CN107828784B - Kit and method for extracting and purifying escherichia coli DNA from complex sample - Google Patents
Kit and method for extracting and purifying escherichia coli DNA from complex sample Download PDFInfo
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Abstract
The invention discloses a kit and a method for extracting and purifying escherichia coli DNA from a complex sample, belonging to the field of molecular biology. The invention relates to a kit for extracting and purifying Escherichia coli DNA from a complex sample, which comprises: 1) carboxylated nano magnetic beads Eget103 coupled with gp37 protein; 2) a sample diluent; 3) sample lysate; 4) nucleic acid cleaning solution; 5) DNA eluent 6) nucleic acid adsorption magnetic beads. The invention specifically binds Escherichia coli by using carboxylated nano magnetic beads Eget103 coupled with gp37 protein, then purifies the Escherichia coli, and then extracts DNA from the purified Escherichia coli by a magnetic bead method. The whole process does not need separation, purification and culture of escherichia coli, and DNA extraction is directly carried out; the experimental process is simplified, and the experimental time is greatly saved.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a kit and a method for extracting and purifying escherichia coli DNA from a complex sample.
Background
Escherichia coli, also known as Escherichia coli (gram-negative Brevibacterium), is a common inhabitant of the human and animal intestinal tracts. According to the pathogenicity, the diarrheagenic Escherichia coli is divided into 5 species of enterotoxigenic Escherichia coli, intestinal invasive Escherichia coli, intestinal pathogenic Escherichia coli, intestinal accumulating Escherichia coli and enterohemorrhagic Escherichia coli.
The T4 bacteriophage is a virus with colibacillus as host and has strong host specificity. The gp37 protein is located at the tail end of a long fiber of a T4 bacteriophage, the C end of the gp37 protein can recognize a host bacterium surface receptor, and the process of causing the bacteriophage to be adsorbed to the surface of escherichia coli is a key protein of a T4 bacteriophage recognition receptor.
The complex samples refer to food, excrement, soil, vomit and other samples, and the samples generally contain inhibitors for inhibiting downstream PCR or NGS detection, such as complex polysaccharide, cholate, lipid, uric acid and the like. The traditional method for extracting the Escherichia coli DNA from a complex sample (food, excrement, soil, vomit and the like) needs to separate and purify a culture strain from the sample and then extract nucleic acid from the purified strain, and the process is complex and takes a long time.
Disclosure of Invention
In order to make up for the defects of the prior art and solve the problems of complex process and long required time of the existing method for extracting the Escherichia coli DNA from the complex sample, the invention provides a kit and a method for extracting and purifying the Escherichia coli DNA from the complex sample.
The technical scheme of the invention is as follows:
a kit for extracting and purifying escherichia coli DNA from a complex sample, comprising:
1) carboxylated nano magnetic beads Eget103 coupled with gp37 protein;
2) a sample diluent;
3) sample lysate;
4) nucleic acid cleaning solution;
5) DNA eluent;
6) the nucleic acid adsorbs the magnetic bead.
As a preferred scheme, the preparation method of the carboxyl nano magnetic bead Eget103 coupled with gp37 protein in 1) comprises the following steps:
a) soluble expression of gp37 protein and purification are carried out by adopting an escherichia coli expression system of gp37 protein;
b) activating carboxyl-activated nano magnetic beads;
c) gp37 protein was coupled to carboxylated nanobeads.
Further, the specific process of coupling c) gp37 protein with carboxylated nano magnetic beads is as follows:
taking a borate solution containing Tween-20 as a coupling buffer solution, and washing the carboxylated nano magnetic beads by using the coupling buffer solution;
mixing the carboxylated nano magnetic beads, the purified gp37 protein and a coupling buffer solution, and coupling for 2-6 hours at 20-30 ℃; removing supernatant by magnetic separation, adding PBST solution to resuspend the magnetic beads, reacting at 20-30 ℃ for 30-90min, and sealing unreacted activated carboxyl groups on the surfaces of the magnetic beads;
the supernatant was magnetically separated, washed with PBS or a preservation solution, and then placed in a preservation solution for preservation at 4 ℃.
Preferably, the pH of the coupling buffer is 8.0 to 9.0. More preferably, the pH of the coupling buffer is 8.5.
Preferably, the composition of the sample diluent is: NaCl 137mM, KCl 2.7mM, Na2HPO44.3mM, KH2PO4 1.4mM, 0.05％Tween-20；
The composition of the sample lysate is: NaCl 0.05mM, EDTA 5mM, Tris-HCl 20mM, pH =8.0, lysozyme 3 mg/ml.
Preferably, the nucleic acid washing solution comprises the following components: NaCl 0.05mM, EDTA 0.05mM, Tris-HCl 20mM, pH =8.0, absolute ethanol 70%.
Preferably, the DNA eluate consists of: EDTA 0.05mM, pH =8.0 Tris-HCl 10 mM.
The method for extracting and purifying the Escherichia coli DNA from the complex sample by adopting the kit comprises the following steps:
1) taking a complex sample, adding the sample diluent, uniformly mixing, and centrifuging to take a supernatant;
2) adding carboxylated nano magnetic beads Eget103 coupled with gp37 protein, and performing shake culture at 36.5-37.5 deg.C for 10-20 min;
3) magnetically separating the magnetic beads combined with the escherichia coli, removing liquid, and cleaning the magnetic beads combined with the escherichia coli by using sample diluent;
4) adding sample lysate into the magnetic beads combined with the escherichia coli, incubating for 15-20min at 36.5-37.5 ℃, and lysing thalli;
5) after cracking, cooling to room temperature, adding nucleic acid adsorption magnetic beads, and uniformly mixing at room temperature;
6) cleaning the magnetic beads adsorbed with the nucleic acid by using a nucleic acid cleaning solution;
7) adding DNA eluent into the magnetic beads adsorbed with nucleic acid, incubating at 36.5-37.5 deg.C, magnetically separating, and storing the supernatant at-20 deg.C.
Preferably, in the step 1), 8-10 mL of sample diluent is added into 1g of complex sample; the volume ratio of the carboxylated nano magnetic beads Eget103 coupled with the gp37 protein in the step 2) to the supernatant in the step 1) is 1: 0.6-1.5.
Preferably, in the step 4), the volume ratio of the magnetic beads combined with the escherichia coli to the sample lysate is 1: 3-5; in the step 5), the volume ratio of the nucleic acid adsorption magnetic beads to the magnetic beads combined with the escherichia coli is 2.5-3.5: 2; in the step 7), the volume ratio of the DNA eluent to the nucleic acid adsorption magnetic beads is 1: 5-7.
The invention has the beneficial effects that:
the invention specifically binds Escherichia coli by using carboxylated nano magnetic beads Eget103 coupled with gp37 protein, then purifies the Escherichia coli, and then extracts DNA from the purified Escherichia coli by a magnetic bead method. The whole process does not need separation, purification and culture of escherichia coli, and DNA extraction is directly carried out; the experimental process is simplified, and the experimental time is greatly saved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is an electrophoretogram of purified gp37 protein;
FIG. 2 is a graph showing the effect of the activation time of the carboxylated nanobead on the gp37 protein coupling amount;
FIG. 3 is a graph showing the effect of pH of the coupling buffer on the amount of gp37 protein coupled;
FIG. 4 is a graph showing the effect of coupling time on the amount of gp37 protein coupled;
FIG. 5 is a graph showing the effect of PCR nucleic acid extraction according to the present invention.
Detailed Description
Example 1
A kit for extracting and purifying escherichia coli DNA from a complex sample, comprising:
1) carboxylated nano magnetic beads Eget103 coupled with gp37 protein;
2) sample diluent (reagent a);
3) sample lysate (reagent B);
4) nucleic acid wash (reagent C);
5) DNA eluent (reagent D);
6) nucleic acid-adsorbing magnetic beads;
wherein,
the composition of the reagent A is as follows: NaCl 137mM, KCl 2.7mM, Na2HPO4 4.3mM, KH2PO4 1.4mM, 0.05％Tween-20。
The composition of the reagent B is as follows: NaCl 0.05mM, EDTA 5mM, Tris-HCl 20mM, pH =8.0, lysozyme 3 mg/ml.
The composition of reagent C was: NaCl 0.05mM, EDTA 0.05mM, Tris-HCl 20mM, pH =8.0, absolute ethanol 70%.
The composition of reagent D was: EDTA 0.05mM, pH =8.0 Tris-HCl 10 mM.
The preparation method of the gp37 protein-coupled carboxyl nano magnetic bead Eget103 comprises the following steps:
a) soluble expression of gp37 protein and purification are carried out by adopting an escherichia coli expression system of gp37 protein; the protein electrophoresis pattern of the purified gp37 protein is shown in FIG. 1.
b) Activating carboxyl-activated nano magnetic beads;
the carboxylated nano magnetic beads are commercialized nano magnetic beads (MagCOOH of Suzhou beaver biomedical engineering Co., Ltd.), after the magnetic beads are uniformly mixed, 100 uL of MagCOOH magnetic beads are put into a 1.5mL centrifuge tube, supernatant is removed by magnetic separation, 200 uL of MEST solution (100 mM MES (2- (N-morpholine) ethanesulfonic acid monohydrate), pH 5.0 and 0.05 percent Tween 20) are used for magnetic separation and washing for 2 times, and then the supernatant is removed;
quickly adding 100 μ L EDC (dichloroethane) solution (10 mg/mL, using the MEST solution as a dispersant) and 100 μ L NHS (10 mg/mL, using the MEST solution as a dispersant) into a centrifuge tube containing magnetic beads, mixing by vortex to fully suspend the magnetic beads, activating at 25 deg.C for 30min, maintaining the suspended state of the magnetic beads during the period, and mixing by a vertical mixer; after the above steps, the carboxyl group on the surface of the magnetic bead is activated, and can be covalently coupled with the biological ligand with primary amino group.
The activation time of the carboxylated nano magnetic beads has a relatively obvious influence on the protein coupling amount, as shown in fig. 2, the optimum activation time of the magnetic beads is 30min, and the gp37 protein coupling amount is influenced by too long or too short activation time.
c) gp37 protein was coupled to carboxylated nanobeads.
The specific process of coupling the gp37 protein and the carboxylated nano magnetic beads in the step c) is as follows:
A. removing supernatant by magnetic separation, using 1mmol/L borate solution containing 0.05g/dL Tween-20 (BST for short) as coupling buffer solution, firstly washing magnetic beads for 2 times by the BST, then adding a proper amount of BST and 150ug of purified gp37, and fixing the volume to 300 uL; wherein the pH of the coupling buffer is 8.0-9.0, most preferably 8.5. The pH of the coupling buffer has a significant effect on the amount of gp37 protein coupled, and as shown in FIG. 3, the amount of gp37 protein coupled is greatest when the pH of the coupling buffer is 8.5.
Coupling for 4 h at the temperature of B.25 ℃, and reversing and uniformly mixing by using a vertical mixer;
the coupling time has a great influence on the coupling amount of gp37 protein, and as shown in FIG. 4, the coupling time is 4 hours, and the coupling amount of gp37 protein is the maximum.
C. The centrifuge tube is placed on a magnetic separation frame for magnetic separation to remove supernatant, 300uL of PBST solution (pH 7.2 and containing 1% BSA) is added for resuspending the magnetic beads, and the reaction is carried out at 25 ℃ for 1 h to block unreacted activated carboxyl groups on the surfaces of the magnetic beads, wherein the suspension state of the magnetic beads is kept.
D. The centrifuge tube is placed on a magnetic separator for magnetic separation to remove supernatant, washed 3 times with 200 uL PBS solution (pH 7.2) or preservation solution each time, and resuspended in preservationSolution (containing 0.02 g/dLNaN)3BST-BSA) to obtain carboxylated nanobead Eget103 coupled with gp37 protein, and storing the coupled nanobead at 4 ℃.
The method for extracting and purifying the Escherichia coli DNA from the complex sample by adopting the kit comprises the following steps:
1) taking 1g of complex sample, adding 9mL of reagent A into a 50mL centrifuge tube, fully and uniformly mixing, centrifuging, and taking 200 mu L of supernate into a 1.5mL centrifuge tube;
2) adding 200 μ L carboxylated nano magnetic bead Eget103 coupled with gp37 protein, and performing shake culture at 37 deg.C and 100rpm for 15 min;
3) placing the magnetic beads combined with the escherichia coli on a magnetic frame for magnetic separation, removing liquid, and cleaning the magnetic beads combined with the escherichia coli for at least 3 times by using 400 mu L of reagent A;
4) adding 800 μ L reagent B into the magnetic beads combined with the Escherichia coli, incubating at 37 deg.C for 15-20min, and lysing thallus;
5) after lysis was complete, the centrifuge tube was removed from the incubation apparatus, cooled to room temperature, and nucleic acid-adsorbing magnetic beads (purchased from BioMag cat #: BMQ300) 300 mu L, and mixing uniformly at room temperature; placing the centrifugal tube on a magnetic frame for magnetic separation, removing waste liquid, and completely sucking residual liquid at the bottom of the centrifugal tube and the tube;
6) cleaning the magnetic beads adsorbed with the nucleic acid by using 1mL of reagent C, performing magnetic separation, and completely sucking the tube cover and the tube bottom residual solution; repeating the steps twice, opening the cover at room temperature and drying;
7) to the magnetic beads with adsorbed nucleic acids, 50. mu.L of reagent D was added to prevent magnetic separation after incubation in a water bath, and the supernatant was pipetted into a new EP tube and stored at-20 ℃.
FIG. 5 is a diagram showing the effect of nucleic acid extraction by the method of the present invention, and it can be seen from FIG. 5 that the present invention can effectively extract the nucleic acid of Escherichia coli, and the extraction process does not require separation and purification of samples, thereby effectively saving the time for extracting nucleic acid from various complex samples.
Claims (7)
1. A kit for extracting and purifying escherichia coli DNA from a complex sample, comprising:
1) carboxylated nanometer magnetic beads Eget103 coupled with gp37 protein of T4 phage;
2) a sample diluent; the composition of the sample diluent is: NaCl 137mM, KCl 2.7mM, Na2HPO4 4.3mM, KH2PO4 1.4mM, 0.05％Tween-20；
3) Sample lysate; the composition of the sample lysate is: NaCl 0.05mM, EDTA 5mM, Tris-HCl 20mM at PH =8.0, lysozyme 3 mg/ml;
4) nucleic acid cleaning solution; the nucleic acid cleaning solution comprises the following components: NaCl 0.05mM, EDTA 0.05mM, Tris-HCl 20mM at PH =8.0, absolute ethanol 70%;
5) DNA eluent; the DNA eluent comprises the following components: 0.05mM EDTA, 10mM Tris-HCl pH = 8.0;
6) the nucleic acid adsorbs the magnetic bead.
2. The kit for extracting and purifying escherichia coli DNA from a complex sample according to claim 1, wherein the preparation method of the carboxyl nanobead Eget103 coupled with gp37 protein in 1) comprises the following steps:
a) soluble expression of gp37 protein and purification are carried out by adopting an escherichia coli expression system of gp37 protein;
b) activating carboxyl-activated nano magnetic beads;
c) gp37 protein was coupled to carboxylated nanobeads.
3. The kit for extracting and purifying escherichia coli DNA from a complex sample as claimed in claim 2, wherein the c) gp37 protein is coupled with carboxylated nanobead by the following specific process:
taking a borate solution containing Tween-20 as a coupling buffer solution, and washing the carboxylated nano magnetic beads by using the coupling buffer solution;
mixing the carboxylated nano magnetic beads, the purified gp37 protein and a coupling buffer solution, and coupling for 2-6 hours at 20-30 ℃; removing supernatant by magnetic separation, adding PBST solution to resuspend the magnetic beads, reacting at 20-30 ℃ for 30-90min, and sealing unreacted activated carboxyl groups on the surfaces of the magnetic beads;
the supernatant was magnetically separated, washed with PBS or a preservation solution, and then placed in a preservation solution for preservation at 4 ℃.
4. The kit for extracting and purifying E.coli DNA from a complex sample according to claim 3, wherein the pH of the coupling buffer is 8.0-9.0.
5. A method for extracting and purifying Escherichia coli DNA from a complex sample by using the kit of claim 1, comprising the steps of:
1) taking a complex sample, adding the sample diluent, uniformly mixing, and centrifuging to take a supernatant;
2) adding carboxylated nano magnetic beads Eget103 coupled with gp37 protein, and performing shake culture at 36.5-37.5 deg.C for 10-20 min;
3) magnetically separating the magnetic beads combined with the escherichia coli, removing liquid, and cleaning the magnetic beads combined with the escherichia coli by using sample diluent;
4) adding sample lysate into the magnetic beads combined with the escherichia coli, incubating for 15-20min at 36.5-37.5 ℃, and lysing thalli;
5) after cracking, cooling to room temperature, adding nucleic acid adsorption magnetic beads, and uniformly mixing at room temperature;
6) cleaning the magnetic beads adsorbed with the nucleic acid by using a nucleic acid cleaning solution;
7) adding DNA eluent into the magnetic beads adsorbed with nucleic acid, incubating at 36.5-37.5 deg.C, magnetically separating, and storing the supernatant at-20 deg.C.
6. The method for extracting and purifying E.coli DNA from a complex sample according to claim 5, wherein: in the step 1), 8-10 mL of sample diluent is added into 1g of complex sample; the volume ratio of the carboxylated nano magnetic beads Eget103 coupled with the gp37 protein in the step 2) to the supernatant in the step 1) is 1: 0.6-1.5.
7. The method for extracting and purifying E.coli DNA from a complex sample according to claim 5, wherein: in the step 4), the volume ratio of the magnetic beads combined with the escherichia coli to the sample lysate is 1: 3-5; in the step 5), the volume ratio of the nucleic acid adsorption magnetic beads to the magnetic beads combined with the escherichia coli is 2.5-3.5: 2; in the step 7), the volume ratio of the DNA eluent to the nucleic acid adsorption magnetic beads is 1: 5-7.
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| CN105531382A (en) * | 2013-06-19 | 2016-04-27 | 六品科技公司 | Phage-based bacterial detection assay |
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| CN105399801A (en) * | 2008-07-04 | 2016-03-16 | 拜奥默里克斯公司 | New bacteriophage adhesion proteins |
| CN105531382A (en) * | 2013-06-19 | 2016-04-27 | 六品科技公司 | Phage-based bacterial detection assay |
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| 《四川部分地区J亚型禽白血病分子流行病学调查及一株ALV-J流行株对ND-IB活疫苗免疫反应的影响》;余松城;《中国优秀硕士学位论文全文数据库 农业科技辑》;20160515(第5期);第D050-304页 * |
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Denomination of invention: Kit and method for extracting and purifying Escherichia coli DNA from complex samples Granted publication date: 20210323 Pledgee: Jinan Chengxi sub branch of Qilu Bank Co.,Ltd. Pledgor: SHANDONG KAIJING BIOTECHNOLOGY CO.,LTD. Registration number: Y2024980012924 |
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