CN109762807A - The kit and application method that DNA of bacteria extracts in a kind of meat - Google Patents
The kit and application method that DNA of bacteria extracts in a kind of meat Download PDFInfo
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- CN109762807A CN109762807A CN201910157397.8A CN201910157397A CN109762807A CN 109762807 A CN109762807 A CN 109762807A CN 201910157397 A CN201910157397 A CN 201910157397A CN 109762807 A CN109762807 A CN 109762807A
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Abstract
The invention belongs to meat products analysis detection fields, and in particular to the kit and application method that DNA of bacteria extracts in a kind of meat.Kit prepared by the present invention can be used for extracting bacterial genomes DNA under conditions of meat sample does not increase bacterium, the core reagent and absorption carrier of kit, including lysate A, the amino magnetic bead in conjunction with liquid B, cleaning solution C, eluent D, after Proteinase K and optimization.Application method includes: first to wash the bacterium in meat sample, is centrifuged with different rates；Take precipitating that lysate A is added, Proteinase K is incubated for；It takes supernatant that the amino magnetic bead optimized liquid B oscillation in conjunction with equivalent is added after centrifugation to mix, Magnetic Isolation is washed and dried with cleaning solution C, adds eluent D to be eluted, obtain DNA of bacteria in meat.The present invention quickly can extract DNA of bacteria from meat sample, be not necessarily to Zengjing Granule.The DNA of bacteria extracted can be used for PCR amplification or LAMP detection.
Description
Technical field
The invention belongs to meat products technical field of analysis and detection, and in particular to the examination that DNA of bacteria extracts in a kind of meat
Agent box and application method.
Background technique
With the improvement of people's life quality, main source of the pork as our people's meat product, consumption figure exist
Constantly rise.So in the entire pork production chain from farm to market, pork can all be caused to be polluted by pathogenic bacteria, influenced
Pork quality safety, can cause the generation of Amphixenosis or food poisoning, endanger human health after being eaten by the mankind.Therefore,
The monitoring for reinforcing pathogenic bacteria in pork, has great importance.Pollution pork causes the main pathogenic bacteria of food poisoning to include sand
Door Salmonella, enteropathogenic E. Coli, listeria monocytogenes etc..
Polymerase chain reaction (PCR) is a kind of general DNA detection technique, extensive since the eighties comes out
Applied to food safety detection and clinical diagnosis.Round pcr is also used for the detection of pathogenic bacteria in pork in recent years, but due to pig
Cell containing meat itself in meat sample product and contain many kinds of substance such as fat, protein and carbohydrate, currently used DNA extracting
The more difficult DNA to bacterium in meat of technology is directly extracted, it usually needs is carried out Zengjing Granule to pork sample, can be obtained
The DNA of wherein bacterium is obtained, Zengjing Granule keeps detection time elongated, and the time for entering market to live fresh pork impacts, also not
It is enough to cope with the food public safety health event of some bursts.
Magnetic Nano material is a kind of novel high polymer material, not only has special magnetic response feature, but also specific surface
Product is big, can modify its surface, and then the property of can choose combines some objects.Amino magnetic bead belongs to magnetic Nano material
One kind, core Fe3O4Magnetic nano-particle, pan coating layer of silicon dioxide, functional amino group, main in connection
Identification absorption is carried out to DNA by electrostatic force and hydrogen bond action, recycle the effect of externally-applied magnetic field can be by DNA from again
It is separated in miscellaneous sample system.The preparation step of amino magnetic bead is easy, low in cost, can be applied to DNA in different samples
Extraction.
Summary of the invention
Present invention aims to overcome that the defect of the prior art, providing one kind can be without Zengjing Granule just by bacterium
The kit and application method that DNA is extracted from pork sample.
To achieve the above objectives, the present invention provides the synthetic method of a kind of nanometer of amino magnetic bead and in pork sample
The method that bacterial nucleic acid extracts, specific technical solution are as described below；
A kind of kit extracting bacterial genomes DNA from meat includes following reagent or carrier, wherein the examination
Agent includes lysate A, in conjunction with liquid B, 20 μ g/ μ L of cleaning solution C, eluent D and Proteinase K, and the carrier is nanometer amino magnetic
Pearl；
Wherein:
The proportion of lysate A are as follows: 4mol/L guanidine hydrochloride, EDTA10mmol/L, NaCl 5mmol/L, 1%Triton X-
100, pH 7.0；
It is dehydrated alcohol in conjunction with liquid B；
The ethanol solution that cleaning solution C is 75%；
Eluent D is the TE buffer of pH8.0；
Nanometer amino magnetic bead is sphere, diameter 80nm, center Fe3O4Magnetic nano-particle, outer layer is by silica institute
Package；One amino magnetic bead can wrap up one to several Fe3O4Magnetic nano-particle wraps up a Fe3O4Magnetic nano-particle
Amino magnetic bead be in sphere, regular shape, wrap up two or more Fe3O4Amino magnetic bead shape be in indefinite form；
The nanometer amino magnetic bead is prepared as follows to obtain:
(a) naked Fe3O4The synthesis of magnetic nano-particle: the FeCl of 15mL 0.8mol/L is taken under the protection of nitrogen3With
The FeSO of 10mL0.8mol/L4·7H2300mL deoxidized water is added in O, is stirred, and the sodium hydroxide of 50mL lmol/L is added, instead
After answering 30min, 67 DEG C are warming up to, 30min is aged, reaction solution is cooled to room temperature, precipitating is washed till neutrality with deoxidized water, is being revolved
Turn to carry out Magnetic Isolation on magnetic frame, naked Fe is made3O4Magnetic nano-particle；
(b) Fe of Silica-coated3O4The synthesis of magnetic nano-particle: the Fe of 270mg is taken3O4Magnetic nano-particle in
In 8.5mL deoxidized water, vortex 2min is ultrasonically treated 10min for the first time；Measure 116mL hexamethylene, 26mL n-hexyl alcohol, 30mL
Triton X-100 forms the microemulsion of Water-In-Oil, is vortexed and carries out second of ultrasonic treatment 10min；Then by Fe3O4Magnetism is received
Grain of rice molecular disperse solution is added in reverse micro emulsion, continues to be vortexed and carry out third time ultrasonic treatment 20min, stand at room temperature
For 24 hours, removal precipitating, obtains the Fe of Silica-coated3O4Magnetic nano-particle；
(c) amido modified silicon packet Fe3O4The synthesis of magnetic nano-particle: obtained microemulsion system is carried out the 4th time
It is ultrasonically treated 5min, then goes to and is stirred at room temperature, 1.2mL ethyl orthosilicate is added dropwise after 30min dropwise, is continued after stirring 30min
25% ammonium hydroxide of 2.5mL is added dropwise, is followed by stirring for 48h, 20mL acetone is added, 10min, dehydrated alcohol weight are centrifuged at 5000r/min
It is outstanding, Magnetic Isolation is carried out on rotation magnetic force frame, dehydrated alcohol washs three times, and 0.1mol/L salt acid soak is used after Magnetic Isolation
36h, carries out Magnetic Isolation on rotation magnetic force frame, and milli-Q water three times, takes the Fe of 200mg Silica-coated3O4Magnetism is received
Rice corpuscles is added in 10mL ultrapure water and 100mL dehydrated alcohol, carries out the 5th ultrasonic treatment 10min, stirs 20min at room temperature
The 3- aminopropyl triethoxysilane of l mL is slowly added dropwise afterwards, continues to be stirred to react 12h；It is alternately washed with dehydrated alcohol and ultrapure water
It washs three times, by Magnetic Isolation, amino magnetic bead is made；
The parameter of above-mentioned ultrasonic treatment are as follows: alternating voltage 220V, output power 100W, supersonic frequency 20KHz, amplitude are adjusted
Range is 60%.
The method that applicant provide a kind of to extract genomic DNA from meat bacterium, the method are included in meat
Sample without increase bacterium in the case where bacterial genomes DNA contained therein is extracted, steps are as follows:
(1) in meat sample bacterium extraction, take 10g meat sample to be placed in valve bag, the sterilizing 0.01mol/L PBS of 10mL be added
Buffer (pH 7.2) closes sack, sufficiently beating 5min, gained meat sample liquid is transferred in 15mL centrifuge tube, 1000r/
Min is centrifuged l min, and gained supernatant is transferred in 10mL centrifuge tube, and it is heavy to collect after 6000r/min centrifugation 20min at 4 DEG C
It forms sediment；
(2) extraction of bacterial genomes DNA: resulting precipitating is walked upwards and is resuspended with the lysate A of 200 μ L, 5 μ L20 are added
The Proteinase K of μ g/ μ L is immediately placed in cooled on ice after incubation, and 12000r/min is centrifuged 5min at 4 DEG C, draws supernatant liquor, adds
Enter the combination liquid B of g nanometers of amino magnetic beads of 40 μ and equivalent volumes, oscillation mixes, and uses after carrying out Magnetic Isolation on rotation magnetic force frame
Cleaning solution C is washed twice, and after drying, 100 μ L eluent D is added, 10min is placed at 65 DEG C, be during which constantly vortexed with abundant
Release is incorporated in nanometer DNA on amino magnetic bead surface, Magnetic Isolation is carried out on rotation magnetic force frame, gained supernatant is bacterium base
Because of a group DNA,
Wherein:
Lysate A are as follows: guanidine hydrochloride 4mol/L, ethylenediamine tetra-acetic acid 10mmol/L, NaCl 5mmol/L, 1%Triton X-
100；pH7.0；
It is dehydrated alcohol in conjunction with liquid B；
The ethanol solution that cleaning solution C is 75%；
Eluent D is the TE buffer of pH8.0；
0.01mol/L PBS buffer solution are as follows: NaCl 8.0g, KCl 0.2g, Na2PO4·12H2O 2.9g, KH2PO4
0.2g, pH value 7.2, ultrapure water is settled to 1000mL.
Above-mentioned warm bath condition are as follows: 56 DEG C of warm bath 30min, then 95 DEG C of 10min.
Concrete application method of the invention are as follows:
Bacterium in meat is preferably extracted in order to reach, the present invention in meat sample bacterium carry out repeatedly be centrifuged into
Row enrichment, concrete operation step are as follows: it is slow that meat piece (such as pork) to be processed is added to the 0.01mol/L PBS containing 60mL sterilizing
In fliud flushing (pH value 7.2), be vortexed mix lmin after impregnate 5min, at 1000r/min be centrifuged l min to remove big impurity,
Supernatant is gone in 1.5mL centrifuge tube, 5min is centrifuged at 10000r/min, collects precipitating.
In order to discharge the nucleic acid in bacterium completely, type, cracking temperature of the present invention using orthogonal experiment to lysate
Degree is explored with pyrolysis time.
Present invention optimizes the best combination times of amino magnetic bead and nucleic acid.
In order to improve the nucleic acid amount of amino magnetic bead absorption, the present invention has also carried out cleaning agent type and cleaning way excellent
Change.
The most suitable additive amount of amino magnetic bead when the present invention also explores bacterial nucleic acid in absorption meat.
Kit of the invention can be extracted in DNA of bacteria in vitro meat and be applied.
More detailed technical solution refers to " specific embodiment ".
Detailed description of the invention
Fig. 1: the transmission electron microscope image of made different amino magnetic beads in the embodiment of the present invention 1.Appended drawing reference
Illustrate: the scale of A figure, D figure in Fig. 1 is 100 μm；The scale of B figure, E figure in Fig. 1 is 200 μm；C figure, F figure in Fig. 1
Scale is 500 μm.
Fig. 2: influence of the different time to amino magnetic bead adsorption of DNA effect in the embodiment of the present invention 2.Description of symbols:
1: first time experimental result；2: second experimental results；3: third time experimental result.
Fig. 3: the glue figure of the most suitable additive amount of amino magnetic bead when adsorbing bacterial nucleic acid in meat in the embodiment of the present invention 2.Attached drawing
Description of symbols: swimming lane M:DL2000DNA Marker；Swimming lane 1: positive control；Swimming lane 2: negative control；Swimming lane 3-10: amino magnetic
Pearl additive amount is successively are as follows: 1,3,5,8,10,13,15,20 μ g/g.
Fig. 4: amino magnetic bead-PCR and heat boil-PCR detection detection of Salmonella artificial contamination pork sample result.Appended drawing reference is said
Bright: the A figure in Fig. 4 is amino magnetic bead-PCR testing result；B figure in Fig. 4 is boiling method-PCR testing result.Swimming lane explanation:
Swimming lane M:DL2000DNA Marker；Swimming lane 1: positive control；Swimming lane 2: negative control；Swimming lane 3-10: Salmonella amount according to
It is secondary are as follows: 2 × 107, 2 × 106, 2 × 105, 2 × 104, 2 × 103, 2 × 102, 2 × 101, 2CFU/g；11: blank control.
Specific embodiment
The present invention is further specifically described combined with specific embodiments below, the features and advantages of the invention can be with description
And it is clearer.But protection scope of the present invention is not limited to following embodiments.Method therefor is such as without spy in following embodiments
Not mentionleting alone bright is laboratory conventional method.
The preparation of 1 nanometer of amino magnetic bead of embodiment
(1) naked Fe is synthesized3O4Magnetic nano-particle
The FeCl of 15mL 0.8mol/L is taken under the protection of nitrogen3With the FeSO of 10mL 0.8mol/L4·7H2O is added
300mL deoxidized water, is stirred, and the sodium hydroxide of 50mL lmol/L is added, and after reacting 30min, is warming up to 67 DEG C, ageing
Reaction solution is cooled to room temperature by 30min, precipitating is washed till neutrality with deoxidized water, in DynaMagTM- 50 rotation magnetic force framves
Magnetic Isolation is carried out in (Thermo Fisher, the U.S.), naked Fe is made3O4Magnetic nano-particle.
(2) in naked Fe3O4Layer of silicon dioxide is wrapped up outside magnetic nano-particle
Take the Fe of 270mg or so3O4Magnetic nano-particle carries out vortex 2min in 8.5mL deoxidized water, with 1500r/min,
Ultrasonic (voltage 220V, output power for the first time are carried out using Ultrasonic Cell Disruptor (Branson Sonifier 450, the U.S.)
100W, supersonic frequency 20KHz, amplitude adjustable range are 60%) to handle 10min；Measure 116mL hexamethylene, 26mL n-hexyl alcohol,
30mL Triton X-100 forms the microemulsion of Water-In-Oil, is vortexed with 1500r/min and carries out second of ultrasonic treatment
(parameter is with ultrasonic treatment for the first time) 10min；Then by Fe3O4Magnetic nano-particle disperses solution and is added in reverse micro emulsion, after
It is continuous to be vortexed and carry out third time ultrasonic treatment (parameter is with ultrasonic treatment for the first time) 20min, it is stored at room temperature for 24 hours, removal precipitating obtains
To Silica-coated Fe3O4Magnetic nano-particle.
(3) amido modified silicon packet Fe3O4The synthesis of magnetic nano-particle
The microemulsion system that upper step obtains is carried out at the 4th ultrasound using Ultrasonic Cell Disruptor (model and manufacturer are same as above)
It goes to and is stirred at room temperature after reason (parameter is with ultrasonic treatment for the first time) 5min, 1.2mL ethyl orthosilicate is added dropwise after 30min dropwise
(TEOS), continue that 25% ammonium hydroxide of 2.5mL is added dropwise after stirring 30min, be followed by stirring for 48h, be added 20mL acetone, 5000r/min from
Heart 10min, dehydrated alcohol are resuspended, Magnetic Isolation, and dehydrated alcohol washs three times, use after carrying out Magnetic Isolation on rotation magnetic force frame
0.1mol/L salt acid soak 36h, carries out Magnetic Isolation on rotation magnetic force frame, and milli-Q water three times, takes 200mg silica
The Fe of package3O4Magnetic nano-particle is added in 10mL ultrapure water and 100mL dehydrated alcohol, carries out the 5th ultrasonic treatment
10min is slowly added dropwise l mL 3- aminopropyl triethoxysilane (APTES) after stirring 20min at room temperature, continues to be stirred to react
12h.Alternately washing three times, carries out Magnetic Isolation on rotation magnetic force frame (condition is same as above) for dehydrated alcohol and ultrapure water, is made and receives
Rice amino magnetic bead.
Using the pattern of transmission electron microscope (H-7650 type, HITACHI, Japan) analysis sample, operating voltage is
200KV.After sample to be tested is diluted 10 times with 95% ethyl alcohol using Ultrasonic Cell Disruptor ultrasonic treatment (at the same ultrasound for the first time of parameter
Reason), keep sample particle evenly dispersed in ethanol, then take it is one after another drop of on copper mesh, after ethyl alcohol volatilization completely after tested.
The transmission electron microscope picture the result is shown in Figure 1 of amino magnetic bead, as seen from the figure, the darker little particle of black in ball center is Fe3O4It is magnetic
The gray shade part of nanoparticle, diameter about 10nm, outer layer covers is silica.
The optimization of the condition of 2 amino magnetic bead adsorption of DNA of embodiment
(1) optimization of lysate and cracking condition
The present embodiment has prepared three kinds of guanidine hydrochloride lysate, guanidinium isothiocyanate lysate and SDS lysate lysates respectively,
Formula is as described below.
Guanidine hydrochloride lysate: 4mol/L guanidine hydrochloride, 10mmol/L ethylenediamine tetra-acetic acid (EDTA), 5mmol/L NaCl, 1%
Triton X-100；pH 7.0.
Guanidinium isothiocyanate lysate: 4mol/L guanidinium isothiocyanate, 10mmol/L ethylenediamine tetra-acetic acid (EDTA), 25mmol/L
Tris-HCl, 1%Triton X-100；pH 7.0.
SDS lysate: 1.5% lauryl sodium sulfate (SDS), 0.5mol/L NaCl；pH 7.0.
Cracking temperature is selected as 37 DEG C, 65 DEG C and 95 DEG C, and pyrolysis time is selected as 10min, 20min and 30min.According to just
Test method(s) is handed over to be grouped, group result is shown in Table 1.
By Salmonella typhimurtum CVCC542 type strain using TSB culture medium (Britain OXOID, according to product description into
Row is prepared) Zengjing Granule 10h is carried out, it takes 500 μ L Salmonella typhimurtum bacterium solutions to carry out centrifugation 5min with 8000r/min later, abandons
Supernatant is added into gained precipitating and is split according to lysate used in difference group shown in table 1, cracking temperature and time
Solution；
Amino magnetic bead particle is sufficiently shaken up, the centrifuge tube for being placed in 1.5ml in right amount is taken, is placed on rotation magnetic force frame and carries out magnetic
Property separation (condition is with embodiment 1), adsorbed completely to amino magnetic bead, discard wherein liquid and (be sure not to touch amino during this
Magnetic bead)；Centrifuge tube is removed, 110 μ L dehydrated alcohols are added, vortex mixing is carried out with 1500r/min；
Cracked sample is transferred in ready amino magnetic bead mixed liquor, vortex mixing is carried out with 1500r/min
Afterwards, 1400r/min oscillation mixes 30min under normal temperature condition, carries out Magnetic Isolation (condition is with embodiment 1), abandons supernatant；
Ready cleaning solution is added, carries out Magnetic Isolation (the same embodiment of condition after carrying out vortex mixing with 1500r/min
1) supernatant, is abandoned, uncap dry 15min at room temperature, and ethyl alcohol is made to volatilize.
Detect DNA concentration using ultramicrospectrophotometer, carry out in group, between group retest three times, as a result such as table 1.
The grouping of 1 cracking condition orthogonal test of table and interpretation of result
The result shows that carrying out cracking 10min using guanidine hydrochloride lysate at 95 DEG C, effect is best.
(2) selection of cleaning solution and cleaning way
The present embodiment has prepared II two kinds of cleaning solution I, cleaning solution cleaning solutions respectively, and specific preparation method is as described below.
Cleaning solution I: being first made into 8mol/L guanidine hydrochloride mother liquor, according still further to cleaning solution mother liquor when use: dehydrated alcohol 1:1.4
Dehydrated alcohol is added in the ratio of (volume ratio).
II: 75% ethyl alcohol of cleaning solution.
Cleaning way is following three kinds: 1. being cleaned 2 times with cleaning solution I；2. being cleaned 2 times with cleaning solution II；3. with cleaning solution I
Cleaning 1 time；It is cleaned 1 time with cleaning solution II.DNA concentration and A260/280 and A260/ are measured using ultramicrospectrophotometer
230 value, in group, between group retest three times, as a result such as table 2.
The interpretation of result of the different cleaning ways of table 2
The result shows that it is best to clean 2 effects using cleaning solution II.
(3) optimization of nanometer amino magnetic bead and DNA binding time
Time of the nanometer amino magnetic bead in conjunction with DNA is optimized in the present embodiment, adsorption time is set in 1~
As a result 30min is shown in Fig. 2.
The result shows that binding time influences adsorption effect less, 10min to be selected as in the present embodiment.
(4) nanometer amino magnetic bead adsorbs the exploration of most suitable additive amount when bacterium in pork
The present embodiment explores most suitable additive amount when bacterium in nanometer amino magnetic bead absorption pork, with Salmonella typhimurium
Bacterium CVCC542 type strain is as experimental strain.
Picking salmonella typhimurium single bacterium colony is inoculated in TSB culture medium, then 37 DEG C of shaking table shaken overnight cultures are pressed
1% inoculum concentration switching TSB culture medium, 37 DEG C of shaken cultivation 2h, the method for plate culture count are counted.
PCR is detected as the pork sample of salmonella feminine gender in superclean bench, with ultraviolet lamp to the positive and negative of sample
15min is respectively irradiated in face, and by sample segment at the meat piece (totally 6 pieces) of 10g, meat piece is added to containing 60mL sterilizing 0.01mol/L pH
In 7.2 PBS buffer solution, be vortexed after mixing lmin with 1500r/min and impregnate 5min, will wherein 45mL liquid be transferred to
In 50mL centrifuge tube, the salmonella typhimurium bacterium solution of 5mL gradient dilution is added, it is big to remove that 1000r/min is centrifuged lmin
Impurity goes to supernatant in 1.5mL centrifuge tube, collects precipitating after 5min is centrifuged at 10000r/min.With 20 μ L pH7.0's
Guanidine hydrochloride lysate is resuspended, and the Proteinase K of 1 μ L, 20 μ g/ μ L is added, sets rapidly after 56 DEG C of incubations 30min, 95 DEG C of effect 10min
In cooled on ice, 12000r/min is centrifuged 5min, be added a certain amount of nanometer of amino magnetic bead (respectively 1,3,5,8,10,13,
15,20 μ g) and equivalent dehydrated alcohol, 1400r/min oscillation mix 10min, carry out Magnetic Isolation (condition with rotation magnetic force frame
With embodiment 1) after washed twice again with cleaning solution II, after drying, be added 50 μ L pH8.0 TE buffer, 65 DEG C placement
During which 10min is constantly vortexed with 1500r/min and is incorporated in nanometer DNA on amino magnetic bead surface with abundant release, with rotation
After magnetic frame carries out Magnetic Isolation (condition is with embodiment 1), gained supernatant is the bacterial genomes DNA that the present invention extracts.
The concentration of gained DNA is measured using ultramicrospectrophotometer, and 2 μ L is taken to carry out PCR detection, Ago-Gel electricity
Swimming map is shown in Fig. 3.
The result shows that the most suitable additive amount in nanometer amino magnetic bead absorption meat when salmonella typhimurium is 5~10 μ g/g.
PBS, that is, phosphate buffer preparation method of sterilizing 0.01mol/L pH 7.2 in the present embodiment are as follows: NaCl
8.0g, KCl 0.2g, Na2PO4·12H2O 2.9g, KH2PO40.2g adjusts pH value to 7.2 with 1mol/L NaOH, adds ultrapure
Water is settled to 1000mL, and 121 DEG C of high pressure steam sterilization 15min are spare.
The TE buffer method of pH8.0 in the present embodiment are as follows: Tris-base 6.057g is taken to be dissolved in 40mL distillation
In water, enriching HCl tune pH to 8.0 is settled to 50mL with ultrapure water, is configured to 1mol/L pH8.0Tris-HCl；Take 9.306g
EDTA·Na2It is scattered in 40mL distilled water, 10mol/L NaOH is added to promote to dissolve and adjusts pH to 8.0, is settled to 50mL (2) with water
Prepare 500mmol/L pH8.0EDTA；Take the 500mmol/L of the 1mol/L pH8.0Tris-HCl and 0.5mL of 2.5mL
PH8.0EDTA solution mixes, and spends and is settled to 250mL from water, be configured to the TE buffer of pH 8.0,121 DEG C of high steams go out
Bacterium 15min is spare.
Salmonella typhimurtum PCR reaction system in the present embodiment: 2 × PCR Mix, 12.5 μ L, upstream primer/5 '-
GCTCTTTCGTC TGGCATTA-3 ' (10 μm of ol/L) 0.5 μ L, downstream primer/5 '-CGGCAATAGCGTCACCTT-3 ' (10 μ
Mol/L) 0.5 μ L, 5 μ L of template DNA, adds aqua sterilisa to 25 μ L.PCR response procedures are as follows: enter after 95 DEG C of initial denaturation 5min and follow
Ring, 95 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 30s, 30 circulations, 72 DEG C of extension 5min, 4 DEG C of preservations.
3 distinct methods of embodiment extract the comparison of salmonella typhimurium DNA in pork sample
The method for repeating embodiment 1 and embodiment 2, i.e., mentioned respectively using nanometer amino magnetic bead method of the invention and boiling method
The DNA for taking salmonella typhimurium in artificial contamination's pork sample, be then added PCR reaction system as described in example 2 into
Row amplification, product are analyzed after agarose gel electrophoresis with gel imaging system, as a result see Fig. 4.
Utilize the pork sample of (nanometer amino magnetic bead method) of the invention detection salmonella typhimurium artificial contamination, sensitivity
Up to 2 × 103CFU/g；And boiling method+PCR detection method is used, sensitivity is 2 × 105CFU/g。
Boiling method extracts the operating procedure of DNA of bacteria in the present embodiment are as follows: artificial contamination's difference prepared by Example 2 is dense
Pork the sample 1mL, 8000r/min for spending salmonella typhimurium are centrifuged 5min, abandon supernatant.With 100 μ L pH8.0TE buffers
Bacterial sediment is resuspended, boiling water bath 10min places 5min on ice, and 8000r/min is centrifuged 5min, and gained supernatant is genome
DNA, -20 DEG C of short-term preservations are spare.
Claims (3)
1. it is a kind of from meat extract bacterial genomes DNA kit, which is characterized in that include following reagent or carrier,
Described in reagent include lysate A, in conjunction with liquid B, 20 μ g/ μ L of cleaning solution C, eluent D and Proteinase K, the carrier is
Nanometer amino magnetic bead；
Wherein:
The proportion of lysate A are as follows: 4mol/L guanidine hydrochloride, 10mmol/L EDTA, 5mmol/L NaCl, 1%Triton X-100,
pH 7.0；
It is dehydrated alcohol in conjunction with liquid B；
Cleaning solution C is 75% ethyl alcohol；
Eluent D is the TE buffer of pH8.0；
The nanometer amino magnetic bead is sphere, diameter 80nm, center Fe3O4Magnetic nano-particle, outer layer is by silica
It is wrapped up；One amino magnetic bead can wrap up one to several Fe3O4Magnetic nano-particle；Wrap up a Fe3O4Magnetic nano particle
The amino magnetic bead of son is in sphere, and regular shape wraps up two or more Fe3O4Amino magnetic bead shape be in indefinite form；
The nanometer amino magnetic bead is prepared in accordance with the following steps:
(a) naked Fe3O4The synthesis of magnetic nano-particle: the FeCl of 15mL 0.8mol/L is taken under the protection of nitrogen3With 10mL
The FeSO of 0.8mol/L4·7H2300mL deoxidized water is added in O, is stirred, and the sodium hydroxide of 50mL lmol/L, reaction is added
After 30min, 67 DEG C are warming up to, 30min is aged, reaction solution is cooled to room temperature, precipitating is washed till neutrality with deoxidized water, is being rotated
Magnetic Isolation is carried out on magnetic frame, and naked Fe is made3O4Magnetic nano-particle；
(b) Fe of Silica-coated3O4The synthesis of magnetic nano-particle: the Fe of 270mg is taken3O4Magnetic nano-particle is in 8.5mL
In deoxidized water, vortex 2min is ultrasonically treated 10min for the first time；Measure 116mL hexamethylene, 26mL n-hexyl alcohol, 30mL Triton
X-100 forms the microemulsion of Water-In-Oil, is vortexed and carries out second of ultrasonic treatment 10min；Then by Fe3O4Magnetic nano-particle
Disperse solution to be added in reverse micro emulsion, continues to be vortexed and third time is ultrasonically treated 20min, stand at room temperature for 24 hours, removal is heavy
It forms sediment, obtains the Fe of Silica-coated3O4Magnetic nano-particle；
(c) amido modified silicon packet Fe3O4The synthesis of magnetic nano-particle: by obtained microemulsion system slightly the 4th ultrasound
It goes to and is stirred at room temperature after processing 5min, 1.2mL ethyl orthosilicate is added dropwise after 30min dropwise, continue to be added dropwise after stirring 30min
25% ammonium hydroxide of 2.5mL is followed by stirring for 48h, and 20mL acetone is added, and 10min is centrifuged at 5000r/min, and dehydrated alcohol is resuspended,
Magnetic Isolation is carried out on rotation magnetic force frame, dehydrated alcohol washs three times, 0.1mol/L salt acid soak 36h is used after Magnetic Isolation,
Magnetic Isolation is carried out on rotation magnetic force frame, milli-Q water three times, takes the Fe of 200mg Silica-coated3O4Magnetic nano particle
Son is added in 10mL ultrapure water and 100mL dehydrated alcohol, the 5th ultrasonic treatment 10min, slowly drips after stirring 20min at room temperature
The 3- aminopropyl triethoxysilane for adding l mL, continues to be stirred to react 12h；It is alternately washed three times with dehydrated alcohol and ultrapure water,
By Magnetic Isolation, amino magnetic bead is made；
The parameter of above-mentioned ultrasonic treatment are as follows: alternating voltage 220V, output power 100W, supersonic frequency 20KHz, amplitude adjustable range
It is 60%.
2. a kind of method for extracting genomic DNA from meat bacterium, which is characterized in that in feelings of the meat sample without increasing bacterium
Bacterial genomes DNA contained therein is extracted under condition, it is characterised in that the following steps:
(1) in meat sample bacterium extraction, take 10g meat sample to be placed in valve bag, be added 10mL sterilizing 0.01mol/L pH 7.2
PBS buffer solution, close sack, sufficiently beating 5min, gained meat sample liquid is transferred in 15mL centrifuge tube, 1000r/min from
Heart l min, gained supernatant is transferred in 10mL centrifuge tube, is collected and is precipitated after 6000r/min centrifugation 20min at 4 DEG C；
(2) extraction of bacterial genomes DNA: resulting precipitating is walked upwards and is resuspended with the lysate A of 200 μ L, 5 μ L, 20 μ g/ is added
The Proteinase K of μ L is immediately placed in cooled on ice after 56 DEG C of incubations 30min, 95 DEG C of effect 10min, and 12000r/min is centrifuged at 4 DEG C
5min draws supernatant liquor, the combination liquid B of g nanometers of amino magnetic beads of 40 μ and equivalent volumes is added, oscillation mixes, in rotation magnetic force
It is washed twice after carrying out Magnetic Isolation on frame with cleaning solution C, after drying, 100 μ L eluent D is added, are placed at 65 DEG C
During which 10min is constantly vortexed and is incorporated in nanometer DNA on amino magnetic bead surface with abundant release, magnetic is carried out on rotation magnetic force frame
Property separation, gained supernatant is bacterial genomes DNA,
Wherein:
Lysate A are as follows: guanidine hydrochloride 4mol/L, ethylenediamine tetra-acetic acid 10mmol/L, NaCl5mmol/L, 1%Triton X-100；
pH7.0；
It is dehydrated alcohol in conjunction with liquid B；
The ethanol solution that cleaning solution C is 75%；
Eluent D is the TE buffer of pH8.0.
3. kit described in claim 1 extracts the application in DNA of bacteria in vitro meat.
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| CN111804280A (en) * | 2020-06-11 | 2020-10-23 | 中国科学院合肥物质科学研究院 | Method for preparing magnetic nano amino compound and nucleic acid extraction method |
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