CN102321762B - 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, 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 at " People's Republic of China's the law of food safety " in February, 2009.It is that the pathogenic bacterium in the environment such as raw material and process for processing, storage of food are separated or in situ detection that food-borne pathogens detects.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, when also causing serious food poisoning after contaminated food.As a kind of pathogenic bacteria of zoonosis, can cause the infection of people, animal, main manifestations is septicemia, meningitis and monocytosis etc.Single Lee of the increasing Salmonella existing in food has danger to the mankind's safety, especially still can growth and breeding in the environment of 4 DEG C, and be one of refrigerated food the main pathogenic fungi of threatening human health.
For the detection of Listeria monocytogenes, there are national standard " inspection of GB 4789.30-2010 Listeria Monocytogenes " and inspection and quarantining for import/export industry standard " Listeria Monocytogenes detection method in SN/T0184.1-2005 import and export food " in China.
Present widely used pathogenic microbes detect method mainly contains four kinds, all be applicable to the detection of Listeria monocytogenes: plate culture of isolated counting process, polymerase chain reaction (polymerase chain reaction, PCR), the fluorescent PCR method of inspection and ELISA enzyme linked immunosorbent assay.Plate culture of isolated identification method is that the methods such as the cultivation of increasing bacterium, separation, biochemical identification of using microbe inspection are carried out the inspection of quantitative and qualitative analysis to the food-borne pathogens that may exist in milk powder.Being characterized in reliable and stable, is the most ripe at present, uses the most extensively, and as the method for inspection of benchmark, but exist detection time long, process is loaded down with trivial details, high and particular serotype is difficult to the problem of isolation identification to operator's technical requirements.PCR polymerase chain reaction (Polymerase Chain Reaction, PCR) is a kind of Protocols in Molecular Biology, for amplifying specific DNA fragmentation.Can regard the special DNA replication dna of in vitro as.Sample (increasing after bacterium through pre-treatment), extracts DNA, carries out pcr amplification taking the DNA that extracts as template.Check PCR product whether to have feature band by agarose gel electrophoresis, thereby carry out quick test to whether polluting food-borne pathogens in sample.Detect with round pcr that harmful microorganism has high specificity, highly sensitive and easy and simple to handle, the advantage such as save time.Fluorescent PCR adds a special oligonucleotide fluorescent probe on regular-PCR basis, PCR product is carried out to mark tracking, 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 by measuring microbe population in radioactive intensity examination sample.But PCR reaction is subject to the impact of sample situation larger, in food source property harmful microorganism checks, the composition (carbohydrate, acids, the materials such as grease) in food is understood normally carrying out of disturbance reponse especially.And the environment detecting, intermediate treatment link also can be with and serve PCR reaction suppressor.Thereby make PCR reaction present higher false positive and false negative rate.ELISA, i.e. enzyme linked immunosorbent assay, has very high susceptibility and repeatability, but can only detect a kind of mark at every turn, can not meet the needs that multiple checking matter detects simultaneously, and ELISA method cost complex operation, test kit is expensive, and a large amount of use will produce very high cost burden.
Summary of the invention
The technical problem to be solved in the present invention is exactly loaded down with trivial details for existing biological detecting 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 method for highly sensitive rapid detection biological species, step comprises below:
(1) biomass cells cracking is discharged to DNA, obtain the solution that contains this biology DNA;
(2) solution that contains biological DNA of step (1) gained and the magnetic nanoparticle that can be combined with DNA are mixed, this magnetic nanoparticle is the Silica-coated γ-Fe of carboxyl modified
2o
3nano particle, under the effect of Virahol, DNA is attached to magnetic nanoparticle surface, under additional the action of a magnetic field, collects magnetic nanoparticle, is the magnetic nanoparticle that combines DNA;
(3) will in the magnetic nanoparticle that combine DNA of step (2) collection gained, add water, magnetic resolution, collects the aqueous solution, obtains the biological DNA solution separating;
(4) DNA solution obtaining taking step (3), as template, utilizes the Auele Specific Primer of target organism, carries out pcr amplification.
In step (1), the method for cracking biomass cells is common conventional lysis method.Preferably, can be pyrolysis method, biological sample to be boiled in water, i.e. lysis, discharges DNA.
In step (2), can adopt the existing various magnetic nanoparticle that can be combined with DNA, preferably the amino Silica-coated γ-Fe of carboxyl modified
2o
3nano particle, is more preferably prepared by the method for the invention.In aqueous dna, add this magnetic nanoparticle and Virahol, can make DNA be attached to magnetic nanoparticle surface, thereby go out this magnetic nanoparticle in conjunction with DNA by magnetic resolution.Quantity of isopropanol is preferably 19 times of aqueous phase solution volume, and DNA is adsorbed in magnetic nanoparticle surface in this case.
Step (3) though in add water DNA to come off from magnetic nanoparticle surface, be dissolved in the water, thereby obtain DNA solution through magnetic resolution.
In step (4), adopting the Auele Specific Primer of existing any biology is primer, carrys out specific amplification target dna fragment.Preferably, for example, adopt listerial Auele Specific Primer to increase, thereby detect in DNA solution whether contain listerial DNA.In the situation that obtaining amplified production, in biology to be measured, contain listeria bacteria, be positive findings.In the situation that not obtaining amplified production, in biology to be measured, do not contain listeria bacteria, be negative findings.Present method can be set up positive control, negative control or blank simultaneously.The preferred Hly of described Auele Specific Primer and Iap.Pcr amplification condition optimization: 95 DEG C of 1min; 95 DEG C of 30s, 58.7 DEG C of 20s, 72 DEG C of 20s, 30 circulations; 72 DEG C of 5min; 4 DEG C 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, comprises the following steps:
(1) testing sample is placed in to centrifuge tube, the centrifugal 1min of 10000r/min, abandoning supernatant; With the deionized water centrifuge washing of sterilizing, remove impurity; With the deionized water suspended sediment of sterilizing, suspension is boiled in boiling water bath to 10min, the centrifugal 1min of 10000r/min, makes biomass cells cracking in sample, discharges DNA, gets its supernatant liquor;
(2) supernatant liquor of getting step (1) gained is in centrifuge tube, add wherein magnetic nanoparticle and the Virahol of specific binding DNA of the present invention, quantity of isopropanol is 19 times of aqueous phase solution volume, and DNA is adsorbed in magnetic nanoparticle surface in this case;
(3) under additional the action of a magnetic field, collect magnetic nanoparticle, with 98% washing with alcohol decon, add deionized water, under additional the action of a magnetic field, collect elutriant, with specified conditions PCR inspection Listeria monocytogenes.
The magnetic nanoparticle of described specific binding DNA is preferably taking ferriferrous oxide nano-particle as kernel, taking silicon-dioxide as shell, and finishing carboxyl.
Described pcr amplification the primer is preferred:
Hly：CCGCCTGCAAGTCCTAA/ACAGGAAGAACATCGGGT；
Iap：GATAAAGCCCAAATAGT/GGACTACTGTTGACGCAA。
Described pcr amplification program is: 95 DEG C of 1min; 95 DEG C of 30s, 58.7 DEG C of 20s, 72 DEG C of 20s, 30 circulations; 72 DEG C of 5min; 4 DEG C of preservations.
The system of described 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
2(10.0 μ l) for O.
The described externally-applied magnetic field in step (2) and (3) is 1000～5000Gs.
The preparation method of the magnetic nanoparticle of specific binding DNA of the present invention, comprises the following steps:
(i) Fe
2+and Fe
3+make γ-Fe with NaOH solution mixing co-precipitation
2o
3nano particle, this particle of Silica-coated is modified amino simultaneously, obtains amido modified magnetic nanoparticle:
Preparation FeSO
47H
2o, FeCl
36H2O and NaOH solution, mix, and under sodium hydroxide solution condition, washing after reaction, dry, pulverizes.Be added to and added tensio-active agent n-hexyl alcohol and hexanaphthene system, ultrasonic to stable, dropping strong aqua, adds tetraethoxy to react.Under externally-applied magnetic field, wash nano particle, after high-temperature calcination, react in alcoholic solution with aminoethyl aminopropyl polydimethylsiloxane (AEAPS), under externally-applied magnetic field, wash, dry.Standing 12h makes particle ageing.After using magnetic resolution post to separate, wash particle with ethanol, and 90 DEG C dry 5h, remove the organism of particle surface, obtain the magnetic nano-particle of Silica-coated.The magnetic nano-particle of Silica-coated and 0.1mL N-(2-amino-ethyl)-3-aminopropyl trimethyl silane are scattered in the mixing solutions of methyl alcohol/glycerol jointly, ultrasonic being uniformly dispersed, after reaction 24h, use magnetic resolution post to separate, use respectively ethanol and distilled water wash, be placed in vacuum drying oven dry, after dry, take out room temperature preservation, obtain amido modified magnetic nanoparticle.
Described amido modified magnetic nanoparticle particle diameter is 320nm～460nm.
Described externally-applied magnetic field is 1000～5000Gs.
The ratio of the mass concentration of the magnetic nanoparticle of described Silica-coated and aminoethyl aminopropyl polydimethylsiloxane (AEAPS) volume is 1mg/ml: 0.025ml to 1mg/ml: 0.05ml.
(ii) carboxylated PEG is combined with amido modified magnetic nanoparticle, obtains the magnetic nanoparticle of carboxyl modified:
Magnetic nanoparticle amido modified 1mg and carboxylated PEG are scattered in the aqueous solution, add N, N-DIC (DIC), stirring is spent the night, separating, washing under externally-applied magnetic field, obtain the magnetic nanoparticle of carboxyl modified, be the magnetic nanoparticle of specific binding DNA.
Described externally-applied magnetic field is 1000～5000Gs.
Described 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) magnetic nanoparticle of specific binding DNA, this magnetic nanoparticle is the Silica-coated 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 Taq archaeal dna polymerase.
The raw material that the present invention is used or reagent except special instruction, all commercially available obtaining.
The required time of the whole testing process of the present invention is only 2 hours, and the detection limit of Listeria monocytogenes is about to 1CFU/25g (mL), and required time is short, and highly sensitive in simple PCR method, result is more reliable.As now methodical supplementary, can be widely used at detection field.
Brief description of the drawings
Below in conjunction with brief description of the drawings feature 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 strain contrasts; 3 is tested Listeria monocytogenes ATCC13932 strain; Wherein hly gene amplification length 308bp, iap gene amplification length 505bp; 4 is streptococcus aureus ATCC 29213 strain contrasts.This results sample 3 is positive.
Embodiment
The invention provides the combined utilization strategy that uses the DNA of DNA specific nano magnetic extraction particle extraction Listeria monocytogenes to detect through PCR again, thus the abundant means that detect Listeria monocytogenes.
First the present invention prepares amido modified nanometer and extracts particle, is then combined with carboxylated PEG, 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 sample, disengage the DNA of Listeria monocytogenes in sample, under the effect of alcohol, making itself and DNA specific nano magnetic extract particle is combined, under additional the action of a magnetic field, collect above-mentioned magnetic particle, discard mixed solution; Water dissolving DNA, obtains after mother liquor, carrying out PCR inspection as template by specified conditions, electrophoresis observation judged result.
Further illustrate the present invention with embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to manufacturer." room temperature " described in embodiment refers to the temperature of the operation room of testing, and is generally 25 DEG C.
The preparation that embodiment 1 carboxyl modified DNA specific nano magnetic is extracted particle
25ml 50mg/ml FeSO
47H
2o solution and 25ml 50mg/ml FeCl
36H
2o solution is dissolved in 500ml distilled water, and ultrasonic limit, 40 DEG C of water bath with thermostatic control limits mechanical stirring drips 50ml 0.1mol/L sodium hydroxide diluting soln, leaves standstill afterwards, pours beaker into, and respectively after water and washing with alcohol, dry 3h at 75 DEG C, grinds.The ratio of 4: 1: 10 is by volume joined hexanaphthene, TritonX-100 and n-hexyl alcohol mixing solutions, ultrasonic, and after the grinding of the above-mentioned solution of 100mL and 50mg, powder mixes, ultrasonic, get 400 μ L 28% strong aquas and drip, add tetraethoxy (TEOS) 2ml, reaction 24h, leaves standstill 48h.Use magnetic resolution post adsorbed product, with ethanol cleaning, dry 5h for 90 DEG C.20mg/ml product and 0.5mL N-(2-amino-ethyl)-3-aminopropyl trimethyl silane are dissolved in to methyl alcohol and glycerol mixed solution (volume ratio of methyl alcohol and glycerol 4: 1), ultrasonic 6h, use magnetic resolution post adsorbed product, clean with ethanol, 40 DEG C dry, obtains amido modified nanometer and extract particle.Amido modified 20mg nanometer is extracted to particle and the carboxylated PEG of 2mg 400Da is scattered in 50ml deionized water, add 2.5mgN, N-DIC (DIC), stirring is spent the night, use magnetic resolution post adsorbed product, with 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 strain 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 25g sample and cultivate by bis-enrichments of LB, 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 DEG C of 150r/min overnight incubation, dilute different gradients with aseptic double-distilled water, as testing sample group 2, plate count press GB 4789.4-2010 and verify simultaneously.Get streptococcus aureus ATCC 29213 strains, inoculation 5ml 7.5%NaCl broth culture, 37 DEG C of 150r/min overnight incubation, dilute different gradients with aseptic double-distilled water, as testing sample group 3, plate count press GB 4789.10-2010 and verify simultaneously.
Testing sample in above-mentioned 1ml 3 groups is operated respectively as follows: be placed in the centrifugal 1min of centrifuge tube 10000r/min, abandoning supernatant; With the deionized water wash of 200 μ l sterilizings 2 times; With the deionized water suspended sediment of 100 μ l sterilizings, suspension is boiled in boiling water bath to 10min, the centrifugal 1min of 10000r/min, gets its supernatant, for subsequent use.Get supernatant liquor in 1.5ml centrifuge tube, add wherein the carboxyl modified DNA specific nano magnetic that embodiment 1 obtains to extract particle 0.1ml, concussion, vortex, fully mixes, then adds 1ml 94% aqueous isopropanol, vortex, concussion, fully mixes, and places 2min; Be placed on magnetic frame, after particle is hunted down completely, discard clear liquid; Add 1ml 98% washing with alcohol one time, discard clear liquid; Add the deionized water of 200 μ l to place 10 minutes, catch magnetic bead with magnetic frame, after particle is hunted down completely, sucking-off clear liquid, is DNA elutriant to be measured.
Embodiment 3 Listeria monocytogenes DNA extraction liquid PCR detect
Directly get DNA elutriant to be measured prepared by embodiment 2 as template, adopt primer Hly and Iap, carry out multiplex PCR amplification, the SK2491-PCR of Sangon Biotech (Shanghai) Co., Ltd. amplification kit that pcr amplification adopts.
Hly：CCGCCTGCAAGTCCTAA/ACAGGAAGAACATCGGGT；
Iap：GATAAAGCCCAAATAGT/GGACTACTGTTGACGCAA。
PCR system:
PCR program:
95 DEG C of 1min; 95 DEG C of 30s, 58.7 DEG C of 20s, 72 DEG C of 20s, 30 circulations; 72 DEG C of 5min; 4 DEG C of preservations.
Embodiment 4 amplified production electrophoresis detection
The PCR product of embodiment 3 gained is carried out to agarose gel electrophoresis detection.Hly primer and Iap primer all detect specific band, positive and probable positive result.Without band detect or only Hly primer detect band or only Iap primer detect band, 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 is salmonella contrast; 3 is Listeria monocytogenes sample; 4 is 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 make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (4)
1. carboxyl modified DNA specific nano magnetic is extracted a preparation method for particle, it is characterized in that, comprises the following steps: 25ml50mg/ml FeSO
47H
2o solution and 25ml50mg/mlFeCl
36H
2o solution is dissolved in 500ml distilled water, and ultrasonic limit, 40 DEG C of water bath with thermostatic control limits mechanical stirring drips 50ml0.1mol/L sodium hydroxide diluting soln, leaves standstill afterwards, pours beaker into, and respectively after water and washing with alcohol, dry 3h at 75 DEG C, grinds; The ratio of 4:1:10 is joined cyclohexane, TritonX-100 and n-hexyl alcohol mixing solutions by volume, ultrasonic, and after the grinding of the above-mentioned solution of 100mL and 50mg, powder mixes, ultrasonic, gets 400 μ L28% strong aquas and drips, and adds tetraethoxy 2ml, and reaction 24h, leaves standstill 48h; Use magnetic resolution post adsorbed product, with ethanol cleaning, dry 5h for 90 DEG C; By 20mg/ml product and 0.5mL N-(2-amino-ethyl)-3-aminopropyl trimethyl silane is dissolved in methyl alcohol and glycerol mixed solution, the volume ratio of described methyl alcohol and glycerol is 4 ︰ 1, ultrasonic 6h, use magnetic resolution post adsorbed product, clean with ethanol, 40 DEG C dry, obtains amido modified nanometer and extract particle; Amido modified 20mg nanometer is extracted to particle and the carboxylated PEG of 2mg400Da is scattered in 50ml deionized water, add 2.5mg N, N-DIC, stirring is spent the night, use magnetic resolution post adsorbed product, with deionized water wash, obtain carboxyl modified DNA specific nano magnetic and extract particle.
2. a method for the highly sensitive rapid detection Listeria monocytogenes of non-diagnostic purpose, is characterized in that, comprises the following steps:
(1) biomass cells cracking is discharged to DNA, obtain the solution that contains this biology DNA;
(2) solution that contains biological DNA of step (1) gained and the magnetic nanoparticle that can be combined with DNA are mixed, this magnetic nanoparticle be preparation method claimed in claim 1 preparation and carboxyl modified DNA specific nano magnetic extract particle, under the effect of Virahol, DNA is attached to magnetic nanoparticle surface, under additional the action of a magnetic field, collect magnetic nanoparticle, be the magnetic nanoparticle that combines DNA;
(3) will in the magnetic nanoparticle that combine DNA of step (2) collection gained, add water, magnetic resolution, collects the aqueous solution, obtains the biological DNA solution separating;
(4) DNA solution obtaining taking step (3), as template, utilizes the Auele Specific Primer of target organism, carries out pcr amplification;
In step (1), the method for cracking biomass cells is pyrolysis method, and it is lysis that biological sample is boiled in water, discharges DNA;
The add-on of the Virahol described in step (2) is 19 times of aqueous phase solution volume, and DNA is adsorbed in magnetic nanoparticle surface in this case;
In step (4), adopt the Auele Specific Primer of Listeria monocytogenes to increase, thereby detect the DNA that whether contains Listeria monocytogenes in DNA solution; Described Auele Specific Primer is Hly and Iap, and pcr amplification condition is: 95 DEG C of 1min; 95 DEG C of 30s, 58.7 DEG C of 20s, 72 DEG C of 20s, 30 circulations; 72 DEG C of 5min; 4 DEG C of preservations;
Hly upstream primer: CCGCCTGCAAGTCCTAA,
Hly downstream primer: ACAGGAAGAACATCGGGT,
Iap upstream primer: GATAAAGCCCAAATAGT,
Iap downstream primer: GGACTACTGTTGACGCAA.
3. a test kit for highly sensitive rapid detection Listeria monocytogenes, is characterized in that, comprising:
(1) magnetic nanoparticle of specific binding DNA, this magnetic nanoparticle be preparation method claimed in claim 1 preparation and carboxyl modified DNA specific nano magnetic extract particle;
(2) primer Hly and Iap,
Hly upstream primer: CCGCCTGCAAGTCCTAA,
Hly downstream primer: ACAGGAAGAACATCGGGT,
Iap upstream primer: GATAAAGCCCAAATAGT,
Iap downstream primer: GGACTACTGTTGACGCAA.
4. test kit as claimed in claim 3, is characterized in that, described test kit also comprises (3) 10 × PCR Buffer; DNTP; With Taq archaeal dna polymerase.
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| CN104313130B (en) * | 2014-09-23 | 2017-10-13 | 华南师范大学 | A kind of functionalized magnetic nano particle of efficiently concentrating microorganism and preparation and application |
| 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 |
| JP6752231B2 (en) | 2015-06-01 | 2020-09-09 | カリフォルニア インスティテュート オブ テクノロジー | Compositions and Methods for Screening T Cells for Specific Populations Using Antigens |
| 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 |
| CN111118182B (en) * | 2019-12-30 | 2022-06-14 | 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 |
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