Glow-type chemiluminescence sensor based on DRCA product DNA hydrogel and application of glow-type chemiluminescence sensor

Abstract

The invention discloses a glow-type chemiluminescence sensor based on DRCA product DNA hydrogel and application of the glow-type chemiluminescence sensor. The glow type chemiluminescence sensor disclosed by the invention comprises an aptamer-primer compound, an annular template 1, an annular template 2, a Phi29 DNA polymerase, dNTPs (deoxyribonucleoside triphosphates), a chemiluminescence molecule and a target object. According to the present invention, the rapid gelling RCA reaction template sequence optimization scheme is established, the DRCA strategy is utilized to synergistically promote the formation of the DNA hydrogel, and the DNA hydrogel is further utilized to delay the reactant diffusion so as to prolong the chemiluminescence reaction time, such that the glow-type chemiluminescence sensor is prepared, the flash-type chemiluminescence defect is effectively overcome, and the application prospect is broad. And a lot of convenience is provided for on-site rapid detection of salmonella typhimurium.

Description

Technical Field

[0001] The invention relates to the technical field of food safety detection, and in particular to a glow-type chemiluminescent sensor based on DRCA product DNA hydrogel and application thereof. Background Art

[0002] Salmonella typhimurium, as a ubiquitous foodborne pathogen, poses a serious threat to public health and can potentially cause serious diseases such as acute gastroenteritis and even sepsis. In view of this, it is particularly important to achieve efficient, accurate and rapid detection of this species in food. Although the microbial culture method has been established as a detection method specified in my country's national standards due to its intuitive principle, mature technology and high detection accuracy, its significant limitation is that the detection cycle is long, usually taking 3-5 days to complete, which is obviously lagging behind the demand for rapid response to foodborne pathogens in modern food safety management. Therefore, exploring and developing new technologies with high sensitivity, high accuracy and the ability to complete detection in a shorter time is of urgent significance for improving the efficiency of food safety detection and protecting public health.

[0003] Based on the limitations of existing detection methods, chemiluminescent biosensors have attracted much attention in the field of foodborne pathogen detection due to their advantages such as fast response speed and no need for external light source excitation. However, most chemiluminescent substrates, such as luminol and acridinium ester, exhibit flash chemiluminescence, that is, luminescence and annihilation are completed within a few seconds. The instantaneous luminescence characteristics often lead to poor detection accuracy and reproducibility.

[0004] DNA hydrogel is a three-dimensional network structure formed by highly cross-linked DNA chains in solution. Its dense network structure can effectively delay the diffusion of chemiluminescent reactants, thereby prolonging the chemiluminescence time and converting flash-type chemiluminescence into glow-type chemiluminescence. At present, there have been reports of glow-type chemiluminescence sensors based on DNA hydrogels. However, these DNA hydrogels mostly rely on the complementary base pairing between a large number of short DNA chains to form. This chemically synthesized DNA requires high-purity raw materials and professional instruments and equipment, and the resulting high cost of DNA synthesis limits its further promotion and application in the field of analysis and detection.

[0005] Rolling circle amplification (RCA) technology is an isothermal nucleic acid amplification technology. With its flexibility in DNA sequence design and constant temperature of reaction conditions, it has become an ideal platform for constructing DNA hydrogels. The ssDNA chains generated by this technology are rich in repeating units composed of RCA template complementary sequences in series, which endows DNA hydrogels with functionality and personalized customization potential. According to the aptamer sequence of the detection object, the corresponding primers and RCA reaction template sequences are designed. By utilizing the competitive binding between the aptamer, primer and target, DNA hydrogels can be generated under the induction of the target, supplemented by the corresponding detection signal output, thereby realizing sensitive detection of the target. However, the formation of RCA product DNA hydrogel usually takes 24 to 72 hours. The long gelation time brings many inconveniences to on-site rapid detection and cannot meet the on-site rapid detection of Salmonella typhimurium. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a glow-type chemiluminescent sensor based on DRCA product DNA hydrogel and its application. The present invention optimizes the RCA reaction template sequence to achieve the rapid construction of DNA hydrogel. On this basis, the double rolling circle amplification (DRAC) strategy is further adopted to work together. The two work together to significantly accelerate the formation process of DNA hydrogel. The unique three-dimensional network structure of DNA hydrogel is used to effectively slow down the diffusion rate of chemiluminescent reactants, thereby extending the chemiluminescent reaction time, and successfully emitting a glow-type chemiluminescent sensor, which effectively overcomes the defects of flash-type chemiluminescence, such as short reaction time and difficult signal capture, and provides many conveniences for on-site rapid detection of Salmonella typhimurium, and has important practical value.

[0007] The technical solution of the present invention is as follows:

[0008] A glow-type chemiluminescent sensor based on DRCA product DNA hydrogel, the glow-type chemiluminescent sensor comprising an aptamer-primer complex, a circular template 1, a circular template 2, a Phi29 DNA polymerase, dNTPs, a chemiluminescent molecule and a target;

[0009] The aptamer sequence in the aptamer-primer complex is shown as SEQ ID No. 1, and the primer sequence is shown as SEQ ID No. 2;

[0010] The sequence of template 1 is shown in SEQ ID No. 3;

[0011] The sequence of template 2 is shown as SEQ ID No.4.

[0012] Furthermore, the chemiluminescent molecule is luminol.

[0013] Furthermore, the preparation method of the aptamer-primer complex is:

[0014] (1) Mix 20 μL of 10 μmol / L aptamer and 20 μL of 10 μmol / L primer in a PCR tube, place in a PCR instrument, anneal at 95°C for 10 min, and then slowly cool to obtain an aptamer-primer complex;

[0015] (2) 14 μL of 5 U / μL exonuclease I was added thereto to hydrolyze the unbound single-stranded DNA to prepare a purified aptamer-primer complex.

[0016] Furthermore, the preparation methods of the cyclic template 1 and the cyclic template 2 are as follows:

[0017] (1) 30 μL of 20 μmol / L circular primer was mixed evenly with the chain template 1 and the chain template 2, and hybridized by gradient cooling method to obtain circular DNA precursors of template 1 and template 2 respectively;

[0018] The loop primer sequence is shown in SEQ ID No.5;

[0019] (2) Then add 7.5 μL of 400 U / μL T 4 DNA ligase was incubated at 37°C for 2 h and incubated at 65°C for 10 min to inactivate the enzyme; then 10 μL of 5U / μL ExoⅠ enzyme and 5 μL of 200U / μL ExoⅢ enzyme were added, incubated at 37°C for 1 h and incubated at 80°C for 20 min to obtain purified circular template 1 and circular template 2, respectively.

[0020] An application of the glow type chemiluminescent sensor, wherein the glow type chemiluminescent sensor is used to detect Salmonella typhimurium.

[0021] Furthermore, the method for detecting Salmonella typhimurium using the glow-type chemiluminescent sensor comprises the following steps:

[0022] (1) preparing DNA hydrogel;

[0023] (2) mixing the DNA hydrogel with the luminol solution, and incubating at 4°C and 600 rpm for 30 min to prepare the luminol@DNA hydrogel;

[0024] (3) After washing, the luminol@DNA hydrogel was transferred to a 96-well plate and 85 μL of luminescence buffer, 10 μL of 1 mmol / L cobalt chloride solution, and 5 μL of 10 mol / L H 2 O 2The solution was kept away from light and then the luminescent image was taken in a dark environment. The RGB value was analyzed by Image J software. The RGB value was substituted into the standard curve to obtain the concentration of the target Salmonella typhimurium in the sample to be tested.

[0025] Furthermore, in step (1), the preparation method of the DNA hydrogel is as follows:

[0026] (1) Take 54 μL of the prepared aptamer-primer complex and 246 μL of the sample to be tested containing the target, incubate at 37°C, 600 rpm for 45 min to allow the aptamer to recognize and bind to the target, then filter through a 0.22 μm filter membrane, collect the filtrate, and obtain the free primer;

[0027] (2) Take 30 μL of free primers, mix thoroughly with 3 μL of prepared circular template 1, 2.5 μL of 25 mmol / L dNTPs, 6 μL of 10× phi29 DNA polymerase reaction buffer, 16.5 μL of water and 2 μL of 10 U / μL phi29 DNA polymerase, and amplify at 37°C and 600 rpm for 45 min to obtain long ssDNA1;

[0028] (3) Take another 30 μL of free primer, mix it thoroughly with 3 μL of prepared circular template 2, 2.5 μL of 25 mmol / L dNTPs, 6 μL of 10× phi29 DNA polymerase reaction buffer, 16.5 μL of water and 2 μL of 10 U / μL phi29 DNA polymerase, and amplify it at 37°C and 600 rpm for 45 min to obtain long ssDNA2;

[0029] (4) After long ssDNA 1 and long ssDNA 2 were mixed, the mixture was vortexed for 20 seconds and centrifuged at 5000 rpm for 3 minutes to obtain a DNA hydrogel.

[0030] Furthermore, in step (2), the luminol solution is prepared by dissolving 17.71 mg of luminol in 1 mL of NaOH; the amount of the luminol solution used is 30 μL.

[0031] Furthermore, in step (3), the luminescent buffer solution is prepared by dissolving 338.181 mg of disodium hydrogen phosphate dihydrate, 1263.6681 mg of sodium dihydrogen phosphate dihydrate and 8 g of sodium hydroxide in 1 L of pure water.

[0032] Furthermore, in step (3), the standard curve is: Y = 20.92 + 2.420 * X, R 2 =0.9927, wherein Y represents the RGB value, and X represents the logarithmic value of the concentration of Salmonella typhimurium.

[0033] The beneficial technical effects of the present invention are:

[0034] The present invention utilizes the competitive binding of the aptamer complementary sequence of Salmonella typhimurium and the aptamer of Salmonella typhimurium to release the free complementary sequence. The free complementary sequence in the system serves as the primer of the RCA reaction, triggering two RCA reactions respectively and producing two long-chain DNAs. Since the RCA template is designed to be partially complementary, the formation of DNA hydrogel is promoted under the joint drive of physical winding and base complementary pairing. Only in the presence of Salmonella typhimurium will the RCA reaction be initiated and the DNA hydrogel will be formed. Thereafter, the DNA hydrogel is mixed and incubated with the luminol solution to form the luminol@DNA hydrogel. Finally, the chemiluminescent reactant H 2 O 2 and chemiluminescent reaction catalyst Co 2+ Slow diffusion in the DNA hydrogel achieves glow-type chemiluminescence. The higher the concentration of Salmonella typhimurium in the test sample, the larger the DNA hydrogel, the more luminol is encapsulated, and the stronger the chemiluminescence intensity is. Therefore, the concentration of Salmonella typhimurium can be determined by taking a chemiluminescent image with a smartphone and analyzing its RGB value with Image J software. In addition, the sensor was also used for actual sample detection, and there was no significant difference between the test results and the plate count results, which proved the excellent performance of the sensor. The constructed detection method has the advantages of simple operation, high selectivity and specificity, and is of great significance for practical applications in the field of food safety.

[0035] The present invention designs and constructs an RCA reaction template sequence that can respond to Salmonella typhimurium and quickly gel, and utilizes the DRCA strategy to synergistically accelerate the formation of DNA hydrogel, which can effectively shorten the detection time.

[0036] The present invention utilizes DNA hydrogel to delay the diffusion time of chemiluminescent reactants, thereby converting flash-type chemiluminescence into glow-type chemiluminescence. No precise and expensive chemiluminescence instrument is required. The chemiluminescence intensity can be determined by taking a photo with a smartphone and analyzing the RGB value with Image J software, which can meet the needs of on-site rapid detection of Salmonella typhimurium. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a schematic diagram of the principle of detecting Salmonella typhimurium using the glow-type chemiluminescent sensor based on DRCA product DNA hydrogel of the present invention.

[0038] Figure 2 The polyacrylamide gel electrophoresis diagrams are of the aptamer-primer complex prepared in Example 1 of the present invention and the circular template 1 and the circular template 2 prepared in Example 2.

[0039] In the figure: A is the polyacrylamide gel electrophoresis of the aptamer-primer complex prepared in Example 1; B is the polyacrylamide gel electrophoresis of the circular template 1 and the circular template 2 prepared in Example 2.

[0040] Figure 3 The fluorescence image, scanning electron microscope image and chemiluminescence kinetic curve of the DRCA product DNA hydrogel prepared in Example 3 of the present invention are shown.

[0041] In the figure: A is a picture of different types of hydrogels; B is a scanning electron micrograph of a DNA hydrogel prepared by mixing long ssDNA1 and long ssDNA2, wherein (a) is a scanning electron micrograph at a magnification of 21,000, and (b) is a scanning electron micrograph at a magnification of 50,000; C is a graph of the chemiluminescence kinetic curve.

[0042] Figure 4 This is the detection result of the glow-type chemiluminescent sensor prepared in Example 4 of the present invention for detecting Salmonella typhimurium.

[0043] In the figure: (a) is the relationship between the concentration of Salmonella typhimurium and the RGB value; (b) is the standard curve; (c) is the specificity diagram of the glow type chemiluminescent sensor. DETAILED DESCRIPTION

[0044] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0045] The term "dNTPs" in the present invention refers to a mixture of four monodeoxyribonucleotides: dATP, dGTP, dTTP and dCTP; "DRCA" refers to double rolling circle amplification.

[0046] The T used in the following examples 4 DNA ligase, exonuclease I (Exo I), exonuclease III (Exo III), and phi29 DNA polymerase can all be purchased through commercial channels.

[0047] The nucleotide sequences in the following examples are as follows:

[0048]

[0049]

[0050] Figure 1Schematic diagram of the glow-type chemiluminescent sensor based on DRCA product DNA hydrogel for detecting Salmonella typhimurium. First, the RCA primer is designed to be partially complementary to the Salmonella typhimurium aptamer to form a duplex structure. In the presence of Salmonella typhimurium, the aptamer tends to bind to the target, causing the duplex structure to be opened and the primer to be released. The released primers hybridize with Circ-DNA 1 (circular template 1) and Circ-DNA 2 (circular template 2), respectively, and then trigger two RCA reactions to generate long ssDNA1 and long ssDNA2. Since Circ-DNA 1 and Circ-DNA 2 are designed to be partially complementary, the generated long ssDNA1 and long ssDNA2 will form a DNA hydrogel under the joint drive of base complementary pairing and physical entanglement after mixing. Then, under the action of physical vibration, luminol is prompted to penetrate into the interior of the DNA hydrogel to form luminol@DNA hydrogel. Since the dense porous structure of DNA hydrogel can effectively slow down the diffusion rate of reactants, the chemiluminescent reactant H 2 O 2 and chemiluminescent catalyst Co 2+ Slowly diffuse into the luminol@DNA hydrogel, achieving the transition from flash chemiluminescence to glow chemiluminescence. The more Salmonella typhimurium there is, the larger the DNA hydrogel formed, the more luminol is absorbed, and the higher the chemiluminescence intensity. By taking luminescent photos with a smartphone and analyzing the RGB values ​​using Image J software, Salmonella typhimurium can be quantified.

[0051] Example 1 Preparation of aptamer-primer complex

[0052] (1) Accurately pipette 20 μL of 10 μmol / L aptamer and 20 μL of 10 μmol / L primer into a PCR tube and mix well, place in a PCR instrument, anneal at 95°C for 10 min, and then slowly cool to obtain an aptamer-primer complex;

[0053] (2) Add 14 μL of exonuclease I (5 U / μL) and 6 μL of matching buffer, mix well, and use a PCR instrument to run under the set program to remove free single strands to prepare a purified aptamer-primer complex. The specific procedure is: incubate at 37°C for 3 hours, then heat at 80°C for 15 minutes to obtain a purified aptamer-primer complex.

[0054] The preparation process of the aptamer-primer complex was characterized by 15% PAGE. Figure 2As shown in Figure A, compared with the single-stranded aptamer (lane 2) and complementary strand (lane 3), the migration rate of the aptamer-primer complex (lane 4) decreases due to the increase in molecular weight. This indicates that the complex was successfully prepared. Considering the interference of free aptamers or primers on the detection results, it is necessary to add ExoⅠ to digest the free aptamers or primers. In lane 5, the disappearance of the single-stranded DNA band indicates that the aptamer-primer complex has been successfully purified.

[0055] Example 2 Preparation of Circular Template 1 and Circular Template 2

[0056] 30 μL of 20 μmol / L circular primer was mixed with 30 μL of 20 μmol / L chain template 1 and chain template 2 respectively, and hybridized by gradient cooling method to obtain circular DNA precursor; then 7.5 μL of T 4 DNA ligase (400U / μL) and 7.5μL of matching buffer were incubated at 37℃ for 2h, and incubated at 65℃ for 10min to inactivate the enzyme, respectively preparing circular template 1 and circular template 2; then 10μL ExoⅠ enzyme (5U / μL), 5μL ExoⅢ enzyme (200U / μL) and 10μL 10×ExoⅠ reaction buffer were added, incubated at 37℃ for 1h, and incubated at 80℃ for 20min to inactivate the enzyme, respectively obtaining purified circular template 1 and circular template 2. The purified circular templates were placed at 4℃ for standby use. Among them, the gradient cooling method was run using a PCR instrument, and the specific program parameters were: heat treatment at 95℃ for 2min, then incubated at 65℃ and 60℃ for 2min and 6min respectively, then cooled to 20℃ at a rate of 0.1℃ per second, and kept at 20℃ for 30s, and finally incubated at 4℃ for 10min.

[0057] The successful synthesis of the circular template was verified by 15% PAGE. Figure 2 As shown in Figure B, compared with the chain template (lane 3, lane 6), adding T 4 After DNA ligase (lane 4, lane 7), the migration rate of the product becomes lower, indicating the successful synthesis of the circular DNA template. In the low molecular weight region, unreacted primer chains are still observed in lanes 4 and 7. Therefore, it is necessary to purify the circularized products (circular template 1, circular template 2) using ExoⅠ and ExoⅢ to remove free single strands and uncircularized double strands, and remove the primers bound to the circular template. Compared with lanes 4 and 7, the free single-stranded bands in lanes 5 and 8 disappeared, and the circularized byproduct bands in the high molecular weight region were greatly reduced, indicating that the purification was successful.

[0058] Example 3 Preparation of Luminol@DNA Hydrogel

[0059] (1) Take 54 μL of the aptamer-primer complex prepared in Example 1 with a concentration of 0.6 μmol / L and 246 μL of the sample to be tested containing the target, and incubate at 37° C. and 600 rpm for 45 min to allow the aptamer to recognize and bind to the target; then filter through a 0.22 μm filter membrane, collect the filtrate, and obtain free primers.

[0060] (2) Take 30 μL of the free primer obtained in step (1), mix it thoroughly with 3 μL of circular template 1, 2.5 μL of dNTPs (25 mmol / L), 6 μL of 10×phi29 DNA polymerase reaction buffer, 16.5 μL of water and 2 μL of phi29 DNA polymerase (10 U / μL), and then amplify it at 37°C and 600 rpm for 45 min to obtain long ssDNA1.

[0061] Take another 30 μL of the free primer obtained in step (1), mix it thoroughly with 3 μL of circular template 2, 2.5 μL of dNTPs (25 mmol / L), 6 μL of 10×phi29 DNA polymerase reaction buffer, 16.5 μL of water and 2 μL of phi29 DNA polymerase (10 U / μL), and then amplify it at 37°C and 600 rpm for 45 min to obtain long ssDNA 2.

[0062] Take three centrifuge tubes and mark them as group (a), group (b), and group (c), respectively. In the centrifuge tube of group (a), 60 μL of long ssDNA1 and 60 μL of long ssDNA1 were added to mix; in the centrifuge tube of group (b), 60 μL of long ssDNA1 and 60 μL of long ssDNA2 were added to mix; in the centrifuge tube of group (c), 60 μL of long ssDNA2 and 60 μL of long ssDNA2 were added to mix; after mixing, the tubes were vortexed for 20 seconds and centrifuged at 5000 rpm for 3 minutes to obtain three groups of DNA hydrogels, group (a), group (b), and group (c), respectively.

[0063] (3) The three groups of DNA hydrogels were mixed with 30 μL of luminol solution (the preparation method of luminol solution was: 17.71 mg of luminol was dissolved in 1 mL of NaOH), placed in a constant temperature mixer, and incubated at 4°C and 600 rpm for 30 min to prepare luminol@DNA hydrogel. The test results are as follows: Figure 3 shown.

[0064] like Figure 3As shown in Figure A, after the same mixing, vortexing and centrifugation treatments were performed on long ssDNA1 and long ssDNA1 (group (a)), long ssDNA1 and long ssDNA2 (group (b)), and long ssDNA2 and long ssDNA2 (group (c)), it was found that the DNA hydrogel obtained in group (b) was larger per unit time, indicating that the DRCA strategy can accelerate the formation of DNA hydrogels through base complementary pairing. In addition, the formation of hydrogels in groups (a) and (c) may be attributed to the excellent design of template chains. Figure 3 The scanning electron microscopy results of Figure B show that the DRCA product DNA hydrogel has a porous structure. This porous structure, like a water-absorbing sponge, has a high loading capacity for liquids and can allow small molecules to enter and exit freely, which may help to delay the diffusion of chemiluminescent molecules and thus prolong the chemiluminescent reaction time. Therefore, the chemiluminescence kinetic curve of the DRCA product DNA hydrogel was further measured, and the results are shown in Figure 3 As shown in Figure C, the chemiluminescence signal of luminol (black line) reaches a peak in a short time and then disappears, showing a typical flash-type chemiluminescence trend. However, the chemiluminescence signal based on DNA hydrogel (red and blue lines) shows a completely different trend: the chemiluminescence signal first increases slowly, and then slowly decreases after a period of stability, indicating that DNA hydrogel can promote the transformation of chemiluminescence from flash type to glow type. Among them, compared with DNA hydrogel (blue line), DRCA product DNA hydrogel (red line) shows a more stable glow-type chemiluminescence trend due to its denser hydrogel structure.

[0065] Example 4 Preparation of glow type chemiluminescent sensor:

[0066] The luminol@DNA hydrogel prepared in Example 3 was washed twice and then transferred to an opaque 96-well plate. 85 μL of luminescence buffer (preparation method: 338.181 mg of disodium hydrogen phosphate dihydrate, 1263.6681 mg of sodium dihydrogen phosphate dihydrate and 8 g of sodium hydroxide were dissolved and fixed to volume in 1 L of pure water), 10 μL of 1 mmol / L cobalt chloride solution and 5 μL of 10 mol / L H 2 O 2 The solution was kept away from light for 10 minutes to allow the chemiluminescence to reach a stable period. The luminescence image was taken using a Huawei Nova7 mobile phone in a dark environment, and its RGB value was analyzed using Image J software.

[0067] Change the concentration of Salmonella typhimurium and repeat the above operation. The test results are as follows Figure 4 (a) and Figure 4As shown in (b), as the concentration of Salmonella typhimurium increases, the RGB value becomes higher. Under the optimal conditions, the RGB value is positively correlated with the logarithmic value of the concentration of Salmonella typhimurium. The linear range is 10 CFU / mL-7.6×10 8 CFU / mL (Y = 20.92 + 2.420 * X, R 2 =0.9927, where Y represents the RGB value and X represents the logarithmic value of the concentration of Salmonella typhimurium). The detection limit of Salmonella typhimurium is 8 CFU / mL.

[0068] Specificity is one of the important characteristics of biosensors. In order to verify the specificity of this experimental method, the response of the constructed glow-type chemiluminescent sensor to Staphylococcus aureus, Escherichia coli, Bacillus cereus and Listeria monocytogenes was measured. Figure 4 As shown in (c), only the Salmonella typhimurium system showed a significant increase in chemiluminescence intensity, while the chemiluminescence signals caused by the other four foodborne pathogens changed less, indicating that this method has good specificity for the detection of Salmonella typhimurium.

[0069] Test example: actual sample testing

[0070] This method was used to determine the concentration of Salmonella typhimurium in commercial chicken samples, and then the plate count method was used to verify the method. The sample was pretreated as follows: first, 25g of commercial chicken was added to 225mL of PBS buffer for homogenization and sterilization. Then centrifuged at 1000rpm for 5min, the precipitate was discarded, and the supernatant was taken. Then 300μL of the supernatant was mixed with 600μL of the bacterial solution to prepare the actual sample to be tested. The results are shown in Table 1. Through SPSS analysis, there was no significant difference between the test results of this method and the test results of the plate count method, indicating that the test results of this method are more reliable.

[0071] Table 1 Results of spiked chicken samples tested by this method and plate count method (n=3)

[0072]

[0073]

[0074] Note: In the same row, the same letters indicate that there is no significant difference between the two test results (p>0.05, n=3), and different lowercase letters indicate significant difference.

[0075] The above is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and changes directly derived or associated by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the protection scope of the present invention.

Claims

1. A glow-type chemiluminescent sensor based on DRCA product DNA hydrogel, characterized in that: The glow-type chemiluminescent sensor comprises an aptamer-primer complex, a circular template 1, a circular template 2, a Phi29 DNA polymerase, dNTPs, a chemiluminescent molecule and a target; The aptamer sequence in the aptamer-primer complex is shown as SEQ ID No.1, and the primer sequence is shown as SEQ ID No.2; The sequence of template 1 is shown in SEQ ID No. 3; The sequence of template 2 is shown as SEQ ID No.

4.

2. The glow type chemiluminescent sensor according to claim 1, characterized in that: The chemiluminescent molecule is luminol.

3. The glow type chemiluminescent sensor according to claim 1, characterized in that: The preparation method of the aptamer-primer complex is: (1) Mix 20 μL of 10 μmol / L aptamer and 20 μL of 10 μmol / L primer in a PCR tube, place in a PCR instrument, anneal at 95°C for 10 min, and then slowly cool to obtain an aptamer-primer complex; (2) 14 μL of 5 U / μL exonuclease I was added thereto to hydrolyze the unbound single-stranded DNA to prepare a purified aptamer-primer complex.

4. The glow type chemiluminescent sensor according to claim 1, characterized in that: The preparation methods of the cyclic template 1 and the cyclic template 2 are as follows: (1) 30 μL of 20 μmol / L circular primer was mixed evenly with the chain template 1 and the chain template 2, and hybridized by gradient cooling method to obtain circular DNA precursors of template 1 and template 2 respectively; The loop primer sequence is shown in SEQ ID No.5; (2) Then, 7.5 μL of 400 U / μL T4 DNA ligase was added, the mixture was incubated at 37°C for 2 h, and the mixture was incubated at 65°C for 10 min to inactivate the enzyme; then, 10 μL of 5 U / μL ExoⅠ enzyme and 5 μL of 200 U / μL ExoⅢ enzyme were added, the mixture was incubated at 37°C for 1 h, and the mixture was incubated at 80°C for 20 min to obtain purified circular template 1 and circular template 2, respectively.

5. An application of the glow type chemiluminescent sensor according to any one of claims 1 to 4, characterized in that: The glow type chemiluminescent sensor is used to detect Salmonella typhimurium.

6. The use according to claim 5, characterized in that: The method for detecting Salmonella typhimurium using the glow-type chemiluminescent sensor comprises the following steps: (1) preparing DNA hydrogel; (2) mixing the DNA hydrogel with the luminol solution, and incubating at 4°C and 600 rpm for 30 min to prepare the luminol@DNA hydrogel; (3) The luminol@DNA hydrogel was washed with water and transferred to a 96-well plate. 85 μL of luminescence buffer, 10 μL of 1 mmol / L cobalt chloride solution, and 5 μL of 10 mol / L H2O2 solution were added. The plate was kept away from light and then the luminescence image was captured in a dark environment. The RGB value was analyzed using Image J software. The RGB value was substituted into the standard curve to obtain the concentration of the target Salmonella typhimurium in the sample to be tested.

7. The use according to claim 6, characterized in that: In step (1), the preparation method of the DNA hydrogel is as follows: (1) Take 54 μL of the prepared aptamer-primer complex and 246 μL of the sample to be tested containing the target, incubate at 37°C, 600 rpm for 45 min to allow the aptamer to recognize and bind to the target, then filter through a 0.22 μm filter membrane, collect the filtrate, and obtain the free primer; (2) Take 30 μL of free primers, mix thoroughly with 3 μL of prepared circular template 1, 2.5 μL of 25 mmol / L dNTPs, 6 μL of 10× phi29 DNA polymerase reaction buffer, 16.5 μL of water and 2 μL of 10 U / μL phi29 DNA polymerase, and amplify at 37°C and 600 rpm for 45 min to obtain long ssDNA1; (3) Take another 30 μL of free primer, mix it thoroughly with 3 μL of prepared circular template 2, 2.5 μL of 25 mmol / L dNTPs, 6 μL of 10× phi29 DNA polymerase reaction buffer, 16.5 μL of water and 2 μL of 10 U / μL phi29 DNA polymerase, and amplify it at 37°C and 600 rpm for 45 min to obtain long ssDNA2; (4) After long ssDNA 1 and long ssDNA 2 were mixed, the mixture was vortexed for 20 seconds and centrifuged at 5000 rpm for 3 minutes to obtain a DNA hydrogel.

8. The use according to claim 6, characterized in that: In step (2), the preparation method of the luminol solution is: dissolving 17.71 mg of luminol in 1 mL of NaOH; the amount of the luminol solution used is 30 μL.

9. The use according to claim 6, characterized in that: In step (3), the luminescent buffer solution is prepared by dissolving 338.181 mg of disodium hydrogen phosphate dihydrate, 1263.6681 mg of sodium dihydrogen phosphate dihydrate and 8 g of sodium hydroxide in 1 L of pure water.

10. The use according to claim 6, characterized in that: In step (3), the standard curve is: Y = 20.92 + 2.420 * X, R 2 =0.9927, wherein Y represents the RGB value, and X represents the logarithmic value of the concentration of Salmonella typhimurium.

Images