CN102399774A - Method for preparing Escherichia coli O157:H7 genome deoxyribonucleic acid (DNA) serving polymerase chain reaction (PCR) standard substance - Google Patents
Method for preparing Escherichia coli O157:H7 genome deoxyribonucleic acid (DNA) serving polymerase chain reaction (PCR) standard substance Download PDFInfo
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- CN102399774A CN102399774A CN2011102827439A CN201110282743A CN102399774A CN 102399774 A CN102399774 A CN 102399774A CN 2011102827439 A CN2011102827439 A CN 2011102827439A CN 201110282743 A CN201110282743 A CN 201110282743A CN 102399774 A CN102399774 A CN 102399774A
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Abstract
The invention relates to a method for preparing Escherichia coli O157:H7 genome deoxyribonucleic acid (DNA) serving a polymerase chain reaction (PCR) standard substance, and belongs to the technical field of biology. The method comprises the following steps of: extracting Escherichia coli O157:H7 genome DNA by using carboxylated magnetic nanometer particles, separating, purifying, measuring a P element by inductively coupled plasma mass spectrometry (ICP-MS) to determine the concentration of the extracted DNA, and performing PCR amplification finally, wherein a target stripe detected by electrophoresis is obvious, the property of the target stripe is unchanged after the target stripe is kept at the temperature of -20 DEG C for half a year, and the stability is high, so the target stripe can be used as the PCR standard substance of the Escherichia coli O157:H7 genome DNA.
Description
Technical field
A kind of Escherichia coli O157: the H7 genomic dna belongs to the biology techniques field as the preparation method of PCR reference material.
Background technology
Escherichia coli O157: H7 extensively is present in occurring in nature; Be a kind of important zoonosis pathogenic bacterium; After being infected, the people often causes serious diarrhoea and septicemia; Serious caused hemolytic uremic syndrome (HUS), thrombocytopenic purpura,thrombotic (TTP), wherein concurrent HUS and TTP person's mortality ratio are up to about 80%, therefore; Infection how to control Escherichia coli O157: H7 is the important topic in the present public hygienics, is the necessary means that guarantees human health to fast, accurately checking of Escherichia coli O157: H7.
The method of traditional bacterial detection has comprised biochemical cultivation, isolation identification, and the testing procedures complicacy is loaded down with trivial details, and the report assay roughly needs 4～7d, and is consuming time oversize, and detection sensitivity is low.
Along with development of molecular biology, round pcr has been widely used in the detection range of food microorganisms, plant-animal eqpidemic disease, forage component and transgene component, makes the quick test of food-borne pathogens develop into a brand-new height.The present invention provides a kind of Escherichia coli O157: the preparation method of positive reference material during H7 PCR detects makes the detected result of PCR more conclusive.
Summary of the invention
The purpose of this invention is to provide a kind of extraction purifying Escherichia coli O157: the H7 genomic dna is as the preparation method of PCR reference material, so that make the PCR test result of samples more accurate.
Technical scheme of the present invention: a kind of extraction purifying Escherichia coli O157: the H7 genomic dna utilizes carboxylated magnetic nano-particle to extract Escherichia coli O157:H7 genomic dna as the preparation method of PCR reference material; Escherichia coli O157: H7 has been that following document is open, document 1: [Food Microbiology 23 (2006) 162 – 168.The use of Fourier transform infrared spectroscopy to differentiate Escherichia coli O157:H7 from other bacteria inoculated into apple juice; Murad A. Al-Holy
a, Mengshi Lin
b, Anna G. Cavinato
c, Barbara A. Rasco
b];
Document 2: [Food Research International 39 (2006) 98 – 105.Inactivation of food spoilage bacteria and Escherichia coli O157:H7 in phosphate buffer and orange juice using dynamic high pressure; Imane Tahiri, Joseph Makhlouf *, Paul Paquin, Ismail Fliss].
(1) cultivation of Escherichia coli O157: H7:
By traditional cultural method Escherichia coli O157: H7 being increased bacterium cultivates;
(2) preparation of surperficial carboxylated magnetic nano-particle:
The magnetic nano-particle of preparation carboxyl modified is used to adsorb Escherichia coli O157: the H7 genomic dna;
(3) pathogenic micro-organism Escherichia coli O157: the extraction of H7 genomic dna;
(4) the Escherichia coli O157 that obtains with step (2): the H7 genomic dna is crossed Sephadex G-200 separator column and is carried out separation and purification;
(5) the Escherichia coli O157 behind the purifying that obtains with step (4): the H7 genomic dna is measured the ICP-MS calculating concentration;
(6) pcr amplification product with the DNA behind step (4) purifying carries out the PCR detection, and agarose electrophoresis detects Escherichia coli O157: the purpose band of H7.
The operation of each step sees specific embodiment for details.
Beneficial effect of the present invention: the present invention adopts carboxylated magnetic nano-particle to extract Escherichia coli O157:H7 genomic dna; And carry out separation and purification; Inductivity coupled plasma mass spectrometry ICP-MS measures the P element to confirm extracting DNA concentration, finally carries out pcr amplification, and electrophoresis detection purpose band is obvious;-20 ℃ of preservation character half a year are constant; Stability is high, can be used as Escherichia coli O157: the PCR reference material of H7 genomic dna, so that make the PCR test result of samples more accurate.
Description of drawings
Fig. 1: Sephadex G-200 separation and purification figure.
Fig. 2: genome DNA sample agarose gel electrophoresis figure.
Fig. 3: genome DNA sample-pcr amplification product agarose gel electrophoresis figure.
Embodiment
Below further specify the present invention through embodiment.
Embodiment 1. Escherichia coli O157: the H7 genomic dna is slightly carried;
(1) bacterium that increases of Escherichia coli O157: H7 is cultivated:
With Escherichia coli O157: the H7 bacterial strain, promptly ATCC 35150, are seeded on the sorbyl alcohol Mai Kangkai SMAC flat board, cultivate 10h for 37 ℃, connect a ring again and are inoculated in 10mL improvement E.C Vulkamycin. PA-93 and increase bacterial context soup, cultivate 10h in 37 ℃.
(2) preparation of magnetic nanometer:
(3) Escherichia coli O157: the extraction of H7 genomic dna:
be middle the deposition with 1mL TE damping fluid dissolving (50mM Tris-Hcl with
; 10mM EDTA; PH8.0), add 100 μ L 10%SDS.
adds the magnetic nanometer of preparation among the 20 μ L (2) in above-mentioned solution.
6. in above-mentioned magnetic particle, add 1mL 70% washing with alcohol.
7. above-mentioned solution is put into the magnetic force frame, remove supernatant.
8. (pH8.0), 65 ℃ are reacted 10min for 10mMTris-HCl, 1mM EDTA in above-mentioned magnetic particle, to add the dissolving of 100 μ L TE damping fluids.
9. above-mentioned solution is put into the magnetic force frame, get supernatant, be the thick Escherichia coli O157 that extracts: the H7 genomic dna.
Embodiment 2.Sephadex G-200 separator column carries out separation and purification
Separator column: Sephadex G-200;
Moving phase: the TE damping fluid (10mM Tris-HCl, 1mM EDTA, pH8.0);
Flow velocity: 0.5mL/min;
Detector: UV-detector (emission wavelength: 260nm);
Applied sample amount: extract DNA sample 1mL;
Begin to collect detached peaks from the sample of 6.5mL, that obtain is the Escherichia coli O157 behind the purifying: the H7 genomic dna.
Embodiment 3. Escherichia coli O157: the H7 genome is measured the P concentration of element through ICP-MS and is measured DNA concentration
1) with the DNA sample that obtains among the embodiment 2, with the pure water 48h that in 4 ℃, dialyses, the small molecules in the removal sample.
2) accurately measure in the 4mL step 1) the good sample of dialysis in the micro-wave digestion pipe, add concentrated nitric acid 6mL, build lid, room temperature is placed and is spent the night, and is put in the microwave dissolver the next morning and clears up.Put into the stink cupboard acid discharge after waiting to clear up completion, then Digestive system is transferred in the 50mL plastic centrifuge tube, be settled to 25mL, mixing is heavy, to be measured surely then.
Clear up program (table 1) as follows:
Table 1 Specimen eliminating program
| Step | Peak power (W) | Rise (falling) temperature time (min) | Temperature (℃) | Hold-time (min) |
| 1 | 1200 | 5 | 120 | 3 |
| 2 | 1200 | 8 | 160 | 5 |
| 3 | 1200 | 5 | 190 | 15 |
| 4 | 1200 | 15 | 15 | Cooling |
Annotate: two circulations of this program run.
Instrument is opened, and DNA sample nitrification liquid is drawn in preheating, under best operating condition, measures, and is quantitative according to typical curve.Do blank simultaneously.
Embodiment 4, with the Escherichia coli O157 that obtains among the embodiment 2: the H7 genome DNA sample, adjustment concentration is 1mg/mL, gets 5 μ L and carries out agarose gel electrophoresis, detection DNA purity.
Deposition condition:
Agarose concentration: 1%;
Voltage: 100V;
Time: 50min.
Embodiment 5, PCR detect Escherichia coli O157: the H7 genomic dna
Pcr amplification uidA, rfbE and fliC H7 gene, detect the Escherichia coli O157 of preparation: the H7 genomic dna can be as the PCR reference material, and carries out specific detection.
UidA gene PCR reaction system:
Extract template 1 μ L (20mg/mL); Add each 1 μ L (final concentration 10 μ M/L) of primer respectively; 1 μ L dNTP (final concentration 20 μ M), 0.5 μ L TaqDNA polysaccharase (enzyme 5U/ μ alive L), 5 μ L, 10 * PCR Buffer (final concentration Tris-HCl pH8.5 10mM, KCl 50nM, MgCl
21.5nM), add water and supply 50 μ L.
RfbE gene PCR reaction system:
Extract template 1 μ L; Add each 1 μ L (final concentration 10 μ M/L) of primer respectively; 1 μ L dNTP (final concentration 20 μ M), 0.5 μ L TaqDNA polysaccharase (enzyme 5U/ μ alive L), 5 μ L, 10 * PCR Buffer (final concentration Tris-HCl pH8.5 10mM, KCl 50nM, MgCl
21.5nM), add water and supply 50 μ L.
FliC
H7The PCR reaction system:
Extract template 1 μ L; Add each 1 μ L (final concentration 10 μ M/L) of primer respectively; 1 μ L dNTP (final concentration 20 μ M), 0.5 μ L TaqDNA polysaccharase (enzyme 5U/ μ alive L), 5 μ L, 10 * PCR Buffer (final concentration Tris-HCl pH8.5 10mM, KCl 50nM, MgCl
21.5nM), add water and supply 50 μ L.
Sex change in advance: 94 ℃ of 3min;
Get into circulation: 94 ℃ of sex change 40s, 57 ℃ of annealing 45s, 72 ℃ are extended 45s, cyclic amplification 35 times;
Stop extending: 72 ℃ of 10min;
Whether the PCR product, detecting has the purpose band if being carried out agarose gel electrophoresis;
The agarose gel electrophoresis condition:
Agarose concentration: 1.5%;
Voltage: 80V;
Time: 1h;
The result shows, all amplifies the purpose band, can be used as the reference material of PCR.
Embodiment 6, stability study
With the Escherichia coli O157 that obtains among the embodiment 2: H7 genomic dna standard substance ,-20 ℃ preserve half a year after, room temperature is got 1 μ L (20mg/mL) and is carried out pcr amplification and agarose gel electrophoresis according to step among the embodiment 5 after melting.The result shows, all amplifies the purpose band, the Escherichia coli O157 of acquisition: the H7 genomic dna is as PCR standard substance stable in properties.
<210> SEQ?ID?NO:?1
<213> uidA
<400> 1
5'-GCGAAAACTGTGGAATTGGG-3'
5'-TGATGCTCCATAACTTCCTG-3'
<210> SEQ?ID?NO:?2
<213> rfbE
<400> 2
5'-TCAAAAGGAAACTATATTCAGAAGTTTGA-3'
5'-CGATATACCTAACGCTAACAAAGCTAA-3'
<210> SEQ?ID?NO:?3
<213> fliC
H7
<400> 3
5'-GCGCTGTCGAGTTCTATCGAGC-3'
5'-CAACGGTGACTTTATCGCCATTCC-3'
Claims (5)
1. one kind is extracted purifying Escherichia coli O157: the H7 genomic dna is characterized in that utilizing carboxylated magnetic nano-particle to extract Escherichia coli O157:H7 genomic dna as the preparation method of PCR reference material;
Process step is:
(1) cultivation of Escherichia coli O157: H7:
By traditional cultural method Escherichia coli O157: H7 being increased bacterium cultivates;
(2) preparation of surperficial carboxylated magnetic nano-particle:
The magnetic nano-particle of preparation carboxyl modified is used to adsorb Escherichia coli O157: the H7 genomic dna;
(3) pathogenic micro-organism Escherichia coli O157: the extraction of H7 genomic dna;
(4) the Escherichia coli O157 that obtains with step (2): the H7 genomic dna is crossed Sephadex G-200 separator column and is carried out separation and purification;
(5) the Escherichia coli O157 behind the purifying that obtains with step (4): the H7 genomic dna is measured the ICP-MS calculating concentration;
(6) pcr amplification product with the DNA behind step (4) purifying carries out the PCR detection, and agarose electrophoresis detects Escherichia coli O157: the purpose band of H7.
2. extraction purifying Escherichia coli O157 according to claim 1: the H7 genomic dna is characterized in that as the preparation method of PCR reference material the bacterium that increases of Escherichia coli O157: H7 is cultivated:
Escherichia coli O157: H7, promptly ATCC 35150, are seeded on the sorbyl alcohol Mai Kangkai SMAC flat board, cultivate 10h for 37 ℃, connect a ring again and are inoculated in 10mL improvement E.C Vulkamycin. PA-93 and increase bacterial context soup, cultivate 10h in 37 ℃.
3. extraction purifying Escherichia coli O157 according to claim 1: the H7 genomic dna is characterized in that the preparation of the magnetic nano-particle that the surface is carboxylated as the preparation method of PCR reference material:
1. get 0.65g FeCl
3, 0.4g trisodium citrate, 20mL terepthaloyl moietie, stirring reaction 10min;
2. get the 1.2g sodium-acetate and add 1. in the gained solution stirring reaction 20min; Again at 230 ℃ of following stirring reaction 10h;
3. in 2. gained solution, add the 20mL absolute ethyl alcohol, ultrasonic 10min, the centrifugal 10min of 6000rpm removes supernatant;
4. in 3. gained precipitates, add the 20mL deionized water, ultrasonic 10min, the centrifugal 10min of 6000rpm removes supernatant;
5. 4. add the 5mL deionized water in the gained deposition to above-mentioned steps, obtaining particle diameter is the surperficial carboxylated magnetic nano-particle of 350nm.
4. extraction purifying Escherichia coli O157 according to claim 1: the H7 genomic dna is characterized in that Escherichia coli O157 as the preparation method of PCR reference material: the extraction of H7 genomic dna:
1. get Escherichia coli O157: H7 improvement E.C Vulkamycin. PA-93 and increase bacterial context soup nutrient solution 1mL, the centrifugal 5min of 10000rpm removes supernatant;
2. 1. middle deposition is dissolved with 1mL TE damping fluid, add 100 μ L 10%SDS; Said TE damping fluid is the 50mM Tris-HCl damping fluid that pH 8.0 contains 10mM EDTA;
3. to 2. adding the surperficial carboxylated magnetic nano-particle for preparing in 20 μ L claim 1 steps (2) or the claim 3 in the gained solution;
4. the 6mol/L NaCl mixed solution that in 3. gained solution, adds 370 μ L w/v, 30% PEG 6000, room temperature reaction 15min;
5. 4. gained solution is put into the magnetic force frame, removes supernatant;
6. to 5. adding 1mL mass concentration 70% ethanol in the gained magnetic particle;
7. 6. gained solution is put into the magnetic force frame, removes supernatant;
8. to 7. adding the dissolving of 100 μ L TE damping fluids in the gained magnetic particle, 65 ℃ are reacted 10min, and said TE damping fluid is the 10mM Tris-HCl damping fluid that pH8.0 contains 1mM EDTA;
9. 8. gained solution is put into the magnetic force frame, gets supernatant, is the thick Escherichia coli O157 that extracts: the H7 genomic dna.
5. extraction purifying Escherichia coli O157 according to claim 1: the H7 genomic dna is characterized in that adopting Sephadex G-200 separator column to carry out separation and purification as the preparation method of PCR reference material:
Separator column: Sephadex G-200;
Moving phase: the TE damping fluid is the 10mM Tris-HCl damping fluid that pH8.0 contains 1mM EDTA;
Flow velocity: 0.5mL/min;
Detector: UV-detector, emission wavelength: 260nm;
Applied sample amount: extract DNA sample 1mL;
Begin to collect detached peaks from the sample of 6.5mL, that obtain is the Escherichia coli O157 behind the purifying: the H7 genomic dna.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103690997A (en) * | 2013-12-05 | 2014-04-02 | 南方医科大学珠江医院 | Kit for preparing acellularized liver scaffold and application method of kit |
| CN107190047A (en) * | 2017-05-22 | 2017-09-22 | 张鹏 | A kind of method of screening bacterium and the method using characteristic element six kinds of pathogenic bacteria of screening |
| CN108950029A (en) * | 2018-06-21 | 2018-12-07 | 齐鲁工业大学 | The multiple PCR detection primer and detection method of Escherichia coli O 157 |
Citations (2)
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|---|---|---|---|---|
| CN101845435A (en) * | 2010-06-11 | 2010-09-29 | 江南大学 | Method for extracting transgenic soybean DNA by using magnetic nanoparticles |
| CN102121002A (en) * | 2010-12-23 | 2011-07-13 | 江南大学 | Method for extracting bacterium genomic DNA by using magnetic nanoparticles |
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2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845435A (en) * | 2010-06-11 | 2010-09-29 | 江南大学 | Method for extracting transgenic soybean DNA by using magnetic nanoparticles |
| CN102121002A (en) * | 2010-12-23 | 2011-07-13 | 江南大学 | Method for extracting bacterium genomic DNA by using magnetic nanoparticles |
Non-Patent Citations (1)
| Title |
|---|
| 薛力刚: "大肠杆菌O157 :H7 核酸探针检测方法的建立", 《生物技术》, vol. 19, no. 6, 31 December 2009 (2009-12-31) * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103690997A (en) * | 2013-12-05 | 2014-04-02 | 南方医科大学珠江医院 | Kit for preparing acellularized liver scaffold and application method of kit |
| CN103690997B (en) * | 2013-12-05 | 2016-06-29 | 南方医科大学珠江医院 | For preparing test kit and the using method thereof of cell liver support |
| CN107190047A (en) * | 2017-05-22 | 2017-09-22 | 张鹏 | A kind of method of screening bacterium and the method using characteristic element six kinds of pathogenic bacteria of screening |
| CN108950029A (en) * | 2018-06-21 | 2018-12-07 | 齐鲁工业大学 | The multiple PCR detection primer and detection method of Escherichia coli O 157 |
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Application publication date: 20120404 |