CN112159854B - Primer composition for detecting CRISPR/Cas12a of escherichia coli O157-H7 and detection method - Google Patents

Primer composition for detecting CRISPR/Cas12a of escherichia coli O157-H7 and detection method Download PDF

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CN112159854B
CN112159854B CN202011173972.2A CN202011173972A CN112159854B CN 112159854 B CN112159854 B CN 112159854B CN 202011173972 A CN202011173972 A CN 202011173972A CN 112159854 B CN112159854 B CN 112159854B
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王淑娟
杨美成
范一灵
秦峰
冯震
蒋波
宋明辉
李琼琼
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SHANGHAI INSTITUTE FOR FOOD AND DRUG CONTROL
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Abstract

The invention provides a primer composition for detecting CRISPR/Cas12a of escherichia coli O157: H7 and a detection method. The primer composition comprises a MIRA amplification primer and crRNA; the MIRA amplification primer comprises a primer selected from the nucleotide sequences of SEQ ID No. 1-2 and a primer selected from the nucleotide sequences of SEQ ID No. 3-5; the nucleotide sequence of crRNA is SEQ ID No.6. The detection method of the escherichia coli O157:H27 by adopting the primer composition has the advantages of high speed, high sensitivity, strong specificity, simple operation and easy observation of reaction results, is suitable for on-site rapid detection, and can provide rapid screening results for supervision and inspection and emergency treatment of products on the market by a supervision department. The invention designs and screens the MIRA amplification primer of the escherichia coli O157H 7 for the first time, optimizes the detection system of CRISPR/Cas12a and combines Fe 3 O 4 @PDA@UiO‑66‑NH 2 The enrichment function of the bacillus coli O157:H2 7 is established, and the blank of the detection field at home and abroad is filled.

Description

Primer composition for detecting CRISPR/Cas12a of escherichia coli O157-H7 and detection method
Technical Field
The invention relates to the technical field of biology, in particular to a primer composition for detecting CRISPR/Cas12a of escherichia coli O157H 7 and a detection method.
Background
Coli O157: H7 (E.coli O157: H7), a gram negative bacterium, is one of the zoonotic pathogens causing human intestinal diseases and even death. It is transmitted to humans mainly by eating contaminated meat or meat products, in particular beef products [1] . Although the traditional national standard-based culture method is a gold standard, the traditional national standard-based culture method is time-consuming and labor-consuming, depends on experienced operators, is easy to generate missed detection, and is difficult to detect in food matrixes with high background flora, such as raw beef. Enzyme-linked immunosorbent assay (ELISA) is highly dependent on the specificity of the antibody. Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) has been widely used in clinical microbiological laboratories in recent years. However, the detection object of the method is a pure colony, and the concentration is required to reach 10 5-7 CFU [2,3] . Therefore, MALDI-TOF MS cannot meet the requirements of rapid detection in terms of sensitivity, specificity and the like.
Detection methods based on nucleic acid, such as Polymerase Chain Reaction (PCR), real-time PCR (RT-PCR) and the like, have been widely applied to detection of pathogens in foods, and have the characteristics of high sensitivity, strong specificity, low detection limit and the like. Rube Gordillo et al established a fliC-based h7 And rfbE gene detection of E.coli O157: H7 in raw meat and ready-to-eat meat products using multiplex Polymerase Chain Reaction (PCR) method [4] . The method uses a pair of amplification primers to detect a target nucleic acid sequence in a sample with or without using a fluorescent dye (or fluorescent probe). However, the nucleic acid amplification steps used in the method need frequent heating and cooling processes, take at least more than 1 hour, and use large auxiliary equipment,the result judgment is carried out with the aid of an electrophoresis apparatus, a DNA amplification apparatus or a sequencer, and the like, so that the method cannot meet the requirements of simple experimental conditions or on-site real-time detection.
Clustered regularly interspaced short palindromic repeats (Clustered regularly interspaced short palindromic repeats, CRISPR) technology has shown great application prospects in clinical diagnostics, drawing great attention. RNA-based CRISPR/Cas nucleic acid detection has been rapidly developed, wherein the trans-cleavage activity of Cas12a and the first application of Cas12a in nucleic acid detection have been patented in China in 2017. Taking the detection principle of Cas12 system as an example: after the Cas12, crRNA (or sgRNA) forms a ternary complex with the target nucleic acid, the side branch ssDNA reporter is trans-cleaved into small fragments. The amount of target nucleic acid is reflected by the amount of reporter. In combination with the recombinase-polymerase amplification (Recombinase Polymerase Amplification, RPA) technique, a detection system for the detection of Cas12a (DNA Endonuclease-Targeted CRISPR Trans Reporter) was developed [5] And SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) detection system based on Cas13 [6] . In addition to RPA pre-amplification, LAMP and PCR methods are also applied to pre-amplification of CRISPR systems. The principle of the multi-enzyme isothermal nucleic acid rapid amplification technology (Multienzyme Isothermal Rapid Amplification, MIRA) is the same as that of the RPA technology, and the technology is also a technology for realizing nucleic acid finger amplification under isothermal conditions. In recent years, the CRISPR technology has been gradually applied to detection of pathogenic microorganisms, such as the preparation method of a Mycobacterium tuberculosis complex detection kit based on CRISPR-Cas12a system in the patent with the application number of 201910735947. X.
Due to the complexity of the actual sample, separating and enriching the target from the sample background is particularly important for improving the sensitivity and accuracy of detection. In recent years, magnetic separation techniques have been widely used for the separation of specific targets such as bacteria, viruses, proteins, toxic chemicals, and cells. Magnetic metal-organic frameworks (MMOFs) are novel functional nano materials which are raised in recent years, and have certain advantages in separation and enrichment [7] Such as CN109759142A, a method for preparing a magnetic composite metal-organic framework material.
However, it is not yet seen that MMOFs binds to CRISPR/Cas12a and is used for detection of E.coli O157:H2 7, and more suitable and efficient primers are not used for CRISPR/Cas12a detection in the field.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a primer composition for CRISPR/Cas12a detection of escherichia coli O157H 7 with high speed, high sensitivity and strong specificity and a detection method for efficiently separating and enriching by combining immune MMOFs.
The invention adopts the following technical scheme to solve the technical problems:
the first aspect of the invention provides a primer composition for detecting CRISPR/Cas12a of escherichia coli O157:H27, comprising a MIRA amplifying primer and crRNA;
wherein, the MIRA amplifying primer comprises a primer selected from the nucleotide sequences of SEQ ID No. 1-2 and a primer selected from the nucleotide sequences of SEQ ID No. 3-5;
the nucleotide sequence of crRNA is SEQ ID No.6.
Further, the nucleotide sequences of the MIRA amplifying primer are SEQ ID No.1 and SEQ ID No. 5.
Further, the primer composition further comprises a probe; the sequence of the probe is 5'-6-FAM-TTATT-BHQ1-3'.
In a second aspect, the present invention provides a kit for detecting Escherichia coli O157:H27, which comprises the above primer composition.
Further, the kit further comprises Fe 3 O 4 @PDA@UiO-66-NH 2 A metal organic framework material.
In a third aspect, the invention provides a method for detecting escherichia coli O157: H7 using the above-described kit, which uses the above-described primer composition, the product of the MIRA amplification reaction, as a target for CRISPR/Cas12a detection.
Further, the conditions of the MIRA amplification reaction are 37-39 ℃ and the temperature is kept constant for 30min.
Further preferably, the conditions for the above MIRA amplification reaction are 37℃and the temperature is kept constant for 30min.
Further, the reaction system of the CRISPR/Cas12a is as follows: 0.6. Mu.L of crRNA (1. Mu.M), 0.8. Mu.L of Cas12a (1. Mu.M), 1.2. Mu.L of probe (10. Mu.M), 1. Mu.L of MIRA amplification product, 16.4. Mu.L of 1 XNEBuffer 2.1; the reaction condition is 37 ℃ and the temperature is kept for 30min.
Further, the above detection method further comprises, prior to amplifying the nucleic acid, using Fe 3 O 4 @PDA@UiO-66-NH 2 The immune metal organic framework material is used for enriching escherichia coli O157 to H7 in a sample to be detected.
Further, the Fe 3 O 4 @PDA@UiO-66-NH 2 The preparation method of the immune metal organic framework material comprises the following steps:
dissolving ferric trichloride, sodium acetate and anhydrous sodium citrate in ethylene glycol, performing ultrasonic treatment until the ferric trichloride, the sodium acetate and the anhydrous sodium citrate are completely dissolved, transferring the mixture into a polytetrafluoroethylene lining of a stainless steel reaction kettle, and sealing and heating the mixture; then cooling to room temperature, collecting the precipitate with magnet, washing, and drying to obtain Fe 3 O 4 A nanoparticle;
step two, the Fe is treated 3 O 4 Dispersing the nano particles in ethanol, and adding a Tris-HCl solution with pH of 8.5; after complete dissolution, adding dopamine hydrochloride solution, and continuously stirring for 15-25 hours at room temperature; collecting the precipitate with magnet, washing, and drying to obtain Fe 3 O 4 PDA nanoparticles;
step three, the Fe is treated 3 O 4 Dispersing the @ PDA nano particles in DMF (dimethyl formamide) solution containing 2-amino terephthalic acid and zirconium tetrachloride, mixing the solution, performing ultrasonic treatment, transferring into a polytetrafluoroethylene lining of a stainless steel reaction kettle, and sealing and heating; collecting the precipitate with magnet, washing, and drying to obtain Fe 3 O 4 @PDA@UiO-66-NH 2 A nanoparticle;
step four, the Fe is treated 3 O 4 @PDA@UiO-66-NH 2 The nanoparticles were activated with glutaraldehyde at a final concentration of 1.25%, after activationWashing off residual glutaraldehyde, adding Escherichia coli O157:H27 monoclonal antibody, binding, blocking with 1% BSA to obtain Fe 3 O 4 @PDA@UiO-66-NH 2 Immune nanoparticles.
Compared with the prior art, the invention has the following technical effects:
(1) The invention detects the Escherichia coli O157:H27 in the sample through the special fluorescent color change of the CRISPR/Cas12a technology.
(2) The method is not limited by detection conditions, and can finish the detection of nucleic acid within 60 minutes.
(3) The invention has the advantage that the qualitative detection sensitivity of the invention to the escherichia coli O157-H7 can reach 0.9 pg.mu.L -1 (DNA concentration) or 6.5X10 4 CFU·mL -1 (bacterial concentration) is superior to the general RT-PCR detection method.
(4) The method is simple to operate, the reaction result is easy to observe, the specificity is good, the method is suitable for on-site rapid detection, and rapid screening results can be provided for supervision and inspection and emergency treatment of products on the market by a supervision department.
(5) The invention designs and screens the MIRA amplification primer of the escherichia coli O157H 7 for the first time, optimizes the detection system of CRISPR/Cas12a and combines Fe 3 O 4 @PDA@UiO-66-NH 2 The enrichment function of the immune nano particles establishes a CRISPR/Cas12a detection method of escherichia coli O157: H7, and fills the gap in the detection field at home and abroad.
Drawings
FIG. 1 shows the results of gel electrophoresis (FIG. 1A), CRISPR/Cas12a reaction (FIG. 1B) of 6 pairs of E.coli O157H 7 MIRA amplification primers according to one embodiment of the present invention, and the results of optimization of CRISPR/Cas12a reaction of two pairs of primers SEQ ID No.1 and SEQ ID No.5 and SEQ ID No.2 and SEQ ID No.5 (FIG. 1C); in fig. 1A and 1B, a: coli (ATCC 25922); b: coli O157: H7 (CICC 21530); c: coli O111; d: pseudomonas aeruginosa; 1-6 represent 6 pairs of primers (SEQ ID No.1 and SEQ ID No.3, SEQ ID No.1 and SEQ ID No.4, SEQ ID No.1 and SEQ ID No.5, SEQ ID No.2 and SEQ ID No.3, SEQ ID No.2 and SEQ ID No.4, and SEQ ID No.2 and SEQ ID No. 5), respectively; in FIG. 1C, 1, 2, 3, 4 represent the volumes of probe (1. Mu.M) of 0.4, 0.8, 1.2, 2.0. Mu.L, "+" and "-" represent the DNA of E.coli O157:H27 as the amplification template and sterile water, respectively, and a and b represent the primer pair of SEQ ID No.1 and SEQ ID No.5, and the primer pair of SEQ ID No.2 and SEQ ID No.5, respectively;
FIG. 2 is an optimized view of a CRISPR/Cas12a reaction system of E.coli O157: H7 in an embodiment of the invention; wherein a: cas12a (Cpf 1); b: crRNA; c: probes (1-8 represent 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0. Mu.L, respectively);
FIG. 3 is a graph showing the detection sensitivity of the DNA concentration of the CRISPR/Cas12a reaction system of E.coli O157: H7 in accordance with one embodiment of the present invention; wherein a: MIRA gel electrophoresis results; b: RT-PCR; c: CRISPR/cas12a;
FIG. 4 is a graph showing the detection sensitivity of the bacterial concentration of the CRISPR/Cas12a reaction system of E.coli O157: H7 in accordance with one embodiment of the present invention; wherein a: MIRA gel electrophoresis results; b: RT-PCR; c: CRISPR/cas12a;
FIG. 5 is a graph of detection specificity results of the CRISPR/Cas12a system of E.coli O157: H7 in an embodiment of the invention; wherein, the strains denoted by numbers 1-21 are shown in Table 2;
FIG. 6 is a graph of the detection results of the CRISPR/Cas12a system of E.coli O157: H7 in a crushed beef sample according to one embodiment of the invention; wherein M represents Fe 3 O 4 @PDA@UiO-66-NH 2 Immune nanoparticles.
Detailed Description
The invention provides a primer composition for detecting CRISPR/Cas12a of escherichia coli O157:H27, which comprises a MIRA amplification primer and crRNA;
wherein, the MIRA amplifying primer comprises a primer selected from the nucleotide sequences of SEQ ID No. 1-2 and a primer selected from the nucleotide sequences of SEQ ID No. 3-5; the nucleotide sequence of crRNA is SEQ ID No.6.
In addition, the primer composition further comprises a probe; the sequence of the probe is 5'-6-FAM-TTATT-BHQ1-3'.
In another aspect, the invention provides a kit for detecting Escherichia coli O157:H27, which comprises the primer composition.
In a preferred embodiment of the present invention, the kit further comprises Fe 3 O 4 @PDA@UiO-66-NH 2 Immune metal organic framework material.
In a preferred embodiment of the present invention, the above Fe 3 O 4 @PDA@UiO-66-NH 2 The preparation method of the immune metal organic framework material comprises the following steps:
(1) 2.43g of ferric trichloride, 6g of sodium acetate and 0.877g of anhydrous sodium citrate were dissolved in 200mL of ethylene glycol. Ultrasonic treatment for 30min, complete dissolution, transferring into polytetrafluoroethylene lining of stainless steel reaction kettle, sealing at 200deg.C, and heating for 10 hr. Cooling to room temperature, collecting the precipitate with magnet, alternately washing with anhydrous ethanol and water for three times, and drying at 80deg.C for 6 hr to obtain Fe 3 O 4 And (3) nanoparticles.
(2) 1g of Fe 3 O 4 Dispersing the nano particles in 200mL of ethanol, adding 200mL of 10mmol.L -1 Tris-HCl (pH 8.5) solution. After complete dissolution, 1g of dopamine hydrochloride (dissolved in 200ml of distilled water) was added and stirring was continued at room temperature for 20 hours. Collecting the precipitate with magnet, alternately washing with anhydrous ethanol and water for three times, and drying at 80deg.C for 6 hr to obtain Fe 3 O 4 PDA nanoparticle.
(3) 0.1g of Fe 3 O 4 The @ PDA nanoparticles were dispersed in 60mL DMF containing 11.04 mmol/L2-amino terephthalic acid and 11.44mmol/L zirconium tetrachloride. The solution was mixed and sonicated for 5min, then transferred to a teflon liner of a stainless steel reactor and heated in a sealed manner at 120 ℃ for 1h. Collecting the precipitate with magnet, alternately washing with anhydrous ethanol and water for three times, and drying at 80deg.C for 6 hr to obtain Fe 3 O 4 @PDA@UiO-66-NH 2 A nanoparticle;
(4)0.05g Fe 3 O 4 @PDA@UiO-66-NH 2 nanoparticles were dispersed in 10mL of 0.01 mol.L glutaraldehyde with a final concentration of 1.25% -1 Shaking in phosphate buffer (pH 7.4) at room temperature for 2 hr, washing with sterile water to remove excessive glutaraldehyde, and re-suspendingIn 10mL 0.01 mol.L -1 To phosphate buffer (pH 7.4), 4mg of an anti-E.coli O157: H7 monoclonal antibody was added and mixed at 4℃for 24 hours. The product was collected using a magnet to remove unbound antibody. Then, in 0.01mol.L containing 1% BSA -1 The mixture was blocked in phosphate buffer (pH 7.4) at 4℃for 2h. After three washes with sterile water, the product was resuspended in 10mL of 0.01 mol.L -1 In phosphate buffer, stored at 4℃for use in the enrichment of E.coli O157: H7.
The present invention will be described in detail and specifically by way of the following specific examples and drawings to provide a better understanding of the present invention, but the following examples do not limit the scope of the present invention.
The methods described in the examples are carried out using conventional methods, if not specified, and the reagents used are, if not specified, conventional commercially available reagents or reagents formulated by conventional methods. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described can be used in the methods of the present invention.
Example 1
The embodiment provides a detection method of escherichia coli O157:H27 based on CRISPR/Cas12a, which comprises the following steps:
1. preparation of primers: the rfbE gene sequence of E.coli O157: H7 obtained from GenBank (Genebank No. S83460.1); MIRA primer was designed with primer 5.0; the primer length is 29-34 bp. The primer sequences used were synthesized by the company Shanghai, inc. of Biotechnology. 5 MIRA primers (comprising two sense strands (F1 and F2) and three antisense strands (R1, R2 and R3)) and 1 crRNA sequence were designed for the target sequences, the nucleotide sequences of which are listed in Table 1. Among them, the amplification and gel electrophoresis were performed using these 6 MIRA amplification primers, and the amplification results are shown in FIG. 1.
TABLE 1 primer information for CRISPR/Cas12a technology
2. By Fe 3 O 4 @PDA@UiO-66-NH 2 The immune metal organic framework material enriches escherichia coli O157 to H7 in a sample to be detected;
3. extracting DNA in the sample to be detected, and carrying out MIRA amplification reaction. Wherein, the MIRA amplification system adopts a DNA constant temperature rapid amplification KIT (basal type) KIT (product number: WLN8203 KIT) of Weifang Anpu future biotechnology Co., ltd; the specific configuration of the MIRA amplification system was 2. Mu.L for each of F1 and R3 at 10. Mu.M; 29.4. Mu.L of buffer A; 2. Mu.L of DNA template; 12.1 μl of double distilled water; 2.5. Mu.L of buffer B; the MIRA amplification reaction conditions were: constant temperature of 37 ℃ for 30min.
4. Probes, cas12a protein, crRNA and MIRA amplification products, and detection reagents were added to the reaction tube, and CRISPR reaction (reaction conditions were constant at 37 ℃ for 30 min) was performed on a fluorescent quantitative PCR instrument, with fluorescent values measured every 30 s. Wherein, the inventors optimize the CRISPR/Cas12a reaction system, as shown in fig. 2, and verified that the CRISPR/Cas12a detection system is specifically configured to be 0.8 μl Cas12a (1 μΜ), 0.6 μl crRNA (1 μΜ), 1.2 μl probe (10 μΜ), 1 μl MIRA product, 16.4 μl1×nebuffer 2.1 after optimization.
The inventors used the above steps 2 to 4 to amplify E.coli (ATCC 25922), E.coli O157:H7 (CICC 21530), E.coli O111 and P.aeruginosa respectively using the above 6 pairs of primers, and then detected by 2.0% agarose gel electrophoresis, and the results are shown in FIG. 1A. Then, CRISPR reaction is carried out, and the obtained detection result is shown in FIG. 1B. Screening out two pairs of primers SEQ ID No.1 and SEQ ID No.5 and SEQ ID No.2 and SEQ ID No.5 according to the results of FIGS. 1A and 1B further gave a CRISPR/Cas12a reaction, the results of which are shown in FIG. 1C. As can be seen from FIG. 1C, the two pairs of primers are found in the optimization process of the CRISPR/Cas12a system, the specificity of SEQ ID No.1 and SEQ ID No.5 is better, and the false positive rate is lower.
Verification example 1
The specificity and sensitivity of the detection method provided in example 1 were measured in this example, and the specific operations and results were as follows:
specificity test
The detection was performed on 4 E.coli strains O157H 7 and 17 other bacterial strains (see Table 2). The CRISPR/Cas12a detection results were determined as follows.
The positive reaction was judged as: real-time fluorescence detection is carried out by adopting a real-time quantitative PCR instrument Light Cycler 480II of Roche diagnostics company; or E-Gel SafeImager by Invitrogen corporation of America TM And a real-time transilluminator for visually observing whether the reaction tube has green fluorescence. And (3) taking sterilized purified water as a negative control test, wherein the result of the negative control test is negative.
1) If the fluorescence value is more than 0 or fluorescence is visible to naked eyes, judging that the CRISPR/Cas12a test result is positive;
2) If the fluorescence value=0 or fluorescence is not visible to naked eyes, judging that the CRISPR/Cas12a test result is negative;
primers SEQ ID No.1 and SEQ ID No.5 showed positive reaction in all 4 E.coli O157:H2 7 strains, and the fluorescence value was greater than 0 by real-time fluorescence detection using real-time quantitative PCR instrument Light Cycler 480II from Roche diagnostics company, and E-Gel SafeImager from Invitrogen Co., USA TM The real-time transilluminator, visible to the naked eye, showed a negative reaction when no fluorescence value (or color change) was detected for the other non-E.coli O157: H7 species for 30min of the reaction (FIG. 5).
TABLE 2 test species and CRISPR/Cas12a detection results
Wherein: CICC refers to China center for type culture collection of Industrial microorganisms; CMCC refers to China center for medical microbiological culture collection center; ATCC refers to the American standard strain collection; ST refers to the food and drug inspection institute of China.
Sensitivity test
Inoculating Escherichia coli O157: H7 strain into mEC +n liquid culture medium, culturing for 48 hr, taking 1mL, preparing 10-time serial gradient dilution with sterile water solution, extracting DNA according to ultra-fast universal DNA nucleic acid releasing agent (product number: WLDR 8202) of Weifang Anpu future biotechnology limited company, and using as template for bacteria concentration sensitivity detection. Taking 1mL of mEC +n cultured bacterial liquid, extracting DNA according to an ultrafast universal DNA nucleic acid releasing agent of Fangan future biotechnology Co., ltd, measuring the concentration of the DNA by adopting a Ai Bende nucleic acid protein tester (Eppendorf Hellma TrayCell), and performing 10 times series gradient dilution on the DNA to be used as a template for detecting the concentration sensitivity of the DNA. The CRISPR/Cas12a test method of the invention is used for measuring the DNA sample. Meanwhile, 0.1mL of bacterial liquid of each dilution level is respectively coated on a TSA agar culture medium and cultured for 48 hours, and the colony number growing on the flat plate is counted.
When the DNA concentration is more than or equal to 0.9 pg.mu.L -1 When the reaction positive result was judged to be less than 15min (fluorescence > 0) (FIG. 3). When the bacterial concentration is more than or equal to 6.5X10 4 CFU·mL -1 When the reaction positive result was judged to be less than 20 min (fluorescence > 0) (FIG. 4).
Therefore, the detection sensitivity of the qualitative test of the CRISPR/Cas12a detection method adopted by the invention can reach 0.9 pg.mu.L -1 (DNA concentration) or 6.5X10 4 CFU·mL -1 (bacterial concentration).
Verification example 2
This example tested the effect of the CRISPR/Cas12a method provided in example 1 on the detection of real samples of crushed beef, 1 crushed beef product was selected on the market for detection. The specific detection process and result are as follows:
preparation of the samples: according to the method of national standard method GB 4789.36-2016 "food microbiology test Escherichia coli O157: H7/NM test", two 25g portions of crushed beef are weighed and inoculated into 225mL of mEC+n liquid culture medium, one portion of the crushed beef is inoculated with 14 CFU.mL -1 Coli O157H 7, and the other was not inoculated as an negative control. Culturing in a 37 ℃ incubator, taking out 1mL of culture solution at 1, 2, 3, 4 and 5 hours respectively, and extracting bacterial genome DNA by using an ultrafast universal DNA nucleic acid releasing agent (product number: WLDR 8202) of Weifang Anpu future biotechnology Co.
Use of Fe in example 1 3 O 4 @PDA@UiO-66-NH 2 Immune nanoparticle enrichmentThe collection and CRISPR/Cas12a method was tested.
The detection result shows that the CRISPR/Cas12a added with the escherichia coli O157: H7 has an amplification curve when detecting real-time fluorescence, the fluorescence value is more than 0, and the fluorescence is visible to naked eyes and is judged to be positive. The fluorescence value=0 in the sample to which Escherichia coli O157: H7 was not added, and fluorescence was not visible to the naked eye, and the sample was judged as a negative sample. And by Fe 3 O 4 @PDA@UiO-66-NH 2 Enrichment of immune nanoparticles, detection time was shortened by 1h (fig. 6).
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for this practical use will also occur to those skilled in the art, and are within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
Reference patent
[1]Kakagianni,M.,&Koutsoumanis,K.Assessment of Escherichia coli O157:H7 growth in ground beef in the Greek chill chain.Food Research International[J].2019,123,590-600.
[2]Ford B A,Burnham C A D.Optimization of Routine Identification of Clinically Relevant Gram-Negative Bacteria by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry and the Bruker Biotyper.Journal of Clinical Microbiology[J].2013, 51(5):1412-1420.
[3] MALDI-TOF MS, a standard operating expert consensus in clinical microbiological identification, journal of Chinese inspection medicine [ J ]. 2019,42 (4): 241-249.
[4]Gordillo R,Juan J.Córdoba,María J.Andrade,et al.Development of PCR assays for detection of Escherichia coli O157:H7 in meat products[J].Meat Science,2011,88(4):767-773.
[5]Chen J S,Ma E,Harrington L B,et al.CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity.Science[J].2018,360(6387).
[6]Gootenberg J S,Abudayyeh O O,Lee J W,et al.Nucleic acid detection with CRISPR-Cas13a/C2c2.Science[J].2017,356(6336):438-442.
[7]Wang L,Lin J.Recent Advances on Magnetic Nanobead Based Biosensors:from Separation to Detection.TrAC Trends in Analytical Chemistry[J].2020:115915.
Sequence listing
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Claims (8)

1. A primer composition for detecting CRISPR/Cas12a of escherichia coli O157: H7 is characterized by comprising a MIRA amplifying primer, crRNA and a probe;
wherein, the nucleotide sequence of the MIRA amplifying primer is SEQ ID No.1 and SEQ ID No.5;
the nucleotide sequence of the crRNA is SEQ ID No.6;
the sequence of the probe is 5'-6-FAM-TTATT-BHQ1-3'.
2. A kit for detecting Escherichia coli O157: H7 comprising the primer composition of claim 1.
3. The kit of claim 2, further comprising Fe 3 O 4 @PDA@UiO-66-NH 2 A metal organic framework material.
4. A method for the detection of e.coli O157: H7 for non-diagnostic purposes using the kit according to any of claims 2-3, characterized in that the primer composition, the product of the amplification reaction by MIRA, is used as target for CRISPR/Cas12a detection.
5. The method according to claim 4, wherein the conditions for the MIRA amplification reaction are 37℃to 39℃and the temperature is kept constant for 30min.
6. The detection method of claim 4, wherein the CRISPR/Cas12a reaction system is: 0.6. Mu.L of 1. Mu.M crRNA, 0.8. Mu.L of 1. Mu.M Cas12a, 1.2. Mu.L of 10. Mu.M probe, 1. Mu.L of LMIRA amplification product, 16.4. Mu.L of 1 XNEBuffer 2.1; the reaction condition is 37 ℃ and the temperature is kept for 30min.
7. The method according to claim 4, further comprising using the Fe before amplification 3 O 4 @PDA@UiO-66-NH 2 The metal organic framework material enriches escherichia coli O157 to H7 in the sample to be detected.
8. The method according to claim 7, wherein the Fe 3 O 4 @PDA@UiO-66-NH 2 The preparation method of the immune metal organic framework material comprises the following steps:
dissolving ferric trichloride, sodium acetate and anhydrous sodium citrate in ethylene glycol, performing ultrasonic treatment until the ferric trichloride, the sodium acetate and the anhydrous sodium citrate are completely dissolved, transferring the mixture into a polytetrafluoroethylene lining of a stainless steel reaction kettle, and sealing and heating the mixture; then cooling to room temperature, collecting the precipitate with a magnet, washing, and drying to obtain Fe 3 O 4 A nanoparticle;
step two, the Fe is treated 3 O 4 Dispersing the nano particles in ethanol, and adding a Tris-HCl solution with pH of 8.5; after complete dissolution, adding dopamine hydrochloride solution, and continuously stirring for 15-25 hours at room temperature; collecting the precipitate with magnet, washing, and drying to obtain Fe 3 O 4 PDA nanoparticles;
step three, the Fe is treated 3 O 4 Dispersing the @ PDA nano particles in DMF (dimethyl formamide) solution containing 2-amino terephthalic acid and zirconium tetrachloride, mixing the solution, performing ultrasonic treatment, transferring into a polytetrafluoroethylene lining of a stainless steel reaction kettle, and sealing and heating; collecting the precipitate with magnet, washing, and drying to obtain Fe 3 O 4 @PDA@UiO-66-NH 2 A nanoparticle;
step four, the Fe is treated 3 O 4 @PDA@UiO-66-NH 2 Nanometer particleActivating glutaraldehyde with final concentration of 1.25%, washing off residual glutaraldehyde, adding Escherichia coli O157:H27 monoclonal antibody, binding, and blocking with 1% BSA to obtain Fe 3 O 4 @PDA@UiO-66-NH 2 Immune nanoparticles.
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