CN112697763A - Method for detecting streptomycin based on dye GelRed label-free aptamer sensor and application - Google Patents
Method for detecting streptomycin based on dye GelRed label-free aptamer sensor and application Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a method for detecting streptomycin based on a dye GelRed label-free nucleic acid aptamer sensor and application thereof. In the presence of streptomycin, the nucleic acid aptamer binds to streptomycin, resulting in a significant decrease in the fluorescence signal, and streptomycin is detected. GelRed is a nucleic acid gel fluorescent dye integrating high sensitivity, low toxicity and ultra-stability, is easy to obtain raw materials, low in cost and large-scale, has high affinity and specificity when being combined with a nucleic acid aptamer, can be used for quickly detecting streptomycin, has the advantages of economy, rapidness, sensitivity, accuracy and the like, and has important significance for controlling food safety and protecting human health.
Description
Technical Field
The invention belongs to the field of drug analysis and food safety, and particularly relates to a method for detecting streptomycin based on a dye GelRed label-free aptamer sensor and application of the method.
Background
Streptomycin is an aminoglycoside antibiotic having antibacterial activity against gram-positive and gram-negative bacteria and mycobacteria, and is widely used for treating diseases such as urinary tract infection, skin infection, respiratory tract infection and mastitis. With the widespread use of streptomycin in animal husbandry, antibiotic residues inevitably occur in animal food, and the presence of these residues is highly appreciated by people, because of their potentially carcinogenic properties, possibly leading to antibiotic resistance and allergic reactions. In recent years, many detection methods, such as high performance liquid chromatography, liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay, have been developed rapidly, and these methods often have the disadvantages of complex operation, expensive instruments, or large detection interference by the environment. With the development of the times, an efficient, low-cost and accurate detection method is sought to meet stricter detection requirements, and the method has important significance for controlling food safety and protecting human health.
Aptamers are selected in vitro from random oligonucleotide pools of DNA or RNA molecules by a method called "systematic evolution of exponentially enriched ligands" (SELEX). Aptamers have the key advantages of thermal stability, chemical stability, large-scale chemical production, controllable in vitro selection, low immunogenicity, targeted versatility, etc., and they can bind to target molecules with high affinity and specificity, which makes aptamers ideal recognition elements in biosensor development. Significant developments in aptamer-based biosensors, such as fluorescence sensors, electrochemical sensors, chemiluminescent sensors, and the like. Among them, the aptamer sensor based on fluorescence is the most commonly used detection method at present mainly because the method is simple to operate, high in sensitivity and low in cost. However, these prior methods require aptamers labeled with fluorophores and quenchers, or fluorescence energy donors and energy acceptors, which are expensive and time consuming to label. Therefore, it is necessary to develop a fluorescence sensor detection technique for label-free aptamers.
Many reports have been made on label-free aptamer fluorescence sensors established by using the principle that the change of spatial conformation after the aptamer is combined with a target substance causes the change of fluorescence intensity of dyes, but the dyes with the characteristics are few, and any aptamer can not form a conformation specifically combined with the fluorescent dye after being combined with the target substance, so the application range is relatively limited. Dyes such as EB and SYBR Green only emit weak fluorescence under the condition of free or coexisting with single-stranded DNA (deoxyribonucleic acid), and the fluorescence intensity is obviously enhanced under the coexisting condition with double-stranded DNA. However, the two dyes have the disadvantages of high toxicity, high price and the like, and are not suitable for large-scale and daily detection.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for detecting streptomycin based on a dye GelRed label-free nucleic acid aptamer sensor and application thereof, which can economically, quickly, accurately and sensitively detect streptomycin and can effectively solve the defects that the existing streptomycin detection operation is complex, or an instrument is expensive or the detection is greatly interfered by the environment.
The invention is realized by the following technical scheme:
a method for detecting streptomycin based on a dye GelRed label-free aptamer sensor comprises the following steps:
step 1) mixing streptomycin aptamers with samples to be detected respectively, and uniformly mixing and incubating for 10-20 min at room temperature;
the streptomycin aptamer has a sequence shown as SEQ ID NO. 1;
step 2) adding a streptomycin aptamer complementary strand into the mixture obtained in the step 1), and carrying out hybridization reaction for 10-20 min at normal temperature;
the sequence of the complementary strand of the streptomycin aptamer is shown as SEQ ID NO. 2;
step 3) adding 1 XGelRed dye into the mixture obtained in the step 2), uniformly mixing, reacting for 5-10 min, and detecting the fluorescence intensity on a computer;
step 4) when streptomycin does not exist in the sample, the streptomycin aptamer and a complementary strand thereof form a double-stranded structure, and the double-stranded structure is combined with GelRed dye, so that the obtained fluorescence intensity is maximum; when the concentration of streptomycin in a sample is increased, the fluorescence intensity caused by adding the GelRed dye is reduced along with the increase of the concentration of streptomycin, and finally the concentration of streptomycin is detected through the change of the intensity of the fluorescence.
Preferably, the concentrations of the streptomycin aptamer and the complementary strand of the streptomycin aptamer are both 4. mu. mol/L.
Preferably, the detection linear range of the streptomycin is 6-100 mu mol/L.
Preferably, the minimum detection limit of streptomycin is 1.2. mu. mol/L.
An application of a method for detecting streptomycin based on a dye GelRed label-free aptamer sensor in detecting the concentration of streptomycin in food.
An application of a method for detecting streptomycin based on a dye GelRed label-free aptamer sensor in detection of antibiotics, toxins and small molecules.
The invention has the following beneficial effects:
(1) the detection method is based on the characteristics that streptomycin binds to the appropriate ligand simply and the dye can also competitively bind to the DNA of the appropriate ligand, has the advantages of simple operation, short time consumption and lower cost, and avoids the problems of high cost of an immunoassay method and liquid phase detection.
(2) In the detection method of the present invention, a labeled aptamer, an expensive enzyme, is unnecessary, which makes the detection method of the present invention truly free from labeling (label-free) and effective.
(3) The mixed detection method is simple and rapid to operate, and can realize real-time monitoring of streptomycin. Furthermore, GelRed was first used in aptamer sensors and detection of streptomycin in milk was successfully achieved.
(4) The detection method is a common detection method, and can be widely applied to detection of other antibiotics, toxins and small molecular substances.
Drawings
FIG. 1 is a schematic diagram of detection of streptomycin based on aptamer recognition label-free fluorescence analysis;
FIG. 2 is a line graph showing the detection of streptomycin at various concentrations and the change in fluorescence;
FIG. 3 is a graph showing fluorescence spectra resulting from detection of streptomycin at different concentrations;
FIG. 4 is a graph of the fluorescence response of the detection of other antibiotics (kanamycin, ampicillin, amoxicillin, cephalexin, penicillin) in example 2;
FIG. 5 is a graph of the fluorescence response of example 3 for the detection of other potential interfering substances (tryptophan, lysine, tyrosine, phenylalanine, glutathione, cysteine);
FIG. 6 is a graph showing the fluorescence response of example 3 for detecting other potential interfering substances (glucose, ionic species).
Detailed Description
The present invention will be described in further detail with reference to the following drawings and specific examples, but the following examples are only for the operation of the present invention and are not intended to limit the present invention.
The invention utilizes GelRed dye which only emits weak fluorescence in a free state or in the presence of single-stranded DNA, the dye has low toxicity, and can be combined with double-stranded DNA when coexisting with the double-stranded DNA so as to obviously enhance a fluorescence signal, and the GelRed dye is utilized to construct a method for detecting streptomycin by using a label-free aptamer sensor. The detection principle of the detection method is as follows: an aptamer and streptomycin are incubated at room temperature for a period of time to form an aptamer-streptomycin complex, and then DNA complementary to the aptamer and a GelRed dye are sequentially added into the complex, so that free aptamer capable of binding to the streptomycin can be hybridized with the complementary DNA to form double-stranded DNA, and the GelRed dye can be inserted into the double-stranded DNA, thereby causing the remarkable enhancement of a fluorescence signal. The higher the concentration of streptomycin, the less free aptamer which can not be bound with streptomycin in the system, the less double-stranded DNA which can be formed with complementary DNA, and the less GelRed dye which is inserted into the double-stranded DNA, resulting in a weakened fluorescence signal. Quantitative analysis was performed according to this principle, and the detection principle is shown in FIG. 1.
Example 1
A method for detecting streptomycin based on a dye GelRed label-free aptamer sensor comprises the following specific steps:
(1) mixing 4 mu mol/L streptomycin aptamer with samples to be detected (containing streptomycin with different concentrations), and uniformly mixing and incubating for 16min at room temperature.
The streptomycin aptamer has a sequence shown as SEQ ID NO.1, and specifically comprises the following components:
5’-TAGGGAATTCGTCGACGGATCCGGGGTCTGGTGTTCTGCTTTGTTCTGTCGGGTCGTCTGCAGGTCGACGCATGCGCCG-3’。
(2) then 4. mu. mol/L of complementary strand of streptomycin aptamer was added and hybridization was carried out at room temperature for 11 min.
The complementary strand of the streptomycin aptamer has a sequence shown as SEQ ID NO.2, and specifically comprises the following components:
5’-CGGCGCATGCGTCGACCTGCAGACGACCCGACAGAACAAAGCAGAACACCAGACCCCGGATCCGTCGACGAATTCCCTA-3’。
(3) and finally adding 1 XGelRed dye, uniformly mixing, reacting for 6min, and detecting the fluorescence intensity on a computer.
(4) When streptomycin is not present in the sample, the streptomycin aptamer forms a double-stranded structure with its complementary strand, binds to the GelRed dye, and the fluorescence intensity obtained is maximal (F0) (ii) a When the streptomycin concentration in a sample is increased, the more aptamer-streptomycin complexes are formed and the less aptamer is free along with the increase of the streptomycin concentration, so that the double-stranded DNA formed by the aptamer and a complementary strand is reduced, the fluorescence intensity (F) caused by adding the GelRed dye is reduced along with the increase of the streptomycin concentration, and finally the streptomycin concentration is detected through the change of the fluorescence intensity.
Under the optimal experimental conditions, as shown in FIGS. 2 and 3, streptomycin is linearly related to the fluorescence intensity (F) within the concentration range of 6-100. mu. mol/L, and the lowest detection limit is 1.2. mu. mol/L.
Example 2 use to detect other antibiotics
Antibiotics commonly found in milk, such as kanamycin (Kana), ampicillin (Amp), amoxicillin (Amx), cephalexin (Cel), penicillin (Pen), were selected, and these samples were assayed according to the detection method of example 1 to observe the change in fluorescence signal. As a result, as shown in FIG. 4, the change in the fluorescence signal caused by the target substance, streptomycin (Str), was very significant, while the change in the fluorescence signals of other antibiotics was small and approximately negligible. The results demonstrate that the method for detecting streptomycin based on aptamer label-free fluorescence analysis has high specificity and specificity.
Example 3 application to detection of other potentially interfering substances
Selecting possible interfering substances in milk such as amino acids (tryptophan Try, lysine Lys, tyrosine Tyr, phenylalanine Phe, glutathione Gsh, cysteine Cys), glucose (Glu) and ionic substances (Na)+、Cl-、K+、Mg2+、SO4 2+、NO3 -) These samples were measured according to the detection method of example 1, and the change in the fluorescence signal was observed. As a result, as shown in FIGS. 5 and 6, the change of the fluorescence signal by the target substance streptomycin (Str) was significant, and the concentrations of amino acids and glucose were 10 times as high as those of streptomycin and Na+、Cl-、K+50 times the streptomycin concentration, Mg2+、SO4 2+、NO3 -Even at a 20-fold higher concentration of streptomycin, only a slight change in fluorescence intensity was observed. The results demonstrate that the method for detecting streptomycin based on aptamer label-free fluorescence analysis has high specificity and specificity.
Example 4 application of detecting spiked streptomycin in milk
Weighing 10mL of milk into a 50mL centrifuge tube, adding 5mL of 5% trichloroacetic acid, uniformly mixing for 3min by vortex, performing water bath ultrasound for 15min, centrifuging for 30min at 10000r/min, transferring supernatant into another 50mL centrifuge tube, filtering the supernatant with a 0.22 mu m filter membrane, and adjusting the pH to about 7 to be used as a milk sample to be detected. The milk sample to be tested was assayed according to the detection method of example 1, and the change in the fluorescence signal was observed. As shown in Table 1 below, the average recovery of streptomycin was between 95.5% and 110.2% when milk samples containing different concentrations of streptomycin (8, 10, 20. mu. mol/L) were analyzed by this method. The result proves that the proposed fluorescence analysis method has good reproducibility and accuracy, and can be used for screening streptomycin in actual samples.
TABLE 1 detection of streptomycin concentration in milk and spiked recovery assay
The invention utilizes a novel fluorescent dye GelRed which can only emit weak fluorescence in a free state or in coexistence with single-stranded DNA. The fluorescent probe can be combined with double-stranded DNA when coexisting with the double-stranded DNA so as to obviously enhance the fluorescent signal, has low toxicity and low price, combines high affinity and high specificity recognition of a proper ligand, and constructs a method for detecting streptomycin by using the label-free aptamer sensor. Experimental results show that the detection method has high specificity, short time consumption and high sensitivity. It is worth noting that the principle of the detection method is only based on DNA hybridization, so that the method can be widely applied to detection of various antibiotics, and provides a new idea and a new method for guaranteeing food safety.
Sequence listing
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Claims (6)
1. A method for detecting streptomycin based on a dye GelRed label-free aptamer sensor is characterized by comprising the following steps:
step 1) mixing streptomycin aptamers with samples to be detected respectively, and uniformly mixing and incubating for 10-20 min at room temperature; the streptomycin aptamer has a sequence shown as SEQ ID NO. 1;
step 2) adding a streptomycin aptamer complementary strand into the mixture obtained in the step 1), and carrying out hybridization reaction for 10-20 min at normal temperature;
the sequence of the complementary strand of the streptomycin aptamer is shown as SEQ ID NO. 2;
step 3) adding 1 XGelRed dye into the mixture obtained in the step 2), uniformly mixing, reacting for 5-10 min, and detecting the fluorescence intensity on a computer;
step 4) when streptomycin does not exist in the sample, the streptomycin aptamer and a complementary strand thereof form a double-stranded structure, and the double-stranded structure is combined with GelRed dye, so that the obtained fluorescence intensity is maximum; when the concentration of streptomycin in a sample is increased, the fluorescence intensity caused by adding the GelRed dye is reduced along with the increase of the concentration of streptomycin, and finally the concentration of streptomycin is detected through the change of the intensity of the fluorescence.
2. The method for detecting streptomycin based on the dye GelRed unlabeled aptamer sensor according to claim 1, wherein the concentrations of the streptomycin aptamer and the complementary strand of the streptomycin aptamer are both 4 μmol/L.
3. The method for detecting streptomycin based on the dye GelRed unlabeled aptamer sensor according to claim 1, wherein the detection linearity range of streptomycin is 6-100 μmol/L.
4. The method for detecting streptomycin based on the dye GelRed unlabeled aptamer sensor according to claim 1, wherein the minimum detection limit of streptomycin is 1.2 μmol/L.
5. The use of the method for detecting streptomycin based on the GelRed unlabeled dye aptamer sensor as claimed in claim 1 for detecting the concentration of streptomycin in food.
6. The method for detecting streptomycin based on the dye GelRed unlabeled aptamer sensor as claimed in claim 1, wherein the method is applied to detection of antibiotics, toxins and small molecules.
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