CN116121345A - Aflatoxin B 1 Fluorescent aptamer sensor, detection method and kit - Google Patents

Aflatoxin B 1 Fluorescent aptamer sensor, detection method and kit Download PDF

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CN116121345A
CN116121345A CN202310067847.0A CN202310067847A CN116121345A CN 116121345 A CN116121345 A CN 116121345A CN 202310067847 A CN202310067847 A CN 202310067847A CN 116121345 A CN116121345 A CN 116121345A
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aflatoxin
fluorescent
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afb
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郑磊
毛瑜
刘长虹
姚丽丽
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Hefei University of Technology
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Abstract

The invention belongs to the field of toxin detection, and relates to aflatoxin B 1 Fluorescent aptamer sensor, detection method and kit. The fluorescent aptamer sensor is SEQ ID NO:1, and a nucleotide sequence shown in the specification. The detection method comprises the following steps: (1) Fluorescence aptamer sensor, RCA circular template, binding buffer and containing aflatoxin B 1 Is mixed with the sample solution of the sample, and is incubated at room temperature; (2) Adding dNTP, RCA reaction buffer and phi29DNA polymerase, incubating the obtained mixture, heating to react, mixing the reaction mixture, binding buffer and fluorescent dye, and measuring fluorescence intensity to obtain aflatoxin B in sample 1 Qualitative and/or quantitative detection is performed. AFB of the invention 1 The fluorescence detection method can realize AFB in complex food matrix 1 Quick, label-free, high-sensitivity and specific detection is performed.

Description

Aflatoxin B 1 Fluorescent aptamer sensor, detection method and kit
Technical Field
The invention belongs to the field of toxin detection, and in particular relates to aflatoxin B 1 Fluorescent aptamer sensor, aflatoxin B based on label-free target inhibition 1 Detection method and aflatoxin B 1 Fluorescent detection kit.
Background
Aflatoxins (AFs) are toxic mutagenic and carcinogenic substances, metabolites of the difurans class, and currently identified AFs are of nearly 20 types. Wherein, aflatoxin B 1 (AFB 1 ) The toxicity is maximum, and the resistance to temperature and humidity is stronger. Furthermore, AFB 1 Has high hepatotoxicity and embryotoxicity to human and animals. The international cancer research institute has established AFB 1 Is representative of a highly toxic substance and is designated as a class I carcinogen. AFB (alpha-fetoprotein) 1 Are widely found in a variety of food and feed products. Thus, an on-site, accurate, hypersensitive AFB was explored 1 Quantitative analysis methods are urgent.
AFB 1 Conventional chromatographic detection methods have been used for detection, including high performance liquid chromatography and liquid chromatography-mass spectrometry. While chromatography-based methods have high detection accuracy and sensitivity, they always require expensive equipment, lengthy analysis times and laborious steps. In addition, immunoassays based methods using antibodies as affinity ligands are more readily available AFB 1 The detection method has advantages in terms of speed, simplicity and portability. However, they may be affected by the complex and costly preparation of antibodies, especially against AFB 1 。
The aptamer is a single-stranded oligonucleotide (ssDNA or RNA) selected by an in vitro screening method called exponential enrichment ligand systematic evolution, forms a well-folded tertiary structure, and can specifically identify a target object. The aptamer is regarded as AFB as a novel recognition element with high affinity, specificity and good thermal stability 1 Better antibody substitutes in the establishment of the detection method. Researchers have designed a variety of aptamer-based AFB 1 Detection methods such as fluorescent, colorimetric or electrochemical aptamer sensors. Among them, fluorescent aptamer sensors are attracting attention because of their simplicity of operation, high sensitivity, and good stability in complex food matrices. However, many of the detection methods reported so far still have some drawbacks, including complex chemical labeling of fluorophores, multiple signal generation steps, or sample detection incubation timeLong. To overcome these drawbacks, aptamer-based unlabeled DNA amplification strategies have been developed, such as Hybridization Chain Reaction (HCR), polymerase Chain Reaction (PCR), strand Displacement Amplification (SDA), helicase-dependent amplification (HDA), and Rolling Circle Amplification (RCA). Of these methods, RCA has received increasing attention because of its mild isothermal enzymatic reaction and high efficiency, and the possibility of generating considerable tandem sequences to achieve signal amplification. RCA is simple to operate, and can be amplified by using only a short linear DNA as a promoter, circular DNA as a template and Phi29DNA polymerase (Phi 29 DP). Thus, many studies have established ultrasensitive detection systems for different target objects using RCA technology as a signal amplifier.
Disclosure of Invention
The invention aims to provide an AFB-based system 1 Simplified fluorescent aptamer sensor in combination with isothermal amplification reaction, which allows ultrasensitive and selective detection of AFB without the use of any labeled or modified DNA probes 1 。
In a first aspect the present invention provides an aflatoxin B 1 A fluorescent aptamer sensor, the fluorescent aptamer sensor being SEQ ID NO:1, the nucleotide sequence shown in the following formula:
5’-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACATAGTCGAGATAT-3’。
the invention uses AFB 1 The bifunctional probe consisting of the aptamer and the primer region is used as a recognition element. When the probe is exposed to AFB 1 When due to AFB 1 Probe complex formation, probe preferential to AFB 1 Binding, rather than binding to the circular template, inhibits initiation of the RCA process. Thus, a significant decrease in fluorescence signal can be monitored to achieve a significant decrease in AFB in complex food matrices 1 Fast, label-free, high sensitivity and selectivity test of (c).
Based on the principle, the second aspect of the invention provides aflatoxin B based on label-free target inhibition 1 The detection method comprises the following steps:
(1) Combining the fluorescent aptamer sensor of claim 1, an RCA circular template, a binding buffer, andcontains aflatoxin B 1 Is mixed with the sample solution of the sample, and is incubated at room temperature;
(2) Adding dNTP, RCA reaction buffer and phi29DNA polymerase, incubating the obtained mixture, heating, mixing the reaction mixture, binding buffer and fluorescent dye, and measuring fluorescence intensity to obtain aflatoxin B 1 Qualitative and/or quantitative detection is performed.
According to a preferred embodiment of the invention, the sequence of the RCA circular template is as follows:
5’-TCGGATATCTCGACTAGTCAAGACCCTAACCCTAACCCTAACCCTACAACATGTCTTTGA-3’，SEQ ID NO：2。
according to a preferred embodiment of the invention, in the system of step (1), the concentration of the fluorescent aptamer sensor is 70-90nM and the concentration of the RCA cyclic template is 40-60nM.
According to a preferred embodiment of the invention, in step (1), the incubation time at room temperature is 25-35min.
According to a preferred embodiment of the invention, in step (2), the incubation is carried out at a temperature of 28-32℃for 55-65min, the heating reaction is carried out at a temperature of 60-70℃for 10-20min.
According to a preferred embodiment of the invention, the fluorescent dye is SYBR TM Gold, fluorescence intensity measurement was performed within 30min after addition of fluorescent dye.
According to a preferred embodiment of the invention, the conditions for the fluorescence intensity determination are λex=498 nm, λem=540 nm.
According to a preferred embodiment of the present invention, the method further comprises the step of preparing aflatoxin B 1 According to the standard curve, calculating aflatoxin B in the sample 1 Is contained in the composition.
In a third aspect the invention provides an aflatoxin B 1 The fluorescence detection kit comprises the following components:
(1) The sequence of the fluorescent aptamer sensor is shown as SEQ ID NO:1 is shown in the specification;
(2) RCA circular template with the sequence shown in SEQ ID NO:2 is shown in the figure;
(3) A binding buffer;
(4)dNTP；
(5) RCA reaction buffer and phi29DNA polymerase;
(6) Fluorescent dyes.
The invention designs the method of AFB 1 Dual-function probe consisting of aptamer and primer region as AFB 1 Fluorescent aptamer sensor and AFB is provided based thereon 1 Fluorescence detection method capable of realizing AFB in complex food matrix 1 Quick, label-free, high-sensitivity and specific detection is performed.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
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The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
Fig. 1 shows the results of optimization of test experimental conditions.
FIG. 2 shows AFB 1 Fluorescence detection standard curve of standard.
FIG. 3 shows AFB 1 And (5) detecting a specific detection result.
FIG. 4 shows rice sample AFB 1 And (5) adding a standard test detection result.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the preferred embodiments of the present invention are described below, it should be understood that the present invention may be embodied in various forms and should not be limited to the embodiments set forth herein.
The sequence adopted by the invention is as follows:
Figure BDA0004073655090000041
1 fluorescence detection of AFB
First, 3. Mu.L of 400nM probe, 2. Mu.L of 400nM CT and 7.5. Mu.L of junctionBuffer (1 XPBS; pH 7.2), 1mM MgCl) 2 And 0.05% Tween-20) with 2.5. Mu.L AFB 1 Incubation was performed at room temperature for 30min, followed by the addition of 2. Mu.L dNTPs, 2. Mu.L of 10 XRCA reaction buffer and 0.2. Mu.L of 10U/. Mu.L phi29DNA polymerase. The reaction mixture was incubated at 30℃for 60min and heated at 65℃for 15min. Finally, the above reaction mixture was added to 70. Mu.L of binding buffer and 10. Mu.L of 10 XSYBR TM In Gold. The resulting mixture was subjected to fluorescence intensity measurement (λex/λem=498/540 nm).
A control group was set, and the reaction procedure of the control group was the same as that of the experimental group, except that no target was added.
In order to obtain optimal conditions, the probe concentration, RCA reaction time, incubation time and SYBR are measured according to the above detection procedure TM Key experimental factors such as Gold stability are optimized.
Optimization of probe concentration
The concentration of the probe is a parameter that directly affects the signal amplification efficiency. The probe concentration was optimized and as a result, as shown in fig. 1 a, the inhibition efficiency was gradually increased with increasing probe concentration, reaching the maximum inhibition intensity at 400nM. When the probe concentration exceeds 400nM, the inhibition efficiency slowly decreases. The most likely reason is that low probe concentrations favor RCA product formation, the higher the probe concentration, the more targets captured. Thus, the optimal concentration of probe was chosen to be 400nM.
Optimization of incubation time at room temperature
Short incubation times are detrimental to binding of the target aptamer. The incubation time was optimized, and as shown in fig. 1 b, when the incubation time was less than 30min, the inhibition efficiency was gradually improved with the increase of time, and the signal response became stable after 30 min. Thus, 30min was determined as the optimal incubation time.
Optimization of RCA reaction time
The RCA reaction time was optimized, and as a result, as shown in fig. 1 c, the inhibition efficiency was significantly improved between 15 and 60min, while the inhibition efficiency was significantly reduced at 120 min. This is probably because the increase in RCA reaction products resulted in an increase in the fluorescent signal captured within 60min, with a gradual increase in the inhibition efficiency. However, after an RCA reaction time of greater than 60 minutes, the resulting product chain length is sufficient to intertwine. Due to the increased steric hindrance, the hybridization of RCA products with fluorescent dyes is not good, resulting in reduced sensitivity. Thus, the optimal reaction time for RCA is 60min.
TM For fluorescent dye SYBR Optimization of Gold stability
The stability of the fluorescent dye was optimized and the results are shown in fig. 1 d. After the addition of the fluorescent dye, the signal response reached a maximum and remained stable for 30 min.
Feasibility verification of detection system
The AFB is adopted 1 Adding different amounts of AFB 1 The standard was analyzed and the change in fluorescence signal intensity was monitored. As shown in FIG. 2, the response of the fluorescent signal is compared with AFB 1 Is closely related, possibly due to the increased target resulting in reduced RCA product yield, and thus enhanced fluorescence signal inhibition. When AFB 1 The concentration is 6.6X10 -3 When the mu M is changed to 66 mu M, the fluorescence signal value is gradually reduced, the inhibition efficiency is increased, and the fluorescence signal value is matched with AFB 1 The concentrations are linearly related. The linear equation is y= -10.35769x-62.45094 (R 2 =0.976), the limit of detection was 6.6nM.
Specificity analysis
To study the detection system and method of the present invention on AFB 1 Specificity of detection AFB is to be detected 1 Is an analog of: mycotoxins OTA, AFM1, ZAE as interfering species and AFB 1 Detection together, AFB 1 、AFM 1 The molecular formulas of ZEN and OTA are shown as a in figure 3. The concentration of mycotoxins analyzed was 500nM, other conditions of investigation and AFB as described above 1 Fluorescence detection was consistent.
As shown in FIG. 3 b, AFM 1 The inhibition efficiency of ZEN and OTA is obviously lower than that of AFB 1 When AFB is present in the sample 1 When (Mix), the signal response intensity does not change significantly. The higher selectivity of the detection method is mainly derived from AFB 1 Specific recognition of the targeted aptamer. The experimental results show that the AFB of the invention 1 The detection system and method have good specificity, sufficient for practical applications.
Marking experiment verification
To verify the utility of fluorescent aptamer sensors based on RCA reactions, the AFB of the present invention was confirmed 1 The performance of the detection method in complex sample matrix is measured by adopting a labeling experiment to determine AFB in rice samples 1 Is contained in the composition.
First, rice samples purchased from the agricultural market were completely crushed using a laboratory mill. 2g of the sample was taken and added to 5mL of methanol-water (80:20, v/v). Then, the mixture was vibrated at 1500rpm for 1h and separated at 5000rpm for 8min. Then 450. Mu.L of the supernatant was transferred to another clean centrifuge tube and diluted with 500. Mu.L of pure water. A further 50. Mu.L of AFB at various concentrations was added 1 Shake it overnight and mix thoroughly. Finally, AFB is detected by using the fluorescent aptamer sensor and the detection method of the invention 1 Quantification was performed and samples were repeatedly tested.
The results are shown in FIG. 4, where the concentration and AFB are normalized 1 Substantially uniform test concentration (recovery in AFB 1 Detection amount/AFB 1 The addition amount indicates). In an actual rice sample, 50nM AFB 1 About 110%,500nM AFB 1 The recovery rate of (2) was about 106%. The result shows that the detection method is accurate and suitable for analyzing actual rice samples.
The foregoing description of embodiments of the invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described.

Claims (10)

1. Aflatoxin B 1 A fluorescent aptamer sensor characterized in that the fluorescent aptamer isThe sensor is SEQ ID NO:1, the nucleotide sequence shown in the following formula:
5’-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAG GCCCACATAGTCGAGATAT-3’。
2. aflatoxin B based on label-free target inhibition 1 The detection method comprises the following steps:
(1) The fluorescent aptamer sensor of claim 1, an RCA circular template, a binding buffer, and a kit containing aflatoxin B 1 Is mixed with the sample solution of the sample, and is incubated at room temperature;
(2) Adding dNTP, RCA reaction buffer and phi29DNA polymerase, incubating the obtained mixture, heating, mixing the reaction mixture, binding buffer and fluorescent dye, and measuring fluorescence intensity to obtain aflatoxin B 1 Qualitative and/or quantitative detection is performed.
3. The assay of claim 2, wherein the RCA circular template has the sequence shown below:
5’-TCGGATATCTCGACTAGTCAAGACCCTAACCCTAACCCTAACCC TACAACATGTCTTTGA-3’，SEQ ID NO：2。
4. the method according to claim 2, wherein in the system of step (1), the final concentration of the fluorescent aptamer sensor is 70-90nM and the final concentration of the RCA circular template is 40-60nM.
5. The detection method according to claim 2, wherein in the step (1), the incubation time at room temperature is 25 to 35min.
6. The detection method according to claim 2, wherein in the step (2), the incubation is performed at 28-32℃for 55-65min, the heating reaction is performed at 60-70℃for 10-20min.
7. The detection method according to claim 2, wherein the fluorescent dye is SYBR TM Gold, addThe fluorescent dye was followed by measurement of fluorescence intensity within 30 min.
8. The detection method according to claim 2, wherein the conditions for the fluorescence intensity measurement are λex=498 nm, λem=540 nm.
9. The method of claim 2, wherein the method further comprises producing aflatoxin B 1 According to the standard curve, calculating aflatoxin B in the sample 1 Is contained in the composition.
10. Aflatoxin B 1 The fluorescence detection kit comprises the following components:
(1) The fluorescent aptamer sensor of claim 1;
(2) RCA circular template with the sequence shown in SEQ ID NO:2 is shown in the figure;
(3) A binding buffer;
(4)dNTP；
(5) RCA reaction buffer and phi29DNA polymerase;
(6) Fluorescent dyes.
CN202310067847.0A 2023-01-12 2023-01-12 Aflatoxin B 1 Fluorescent aptamer sensor, detection method and kit Pending CN116121345A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116908432A (en) * 2023-07-07 2023-10-20 岭南师范学院 Method for detecting aflatoxin B1 content by fluorescence analysis based on optical cutting

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116908432A (en) * 2023-07-07 2023-10-20 岭南师范学院 Method for detecting aflatoxin B1 content by fluorescence analysis based on optical cutting

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