CN102321762A - Method for rapid detection of listeria monocytogenes with high sensitivity and kit thereof - Google Patents
Method for rapid detection of listeria monocytogenes with high sensitivity and kit thereof Download PDFInfo
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Abstract
The invention discloses a method for rapid detection of listeria monocytogenes with high sensitivity and a kit thereof, and the method comprises the following steps: (1) lysing biological cells to release DNA; (2) mixing the solution containing the biological DNA and magnetic nano-particles which can be bonded with the DNA, wherein the magnetic nano-particles are gamma-Fe2O3 nano-particles coated by carboxyl-modified silica, allowing the DNA to be bonded onto the surfaces of the magnetic nano-particles under the action of isopropanol, collecting the magnetic nano-particles under the action of an external magnetic field to obtain DNA-bonded magnetic nano-particles; (3) adding water, performing magnetic separation, collecting the aqueous solution to obtain the separated biological DNA solution; (4) performing PCR amplification by a specific primer of a target organism with the DNA solution as a template. The whole detection process of the invention only need 2 hours; the detection limit of listeria monocytogenes is about 1 CFU/25 g (mL); the time required is short; the sensitivity is high; and the results are reliable.
Description
Technical field
The present invention relates to field of biological detection, specifically disclose a kind of method and test kit thereof of highly sensitive rapid detection Listeria monocytogenes.
Background technology
In recent years, the food-borne pathogens Fast Detection Technique just develops rapidly, and passed through by ten one Standing Committees of the National People's Congress seven meeting in " the People's Republic of China's food safety method " in February, 2009.The food-borne pathogens detection is to the raw material of food and the pathogenic bacterium in the environment such as process for processing, storage separate or in situ detection.Food-borne pathogens rapid detection method therefor comprises it being immunology, biological chemistry, biophysics etc.
Listeria monocytogenes is unique generally acknowledged listeria bacteria that can cause human diseases.It is a kind of common soil bacteria, behind contaminated food, also can cause serious food poisoning.As a kind of pathogenic bacteria of zoonosis, can cause the infection of people, animal, mainly show as septicemia, meningitis and monocytosis etc.Lee Salmonella that singly increases that exists in the food has danger to the mankind's safety, but growth and breeding still in 4 ℃ environment especially is one of refrigerated food The main pathogenic fungi of threatening human health.
For the detection of Listeria monocytogenes, there are national standard " check of GB 4789.30-2010 monocyte hyperplasia listeria spp " and inspection and quarantining for import/export industry standard " monocyte hyperplasia listeria spp detection method in the SN/T0184.1-2005 import and export food " in China.
Present widely used pathogenic bacterium detection method mainly contains four kinds; All be applicable to the detection of Listeria monocytogenes: plate culture of isolated counting process; The polymerase chain reaction (polymerase chain reaction, PCR), the fluorescent PCR method of inspection and ELISA EUSA.Plate isolated and identified method is that the methods such as bacterium cultivation, separation, biochemical identification that increase of using microbe check are carried out qualitative and quantitative check to the food-borne pathogens that possibly exist in the milk powder.Being characterized in reliable and stable, is the most ripe at present, uses the most extensively, as the method for inspection of benchmark, but exists detection time long, and process is loaded down with trivial details, is difficult to the problem of isolation identification to operator's technical requirements height and to particular serotype.(Polymerase Chain Reaction is a kind of Protocols in Molecular Biology PCR), is used to amplify specific dna fragmentation in the PCR polymerase chain reaction.Can regard the outer special dna replication dna of organism as.Sample (after pre-treatment increases bacterium) extracts DNA, is that template is carried out pcr amplification with the DNA that extracts.Through agarose gel electrophoresis check PCR product whether the characteristic band is arranged, thereby carry out quick test whether polluting food-borne pathogens in the sample.With round pcr detect harmful microorganism have high specificity, highly sensitive and easy and simple to handle, advantage such as save time.Fluorescent PCR adds a special oligonucleotide fluorescent probe on the regular-PCR basis; The PCR product is carried out mark to be followed the tracks of; The real time and on line monitoring reaction process; Can analyze product in conjunction with corresponding software, calculate the original template of testing sample, can be through measuring microbe population in the radioactive intensity examination sample.But the PCR reaction receives the influence of sample situation bigger, and in the inspection of food source property harmful microorganism, the composition in the food (carbohydrate, acids, materials such as grease) is understood normally carrying out of disturbance reponse especially.And the environment that detects, the intermediate treatment link also can be brought some PCR reaction suppressors.Thereby make the PCR reaction present higher false positive and false negative rate.ELISA, i.e. EUSA has very high susceptibility and repeatability; But can only detect a kind of mark at every turn, can not satisfy the needs that multiple checking matter detects simultaneously, and ELISA method cost complex operation; Test kit costs an arm and a leg, and a large amount of uses will produce very high cost burden.
Summary of the invention
The technical problem that the present invention will solve is exactly loaded down with trivial details to prior biological detection method testing process, and the deficiency that sensitivity is not high provides a kind of method and test kit thereof of highly sensitive rapid detection Listeria monocytogenes.
The present invention aims to provide a kind of method of highly sensitive rapid detection Listeria monocytogenes.
The technical scheme that the present invention solves the problems of the technologies described above is:
A kind of method of highly sensitive rapid detection biological species comprises below the step:
(1) the biomass cells cracking is discharged DNA, obtain containing the solution of this biology DNA;
(2) with the solution that contains biological DNA of step (1) gained with can mix with DNA bonded magnetic nanoparticle, this magnetic nanoparticle is that the silicon-dioxide of carboxyl modified wraps up γ-Fe
2O
3Nano particle, under the effect of Virahol, DNA is attached to the magnetic nanoparticle surface, is adding under the action of a magnetic field, collects magnetic nanoparticle, is the magnetic nanoparticle that has combined DNA;
(3) with step (2) collect gained combination add water in the magnetic nanoparticle of DNA, magnetic resolution is collected the aqueous solution, promptly obtains the dna solution of isolating biology;
(4) dna solution that obtains with step (3) is a template, utilizes the Auele Specific Primer of target organism, carries out pcr amplification.
The method of cracking biomass cells is common conventional lysis method in the step (1).Preferable, can be pyrolysis method, biological sample to be boiled in water, i.e. lysis discharges DNA.
Can adopt in the step (2) existing various can with DNA bonded magnetic nanoparticle, the preferably amino silicon-dioxide of carboxyl modified parcel γ-Fe
2O
3Nano particle, better is prepared by the method for the invention.In aqueous dna, add this magnetic nanoparticle and Virahol, can make DNA be attached to the magnetic nanoparticle surface, thereby go out the magnetic nanoparticle of this combination DNA through magnetic resolution.Quantity of isopropanol is preferable is 19 times of aqueous phase solution volume, and DNA is adsorbed in the magnetic nanoparticle surface in this case.
Step (3) though in add entry DNA and come off from the magnetic nanoparticle surface, be dissolved in the water, thereby obtain dna solution through magnetic resolution.
Adopting the Auele Specific Primer of existing any biology in the step (4) is primer, comes specific amplification target dna fragment.Preferable, for example adopt listerial Auele Specific Primer to increase, thereby detect in the dna solution whether contain listerial DNA.Obtaining containing the listeria bacteria in the biology then to be measured under the situation of amplified production, be positive findings.Under the situation that does not obtain amplified production, do not contain the listeria bacteria in the biology then to be measured, be negative findings.Present method can be set up positive control, negative control or blank simultaneously.Preferred Hly of said Auele Specific Primer and Iap.Pcr amplification condition optimization: 95 ℃ of 1min; 95 ℃ of 30s, 58.7 ℃ of 20s, 72 ℃ of 20s, 30 circulations; 72 ℃ of 5min; 4 ℃ of preservations.
Hly：CCGCCTGCAAGTCCTAA/ACAGGAAGAACATCGGGT，
Iap：GATAAAGCCCAAATAGT/GGACTACTGTTGACGCAA。
A preferred version of the present invention is: a kind of method of highly sensitive rapid detection Listeria monocytogenes may further comprise the steps:
(1) testing sample is placed centrifuge tube, the centrifugal 1min of 10000r/min, abandoning supernatant; With the deionized water centrifuge washing of sterilization, remove impurity; Deionized water suspended sediment with sterilization boils 10min with suspension-s in boiling water bath, the centrifugal 1min of 10000r/min makes biomass cells cracking in the sample, discharges DNA, gets its supernatant;
(2) supernatant of getting step (1) gained is in centrifuge tube; To the magnetic nanoparticle and the Virahol that wherein add specificity combination DNA of the present invention; Quantity of isopropanol is 19 times of aqueous phase solution volume, and DNA is adsorbed in the magnetic nanoparticle surface in this case;
(3) adding under the action of a magnetic field, collecting magnetic nanoparticle,, adding deionized water, adding under the action of a magnetic field, collecting elutriant, with specified conditions PCR check Listeria monocytogenes with 98% washing with alcohol decon.
It is kernel with the ferriferrous oxide nano-particle preferably that described specificity combines the magnetic nanoparticle of DNA, is shell with silicon-dioxide, the finishing carboxyl.
Said pcr amplification the primer is preferred:
Hly：CCGCCTGCAAGTCCTAA/ACAGGAAGAACATCGGGT；
Iap：GATAAAGCCCAAATAGT/GGACTACTGTTGACGCAA。
Said pcr amplification program is: 95 ℃ of 1min; 95 ℃ of 30s, 58.7 ℃ of 20s, 72 ℃ of 20s, 30 circulations; 72 ℃ of 5min; 4 ℃ of preservations.
The system of said pcr amplification is: 10 * PCR Buffer (2.5 μ l), Blend Taq-Plus (0.5 μ l), template (2.0 μ l), primer (totally 8.0 μ l), dNTPs (2.0 μ l), ddH
2O (10.0 μ l).
Step (2) and (3) described externally-applied magnetic field are 1000～5000Gs.
Specificity of the present invention combines the preparation method of the magnetic nanoparticle of DNA, may further comprise the steps:
(i) Fe
2+And Fe
3+Make γ-Fe with NaOH solution mixing co-precipitation
2O
3Nano particle, this particle of silicon-dioxide parcel is modified amino simultaneously, gets amido modified magnetic nanoparticle:
Preparation FeSO
47H
2O, FeCl
36H2O and NaOH solution mix, and under the sodium hydroxide solution condition, react after scouring, and drying is pulverized.Be added to and added tensio-active agent n-hexyl alcohol and hexanaphthene system, ultrasonic to stable, the dropping strong aqua adds the tetraethoxy reaction and accomplishes.The washing nano particle after the high-temperature calcination, reacts in alcoholic solution with aminoethyl aminopropyl YSR 3286 (AEAPS) under externally-applied magnetic field, washing under externally-applied magnetic field, drying.Leave standstill 12h and make the particle ageing.After using the magnetic resolution post to separate, wash particle with ethanol, and 90 ℃ of oven dry 5h, the organism of removal particle surface obtains the magnetic nano-particle that silicon-dioxide wraps up.With the magnetic nano-particle of silicon-dioxide parcel and 0.1mL N-(2-amino-ethyl)-3-aminopropyl trimethyl silane co-dispersed in the mixing solutions of methyl alcohol/USP Kosher; Ultra-sonic dispersion is even; Use the magnetic resolution post to separate behind the reaction 24h, use ethanol and distilled water wash respectively, place vacuum drying oven dry; Room temperature preservation is taken out in dry back, obtains amido modified magnetic nanoparticle.
Described amido modified magnetic nanoparticle particle diameter is 320nm～460nm.
Described externally-applied magnetic field is 1000～5000Gs.
The mass concentration of the magnetic nanoparticle of described silicon-dioxide parcel and the ratio of aminoethyl aminopropyl YSR 3286 (AEAPS) volume are 1mg/ml: 0.025ml to 1mg/ml: 0.05ml.
(ii) carboxylated PEG combines with amido modified magnetic nanoparticle, gets the magnetic nanoparticle of carboxyl modified:
Magnetic nanoparticle and carboxylated PEG that 1mg is amido modified are scattered in the aqueous solution, add N, N-DIC (DIC); Stirred overnight; Separating, washing under externally-applied magnetic field obtains the magnetic nanoparticle of carboxyl modified, is the magnetic nanoparticle that specificity combines DNA.
Described externally-applied magnetic field is 1000～5000Gs.
Said carboxylated PEG molecular weight is 400Da to 700Da..
Described amido modified magnetic nanoparticle and carboxylated PEG mass ratio are 10: 1 to 20: 1.
The present invention also provides a kind of test kit of highly sensitive rapid detection Listeria monocytogenes, comprising:
(1) specificity combines the magnetic nanoparticle of DNA, and this magnetic nanoparticle is the silicon-dioxide parcel γ-Fe of carboxyl modified
2O
3Nano particle;
(2) primer Hly and Iap,
Hly：CCGCCTGCAAGTCCTAA/ACAGGAAGAACATCGGGT，
Iap：GATAAAGCCCAAATAGT/GGACTACTGTTGACGCAA。
This test kit also comprises (3) 10 * PCR Buffer; DNTP; With the Taq archaeal dna polymerase.
Raw material that the present invention is used or reagent except that specifying, all commercially available getting.
The required time of the whole testing process of the present invention is merely 2 hours, and the detection limit of Listeria monocytogenes is about 1CFU/25g (mL), and required time is short, and highly sensitive in simple PCR method, the result is more reliable.As replenishing of existing method, can be widely used at detection range.
Description of drawings
Below in conjunction with description of drawings characteristic of the present invention and beneficial effect.
Fig. 1 is that Listeria monocytogenes 1CFU/25g (mL) genomic dna is through present method detected result electrophorogram.1 is 1000bp DNA Mark; 2 is salmonella NCTC 6017 strains contrasts; 3 is seized Listeria monocytogenes ATCC13932 strain; Hly gene amplification length 308bp wherein, iap gene amplification length 505bp; 4 is streptococcus aureus ATCC 29213 strains contrasts.This results sample 3 is positive.
Embodiment
The invention provides use DNA specific nano magnetic and extract the combined utilization strategy that the DNA of particle extraction Listeria monocytogenes detects through PCR again, thus the means of abundant detection Listeria monocytogenes.
The present invention at first prepares amido modified nanometer and extracts particle, combines with carboxylated PEG then, and the DNA specific nano magnetic that obtains carboxyl modified is extracted particle, and this material has magnetic and has biocompatibility, can specific adsorption DNA.
Adopt pyrolysis method to make Listeria monocytogenes cracking in the sample, disengage the DNA of Listeria monocytogenes in the sample, under the effect of alcohol; Making itself and DNA specific nano magnetic extract particle combines; Adding under the action of a magnetic field, collecting above-mentioned magnetic particle, discarding mixed solution; The water dissolving DNA obtains carrying out PCR check, electrophoresis observation judged result as template by specified conditions behind the mother liquor.
Further specify the present invention with embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer." room temperature " described in the embodiment is meant the temperature of the operation room that makes an experiment, and is generally 25 ℃.
25ml 50mg/ml FeSO
47H
2O solution and 25ml 50mg/ml FeCl
36H
2O solution is dissolved in 500ml zero(ppm) water, ultrasonic limit, 40 ℃ of water bath with thermostatic control limits mechanical stirring, and Dropwise 5 0ml 0.1mol/L sodium hydroxide diluting soln leaves standstill afterwards, pours beaker into, and after water and the washing with alcohol, 75 ℃ of following dry 3h grind respectively.4: 1: 10 by volume ratio is joined hexanaphthene, TritonX-100 and n-hexyl alcohol mixing solutions, and is ultrasonic, powder after the grinding of above-mentioned solution of 100mL and 50mg; Ultrasonic, get 400 μ L, 28% strong aqua and drip, add tetraethoxy (TEOS) 2ml; Reaction 24h leaves standstill 48h.Use magnetic resolution post adsorbed product, clean, 90 ℃ of oven dry 5h with ethanol.20mg/ml product and 0.5mL N-(2-amino-ethyl)-3-aminopropyl trimethyl silane are dissolved in methyl alcohol and USP Kosher mixed solution (volume ratio of methyl alcohol and USP Kosher 4: 1); Ultrasonic 6h; Use magnetic resolution post adsorbed product; Clean with ethanol, 40 ℃ of dryings promptly get amido modified nanometer and extract particle.The nanometer that 20mg is amido modified is extracted particle and the carboxylated PEG of 2mg 400Da is scattered in the 50ml deionized water; Add 2.5mgN; N-DIC (DIC), stirred overnight is used magnetic resolution post adsorbed product; Use deionized water wash, obtain carboxyl modified DNA specific nano magnetic and extract particle.
Embodiment 2DNA magnetic resolution is extracted
Material: Listeria monocytogenes ATCC13932 strain, Salmonellas NCTC 6017 strains, the above bacterial strain of streptococcus aureus ATCC 29213 strains is to be bought and preserves in microbial room of Shanghai Deno Testing Service Co., Ltd..Milk powder is provided by Shanghai Deno Testing Service Co., Ltd..LB1 substratum, LB2 substratum and broth medium are provided by Shanghai Deno Testing Service Co., Ltd..
Add and detect: get Listeria monocytogenes ATCC13932 strain and pollute milk powder, get the 25g sample and cultivate by LB secondary enrichment, dilute different gradients, as testing sample group 1, plate count is also pressed GB/T 4789.30.2010 checking simultaneously.Get Salmonellas NCTC 6017 strains, inoculation 5ml nutrient broth, 37 ℃ of 150r/min overnight cultures are diluted different gradients with aseptic double-distilled water, and as testing sample group 2, plate count is also pressed GB 4789.4-2010 checking simultaneously.Get streptococcus aureus ATCC 29213 strains, inoculation 5ml 7.5%NaCl broth culture, 37 ℃ of 150r/min overnight cultures are diluted different gradients with aseptic double-distilled water, and as testing sample group 3, plate count is also pressed GB 4789.10-2010 checking simultaneously.
With the following respectively operation of testing sample among above-mentioned 3 groups of the 1ml: place the centrifugal 1min of centrifuge tube 10000r/min, abandoning supernatant; With the deionized water wash of 200 μ l sterilization 2 times; Deionized water suspended sediment with 100 μ l sterilization boils 10min with suspension-s in boiling water bath, the centrifugal 1min of 10000r/min gets its supernatant, and is subsequent use.Get supernatant in the 1.5ml centrifuge tube,, shake to wherein adding the carboxyl modified DNA specific nano magnetic extraction particle 0.1ml that embodiment 1 obtains, vortex, fully mixing adds 1ml 94% aqueous isopropanol again, vortex, concussion, abundant mixing, placement 2min; Place on the magnetic force frame, treat that particle is hunted down fully after, discard clear liquid; Add 1ml 98% washing with alcohol one time, discard clear liquid; The deionized water that adds 200 μ l was placed 10 minutes, caught magnetic bead with the magnetic force frame, treat that particle is hunted down fully after, the sucking-off clear liquid is DNA elutriant to be measured.
The DNA elutriant to be measured of directly getting embodiment 2 preparations adopts primer Hly and Iap as template, carries out the multiplex PCR amplification, the SK2491-PCR of Sangon Biotech (Shanghai) Co., Ltd. amplification kit that pcr amplification adopts.
Hly：CCGCCTGCAAGTCCTAA/ACAGGAAGAACATCGGGT；
Iap：GATAAAGCCCAAATAGT/GGACTACTGTTGACGCAA。
The PCR system:
The PCR program:
95 ℃ of 1min; 95 ℃ of 30s, 58.7 ℃ of 20s, 72 ℃ of 20s, 30 circulations; 72 ℃ of 5min; 4 ℃ of preservations.
The PCR product of embodiment 3 gained is carried out the agarose gel electrophoresis detection.Hly primer and Iap primer all detect specific band, then positive and probable positive result.No band detect or only the Hly primer detect band or only the Iap primer detect band, then negative result.Detected result is referring to Fig. 1.Fig. 1 is the electrophorogram of Listeria monocytogenes 1CFU/25g (mL) genomic dna through present method detected result.1 is 1000bp DNA Marker; 2 are the salmonella contrast; 3 is the Listeria monocytogenes sample; 4 are the streptococcus aureus contrast.As a result, sample 3 is positive.
Should be understood that after having read foregoing of the present invention those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. the method for a highly sensitive rapid detection biological species is characterized in that, comprises below the step:
(1) the biomass cells cracking is discharged DNA, obtain containing the solution of this biology DNA;
(2) with the solution that contains biological DNA of step (1) gained with can mix with DNA bonded magnetic nanoparticle, this magnetic nanoparticle is that the silicon-dioxide of carboxyl modified wraps up γ-Fe
2O
3Nano particle, under the effect of Virahol, DNA is attached to the magnetic nanoparticle surface, is adding under the action of a magnetic field, collects magnetic nanoparticle, is the magnetic nanoparticle that has combined DNA;
(3) with step (2) collect gained combination add water in the magnetic nanoparticle of DNA, magnetic resolution is collected the aqueous solution, promptly obtains the dna solution of isolating biology;
(4) dna solution that obtains with step (3) is a template, utilizes the Auele Specific Primer of target organism, carries out pcr amplification.
2. the method for claim 1 is characterized in that, the method for cracking biomass cells is a pyrolysis method in the step (1), and it is lysis that biological sample is boiled in water, discharges DNA;
The add-on of the Virahol described in the step (2) is 19 times of aqueous phase solution volume, and DNA is adsorbed in the magnetic nanoparticle surface in this case.
3. the method for claim 1 is characterized in that, adopts listerial Auele Specific Primer to increase in the step (4), thereby detects in the dna solution whether contain listerial DNA; Said Auele Specific Primer is Hly and Iap, and the pcr amplification condition is: 95 ℃ of 1min; 95 ℃ of 30s, 58.7 ℃ of 20s, 72 ℃ of 20s, 30 circulations; 72 ℃ of 5min; 4 ℃ of preservations;
Hly：CCGCCTGCAAGTCCTAA/ACAGGAAGAACATCGGGT，
Iap：GATAAAGCCCAAATAGT/GGACTACTGTTGACGCAA。
4. the method for a highly sensitive rapid detection Listeria monocytogenes is characterized in that, may further comprise the steps:
(1) testing sample is placed centrifuge tube, the centrifugal 1min of 10000r/min, abandoning supernatant; With the deionized water centrifuge washing of sterilization, remove impurity; Deionized water suspended sediment with sterilization boils 10min with suspension-s in boiling water bath, the centrifugal 1min of 10000r/min makes biomass cells cracking in the sample, discharges DNA, gets its supernatant;
(2) supernatant of getting step (1) gained is in centrifuge tube; To the magnetic nanoparticle and the Virahol that wherein add specificity combination DNA of the present invention; Quantity of isopropanol is 19 times of aqueous phase solution volume, and DNA is adsorbed in the magnetic nanoparticle surface in this case;
(3) adding under the action of a magnetic field, collecting magnetic nanoparticle,, adding deionized water, adding under the action of a magnetic field, collecting elutriant, with specified conditions PCR check Listeria monocytogenes with 98% washing with alcohol decon.
5. method as claimed in claim 4 is characterized in that, it is kernel with the ferriferrous oxide nano-particle that described specificity combines the magnetic nanoparticle of DNA, is shell with silicon-dioxide, the finishing carboxyl.
6. method as claimed in claim 4 is characterized in that, said pcr amplification the primer is Hly and Iap:
Hly：CCGCCTGCAAGTCCTAA/ACAGGAAGAACATCGGGT，
Iap：GATAAAGCCCAAATAGT/GGACTACTGTTGACGCAA；
Said pcr amplification condition is: 95 ℃ of 1min; 95 ℃ of 30s, 58.7 ℃ of 20s, 72 ℃ of 20s, 30 circulations; 72 ℃ of 5min; 4 ℃ of preservations.
7. a specific specificity combines the preparation method of the magnetic nanoparticle of DNA, it is characterized in that, may further comprise the steps:
(i) Fe
2+And Fe
3+Make γ-Fe with NaOH solution mixing co-precipitation
2O
3Nano particle, this particle of silicon-dioxide parcel is modified amino simultaneously, gets amido modified magnetic nanoparticle:
Preparation FeSO
47H
2O, FeCl
36H
2O and NaOH solution mix, and under the sodium hydroxide solution condition, react after scouring, and drying is pulverized; Be added to and added tensio-active agent n-hexyl alcohol and hexanaphthene system, ultrasonic to stable, the dropping strong aqua adds the tetraethoxy reaction and accomplishes; The washing nano particle after the high-temperature calcination, reacts in alcoholic solution with aminoethyl aminopropyl YSR 3286 (AEAPS) under externally-applied magnetic field, washing under externally-applied magnetic field, drying; Leave standstill 12h and make the particle ageing; After using the magnetic resolution post to separate, wash particle with ethanol, and 90 ℃ of oven dry 5h, the organism of removal particle surface obtains the magnetic nano-particle that silicon-dioxide wraps up; With the magnetic nano-particle of silicon-dioxide parcel and 0.1mL N-(2-amino-ethyl)-3-aminopropyl trimethyl silane co-dispersed in the mixing solutions of methyl alcohol/USP Kosher; Ultra-sonic dispersion is even; Use the magnetic resolution post to separate behind the reaction 24h, use ethanol and distilled water wash respectively, place vacuum drying oven dry; Room temperature preservation is taken out in dry back, obtains amido modified magnetic nanoparticle;
(ii) carboxylated PEG combines with amido modified magnetic nanoparticle, gets the magnetic nanoparticle of carboxyl modified:
Magnetic nanoparticle and carboxylated PEG that 1mg is amido modified are scattered in the aqueous solution, add N, N-DIC (DIC); Stirred overnight; Separating, washing under externally-applied magnetic field obtains the magnetic nanoparticle of carboxyl modified, is the magnetic nanoparticle that specificity combines DNA.
8. method as claimed in claim 7 is characterized in that, described amido modified magnetic nanoparticle particle diameter is 320nm～460nm; Described externally-applied magnetic field is 1000～5000Gs;
The mass concentration of the magnetic nanoparticle of described silicon-dioxide parcel and the ratio of aminoethyl aminopropyl YSR 3286 (AEAPS) volume are 1mg/ml: 0.025ml to 1mg/ml: 0.05ml;
Said carboxylated PEG molecular weight is 400Da to 700Da;
Described amido modified magnetic nanoparticle and carboxylated PEG mass ratio are 10: 1 to 20: 1.
9. the test kit of a highly sensitive rapid detection Listeria monocytogenes is characterized in that, comprising:
(1) specificity combines the magnetic nanoparticle of DNA, and this magnetic nanoparticle is the silicon-dioxide parcel γ-Fe of carboxyl modified
2O
3Nano particle;
(2) primer Hly and Iap,
Hly：CCGCCTGCAAGTCCTAA/ACAGGAAGAACATCGGGT，
Iap：GATAAAGCCCAAATAGT/GGACTACTGTTGACGCAA。
10. test kit as claimed in claim 9 is characterized in that, described test kit also comprises (3) 10 * PCR Buffer; DNTP; With the Taq archaeal dna polymerase.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602739A (en) * | 2013-11-13 | 2014-02-26 | 浙江省水产技术推广总站 | Quick typing kit for major serotypes of listeria monocytogenes and application of quick typing kit |
| CN104313130A (en) * | 2014-09-23 | 2015-01-28 | 华南师范大学 | Functionalized magnetic nanoparticles for efficiently enriching microorganisms, preparation and applications thereof |
| CN104328187A (en) * | 2014-11-05 | 2015-02-04 | 上海大学 | Primer pair for detecting listeria monocytogenes and method for detecting listeria monocytogenes |
| CN104593501A (en) * | 2015-01-17 | 2015-05-06 | 渤海大学 | PCR method and kit for detecting listeria monocytogenes in food |
| CN106248933A (en) * | 2016-08-19 | 2016-12-21 | 浙江工商大学 | The method quickly detecting the rugged Cronobacter sakazakii of slope based on PGM and magnetic Nano microsphere |
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| CN111118182A (en) * | 2019-12-30 | 2020-05-08 | 广东省微生物研究所（广东省微生物分析检测中心） | Specific novel molecular target of serotype listeria monocytogenes and rapid detection method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1995052A (en) * | 2006-01-06 | 2007-07-11 | 上海柏汇申生物科技有限公司 | Magnetic nano particle compound for nucleic acid separation and its preparation method and uses |
| CN101268189A (en) * | 2003-04-02 | 2008-09-17 | 亚钧阔特生命科学公司 | Method for isolating nucleic acids |
| CN101824450A (en) * | 2010-04-23 | 2010-09-08 | 北京博迈世纪生物技术有限公司 | Kit for extracting bacterial genome based on magnetic bead and extraction method thereof |
-
2011
- 2011-09-14 CN CN201110270600.6A patent/CN102321762B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101268189A (en) * | 2003-04-02 | 2008-09-17 | 亚钧阔特生命科学公司 | Method for isolating nucleic acids |
| CN1995052A (en) * | 2006-01-06 | 2007-07-11 | 上海柏汇申生物科技有限公司 | Magnetic nano particle compound for nucleic acid separation and its preparation method and uses |
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