpin Element Length and Sequence Filtering The nucleic acid chains shown in Table 2 were synthesized, and the truncated and extended aptamer sequences were synthesized to obtain recognition elements, named a1, b4, c6, d7, e10, and f11, respectively. d7 corresponds to a reference original aptamer sequence with a length of 76 nt bases. a1, b4, and c6 were truncated by 6, 3, and 1 bases respectively, while e10 and f11 were extended by 3 and 8 bases respectively. The nucleic acid chains were annealed according to the method in Example 2 and incubated at 37°C for 30 minutes with 10 mM C4-HSL standard aqueous solution or an equal volume of enzyme-free water, respectively, as positive and negative binding samples. The specific method is as follows: 100 μM of aptamer was prepared with sterile water, unwound at 95°C for 10 minutes, and then slowly cooled to 25°C at a rate of 1°C per minute to form a stable stem-loop secondary structure. The recognition elements were mixed with 10 mM target small molecule C4-HSL standard aqueous solution at a volume ratio of 2:3 to form the positive experimental group. A negative control group was also included, with an equal volume of enzyme-free water added. The mixture was incubated at 37°C for 30-60 minutes. Under these conditions, C4-HSL in the positively bound samples specifically binds to the recognition element, thereby inhibiting the formation of the aptamer stem-loop structure; while in the negatively bound samples, because no target is present, the aptamer retains its stem-loop structure.

[0040] The specific system and procedure for the T4 / T7 one-step reaction and CRISPR detection system provided in Example 2 are as follows: First, add 0.6 μL of the bound sample or an equal volume of enzyme-free water to the T4 / T7 one-step reaction system. The T4 / T7 one-step reaction system includes 0.2 μL of T4 DNA polymerase, 0.5 μL of dNTP mixture, 0.2 μL of T7 RNA polymerase, 0.5 μL of NTPs mixture, and 2 μL of NEBuffer 2.1, and bring the total volume to 20 μL with enzyme-free water. After reacting at 37°C for 60 minutes, heat at 60°C for 3 minutes to inactivate the T4 / T7 enzymes. Subsequently, CRISPR / enAsuCas12a detection was performed. 10 μL of the reaction product obtained from the T4 / T7 one-step method and enzyme-free water (as a blank control for CRISPR, NC) were added to the CRISPR detection system. This CRISPR detection system contained: 0.4 μL of enAsuCas12a protein (1 mg / mL, the enAsuCas12a protein used was expressed and purified in our laboratory), 1 μL of PAM-DO4 (16.6 nM), 1 μL of fluorescent reporter molecule (25 μM, FAM-TTATT-BHQ1), and 2 μL of NEB2.1 reaction buffer, with enzyme-free water added to bring the volume to 20 μL. Finally, after mixing, the system was placed in a microplate reader, and the fluorescence signal (excitation wavelength 485 nm, emission wavelength 520 nm) was monitored at 39℃ for 60 minutes. The signal intensities of positive and negative binding samples and the blank control group (NC) of the CRISPR reaction were recorded. The results are as follows: Figure 3 As shown in Figure A, compared to other nucleic acid chains, the fluorescence intensity difference between the targetless and target groups of d7 is the greatest. Figure 3 The real-time fluorescence curves of BG correspond to a1, b4, c6, d7, e10, and f11, respectively, and the number of sticky terminal base pairs formed are 1, 4, 6, 7, 10, and 11, respectively. Therefore, d7 was determined to be the optimal recognition element for subsequent construction of the aptamer switch sequence.

[0041] Example 4: Signal Comparison of Different Magnetic Bead Sizes Step 1: Optimal magnetic bead particle size screening An improvement to the detection system of Example 3 was made by introducing magnetic beads to achieve the separation and enrichment of AHL in the sample. The biotin-modified aptamer switch nucleic acid chain (d7-BIO) was annealed according to the methods in Examples 2 and 3 to promote the formation of a stable stem-loop secondary structure. Streptavidin magnetic beads of different sizes (300 nm, 1 μm, 2.8 μm, 5 μm) were washed and reconstituted in Buffer I (10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, 0.01%-0.1% Tween-20, pH 7.5), and then thoroughly resuspended with the annealed aptamer. The mixture was then slowly incubated with shaking at room temperature to allow the biotin-modified nucleic acid chain d7-BIO to specifically bind to the streptavidin-modified magnetic beads. The prepared magnetic aptamer switch was stored at -4°C. Before use, clean the magnetic aptamer switch, remove the supernatant by magnetic attraction, mix with 10mM target small molecule C4-HSL standard aqueous solution or an equal volume of enzyme-free water, and incubate at 37℃ for 60 minutes. These samples are then used as positive and negative binding samples for subsequent T4 / T7 CRISPR detection.

[0042] The detection was performed according to the method provided in Example 3. First, 0.6 μL of the bound sample or an equal volume of enzyme-free water was added to the T4 / T7 one-step reaction system. The T4 / T7 one-step reaction system included 0.2 μL of T4 DNA polymerase, 0.5 μL of dNTP mixture, 0.2 μL of T7 RNA polymerase, 0.5 μL of NTPs mixture, and 2 μL of NEBuffer 2.1, and the total volume was brought to 20 μL with enzyme-free water. After reacting at 37°C for 60 minutes, the mixture was heated at 60°C for 3 minutes to inactivate the T4 / T7 enzymes. Subsequently, CRISPR / enAsuCas12a detection was performed. 10 μL of the reaction product obtained from the extended transcription was added to the CRISPR detection system, which contained: 0.4 μL of enAsuCas12a protein (1 mg / mL, the enAsuCas12a protein used was expressed and purified in our laboratory), 1 μL of PAM-D04 (16.6 nM), 1 μL of fluorescent reporter molecule (25 μM, FAM-TTATT-BHQ1), and 2 μL of NEB2.1 reaction buffer. Enzyme-free water was added to bring the volume to 20 μL. Finally, after mixing, the mixture was placed in a microplate reader, and the fluorescence signal (excitation wavelength 485 nm, emission wavelength 520 nm) was monitored at 39℃ for 60 minutes. The signal intensities of the positive, negative, and blank control (NC) groups of the CRISPR reaction were recorded, and the fluorescence intensity was statistically analyzed. Results are as follows: Figure 5As shown in Figure A, it can be seen that all four particle sizes of magnetic beads can achieve sensitive target detection. When the particle size of the magnetic beads is 5 μm, the difference in fluorescence signal intensity between the target-positive group and the target-negative group is the largest, at 72.69%, confirming that 5 μm magnetic beads should be used for subsequent experiments.

[0043] Step 2: Magnetic aptamer switch sequence compatibility test The target molecule C4-HSL was dissolved in enzyme-free water to prepare test solutions with concentrations of 1 μM, 10 μM, 100 μM, and 1 mM. Following the same modification and synthesis method as step 1 in Example 4, 5 μm magnetic aptamer switch d7-BIO and d7-5-BIO with 5'-extended nucleic acid strands (as shown in Table 2) were prepared. These were mixed with different concentration gradients of target molecule C4-HSL standard solutions to form the positive binding group. A negative binding group was also prepared by adding an equal volume of enzyme-free water. The mixtures were incubated at 37°C for 30-60 minutes. The detection process was the same as step 1 in Example 4, and the results are as follows: Figure 5 As shown in Figure B, AHL sample recognition at concentrations as low as 1 nM can be achieved both before and after 5' end extension. This indicates that the modified magnetic beads have no significant impact on aptamer binding, DNA extension, or crRNA transcription. Experimental results show that both the magnetic aptamer switches d7-BIO and d7-5-BIO can achieve AHL recognition at concentrations as low as 1 nM, regardless of whether 5' end extension is performed. Although the absolute signal dynamic range of d7-5-BIO is slightly lower than that of d7-BIO, the detection sensitivity of both magnetic aptamer switches is superior to the detection limit for AHL (2.3 ng / mL, approximately equivalent to 13.4 nM) of the commonly used gas chromatography-mass spectrometry (GC-MS) method. These results collectively demonstrate that the magnetic modification strategy of this invention can maintain high sensitivity while providing flexible space for sequence optimization, which is beneficial for further improving the applicability of the detection system in complex samples.

[0044] Example 5, Method Validation and Sensitivity Testing: Signal Comparison of Target Substrate at Different Concentrations First, a 100 μM C4-HSL standard solution was prepared. The effects of positive and negative experimental groups, as well as the absence of key elements in the CRISPR detection (binding, extension, and transcription) on the detection signal were verified sequentially. Otherwise, the detection was performed according to the method provided in Example 4. 0.6 μL of positive / negative binding sample or an equal volume of enzyme-free water was added to the T4 / T7 one-step reaction system. The T4 / T7 one-step reaction system included 0.2 μL / 0 μL T4 DNA polymerase, 0.2 μL / 0 μL T7 RNA polymerase, 0.5 μL dNTP mixture, 0.5 μL NTPs mixture, and 2 μL NEBuffer 2.1, with the total volume brought to 20 μL with enzyme-free water. After reacting at 37°C for 60 minutes, the mixture was heated at 60°C for 3 minutes to inactivate the T4 / T7 enzymes. Subsequently, CRISPR / enAsuCas12a detection was performed. 10 μL of the above reaction product was added to the CRISPR detection system, mixed thoroughly, and placed in a microplate reader. Fluorescence signals were monitored at 39℃ for 60 minutes (excitation wavelength 485 nm, emission wavelength 520 nm), and the fluorescence intensity was recorded and calculated. The CRISPR detection system contained: 0.4 μL of enAsuCas12a protein (1 mg / mL, the enAsuCas12a protein used was expressed and purified in our laboratory), 1 μL of PAM-D04 (16.6 nM), 1 μL of fluorescent reporter molecule (25 μM, FAM-TTATT-BHQ1), and 2 μL of NEB2.1 reaction buffer, with enzyme-free water added to bring the volume to 20 μL.

[0045] The results are as follows Figure 6 As shown in Figure A, there are significant differences in fluorescence intensity signals between the positive and negative experimental groups and those lacking recognition elements.

[0046] To further investigate the role of the T4 / T7 one-step inactivation step in eliminating subsequent CRISPR false positive tests, the specific process is as follows: Following the method provided in Example 4, the relationship between the fluorescence signal and inactivation in the non-magnetic aptamer-switched CRISPR system was tested. First, 0.6 μL of enzyme-free water (without positive or negative binding samples) was added to the T4 / T7 one-step reaction system. The T4 / T7 one-step reaction system included 0.2 μL of T4 DNA polymerase, 0.5 μL of dNTP mixture, 0.2 μL of T7 RNA polymerase, 0.5 μL of NTPs mixture, and 2 μL of NEBuffer 2.1, and the total volume was brought to 20 μL with enzyme-free water. After reacting at 37°C for 60 minutes, no heating treatment or inactivation temperature and time testing was performed; instead, heating was performed at 55-70°C for 2-10 minutes. Subsequently, CRISPR / enAsuCas12a detection was performed. 10 μL of the reaction product obtained from the extended transcription and an equal volume of enzyme-free water were added to the CRISPR detection system. The CRISPR detection system contained: 0.4 μL of enAsuCas12a protein (1 mg / mL, the enAsuCas12a protein used was expressed and purified in our laboratory), 1 μL of PAM-DO4 (16.6 nM), 1 μL of fluorescent reporter molecule (25 μM, FAM-TTATT-BHQ1), and 2 μL of NEB2.1 reaction buffer, with enzyme-free water added to bring the volume to 20 μL. Finally, after mixing, the mixture was placed in a microplate reader, and the fluorescence signal (excitation wavelength 485 nm, emission wavelength 520 nm) was monitored at 39℃ for 60 minutes. The signal intensity of the test group and the blank control group (NC) of the CRISPR reaction was recorded. The results are as follows: Figure 6 As shown in Figure B, uninactivated T4 / T7 enzymes severely interfere with subsequent CRISPR reactions, leading to significant false positives and producing high fluorescence signals. However, after inactivation by heating at 55℃, 65℃, or 70℃ for 2-10 minutes, the fluorescence signal remains at a low level and is not significantly different from the blank control group (NC) of the CRISPR reaction, effectively eliminating false positives.

[0047] Subsequently, sensitivity testing was performed. The target molecule C4-HSL was dissolved in enzyme-free water to prepare standard solutions with concentrations of 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, and 1 μM. These solutions were added to a magnetic d7-BIO-5 μM recognition element tube as the experimental group. An equal volume of enzyme-free water was added as the negative control group (target concentration 0). Simultaneously, the T4T7 product in the CRISPR reaction was replaced with the same volume of enzyme-free water as the NC. The samples were incubated at 37°C for 60 minutes. Under these conditions, C4-HSL in the positive group specifically bound to the aptamers on the magnetic beads, thus inhibiting the formation of the aptamer stem-loop structure; while in the negative group, due to the absence of the target, the aptamers maintained their stem-loop structure.

[0048] The detection was performed according to the method provided in Example 4. By comparing the fluorescence intensity of the positive and negative groups, the inhibitory effect of C4-HSL binding on the cleavage activity of the CRISPR / enAsuCas12a system was evaluated, thus achieving the detection of the target molecule. The results are as follows: Figure 7 As shown, AHL signal detection as low as 1 pM can be achieved under laboratory conditions.

[0049] Example 6: Specificity verification of multiple AHLs Following the method described in step 1 of Example 4, the magnetic C4 / C12 hairpin switches, magnetic C4 hairpin switches, and magnetic C12 hairpin switches shown in Table 1 were synthesized and prepared respectively. After annealing, the aptamers were diluted with Buffer I (10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, 0.01%-0.1% Tween-20, pH 7.5) and placed in streptavidin magnetic beads with a particle size of 5 μm. The beads were vortexed for 20 seconds to fully resuspend the aptamers and then slowly incubated with shaking at room temperature. After preparation, each switch was stored in [location missing]. Store at 4℃ for later use. Clean as described in Example 4 before use.

[0050] C4-HSL, C5-HSL, C8-HSL, and C12-HSL were dissolved in enzyme-free water to prepare standard solutions with concentrations of 5 μM and 500 nM, respectively, for later use. The switch specificity tests for each magnetic aptamer were grouped as follows: (1) Specificity test of magnetic C4 / C12 hairpin switch: C4-HSL positive group and C12-HSL positive group were set up, and C4-HSL or C12-HSL standard solution with a concentration of 500 nM was added respectively; non-target interference group was set up, and C5-HSL or C8-HSL standard solution with a concentration of 10 times (5 μM) was added respectively; a negative control group was set up, and an equal volume of enzyme-free water was added to replace the target solution.

[0051] (2) Specificity test of magnetic C4 hairpin switch: a target positive group was set up, and a C4-HSL standard solution with a concentration of 500 nM was added; a non-target interference group was set up, and a C5-HSL, C8-HSL or C12-HSL standard solution with a concentration of 10 times (5 μM) was added respectively; a negative control group was set up, and an equal volume of enzyme-free water was added to replace the target solution.

[0052] (3) Specificity test of magnetic C12 hairpin switch: a target positive group was set up, and a C12-HSL standard solution with a concentration of 500 nM was added; a non-target interference group was set up, and a C4-HSL, C5-HSL or C8-HSL standard solution with a concentration of 10 times (5 μM) was added respectively; a negative control group was set up, and an equal volume of enzyme-free water was added to replace the target solution.

[0053] In all three sets of tests, a blank control group (NC) was also set up: in the subsequent CRISPR detection steps, an equal volume of enzyme-free water was added to the CRISPR reaction system instead of the T4 / T7 extended transcription products to reflect the background fluorescence level of the CRISPR detection system itself, thereby eliminating the interference of non-specific activation of the CRISPR system on the interpretation of results. The non-target interference group used a concentration ten times that of the target to evaluate the selection specificity of each magnetic aptamer switch for similar AHL molecules with similar chain lengths under harsh conditions.

[0054] Samples from all groups were incubated at 37°C for 60 minutes. Under these conditions, in the positive group, the corresponding target molecules (C4-HSL or C12-HSL) specifically bound to the aptamers on the magnetic beads, causing the stem-loop structure of the aptamers to unwind; in the negative control group, because there was no target present, the aptamers maintained a stable stem-loop secondary structure; in the non-target interference group, non-target AHL molecules should not specifically bind to the corresponding aptamers even at tenfold concentrations. After incubation, T4 / T7 extension transcription and subsequent CRISPR / enAsuCas12a fluorescence detection were performed according to the method provided in Example 4, and the signal intensity of each group was recorded to assess specificity. 0.6 μL of positive / negative group sample or an equal volume of enzyme-free water was added to the T4 / T7 one-step reaction system. The T4 / T7 one-step reaction system consisted of 0.2 μL / 0 μL T4 DNA polymerase, 0.2 μL / 0 μL T7 RNA polymerase, 0.5 μL dNTP mixture, 0.5 μL NTPs mixture, and 2 μL NEBuffer 2.1, with the total volume brought to 20 μL by adding enzyme-free water. The reaction was carried out at 37°C for 60 minutes, followed by heating at 60°C for 3 minutes to inactivate the T4 / T7 enzymes. Subsequently, CRISPR / enAsuCas12a detection was performed. 10 μL of the above reaction product was added to the CRISPR detection system, which contained: 0.4 μL of enAsuCas12a protein (1 mg / mL, the enAsuCas12a protein used was expressed and purified in our laboratory), 1 μL of PAM-DO4 (16.6 nM), 1 μL of fluorescent reporter molecule (25 μM, FAM-TTATT-BHQ1), and 2 μL of NEB2.1 reaction buffer. Enzyme-free water was added to bring the volume to 20 μL. Results are as follows: Figure 8 As shown in AC, when multiple quorum sensing signals are present, there are specific signal differences only in the corresponding C4-HSL or C12-HSL detection samples. This design has sequence editability and specificity.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting N-acylhomoserine lactone in a sample, characterized in that, include: S1. Configure the reaction system; the reaction system includes a detection unit, a first signal amplification unit, and a second signal amplification unit, wherein the detection unit includes an aptamer sensor with viscous ends, and the aptamer sensor has the structure shown in Formula 1. 5'-n(a)-n(b)-n(c)-3' Equation 1; In Formula 1, n(a) represents the reverse complementary sequence of the polynucleotide fragment shown in SEQ ID NO:4, n(b) represents the positive strand sequence of the T7 promoter, and n(c) represents the aptamer sequence that can specifically bind to the target substance N-acylhomoserine lactone that may be present in the sample to be tested. The first signal amplification unit includes a T4 DNA polymerase, a T7 RNA polymerase, and a crRNA backbone sequence, wherein the nucleotide sequence of the crRNA backbone sequence is SEQ ID NO:46; The second signal amplification unit includes a double-stranded DNA substrate, a Cas enzyme, and a fluorescent signal molecule, wherein the nucleotide sequence of the double-stranded DNA substrate is SEQ ID NO:19; S2. The detection unit and the sample to be tested are mixed. After the aptamer sensor binds to the N-acyl homoserine lactone that may exist in the sample to be tested and undergoes a conformational change, the first signal amplification unit is added to the reaction system. The sticky ends in the aptamer sensor that may contain sticky ends are extended and transcribed by T4 DNA polymerase and T7 RNA polymerase to obtain the spacer sequence of crRNA. S3. After the reaction in S2 is completed, the reaction system is subjected to enzyme inactivation. S4. The reaction system after enzyme inactivation in S3 is mixed with the second signal amplification unit, and the possible crRNA spacer sequence and crRNA backbone sequence are self-assembled to obtain crRNA, and the cleavage activity of Cas enzyme is activated, thereby cleaving the double-stranded DNA substrate and fluorescent signal molecules. S5. By detecting the fluorescence signal of the reaction system, confirm whether the sample to be tested contains N-acylhomoserine lactone.

2. The method according to claim 1, characterized in that, The N-acylhomoserine lactone includes C4-HSL and / or C12-HSL, the nucleotide sequence of the aptamer that specifically binds to C4-HSL is SEQ ID NO:44, the nucleotide sequence of the aptamer that specifically binds to C12-HSL is SEQ ID NO:45, and the nucleotide sequences of the aptamers that specifically bind to both C4-HSL and C12-HSL are at least one of SEQ ID NO:37-43.

3. The method according to claim 1, characterized in that, The aptamer sensor has a labeling group modified at its 5' end, and the detection unit also includes magnetic beads modified with antibodies against the labeling group.

4. The method according to claim 3, characterized in that, The magnetic beads have a particle size of 300nm-5μm.

5. The method according to claim 1, characterized in that, In S3, the enzyme is inactivated at a temperature of 55-70°C for 2-10 minutes.

6. The method according to claim 1, characterized in that, The Cas enzyme is the Cas12a protein derived from amino acid cocci.

7. The composition, characterized in that, The system includes a detection unit, a first signal amplification unit, and a second signal amplification unit. The detection unit includes an aptamer sensor with a sticky end, and the aptamer sensor has the structure shown in Formula 1. 5'-n(a)-n(b)-n(c)-3' Equation 1; In Formula 1, n(a) represents the reverse complementary sequence of the polynucleotide fragment shown in SEQ ID NO:4, n(b) represents the positive strand sequence of the T7 promoter, and n(c) is the aptamer sequence that can specifically bind to the target substance N-acylhomoserine lactone that may be present in the sample to be tested. The first signal amplification unit includes a T4 DNA polymerase, a T7 RNA polymerase, and a crRNA backbone, wherein the nucleotide sequence of the crRNA backbone is SEQ ID NO:46; The second signal amplification unit includes a double-stranded DNA substrate, a Cas enzyme, and a fluorescent signal molecule, wherein the nucleotide sequence of the double-stranded DNA substrate is SEQ ID NO:

19.

8. The composition according to claim 7, characterized in that, The aptamer sensor containing the sticky end is selected from at least one of A1)-A9): A1) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:37; A2) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:38; A3) The nucleotide sequence of the DNA molecule is shown in SEQ ID NO:39; A4) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:40; A5) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:41; A6) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:42; A7) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:43; A8) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:44; A9) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:

45.

9. The composition according to claim 7 or 8, characterized in that, The aptamer sensor has a labeling group modified at its 5' end, and the detection unit also includes magnetic beads modified with antibodies against the labeling group.

10. A reagent kit, characterized in that, Includes the composition according to any one of claims 7-9.

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