A functional nucleic acid probe, and a preparation method and application thereof

Abstract

The application discloses a functional nucleic acid probe and a preparation method and application thereof. The preparation method comprises the following steps: forming a DNA sheet by self-assembly of a first DNA single strand and a second DNA single strand, and then combining the DNA sheet with a molecular beacon probe to prepare the functional nucleic acid probe; wherein the end of the first DNA single strand and the end of the second DNA single strand are complementary to each other; and the molecular beacon probe comprises an aptamer sequence of foodborne pathogenic bacteria. The fluorescence polarization method based on the functional nucleic acid probe provided by the application has the advantages of high sensitivity, strong specificity, short detection time and the like, and can be used for self-immediate monitoring of the content of foodborne pathogenic bacteria in drinking water or the content of foodborne pathogenic bacteria in environmental water, and has great application value and market value in family health and environmental protection and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of fluorescent analysis and detection, and particularly relates to a functional nucleic acid probe and a preparation method and application thereof. BACKGROUND

[0002] Food hygiene refers to the comprehensive conditions and measures to prevent food from being contaminated from production to consumption. Unhealthy habits in the food chain from slaughter or harvesting, processing, storage, distribution, transportation to preparation can expose consumers to potentially fatal foodborne infections. With the world economic globalization and trade internationalization, food safety has attracted much attention in developed and developing countries. Among them, food safety caused by pathogens is a major concern of public health worldwide, and countries spend a lot of resources to overcome this problem. Bacterial food infection is the source of concern for both developed and developing countries, in addition to the value of life and health, foodborne diseases also have a negative impact on the economy, trade and industry of the affected countries. Food safety needs to ensure that there are no microorganisms and chemical contaminants in food that pose a significant threat to human health, thereby leading to an increasing outbreak of foodborne diseases (FBD), which is one of the most important public health problems, resulting in a large number of hospitalizations and deaths each year. In this regard, the consumption of contaminated food and water leads to more than 600 million cases of FBD and 420,000 deaths each year [7] , which requires more attention to food safety issues worldwide.

[0003] In recent years, contamination of bacteria and their metabolites in food processing processes has caused serious harm to human health and the economy. According to the data of the Centers for Disease Control and Prevention (CDC), 70% of diarrhea is related to various pathogenic microorganisms commonly found in food, such as Staphylococcus aureus, Salmonella and E. coli O157:H7, and other common foodborne pathogens, especially on the surface of sashimi such as common raw fish and raw shrimp slices. These bacteria can produce toxins, which are usually very stable in food matrices, and food cannot kill pathogenic bacteria without long cooking. At the same time, with the passage of time, several bacterial strains begin to show resistance to commonly used antibiotics, which poses a serious threat to global public health. Staphylococcus aureus is one of the most common causes of hospital-acquired infections. It can cause a wide range of infections, including mild wound infections such as skin pockets, scalded skin syndrome, and severe conditions that can be life-threatening. Stanaway et al. found that Salmonella is still a major cause of illness and death worldwide. In particular, malnourished children, the elderly, immunocompromised adults (such as HIV-infected individuals), patients with acute malaria, and those with pre-existing debilitating diseases are at greater risk. This infection can attack healthy hosts and cause diarrhea, and even cause bacteremia, meningitis and tonsillitis. Therefore, for foodborne pathogens, prevention is better than cure, and the spread of contaminated food needs to be addressed from the source, which requires a quick and simple detection method to detect and prevent.

[0004] The conventional detection method of foodborne pathogens is based on culture and standard biochemical identification. However, it usually takes more than a week to complete the culture and identification of pathogenic bacteria. In addition, the traditional method may be limited by limited sensitivity and false negative results. Molecular amplification methods (such as PCR) and immunological methods (such as ELISA) as alternatives to standard methods have opened up countless new possibilities for pathogen detection and have made remarkable achievements. However, PCR technology and its derivatives such as RT-PCR require professional personnel to use sophisticated instruments, and at least 6-12 hours of DNA extraction, amplification and detection. Although PCR can ensure high sensitivity, false negatives and false positives are common problems. The antibody of ELISA method is expensive, susceptible to environmental influence, high cost, and needs to be transported and stored in the refrigerator. In addition, the complex operation of antibody immobilization, target recognition, catalytic reaction and the like increases the processing time, which is not conducive to the instantaneity of rapid detection. In addition, ELISA has insufficient sensitivity, and there are many problems of false positives and false negatives. The analysis method needs to be further improved in terms of analysis time, sensitivity, specificity, reliability and applicability of methodological parameters. In this invention, we use DNA sheet-MB to enhance the fluorescence polarization signal, and detect the target pathogenic bacteria in one step. Even quantitative detection of pathogenic bacteria in lake water and tap water can be achieved within 45 min. To our knowledge, this is the first time to use DNA sheet-MB to enhance the fluorescence polarization signal to improve the detection ability of common pathogenic bacteria, which is expected to provide a simple method for rapid detection of foodborne pathogenic bacteria. SUMMARY

[0005] The main purpose of the present application is to provide a functional nucleic acid probe and its preparation method and application to overcome the shortcomings of the prior art.

[0006] To achieve the above-mentioned purposes, the technical scheme adopted by the present application comprises:

[0007] The present application provides a preparation method of a functional nucleic acid probe, which comprises:

[0008] The first DNA single strand and the second DNA single strand are self-assembled to form a DNA sheet, and then the DNA sheet is combined with a molecular beacon probe to prepare a functional nucleic acid probe;

[0009] The end of the first DNA single strand is complementary to the end of the second DNA single strand; the sequence of the first DNA single strand is shown as SEQ ID NO: 1, and the sequence of the second DNA single strand is shown as SEQ ID NO: 2; the molecular beacon probe comprises an aptamer sequence of a foodborne pathogenic bacterium, and the sequence of the molecular beacon probe is shown as SEQ ID NO: 3.

[0010] The embodiment of the present application also provides the functional nucleic acid probe prepared by the preparation method.

[0011] The embodiment of the present application also provides the use of the functional nucleic acid probe in the preparation of a foodborne pathogenic bacteria detection sensor or a product with a foodborne pathogenic bacteria detection function.

[0012] The embodiment of the present application also provides a foodborne pathogenic bacteria detection sensor comprising the functional nucleic acid probe.

[0013] The embodiment of the present application also provides a fluorescence polarization detection method of foodborne pathogenic bacteria, comprising:

[0014] A series of standard foodborne pathogenic bacteria with different concentrations are mixed with the functional nucleic acid probe, the fluorescence polarization signal probe, deoxyribonucleotides and Klenow enzyme, and then the fluorescence polarization value is detected, so as to establish a standard curve of the concentration of foodborne pathogenic bacteria and the fluorescence polarization value, wherein the sequence of the fluorescence polarization signal probe is shown as SEQ ID NO: 4.

[0015] And the test sample containing foodborne pathogenic bacteria is mixed with the functional nucleic acid probe, the fluorescence polarization signal probe, deoxyribonucleotides and Klenow enzyme, and the fluorescence polarization value of the test sample is obtained by testing, and then the standard curve is compared, so as to obtain the concentration of foodborne pathogenic bacteria in the test sample.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] (1) In the present application, DNA sheet is first introduced as a fluorescence polarization detection element into simple engineered DNA nanometer assembly, and the first DNA single strand and the second DNA single strand maximize the assembly simplicity of only two oligonucleotides;

[0018] (2) The unique nanostructure of the foodborne pathogenic bacteria sensor based on the functional nucleic acid probe in the present application greatly enhances the fluorescence polarization signal and the signal-to-noise ratio, which is crucial for constructing any analysis method;

[0019] (3) The detection mode of the fluorescence polarization method in the present application can be used as a foodborne pathogenic bacteria sensing detection platform based on the functional nucleic acid probe, which will open up a new way for the design of functional nucleic acids and their application in the fields of food safety monitoring, disease diagnosis and biomedical research. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings required by the embodiments or the prior art description will be briefly introduced below. Obviously, the accompanying drawings in the following description only represent some embodiments of the present application, and all other drawings obtained by those of ordinary skill in the art without creative effort based on these drawings also belong to the protection scope of the present application.

[0021] Figure 1 A schematic diagram of the principle of the fluorescence polarization method based on the functional nucleic acid probe for the ultra-sensitive detection of Salmonella in a typical embodiment of the present application;

[0022] Figures 2a-2b A PAGE electrophoresis diagram and a fluorescence polarization diagram for the detection of Salmonella in a typical embodiment of the present application;

[0023] Figures 3a-3b A sensitivity detection diagram of the fluorescence polarization method based on the functional nucleic acid probe for Salmonella in a typical embodiment of the present application. DETAILED DESCRIPTION

[0024] In view of the defects of the prior art, the present inventors have obtained the technical solutions of the present application through long-term research and a large number of practices. The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort also belong to the protection scope of the present application.

[0025] In order to solve the problems of sensitive and accurate detection of heavy metal ions in drinking water and environmental water, the present application provides a functional nucleic acid probe based on the enhancement of fluorescence polarization signal for the detection of foodborne pathogenic bacteria and its application in actual sample detection.

[0026] Specifically, as one aspect of the technical solutions of the present application, a preparation method of a functional nucleic acid probe includes:

[0027] The first DNA single strand and the second DNA single strand are self-assembled to form a DNA sheet, and then the DNA sheet is combined with a molecular beacon probe (denoted as: molecular beacon MB) to obtain a functional nucleic acid probe (denoted as: DNA sheet-MB);

[0028] The end of the first DNA single strand is complementary to the end of the second DNA single strand; the sequence of the first DNA single strand is shown as SEQ ID NO: 1, and the sequence of the second DNA single strand is shown as SEQ ID NO: 2; the molecular beacon probe comprises an aptamer sequence of a foodborne pathogenic bacteria, and the sequence of the molecular beacon probe is shown as SEQ ID NO: 3.

[0029] In some preferred embodiments, the preparation method specifically comprises: self-assembling a first DNA single strand (denoted as: Tile 1) and a second DNA single strand (denoted as: Tile 2) to form a DNA tile (denoted as: DNA Tile), and then self-assembling the DNA tile again to form a DNA sheet.

[0030] Further, the DNA sheet has a hanging overhang.

[0031] In some preferred embodiments, the sequence of the first DNA single strand is, for example, TGGAC TAGGA CAAGA TGCTTAGCTC CGTAG ATGGT AGATG CGTAT GATCT TT.

[0032] In some preferred embodiments, the sequence of the second DNA single strand is, for example, (20A)-TGTCC TAGTC CACTACCATC TACGG AGCTA AGCAT CTGAT CATAC GCATT TT.

[0033] In some preferred embodiments, the sequence of the molecular beacon probe is, for example, (20T)-AGAAC TAGCA TATGGCGGCG TCACC CGACG GGGAC TTGAC ATTAT GACAG CGCCG CCATA TGCTA GTTCT TTT.

[0034] In some preferred embodiments, the preparation method specifically comprises:

[0035] heating and denaturing the first DNA single strand (denoted as: Tile 1) and the second DNA single strand (denoted as: Tile 2) at 90-95°C for 5-10 min, and then cooling to 20-25°C at a cooling rate of 1-2°C / min and maintaining for 30-60 min to obtain the DNA sheet;

[0036] and incubating the DNA sheet with a molecular beacon probe (denoted as: molecular beacon MB) at 37°C for 10-20 min to obtain the functional nucleic acid probe.

[0037] Further, the molar ratio of the first DNA single strand, the second DNA single strand, and the molecular beacon probe is 1-2:1:1-1.5.

[0038] Further, the concentration of the Tile 1 is 1 μmol / L.

[0039] Further, the concentration of the Tile 2 is 1 μmol / L.

[0040] Further, the concentration of the MB is 1 μmol / L.

[0041] In some preferred embodiments, the foodborne pathogenic bacteria include any one of Staphylococcus aureus, Salmonella, and Escherichia coli, and the like, but are not limited thereto.

[0042] Further, the foodborne pathogenic bacteria are Salmonella.

[0043] Another aspect of the embodiments of the present application also provides the functional nucleic acid probe prepared by the aforementioned preparation method.

[0044] Another aspect of the embodiments of the present application also provides the use of the aforementioned functional nucleic acid probe in the preparation of a foodborne pathogenic bacteria detection sensor or a product having a foodborne pathogenic bacteria detection function.

[0045] Another aspect of the embodiments of the present application also provides a foodborne pathogenic bacteria detection sensor, which includes the aforementioned functional nucleic acid probe.

[0046] Another aspect of the embodiments of the present application also provides a fluorescence polarization detection method of foodborne pathogenic bacteria, which includes:

[0047] A series of standard foodborne pathogenic bacteria with different concentrations are mixed with the aforementioned functional nucleic acid probe, fluorescence polarization signal probe, deoxyribonucleotides (dNTPs, including dTTP, dATP, dCTP, dGTP), Klenow enzyme (KF-Poly) for reaction, and then the fluorescence polarization value is detected, so as to establish a standard curve of the concentration of foodborne pathogenic bacteria and the fluorescence polarization value, wherein the sequence of the fluorescence polarization signal probe is shown in SEQ ID NO: 4.

[0048] Further, a test sample containing foodborne pathogenic bacteria is mixed with the functional nucleic acid probe, fluorescence polarization signal probe, deoxyribonucleotides, Klenow enzyme for reaction, and then the fluorescence polarization value of the test sample is obtained by testing, and then the standard curve is compared, so as to obtain the concentration of foodborne pathogenic bacteria in the test sample.

[0049] In some preferred embodiments, the sequence of the fluorescence polarization signal probe is FAM-TTTTT TTTTTTTTTT TTTTT.

[0050] In some preferred embodiments, the foodborne pathogenic bacteria in the test sample containing foodborne pathogenic bacteria include any one of Staphylococcus aureus, Salmonella, and Escherichia coli, and the like, but are not limited thereto.

[0051] Further, the foodborne pathogenic bacteria in the test sample containing foodborne pathogenic bacteria are Salmonella.

[0052] Further, the test sample of the Salmonella is derived from tap water or lake water.

[0053] In some preferred embodiments, the schematic diagram of the super-sensitive detection of Salmonella based on the functional nucleic acid probe and the fluorescence polarization method in the present application is shown in Figure 1

[0054] In some preferred embodiments, in the detection of foodborne pathogenic bacteria, the foodborne pathogenic bacteria are first mixed with the functional nucleic acid probe, the fluorescence polarization signal probe, the deoxyribonucleotide and the Klenow enzyme to form a foodborne pathogenic bacteria detection sensing system, and incubated at 37°C for 45 min.

[0055] Further, when the foodborne pathogenic bacteria detection sensing system is 25 μL, it contains 5.0 μL of DNA sheet-MB (1 μM), 2.5 μL of the foodborne pathogenic bacteria with the desired concentration, 2.5 μL of the fluorescence polarization signal probe (denoted as: FSP-X) (1 μM), 1.0 μL of the deoxyribonucleotide (10 mM), 0.5 μL of the Klenow enzyme (5 U / μL), and the rest of the volume is made up with double distilled water.

[0056] ​In some preferred embodiments, when the target foodborne pathogen is present, the aptamer sequence on the MB specifically recognizes and captures the target foodborne pathogen, at the same time, the conformation of the MB changes, the MB hairpin structure opens, and the AP binding sequence originally enclosed in the MB hairpin stem is released, the free AP primer (sequence such as: AGAAC TAGCATAT) in the system will bind to it, under the action of KF polymerase, the KF polymerase performs isothermal amplification with the opened MB as the template and the AP as the primer, and with the continuous amplification, a double-stranded DNA molecule (dsDNAs) is gradually formed, and the target foodborne pathogen originally combined with the aptamer sequence on the template is replaced. The replaced target foodborne pathogen will bind to the next MB molecule and cause a conformational change, and then bind to the primer for amplification. Therefore, the detection system achieves the purpose of sensitivity through the recycling of the target foodborne pathogen. Isothermal amplification not only enables the recycling of the target foodborne pathogen in the system, but also enables a complete dsDNA to fall off from the DNA sheet after each amplification, so that the suspended chain on the DNA sheet, which is enclosed because it is combined with the MB, is released again. At this time, the fluorescence polarization signal probe modified with the fluorescence group FAM will quickly bind to the suspended chain, and with the progress of the amplification, more and more suspended chains will bind to the fluorescence polarization signal probe. At this time, the complex formed by the fluorescence polarization signal probe and the DNA sheet has a very strong fluorescence polarization signal compared with the fluorescence polarization signal probe alone. The portable fluorescence polarization instrument is used to detect the fluorescence polarization signal of the sensing system, and the content of the pathogen in the system is proportional to the size of the detected fluorescence polarization signal, so that the quantitative detection of the pathogen can be realized.

[0057] In some more specific embodiments, the specific operation steps of the fluorescence polarization method for detecting foodborne pathogens based on the DNA sheet-MB enhanced fluorescence polarization signal in the present application are as follows:

[0058] (1) Preparation of DNA sheet and DNA sheet-MB: (i) Preparation of DNA sheet: mix equal volumes of Tile-1 and Tile-2, add MgCl2 solution with a final concentration of 15 mM, then heat at 95°C for 5 min, and slowly cool to room temperature to form a DNA sheet, which is stored at 4°C for standby. (ii) Preparation of DNA sheet-MB: The MB probe should first be reacted at 95°C for 5 min, then slowly annealed to room temperature to form a hairpin structure. Take a certain volume of DNA sheet solution, then add MB probe with the same concentration, and finally incubate at 37°C for 15 min to form a DNA sheet-MB complex, which is stored at 4°C for standby.

[0059] (2) DNA sheet-MB sensor detection of foodborne pathogenic bacteria: in the feasibility verification of foodborne pathogenic bacteria detection, 25 μL of foodborne pathogenic bacteria detection sensor system contains 5.0 μL of DNA sheet-MB (1 μM), 2.5 μL of foodborne pathogenic bacteria of the required concentration, 2.5 μL of fluorescence polarization signal probe (1 μM), 1.0 μL of deoxyribonucleotide (10 mM), 0.5 μL of Klenow enzyme (5 U / μL), the rest of the volume is made up with double distilled water, and then shaken and mixed. Incubate at 37℃ for 45 min. The blank control group is replaced with the same volume of sterile water instead of the detection target. Finally, use a portable fluorescence polarization instrument to measure the fluorescence polarization value.

[0060] (3) Detection of fluorescence polarization signal: the fluorescence polarization measurement of FAM fluorescence uses a portable fluorescence polarization instrument. First, 1 mL of Tris-buffer (pH 8.0) is sucked into the fluorescence polarization glass tube to remove the background, then the sample mixture is transferred into the Tris-buffer and mixed thoroughly, and finally the fluorescence polarization value of the mixture is measured and recorded.

[0061] (4) Establishment of standard curve: the same kind of different concentrations of foodborne pathogenic bacteria are added in (2), and the fluorescence polarization signal is detected after the reaction. The detection of fluorescence polarization is the same as (3). We record the fluorescence polarization signal generated by foodborne pathogenic bacteria of different concentrations in detail, and on this basis, we establish a standard curve for the detection of foodborne pathogenic bacteria samples of different concentrations, with different concentrations of foodborne pathogenic bacteria as the horizontal coordinate and the signal value of the fluorescence polarization output as the vertical coordinate.

[0062] (5) Quantitative detection of actual samples: unknown concentration of foodborne pathogenic bacteria actual samples are used as detection targets, and fluorescence polarization signal detection is carried out according to the same processing method in (1), (2) and (3). The obtained fluorescence polarization detection value is brought into the standard curve for the detection of foodborne pathogenic bacteria samples of different concentrations established in (4), and the concentration of the detected foodborne pathogenic bacteria in the actual sample is calculated.

[0063] The fluorescence polarization method based on functional nucleic acid probes provided in the present application has the advantages of high sensitivity, strong specificity, short detection time, etc., and can be used for self-monitoring of foodborne pathogenic bacteria content in drinking water or foodborne pathogenic bacteria content in environmental water at home, and has great application value and market value in family health and environmental protection.

[0064] The technical solutions of the present application will be further described in detail below in combination with several preferred embodiments and drawings. The present embodiment is implemented on the premise of the inventive technical solutions, and detailed implementation methods and specific operation processes are given, but the protection scope of the present application is not limited to the following examples.

[0065] The experimental materials used in the examples below, unless otherwise specified, can be purchased from conventional biochemical reagent companies.

[0066] Example 1

[0067] This example is the detection of Salmonella in spiked tap water (the concentration of Salmonella is 10 1 CFU / mL):

[0068] (1) Preparation of DNA sheet and DNA sheet-MB: (i) Preparation of DNA sheet: equal concentrations of Tile-1 and Tile-2 were mixed in equal volumes, 15 mM MgCl2 solution was added, then heated at 95°C for 5 min, slowly cooled to room temperature to form DNA sheet, and stored at 4°C for standby. (ii) Preparation of DNA sheet-MB: the MB probe should first be reacted at 95°C for 5 min, then slowly annealed to room temperature to form a hairpin structure. A certain volume of DNA sheet solution was taken, then equal concentration of MB probe was added, and finally incubated at 37°C for 15 min to form DNA sheet-MB complex, and stored at 4°C for standby;

[0069] (2) Feasibility steps of DNA sheet-MB sensor for detecting Salmonella: In the feasibility verification of Salmonella detection, 25 μL of Salmonella detection sensor system contains 5.0 μL of DNA sheet-MB (1 μM), 2.5 μL of spiked Salmonella solution, 2.5 μL of fluorescence polarization signal probe (1 μM), 1.0 μL of deoxyribonucleotide (10 mM), 0.5 μL of Klenow enzyme (5 U / μL), and the rest is made up with double distilled water, shaken and mixed, then incubated at 37°C for 45 min. The blank control group is replaced with an equal volume of sterile water instead of the detection target. Finally, the portable fluorescence polarization instrument is used to measure the fluorescence polarization value.

[0070] (3) Detection of fluorescence polarization signal: The fluorescence polarization measurement of FAM fluorescence uses a portable fluorescence polarization instrument. First, 1 mL of Tris-buffer (pH 8.0) is sucked into the fluorescence polarization glass tube to remove the background, then the sample mixture is all transferred into the Tris-buffer and mixed thoroughly, and finally the fluorescence polarization value of the mixture is measured and recorded;

[0071] (4) Establishment of standard curve: different concentrations of Salmonella (0, 1 × 10 0 , 1 × 10 1 , 1 × 10 2 , 1 × 10 3 , 1 × 10 4 , 1 × 105 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 CFU / mL), and the fluorescence polarization signal was detected after the reaction. The detection of fluorescence polarization was the same as (3). We recorded the fluorescence generated by Salmonella at different concentrations in detail, and on this basis, we established a standard curve for the detection of Salmonella samples at different concentrations, taking the concentration of Salmonella as the horizontal coordinate and the signal value of the fluorescence polarization output as the vertical coordinate. Through experiments, the standard curve we established was F = 29.58lg[C Salmonella / (CFU / mL)] + 22.80, with a correlation coefficient R 2 = 0.9907. The PAGE electropherogram and fluorescence polarization chart for the detection of Salmonella in this embodiment are shown in Figures 2a-2b ;

[0072] (5) Quantitative detection of actual samples: The tap water actual sample with added Salmonella was taken as the detection target, and the fluorescence polarization signal was detected according to the same processing method in (1), (2) and (3). The Salmonella concentration in the actual sample with added standard was calculated to be 9.45 x 10 0 CFU / mL by bringing the fluorescence polarization detection value into the standard curve for the detection of Salmonella samples established in (4). The recovery rate of Salmonella was 94.5%, and the relative standard deviation RSD was 4.71%.

[0073] Example 2

[0074] This embodiment is the detection of Salmonella in tap water (the concentration of Salmonella was 10 4 CFU / mL):

[0075] (1) Preparation of DNA sheet and DNA sheet-MB: (i) Preparation of DNA sheet: equal concentrations of Tile-1 and Tile-2 were mixed in equal volumes, 15 mM MgCl2 solution was added, and then heated at 95°C for 5 min and slowly cooled to room temperature to form a DNA sheet, which was stored at 4°C for standby use. (ii) Preparation of DNA sheet-MB: the MB probe should first be reacted at 95°C for 5 min, and then slowly annealed to room temperature to form a hairpin structure. A certain volume of DNA sheet solution was taken, and then equal concentration of MB probe was added. Finally, incubate at 37°C for 15 min to form a DNA sheet-MB complex, which is stored at 4°C for standby use;

[0076] (2) The feasibility steps of DNA sheet-MB sensor detecting Salmonella: In the feasibility verification of detecting Salmonella, 25 μL of the detection sensor system of Salmonella contained 5.0 μL of DNA sheet-MB (1 μM), 2.5 μL of the labeled Salmonella solution, 2.5 μL of the fluorescence polarization signal probe (1 μM), 1.0 μL of deoxyribonucleotide (10 mM), 0.5 μL of Klenow enzyme (5 U / μL), and the rest of the volume was supplemented with double distilled water, and after shaking and mixing, it was incubated at 37°C for 45 min. The blank control group was replaced with the same volume of sterile water instead of the detection target. Finally, the portable fluorescence polarization instrument was used to measure the fluorescence polarization value;

[0077] (3) The detection of fluorescence polarization signal: The fluorescence polarization measurement of FAM fluorescence used a portable fluorescence polarization instrument. First, 1 mL of Tris-buffer (pH 8.0) was sucked into the fluorescence polarization glass tube to remove the background, then the sample mixture was all transferred into the Tris-buffer and mixed well, and finally the fluorescence polarization value of the mixture was measured and recorded;

[0078] (4) The establishment of standard curve: In (2), different concentrations of Salmonella (0, 1×10 0 , 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 and 1×10 10 CFU / mL) were added, and after the reaction, the fluorescence polarization signal was detected. The detection of fluorescence polarization was the same as (3). We recorded the fluorescence generated by different concentrations of Salmonella in detail, and on this basis, we established the standard curve for detecting different concentrations of Salmonella samples, with different concentrations of Salmonella as the horizontal coordinate and the signal value of fluorescence polarization output as the vertical coordinate. Through the experiment, the standard curve we established was F = 29.58 lg[C Salmonella / (CFU / mL)] + 22.80, and the correlation coefficient R 2 = 0.9907; the standard curve is shown in Figures 3a-3b ;

[0079] (5) Quantitative detection of actual samples: The lake water sample spiked with Salmonella was used as the detection target, and the fluorescence polarization signal was detected according to the same treatment method in (1), (2) and (3). The obtained fluorescence polarization detection value was brought into the standard curve for Salmonella sample detection established in (4) to calculate the concentration of Salmonella in the spiked actual sample, which was 9.57 x 10 3 CFU / mL, the recovery rate of Salmonella was 95.7%, and the relative standard deviation RSD was 3.15%.

[0080] Example 3

[0081] This example is the detection of Salmonella in spiked tap water (the concentration of Salmonella is 10 7 CFU / mL):

[0082] (1) Preparation of DNA sheet and DNA sheet-MB: (i) Preparation of DNA sheet: equal concentrations of Tile-1 and Tile-2 were mixed in equal volumes, 15 mM MgCl2 solution was added, then heated at 95°C for 5 min, slowly cooled to room temperature to form DNA sheet, and stored at 4°C for standby. (ii) Preparation of DNA sheet-MB: the MB probe should first be reacted at 95°C for 5 min, then slowly annealed to room temperature to form a hairpin structure. A certain volume of DNA sheet solution was taken, then equal concentration of MB probe was added, and finally incubated at 37°C for 15 min to form DNA sheet-MB complex, which was stored at 4°C for standby;

[0083] (2) Feasibility steps of DNA sheet-MB sensor for detecting Salmonella: In the feasibility verification of Salmonella detection, 25 μL of Salmonella detection sensor system contained 5.0 μL of DNA sheet-MB (1 μM), 2.5 μL of spiked Salmonella solution, 2.5 μL of fluorescence polarization signal probe (1 μM), 1.0 μL of deoxyribonucleotide (10 mM), 0.5 μL of Klenow enzyme (5 U / μL), and the rest was made up with double distilled water. After shaking and mixing, it was incubated at 37°C for 45 min. The blank control group used equal volume of sterile water instead of the detection target. Finally, the portable fluorescence polarization instrument was used to measure the fluorescence polarization value.

[0084] (3) Detection of fluorescence polarization signal: The fluorescence polarization measurement of FAM fluorescence used a portable fluorescence polarization instrument. First, 1 mL of Tris-buffer (pH 8.0) was sucked into the fluorescence polarization glass tube to remove the background, then the sample mixture was transferred into the Tris-buffer for thorough mixing, and finally the fluorescence polarization value of the mixture was measured and recorded.

[0085] (4) Standard curve establishment: Salmonella of different concentrations (0, 1 x 10 0 , 1 x 10 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 and 1 x 10 10 CFU / mL) were added in (2), and the fluorescence polarization signal was detected after reaction. The detection of fluorescence polarization was the same as (3). The fluorescence generated by Salmonella of different concentrations was recorded in detail, and on this basis, the standard curve for detecting Salmonella samples of different concentrations was established by taking Salmonella of different concentrations as the horizontal coordinate and the signal value of fluorescence polarization output as the vertical coordinate. Through experiments, the standard curve established by us was F = 29.58 lg[C Salmonella / (CFU / mL)] + 22.80, and the correlation coefficient R 2 = 0.9907. The PAGE electropherogram and fluorescence polarization chart for detecting Salmonella in this embodiment are shown in Figures 2a-2b ;

[0086] (5) Quantitative detection of actual samples: The tap water actual sample added with Salmonella was taken as the detection target, and the fluorescence polarization signal was detected according to the same treatment method in (1), (2) and (3). The Salmonella concentration in the actual sample added with standard was calculated to be 9.67 x 10 6 CFU / mL by taking the fluorescence polarization detection value into the standard curve for detecting Salmonella samples established in (4), the recovery rate of Salmonella was 96.7%, and the relative standard deviation RSD was 3.62%.

[0087] Example 4

[0088] This embodiment is for detecting Salmonella in tap water (the concentration of Salmonella was 10 1 CFU / mL):

[0089] (1) Preparation of DNA sheet and DNA sheet-MB: (i) Preparation of DNA sheet: Tile-1 and Tile-2 were mixed in equal volume, then MgCl2 solution was added to a final concentration of 15 mM, and heated at 95 °C for 5 min, and then slowly cooled to room temperature to form DNA sheet, which was stored at 4 °C for later use. (ii) Preparation of DNA sheet-MB: MB probe should be first reacted at 95 °C for 5 min, and then slowly annealed to room temperature to form a hairpin structure. A certain volume of DNA sheet solution was taken, then equal concentration of MB probe was added, and finally incubated at 37 °C for 15 min to form DNA sheet-MB complex, which was stored at 4 °C for later use;

[0090] (2) Feasibility steps of DNA sheet-MB sensor for detecting Salmonella: In the feasibility verification of Salmonella detection, 25 μL of Salmonella detection sensor system contained 5.0 μL of DNA sheet-MB (1 μM), 2.5 μL of spiked Salmonella solution, 2.5 μL of fluorescence polarization signal probe (1 μM), 1.0 μL of deoxyribonucleotide (10 mM), 0.5 μL of Klenow enzyme (5 U / μL), and the rest was made up with double distilled water. After shaking and mixing, it was incubated at 37 °C for 45 min. The blank control group used equal volume of sterile water instead of the detection target. Finally, the fluorescence polarization value was measured using a portable fluorescence polarization instrument;

[0091] (3) Detection of fluorescence polarization signal: The fluorescence polarization measurement of FAM fluorescence used a portable fluorescence polarization instrument. First, 1 mL of Tris-buffer (pH 8.0) was sucked into the fluorescence polarization glass tube to remove the background, then the sample mixture was transferred into the Tris-buffer for thorough mixing, and finally the fluorescence polarization value of the mixture was measured and recorded;

[0092] (4) Establishment of standard curve: Different concentrations of Salmonella (0, 1 × 10 0 , 1 × 10 1 , 1 × 10 2 , 1 × 10 3 , 1 × 10 4 , 1 × 10 5 , 1 × 10 6 , 1 × 10 7 , 1 × 10 8 , 1 × 10 9 and 1 × 10 10CFU / mL), and the fluorescence polarization signal was detected after the reaction. The detection of fluorescence polarization was the same as (3). We recorded the fluorescence of Salmonella produced at different concentrations in detail, and on this basis, we established a standard curve for the detection of Salmonella samples at different concentrations, with the concentration of Salmonella as the horizontal coordinate and the signal value of the fluorescence polarization output as the vertical coordinate. Through experiments, the standard curve we established was F = 29.58lg[C Salmonella / (CFU / mL)] + 22.80, the correlation coefficient R 2 = 0.9907; the standard curve is shown in Figures 3a-3b ;

[0093] (5) Quantitative detection of actual samples: The lake water actual sample with added Salmonella was taken as the detection target, and the fluorescence polarization signal was detected according to the same processing method in (1), (2) and (3). The Salmonella concentration in the actual sample with added Salmonella was calculated to be 9.36 x 10 0 CFU / mL by bringing the obtained fluorescence polarization detection value into the standard curve for the detection of Salmonella samples established in (4). The recovery rate of Salmonella was 93.6%, and the relative standard deviation RSD was 2.36%.

[0094] In addition, the inventors of the present case also conducted experiments with other raw materials, process operations and process conditions described in the specification in reference to the foregoing examples, and all obtained relatively ideal results.

[0095] It should be understood that the technical solutions of the present application are not limited to the above specific implementation cases, and any technical modification made according to the technical solutions of the present application without departing from the purpose of the present application and the scope protected by the claims falls within the protection scope of the present application.

Claims

1. A method for preparing a functional nucleic acid probe, characterized by The method comprises the following steps: The first DNA single strand and the second DNA single strand are self-assembled to form a DNA sheet, and then the DNA sheet is combined with a molecular beacon probe to obtain a functional nucleic acid probe; The end of the first DNA single strand is complementary to the end of the second DNA single strand; the sequence of the first DNA single strand is shown as SEQ ID NO: 1, and the sequence of the second DNA single strand is a sequence obtained by modifying 20 consecutive A bases at the 5' end of SEQ ID NO: 2; the molecular beacon probe comprises an aptamer sequence of a foodborne pathogenic bacterium, and the sequence of the molecular beacon probe is a sequence obtained by modifying 20 consecutive T bases at the 5' end of SEQ ID NO: 3; and the foodborne pathogenic bacterium is Salmonella.

2. The production method according to claim 1, characterized by Specifically, the method comprises the following steps: The first DNA single strand and the second DNA single strand are self-assembled to form a DNA sheet.

3. The method of claim 2, wherein: The DNA sheet has a hanging overhanging end.

4. The method of claim 1, wherein Specifically, the method comprises the following steps: The first DNA single strand and the second DNA single strand are heated and denatured at 90-95 °C for 5-10 min, and then cooled to 20-25 °C at a cooling rate of 1-2 °C / min and incubated for 30-60 min to obtain the DNA sheet; And the DNA sheet is incubated with the molecular beacon probe at 37 °C for 10-20 min to obtain the functional nucleic acid probe.

5. The method of claim 4, wherein: The molar ratio of the first DNA single strand, the second DNA single strand and the molecular beacon probe is 1-2:1:1-1.

5.

6. The functional nucleic acid probe prepared by the method of any one of claims 1-5.

7. Use of the functional nucleic acid probe of claim 6 in the preparation of a product having a Salmonella detection function.

8. A Salmonella detection sensor characterized by The functional nucleic acid probe of claim 6.

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