A kit and method for closed-tube visualization detection of Escherichia coli O157:H7

Abstract

The application discloses a kit and a method for closed-tube visual detection of E.coli O157:H7. The kit comprises a mixed reaction system in a detection tube and thiol DNA modified gold nanoparticles on a cover of the detection tube. The mixed reaction system in the tube comprises an RPA amplification system and a CRISPR / Cas12a reaction system mixed with each other. The thiol DNA modified gold nanoparticle mixture on the cover of the detection tube can be added into the mixed reaction system in a closed tube. The application places the RPA system, the CRISPR system and the gold nanoparticles in the same tube through the design of a DNA-crRNA complex, and forms a closed-tube detection system to prevent the generation of cross contamination.

Description

Technical Field

[0001] This invention belongs to the fields of biology and chemistry, and specifically relates to a kit and method for closed-tube visualization detection of Escherichia coli O157:H7. Background Technology

[0002] With the continuous development and impact of society and industrialization, people's living standards have significantly improved, and the issue of public safety has become a major concern, making food safety one of the most discussed social issues. Food safety is not only related to consumers' vital interests and health, but also closely linked to social and economic development and national prosperity. In recent years, the frequency of foodborne illness incidents has been increasing, and foodborne illness has become one of the most widespread public health problems in the world today. With the continuous development of express delivery and food delivery platforms, food can be transmitted during transportation, leading to various food quality problems caused by foodborne pathogens, increasing the risk of outbreaks of certain foodborne illnesses. Escherichia coli O157:H7 is one type of E. coli, commonly found in the intestines of warm-blooded animals such as cattle and sheep. This type of E. coli releases a potent toxin and can cause severe intestinal symptoms, such as bloody diarrhea. This disease can be contracted through drinking contaminated water or eating undercooked food (especially minced beef, hamburgers, and roast beef). Cases of illness caused by consuming unpasteurized milk, cheese, vegetables, fruit juices, and dairy products have also been reported. Furthermore, poor personal hygiene can lead to human-to-human transmission or infection through consuming food contaminated with feces. The health problems caused by E. coli O157:H7 cannot be ignored, as food safety incidents caused by E. coli O157:H7 continue to occur. Therefore, detecting E. coli O157:H7 in food is a primary means of preventing food safety problems.

[0003] Currently, detection techniques for E. coli O157:H7 mainly include plate culture, various amplification techniques such as PCR, and immunoassay. These detection methods are time-consuming and require professional operation, necessitating strict laboratory procedures and increasing the risk of contamination, potentially introducing pathogens into the environment and posing a threat to human health. Temperature-dependent or isothermal nucleic acid amplification techniques, such as polymerase chain reaction (RPA), multiplex RPA, quantitative real-time RPA, and loop-mediated isothermal amplification (LAMP), amplify the target gene and then detect it using sequencing or specific fluorescent signals. Immunological methods rely on the specific reaction between antigens and antibodies, such as enzyme-linked immunosorbent assay (ELISA) and immunochromatography (IC). While these methods offer high specificity, they suffer from low sensitivity, are time-consuming, and costly, making them unsuitable for large-scale testing at the grassroots level. Therefore, developing a time-saving, labor-saving, low-cost, and accurate one-step detection method has become the main research direction.

[0004] Clustered regularly interspaced short palindromic repeats (CRISPR) are an immune mechanism used by many bacteria and archaea to combat invasion. In nature, a fierce competition for survival exists between bacteria / archaea and bacteriophages. To resist invasion by foreign bacteriophages, bacteria and archaea have evolved the CRISPR-Cas system to recognize and cleave invading nucleic acids, thus protecting themselves. Cas12a is a type 2 V Cas protein. When inactive, it has no cleavage activity. When it binds to specific crRNA, Cas12a undergoes a conformational change, forming a Cas12a-crRNA binary complex. This complex specifically recognizes target DNA and activates its endonuclease activity. After Cas12a sequentially cleaves the DNA double strand in a cis-cleavage manner, this active site remains exposed, exhibiting trans-cleavage activity and non-specifically cleaving the surrounding ssDNA. Scientists have designed various highly sensitive and specific nucleic acid detection platforms to leverage the characteristics of Cas12a, and are also continuously developing portable, low-cost, and rapid assay platforms that do not require large instruments. However, because the CRISPR-Cas12 enzyme is highly temperature-sensitive, the amplification step of the strain-specific fragment is usually performed at a different reaction temperature than the CRISPR-Cas12 system, leading to the need for multiple tube opening and sample loading processes, which can cause aerosol contamination and false positives. Furthermore, the fluorescence generated by Cas12 protein cleaving DNA probes requires specialized instruments for observation, further increasing detection costs. Therefore, a closed-tube visualization detection system is needed. Summary of the Invention

[0005] To address the problems in existing technologies, this invention proposes a kit and method for closed-tube visualization detection of Escherichia coli O157:H7. This invention overcomes the technical deficiencies of traditional E. coli O157:H7 detection methods by targeting specific gene fragments, providing a simple, rapid, sensitive, specific, and portable single-tube detection method.

[0006] Therefore, the technical solution adopted by the present invention is as follows:

[0007] This invention first provides a kit for closed-tube visualization detection of Escherichia coli O157:H7, which includes a mixed reaction system located inside the detection tube and a mixture of thiol-DNA-modified gold nanoparticles located on the cap of the detection tube.

[0008] The mixed reaction system in the detection tube includes a mixed RPA amplification system and a CRISPR / Cas12a reaction system with protective DNA modification.

[0009] The RPA amplification system is used to amplify specific gene fragments in the genome of Escherichia coli O157:H7 using RPA, and includes primer RPA-F, primer RPA-R, Primer-Free-R buffer, DNA template, RNAase-free water, and magnesium acetate.

[0010] The CRISPR / Cas12a reaction system includes: Cas12a, a DNA-crRNA complex, a Linker-ssDNA, buffer C, and ddH2O. The DNA-crRNA complex is formed by the pairing of a protective DNA strand with crRNA. It can unwind at 37°C and slowly release crRNA to bind to the target DNA on a specific gene fragment in the *E. coli* O157:H7 genome. The sequence of the protective DNA strand is shown in SEQ ID No. 3, and the protective DNA is designed with a stem-loop structure. The first 16 bases of the protective DNA pair complementaryly with the 16 bases of the crRNA-specific sequence, and the last 8 bases, after unwinding, can complement the first 8 bases of the protective DNA strand to form a stem-loop structure. When the system reacts at 37°C for a certain time (e.g., 10 min), the DNA-crRNA complex gradually unwinds, releasing crRNA. The DNA strand itself forms a stem-loop structure, ensuring that it does not form a complex again after unwinding.

[0011] The gold nanoparticles modified with thiol DNA on the detection tube cap include Au NPs-DNA1, Au NPs-DNA2, and a saturated sodium chloride solution; wherein Au NPs-DNA1 and Au NPs-DNA2 are formed by modifying the gold nanoparticles with two different thiol DNA sequences, wherein the two thiol DNA sequences are complementary to the Linker-ssDNA in the mixed reaction system;

[0012] The mixture of thiol-DNA-modified gold nanoparticles on the tube cap is disposed on the lower surface of the tube cap, allowing it to be added to the mixed reaction system while the detection tube is closed.

[0013] As a preferred embodiment of the present invention, the volume of the in-tube mixing reaction system is equal to that of the mixture of gold nanoparticles modified with thiol DNA;

[0014] The total volume of the in-tube mixing reaction system was 10 μL, the RPA amplification system was 2.5 μL, and the CRISPR / Cas12a reaction system after DNA protection modification was 7.5 μL. The concentrations of each component were as follows: In the 2.5 μL RPA amplification system, primers RPA-F and RPA-R were 4.8 μM, magnesium acetate was 14 mM, primer-free-R buffer was 1.475 μL, DNA template was 0.1 μL, and RNAase-free water was 0.56 μL; In the 7.5 μL CRISPR / Cas12a reaction system, Cas12a was 250 nM, DNA-crRNA complex was 75 nM, and linker-ssDNA was 1 μM; the volume of buffer C added was 1 μL.

[0015] The buffer C is composed of the following components: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM MTT; the pH of buffer C is 7.9.

[0016] As a preferred embodiment of the present invention, the total volume of the gold nanoparticle mixture modified with thiol DNA is 10 μL, wherein the concentration of Au NPs-DNA1 is 2.7 μM and the concentration of Au NPs-DNA2 is 2.7 μM.

[0017] As a preferred embodiment of the present invention, the sequence of RPA-F is shown in SEQ ID No. 1, and the sequence of RPA-R is shown in SEQ ID No. 2.

[0018] As a preferred embodiment of the present invention, the sequence of the crRNA is shown in SEQ ID No. 4.

[0019] As a preferred embodiment of the present invention, the sequence of the Linker-ssDNA is shown in SEQ ID No. 5.

[0020] As a preferred embodiment of the present invention, the sequence of NPs-DNA1 is shown in SEQ ID No. 6; the sequence of NPs-DNA2 is shown in SEQ ID No. 7.

[0021] The sequences designed in this invention correspond as follows:

[0022]

[0023] The present invention also provides an application of the above-mentioned kit in the detection of Escherichia coli O157:H7.

[0024] This invention also provides a method for closed-tube visualization detection of Escherichia coli O157:H7 using the above-mentioned kit, which includes the following steps:

[0025] 1) Add the sample to be tested into the mixed reaction system in the tube, and perform RPA amplification of the specific gene fragment in the genome of Escherichia coli O157:H7 using the amplification primers in the reaction system at 37℃;

[0026] Simultaneously, the DNA-crRNA complex in the reaction system unwinds at 37°C and slowly releases crRNA to bind to the target DNA on the specific gene fragment; under the mediation of crRNA, Cas12a specifically recognizes the amplified fragment of the specific gene fragment and activates nuclease activity to cleave the Linker-ssDNA that complements the two thiol DNAs in the reaction system.

[0027] 2) After the reaction in step 1) is complete, add the mixture of gold nanoparticles modified with thiol DNA to the system in the tube and observe the color change of the system with the naked eye. When the sample contains E. coli O157:H7, the gold nanoparticles do not aggregate after Linker-ssDNA is cleaved, and the system is red. When the sample does not contain E. coli O157:H7, the gold nanoparticles aggregate, and the red color of the system fades. This achieves closed-tube detection of E. coli O157:H7.

[0028] As a preferred embodiment of the present invention, in step 2), after adding the mixture of gold nanoparticles modified with thiol DNA, the mixture is reacted for 5 minutes, then centrifuged, and the color change is observed.

[0029] Compared with existing technologies, the method of this invention has advantages such as high sensitivity, strong specificity, rapid and efficient operation, convenient operation, user-friendliness, and low cost, and does not require any detection or observation instruments. This invention utilizes a DNA strand protection design. The first 16 bases of the protected DNA strand are designed to pair complementaryly with the 16 bases of the crRNA-specific sequence. Under specific conditions, this complementary pairing forms a DNA-crRNA complex, thereby inhibiting the CRISPR system reaction. The last 8 bases of the protected DNA strand are designed to pair complementaryly with the first 8 bases at the 5' end after unwinding, forming a stem-loop structure and preventing the DNA strand from rebinding to crRNA. The presence of the DNA-crRNA complex ensures that the RPA system reacts completely independently in the overall system. After mixing the RPA and CRISPR systems and placing them at the bottom of a tube, with gold nanoparticles on the tube cap, the system is reacted at 37°C for 10 minutes. During this time, the DNA-crRNA complex gradually unwinds, releasing crRNA, and the DNA strand forms a stem-loop structure, preventing further complementary formation of the complex with crRNA after unwinding. After reacting the system at 37°C for 40 minutes, the Au-DNA1 / Au-DNA2 from the tube cap is dropped and mixed with the RPA-CRISPR system. The color reaction change is observed, thus forming a closed-tube detection system to prevent cross-contamination. This invention provides a novel point-of-care detection platform for Escherichia coli O157:H7 with broad application value. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the working principle of the present invention.

[0031] Figure 2 The figure shows the results of the closed-tube detection sensitivity experiment of Escherichia coli O157:H7 according to the present invention.

[0032] Figure 3 This is a graph showing the results of the sensitivity detection of Escherichia coli O157:H7 in food samples using closed-tube methods according to the present invention.

[0033] Figure 4 This is a colorimetric result diagram of the study on the concentration of Linker-ssDNA in the system according to the present invention.

[0034] Figure 5 This is a diagram showing the results of the detection specificity experiment of Escherichia coli O157:H7 in this invention. Detailed Implementation

[0035] The present invention will be further described and illustrated below with reference to specific embodiments. The embodiments described are merely examples of the content of this disclosure and do not limit the scope of the invention. The technical features of each embodiment in the present invention can be combined accordingly, provided that there is no mutual conflict.

[0036] I. Visual Detection of Escherichia coli O157:H7 using the CRISPR / Cas12a System

[0037] Reference Figure 1 The procedure involved pretreating the actual samples using thermal lysis. Under aseptic conditions, 1 mL of the homogenized sample was transferred to a sterile centrifuge tube and centrifuged at 8000×g for 2 min, discarding the supernatant. The sample was resuspended in 200 μL of sterile deionized water, centrifuged at 8000×g for 2 min, and the supernatant was discarded. 100 μL of sterile deionized water was added, and the sample was boiled at 100℃ for 10 min and then cooled to serve as an RPA template for amplification. A protective DNA strand was designed, and the optimal fragment length of the protective DNA strand (i.e., SEQ ID No. 3) was selected by PAGE gel electrophoresis. The DNA strand was reacted with crRNA (i.e., SEQ ID No. 4) at 85℃ for 5 min, and the temperature was slowly reduced to 4℃ at a rate of 0.1℃ / s to obtain the DNA-crRNA complex. The 2.5 μL RPA amplification system was constructed as follows: Primer RPA-F (SEQ ID No. 1) and primer RPA-R (SEQ ID No. 2) were at 4.8 μM each; magnesium acetate was at 14 mM; primer-free-R buffer was at 1.475 μL; DNA template was at 0.1 μL; and RNAase-free water was at 0.56 μL. A 7.5 μL CRISPR / Cas12a reaction system was constructed, including 250 nM Cas12a, 75 nM DNA-crRNA, 1 μM Linker-ssDNA (SEQ ID No. 4), 2.5 μL amplification product, and 1 μL buffer C (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH...). 7.9) Add RNase-free water to a final volume of 10 μL, and thoroughly mix the RPA and CRISPR systems at the bottom of the tube. Place 10 μL of Au-DNA1 / Au-DNA2 and an appropriate amount of saturated sodium chloride on the tube cap (Au NPs-DNA1 concentration is 2.7 μM, Au NPs-DNA2 concentration is 2.7 μM). Add the sample to be tested to the mixed reaction system in the tube and incubate at 37 °C for 40 min. After 40 min, shake off the gold nanoparticle mixture from the tube cap and mix it with the reaction system, then observe the colorimetric results.

[0038] II. Sensitivity Characterization of the Method of the Invention for Detecting Escherichia coli O157:H7

[0039] Genomic DNA of foodborne pathogens was extracted using a bacterial genomic DNA extraction kit. To investigate the detection sensitivity of this invention for *E. coli* O157:H7 in DNA of different concentrations, DNA samples with different concentrations of *E. coli* O157:H7 (69.7 pg / μL, 6.97 pg / μL, 697 fg / μL, 69.7 fg / μL, 6.97 fg / μL, 697 ag / μL, 69.7 ag / μL, and 6.97 ag / μL) were added to a mixed system containing RPA and CRISPR. The specific gene fragment system was analyzed using the closed-tube visualization detection method of the CRISPR / Cas12a system of this invention. Figure 2 In Figure A, the effect of different concentrations of E. coli O157:H7 on the colorimetric changes of the samples was demonstrated. E. coli O157:H7 as low as 6.97 ag / μL could be directly identified by naked-eye observation. Figure 2 Part B demonstrates the use of a UV spectrophotometer and microplate reader to detect different DNA concentrations of *E. coli* O157:H7, plotting UV absorption peaks and error bar charts. *E. coli* O157:H7 with different concentrations (69.7 pg / μL, 6.97 pg / μL, 697 fg / μL, 69.7 fg / μL, 6.97 fg / μL, 697 ag / μL, 69.7 ag / μL, and 6.97 ag / μL) were subjected to RPA isothermal amplification and analyzed using the CRISPR / Cas12a visualization detection system. Visual observation showed that *E. coli* O157:H7 concentrations as low as 6.97 ag / μL were detected. Therefore, the detection limit of the method of this invention is 6.97 ag / μL.

[0040] This invention further investigated the concentration of Linker-ssDNA in the system, and the colorimetric results are as follows: Figure 4 As shown, Figure 4 The results showed that the colorimetric effect was best when the Linker-ssDNA concentration was 1 μM.

[0041] III. The method of this invention for detecting Escherichia coli O157:H7 in various food samples

[0042] The sensitivity of Escherichia coli O157:H7 in milk with different bacterial concentrations was detected using the closed-tube visualization detection method of the CRISPR / Cas12a system. Figure 3 Image A shows the effect of milk samples with different levels of E. coli O157:H7 on colorimetric assays using gold nanoparticle probes. The results can be directly observed with the naked eye down to the limit of detection (1×10⁻⁶). 0CFU / mL. Figure 3 Figure B demonstrates the sensitivity detection of different bacterial concentrations of *E. coli* O157:H7 in milk using a UV spectrophotometer and ELISA reader, plotting UV absorption peaks and error bar charts. This invention uses concentrations of 1×10⁻⁶... 6 CFU / mL, 1×10 5 CFU / mL, 1×10 4 CFU / mL, 1×10 3 CFU / mL, 1×10 2 CFU / mL, 1×10 1 CFU / mL, 1×10 0 CFU / mL of E. coli O157:H7 was added to milk samples for pretreatment and followed by RPA isothermal amplification. Analysis was performed using a closed-tube visualization detection system based on the CRISPR / Cas12a framework. Colorimetric results observed with the naked eye showed identification as low as 1×10⁻⁶. 0 The relationship between the content of E. coli O157:H7 (CFU / mL) in milk samples and colorimetric changes was investigated using a UV spectrophotometer.

[0043] IV. Characterization of the Detection Specificity of the Method of the Invention

[0044] To characterize the specificity of this CRISPR / Cas12a system-based visual detection method for detecting *Escherichia coli* O157:H7, clinical isolates of *E. coli* O157:H7, *Salmonella*, *Staphylococcus aureus*, *Listeria monocytogenes*, and *Vibrio parahaemolyticus* were used as samples for testing. Colorimetric results showed that... Figure 5 As shown, the clinical isolate of Escherichia coli O157:H7 appears purple-red; the specific system for Listeria monocytogenes (LM), Salmonella, Vibrio parahaemolyticus (VP), and Staphylococcus aureus (SA) appears colorless, and the gold nanoparticles aggregate; the above indicates that the CRISPR / Cas12a system closed-tube visualization detection method has good accuracy and specificity.

[0045] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A closed-tube visual detection method for Escherichia coli E. coli The O157:H7 kit is characterized by... This includes a mixed reaction system located inside the detection tube and a mixture of thiol-DNA-modified gold nanoparticles located on the cap of the detection tube; The mixed reaction system in the detection tube includes a mixed RPA amplification system and a CRISPR / Cas12a reaction system with protective DNA modification. The RPA amplification system is used for Escherichia coli. E. coli RPA amplification of a specific gene fragment in the O157: H7 genome includes primer RPA-F, primer RPA-R, Primer-Free-R buffer, DNA template, RNAase-free water, and magnesium acetate; the sequence of RPA-F is shown in SEQ ID No. 1, and the sequence of RPA-R is shown in SEQ ID No. 2; The CRISPR / Cas12a reaction system comprises: Cas12a, DNA-crRNA complex, Linker-ssDNA, buffer C, and ddH2O; the buffer C is composed of the following components: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM MTT; the pH of the buffer C is 7.9; the sequence of the crRNA is shown in SEQ ID No. 4; the sequence of the Linker-ssDNA is shown in SEQ ID No.

5. The DNA-crRNA complex is formed by the pairing of a protective DNA strand with crRNA, and can unwind at 37 °C to slowly release crRNA, which can bind to E. coli. E. coli The protective DNA strand binds to a specific gene fragment in the O157:H7 genome; the sequence of the protective DNA strand is shown in SEQ ID No. 3, and the protective DNA is designed with a stem-loop structure. The first 16 bases of the 3' end of the protective DNA pair complementaryly with the 16 bases of the crRNA specific sequence. After unwinding, the last 8 bases of the protective DNA can complement the first 8 bases of the protective DNA strand to form a stem-loop structure. When the system reacts at 37 °C for a certain time, the DNA-crRNA complex will gradually unwind and release crRNA. The protective DNA strand itself forms a stem-loop structure and ensures that it does not complement crRNA again to form a complex after unwinding. The gold nanoparticles modified with thiol DNA on the detection tube cap comprise Au NPs-DNA1, Au NPs-DNA2, and a saturated sodium chloride solution; wherein Au NPs-DNA1 and Au NPs-DNA2 are formed by modifying the gold nanoparticles with two different thiol DNA sequences, wherein the two thiol DNA sequences are complementary to the Linker-ssDNA in the mixed reaction system; the sequence of Au NPs-DNA1 is shown in SEQ ID No. 6; the sequence of Au NPs-DNA2 is shown in SEQ ID No. 7; The mixture of thiol-DNA-modified gold nanoparticles on the tube cap is disposed on the lower surface of the tube cap, allowing it to be added to the mixed reaction system while the detection tube is closed.

2. The reagent kit according to claim 1, characterized in that, The volume of the in-tube mixing reaction system is equal to that of the thiol DNA-modified gold nanoparticle mixture. The total volume of the in-tube mixing reaction system was 10 μL, the RPA amplification system was 2.5 μL, and the CRISPR / Cas12a reaction system after DNA protection modification was 7.5 μL. The concentrations of each component were as follows: In the 2.5 μL RPA amplification system, primers RPA-F and RPA-R were 4.8 μM, magnesium acetate was 14 mM, primer-free-R buffer was 1.475 μL, DNA template was 0.1 μL, and RNAase-free water was 0.56 μL; In the 7.5 μL CRISPR / Cas12a reaction system, Cas12a was 250 nM, DNA-crRNA complex was 75 nM, and linker-ssDNA was 1 μM; the volume of buffer C added was 1 μL.

3. The reagent kit according to claim 1, characterized in that, The total volume of the gold nanoparticle mixture modified with thiol DNA was 10 μL, wherein the concentration of Au NPs-DNA1 was 2.7 μM and the concentration of Au NPs-DNA2 was 2.7 μM.

4. The kit according to any one of claims 1-3 for detecting Escherichia coli in food samples E. coli O157: Applications in H7.

5. A closed-tube visual detection method for Escherichia coli in food samples based on the kit described in any one of claims 1-3. E. coli The method of O157: H7 is characterized by... Includes the following steps: 1) Add the sample to be tested to the mixed reaction system in the tube. The amplification primers in the reaction system are for E. coli. E. coli O157: A specific gene fragment in the H7 genome was amplified by RPA at 37 ℃; Simultaneously, the DNA-crRNA complex in the reaction system unwinds at 37°C and slowly releases crRNA to bind to the target DNA on the specific gene fragment; under the mediation of crRNA, Cas12a specifically recognizes the amplified fragment of the specific gene fragment and activates nuclease activity to cleave the Linker-ssDNA that complements the two thiol DNAs in the reaction system. 2) After the reaction in step 1) is complete, add the mixture of gold nanoparticles modified with thiol DNA to the system in the tube, and observe the color change of the system with the naked eye; among them, when the sample contains E. coli E. coli At O157: H7, after Linker-ssDNA cleavage, the gold nanoparticles do not aggregate, and the system color is red; when there is no E. coli in the sample. E. coli At O157: H7, gold nanoparticles aggregate, and the system loses its red color; thus achieving the effect of targeting Escherichia coli. E. coli O157: H7 closed tube detection.

6. The method according to claim 5, characterized in that, In step 2), after adding the mixture of gold nanoparticles modified with thiol DNA, the mixture is reacted for 5 minutes, then centrifuged, and the color change is observed.

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