CN112159854A - Primer composition for CRISPR/Cas12a detection of Escherichia coli O157: H7 and detection method - Google Patents
Primer composition for CRISPR/Cas12a detection of Escherichia coli O157: H7 and detection method Download PDFInfo
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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 with a nucleotide sequence of SEQ ID No. 1-2 and a primer with a nucleotide sequence of SEQ ID No. 3-5; the nucleotide sequence of the crRNA is SEQ ID No. 6. The detection method of the Escherichia coli O157: H7 using the primer composition has the advantages of high speed, high sensitivity, strong specificity, simple operation, easy observation of reaction results, suitability for on-site rapid detection and capability of providing rapid screening results for supervision and inspection and emergency treatment of products on the market by supervision departments. The MIRA amplification primers of Escherichia coli O157: H7 are designed and screened for the first time, a CRISPR/Cas12a detection system is optimized, and Fe is combined3O4@PDA@UiO‑66‑NH2The enrichment function of the bacillus coli establishes a CRISPR/Cas12a detection method of the escherichia coli O157: H7, and fills the blank in the detection field at home and abroad.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer composition for detecting CRISPR/Cas12a of Escherichia coli O157: H7 and a detection method.
Background
Escherichia coli O157: H7(e.coli O157: H7), a gram-negative bacterium, is one of the human and veterinary co-morbid pathogens that cause human intestinal disease and even death. It is mainly transmitted to human beings by eating contaminated meat or meat products, especially beef products[1]. Although the traditional culture method based on national standards is a gold standard, the traditional 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 assays (ELISAs) are highly dependent on the specificity of the antibody. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in recent yearsSo that the reagent can be widely applied to clinical microbiological laboratories. However, the test object of this method is a pure colony, and the concentration needs to be 105-7CFU[2,3]. Therefore, MALDI-TOF MS cannot satisfy the requirement of rapid detection in terms of sensitivity, specificity, and the like.
Nucleic acid-based detection methods, such as Polymerase Chain Reaction (PCR), real-time PCR (RT-PCR), and the like, have been widely used for detection of pathogens in food, and have the characteristics of high sensitivity, strong specificity, low detection limit, and the like. RubnGordillo et al established a method based on fliCh7And rfbE gene by multiplex Polymerase Chain Reaction (PCR) for detecting Escherichia coli O157: H7 in raw meat and instant meat product[4]. Such methods employ a pair of amplification primers to detect a target nucleic acid sequence in a sample, with or without the use of a fluorescent dye (or fluorescent probe). However, the nucleic acid amplification step used in the method needs frequent heating and cooling processes, takes at least more than 1h, requires large auxiliary equipment such as an electrophoresis apparatus, a DNA amplification apparatus or a sequencer for auxiliary result judgment, and cannot meet the requirements of simple experimental conditions or on-site real-time detection.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology shows a huge application prospect in clinical diagnosis, and attracts extensive attention. The CRISPR/Cas nucleic acid detection based on RNA is rapidly developed, wherein the trans-cleavage activity of Cas12a and the first application thereof in nucleic acid detection are invented in China in 2017. Taking the detection principle of the Cas12 system as an example: after Cas12, crRNA (or sgRNA) forms a ternary complex with the target nucleic acid, the collateral ssDNA reporter is cleaved in trans into small fragments. The amount of target nucleic acid is reflected by the amount of reporter. A detection system of DNA Endonuclease-Targeted CRISPR Trans Reporter based on Cas12a is developed by combining Recombinase-Polymerase Amplification (RPA) technology[5]And Cas 13-based SHERLLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) detection system[6]. In addition to RPA pre-amplification, LAMP and PCR methods are also applied to pre-amplification of CRISPR systems. The principle of the Multienzyme Isothermal nucleic acid Rapid Amplification technology (MIRA) is the same as that of the RPA technology, and the technology is also a technology for realizing the nucleic acid exponential Amplification under the Isothermal condition. In recent years, CRISPR technology has been gradually applied to detection of pathogenic microorganisms, such as patent application No. 201910735947.X, manufacturing method of mycobacterium tuberculosis complex detection kit based on CRISPR-Cas12a system.
Due to the complexity of the actual sample, the separation and enrichment of the target from the sample background is very important for improving the sensitivity and accuracy of the 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 a new type of functional nanomaterials emerging in recent years, and show certain advantages in separation and enrichment[7]For example, the patent publication No. CN109759142A entitled "preparation method of magnetic composite metal organic framework material".
However, the combination of MMOFs and CRISPR/Cas12a and the detection of Escherichia coli O157: H7 are not found at present, and no suitable efficient primer is used for the 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 detecting CRISPR/Cas12a of Escherichia coli O157: H7, which has high speed, high sensitivity and strong specificity, and a detection method for efficiently separating and enriching combined immune MMOFs.
In order to solve the technical problems, the invention adopts the following technical scheme:
the first aspect of the invention provides a primer composition for CRISPR/Cas12a detection of Escherichia coli O157: H7, which comprises an MIRA amplification primer and crRNA;
wherein the MIRA amplification primer comprises a primer with a nucleotide sequence of SEQ ID No. 1-2 and a primer with a nucleotide sequence of SEQ ID No. 3-5;
the nucleotide sequence of the crRNA is SEQ ID No. 6.
Further, the nucleotide sequences of the MIRA amplification primers are SEQ ID No.1 and SEQ ID number 5.
Further, the primer composition also comprises a probe; the sequence of the probe is 5 '-6-FAM-TTATT-BHQ 1-3'.
The second aspect of the invention provides a kit for detecting Escherichia coli O157: H7, which comprises the primer composition.
Further, the kit also comprises Fe3O4@PDA@UiO-66-NH2A metal organic framework material.
The third aspect of the invention provides a detection method of Escherichia coli O157: H7 by using the kit, wherein the primer composition is used, and a product obtained by MIRA amplification reaction is used as a target for CRISPR/Cas12a detection.
Further, the MIRA amplification reaction is carried out under the conditions of 37-39 ℃ and constant temperature for 30 min.
Further preferably, the MIRA amplification reaction is carried out at 37 ℃ for 30 min.
Further, the reaction system of CRISPR/Cas12a is as follows: 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 was 37 ℃ and the temperature was kept constant for 30 min.
Further, the detection method may further comprise the step of using Fe before amplifying the nucleic acid3O4@PDA@UiO-66-NH2The immune metal organic framework material enriches Escherichia coli O157H 7 in a sample to be detected.
Further, Fe as described above3O4@PDA@UiO-66-NH2The 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 solution into a polytetrafluoroethylene lining of a stainless steel reaction kettle, and sealing and heating the solution; cooling to room temperature, collecting the precipitate with magnet, washing, and drying to obtainTo Fe3O4A nanoparticle;
step two, the Fe is added3O4Dispersing the nanoparticles in ethanol, and adding a Tris-HCl solution with the pH value of 8.5; after complete dissolution, adding a dopamine hydrochloride solution, and continuously stirring for 15-25 hours at room temperature; collecting the precipitate with magnet, washing, and drying to obtain Fe3O4@ PDA nanoparticles;
step three, mixing the Fe3O4The method comprises the following steps of (1) dispersing @ PDA nano particles in a DMF (dimethyl formamide) solution containing 2-amino terephthalic acid and zirconium tetrachloride, mixing the solution and carrying out ultrasonic treatment, then transferring the solution to a polytetrafluoroethylene lining of a stainless steel reaction kettle, and sealing and heating the stainless steel reaction kettle; collecting the precipitate with magnet, washing, and drying to obtain Fe3O4@PDA@UiO-66-NH2A nanoparticle;
step four, adding the Fe3O4@PDA@UiO-66-NH2Activating the nanoparticles with 1.25% glutaraldehyde, washing off residual glutaraldehyde, adding Escherichia coli O157: H7 monoclonal antibody, combining, and blocking with 1% BSA to obtain Fe3O4@PDA@UiO-66-NH2An immunonanoparticle.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
(1) the method detects the enterobacter coli O157: H7 in a sample through the unique fluorescence color change of the CRISPR/Cas12a technology.
(2) The method is not limited by detection conditions, and can complete the detection of nucleic acid within 60 min.
(3) The invention has the qualitative detection sensitivity of 0.9 pg. mu.L to the Escherichia coli O157H 7-1(DNA concentration) or 6.5X 104CFU·mL-1(bacterial concentration) is superior to the general RT-PCR detection method.
(4) The method has the advantages of simple operation, easy observation of reaction results and good specificity, 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 supervision departments.
(5) Hair brushThe MIRA amplification primer of Escherichia coli O157: H7 is designed and screened for the first time, the detection system of CRISPR/Cas12a is optimized, and Fe is combined3O4@PDA@UiO-66-NH2The enrichment function of the immune nanoparticles establishes a CRISPR/Cas12a detection method of Escherichia coli O157: H7, and fills the blank of the detection field at home and abroad.
Drawings
FIG. 1 shows the results of gel electrophoresis (FIG. 1A) and CRISPR/Cas12a reactions (FIG. 1B) of 6 pairs of MIRA amplification primers of Escherichia coli O157: H7 and the results of the CRISPR/Cas12a reaction optimization (FIG. 1C) of two pairs of primers of SEQ ID No.1 and SEQ ID No.5 and SEQ ID No.2 and SEQ ID No.5 in one embodiment of the present invention; in fig. 1A and 1B, a: escherichia coli (ATCC 25922); b: escherichia coli O157: H7(CICC 21530); c: escherichia 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 probe (1. mu.M) with a volume of 0.4, 0.8, 1.2, 2.0. mu.L, "+" and "-" represent the amplification template of E.coli O157: H7 DNA 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 a diagram of the CRISPR/Cas12a reaction system of Escherichia coli O157: H7 in an embodiment of the present 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 results of detecting the DNA concentration of the CRISPR/Cas12a reaction system of Escherichia coli O157: H7 in one embodiment of the present invention; wherein, a: the result of MIRA gel electrophoresis; b: RT-PCR; c: CRISPR/cas12 a;
FIG. 4 is a graph showing the results of the detection sensitivity of the bacteria concentration in the CRISPR/Cas12a reaction system of Escherichia coli O157: H7 in one embodiment of the present invention; wherein, a: the result of MIRA gel electrophoresis; b: RT-PCR; c: CRISPR/cas12 a;
FIG. 5 is a diagram showing the detection specificity of the CRISPR/Cas12a system of Escherichia coli O157: H7 in one embodiment of the present invention; wherein, the strains numbered from 1 to 21 are shown in Table 2;
FIG. 6 is a graph showing the results of detection of CRISPR/Cas12a system of Escherichia coli O157: H7 in a ground beef sample according to an embodiment of the present invention; wherein M represents Fe3O4@PDA@UiO-66-NH2An immunonanoparticle.
Detailed Description
The invention provides a primer composition for detecting CRISPR/Cas12a of Escherichia coli O157: H7, which comprises an MIRA amplification primer and crRNA;
wherein the MIRA amplification primer comprises a primer with a nucleotide sequence of SEQ ID No. 1-2 and a primer with a nucleotide sequence of SEQ ID No. 3-5; the nucleotide sequence of the crRNA is SEQ ID No. 6.
In addition, the primer composition also comprises a probe; the sequence of the probe is 5 '-6-FAM-TTATT-BHQ 1-3'.
In another aspect, the invention provides a kit for detecting Escherichia coli O157: H7, comprising the primer composition.
In a preferred embodiment of the present invention, the kit further comprises Fe3O4@PDA@UiO-66-NH2An immuometalorganic framework material.
In a preferred embodiment of the present invention, the Fe3O4@PDA@UiO-66-NH2The preparation method of the immune metal organic framework material comprises the following steps:
(1)2.43g of ferric chloride, 6g of sodium acetate and 0.877g of anhydrous sodium citrate were dissolved in 200mL of ethylene glycol. Ultrasonic treating for 30min, dissolving completely, transferring into polytetrafluoroethylene lining of stainless steel reaction kettle, sealing at 200 deg.C, and heating for 10 hr. Cooling to room temperature, collecting the precipitate with magnet, washing with anhydrous ethanol and water alternately for three times, and drying at 80 deg.C for 6 hr to obtain Fe3O4And (3) nanoparticles.
(2) Mixing 1g of Fe3O4Dispersing the nano particles in 200mL of ethanol, adding 200mL of 10 mmol.L-1Tris-HCl (pH 8.5) solution. After complete dissolution1g dopamine hydrochloride (dissolved in 200ml distilled water) was added and stirring was continued at room temperature for 20 hours. Collecting the precipitate with magnet, washing with anhydrous ethanol and water alternately for three times, and drying at 80 deg.C for 6 hr to obtain Fe3O4@ PDA nanoparticles.
(3) 0.1g of Fe3O4@ PDA nanoparticles were dispersed in 60mL of DMF solution containing 11.04 mmol/L2-amino terephthalic acid and 11.44mmol/L zirconium tetrachloride. Mixing the solution and performing ultrasonic treatment for 5min, transferring the solution into a polytetrafluoroethylene lining of a stainless steel reaction kettle, and sealing and heating the solution at 120 ℃ for 1 h. Collecting the precipitate with magnet, washing with anhydrous ethanol and water alternately for three times, and drying at 80 deg.C for 6 hr to obtain Fe3O4@PDA@UiO-66-NH2A nanoparticle;
(4)0.05g Fe3O4@PDA@UiO-66-NH2the nanoparticles are dispersed in 10mL of 0.01 mol.L of glutaraldehyde with the final concentration of 1.25%-1Shaking in phosphate buffer (pH7.4) at room temperature for 2 hr, washing with sterile water to remove excess glutaraldehyde, and suspending in 10mL of 0.01 mol/L-14mg of monoclonal antibody against Escherichia coli O157: H7 was added to a phosphate buffer (pH7.4) and mixed at 4 ℃ for 24 hours. The product was collected using a magnet to remove unbound antibody. Then, the mixture was stirred at 0.01 mol. L in the presence of 1% BSA-1Blocking was performed in phosphate buffer (pH7.4) at 4 ℃ for 2 h. After three times of washing with sterile water, the product was resuspended in 10mL of 0.01 mol. L-1In phosphate buffer, stored at 4 ℃ for use in enrichment of E.coli O157: H7.
The present invention will be described in detail and specifically with reference to the following examples and drawings so as to better understand the present invention, but the following examples do not limit the scope of the present invention.
In the examples, the conventional methods were used unless otherwise specified, and the reagents used were, for example, conventional commercially available reagents or reagents prepared by conventional methods without specifically specified. 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 herein can be used in the methods of the present invention.
Example 1
The present embodiment provides a CRISPR/Cas12 a-based detection method of escherichia coli O157: H7, which comprises the following steps:
1. preparation of primers: the rfbE gene sequence of Escherichia coli O157: H7 (Genebank No. S83460.1) obtained from GenBank; designing an MIRA primer by using a primer 5.0; the length of the primer is 29-34 bp. The primer sequences used were synthesized by Biotechnology engineering (Shanghai) GmbH. 5 MIRA primers (including two sense strands (F1 and F2) and three antisense strands (R1, R2 and R3)) and 1 crRNA sequence were designed for the target sequence, and their nucleotide sequences are listed in Table 1. The results of amplification using these 6 MIRA amplification primers and gel electrophoresis are shown in FIG. 1.
TABLE 1 primer information for CRISPR/Cas12a technology
2. Using Fe3O4@PDA@UiO-66-NH2Enriching Escherichia coli O157H 7 in a sample to be detected by using the immune metal organic framework material;
3. and extracting DNA in a sample to be detected, and carrying out MIRA amplification reaction. Wherein, the MIRA amplification system adopts a DNA constant-temperature rapid amplification KIT (basic type) of Weifang Anpu future biotechnology limited company (product number: WLN8203 KIT); the specific configuration of the MIRA amplification system was 2. mu.L each of 10. mu.M F1 and R3; 29.4. mu.L buffer A; 2 μ L of DNA template; 12.1 μ L of double distilled water; 2.5. mu.L of buffer B; the MIRA amplification reaction conditions are as follows: keeping the temperature at 37 ℃ for 30 min.
4. The probe, the Cas12a protein, the crRNA, the MIRA amplification product and the detection reagent are added into the reaction tube, the CRISPR reaction is carried out on a fluorescent quantitative PCR instrument (the reaction condition is constant temperature of 37 ℃ for 30min), and the fluorescence value is measured every 30 s. The CRISPR/Cas12a reaction system is optimized by the inventor, as shown in FIG. 2, the CRISPR/Cas12a detection system is optimized and then is specifically configured to be 0.8 muL Cas12a (1 muM), 0.6 muL crRNA (1 muM), 1.2 muL probe (10 muM), 1 muL MIRA product and 16.4 muL 1 XNEBuffer 2.1.
The inventors of the present invention, using the above steps 2 to 4, amplified the E.coli (ATCC25922), E.coli O157: H7(CICC 21530), E.coli O111 and Pseudomonas aeruginosa by the above 6 primer pairs, respectively, and detected them by 2.0% agarose gel electrophoresis, and the results are shown in FIG. 1A. Then CRISPR reaction was performed, and the detection result obtained is shown in fig. 1B. The two pairs of primers of SEQ ID No.1 and SEQ ID No.5 and SEQ ID No.2 and SEQ ID No.5 screened according to the results of FIG. 1A and FIG. 1B are further subjected to CRISPR/Cas12a reaction, and the result is shown in FIG. 1C. As can be seen from FIG. 1C, the two pairs of primers are used in the optimization process of the CRISPR/Cas12a system, and the specificity of SEQ ID No.1 and SEQ ID No.5 is better, and the false positive rate is lower.
Verification example 1
This example detects the specificity and sensitivity of the detection method provided in example 1, with the following specific operations and results:
specificity test
The detection was carried out by means of 4 strains of Escherichia coli O157: H7 and 17 other bacterial species (see Table 2). The CRISPR/Cas12a detection result was determined as follows.
The phenomenon that the result is judged to be positive reaction is as follows: real-time fluorescence detection is carried out by adopting a real-time quantitative PCR instrument Light Cycler 480II of Roche diagnostics; or E-Gel SafeImager from Invitrogen corporation, USATMAnd a real-time transilluminator for observing whether the reaction tube has green fluorescence or not by naked eyes. And (4) performing a negative control test by using the sterilized purified water, wherein the negative control test result is negative.
1) If the fluorescence value is greater than 0 or fluorescence is visible with naked eyes, determining that the test result of the CRISPR/Cas12a is positive;
2) if the fluorescence value is 0 or no fluorescence can be seen by naked eyes, determining that the CRISPR/Cas12a test result is negative;
the primers SEQ ID No.1 and SEQ ID No.5 showed positive reactions in all 4 Escherichia coli strains O157: H7, the fluorescence value of the primers was larger than 0 by real-time fluorescence detection using a real-time quantitative PCR instrument Light Cycler 480II of Roche diagnostics, and the primers were E-Gel safeImager of InvitrogenTMThe real-time transmission instrument can detect green fluorescence (figure 5) by naked eyes, and the fluorescence value (or color change) of other non-Escherichia coli O157: H7 strains is not detected within 30min of the reaction, and the reaction is shown as negative reaction.
TABLE 2 detection results of test strains and CRISPR/Cas12a
Wherein: CICC means China center for preservation and management of industrial microorganism strains; CMCC means China medical microbiological culture collection management center; ATCC refers to the American Standard Strain Collection; ST is provided by the Chinese food and drug testing research institute.
Sensitivity test
Escherichia coli O157: H7 strain is inoculated in mEC + n liquid medium, after 48H of culture, 1mL of 10-fold series gradient diluent is prepared by sterile aqueous solution, and DNA is extracted by an ultra-fast universal DNA nucleic acid releasing agent (product number: WLDR8202) of Weifang' an Pupu future Biotech Co., Ltd, and is used as a template for bacteria concentration sensitivity detection. Taking 1mL of mEC + n cultured bacterial solution, extracting DNA by an ultrafast universal DNA nucleic acid releasing agent of Weifang Anpu future limited biotechnology company, determining the DNA concentration by an Ebend nucleic acid protein determinator (Eppendorf Hellma TryCell), and performing 10-time series gradient dilution on the DNA to be used as a template for DNA concentration sensitivity detection. The CRISPR/Cas12a test method of the invention assays the DNA samples described above. Meanwhile, 0.1mL of the bacterial liquid of each dilution grade is taken and respectively coated on a TSA agar medium, the culture is carried out for 48h, and the number of colonies growing on the plate is counted.
When the DNA concentration is more than or equal to 0.9 pg. mu.L-1The time for judging the positive result is less than 15min (the fluorescence value is more than 0) (figure 3). When the concentration of bacteria is more than or equal to 6.5 multiplied by 104CFU·mL-1The interpretation time of the reaction positive result is less than 20 min (fluorescence value is more than 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 6.5X 104CFU·mL-1(bacterial concentration).
Verification example 2
This example tests the effect of the CRISPR/Cas12a method provided in example 1 on the detection of actual samples of ground beef, and 1 ground beef product was selected for detection in the market. The specific detection process and results are as follows:
preparation of a sample: according to the method of GB 4789.36-2016 (food microbiology test for Escherichia coli O157: H7/NM test), two 25g of crushed beef is weighed and inoculated into 225mL of mEC + n liquid culture medium, wherein one part is inoculated with 14CFU mL-1Coli O157H 7, and another aliquot was not inoculated as a negative control. Culturing in an incubator at 37 ℃, 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: WLDR8202) of Weifang Anpu future Biotech Co.
Using Fe in example 13O4@PDA@UiO-66-NH2Immune nanoparticle enrichment and detection by the CRISPR/Cas12a method.
The detection result shows that the CRISPR/Cas12a sample added with the Escherichia coli O157: H7 has an amplification curve in real-time fluorescence detection, the fluorescence value is greater than 0, and the sample is judged to be positive if fluorescence is visible with naked eyes. The sample to which no E.coli O157H 7 was added had a fluorescence value of 0, and was judged as a negative sample when fluorescence was not observed with the naked eye. And by Fe3O4@PDA@UiO-66-NH2Enrichment of immune nanoparticles, detection time was shortened by 1h (fig. 6).
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications or alterations to this practice will occur to those skilled in the art and are intended to be within the scope of this invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
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 is commonly recognized by experts in the standardization of microbial identification in China journal of laboratory medicine [ J ]. 2019,42(4): 241-.
[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.
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Claims (10)
1. A primer composition for detecting CRISPR/Cas12a of Escherichia coli O157: H7 is characterized by comprising an MIRA amplification primer and crRNA;
wherein the MIRA amplification primer comprises a primer with a nucleotide sequence of SEQ ID No. 1-2 and a primer with a nucleotide sequence of SEQ ID No. 3-5;
the nucleotide sequence of the crRNA is SEQ ID No. 6.
2. The primer composition of claim 1, wherein the nucleotide sequences of the MIRA amplification primers are SEQ ID No.1 and SEQ ID No. 5.
3. The primer composition of claim 1, further comprising a probe; the sequence of the probe is 5 '-6-FAM-TTATT-BHQ 1-3'.
4. A kit for detecting Escherichia coli O157: H7 comprising the primer composition according to any one of claims 1 to 3.
5. The kit of claim 4, further comprising Fe3O4@PDA@UiO-66-NH2A metal organic framework material.
6. A detection method of Escherichia coli O157H 7 using the kit according to any one of claims 4 to 5, wherein the product of MIRA amplification reaction using the primer composition is used as a target for CRISPR/Cas12a detection.
7. The detection method according to claim 6, wherein the MIRA amplification reaction is performed under conditions of 37 ℃ to 39 ℃ for 30min at constant temperature.
8. The detection method according to claim 6, wherein the reaction system of CRISPR/Cas12a is as follows: 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 MIRA amplification product, 16.4. mu.L of 1 XNEBuffer 2.1; the reaction condition was 37 ℃ and the temperature was kept constant for 30 min.
9. The detection method according to claim 6, further comprising, prior to the amplification,using said Fe3O4@PDA@UiO-66-NH2The metal organic framework material enriches Escherichia coli O157H 7 in a sample to be detected.
10. The detection method according to claim 9, wherein the Fe is3O4@PDA@UiO-66-NH2The 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 solution into a polytetrafluoroethylene lining of a stainless steel reaction kettle, and sealing and heating the solution; then cooling to room temperature, collecting the obtained precipitate with a magnet, washing and drying to obtain Fe3O4A nanoparticle;
step two, mixing the Fe3O4Dispersing the nanoparticles in ethanol, and adding a Tris-HCl solution with the pH value of 8.5; after complete dissolution, adding a dopamine hydrochloride solution, and continuously stirring for 15-25 hours at room temperature; collecting the precipitate with magnet, washing, and drying to obtain Fe3O4@ PDA nanoparticles;
step three, mixing the Fe3O4The method comprises the following steps of (1) dispersing @ PDA nano particles in a DMF (dimethyl formamide) solution containing 2-amino terephthalic acid and zirconium tetrachloride, mixing the solution and carrying out ultrasonic treatment, then transferring the solution to a polytetrafluoroethylene lining of a stainless steel reaction kettle, and sealing and heating the stainless steel reaction kettle; collecting the precipitate with magnet, washing, and drying to obtain Fe3O4@PDA@UiO-66-NH2A nanoparticle;
step four, mixing the Fe3O4@PDA@UiO-66-NH2Activating the nanoparticles with 1.25% glutaraldehyde, washing off residual glutaraldehyde, adding Escherichia coli O157: H7 monoclonal antibody, combining, and blocking with 1% BSA to obtain Fe3O4@PDA@UiO-66-NH2An immunonanoparticle.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106512965A (en) * | 2016-11-28 | 2017-03-22 | 复旦大学 | Synthetic method and application of metal-organic framework composite nanomaterial |
| CN108531631A (en) * | 2018-06-19 | 2018-09-14 | 郑州轻工业学院 | One kind is for detecting enteropathogenic E. Coli O157:The RPA-LFD primer pairs and probe of H7 and its application |
| CN111118218A (en) * | 2020-01-20 | 2020-05-08 | 杭州奥盛仪器有限公司 | CRISPR-Cas12a protease isothermal detection primer group and kit for prawn iridovirus and detection method thereof |
| CN111621579A (en) * | 2020-05-30 | 2020-09-04 | 潍坊安普未来生物科技有限公司 | Primer, probe, reagent and method for quickly detecting vibrio parahaemolyticus at normal temperature and isothermal temperature |
-
2020
- 2020-10-28 CN CN202011173972.2A patent/CN112159854B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106512965A (en) * | 2016-11-28 | 2017-03-22 | 复旦大学 | Synthetic method and application of metal-organic framework composite nanomaterial |
| CN108531631A (en) * | 2018-06-19 | 2018-09-14 | 郑州轻工业学院 | One kind is for detecting enteropathogenic E. Coli O157:The RPA-LFD primer pairs and probe of H7 and its application |
| CN111118218A (en) * | 2020-01-20 | 2020-05-08 | 杭州奥盛仪器有限公司 | CRISPR-Cas12a protease isothermal detection primer group and kit for prawn iridovirus and detection method thereof |
| CN111621579A (en) * | 2020-05-30 | 2020-09-04 | 潍坊安普未来生物科技有限公司 | Primer, probe, reagent and method for quickly detecting vibrio parahaemolyticus at normal temperature and isothermal temperature |
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|---|---|---|---|---|
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| CN116676407A (en) * | 2023-06-25 | 2023-09-01 | 新乡医学院 | Detection reagent for trichomonas vaginalis, kit and detection method thereof |
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