Hairpin probe combination and kit for detecting self-hybridization chain reaction of AFB1

Abstract

The invention provides a hairpin probe combination and a kit for self-hybridization chain reaction for AFB1 detection, and belongs to the field of DNA amplification and detection. According to the self-hybridization chain reaction SHCR provided by the invention, the palindromic sequence is introduced into the stem part of the DNA hairpin probe, a traditional HCR system needing at least two DNA hairpin probes is simplified into an SHCR system needing only one DNA hairpin probe, and the SHCR system is used for detecting AFB1, so that the cost and the complexity of sequence design and operation are reduced; the aptamer can be further expanded to detection of other non-nucleic acid target substances by simply integrating corresponding aptamer sequences, and the application prospect is wide.

Description

Technical Field

[0001] The present application relates to the field of DNA amplification and detection, and in particular to a hairpin probe combination and a kit and application thereof for detecting AFB1 by self-hybridization chain reaction. Background Art

[0002] Aflatoxin B1 (AFB1) is a secondary metabolite produced by the Aspergillus flavus and Aspergillus parasiticus fungi. It has a stable structure, mutagenic and teratogenic properties, and is highly toxic. AFB1 can be produced and multiplied in food, posing serious risks to humans and animals along the food chain. Therefore, the development of highly sensitive and selective AFB1 detection methods is crucial.

[0003] The hybridization chain reaction (HCR) is an isothermal, enzyme-free, and highly efficient amplification strategy proposed by Pierce et al. in 2004. The system generally consists of two DNA probes (H1 and H2) with hairpin structures and a trigger strand. The DNA hairpin probes contain three domains: a stem, a loop, and a sticky end. Because the complementary sequences between the hairpin probes H1 and H2 are trapped in a secondary structure, they can coexist metastable in solution in the absence of trigger DNA. Once the trigger strand is added, it drives the opening of hairpin H1, and the exposed domains open hairpin H2. The newly exposed H2 domain then shares the same sequence as the promoter strand, triggering the alternating opening of the two hairpins, ultimately forming a long double-stranded DNA with a gap.

[0004] The DNA hairpin probe used in traditional HCR is generally 48 bases, including 6 hairpin loop bases, 6 sticky end bases and 36 hairpin stem bases, which brings high sequence design complexity and cost. In order to simplify the HCR system, people have designed DNA hairpin probes with 36-42 bases, but because this simplified strategy excessively reduces the length of the stem, it leads to significant background signal leakage. To solve this problem, Winssinger et al. developed an HCR system based on peptide nucleic acid (PNA), shortening the hairpin probe to 20 bases. Asanuma et al. reported an HCR system based on left-handed acyclic D-threitol nucleic acid (D-aTNA). The hairpin probe of this strategy only needs 26 bases to coexist stably in solution.

[0005] While the aforementioned methods simplify the HCR system to some extent, they all require two essential DNA hairpin probes, which limits the reduction in cost and design complexity brought about by this simplification. Therefore, further simplification of the HCR system is still needed. Summary of the Invention

[0006] Current HCR architectures are complex and involve numerous sequences. These systems typically consist of two hairpin DNA probes (H1 and H2) and a trigger strand. The DNA hairpin probes contain three structural domains: a stem, a loop, and a sticky end. Because the complementary sequences between the hairpin probes H1 and H2 are trapped in a secondary structure, they can coexist in a metastable state in solution in the absence of trigger DNA.

[0007] Since the traditional HCR system requires two DNA hairpin probes, the structural design is complex, and the sequence synthesis cost and reaction cost increase, resulting in an increase in the overall cost. To address the current problem, the present application discloses a hairpin probe combination for self-hybridization chain reaction of AFB1 detection. By designing a single DNA hairpin probe and initiating the HCR reaction with a trigger chain, the structure is simplified and the cost is reduced.

[0008] The present application discloses a hairpin probe combination for detecting AFB1 by self-hybridization chain reaction, wherein the hairpin probe combination comprises an aptamer trigger chain and a hairpin probe; The hairpin probe consists of two unpaired sticky ends, a hairpin stem consisting of a palindromic sequence, and a hairpin loop connected to the hairpin stem.

[0009] In certain preferred embodiments, the sum of the number of cytosine bases and the number of guanine bases in the hairpin stem sequence consisting of a palindromic sequence is not less than two-thirds of the total number of bases; Preferably, the sum of cytosine bases and guanine bases of the hairpin stem sequence using a palindromic sequence is two-thirds of the total number of bases.

[0010] In certain preferred embodiments, the hairpin stem employing a palindromic sequence is 18 bases in length.

[0011] In certain preferred embodiments, the total number of bases at the two sticky ends is equal to the total number of bases in the hairpin loop, and the length of the hairpin loop is 12 bases.

[0012] In certain preferred embodiments, the two non-paired sticky end sequences are identical and complementary to the hairpin loop sequence.

[0013] In certain preferred embodiments, the aptamer trigger chain comprises a trigger chain sequence and an AFB1 aptamer sequence; The trigger chain sequence includes a sticky end complementary region complementary to the sticky end, a hairpin stem complementary region complementary to the hairpin stem, and a free segment; The complementary region of the sticky end is identical to the sequence of the free segment. The complementary region of the sticky end can be reversely complementary paired with the sticky end. The complementary region of the hairpin stem can be reversely complementary paired with the hairpin stem sequence.

[0014] In certain preferred embodiments, the sequence of the hairpin probe is shown as SEQ ID NO.1, and the sequence of the aptamer trigger chain is shown as SEQ ID NO.2.

[0015] In traditional hybridization chain reaction (HCR), Figure 1 As shown, when the trigger strand DNA is present, the sticky end (a) of hairpin H1 hybridizes with the complementary sequence a* in the trigger strand (I). This process causes the hairpin structure of H1 to open, forming the I·H1 intermediate. The newly exposed sequence c on H1 then complementarily pairs with the sticky end (c*) of H2, thereby opening the hairpin structure of H2 and forming the I·H1·H2 complex. This step exposes a sequence (a*b*) in H2 that is identical to the trigger strand, triggering the opening of another hairpin structure in H1, triggering a series of cascade reactions, and ultimately generating a long DNA nanowire with a nick.

[0016] In a specific embodiment of the present application, the stem of the hairpin probe adopts a palindromic sequence, and the sum of cytosine bases and guanine bases is exactly two-thirds of the total number of bases.

[0017] At the same time, the two sticky ends of the hairpin probe are designed to complement the hairpin loop region. For example, the two sticky end regions are composed of 6 thymine bases (T), and the hairpin loop region is composed of 12 adenine bases (A). This poly-T or A design not only enables a single H to form a stable hairpin structure, effectively preventing the hairpin loop and stem region from pairing to form double-stranded DNA, but also enables hybridization between them to form a double-stranded DNA nanowire structure. The newly exposed domains serve as the trigger sequence for HCR and continue to hybridize with H, resulting in continuous growth. Therefore, the probe combination designed in this application is named the self-hybridization chain reaction based on a single hairpin (SHCR).

[0018] The reaction principle of the AFB1 detection based on the aforementioned single-hair card probe SHCR system is as follows: Figure 2 The symmetrical structural design of the hairpin probe H of the present application can also allow the 5' end or 3' end of the trigger strand T to hybridize with the 3' end or 5' end of the hairpin H at the same time, thereby improving the hybridization efficiency.

[0019] In another aspect, the present application provides a detection kit comprising the aforementioned hairpin probe combination for self-hybridization chain reaction for detecting AFB1.

[0020] In certain preferred embodiments, the buffer solution used in the self-hybridization chain reaction (SHCR) is 20 mM Tris (comprising 140 mM sodium chloride, 50 mM magnesium chloride, pH 7.5).

[0021] The reaction system using buffer was as follows: the trigger and blocker strands of the aptamer were premixed in buffer. The mixture and the hairpin probe were then annealed at 95°C for 5 minutes and cooled to room temperature for 3 hours before use. Different concentrations of the trigger-blocker strand mixture, AFB1, and the hairpin probe were mixed in buffer and incubated at room temperature.

[0022] In another aspect, the present application may also use the aforementioned hairpin probe combination for the self-hybridization chain reaction for detecting AFB1 or the aforementioned detection kit in detecting AFB1.

[0023] Compared with the existing technology, the beneficial effects of the present application include: the self-hybridization chain reaction (SHCR) provided by the present application simplifies the HCR system that traditionally requires at least two DNA hairpin probes into a SHCR system that only requires one DNA hairpin probe by introducing a palindromic sequence into the stem of the DNA hairpin probe, and uses it for the detection of AFB1, reducing the cost and complexity of sequence design and operation; it can also be further expanded to the detection of other non-nucleic acid target substances by simply integrating the corresponding aptamer sequence, and has broad application prospects.

[0024] Figure 1 Schematic diagram of the traditional hybridization chain reaction (HCR) provided in this application; Figure 2 This is a schematic diagram of the principle of detecting AFB1 based on self-hybridization chain reaction (SHCR) provided in this application; Figure 3 Schematic diagram of the simulated structure of the hairpin probe H in the self-hybridization chain reaction SHCR provided in Example 1; Figure 4 Schematic diagram of the analysis results of SHCR before and after the reaction using fluorescence detection, gel electrophoresis, and atomic force microscopy provided in Example 3; Figure 5 Schematic diagram of the results of analyzing the SHCR reaction using the optimized experimental conditions provided in Example 5; Figure 6 Schematic diagram of the results of AFB1 sensitivity detection using self-hybridization chain reaction (SHCR) provided in Example 6; Figure 7 Schematic diagram of the results of AFB1-specific detection using self-hybridization chain reaction (SHCR) provided in Example 7; DETAILED DESCRIPTION

[0025] The embodiments of the present application will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, the conditions are carried out according to conventional conditions or manufacturer recommendations. The reagents or instruments used are not specified by the manufacturer and are conventional products that can be purchased commercially.

[0026] The features and performance of the present application are further described in detail below with reference to the embodiments.

[0027] In the first embodiment, a self-hybridization chain reaction (SHCR) sequence based on a single hairpin is designed.

[0028] In this example, aflatoxin B1 (AFB1) was selected as a model molecule, and an aptamer specifically binding to AFB1 was introduced into the SHCR system. The designed hairpin probe assembly includes an aptamer trigger strand and a hairpin probe. The sequence of the hairpin probe is shown in SEQ ID NO. 1, and the sequence of the aptamer trigger strand is shown in SEQ ID NO. 2.

[0029] By simulating the DNA sequence of the hairpin probe, the results are as follows Figure 3 As shown in Figure 2, the predicted hairpin probe structure consists of 18 bases in the stem and 12 bases in the loop. This single hairpin structure is relatively stable and is the primary product formed after annealing. The simulation results are consistent with expectations.

[0030] In the second example, based on the hairpin probe combination designed in Example 1, a fluorescent group FAM and a quencher group BHQ1 were modified on either side of the hairpin probe stem. The sequence of the hairpin probe with the fluorescent group and the quencher group is shown in SEQ ID NO. 3. The present invention uses fluorescence intensity as the signal output. The proximity and separation of FAM and BHQ1 lead to quenching and recovery of the fluorescence signal, thus designing an AFB1 signal amplification aptamer biosensor based on a single hairpin self-hybridization chain reaction.

[0031] In the third embodiment, in order to compare the effects of the hairpin probe and aptamer trigger chain of the hairpin probe combination designed in this application, aflatoxin B1 (AFB1) was selected as a model molecule in this embodiment, and the AFB1 aptamer was combined with the SHCR system. With fluorescence intensity as the signal output mode, the fluorescent group FAM and the quencher group BHQ1 were modified on both sides of the hairpin probe stem, respectively, to design an AFB1 signal amplification aptamer biosensor based on the SHCR system; in addition, a blocking chain partially hybridized with the aptamer trigger chain was designed to block nonspecific reactions; the sequence of the blocking chain is shown in SEQ ID NO. 4.

[0032] The principle of SHCR system detection of AFB1 is as follows Figure 2 As shown, the designed aptamer trigger chain T A Contains the trigger chain sequence T D and AFB1 aptamer sequences, while the blocking strand B partially hybridizes with the aptamer trigger strand to avoid T in the absence of AFB1. D Hybridization between H and AFB1. When AFB1 is added, the AFB1 aptamer is recognized by AFB1, and the trigger chain T is released. D The H is opened through a strand displacement reaction, thereby triggering the SHCR reaction.

[0033] The buffer solution used in this system is: 20 mM Tris, 140 mM NaCl, 50 mM MgCl, pH 7.5. The blocking chain B and the aptamer trigger chain T are mixed in the buffer. A The mixture was pre-mixed together, and then the mixture was annealed with the hairpin probe H at 95°C for 5 minutes and slowly cooled to room temperature over 3 hours to form a double-stranded complex T A -B and stable hairpin H. A one-pot method was used to detect AFB1, that is, different concentrations of T A -B, AFB1 and H were mixed and incubated at room temperature. According to the fluorescence characteristics of FAM, the excitation wavelength was set to 487 nm, the emission spectrum range was set to 500-600 nm, and the slit width was set to 5 nm when measuring with a fluorescence spectrophotometer. F / F 0 AFB1 was quantitatively determined, F and F 0 Represent the fluorescence intensity in the presence and absence of AFB1, respectively.

[0034] In the fourth example, to verify the feasibility of the SHCR system, we used fluorescence detection, gel electrophoresis, and atomic force microscopy to characterize it. In the fluorescence detection, we used a hairpin labeled with the fluorophore FAM and the quencher BHQ1. In the absence of AFB1, the probe H exists stably in the form of a hairpin, which brings FAM and BHQ1 into close proximity, resulting in fluorescence quenching. After the addition of AFB1, the AFB1 aptamer is recognized by AFB1, and the released trigger chain T D Opening H triggers the SHCR reaction, causing FAM and BHQ1 to move away from each other and the fluorescence signal to recover. Figure 4 As shown in Figure A, in the absence of AFB1, the fluorescence signal of the SHCR system is weak, while the fluorescence signal of the system with the addition of AFB1 is significantly enhanced, indicating that the hairpin H is opened after AFB1 binds to its aptamer, which shows that the designed SHCR system can provide a feasible fluorescence signal for the detection of AFB1. In addition, we further performed gel electrophoresis to support the results, as shown in Figure 4. Figure 4 As shown in B. The results show that in the SHCR system with the introduction of AFB1, DNA nanowires of different lengths can be observed, which is similar to the fluorescence results. At the same time, the morphology of the SHCR system was characterized by AFM, and it was observed that no DNA nanowires were generated when AFB1 was not added ( Figure 4 C), while the introduction of AFB1 produced longer nanowires ( Figure 4 D).

[0035] In the fifth embodiment, in order to obtain the best experimental effect of the SHCR system, we conducted an optimization experiment.

[0036] AFB1 was used as the detection object to analyze the length of the blocking chain B, B and T A Ratio (B / T A )、T A concentration, H concentration, Mg in the buffer 2+ The experimental conditions such as concentration, reaction time, etc. Figure 5 As shown, the results show that in B8, B / T A =2 / 1, 100 nM T A 、300 nM H1、50 mM Mg 2+ The maximum relative fluorescence intensity was obtained under the conditions of 20 min reaction. F / F 0 ( F and F 0 are the fluorescence intensities at 520 nm in the presence and absence of AFB1, respectively).

[0037] In the sixth embodiment, the detection performance of the SHCR system for detecting AFB1 was evaluated. Under the optimal experimental conditions of Example 5, the sensitivity of the SHCR system was evaluated.

[0038] When AFB1 is used as a detection target for research, Figure 6 As shown in A, with the increase of AFB1 concentration, the fluorescence intensity gradually increased and then reached a stable period. F / F 0 A good linear relationship was obtained in the range of 5.0-300 ng / mL (y=0.00496x+1.077, R 2 =0.992)( Figure 6 B). Based on the 3σ / slope principle, the detection limit was calculated to be 1.93 ng / mL.

[0039] In the seventh example, the selectivity of the SHCR system for AFB1 structural analogs and other mycotoxins that may coexist with AFB1 was evaluated.

[0040] like Figure 7 As shown, taking ZEN, AFG1, AFG2, OTA, AFM1, AFM2 and AFB2 as controls, at the same concentration, the relative fluorescence intensity of AFB1 was more than twice that of other similar mycotoxins, indicating that the SHCR system had good selectivity for AFB1.

[0041] In the eighth embodiment, the SHCR system can also be used to detect the concentration of AFB1 in actual samples.

[0042] To evaluate the practicality of the SHCR system, samples of liquor, corn, chili peppers, and pepper purchased from a local supermarket were used. After simple processing, the samples were spiked with different concentrations of AFB1 and the linear equation was substituted into the above equation for calculation. The test results for the above samples are shown in Table 1: The specific results are shown in Table 1 Table 1 Test results of four different samples

[0043] Calculated AFB1 recoveries ranged from 99.30% to 107.22%, with a relative standard deviation (RSD) less than 3.17%. This demonstrates the practicality and versatility of the SHCR system proposed in this application, enabling successful application in the detection of AFB1 in real samples and providing a potential method for AFB1 detection in food safety.

[0044] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

Claims

1. A hairpin probe assembly for detecting AFB1 by self-hybridization chain reaction, characterized in that: The hairpin probe combination includes an aptamer trigger chain and a hairpin probe; The hairpin probe comprises two unpaired sticky ends, a hairpin stem consisting of a palindromic sequence, and a hairpin loop connected to the hairpin stem.

2. The hairpin probe assembly for detecting AFB1 by self-hybridization chain reaction according to claim 1, characterized in that: The sum of the number of cytosine bases and the number of guanine bases in the hairpin stem sequence consisting of a palindromic sequence is not less than two-thirds of the total number of bases; Preferably, the sum of cytosine bases and guanine bases of the hairpin stem sequence using a palindromic sequence is two-thirds of the total number of bases.

3. The hairpin probe assembly for self-hybridization chain reaction for detecting AFB1 according to claim 1, characterized in that: The length of the hairpin stem using a palindromic sequence is 18 bases.

4. The hairpin probe assembly for detecting AFB1 by self-hybridization chain reaction according to claim 1, characterized in that: The total number of bases of the two unpaired sticky ends is equal to the total number of bases of the hairpin loop, and the length of the hairpin loop is 12 bases.

5. The hairpin probe assembly for self-hybridization chain reaction for detecting AFB1 according to claim 1, characterized in that: The two non-paired sticky end sequences are identical and complementary to the hairpin loop sequence.

6. The hairpin probe assembly for detecting AFB1 by self-hybridization chain reaction according to claim 1, characterized in that: The aptamer trigger chain comprises a trigger chain sequence and an AFB1 aptamer sequence; The trigger chain sequence includes a sticky end complementary region complementary to the sticky end, a hairpin stem complementary region complementary to the hairpin stem, and a free segment; The sticky end complementary region and the free segment sequence are identical, the sticky end complementary region and the sticky end can be reversely complementary paired, and the hairpin stem complementary region and the hairpin stem sequence can be reversely complementary paired.

7. The hairpin probe assembly for self-hybridization chain reaction for detecting AFB1 according to any one of claims 1 to 6, characterized in that: The sequence of the hairpin probe is shown in SEQ ID NO.1, and the sequence of the aptamer trigger chain is shown in SEQ ID NO.

2.

8. A detection kit, characterized in that The detection kit comprises a hairpin probe combination for self-hybridization chain reaction for detecting AFB1 according to any one of claims 1 to 7.

9. The detection kit according to claim 8, characterized in that The self-hybridization chain reaction used a buffer solution including 20 mM Tris (including 140 mM sodium chloride, 50 mM magnesium chloride, pH 7.5).

10. Use of the hairpin probe combination for self-hybridization chain reaction for detecting AFB1 according to any one of claims 1 to 7 or the detection kit according to any one of claims 8 to 9 in detecting AFB1.

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