CN114292934A - Method for rapidly detecting tetracycline drug-resistant gene tetL of bacillus by immunomagnetic separation-loop-mediated isothermal amplification - Google Patents
Method for rapidly detecting tetracycline drug-resistant gene tetL of bacillus by immunomagnetic separation-loop-mediated isothermal amplification Download PDFInfo
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Abstract
The invention provides a method for rapidly detecting a tetracycline drug-resistant gene tetL of bacillus by immunomagnetic separation-loop-mediated isothermal amplification. The method comprises the steps of coupling carboxyl magnetic beads and bacillus polyclonal antibodies to prepare immunomagnetic beads, and then adding the immunomagnetic beads to a sample to be detected to obtain an immunomagnetic bead-bacillus compound (magnetic bacteria); extracting bacillus nucleic acid by boiling method; designing specific LAMP primers aiming at the tetracycline drug-resistant gene tetL, adding the nucleic acid into an LAMP reaction system, and amplifying and detecting a fluorescent signal in a real-time fluorescent quantitative PCR instrument. The method can be used for rapidly detecting the tetracycline resistance gene tetL of the bacillus in the pasteurized milk, and reducing the harm caused by the horizontal transfer of the resistance gene. The method has the advantages of strong specificity, high sensitivity and short detection time, and realizes the rapid detection of the drug-resistant bacillus.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a method for rapidly detecting a tetracycline resistance gene tetL of bacillus by immunomagnetic separation-loop-mediated isothermal amplification.
Background
Antibiotics are widely applied to treat diseases such as cow mastitis and the like, so that drug-resistant bacteria in raw milk are accumulated, and heat-resistant bacillus still exists in milk after pasteurization treatment, so that pasteurized milk and products produced by using pasteurized milk contain the drug-resistant bacillus. Drug-resistant genes carried by drug-resistant bacillus, especially tetracycline drug-resistant genes can be transferred to staphylococcus aureus and enterococcus through plasmids or movable elements, and finally can be transferred to human bodies through food chains, so that the threat to human health is brought. Therefore, it is necessary to enhance the detection of drug-resistant bacillus in dairy products and reduce the possible harm caused by the level transfer of drug-resistant genes.
The detection of drug-resistant bacillus comprises two means of a culture method and molecular biology at present. The detection based on the culture method cannot realize rapid detection due to poor sensitivity and long time consumption. Loop-mediated isothermal amplification (LAMP) in molecular biology is an in vitro nucleic acid amplification technology, can amplify a large number of target genes under a constant temperature condition, and has the advantages of rapid reaction, simple operation, strong specificity and the like. At present, LAMP detection and enrichment culture are combined generally in research, so that the sensitivity of the LAMP detection method is improved, but the enrichment culture generally needs 18-24h, the overall detection time is prolonged, and rapid detection cannot be realized. In addition, when drug-resistant bacillus in pasteurized milk is actually detected, due to the complex matrix and the possible interference of other bacteria, the high-sensitivity and high-specificity detection of the drug-resistant bacillus is difficult. Therefore, how to establish a method for rapidly detecting drug-resistant bacillus in pasteurized milk with strong specificity and high sensitivity still remains a technical problem to be solved urgently at present.
Disclosure of Invention
The invention aims to provide a method for rapidly detecting a tetracycline resistance gene tetL of bacillus by immunomagnetic separation-loop-mediated isothermal amplification (IMS-LAMP).
In order to realize the purpose of the invention, in a first aspect, the invention provides a set of LAMP detection primer combination of a bacillus tetracycline resistance gene tetL, wherein the primer combination consists of primers F3, B3, FIP, BIP and LB, and the sequences of the primers are respectively shown in SEQ ID NO. 1-5.
In a second aspect, the invention provides an immunomagnetic bead for separating and enriching bacillus, wherein the immunomagnetic bead is prepared by coupling bacillus specific antibodies on the surface of a magnetic bead after activating carboxyl magnetic beads.
Specifically, the preparation method of the immunomagnetic beads comprises the following steps:
taking 0.8-1mg of carboxyl magnetic beads into 3mL of PB, carrying out magnetic separation for 10min, then removing the supernatant, repeatedly washing for 3 times, redissolving in 3mL of PB, and carrying out ultrasonic treatment for 10s after redissolving each time; respectively weighing 1mg of Ethyl Dimethylamino Carbodiimide (EDC) and N-hydroxy thiosuccinimide (NHSS), dissolving with 100 μ L of PB, immediately adding the two solutions into the magnetic bead solution, and performing shaking table reaction at 37 ℃ and 180-220rpm for 1-2 h; performing magnetic separation for 10min, removing supernatant, washing with PB for 2 times, and re-dissolving with 3mL PB; adding 100 mu g of bacillus specific antibody into the magnetic bead solution, uniformly mixing by vortex, and reacting for 2h at 37 ℃ by a shaking table at 220 rpm; weighing 40mg of skim milk, dissolving in 400 mu L of PB, adding the solution, and carrying out closed reaction for 4 h; after magnetic separation for 10min, the supernatant was removed, washed 2 times with PB, redissolved with 1mL of redissolved solution, and stored at 4 ℃.
Preferably, the average particle size of the magnetic beads is 180 nm. The magnetic beads are made of Fe3O4。
The concentration of PB was 0.01M, pH 6.0.
The compound solution is 25% of sucrose, 1% of skim milk and 0.7% of Procllin.
The bacillus specific antibody is a polyclonal antibody.
The polyclonal antibody is prepared by the following method: 1) preparing an immunogen; 2) immunizing an animal; 3) measuring the serum titer; 4) purifying the antiserum; 5) and (5) detecting the specificity of the antibody.
Preferably, the rabbit is immunized with Bacillus cereus and Bacillus subtilis spores as immunogens, and antiserum is collected.
In a third aspect, the invention provides a kit for detecting a tetracycline resistance gene tetL of bacillus, comprising the primer combination and the immunomagnetic beads.
In a fourth aspect, the invention provides a method for rapidly detecting a tetracycline resistance gene tetL of bacillus by immunomagnetic separation-loop-mediated isothermal amplification, wherein the kit is adopted for detection;
the method comprises the steps of immunomagnetic separation, nucleic acid extraction, loop-mediated isothermal amplification and the like (figure 1).
Preferably, the addition amount of the immunomagnetic beads is 20 μ L.
Preferably, the bacillus nucleic acid is extracted by boiling; the reaction conditions are as follows: 100 ℃ for 10 min.
The LAMP reaction system used for the loop-mediated isothermal amplification is as follows: mu.L LAMP mixture, 2.5. mu.L primer mixture, 5. mu.L template, 0.5. mu.L dye, and 25. mu.L ultrapure water.
Preferably, the LAMP mixture contains 8000U/mL Bst 2.0 polymerase.
Preferably, the primer mixture contains 0.2. mu. mol/L of each of the primers F3 and B3, 1.6. mu. mol/L of each of the primers FIP and BIP, and 0.4. mu. mol/L of each of the primers LF and LB.
The LAMP reaction program is as follows: at 65 ℃ for 1min, for 45-49 cycles, and after each cycle, collecting the fluorescence signal.
In a fifth aspect, the invention provides the use of said kit or said method for the detection of bacillus in a sample (food, milk).
Such bacilli include, but are not limited to, bacillus cereus, bacillus licheniformis, bacillus pumilus.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the method has the advantages that (I) the bacillus in pasteurized milk is rapidly enriched by utilizing an immunomagnetic separation technology, the interference of food substrates and escherichia coli is eliminated, the sensitivity of the detection method is improved, and the detection time is shortened.
And (II) the LMAP method for detecting the tetracycline drug resistance gene tetL of the bacillus has the characteristics of rapidness, specificity, sensitivity, simple operation and the like compared with PCR and qPCR.
And (III) the IMS-LAMP technology is adopted to detect the tetracycline drug-resistant gene tetL in the bacillus, the required time is only 2 hours, the detection time is greatly shortened, and the sensitivity is 10 times higher than that of a qPCR detection method.
(IV) the IMS-LAMP technology provided by the invention can detect 2.15 x 10 in pasteurized milk1CFU/mL of drug-resistant Bacillus.
Drawings
FIG. 1 is a flow chart of the IMS-LAMP technology for detecting drug-resistant bacillus in pasteurized milk.
FIG. 2 is a diagram illustrating the optimization of the amount of immunomagnetic beads in a preferred embodiment of the present invention. Indicates that the differences between the different treatment groups had statistical significance, indicates P <0.01, indicates P <0.001, and indicates P < 0.0001. NS means no significant difference.
FIG. 3 is a diagram of an immunomagnetic bead specificity assay in a preferred embodiment of the invention.
FIG. 4 is a diagram illustrating the observation of immunomagnetic bead-Bacillus complexes in a preferred embodiment of the present invention.
FIG. 5 is a comparison of the extraction of Bacillus nucleic acid in the preferred embodiment of the present invention.
FIG. 6 shows LAMP primer optimization in a preferred embodiment of the present invention.
FIG. 7 shows the temperature optimization of LAMP reaction in the preferred embodiment of the present invention.
FIG. 8 shows the optimization of the amount of LAMP reaction template in the preferred embodiment of the present invention.
FIG. 9 shows the specificity test of the IMS-LAMP method in the preferred embodiment of the present invention.
FIG. 10 shows the sensitivity test of IMS-LAMP method in the preferred embodiment of the present invention.
FIG. 11 is a qPCR method sensitivity test in a preferred embodiment of the invention.
FIG. 12 shows the detection of artificially contaminated milk by IMS-LAMP method in the preferred embodiment of the present invention.
Detailed Description
The invention provides a method for rapidly detecting a tetracycline drug-resistant gene tetL of bacillus by immunomagnetic separation-loop-mediated isothermal amplification (IMS-LAMP), which comprises the steps of coupling carboxyl magnetic beads and a bacillus polyclonal antibody to prepare immunomagnetic beads, and then adding the immunomagnetic beads to a sample to be detected to obtain an immunomagnetic bead-bacillus compound (magnetobacteria); extracting bacillus nucleic acid by boiling method; designing specific LAMP primers aiming at the tetracycline drug-resistant gene tetL, adding the nucleic acid into an LAMP reaction system, and amplifying and detecting a fluorescent signal in a real-time fluorescent quantitative PCR instrument. The method can quickly detect the tetracycline resistance gene tetL of the bacillus in the pasteurized milk and reduce the harm caused by the horizontal transfer of the resistance gene. The method has the advantages of strong specificity, high sensitivity and short detection time, and realizes the rapid detection of the drug-resistant bacillus.
Specifically, the invention adopts the following technical scheme:
the invention provides a method for rapidly detecting a tetracycline drug-resistant gene tetL of bacillus by IMS-LAMP, which adopts an immunomagnetic separation technology to enrich the bacillus in pasteurized milk.
The invention takes the immunomagnetic separation technology as a pretreatment method, realizes the rapid enrichment of bacillus without microbial enrichment, and eliminates the interference of milk matrix and escherichia coli, thereby improving the sensitivity of the detection method and shortening the detection time.
Preferably, the addition amount of the immunomagnetic beads is 20 μ L;
preferably, the immunomagnetic beads are prepared by mixing carboxyl magnetic beads with the particle size of 180nm and bacillus polyclonal antibodies, and the mixture is added into the pasteurized milk containing bacillus after the specificity is detected by escherichia coli; preferably, the escherichia coli is ATCC 80739.
Preferably, the bacillus includes, but is not limited to bacillus cereus BA117, bacillus cereus S7, bacillus cereus S11, bacillus cereus ATCC11778, bacillus cereus BA128, bacillus licheniformis BA130, bacillus pumilus BA 102.
The invention also provides a method for extracting the bacillus nucleic acid by a boiling method.
Preferably, the bacillus nucleic acid is extracted by a boiling method under the following reaction conditions: 100 ℃ for 10min (heating block).
The invention also provides a method for detecting the tetracycline resistance gene tetL of the bacillus by LAMP.
Preferably, LAMP Primer design software Primer Explorer V4 is used to design a Primer of the tetracycline resistance gene tetL, which is synthesized by Biotechnology engineering (Shanghai) GmbH, and the LAMP Primer sequence is as follows (5 '-3'):
F3：AGTTCTTTGTGGGGGAATT
B3：AGCAGTTAAAAAGCTAACAGAA
FIP：ACTTAGCTGGTGAACATCTTTCATCTTGGAACAGTAGCAGGGT
BIP：TGAGTGTCATTATTTTCGGCTACACGATGTTTAACACGTATAAAGGAC
LB：GGTGGGATACTTGTTGATAGAAGAG
preferably, the LAMP reaction system is: 12.5. mu.L LAMP mixture (containing 8000U/mL Bst 2.0 polymerase), 2.5. mu.L primer mixture (0.2. mu. mol/L for each of outer primers F3/B3, 1.6. mu. mol/L for each of inner primers FIP/BIP, and 0.4. mu. mol/L for loop primer LF/LB), 5. mu.L template, 0.5. mu.L dye, and 25. mu.L ultrapure water.
Preferably, the LAMP reaction program is: at 65 ℃ for 1min, 49 cycles were performed, and the fluorescence signal was collected after the end of each cycle.
The invention also provides application of the IMS-LAMP technology in detection of the tetracycline resistance gene tetL of the bacillus.
The invention also provides application of the method in detection of food microorganisms (particularly drug-resistant bacillus).
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.
The bacillus antibodies used in the examples below were bacillus cereus polyclonal antibodies. Purchased from abcam, cat # ab 20556.
Example 1 preparation of immunomagnetic beads
Taking 1mg (average particle size of 180nm) of carboxyl magnetic beads into 3mL PB (pH 6.0, 0.01M), carrying out magnetic separation for 10min, then removing the supernatant, repeatedly washing for 3 times, re-dissolving in 3mL PB (pH 6.0, 0.01M), and carrying out ultrasonic treatment for 10s after re-dissolving each time; 1mg of Ethyldimethylaminocarbodiimide (EDC) and N-hydroxysulfosuccinimide (NHSS) were weighed out separately and dissolved with 100. mu.L of PB (pH 6.0, 0.01M), and then both solutions were immediately added to the magnetic bead solution and reacted for 1 hour in a shaker (37 ℃ C.; 220 rpm); performing magnetic separation for 10min, removing supernatant, washing with PB (pH 6.0, 0.01M) twice, and re-dissolving with 3mL PB (pH 8.0, 0.01M); adding 100 mu g of bacillus antibody into the magnetic bead solution, fully and uniformly mixing by vortex, and reacting for 2h in a shaking table (37 ℃ C.; 220 rpm); weighing 40mg of skim milk, dissolving in 400 μ L PB (pH 8.0, 0.01M), adding the above solution, and blocking for reaction for 4 h; after magnetic separation for 10min, the supernatant was removed, washed twice with PB (pH 8.0, 0.01M), and finally reconstituted with 1mL of a special reconstitution solution (25% sucrose, 1% skim milk, 0.7% Procllin) and placed in a refrigerator at 4 ℃ for further use.
Example 2 method for enriching bacillus using immunomagnetic beads
1. Optimization of immunomagnetic bead dosage
The immunomagnetic beads used in this example were the same as those used in example 1. Adding immunomagnetic beads to 105And (3) carrying out immunomagnetic capture on the CFU/mL bacillus cereus BA117 bacterial solution for 1h, carrying out magnetic separation on a magnet for 3min, smearing the heavy suspension precipitate of the stock solution and the PBS on a Nutrient Agar (NA) culture medium, culturing at 30 ℃ for 24h, counting, and calculating the capture efficiency according to the following formula. The addition amounts of immunomagnetic beads were 1, 2, 5, 10, 15, 20, 25, and 30. mu.L, respectively. As shown in FIG. 2, when the amount of immunomagnetic beads was increased from 1. mu.L to 20. mu.L, the capture efficiency was significantly increased from 2.3% to 89.5% (P)<0.05), the dosage of the immunomagnetic beads is continuously added to 30 mu L, and the capture efficiency is not obviously improved (P)>0.05)。
2. Immunomagnetic bead specificity assay
The immunomagnetic beads used in this example were the same as those used in example 1. Adding 20 μ L of immunomagnetic beads into 6 strains of 10-strain mixture5CFU/mL bacillus and Escherichia coli liquid. Performing magnetic separation on a magnet for 3min after 1h of immunomagnetic capture, smearing the heavy-suspended precipitate of the stock solution and PBS on an NA or LB culture medium, and calculating the capture efficiency after culturing for 24h at 30 ℃. In addition, 20. mu.L of immunomagnetic beads were added to a concentration of 107Carrying out immunomagnetic capture on the CFU/mL Bacillus cereus BA117 bacterial solution for 1h, then carrying out magnetic separation on a magnet for 3min, and precipitating with ddH2And O, resuspending, sucking 10 mu L of magnetobacteria, dropping on a sample-carrying copper net, and standing and drying at room temperature overnight for subsequent transmission electron microscope observation.
The experimental results are shown in fig. 3, the capture efficiency of the immunomagnetic beads on bacillus is respectively bacillus cereus S7 (94.8%), bacillus cereus S11 (98.0%), bacillus cereus BA128 (94.1%), bacillus cereus ATCC11778 (81.2%), bacillus licheniformis BA130 (61.9%), bacillus pumilus BA102 (60.5%), and the capture rate on escherichia coli ATCC80739 is only 3.8%, and the results show that the immunomagnetic beads prepared by the experiment have high specificity. In addition, in order to further confirm the immunological binding between the immunomagnetic beads and bacillus cereus, the prepared magnetic bacteria were observed by a transmission electron microscope (fig. 4), confirming that the immunomagnetic beads captured bacillus.
Example 3 Bacillus nucleic acid extraction
The nucleic acid of the bacillus cereus BA117 is extracted by a boiling method and a kit method respectively, and the feasibility of extracting the nucleic acid by a thermal cracking method is proved.
1. Boiling method
Taking Bacillus cereus BA117 bacterial liquid (OD)6000.8)2mL, centrifuging at 12000rpm for 1min, discarding the supernatant, redissolving with 200. mu.L of precipitation Buffer, treating in a metal bath at 100 ℃ for 10min, centrifuging at 10000rpm for 1min, and collecting the supernatant as a template and storing at-20 ℃.
2. Reagent kit method
Taking Bacillus cereus BA117 bacterial liquid (OD)6000.8)2mL, centrifuging at 12000rpm for 1min, discarding the supernatant, adding 500 μ L TES for resuspension, centrifuging at 10000rpm for 1min, discarding the supernatant, repeating the above steps, and removing the supernatant; adding 200 mu L of lysozyme, blowing and beating a gun head until the lysozyme is fully and uniformly mixed, and treating for 1h at 37 ℃; centrifuging to remove supernatant, adding 100 μ L BTL Buffer and 20 μ L lipoprotein kinase K Solution, mixing by vortex, incubating at 55 deg.C for 1h, and mixing by vortex every 20min for 30 s. Add 5. mu.L RNase A and mix by turning upside down, incubate for 5min at room temperature. Then centrifuged (24 ℃, 10000 Xg) for 2min, the supernatant was aspirated into a new 1.5mL centrifuge tube, 220. mu.L BDL Buffer was added, and vortexedAfter mixing, incubation is carried out for 10min at 65 ℃. Add 220. mu.L of absolute ethanol and vortex at maximum speed for 20s to mix well. Transferring all liquid to a collection tube
The DNA Mini column was centrifuged (10000 Xg) at room temperature for 1min, and the collection tube and filtrate were discarded. Will be provided with
The DNA Mini column was fitted to a new 2mL collection tube, 500. mu.L HBC Buffer was added, centrifuged (10000 Xg) at room temperature for 1min, and the filtrate was discarded. Add 700. mu.L of DNA Wash Buffer, centrifuge (10000 Xg) at room temperature for 1min, and discard the filtrate. Repeating the above steps, discarding the filtrate, placing the adsorption column back to the collection tube, centrifuging at room temperature (10000 Xg) for 2min, and discarding the waste liquid. The adsorption column was transferred to a 1.5mL centrifuge tube, suspended in the air and 200. mu.L solution Buffer was added dropwise to the center of the membrane, left at room temperature for 3min, followed by centrifugation (10000 Xg) at room temperature for 1min, and the DNA solution was collected and stored at-20 ℃.
The LAMP amplification is carried out by respectively taking the nucleic acids extracted by the two methods as templates, the experimental result is shown in figure 5, the generation time of the amplification curve corresponding to the kit method is 10.3min, and the boiling method is 12.6 min. Although the boiling method is a method in which the amplification curve is generated later than the kit method, the same amplification effect is obtained, and the nucleic acid extraction time is shortened from 3 hours to 10 minutes, which greatly shortens the overall detection time, so that the boiling method is preferable for nucleic acid extraction.
Example 4 LAMP method for detecting tetracycline-resistant gene tetL of Bacillus
1. Primer optimization
3 sets of primers are designed aiming at six specific regions of a tetracycline drug resistance gene tetL by using Primer Explorer 4 software, each set of primers comprises an inner Primer BIP/FIP, an outer Primer F3/B3 and a loop Primer LF/LB, and the Primer sequences are shown in Table 1.
TABLE 1 LAMP primer information
Nucleic acids of Bacillus cereus BA117 were extracted by boiling as templates, and LAMP amplification was carried out using the primers shown in Table 1, respectively. Wherein the LAMP reaction system is as follows: 12.5. mu.L LAMP mixture (containing 8000U/mL Bst 2.0 polymerase), 2.5. mu.L primer mixture (0.2. mu. mol/L for each of outer primers F3/B3, 1.6. mu. mol/L for each of inner primers FIP/BIP, and 0.4. mu. mol/L for loop primer LF/LB), 1. mu.L template, 0.5. mu.L dye, and a supplemental volume of ultrapure water to 25. mu.L. In addition, the LAMP reaction program was: at 65 ℃ for 1min, 49 cycles were performed, and the fluorescence signal was collected after the end of each cycle. As a result, as shown in FIG. 6, the tetL-1 primer generated an amplification curve at 17.1min, and the tetL-2 and tetL-3 primers generated amplification curves at 33.9min and 44.2min, respectively, and thus the tetL-1 primer was selected for the subsequent experiments.
2. Reaction temperature optimization
The tetL-1 primer was selected for LAMP amplification at 59 ℃, 62 ℃, 65 ℃, 68 ℃, 71 ℃ to determine the optimal reaction temperature. As shown in FIG. 7, the amplification reaction was accelerated when the reaction temperature was increased from 59 ℃ to 65 ℃ and the amplification curve generation time was decreased from 23.2min to 15.64min, and the amplification reaction was slowed when the reaction temperature was further increased and showed no fluorescence signal when the reaction temperature was 71 ℃. Therefore, 65 ℃ was selected as the LAMP reaction temperature in this experiment.
3. Reaction template quantity optimization
The nucleic acid of Bacillus cereus BA117 was extracted by boiling method, and LAMP amplification was carried out using 1, 2, 3, 4, and 5. mu.L of crude DNA as templates, respectively, to obtain the optimum amount of template added. As a result, as shown in FIG. 8, when the template addition amount was increased from 1. mu.L to 4. mu.L, the time threshold was shortened from 48.04min to 25.96min, and the template amount was increased continuously, and the time threshold was shortened but not significant (P >0.05), and 5. mu.L was selected as the optimal template addition amount for the experiment in consideration that the target bacteria at a low concentration may not have an amplification product due to the low template amount.
Example 5 method for detecting tetracycline-resistant gene tetL of Bacillus by IMS-LAMP
1. IMS-LAMP method specificity test
20 mu.L of immunomagnetic beads are used for capturing the bacillus in the table 2, and then the tetracycline resistance gene tetL is amplified by LAMP, and the specificity of the method is judged according to an amplification curve.
TABLE 2 Experimental strains for testing the specificity of the IMS-LAMP method
As shown in FIG. 9, only Bacillus cereus BA117, Bacillus cereus S7 and Bacillus cereus S11 carrying tetracycline resistance gene tetL had amplification curves, while Bacillus cereus ATCC11778, Bacillus cereus BA128, Bacillus licheniformis BA130 and Bacillus pumilus BA102 all had no amplification curves, demonstrating that the IMS-LAMP method had good specificity.
2. IMS-LAMP method sensitivity test
Prepared at a concentration of 1.16X 105-1.16×100The CFU/mL Bacillus cereus BA117 bacterial solution was detected by the IMS-LAMP method established in this study. As a result, as shown in FIG. 10, the method can detect 1.16X 101CFU/mL of Bacillus cereus BA 117. The minimum detection limit of Bacillus cereus BA117 detected by qPCR method is 2.12 × 102CFU/mL, so IMS-LAMP method sensitivity than qPCR method is 10 times higher. The results of the sensitivity test of the qPCR method are shown in figure 11.
Example 6 method for detecting the tetracycline-resistant gene tetL of Bacillus in artificially contaminated milk
Adding Bacillus cereus BA117 bacterial solution into sterilized pasteurized milk to final concentration of 2.15 × 105-2.15×100CFU/mL. Adding 20 μ L immunomagnetic beads into artificial contaminated milk, reacting for 1h, magnetically separating for 3min to separate milk matrix from magnetic bacteria, removing supernatant, and adding 50 μ L ddH2O re-dissolving, boiling (100 deg.C; 10min) to extract the nucleic acid of bacillus. And (3) carrying out magnetic separation for 3min, absorbing the supernatant to an LAMP reaction system, and amplifying and detecting a fluorescent signal in a real-time fluorescent quantitative PCR instrument. As shown in FIG. 12, the method can detect 2.15X 10 pasteurized milk1CFU/mL of drug-resistant Bacillus cereus BA 117.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Claims (10)
1. The LAMP detection primer combination of the tetracycline resistance gene tetL of the bacillus is characterized in that the primer combination consists of primers F3, B3, FIP, BIP and LB, and the sequences of the primers are respectively shown in SEQ ID NO. 1-5.
2. The immunomagnetic bead for separating and enriching the bacillus is characterized in that the immunomagnetic bead is prepared by coupling bacillus specific antibodies on the surface of the magnetic bead after activating carboxyl magnetic beads.
3. The immunomagnetic bead according to claim 2, wherein the immunomagnetic bead is prepared by a method comprising:
taking 0.8-1mg of carboxyl magnetic beads into 3mL of PB, carrying out magnetic separation for 10min, then removing the supernatant, repeatedly washing for 3 times, redissolving in 3mL of PB, and carrying out ultrasonic treatment for 10s after redissolving each time; respectively weighing 1mg of ethyl dimethylamino carbodiimide and N-hydroxy thiosuccinimide, dissolving by using 100 mu L of PB, immediately adding the two solutions into a magnetic bead solution, and carrying out shaking table reaction at 37 ℃ and 180-220rpm for 1-2 h; performing magnetic separation for 10min, removing supernatant, washing with PB for 2 times, and re-dissolving with 3mL PB; adding 100 mu g of bacillus specific antibody into the magnetic bead solution, uniformly mixing by vortex, and reacting for 2h at 37 ℃ by a shaking table at 220 rpm; weighing 40mg of skim milk, dissolving in 400 mu L of PB, adding the solution, and carrying out closed reaction for 4 h; performing magnetic separation for 10min, removing supernatant, washing with PB for 2 times, re-dissolving with 1mL of re-dissolving solution, and storing at 4 deg.C;
wherein the average particle size of the magnetic beads is 180 nm;
the concentration of the PB is 0.01M, and the pH value is 6.0;
the compound solution is 25% of sucrose, 1% of skim milk and 0.7% of Procllin.
4. The immunomagnetic bead of claim 2, wherein the bacillus-specific antibody is a polyclonal antibody;
the polyclonal antibody is prepared by the following method: 1) preparing an immunogen; 2) immunizing an animal; 3) measuring the serum titer; 4) purifying the antiserum; 5) and (5) detecting the specificity of the antibody.
5. A kit for detecting a tetracycline resistance gene tetL of Bacillus, comprising the primer combination of claim 1 and the immunomagnetic beads of any one of claims 2 to 4.
6. A method for rapidly detecting a tetracycline resistance gene tetL of bacillus by immunomagnetic separation-loop-mediated isothermal amplification is characterized in that the kit of claim 5 is adopted for detection;
the method comprises the steps of immunomagnetic separation, nucleic acid extraction and loop-mediated isothermal amplification.
7. The method of claim 6, wherein the amount of immunomagnetic beads added is 20 μ L.
8. The method according to claim 6, wherein the Bacillus nucleic acid is extracted by boiling; the reaction conditions are as follows: 100 ℃ for 10 min.
9. The method of claim 6, wherein the LAMP reaction system used for loop-mediated isothermal amplification is: 12.5 microliter LAMP mixed solution, 2.5 microliter primer mixture, 5 microliter template, 0.5 microliter dye and 25 microliter ultrapure water;
wherein the LAMP mixed solution contains 8000U/mL Bst 2.0 polymerase;
the primer mixture contains 0.2 mu mol/L of each primer F3/B3, 1.6 mu mol/L of each primer FIP/BIP and 0.4 mu mol/L of each primer LF/LB;
the LAMP reaction program is as follows: at 65 ℃ for 1min, for 45-49 cycles, and after each cycle, collecting the fluorescence signal.
10. Use of a kit according to claim 5 or a method according to any one of claims 6 to 9 for the detection of bacillus in a sample;
the bacillus comprises bacillus cereus, bacillus licheniformis and bacillus pumilus.
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| CN106947756A (en) * | 2017-02-20 | 2017-07-14 | 南昌大学 | The method of quick Magneto separate bacillus cereus |
| CN109825554A (en) * | 2017-11-23 | 2019-05-31 | 东北农业大学 | A method of the quickly detection rugged Cronobacter sakazakii of slope is captured using immune magnetic |
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| CN106947756A (en) * | 2017-02-20 | 2017-07-14 | 南昌大学 | The method of quick Magneto separate bacillus cereus |
| CN109825554A (en) * | 2017-11-23 | 2019-05-31 | 东北农业大学 | A method of the quickly detection rugged Cronobacter sakazakii of slope is captured using immune magnetic |
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