A fluorescent-on nucleic acid aptamer beacon probe, kit, detection method and application

Abstract

The present invention relates to the field of biotechnology, and in particular to a fluorescence-on type nucleic acid aptamer beacon probe, a kit, a detection method and an application. The fluorescence-on type nucleic acid aptamer beacon probe comprises a nucleic acid aptamer, a short cDNA, a polyT connecting arm, a fluorescent group and a quenching group; the polyT connecting arm is connected to the 3' end of the nucleic acid aptamer and the 5' end of the short cDNA, and the quenching group and the fluorescent group are respectively marked at the 5' end of the nucleic acid aptamer and the 3' end of the short cDNA, or the fluorescent group and the quenching group are respectively marked at the 5' end of the nucleic acid aptamer and the 3' end of the short cDNA; the 3' end sequence of the short cDNA is complementary to the 5' end sequence of the nucleic acid aptamer so that the quenching group and the fluorescent group are adjacent. The fluorescence-on type nucleic acid aptamer beacon probe is used for the detection of aflatoxin B1, and realizes rapid, sensitive and specific detection through fluorescence intensity measurement.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a fluorescence-on type nucleic acid aptamer beacon probe, a kit, a detection method and an application thereof. Background Art

[0002] Aptamers are single-stranded DNA or RNA that can specifically bind to a target. Compared with antibodies, aptamers have the advantages of low immunogenicity, good biocompatibility, chemical synthesis, low cost, and easy modification. Aptamers have been applied in the fields of chemistry, biology, and medicine, and are widely favored by researchers. Especially in the field of analytical detection, researchers have used aptamers as biorecognition elements to develop many aptamer sensors for detecting specific targets. According to the signal transduction method, these aptamer sensors can be divided into colorimetric method, fluorescence method, electrochemical method, surface enhanced Raman scattering method, surface plasmon resonance method and other types. Among them, fluorescent aptamer sensors have attracted much attention because of their high sensitivity, easy operation, and fast response speed.

[0003] Fluorescent aptamer sensors are usually constructed based on the principle of fluorescence resonance energy transfer (FRET), which indicates the presence of the target by turning on or off the fluorescence signal. There are two main types of FRET-based nucleic acid aptamer sensors: (1) DNA chain substitution type and (2) aptamer beacon type. The working principle of the DNA chain substitution type is as follows: Figure 1 a and Figure 1 As shown in Figure 2, multiple DNA strands are usually labeled with fluorescent groups (fluorophore, F) or quencher groups (quencher, Q). In the absence of a target, hybridization reactions occur between the DNA strands, causing the fluorescent group F and the quencher group Q to approach each other, reaching the FRET action distance, and fluorescence quenching occurs. When the target is present, the DNA strand with aptamer function will bind to the target instead of hybridizing with other DNA strands, causing the fluorescent group F and the quencher group Q to be far apart, exceeding the FRET action distance, and the system fluorescence intensity is high. This DNA chain replacement design strategy does not require knowing the specific structural characteristics of the aptamer and is applicable to any given aptamer. However, the actual effect of this design strategy depends to a large extent on the efficiency of the hybridization reaction between the DNA strands. The fact is that since the hybridization efficiency between DNA molecules is much lower than the hybridization efficiency within the molecule, when the concentration ratio (C aptamer :C cDNA) is 1:1, most (~90%) of the aptamers or cDNAs are in a non-hybridized state, resulting in a very high background fluorescence intensity. In order to increase intermolecular hybridization and thus reduce the background value, an excess of DNA single strands labeled with the quencher group Q are usually used, which increases the reagent cost. In addition, during the hybridization process, the target tends to bind to the free aptamer first rather than the aptamer in the hybridized state. Therefore, if the DNA single strand labeled with the quencher group Q is an aptamer and is in excess relative to cDNA, it may lead to a decrease in the sensitivity of this DNA chain-substituted aptamer sensor, or even no target response signal at all. In contrast, the aptamer beacon design strategy (principle such as Figure 1 c and Figure 1 d) only requires a single DNA strand, which is simpler in structure and more efficient. However, the versatility of this aptamer beacon design strategy is poor. This is because this strategy requires the aptamer molecule used to have a hairpin (stem-loop) structure, and the target-aptamer binding reaction can lead to the opening and closing of the hairpin (stem-loop) structure, thereby causing fluorescence recovery or fluorescence quenching. Obviously, not all aptamers have such a suitable structure and change, which depends entirely on the individual structural characteristics of the aptamer.

[0004] In this application, we covalently coupled nucleic acid aptamers and short cDNA chains to a DNA molecule through a polyT linker unit to prepare a new aptamer beacon probe, which was successfully used for the high-efficiency detection of the target aflatoxin B1. Summary of the invention

[0005] The present invention provides a fluorescence-on type nucleic acid aptamer beacon probe, a kit, a detection method and an application. The prepared novel nucleic acid aptamer beacon probe is used for the detection of aflatoxin B1, has high sensitivity and specificity, and solves the problems existing in the prior art.

[0006] One of the technical solutions adopted by the present invention is:

[0007] Provided is a fluorescence-on type nucleic acid aptamer beacon probe, comprising a nucleic acid aptamer, a short cDNA, a polyT connecting arm, a fluorescent group and a quenching group; the polyT connecting arm is connected to the 3' end of the nucleic acid aptamer and the 5' end of the short cDNA, the quenching group and the fluorescent group are respectively marked at the 5' end of the nucleic acid aptamer and the 3' end of the short cDNA, or the fluorescent group and the quenching group are respectively marked at the 5' end of the nucleic acid aptamer and the 3' end of the short cDNA; the 3' end sequence of the short cDNA is complementary to the 5' end sequence of the nucleic acid aptamer so that the quenching group and the fluorescent group are adjacent to each other;

[0008] The aptamer sequence is a DNA with specific recognition ability for aflatoxin B1, and its nucleotide sequence is shown in SEQ ID No. 1; the short cDNA sequence is shown in SEQ ID No. 2; and the polyT connecting arm is polymerized from 48 to 72 thymine deoxynucleotides.

[0009] Furthermore, the polyT linker is polymerized from 48 thymidine deoxynucleotides, and its nucleotide sequence is shown in SEQ ID No3.

[0010] SEQ ID No1: 5'-BHQ1-ACGTGTTGTCTCTCTGTGTCTCGT-3';

[0011] SEQ ID No2: 5'-AACACGT-FAM-3';

[0012] SEQ ID No3:5'

[0013] TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3'.

[0014] In the above SEQ ID No. 1 and SEQ ID No. 2, BHQ1 and FAM are respectively the fluorescent quencher and fluorescent group modified at the end. (The end fluorescent group or quenching group is omitted in each sequence in the sequence table.)

[0015] Furthermore, the full sequence of the fluorescent on-type nucleic acid aptamer beacon probe is shown in the following SEQ ID No. 4:

[0016] 【5'-BHQ1-ACGTGTTGTCTCTCTGTGTCTCGT

[0017] TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAACACGT-FAM-3'].

[0018] Furthermore, the quenching group is black hole quencher Ⅰ (black hole quencher Ⅰ, BHQ1), and the fluorescent group is 6-carboxyfluorescein (6-carboxyfluorescein, FAM).

[0019] The second technical solution adopted by the present invention is:

[0020] A kit for detecting aflatoxin B1 is provided, comprising the fluorescent on-type nucleic acid aptamer beacon probe as described above.

[0021] The present invention also provides a sensor system for detecting aflatoxin B1, comprising the fluorescence-on type nucleic acid aptamer beacon probe or the kit as described above; in the sensor system, the concentration of the nucleic acid aptamer beacon probe is 20 nmol / L.

[0022] Furthermore, the sensor system further includes a buffer solution, which is Buffer-A with a pH of 8.0, containing 20 mmol / L phosphate, 150 mmol / L NaCl, 20 mmol / L MgCl2 and 0.01% (v / v) Tween 20.

[0023] The third technical solution adopted by the present invention is:

[0024] Provided is a method for detecting aflatoxin B1, comprising the step of detecting aflatoxin B1 using the fluorescent on-type nucleic acid aptamer beacon probe as described above.

[0025] Furthermore, the above method comprises the following steps:

[0026] S1: mixing the fluorescent-on type nucleic acid aptamer beacon probe and a certain concentration of the sample to be tested in a corresponding buffer solution, and incubating at room temperature to obtain a mixed incubation solution;

[0027] S2 measures the fluorescence intensity of the mixed incubation solution. If the fluorescence intensity increases, it means that the sample to be tested contains aflatoxin B1.

[0028] Furthermore, S1 was incubated at room temperature for 3 min; the buffer solution was Buffer-A with a pH of 8.0; and the excitation / emission wavelengths in S2 were 485 / 525 nm.

[0029] The fourth technical solution adopted by the present invention is:

[0030] Provided is the use of the probe or kit described above in detecting aflatoxin B1.

[0031] Beneficial effects of the present invention:

[0032] The working principle of the fluorescent on-type aptamer beacon probe of the present invention is based on the hybridization / dissociation reaction within the DNA molecule, rather than the reaction between DNA molecules, so it is more efficient and requires shorter cDNA. The newly prepared nucleic acid aptamer beacon probe has the advantages of simple structure, low background, large signal change amplitude, rapid response, and easy operation.

[0033] The polyT linker of the fluorescence-on type aptamer beacon probe of the present invention is used to connect the 3' end of the aptamer and the 5' end of the short cDNA to form a whole DNA molecule. The fluorescent group F is labeled at the 3' end of the cDNA sequence, and the quencher group Q is labeled at the 5' end of the aptamer sequence. When there is no target molecule, the short cDNA will hybridize with the aptamer, so that the fluorescent group F and the quencher group Q are adjacent to each other, and fluorescence quenching occurs, and the fluorescence intensity is low. After adding the target, the aptamer is more inclined to bind to the target, so that the cDNA dissociates, the quencher group Q is away from the fluorescent group F, and the fluorescence is restored. It is used for the detection of aflatoxin B1 concentration, and rapid and high-specificity detection is achieved through the determination of fluorescence intensity. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the working principle of the common FRET-based nucleic acid aptamer sensor;

[0035] Figure 2 This is a schematic diagram of the principle of the present invention for rapidly detecting a target object based on the structural change of an aptamer beacon probe;

[0036] Figure 3 is the fluorescence intensity of samples containing different reagents;

[0037] Figure 4 The effect of the length of the PolyT linker arm on the fluorescence intensity of samples containing or not containing AFB1;

[0038] Figure 5 is the effect of cDNA length on the fluorescence intensity of samples containing or not containing AFB1;

[0039] Figure 6 is the effect of buffer pH on the fluorescence intensity of AA-48T-7 with or without AFB1 (500nmol / L);

[0040] Figure 7 The effect of MgCl2 concentration in the buffer on the fluorescence intensity of AA-48T-7 with or without AFB1 (500nmol / L);

[0041] Figure 8 To detect different concentrations of AFB1 using nucleic acid aptamer beacon probes AA-0T-0 and AA-48T-7 respectively;

[0042] Fig. 9 This is the specificity of the nucleic acid aptamer beacon probe AA-48T-7 of the present invention to AFB1.

[0043] in, Figure 1 In the figure, a and b are DNA chain replacement type aptamer sensors; c and d are aptamer beacon type aptamer sensors. DETAILED DESCRIPTION

[0044] In order to clearly illustrate the technical features of the present invention, the present invention is described in detail below through specific implementation methods in conjunction with the accompanying drawings.

[0045] In the present invention, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0046] 1. Materials and Reagents

[0047] The DNA chains (Table 1) used in the present invention were synthesized and HPLC purified by Shanghai Shenggong Biotechnology Co., Ltd. Aflatoxin B1 (AFB1), ochratoxin A (OTA), zearalenone (ZAE) and deoxynivalenol (DON) standards were purchased from Shanghai Shenggong Biotechnology Co., Ltd. Beer and corn flour were purchased from a local supermarket. Sodium chloride (NaCl), calcium chloride (CaCl2), magnesium chloride (MgCl2) and Tween 20 were purchased from Shanghai McLean Biochemical Reagent Co., Ltd. All aqueous solutions were prepared with ultrapure water having a resistivity greater than 18.2 MΩ·cm. Buffer-A (containing 20 mmol / L phosphate, 150 mmol / L NaCl and 20 mmol / L MgCl2 and 0.01% (v / v) Tween 20) with a pH of 8.0 was used as a buffer system to determine AFB1.

[0048] Table 1 Information of different DNA chains

[0049]

[0050] 2. Detection Method

[0051] The fluorescence-on aptamer beacon probe of the present invention and a certain concentration of the target (AFB1) were mixed in a corresponding buffer solution, the final concentration of the nucleic acid aptamer beacon probe was 20nmol / L, and incubated at room temperature for 3min. Then, the mixed incubation solution was transferred to a microvolume quartz dish with an optical path of 1cm. The fluorescence intensity was measured using a fluorescence spectrometer (Hitachi F-4700, Japan). The excitation / emission wavelength was set to 485 / 525nm. Each sample was measured three times, and the average value was used.

[0052] The design principle of the fluorescence-on aptamer beacon probe of the present invention can be found in Figure 2 The single-molecule system is composed of five elements: a nucleic acid aptamer with a specific sequence, a short cDNA, a polyT connecting arm, a fluorescent group F, and a quenching group Q.

[0053] Example 1 Detection feasibility study

[0054] To test the feasibility of this assay, the fluorescence intensity of samples containing different reagents was measured. Figure 3 As shown, sample a is a buffer solution containing only FAM-labeled cDNA, and its fluorescence intensity is high. Sample b contains FAM-labeled cDNA and BHQ1-labeled AFB1 aptamer (AA), and its fluorescence intensity is comparable to that of sample a. This indicates that AA and cDNA do not hybridize effectively, which may be because the cDNA is too short to support stable intermolecular hybridization. Therefore, the FAM labeled at the 3' end of the cDNA and the BHQ1 labeled at the 5' end of the AA are still far apart, exceeding the fluorescence resonance energy transfer distance, and the FAM fluorescence cannot be quenched by BHQ1. Sample c is a buffer solution containing the aptamer beacon probe AA-48T-7 of the present invention. Compared with sample a and sample b, the fluorescence intensity of sample c is much lower. This is because the polyT linker (48T) connects AA and short cDNA in the same molecule, so that the hybridization reaction between AA and cDNA is transformed from inefficient intermolecular hybridization to efficient intramolecular hybridization, and the fluorescence quenching effect is greatly improved. After adding AFB1, the fluorescence intensity of sample d increased significantly compared with sample c, indicating that fluorescence recovery occurred. These results prove that this method is feasible for AFB1 detection.

[0055] Example 2 Investigating the effect of the length of the polyT linker on the detection effect

[0056] See also Figure 4 , showing the effect of the length of the polyT linker on the fluorescence intensity. 0nt means that the aptamer is directly linked to the short cDNA. 24nt, 48nt and 72nt mean that the aptamer is indirectly linked to the short cDNA through 24, 48 and 72 thymine (T) deoxyribonucleotides, respectively. It can be seen that as the length of the polyT linker increases, the fluorescence intensity (F blank ) and the fluorescence intensity of samples containing AFB1 (F AFB1 ) were increased. In order to maintain a low background, reduce synthesis costs, and increase response signal changes, the optimal length of the polyT linker was determined to be 48nt (i.e., 48T).

[0057] Example 3 Investigating the effect of cDNA length on detection results

[0058] Aptamer beacon probes containing cDNAs of different lengths (5nt, 6nt, 7nt, 8nt, and 9nt) were used to detect AFB1. Figure 5 As a result, as the length of cDNA increased, F blank and F AFB1This indicates that longer cDNA is conducive to hybridization with the aptamer sequence and can reduce the signal background. However, too long cDNA will produce an overly stable aptamer-cDNA hybrid structure, which makes it difficult for cDNA to be released from the aptamer-cDNA hybrid structure, reducing the sensitivity of the method. The optimal length of cDNA is 7 nt. At this time, the signal gain (F AFB1 / F blank )-1)×100% reaches the maximum value and the sensitivity is higher.

[0059] Example 4 Investigating the effect of buffer on detection results

[0060] 1. Investigate the effect of buffer pH on the detection effect

[0061] The pH value of the binding buffer may affect the binding of the aptamer to the target and the fluorescence intensity of FAM. The fluorescence response of the aptamer beacon probe AA-48T-7 to the same concentration of AFB1 was tested in a buffer with a pH value of 5.0 to 10.0. Figure 6 As shown in Figure 2, the fluorescence intensity of AA-48T-7 increases with the increase of pH value and reaches the maximum when the pH value is 8.0. In the pH range of 6.0 to 8.0, the fluorescence intensity change caused by AFB1 binding (ΔF = F AFB1 -F blank ) increases with the increase of pH. When the pH value increases from 8.0 to 10.0, the ΔF value does not increase further. Finally, it is determined to use a buffer of pH 8.0 for detection.

[0062] 2. Investigate the effect of salt ion concentration in buffer on detection effect

[0063] like Figure 7 As shown, with the increase of MgCl2, the blank sample (F blank ) decreased in fluorescence intensity, and the sample containing AFB1 (F AFB1 ) first increased and then decreased. The fluorescence change caused by AFB1 (ΔF = F AFB1 -F blank ) reached its maximum value when the MgCl2 concentration was 20mmol / L, with good sensitivity.

[0064] Example 5 Advantages of probe AA-48T-7 in detecting targets of different concentrations

[0065] Different concentrations of AFB1 were detected using the two probes AA-0T-0 and AA-48T-7 in Table 1. The working principles of these two probes are as follows: Figure 8As shown in the inset. For probe AA-48T-7, the fluorescence intensity increased with the increase of AFB1 concentration, and the detection limit LOD reached 0.9nmol / L. In the concentration range of 8 to 125nmol / L, the logarithm of the fluorescence intensity (F) and the concentration of AFB1 (log AFB1 ) is linearly related: F = 3.0738logC AFB1 +483.37,R 2 =0.98804. For probe AA-0T-0, the fluorescence intensity decreases with the increase of AFB1 concentration. Moreover, for the maximum change value of fluorescence intensity (|F AFB1 -F blank |), AA-0T-0 is much smaller than the probe AA-48T-7, indicating that the probe design of the present invention has a larger signal variation amplitude and a significant advantage.

[0066] Example 6 Specificity Test

[0067] In order to evaluate the specificity of the aptamer beacon probe prepared by the present invention, some non-target substances were detected using the probe. In this part of the experiment, the concentration of the target substance used was 500nmol / L, and the concentration of the non-target substance was 1μmol / L. The experimental steps and experimental conditions for detecting non-target substances were the same as those for detecting target substances.

[0068] The probe of the present invention was used to detect some non-target analytes (ZAE, OTA and DON). Fig. 9 As shown. The concentration of AFB1 is 500nmol / L, and the concentration of non-target analytes is 1μmol / L. The blank sample blank is a buffer solution without any analyte, and the mixture sample mixture is a mixed solution containing AFB1 and all non-target substances (ZAE, OTA and DON). It can be seen that the fluorescence intensity will increase significantly only when AFB1 is contained in the sample. Moreover, the presence of non-target analytes will not affect the response of probe AA-48T-7 to AFB1.

[0069] Example 7 Detection of target substances in complex matrix samples

[0070] Beer sample: The beer purchased from the supermarket was ultrasonically degassed at room temperature for 30 minutes, and then diluted 10 times with a buffer. Then, a certain concentration of the target AFB1 standard was added to the diluted beer. Then, the target concentration was determined by the detection method of the present invention, and the recovery rate was calculated.

[0071] Corn liquid sample: Take 1g corn flour and dissolve it in 5mL methanol / water (v / v=7:3) solution, vortex and oscillate for 20min to obtain corn flour dissolution. Then, centrifuge the corn flour dissolution at 12000rpm for 15min, take the supernatant, and filter it through a 0.22μm filter to obtain corn flour extract. Then, dilute the corn flour extract 10 times with buffer solution, and add a certain concentration of target AFB1 standard. Determine the concentration of the target substance therein using the method of the present invention, and calculate the recovery rate.

[0072] The nucleic acid aptamer beacon probe AA-48T-7 of the present invention was used to detect AFB1 in beer diluted 10 times and corn flour extract diluted 10 times. As shown in Table 2, the recovery rate of AFB1 in beer samples was 93.8% to 109.2%; the recovery rate of AFB1 in corn flour extract samples was 95.7% to 110.2%. The above results prove that the above probe has good resistance to complex matrix interference and can be used for actual sample detection.

[0073] Table 2 Recovery of spiked AFB1 in complex matrix samples

[0074]

[0075] The present invention successfully prepares a fluorescence-on type nucleic acid aptamer beacon probe by integrating a specific nucleic acid aptamer with a short cDNA, a fluorescent group and a quenching group in the same molecule, and uses it for the rapid detection of aflatoxin B1 (AFB1). The nucleic acid aptamer beacon probe has a simple structure, low background, large signal amplification, rapid response, and easy regeneration. The preparation method of the nucleic acid aptamer beacon probe has no special requirements on the aptamer structure, is theoretically applicable to any aptamer, and has a good prospect for promotion and application.

[0076] The above is a detailed introduction to the fluorescent on-type nucleic acid aptamer beacon probe, kit, detection method and application provided by the present invention. The above specific implementation cannot be used as a limitation on the protection scope of the present invention. For those skilled in the art, any replacement, improvement or change made to the implementation of the present invention falls within the protection scope of the present invention.

[0077] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

Claims

1. A fluorescent-on type nucleic acid aptamer beacon probe, characterized in that: The probe is composed of a nucleic acid aptamer, a short cDNA, a polyT connecting arm, a fluorescent group and a quenching group; the two ends of the polyT connecting arm are respectively connected to the 3 ， end and the 5 ， At the 5 end of the aptamer ， end and the 3 ， The quenching group and the fluorescent group are labeled at the 5 ends of the nucleic acid aptamer, respectively. ， end and the 3 ， The ends of the short cDNA are labeled with a fluorescent group and a quenching group respectively; ， The end sequence of the nucleic acid aptamer is ， The end sequences are complementary so that the quencher and the fluorescent group are adjacent; The nucleic acid aptamer is a section of DNA with specific recognition ability for aflatoxin B1, and its nucleotide sequence is shown in SEQ ID No.1; the sequence of the short cDNA is shown in SEQ ID No.2; and the polyT connecting arm is polymerized from 48-72 thymine deoxynucleotides.

2. The fluorescence-on type nucleic acid aptamer beacon probe according to claim 1, characterized in that: The polyT linker is formed by polymerization of 48 thymidine deoxynucleotides, and its nucleotide sequence is shown in SEQ ID No.

3.

3. The fluorescence-on type nucleic acid aptamer beacon probe according to claim 1, characterized in that: The full sequence of the fluorescent on-type nucleic acid aptamer beacon probe is shown in the following SEQ ID No.4: 5-BHQ1-ACGTGTTGTCTCTCTGTGTCTCGT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT AACACGT-FAM -3 ， 。 4. The fluorescence-on type nucleic acid aptamer beacon probe according to claim 1, characterized in that: The quenching group is black hole quencher I, and the fluorescent group is 6-carboxyfluorescein.

5. A kit for detecting aflatoxin B1, characterized in that: The kit comprises the fluorescent-on type nucleic acid aptamer beacon probe according to any one of claims 1 to 4.

6. A method for detecting aflatoxin B1, characterized in that: The method comprises the step of detecting aflatoxin B1 by using the fluorescent on-type nucleic acid aptamer beacon probe described in any one of claims 1 to 4.

7. The method according to claim 6, characterized in that The steps are as follows: S1: mixing the fluorescent open-type nucleic acid aptamer beacon probe according to any one of claims 1 to 4 and a sample to be tested at a certain concentration in a corresponding buffer solution, and incubating at room temperature to obtain a mixed incubation solution; S2 measures the fluorescence intensity of the mixed incubation solution. If the fluorescence intensity increases, it means that the sample to be tested contains aflatoxin B1.

8. Use of the probe according to any one of claims 1 to 4 or the kit according to claim 5 in detecting aflatoxin B1.

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