Endogenous protein activated deoxyribozyme-based pathogen viable bacteria detection kit

Abstract

The invention provides a pathogen viable bacteria detection kit based on endogenous protein activated deoxyribozyme. The kit comprises a deoxyribozyme recognition element and a signal transduction element. The deoxyribozyme recognition element is composed of a substrate chain and an enzyme chain, and the signal transduction element is composed of two DNA reporter probes (Sub-F and Sub-Q), wherein the tail ends of the two DNA reporter probes are respectively marked with a fluorescence staining group and a fluorescence quenching group. The deoxyribozyme recognition element can recognize specific endogenous protein of target pathogenic viable bacteria and activate an enzyme chain to cut a substrate chain to generate two independent substrate chain fragments. And then a signal transduction element is introduced to realize specific output of a fluorescence signal. The kit provided by the invention can realize detection of living salmonella by targeting endogenous protein, can sensitively detect 190 CFU / mL of living salmonella with abundance as low as 0.1%, and has a good application prospect in the directions of food safety detection and clinical examination.

Description

Technical Field

[0001] This invention belongs to the field of functional nucleic acid detection technology, specifically relating to a pathogen live bacteria detection kit based on endogenous protein-activated deoxyribonuclease. Background Technology

[0002] Foodborne and medically significant pathogens cause millions of deaths and hospitalizations each year. Salmonella infection has become a major bacterial disease due to its widespread presence in the food supply chain. Salmonella can survive in various foods for anywhere from 5 months to 2 years, easily spreading to humans and causing diseases such as gastroenteritis, sepsis, and typhoid fever. Almost all types of food can serve as carriers of foodborne outbreaks of salmonellosis, with animal-based foods accounting for approximately 75% of Salmonella infections. Live bacteria are the primary cause of infection, and the lack of technology to distinguish between live and dead bacteria is considered one of the biggest limitations in food safety regulation and clinical diagnosis. Currently, live bacteria are mainly detected through plate counts and DNA testing. Plate counts (ISO 6579-1) are the standard method for live bacteria detection, but they take 3-4 days. Compared to DNA, RNA degrades rapidly after cell death, and RNA detection holds promise for accurate quantification of live bacteria. RNA-targeted detection primarily utilizes quantitative reverse transcription polymerase chain reaction (RT-qPCR), which can detect trace levels of target bacteria within 2-4 hours. RNA detection requires nucleic acid extraction and amplification, which poses a challenge for on-site testing.

[0003] Deoxyribonucleases (DNAzymes), as short, artificially catalytic nucleic acid molecules, contain a functional domain responsible for recognizing target cofactors such as metal ions and endogenous proteins. Deoxyribonucleases have great potential for developing biosensors targeting various pathogens; fluorescent deoxyribonuclease sensors targeting pathogens such as *Escherichia coli*, *Legionella pneumophila*, *Clostridium difficile*, and *Staphylococcus aureus* have already been developed. However, in many studies, the mechanism of action between deoxyribonucleases and their target cofactors remains unclear, detection procedures are overly lengthy, and methods that directly modify the fluorescent and quenching groups at the ends of the deoxyribonuclease substrate chain result in excessively long detection times, thus failing to meet the requirements for reliable detection in real-world environments. Therefore, developing fluorescent deoxyribonuclease sensors with clear mechanisms of action, simple and rapid procedures, high sensitivity, and strong specificity is urgently needed and meaningful. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a pathogen live bacteria detection kit based on endogenous protein-activated deoxyribonuclease. This method, through the design of a sequential detection mechanism, avoids internal modification within the deoxyribonuclease active domain, thereby enhancing its cleavage activity. Simultaneously, based on the activation of the DNAzyme by the endogenous protein RNase H2, nucleic acid extraction and amplification are unnecessary. This method exhibits high sensitivity and specificity, enabling accurate detection of pathogen live bacteria.

[0005] This invention first provides a pathogen live bacteria detection kit based on endogenous protein-activated deoxyribonuclease, comprising the following parts: (1) Deoxyribozyme recognition element, including deoxyribozyme substrate chain and enzyme chain; (2) Signal transduction elements, including DNA reporter probe Sub-F with terminal fluorescent staining group and DNA reporter probe Sub-Q with terminal fluorescent quenching group.

[0006] The endogenous protein-activated deoxyribonuclease enzyme chain can be activated by the Salmonella-specific endogenous protein RNase H2, which in turn catalyzes the cleavage of the endogenous protein-activated deoxyribonuclease substrate chain at the 3' end of the RNA base site, producing two isolated fragments. The nucleotide sequence of the deoxyribonuclease enzyme chain is shown in SEQ ID No. 2 of the sequence listing, and the nucleotide sequence of the deoxyribonuclease substrate chain is shown in SEQ ID No. 1 of the sequence listing.

[0007] The DNA reporter probe Sub-F, terminally labeled with a fluorescent staining group, and the DNA reporter probe Sub-Q, terminally labeled with a fluorescent quenching group, can hybridize complementaryly with two isolated fragments generated by catalytic cleavage, thereby achieving signal output. The nucleotide sequences of the DNA reporter probe Sub-F and the DNA reporter probe Sub-Q terminally labeled with a fluorescent staining group are shown in SEQ ID No. 3 and SEQ ID No. 4 of the sequence listing.

[0008] Furthermore, the application of the pathogen live bacteria detection kit for endogenous protein-activated deoxyribonuclease in live bacteria detection.

[0009] The pathogen live bacteria detection kit based on endogenous protein-activated deoxyribonuclease according to claim 2 is characterized by the following steps in its application for quantitative detection of pathogen live bacteria: Step 1: Dissolve the deoxyribonuclease substrate chain and deoxyribonuclease chain from the kit in buffer solution, incubate at 70-90℃ for 3-5 min, cool naturally to 15-30℃, and cool at room temperature for at least 10 min to obtain the deoxyribonuclease solution. Step 2: Add the sample to be tested to the reaction mixture from Step 1, and incubate at 20-35 °C for 15-30 min to activate the deoxyribozyme cleavage activity, which in turn catalyzes the cleavage of the 3' end of a single RNA linker in the deoxyribozyme substrate chain, generating two isolated fragments. Step 3: Add the DNA reporter probes Sub-F and Sub-Q, which are labeled with fluorescent and quenching groups respectively, to the reaction mixture from Step 2. After mixing, incubate at 20-35 °C for 10-60 min to obtain the reaction mixture. Measure the fluorescence intensity of the reaction mixture.

[0010] Furthermore, the application of the endogenous protein-activated deoxyribonuclease pathogen live bacteria detection kit in live bacteria detection is characterized by the following: the total volume of the reaction system is 20-40 μL, the concentration of the endogenous protein-activated deoxyribonuclease substrate chain is 50 nM-1 μM, the concentration of the endogenous protein-activated deoxyribonuclease enzyme chain is 50 nM-1 μM, the concentration of Sub-F is 50 nM-1 μM, and the concentration of Sub-Q is 50 nM-1 μM.

[0011] Furthermore, the application of the endogenous protein-activated deoxyribonuclease pathogen live bacteria detection kit in live bacteria detection is characterized in that the buffer solution is obtained by dissolving 4-hydroxyethylpiperazine ethanesulfonic acid, NaCl, and MgCl2 in nuclease-free water and adjusting the pH value to 7-8, wherein the concentration of 4-hydroxyethylpiperazine ethanesulfonic acid is 40-60 mmol / L, the concentration of NaCl is 140-160 mmol / L, and the concentration of MgCl2 is 10-20 mmol / L.

[0012] Furthermore, the pathogen live bacteria detection kit based on endogenous protein-activated deoxyribonuclease, in its application for quantitative detection of pathogen live bacteria, is characterized in that, when measuring fluorescence intensity, the excitation and emission wavelengths should be determined according to the characteristic excitation and emission wavelengths of the selected fluorescent group.

[0013] The present invention demonstrates through experiments that the pathogen live bacteria detection kit based on endogenous protein-activated deoxyribonuclease provided by the present invention has excellent detection rate, sensitivity and specificity, and can distinguish between the live and dead states of pathogens.

[0014] The detection principle of this invention is as follows: Figure 1As shown, RNase H2 is a specific endogenous protein secreted by live Salmonella, which is rapidly degraded and inactivated in dead bacteria. In the absence of RNase H2, the deoxyribonuclease enzyme chain lacks cleavage activity, allowing the intact deoxyribonuclease substrate chain to facilitate close proximity of DNA probes modified with fluorescent and quenching groups, respectively, achieving ultra-low background detection. In the presence of RNase H2, the catalytic activity of the deoxyribonuclease enzyme chain is activated, catalytically cleaving the 3' end of the deoxyribonuclease substrate RNA bases to generate two separate fragments. DNA probes with complementary ends labeled with fluorescent and quenching groups are introduced, activating the signal. Finally, rapid and sensitive analysis of live Salmonella bacteria is achieved through fluorescence detection. In the sequence design, the deoxyribonuclease does not contain internal group modifications, allowing the target cofactor to bind more stably to the deoxyribonuclease, thereby significantly improving the cleavage efficiency of the deoxyribonuclease enzyme chain. Based on this mechanism, live bacteria can be more efficiently recognized by the deoxyribonuclease, improving the detection rate and sensitivity.

[0015] Compared with the prior art, the present invention has the following advantages: (1) This method eliminates the need for pretreatment steps such as nucleic acid extraction and amplification of the samples to be tested, which saves costs and simplifies the operation process; (2) This method can complete the detection within 25 minutes, which is an advantage in the rapid detection of live foodborne pathogens; (3) The detection limit of this method is 190 CFU / mL, and the sensitivity is high, which can meet the needs of clinical or environmental testing. (4) This method uses Salmonella RNase H2 as a target, and can identify Salmonella viable bacteria with an abundance as low as 0.1%, thus avoiding false positives in the detection. This invention exhibits good detection specificity for live Salmonella bacteria and can still specifically detect the target pathogen live bacteria in the presence of other interfering bacteria. Attached Figure Description

[0016] Figure 1 The working principle of the detection kit provided by this invention.

[0017] Figure 2 This is to establish a standard curve for the relationship between Salmonella concentration and fluorescence intensity in Example 1.

[0018] Figure 3 The results are from the test in Example 2 on actual samples of simulated contamination, such as chicken, beef, milk, and blood.

[0019] Figure 4 This is the result of a specific test for Salmonella in the presence of other interfering bacteria in Example 3.

[0020] Figure 5This shows the relationship between the abundance of viable Salmonella and fluorescence intensity in Example 4.

[0021] Figure 6 This is a schematic diagram illustrating the principle of Salmonella detection in Comparative Example 1.

[0022] Figure 7 This is a kinetic analysis of the detection of Salmonella in Comparative Example 1. Detailed Implementation

[0023] The specific method for preparing Salmonella samples as described in the following examples is as follows: First, Salmonella is inoculated into LB medium and cultured at 37°C until its OD value reaches 100%. 600 Approximately 1, this is collected to obtain a coarse bacterial mixture (CBM).

[0024] First, the reaction was carried out in a 30 μL solution containing 1× buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 15 mM MgCl2), 400 nM deoxyribonuclease substrate (Sub), and 400 nM deoxyribonuclease enzyme (Dz). The reaction was carried out at 80 °C for 2 min to denature Sub and Dz, followed by cooling to room temperature for 10 min to hybridize Sub and Dz. Then, 7.5 μL of the sample to be tested was added, and the mixture was incubated at 30 °C for 15 min. Finally, 300 nM DNA probes with fluorescently labeled ends (Sub-F) and 300 nM DNA probes with quenching ends (Sub-Q) were added, and the mixture was reacted at room temperature for 10 min. Fluorescence intensity was then measured at an excitation wavelength of 480 nm, with an emission range of 510–600 nm and a step size of 2 nm.

[0025] The characteristic feature is that experiments were conducted using Salmonella solutions of different concentrations, specifically 10... 8 10 7 10 6 10 5 10 4 10 3 10 2 10 1 0 CFU / mL, fluorescence intensity data were measured.

[0026] The fluorescence intensity of Salmonella solutions at different concentrations was measured as follows: Figure 2 As shown, the fluorescence intensity gradually increases with increasing Salmonella concentration. When the Salmonella concentration is at 10... 2 -10 6 Within the specified range, the concentration of Salmonella showed a linear relationship with fluorescence intensity. The linear regression equation was: y = 306.8x - 120.73, R0 2=0.9959, where x represents the concentration of Salmonella (lg CFU / mL), y represents the fluorescence intensity, and R 2 The value represents the correlation coefficient. The limit of detection (LOD) is 190 CFU / mL, indicating good detection sensitivity, and it can be applied to pathogen detection in various scenarios.

[0027] First, the chicken and beef samples were ground and sterilized together with the milk and blood samples. Then, 5 g of chicken and beef samples were taken, and 45 mL of water was added to each sample. The mixture was shaken for 2 minutes and centrifuged at 2000 g for 10 minutes. The supernatant was collected. Finally, Salmonella solutions of different concentrations were added to the chicken, beef, milk, and blood samples to achieve final concentrations of 0 and 10, respectively. 4 and 10 6 CFU / mL. This resulted in 8 negative controls and 8 positive controls for each actual sample.

[0028] The detection steps described in Example 1 are characterized by a designed concentration of 10. 8 Five common foodborne pathogens, namely Pseudomonas aeruginosa, Staphylococcus epidermidis, Acinetobacter baumannii, Staphylococcus aureus, and Escherichia coli, were mixed at a 1:1 ratio with different concentrations (0, 10... 6 10 7 10 8 A mixture of Salmonella (CFU / mL) was prepared, and fluorescence data were measured.

[0029] For a fluorescent sensor of deoxyribonuclease directly modified with fluorescent and quenching groups, the detection procedure is as follows: First, the reaction is carried out in a 30 μL solution containing 1× buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 15 mM MgCl2) and 400 nM of deoxyribonuclease directly modified with fluorescent and quenching groups. Second, 7.5 μL of crude bacterial mixture (CBM) is added, and the cleavage reaction is carried out at 30 °C. Finally, the fluorescence intensity is measured with an excitation wavelength of 480 nm, an emission range of 510-600 nm, and a step size of 2 nm.

[0030] The kinetics of the direct effect of fluorescent group and quenching group modification are shown in Figure 7. The deoxyribozyme fluorescent sensor without internal modification provided by this invention can complete the cleavage reaction within 15 min, while its first-order reaction rate reaches 0.373 min. -1 It is approximately 20 times faster than the first-order reaction rate of directly modified fluorescent sensors.

Claims

1. A pathogen live bacteria detection kit based on endogenous protein-activated deoxyribonuclease, characterized in that: The kit includes: (1) Deoxyribozyme recognition element, including deoxyribozyme substrate chain and enzyme chain; (2) Signal transduction elements, including DNA reporter probe Sub-F with terminal fluorescent staining groups and DNA reporter probe Sub-Q with terminal fluorescent quenching groups; The deoxyribonuclease substrate chain and the deoxyribonuclease chain bind through complementary base pairing to form a deoxyribonuclease, i.e., a recognition element. Salmonella-specific endogenous proteins can recognize the deoxyribonuclease and activate its cleavage activity, thereby catalyzing the cleavage of the 3' end of a single RNA linker in the deoxyribonuclease substrate chain, generating two isolated fragments. Subsequently, signal transduction elements, namely DNA reporter probes Sub-F and Sub-Q complementary to the two isolated fragments, are introduced to achieve signal output.

2. The kit according to claim 1, characterized in that, The nucleotide sequence of the endogenous protein-activated deoxyribonuclease substrate chain is shown in SEQ ID No. 1 of the sequence listing; the nucleotide sequence of the endogenous protein-activated deoxyribonuclease enzyme chain is shown in SEQ ID No. 2 of the sequence listing; the nucleotide sequence of Sub F is shown in SEQ ID No. 3 of the sequence listing; and the nucleotide sequence of Sub Q is shown in SEQ ID No. 4 of the sequence listing.

3. The pathogen live bacteria detection kit based on endogenous protein-activated deoxyribonuclease as described in claim 1, and its application in the quantitative detection of pathogen live bacteria.

4. The pathogen live bacteria detection kit based on endogenous protein-activated deoxyribonuclease according to claim 2, characterized in its application in the quantitative detection of pathogen live bacteria, is as follows: Includes the following steps: Step 1: Dissolve the deoxyribonuclease substrate chain and deoxyribonuclease chain from the kit in buffer solution, incubate at 70-90 °C for 3-5 min, cool naturally to 15-30 °C, and cool at room temperature for at least 10 min to obtain the deoxyribonuclease solution. Step 2: Add the sample to be tested to the reaction mixture from Step 1, and incubate at 20-35 °C for 15-30 min to activate the deoxyribozyme cleavage activity, which in turn catalyzes the cleavage of the 3' end of a single RNA linker in the deoxyribozyme substrate chain, generating two isolated fragments. Step 3: Add the DNA reporter probes Sub-F and Sub-Q, which are labeled with fluorescent and quenching groups at the ends, respectively, to the kit, mix well, and incubate at 20-35 °C for 10-60 min to obtain the reaction mixture. Measure the fluorescence intensity of the reaction mixture.

5. The pathogen live bacteria detection kit based on endogenous protein-activated deoxyribonuclease according to claim 3, characterized in its application in the quantitative detection of pathogen live bacteria, is as follows: The total volume of the reaction system was 20–40 μL, the concentration of the endogenous protein-activated deoxyribonuclease substrate chain was 50 nM–1 μM, the concentration of the endogenous protein-activated deoxyribonuclease enzyme chain was 50 nM–1 μM, the concentration of Sub-F was 50 nM–1 μM, and the concentration of Sub-Q was 50 nM–1 μM.

6. The pathogen live bacteria detection kit based on endogenous protein-activated deoxyribonuclease according to claim 3, characterized in its application in the quantitative detection of pathogen live bacteria, is as follows: The buffer solution is prepared by dissolving 4-hydroxyethylpiperazine ethanesulfonic acid, NaCl, and MgCl2 in nuclease-free water and adjusting the pH to 7-8. The concentration of 4-hydroxyethylpiperazine ethanesulfonic acid is 40-60 mmol / L, the concentration of NaCl is 140-160 mmol / L, and the concentration of MgCl2 is 10-20 mmol / L.

7. The pathogen live bacteria detection kit based on endogenous protein-activated deoxyribonuclease according to claim 3, characterized in its application in the quantitative detection of pathogen live bacteria, is as follows: When measuring fluorescence intensity, the excitation and emission wavelengths should be determined based on the characteristic excitation and emission wavelengths of the selected fluorophore.

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