Salmonella typhimurium fluorescence detection method based on double signal amplification

Abstract

The invention discloses a salmonella typhimurium fluorescence detection method based on dual signal amplification, and relates to the technical field of microbiological detection.The method comprises the specific steps that firstly, SNA1 and SNA2 probes are constructed, related chains are co-modified on the surfaces of gold nanoparticles through a freezing method, and part of sequences are closed; then preparing an Apt.-F compound; then incubating the sample and the compound; then starting a dual signal amplification reaction; and finally, detecting the fluorescence intensity by using a fluorospectro photometer, and quantifying the salmonella typhimurium according to the relationship between the fluorescence intensity and the bacterium concentration. According to the invention, two kinds of functionalized SNA probes are constructed, the aptamer and DNAzyme are arranged, a dual signal amplification pathway is established, target bacteria are accurately identified through the aptamer, a trigger chain is released to start chain replacement, a fluorophore is released, the detection sensitivity is improved, the probe design is optimized to ensure specificity, the process is simplified, and rapid quantification is realized; an efficient scheme is provided for trace pathogenic bacterium detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganism detection, and particularly to a fluorescence detection method for Salmonella typhimurium based on double signal amplification. BACKGROUND

[0002] Salmonella is a typical foodborne pathogenic bacteria, which often contaminates food such as meat, eggs, dairy products, etc. It is easy to cause acute gastroenteritis in the human body, and symptoms such as vomiting, diarrhea, fever, etc. The health of the elderly, children and people with low immunity is particularly significant. Therefore, establishing a high-efficiency detection technology for Salmonella in food is the core demand to protect food safety and public health. With the iteration of detection technology, it gradually develops from traditional culture method to molecular biology technology. The detection method based on nucleic acid recognition has become a research focus due to its strong specificity and fast response speed. Aptamer is a nucleic acid molecule that can specifically bind to the target, and has the characteristics of high stability and accurate recognition. DNAzyme has high enzyme cutting activity, and can realize signal output by cutting the substrate. The combination of the target recognition of aptamer and the signal regulation function of DNAzyme becomes an important direction to break through the bottleneck of trace Salmonella detection, and lays a technical foundation for constructing a high-sensitivity detection system.

[0003] The current traditional technology for detecting Salmonella still has obvious limitations, and it is difficult to adapt to the diversified needs of actual detection scenes. Although the traditional culture separation method is regarded as the gold standard for detection, it needs to go through several steps such as enrichment, separation and purification, biochemical identification, etc. The overall process is time-consuming, and it cannot meet the needs of instant detection or rapid response to sudden pollution incidents in food production and circulation. The immune detection technology such as enzyme-linked immunosorbent assay shortens the detection time, but the signal intensity is insufficient for low-concentration Salmonella, and it is easily disturbed by impurities in the sample matrix, resulting in false negative or false positive results. The conventional fluorescence detection technology relies on a single signal mechanism, and the signal amplification capacity is limited, making it difficult to realize accurate quantification of trace target bacteria. Moreover, some methods need to rely on complex instruments and professional operation, which not only increases the detection cost, but also limits the popularization in basic laboratories or on-site detection scenes, and cannot efficiently cover the food safety monitoring needs throughout the whole chain. SUMMARY

[0004] The purpose of the present application is to make up for the shortcomings of the prior art, and provide a fluorescence detection method for Salmonella typhimurium based on double signal amplification. The method constructs SNA1 and SNA2 probes, combines Apt.-F complex, and uses double signal amplification mechanism to improve the detection sensitivity. The specific steps include probe construction, complex preparation, sample incubation, double signal amplification reaction initiation, fluorescence detection and quantification. The fluorescence intensity of the system after reaction is detected by fluorescence spectrophotometer, realizing high-sensitivity quantitative detection of Salmonella typhimurium. The method has the characteristics of simple operation and accurate results.

[0005] The application provides a Salmonella typhimurium fluorescence detection method based on double signal amplification.

[0006] S100, constructing SNA1 and SNA2 probes: SNA1 probe is obtained by co-modifying 8-17 DNAzyme walking chain D1 and hairpin structure substrate chain S1 on the surface of gold nanoparticles through a freezing method, part of the sequence of the D1 chain is closed by hybridization with a locking chain L1, the loop part of the S1 chain contains a DNAzyme cleavage site, and the end of the S1 chain is modified with a fluorescent group; SNA2 probe is obtained by co-modifying 8-17 DNAzyme walking chain D2 and hairpin structure substrate chain S2 on the surface of gold nanoparticles through a freezing method, part of the sequence of the D2 chain is closed by hybridization with a locking chain L2, and the end of the S2 chain is modified with a fluorescent group.

[0007] S200, preparing an Apt.-F complex: a trigger chain F is mixed with Salmonella typhimurium aptamer, and an Apt.-F complex is formed through annealing treatment;

[0008] S300, incubating a sample with the Apt.-F complex: the Apt.-F complex is added to the sample to be detected, and incubation is performed at a set temperature for a set time;

[0009] S400, starting a double signal amplification reaction: the SNA1 probe, the SNA2 probe, a DNAzyme reaction buffer and a MgCl2 solution are added to the system after incubation, and incubation is performed at a set temperature for a set time;

[0010] S500, fluorescence detection and quantification: the fluorescence intensity of the system after reaction is detected by using a fluorescence spectrophotometer, and the Salmonella typhimurium in the sample to be detected is quantified according to the corresponding relationship between the fluorescence intensity and the Salmonella typhimurium concentration.

[0011] Further, in the S100, the molar ratio of the mixed annealing of the D1 chain and the L1 chain is 1:5, the molar ratio of the mixed annealing of the D2 chain and the L2 chain is 1:5, the molar concentration ratio of the D1 chain and the S1 chain in the SNA1 probe is 1:20, the molar concentration ratio of the D2 chain and the S2 chain in the SNA2 probe is 1:20, and the sequence of the S1 chain and the S2 chain both contain consecutive thymine nucleotides as a connecting arm.

[0012] Further, in the S100, when constructing the SNA1 and SNA2 probes, the D1 chain, the L1 chain, the D2 chain, the L2 chain, the S1 chain and the S2 chain are subjected to thiol reduction treatment: 10 μL of TCEP solution with a concentration of 10 mmol / L is added to 40 μL of D1-L1 chain annealing solution or D2-L2 chain annealing solution; 10 μL of TCEP solution with a concentration of 0.5 mol / L is added to 80 μL of S1 chain annealing solution or S2 chain annealing solution; all thiol reduction treatments are carried out at room temperature for 1 h; the specific conditions of the freezing method are freezing at -20℃ for 2 h, and then thawing at room temperature; after thawing, the mixed solution is subjected to centrifugal washing, the centrifugal speed is 12000 rpm, the centrifugal time is 15 min, and the centrifugal washing is repeated for 3 times.

[0013] Further, in the S100, when constructing the SNA1 and SNA2 probes, 10 μL of PEG20000 solution with a concentration of 0.1 g / mL is added; the final concentration of the AuNPs solution in the reaction system is 2 nmol / L; the total volume of the reaction system is supplemented to 1 mL by adding water, and the water is mixed thoroughly after being added.

[0014] Further, in the S200, when preparing the Apt.-F complex, the mixed molar ratio of the F chain and the Apt. chain is 1:1; the specific conditions of the annealing treatment are as follows: the mixed F chain and Apt. chain solution is incubated at 95℃ for 5 min, and then slowly cooled to room temperature in a water bath; the final concentration of the Apt.-F complex is 10 μmol / L.

[0015] Further, in the S300, when incubating the sample and the Apt.-F complex, the addition volume of the sample to be tested is 50 μL, and the addition volume of the Apt.-F complex is 10 μL; the set temperature of the incubation is 37℃, and the set time is 1 h; the incubation process needs to be subjected to shaking treatment, and the shaking rate is 180 r / min; when the sample to be tested is a solid food sample, the sample is first made into a homogenate, and then the supernatant is taken after centrifugation as the sample to be tested.

[0016] Further, in the S400, when starting the double signal amplification reaction, the SNA1 probe needs to be diluted by 20 times before use, and the SNA2 probe needs to be diluted by 10 times before use; the concentration of the DNAzyme reaction buffer is 5×, and the addition volume is 20 μL; the composition of the buffer is 20 mmol / L Tris-HCl and 50 mmol / L KCl, and the pH value is 7.5; the concentration of the MgCl2 solution is 50 mmol / L, and the addition volume is 20 μL; the set temperature of the incubation is 37℃, and the set time is 1.5 h; the incubation process needs to be subjected to shaking treatment, and the shaking rate is 180 r / min.

[0017] Further, in the step S400, the specific process of the dual signal amplification reaction is as follows: in step S300, the F strand released from the Apt.-F complex and the L1 strand on the SNA1 probe have a strand displacement reaction, and the D1 strand is displaced; in the presence of Mg2+, the D1 strand combines with the S1 strand on the SNA1 probe and cuts the rA site of the S1 strand, and a S1' fragment with FAM is released; the S1' fragment and the L2 strand on the SNA2 probe have a strand displacement reaction, and the D2 strand is displaced; in the presence of Mg2+, the D2 strand combines with the S2 strand on the SNA2 probe and cuts the rA site of the S2 strand, and a S2' fragment with FAM is released.

[0018] Further, in the step S500, the excitation wavelength of the fluorescence spectrophotometer is set to 489 nm, and the emission wavelength is set to 520 nm; the corresponding relationship between the fluorescence intensity and the concentration of Salmonella typhimurium is that the fluorescence intensity increase value and the logarithmic value of the concentration of Salmonella typhimurium have a linear relationship, the linear equation is y = 7553.5589x - 20262.8384, and the correlation coefficient R 2 = 0.963; wherein, y represents the fluorescence intensity increase value, x represents the logarithmic value of the concentration of Salmonella typhimurium, and the fluorescence intensity increase value is the difference between the fluorescence intensity of the sample to be tested and the fluorescence intensity of the blank control group.

[0019] Further, in the step S100, the specific sequences of the oligonucleotides used in the steps SNA1 and SNA2 to S500 are as follows:

[0020] F strand: 5'-CGGTCGAAACATAATGTCAAGTCC-3';

[0021] Apt. strand: 5'-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG-3';

[0022] D1 strand: 5'-HS-TTTTTTTTTTTTTTTTTTTTTTTTTTAACGACAGCACTCCGAGCCGGTCGAAACATAATGG-3';

[0023] L1 strand: 5'-GGACTTGACATTATGTTTCGACCG-3';

[0024] S1 strand: 5'-HS-SH-TTTTTTTTTTATACACGAATTTTTTCCATTATGTrAGGTGCTGTCGTGTCTCTTCGTGTATG-FAM-3';

[0025] D2 strand: 5'-HS-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTGCTGTCGTGTCTCTTCTCCGAGCCGGTCGAAATAGTGTCG-3';

[0026] L2 strand: 5'-CATACACGAAGAGACACGACAGCAC-3';

[0027] S2 strand: 5'-HS-SH-TTTTTTTTTTTCAGTCAACTTTTTTTCGACACTATrAGGAAGAGACATTTTTTAGTTGACTG-FAM-3'; in the above sequence, HS represents a 5' end modified mercapto group, HS-SH represents a 5' end modified bimer capto group, FAM represents a 3' end modified fluorescent group FAM, and rA represents a ribo-adenine nucleotide.

[0028] Compared with the prior art, the Salmonella typhimurium fluorescence detection method based on double signal amplification has the following beneficial effects:

[0029] Firstly, the present application builds two kinds of functionalized SNA probes, integrates aptamer specific recognition and DNAzyme enzyme cutting activity, builds a cascade double signal amplification path, and releases the trigger chain after the aptamer is precisely combined with the target bacteria. The trigger chain starts the strand displacement reaction to release the DNAzyme walking chain, and then cuts the substrate chain to release the fluorescent group. Through two-stage signal amplification effect, the detection sensitivity is significantly improved, the problem of insufficient response of the traditional detection method to low concentration target bacteria is effectively solved, specific sequence complementary relationship and functional group modification are used in the probe design to ensure the specificity of the reaction process, reduce the interference of non-target substances, make the detection result more accurate, and at the same time, the whole detection process does not need complex sample pretreatment and instrument operation, simplifies the detection steps, shortens the detection period, realizes the rapid quantitative analysis of the target bacteria, and provides an efficient technical scheme for trace pathogenic bacteria detection.

[0030] Secondly, the application strengthens the stability and practicability of the detection system through the synergistic effect of the optimization of the structure design of the SNA probe and the reaction system, the hairpin structure on the surface of the probe and the walking chain form a closed state through accurate sequence design, the signal amplification reaction is started only when the target bacteria exist, the background signal interference is reduced, the signal-to-noise ratio of detection is improved, the complex formed by the aptamer and the trigger chain has high efficient recognition and signal triggering capacity, cooperates with the high efficient cutting activity of the DNAzyme, so that the fluorescence signal output forms a stable linear corresponding relationship with the concentration of the target bacteria, the accuracy and repeatability of quantitative detection are guaranteed, the design does not need to rely on expensive instruments and complex labeling process, reduces the detection cost, meanwhile, adapts to complex sample matrix, can meet the on-site detection demand in the field of food and the like, provides rapid and reliable technical support for food safety risk prevention and control, and has important practical application value.

[0031] Other advantages, objects, and features of the application will be apparent from the following specification, and in some cases, will be apparent from the practice of the application. It is intended to cover all such variations and modifications as come within the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creating any creative labor.

[0033] Figure 1 Principle diagram for detecting Salmonella based on spherical nucleic acid fluorescence signal amplification method;

[0034] Figure 2 For probe characterization, including (a) UV-visible spectrum of AuNPs, SNA1 probe, SNA2 probe; (b) particle size distribution graph of SNA1 probe; (c) particle size distribution graph of SNA2 probe;

[0035] Figure 3 For feasibility verification, including (a) fluorescence spectrum; (b) chain reaction verification agarose gel electrophoresis graph; (c) probe reaction verification agarose gel electrophoresis graph;

[0036] Figure 4 Including (a) DNAzyme chain and S chain concentration ratio optimization; (b) probe DNA chain density optimization;

[0037] Figure 5 Including (a) probe dilution multiple optimization; (b) probe addition sequence optimization; (c) reaction time optimization;

[0038] Figure 6 For the protocol sensitivity, including (a) linear regression equation; (b) fluorescence spectrum;

[0039] Figure 7 For the method validation, including (a) specificity; (b) stability. DETAILED DESCRIPTION

[0040] In order to further illustrate the technical means and effects taken by the present application to achieve the predetermined object of the application, the specific embodiments, structures, features and effects thereof according to the present application are described in detail below in combination with the drawings and preferred embodiments.

[0041] Example 1:

[0042] The basic process of fluorescence detection of Salmonella typhimurium based on double signal amplification is implemented.

[0043] I. Experimental purpose:

[0044] Specific fluorescence quantitative detection of Salmonella typhimurium is carried out to verify the basic feasibility of the method.

[0045] II. Experimental materials and reagents:

[0046] Oligonucleotide chain: F chain, Apt. chain, D1 chain, L1 chain, S1 chain, D2 chain, L2 chain, S2 chain (all containing corresponding modified groups), as shown in Figure 1

[0047] Key reagents: gold nanoparticles (AuNPs), TCEP solution (10 mmol / L, 0.5 mol / L), PEG20000 solution (0.1 g / mL), DNAzyme reaction buffer (20 mmol / L Tris-HCl, 50 mmol / L KCl, pH=7.5), MgCl2 solution (50 mmol / L), annealing buffer;

[0048] Instruments: fluorescence spectrophotometer, centrifuge, constant temperature shaker, pipette, vortex mixer.

[0049] III. Experimental steps:

[0050] S100, construction of SNA1 and SNA2 probes:

[0051] DNA chain annealing: mix D1 chain and L1 chain at a molar ratio of 1:5, mix D2 chain and L2 chain at a molar ratio of 1:5, dilute D1 and D2 chains to a concentration of 2 μmol / L with annealing buffer; dilute S1 and S2 chains to 10 μmol / L with annealing buffer. All DNA solutions are incubated at 95℃ for 5 min to destroy the intrachain secondary structure, and then slowly cooled to room temperature in a water bath to promote specific interchain hybridization.​

[0052] Thiol reduction: 10 μL of 10 mmol / L TCEP solution was added to 40 μL of D1-L1 annealing solution or D2-L2 annealing solution; 10 μL of 0.5 mol / L TCEP solution was added to 80 μL of S1 strand or S2 strand annealing solution, and it was left at room temperature for 1 h to ensure that the thiol group was fully activated, laying a foundation for stable combination with the surface of gold nanoparticles in the subsequent reaction.

[0053] Probe assembly: the treated D1-L1 strand was mixed with the S1 strand (molar concentration ratio 1:20), and the D2-L2 strand was mixed with the S2 strand (molar concentration ratio 1:20). Then, 10 μL of 0.1 g / mL PEG20000 solution and AuNPs solution (final concentration 2 nmol / L) were added, and water was added to make up to 1 mL. The mixture was vortexed to ensure uniform contact of each component.

[0054] Freeze-thaw and purification: the mixture was frozen at -20 °C for 2 h to promote the formation of uniform assembly structure of DNA strands and AuNPs, and the spatial configuration of the probe was further consolidated during the thawing process at room temperature, reducing assembly defects. After thawing, the unbound free DNA strands were removed by centrifugation at 12000 rpm for 15 min, and the precipitate was collected after repeating the washing process for 3 times, which was the SNA1 and SNA2 probes, improving the specificity of the subsequent reaction.

[0055] S200, preparation of Apt.-F complex:

[0056] The F strand and the Apt. strand were mixed at a molar ratio of 1:1, and incubated at 95 °C for 5 min to break the initial disordered structure. Then, the mixture was slowly cooled to room temperature in a water bath to promote specific complementary pairing between the two, and an Apt.-F complex with a final concentration of 10 μmol / L was prepared. The complex was stored at 4 °C to maintain its stable structure and avoid dissociation.

[0057] S300, incubation of sample with Apt.-F complex:

[0058] 50 μL of the liquid sample (containing Salmonella typhi) to be tested was added to a centrifuge tube, and 10 μL of the Apt.-F complex was added. The mixture was incubated at 37 °C and 180 r / min for 1 h. The shaking rate was controlled at 180 r / min to ensure that the sample and the complex were in full contact, and to avoid denaturation of the complex caused by vigorous shaking.

[0059] If it is a solid sample (such as meat), the sample was first homogenized to release the target bacteria. The supernatant was obtained by centrifugation at 8000 rpm for 10 min to remove solid impurities, and then the sample was incubated according to the above steps to reduce the interference of impurities in the subsequent reaction.

[0060] S400, initiation of double signal amplification reaction:

[0061] SNA1 probe was diluted 20 times, and SNA2 probe was diluted 10 times. The dilution ratio was verified in the previous stage to balance the signal intensity and reagent cost. The diluted probes, 20 μL of 5×DNAzyme reaction buffer, and 20 μL of 50 mmol / MgCl2 solution were added to the incubated system, and the system was incubated at 37°C and 180 r / min for 1.5 h.

[0062] During the reaction, the released F strand undergoes strand displacement with the L1 strand of SNA1 to remove the D1 strand block, the activated D1 strand cuts the rA site of the S1 strand to release the S1' fragment; the S1' fragment continues to displace the L2 strand of SNA2 to activate the D2 strand, and the D2 strand cuts the rA site of the S2 strand to release the S2' fragment (both carrying FAM fluorescent groups), thereby realizing double enhancement of the fluorescence signal through cascade reaction.

[0063] S500, fluorescence detection and quantification:

[0064] The fluorescence spectrophotometer was set to an excitation wavelength of 489 nm and an emission wavelength of 520 nm, which could accurately capture the emission signal of the FAM fluorescent group and reduce the fluorescence interference of other substances. The fluorescence intensity F of the detection system was detected, and the fluorescence intensity F0 of the blank control group (without Salmonella) was also detected. The fluorescence intensity increase value ΔF was calculated, and the formula was: ΔF=F-F0 to eliminate the background signal effect.

[0065] According to the linear equation: y=7553.5589x-20262.8384, the formula for calculating the fluorescence intensity increase value ΔF was substituted to calculate the concentration of Salmonella in the sample, wherein x was the logarithmic value of the concentration of Salmonella, and R 2 =0.963. The equation was verified by multiple experiments to ensure the reliability of the quantitative results.

[0066] Four, experimental results

[0067] The fluorescence signal was successfully detected, and the fluorescence intensity increase value had a good linear relationship with the logarithmic value of the concentration of Salmonella, which verified the feasibility of the basic process and provided a basis for subsequent actual sample detection.

[0068] Example two

[0069] SNA probe preparation parameter optimization implementation.

[0070] I. Experimental purpose:

[0071] The key parameters in the preparation of SNA1 and SNA2 probes were optimized to improve the stability and reactivity of the probes, and to provide probes with better performance for subsequent detection processes.

[0072] II. Experimental materials and reagents:

[0073] The same as Example One, additionally prepare oligonucleotide chains of different proportions, gradient concentration PEG20000 solution (5 μL, 10 μL, 15 μL, 20 μL).

[0074] III. Experimental steps:

[0075] Optimization of the molar ratio of D chain to L chain:

[0076] Set the D1:L1, D2:L2 molar ratio gradient (1:3, 1:5, 1:7), and prepare the probe according to the S100 steps of Example One. Detect the DNA assembly efficiency on the surface of AuNPs by UV-visible spectroscopy, and observe the characteristic absorption peak shift: when the ratio is 1:3, the insufficient use of L chain leads to the partial blocking of D chain, and subsequent non-specific signals are prone to occur; when the ratio is 1:7, the excess L chain is prone to be free in the system, interfering with the subsequent strand displacement reaction; when the ratio is 1:5, the characteristic absorption peak shift is moderate, the DNA chain is uniformly covered, and the blocking effect is best, so the ratio is determined as the optimal.

[0077] Optimization of the molar concentration ratio of D chain to S chain:

[0078] Set the D1:S1, D2:S2 molar concentration ratio gradient (1:10, 1:15, 1:20, 1:25), and prepare the probe. After reacting with Apt.-F complex and Salmonella, detect the fluorescence intensity increase value. When the ratio is 1:10, the insufficient use of S chain leads to less cuttable substrate, resulting in weak fluorescence signal; when the ratio is 1:25, the excess S chain will hinder the binding of D chain and substrate, slowing down the reaction rate; when the ratio is 1:20, the fluorescence intensity increase value reaches the peak, and the substrate amount and catalytic efficiency are best matched, so the ratio is determined as the optimal.

[0079] Optimization of the PEG20000 addition amount:

[0080] Set the PEG20000 addition amount gradient (5 μL, 10 μL, 15 μL, 20 μL), and keep the rest of the parameters unchanged. Detect the probe particle size uniformity by particle size analyzer: when the addition amount is 5 μL, the probe is prone to agglomeration and poor dispersion; when the addition amount is 15 μL and 20 μL, excess PEG will be adsorbed on the surface of the probe, affecting the activity of the DNA chain; when the addition amount is 10 μL, the probe particle size is uniform (about 78 nm), the dispersion is good, and the activity of the DNA chain is not affected, so the addition amount is determined as the optimal in combination with the fluorescence signal intensity.

[0081] Freeze-thaw condition verification:

[0082] Comparison of the performance of the probe after freezing at -20°C for 1 h, 2 h, and 3 h: when frozen for 1 h, the DNA strands are not fully assembled with the AuNPs, and the probe structure is loose; when frozen for 3 h, excessive freezing can easily lead to aggregation of the AuNPs, affecting the stability of the probe; when frozen for 2 h, the probe structure is tight and uniformly dispersed, and the fluorescence signal intensity is the highest, so the freezing time is determined to be the optimal.

[0083] IV. Experimental results:

[0084] The optimal parameter combination is D strand: L strand = 1:5, D strand: S strand = 1:20, PEG20000 addition amount 10 μL, and freezing time 2 h, as described in Figure 4 . Under this parameter, the probe prepared has uniform particle size, as shown in Figure 2 , the DNA assembly efficiency is high, and the fluorescence signal amplification effect in the subsequent reaction is optimal, which provides a guarantee for the sensitivity improvement of the detection method.

[0085] Example Three:

[0086] Optimization of Apt.-F complex preparation and incubation conditions.

[0087] I. Experimental purpose:

[0088] Optimize the preparation process of the Apt.-F complex and the incubation conditions with the sample, improve the target bacteria recognition efficiency, reduce non-specific binding, and ensure the accurate initiation of the subsequent signal amplification reaction.

[0089] II. Experimental materials and reagents:

[0090] The same as in Example One, except that different proportions of F strand and Apt. strand (1:0.8, 1:1, 1:1.2, 1:1.5), gradient incubation temperatures (35°C, 37°C, 39°C), and times (30 min, 60 min, 90 min) are added.

[0091] III. Experimental steps:

[0092] Optimization of the molar ratio of F strand to Apt. strand:

[0093] Set the molar ratio gradient (1:0.8, 1:1, 1:1.2, 1:1.5) and prepare the complex according to the steps in Example One. Verify the complex formation efficiency by agarose gel electrophoresis: when the ratio is 1:0.8, the lack of F strand leads to partial Apt. strand being free, which is prone to bind to non-target bacteria in the subsequent reaction; when the ratio is 1:1.2 or 1:1.5, excessive F strand can trigger the signal reaction in advance, increasing the background value; when the ratio is 1:1, the electrophoresis band is single and clear, and the complex is completely formed, so the ratio is determined to be the optimal.

[0094] Optimization of annealing cooling rate:

[0095] The stability of the complex was detected by comparing the two methods of rapid cooling (room temperature standing) and slow cooling (1 °C / min cooling to room temperature). When rapidly cooled, the interchain hybridization speed is fast, but mismatched structures are easily formed, the complex stability is poor, and it is easy to dissociate early during subsequent incubation. When slowly cooled, the F chain and the Apt. chain are fully complementary to each other, forming a more stable double-stranded structure, which can accurately dissociate and release the F chain when contacted with the target bacteria, so slow cooling is the optimal method.

[0096] Incubation temperature and time optimization:

[0097] The incubation temperature gradient (35 °C, 37 °C, 39 °C) and time gradient (30 min, 60 min, 90 min) were set, and the shaking rate was maintained at 180 r / min. The reaction rate is slow at 35 °C, and the complex does not fully bind to the target bacteria at 30 min, and although it fully binds at 90 min, it takes too long. Incubation at 39 °C can easily lead to denaturation of the complex, increasing non-specific binding. Under the condition of 37 °C and 60 min, the complex has the highest binding efficiency to the target bacteria, less non-specific binding, and moderate time consumption, so this condition is determined to be the optimal condition.

[0098] Shaking rate effect verification:

[0099] The shaking rate gradient (150 r / min, 180 r / min, 210 r / min) was set, and the other conditions remained unchanged. When the shaking rate is 150 r / min, the sample and the complex do not mix well, and the binding efficiency is low. When the shaking rate is 210 r / min, the vigorous shaking leads to the dissociation of part of the complex, and the signal stability is poor. When the shaking rate is 180 r / min, the mixing is uniform and the complex structure is stable, the fluorescence signal intensity is high and the repeatability is good, so this rate is determined to be the optimal rate.

[0100] Four, experimental results:

[0101] The optimal conditions are F chain:Apt. chain = 1:1, slow cooling annealing, 37 °C incubation for 60 min, and shaking rate of 180 r / min. Under these conditions, the Apt.-F complex prepared has good stability, high recognition efficiency to the target bacteria, and less non-specific binding, providing a precise trigger basis for the subsequent double signal amplification reaction.

[0102] Example four:

[0103] Double signal amplification reaction condition optimization implementation.

[0104] I. Experimental purpose:

[0105] Optimize the key parameters of the signal amplification reaction, shorten the reaction time and improve the signal amplification multiple, while controlling the amount of reagents to reduce costs and improve the practicality of the method.

[0106] II. Experimental materials and reagents:

[0107] As in Example One, increase the probe dilution gradient (SNA1: 10-fold, 20-fold, 30-fold; SNA2: 5-fold, 10-fold, 15-fold), buffer pH gradient (7.0, 7.5, 8.0), MgCl2concentration gradient (5 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L).

[0108] III. Experimental Steps:

[0109] Probe dilution optimization:

[0110] Set the SNA1 probe dilution gradient (10-fold, 20-fold, 30-fold) and the SNA2 probe dilution gradient (5-fold, 10-fold, 15-fold) and combine them using the chessboard method. When the dilution is too low, the probe concentration is too high, which can easily lead to aggregation and strong background signal. When the dilution is too high, the probe concentration is insufficient, and the signal amplification effect is weak. When SNA1 is diluted 20-fold and SNA2 is diluted 10-fold, the background signal is low and the fluorescence intensity increases the most, indicating that the concentrations of the two are best matched. Therefore, this combination is determined to be the optimal one.

[0111] Reaction buffer optimization:

[0112] Set the pH gradient (7.0, 7.5, 8.0) while keeping the other components of the buffer unchanged. When pH = 7.0, the DNAzyme catalytic activity is insufficient and the substrate cutting efficiency is low. When pH = 8.0, it is easy to cause DNA chain denaturation and reduce hybridization specificity. When pH = 7.5, the DNAzyme catalytic activity is the highest and the DNA chain hybridization specificity is good, with complete substrate cutting. Therefore, this pH is determined to be the optimal one.

[0113] MgCl2concentration optimization:

[0114] Set the MgCl2final concentration gradient (5 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L) while keeping the other conditions unchanged. Mg 2+ As a catalytic cofactor of DNAzyme, when the concentration is 5 mmol / L, the activity is insufficient and the substrate chain cannot be effectively cut. When the concentration is 15 mmol / L or 20 mmol / L, it is easy to cause non-specific polymerization of DNA chains and interfere with the reaction. When the concentration is 10 mmol / L, the DNAzyme catalytic efficiency is the highest, the substrate is completely cut, and there is no obvious non-specific reaction. Therefore, this concentration is determined to be the optimal one.

[0115] Reaction time and temperature optimization:

[0116] Set the reaction time gradient (60 min, 90 min, 120 min), temperature gradient (35℃, 37℃, 39℃), keep the shaking rate 180r / min. The reaction rate is slow at 35℃, and the signal platform period is not reached at 120 min; although the reaction is fast at 39℃, the stability of DNAzyme decreases, and the signal decays at 120 min; under the condition of 37℃, 90 min, the reaction reaches the platform period, the substrate is fully cut, the signal is stable and the time is moderate, so the condition is determined as the optimal condition.

[0117] Four, experimental results:

[0118] The optimal conditions are SNA1 diluted 20 times, SNA2 diluted 10 times, buffer pH = 7.5, MgCl2 final concentration 10 mmol / L, 37℃ reaction for 90 min. Under this condition, the signal amplification factor is the highest, the reaction efficiency is optimal, and the reagent consumption and reaction time are controlled, which improves the practicability and economy of the method, such as Figure 5 .

[0119] Example five:

[0120] Detection of salmonella typhi in liquid food samples.

[0121] I. Experimental purpose:

[0122] Verify the detection applicability and accuracy of this method for liquid food samples (such as vegetable soup), simulate the actual detection scene, and provide technical support for the safety monitoring of liquid food.

[0123] II. Experimental materials and reagents:

[0124] The same as example one, the sample to be tested is a commercially available vegetable soup, salmonella typhi standard strain (concentration 10 8 CFU / mL), sterile normal saline.

[0125] III. Experimental steps:

[0126] Sample pretreatment:

[0127] Take vegetable soup, filter to remove solid impurities to avoid adsorption of probes or target bacteria, which will affect the reaction efficiency; centrifuge at 8000 rpm for 10 min to remove the oil layer and reduce the quenching effect of oil on the fluorescence signal; take the supernatant, sterilize at 121℃ for 20 min as a blank matrix to exclude the interference of the components of vegetable soup on the detection.

[0128] Dilute the salmonella standard strain to 10 3 , 10 4 , 10 5 , 10 6The spiked samples, with the concentration of 0, 10, 100, 1000, 10000 CFU / mL, covered the range of possible contamination concentration, ensuring the detection ability of the method for samples with different contamination levels.

[0129] Detection procedure:

[0130] Each spiked sample was detected according to the complete steps of Example 1: the SNA probe was prepared using the optimized parameters, the Apt.-F complex was prepared according to the optimal conditions, and the incubation and signal amplification reaction were performed according to the optimized conditions. Three parallel samples were set for each concentration, and a blank control group (blank matrix) was also set to reduce accidental errors and ensure the reliability of the results.

[0131] Accuracy and precision analysis:

[0132] The recovery rate (recovery rate = measured concentration / spiked concentration x 100%) and relative standard deviation (RSD) of each concentration were calculated, as shown in Table 1. The recovery rate reflects the degree of agreement between the detection results and the actual concentration, and the RSD reflects the repeatability of the method. A recovery rate in the range of 85%-105% indicates that the vegetable soup matrix does not significantly inhibit the reaction, and the detection results can truly reflect the concentration of the target bacteria in the sample; an RSD < 2% indicates that the method has good repeatability and the detection results are stable.

[0133] Spiked concentration (LogC, CFU / mL) Measured concentration (LogC, CFU / mL) Recovery RSD (n=3) 3 3.07±0.04 102.3±1.3% 1.25% 4 3.65±0.05 91.2±1.1% 1.25% 5 4.28±0.06 85.6±1.2% 1.38% 6 5.85±0.11 97.4±1.9% 1.93%

[0134] Table 1: Spiked detection of Salmonella typhi in vegetable soup

[0135] Four, experimental results:

[0136] All spiked samples were successfully detected, with a recovery rate of 85%-105% and an RSD < 2%. This indicates that the method has high detection accuracy and good reproducibility for liquid food samples, can effectively eliminate the interference of liquid food matrix, and is suitable for the detection of Salmonella typhi in liquid food such as vegetable soup and beverages.

[0137] Example six:

[0138] Detection of Salmonella typhi in solid food samples.

[0139] I. Experimental purpose:

[0140] To verify the applicability of the method for solid food samples (such as cured meat and vegetables), optimize the pretreatment process of solid samples, and solve the problem of difficulty in releasing target bacteria from solid matrix.

[0141] II. Experimental materials and reagents:

[0142] The same as in Example 1, the sample to be tested is a commercially available cured meat, the Salmonella typhi standard strain, sterile physiological saline, a homogenizer, and absorbent cotton.

[0143] III. Experimental steps:

[0144] Sample pretreatment optimization:

[0145] Take 25g of cured meat sample and add 225mL of sterile physiological saline (diluted at a ratio of 1:9, in line with the conventional dilution ratio for microbial detection). Homogenize for 2min to prepare a homogenate. The homogenization process can break down the meat structure and allow the target bacteria to be fully released into the physiological saline, avoiding detection errors caused by uneven samples. Centrifuge at 8000rpm for 10min to remove the supernatant and remove solid impurities such as unbroken meat residue.

[0146] Add sterile absorbent cotton to the supernatant and filter to further remove fat and suspended impurities. Fat is prone to form an oil film on the surface of the solution, hindering the contact between the probe and the target bacteria. Suspended impurities may adsorb the probe, causing a decrease in signal. The filtration step can significantly reduce these interferences. Obtain the sample matrix to be tested, sterilize at 121℃ for 20min as a blank matrix to exclude the influence of cured meat components on the reaction.

[0147] Dilute the standard Salmonella strain to 10 3 -10 8 CFU / mL of spiked samples using the blank matrix, covering the actual possible contamination concentration to ensure the practicality of the method.

[0148] Detection process:

[0149] Detect according to the complete steps of Example 1: take 50μL of spiked sample supernatant and incubate with Apt.-F complex. Incubation temperature is 37℃, incubation time is 60min, and shaking speed is 180r / min. The subsequent signal amplification reaction uses the optimized parameters (SNA1 is diluted 20 times, SNA2 is diluted 10 times, and the reaction is carried out at 37℃ for 90min). Three parallel samples are set for each concentration to reduce accidental errors.

[0150] Matrix interference verification:

[0151] Compare the fluorescence intensity increase value of the spiked sample and the pure bacterial liquid sample (same concentration): the pure bacterial liquid sample has no matrix interference and the fluorescence signal is stable. If the spiked sample is interfered by the matrix, the fluorescence signal will show obvious deviation. The difference between the two is within 5%, indicating that the protein, salt and other components in the solid food matrix do not have obvious interference on the hybridization and cleavage reaction of DNA chain, and the detection method is suitable for such samples.

[0152] Four, experimental results:

[0153] The solid sample pretreatment has small matrix interference, the recovery of the spiked sample is between 88% and 103%, and the RSD is less than 1.8%. It is shown that the method can effectively solve the problems of many impurities and difficult release of target bacteria in solid food by optimizing the pretreatment process, and is suitable for the detection of Salmonella typhimurium in cured meat, vegetables and other solid food.

[0154] Example Seven:

[0155] Methodology verification (sensitivity, specificity, stability) implementation:

[0156] I. Experimental purpose:

[0157] The sensitivity, specificity and stability of the detection method are verified to provide comprehensive data support for the practical application of the method and ensure the reliability of the method in different scenarios.

[0158] II. Experimental materials and reagents:

[0159] The same as in Example One, additionally prepare E. coli, S. aureus, P. fluorescens, L. monocytogenes and V. parahaemolyticus standard strains (all are common foodborne pathogens).

[0160] III. Experimental steps:

[0161] Sensitivity verification:

[0162] Dilute the Salmonella typhimurium standard strain with sterile physiological saline to 10 2 , 5 x 10 2 , 10 3 , 10 4 , …, 10 8 CFU / mL, covering low, medium and high contamination concentrations. Detect according to the steps in Example One, set 3 parallel samples for each concentration to ensure data repeatability.

[0163] The lowest detection limit (LOD) is calculated based on 3 times the standard deviation of the fluorescence intensity of the blank control group. This calculation method meets the conventional standard of microbial detection and can effectively avoid false negative results. Draw the standard curve of the increase in fluorescence intensity and the logarithmic value of the concentration, and the linear relationship is good, which shows that the method is accurate in quantification within the concentration range and meets the detection needs of samples with different contamination levels.

[0164] Specificity verification:

[0165] Prepare 10 6CFU / mL of Salmonella Typhimurium, Escherichia coli, Staphylococcus aureus, Pseudomonas fluorescens, Listeria monocytogenes, and Vibrio parahaemolyticus bacterial suspensions were tested. This concentration is near the critical concentration for common foodborne pathogens. Following the steps in Example 1, the fluorescence signal intensity of each strain was compared: methods with good specificity only produce strong fluorescence signals for the target bacteria, while other strains show weak or no signals, effectively avoiding false positive results.

[0166] Stability verification:

[0167] The prepared SNA1 and SNA2 probes were stored at 4°C (normal laboratory refrigeration conditions) and removed on days 1, 3, 5, and 7, respectively. The same concentration (10...) was then tested. 5 The fluorescence intensity of Salmonella samples (CFU / mL) was analyzed, and the signal change rate was analyzed. A small signal change rate indicates that the probe is structurally stable and its activity does not decay under the storage conditions. This can reduce the frequent operation of probe preparation and improve the convenience of practical application of the method.

[0168] IV. Experimental Results:

[0169] Sensitivity: at 10 3 -10 8 Good linearity was observed within the CFU / mL range (R0). 2 =0.963), LOD is 5.75×10 2 CFU / mL, capable of detecting low concentrations of contamination, meeting the sensitivity requirements for food safety monitoring, such as Figure 3 As shown;

[0170] Specificity: The fluorescence signal of *Salmonella typhimurium* was significantly higher than that of other strains (difference > 80%), while the fluorescence signals of other strains were close to those of the control group. This demonstrates good specificity and effectively distinguishes the target bacterium from other common pathogens. Figure 5 As shown;

[0171] Stability: After 7 days of storage, the fluorescence signal change rate is ≤5%, maintaining good detection performance. It can be stably stored at 4℃, facilitating practical laboratory operations. Figure 6 As shown.

[0172] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for detecting Salmonella typhimurium based on dual signal amplification, characterized in that, The specific steps of the method are: S100, constructing SNA1 and SNA2 probes: on the surface of gold nanoparticles, SNA1 probe is obtained by co-modifying 8-17 DNAzyme walking chain D1 and hairpin structure substrate chain S1 through freezing method, part of the sequence of D1 chain is closed by hybridizing with locking chain L1, the loop part of S1 chain contains DNAzyme cleavage site, and the end of S1 chain is modified with a fluorescent group; on the surface of gold nanoparticles, SNA2 probe is obtained by co-modifying 8-17 DNAzyme walking chain D2 and hairpin structure substrate chain S2 through freezing method, part of the sequence of D2 chain is closed by hybridizing with locking chain L2, and the end of S2 chain is modified with a fluorescent group; S200, preparing Apt.-F complex: mixing trigger chain F and Salmonella typhimurium aptamer, and forming Apt.-F complex through annealing treatment; S300, incubating sample with Apt.-F complex: adding Apt.-F complex into the sample to be tested, and incubating at a set temperature for a set time; S400, starting double signal amplification reaction: adding SNA1 probe, SNA2 probe, DNAzyme reaction buffer and MgCl2 solution into the incubated system, and incubating at a set temperature for a set time; S500, fluorescence detection and quantification: detecting the fluorescence intensity of the system after reaction by using a fluorescence spectrophotometer, and quantifying Salmonella typhimurium in the sample to be tested according to the corresponding relationship between the fluorescence intensity and the concentration of Salmonella typhimurium.

2. The Salmonella typhimurium fluorescent detection method based on double signal amplification according to claim 1, characterized in that, In the S100, constructing SNA1 and SNA2 probes, the molar ratio of D1 chain and L1 chain mixed annealing is 1:5, and the molar ratio of D2 chain and L2 chain mixed annealing is 1:5; the molar concentration ratio of D1 chain and S1 chain in SNA1 probe is 1:20, and the molar concentration ratio of D2 chain and S2 chain in SNA2 probe is 1:20; both S1 chain and S2 chain contain continuous thymine nucleotides as a connecting arm in the sequence.

3. The Salmonella typhimurium fluorescent detection method based on double signal amplification according to claim 1, characterized in that, In the S100, constructing SNA1 and SNA2 probes, when constructing SNA1 and SNA2 probes, the D1 chain, the L1 chain, the D2 chain, the L2 chain, the S1 chain and the S2 chain are subjected to thiol reduction treatment: 10 μL of TCEP solution with a concentration of 10 mmol / L is added to 40 μL of D1-L1 chain annealing solution or D2-L2 chain annealing solution; 10 μL of TCEP solution with a concentration of 0.5 mol / L is added to 80 μL of S1 chain annealing solution or S2 chain annealing solution; all thiol reduction treatments are carried out at room temperature for 1 h; the specific conditions of the freezing method are freezing at-20℃ for 2 h, and then thawing at room temperature; after thawing, the mixed solution needs to be centrifuged and washed, the centrifugal speed is 12000 rpm, the centrifugal time is 15 min, and the centrifugal washing is repeated for 3 times.

4. The Salmonella typhimurium fluorescent detection method based on double signal amplification according to claim 1, characterized in that, In the S100, constructing SNA1 and SNA2 probes, 10 μL of PEG20000 solution with a concentration of 0.1 g / mL is added; the final concentration of AuNPs solution in the reaction system is 2 nmol / L; the total volume of the reaction system is supplemented to 1 mL by adding water, and the mixture needs to be fully mixed after adding water.

5. The Salmonella typhimurium fluorescent detection method based on double signal amplification according to claim 1, characterized in that, In the S200, the mixing molar ratio of the F strand and the Apt. strand is 1:1 during preparation of the Apt.-F complex; the specific conditions of the annealing treatment are as follows: the mixed F strand and Apt. strand solution is incubated at 95℃ for 5 min, and then slowly cooled to room temperature in a water bath; and the final concentration of the Apt.-F complex is 10 μmol / L.

6. The Salmonella typhimurium fluorescent detection method based on double signal amplification according to claim 1, characterized in that, In the S300, the adding volume of the sample to be tested is 50 μL, and the adding volume of the Apt.-F complex is 10 μL; the setting temperature of the incubation is 37℃, and the setting time is 1 h; the shaking rate during the incubation is 180 r / min; when the sample to be tested is a solid food sample, the sample is first made into a homogenate, and then the supernatant is taken after centrifugation as the sample to be tested.

7. The Salmonella typhimurium fluorescent detection method based on double signal amplification according to claim 1, characterized in that, In the S400, the SNA1 probe is diluted 20 times before use, and the SNA2 probe is diluted 10 times before use; the concentration of the DNAzyme reaction buffer is 5×, and the adding volume is 20 μL; the composition of the buffer is 20 mmol / L Tris-HCl and 50 mmol / L KCl, and the pH value is 7.5; the concentration of the MgCl2 solution is 50 mmol / L, and the adding volume is 20 μL; the setting temperature of the incubation is 37℃, and the setting time is 1.5 h; the shaking rate during the incubation is 180 r / min.

8. The Salmonella typhimurium fluorescent detection method based on double signal amplification according to claim 1, characterized in that, In the S400, the specific process of the double signal amplification reaction is as follows: in the S300, the F strand released from the incubation of the sample and the Apt.-F complex and the L1 strand on the SNA1 probe undergoes strand displacement reaction, and the D1 strand is displaced; in the presence of Mg2+, the D1 strand combines with the S1 strand on the SNA1 probe and cuts the rA site of the S1 strand, and the S1' fragment with FAM is released; the S1' fragment and the L2 strand on the SNA2 probe undergo strand displacement reaction, and the D2 strand is displaced; in the presence of Mg2+, the D2 strand combines with the S2 strand on the SNA2 probe and cuts the rA site of the S2 strand, and the S2' fragment with FAM is released.

9. The Salmonella typhimurium fluorescent detection method based on double signal amplification according to claim 1, characterized in that, In the fluorescence detection and quantification, the excitation wavelength of the fluorescence spectrophotometer is set to 489 nm, and the emission wavelength is set to 520 nm; the corresponding relationship between the fluorescence intensity and the concentration of Salmonella typhimurium is that the fluorescence intensity increase value is linearly related to the logarithmic value of the concentration of Salmonella typhimurium, the linear equation is y = 7553.5589x - 20262.8384, and the correlation coefficient R 2 = 0.963; wherein y represents the fluorescence intensity increase value, x represents the logarithmic value of the concentration of Salmonella typhimurium, and the fluorescence intensity increase value is the difference between the fluorescence intensity of the sample to be tested and the fluorescence intensity of the blank control group.

10. The Salmonella typhimurium fluorescent detection method based on double signal amplification according to claim 1, characterized in that, In the S100, the specific sequences of the oligonucleotides used are as follows: F strand: 5'-CGGTCGAAACATAATGTCAAGTCC-3'; Apt. strand: 5'-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG-3'; D1 strand: 5'-HS-TTTTTTTTTTTTTTTTTTTTTTTTTTAACGACAGCACTCCGAGCCGGTCGAAACATAATGG-3'; L1 strand: 5'-GGACTTGACATTATGTTTCGACCG-3'; S1 strand: 5'-HS-SH-TTTTTTTTTTATACACGAATTTTTTCCATTATGTrAGGTGCTGTCGTGTCTCTTCGTGTATG-FAM-3'; D2 strand: 5'-HS-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTGCTGTCGTGTCTCTTCTCCGAGCCGGTCGAAATAGTGTCG-3'; L2 strand: 5'-CATACACGAAGAGACACGACAGCAC-3'; S2 strand: 5'-HS-SH-TTTTTTTTTTTCAGTCAACTTTTTTTCGACACTATrAGGAAGAGACATTTTTTAGTTGACTG-FAM-3'; In the above sequences, HS represents a 5' end modified thiol group, HS-SH represents a 5' end modified disulfide group, FAM represents a 3' end modified fluorescent group FAM, and rA represents a ribo-adenine nucleotide.

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