CN104459126A - Method of gathering salmonella based on immunomagnetic beads - Google Patents
Method of gathering salmonella based on immunomagnetic beads Download PDFInfo
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- CN104459126A CN104459126A CN201410772937.0A CN201410772937A CN104459126A CN 104459126 A CN104459126 A CN 104459126A CN 201410772937 A CN201410772937 A CN 201410772937A CN 104459126 A CN104459126 A CN 104459126A
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- salmonella
- sample
- phosphate buffer
- immunomagnetic beads
- enrichment
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
Abstract
The invention relates to a method of gathering salmonella based on immunomagnetic beads, belonging to the field of microbiological detection. The method of gathering salmonella based on immunomagnetic beads specifically comprises the following steps: (1) preparing a to-be-tested sample into a to-be-tested sample uniform solution by a phosphate buffer solution, absorbing and adding an appropriate amount of the to-be-tested sample uniform solution into 150-300 mu l of magnetic beads to react at 37 DEG C for 30-60 minutes to obtain a solution; (2) placing the solution on magnets for acting for 2-3 minutes, removing a suspension liquid after the magnets sufficiently adsorb the solution, adding 1ml of PBS and repeatedly washing for 3 times; and (3) culturing the gathered salmonella on an XLT4 agar culture-medium to culture for 24 hours, and counting to determine the quantity of the salmonella in the sample. The method disclosed by the invention is strong in specificity, high in accuracy, short in detection time, simple to operate; large-scale equipment and specially trained professionals are not needed, and the sample is not needed to be specially pre-treated; and the method can be applied to detecting salmonella in eggs, livestock tissues, feed, water and livestock fecal samples.
Description
Technical field
The invention belongs to technical field of microbial detection, be specifically related to a kind of method based on immunomagnetic beads enrichment salmonella.
Background technology
Salmonella (
salmonella) be a kind of important infecting both domestic animals and human, gram negative pathogenic bacterium in enterobacteriaceae.Animal derived salmonella easily causes ingester's acute diarrhea, even breaks out food poisoning, causes gastroenteritis, typhoid fever and paratyphoid, and wherein meat and poultry are the main medias that salmonella is propagated.In recent years, the harm that salmonella causes in the world is continuous ascendant trend, and on public hygienics, tool is of great significance.
The detection difficult point of salmonella is that content is low, the matrix composition of interference measurement complicated, causes very large difficulty to Accurate Determining.Therefore, in the detection techniques of food microorganism of GB, adopt two step enrichments to improve the recall rate of salmonella, after increasing bacterium in advance as utilized buffered peptone water (BPW), adopt selenite cystine broth (SC) to carry out selective enrichment, and line on xylose lysine deoxidation agar,bile salt (XLD).Although this method Selective Separation can identify salmonella to a certain extent, length consuming time (about 72h), complex operation, is unfavorable for detecting fast and applying.
Immunomagnetic beads (Imrnunomagnetiebead, IMB) magnetic bead is called for short, it is that a kind of Novel immune that 20 century 70s grow up mid-term learns a skill, it is based on immunology, penetrate into the every field such as pathology, pharmacology, physiology, microorganism, biochemistry and molecular genetics, its application is increasingly extensive.Had immunomagnetic bead technique in the last few years for the report of bacterial detection, its principal feature is: velocity of separation is fast, and efficiency is high, favorable repeatability; Simple to operate and do not need expensive equipment; Biological characteristics and the function of separated material can not be affected.The present invention adopts salmonella specific immunity magnetic bead gathering trace salmonella from sample, amount of samples is few and can not injure bacterium, compared with isolated culture traditional in GB, eliminate pre-increasing bacterium and the selective enrichment process of loaded down with trivial details length consuming time, directly salmonella is separated, has saved 42 ~ 48h detection time.In addition, because immunomagnetic beads can very similar other bacterial strains of recognition property, as proteus mirabilis, specific adsorption salmonella, avoids the interference of the complicated ingredient in sample to testing result, improves detection accuracy.
Summary of the invention
The present invention is intended to for prior art not enough, provides a kind of salmonella enrichment method based on immunomagnetic beads.Magnetic bead carries out coupling with salmonella specific antibody under suitable conditions, makes salmonella specific immunity magnetic bead, joins in the testing sample of pre-treatment, catch salmonella under certain condition by salmonella specific immunity magnetic bead.
For achieving the above object, the technical scheme that the present invention takes is: this preparation method being used for the specific immunity magnetic bead of enrichment salmonella take magnetic bead as carrier, coupling salmonella specific antibody, and being prepared into can the immunomagnetic beads of enrichment salmonella.
For a preparation method for the specific immunomagnetic beads of enrichment salmonella, the method take magnetic bead as carrier, coupling salmonella specific antibody, and being prepared into can the immunomagnetic beads of enrichment salmonella;
Concrete grammar is:
(1) get the magnetic carrier of 500 μ l, be placed in centrifuge tube, discard conserving liquid, add the PBS damping fluid of 0.01 ~ 0.1M PH6.0 ~ 7.4;
(2) in above-mentioned mixed liquor, add 500 μ l salmonella specific antibodies, under room temperature, after reacting 6 h on the oscillator, be placed in magnetic field, separation of supernatant.
The preparation method of magnetic carrier involved in said method is as follows:
(1) FeCl of 27% is configured
36H
2o solution and FeCl
2(7.2g FeCl
24H
2o+20 ml H
2o+4 ml HCl) solution.
(2) after FeCl2 solution becomes light green color, by the FeCl of 27%
36H
2o solution 29 ml joins wherein, and moisturizing to 100 ml, stir and make it even;
(3) in above-mentioned mixed solution, 40 ml ammoniacal liquor are added, vigorous stirring 20 min.Magnet is placed in beaker bottom, after magnetic-particle precipitation in solution, removes supernatant, then add 40ml water, after stirring 10min, except anhydrating;
(4) oleic acid 6g is placed in 60 DEG C of water-baths and heats 10min, then added except in the magnetic-particle anhydrated, and in 60 DEG C of stirred in water bath 20min.Remove unnecessary oleic acid, add 100ml ethanol agitator treating 10min, wash 2 times;
(5) 10ml octane is measured, first in above-mentioned magnetic-particle, slowly drip octane with dropper and be about 2ml, mixing, then, each slowly dropping octane 1-2ml, after adding octane at every turn, magnet can be placed in beaker bottom, observe whether occur magnetic fluid phenomenon, as occurred, then be successfully prepared, put into Refrigerator store; As do not occurred, then continue to drip octane, until there is magnetic fluid phenomenon;
(6) get 1ml magnetic fluid, add 20ml acetone, acutely shake.Magnet is placed in beaker bottom, after 5min, removes supernatant, repeat 3 times.60 DEG C of vacuum drying 20min, add 20ml toluene afterwards, ultrasonic 1h;
(7) get the magnetic particle of 1ml 4.65 mg/ml, join in the potpourri containing 20ml isopropyl alcohol, 2ml deionized water and 1 ml ammoniacal liquor, 300rpm mechanical raking 10min.Afterwards, add 2ml ethyl orthosilicate, under room temperature, magnetic stirring 24h.With ethanol washing several, until PH is 7;
(8) take dry magnetic particle 300mg, be dispersed in 600ml ethanol, add 3ml deionized water, ultrasonic 20min.Afterwards, add 120 μ l APTES(3-aminopropyl triethoxysilanes), magnetic agitation 7h.The centrifugal 30min of 8000rpm, abandons supernatant, by precipitation with ultrasonic disperse in ethanol, Magneto separate 5 times.4 DEG C of Refrigerator stores, for subsequent use.
For a preparation method for the specific immunomagnetic beads of enrichment salmonella, involved salmonella specific antibody preparation method is specific as follows:
(1) clone of salmonella outer membrane protein OmpC
Boiling method is adopted to extract S. pullonum DNA profiling, according to S. pullonum gene order in GenBank, the primer of restriction enzyme Sac I and Hind III restriction enzyme site is contained at design two ends, and primer sequence (OmpC-F, OmpC-R) is as follows:
OmpC-F：5’-CGAGCTCATGGTTAAAGTACTGTCCCTC-3’
OmpC-R：5’-CCCAAGCTTGGTGGACATGTTTTTGTT-3’
With S. pullonum reference culture (CVCC 533) template, pcr amplification is carried out to OmpC gene, its reaction system (total 25 μ L): 5 × MIX 10 μ L, upstream primer (20 μm of ol/L): 0.5 μ L, downstream primer (20 μm of ol/L): 0.5 μ L, DNA profiling: 2 μ L, ddH2O:12 μ L.The rearmounted PCR instrument of brief centrifugation increases.Reaction conditions is: 95 DEG C of 5min; 94 DEG C of 1min, 57 DEG C of 1min, 72 DEG C of 1min, totally 30 circulations; Last 72 DEG C of 10min.After reaction terminates, observe qualification PCR reaction product with 1% agarose gel electrophoresis.PCR primer after purifying is connected with PMD19-T carrier.Get PMD19-T carrier 0.8 μ l, cloned sequence 4.2 μ l and 5 μ l solution1 respectively, add in PCR thin walled tube, 16 DEG C connect 3 h.Transform afterwards, and positive colony is verified.
(2) structure of prokaryotic expression plasmid pET32 a (+)-OmpC
Positive colony PMD19-T-OmpC and pET-32a(+ by correct for order-checking) Zengjing Granule 12-16 h in the LB fluid nutrient medium containing Amp.Positive recombinant plasmid and expression vector carry out plasmid extraction, and adopt 1% agarose gel electrophoresis to detect the effect of plasmid enzyme restriction, electrophoresis terminates rear gel imaging system analysis.Plasmid PMD19-T-OmpC and pET-32a(+ to extracting) carry out double digestion, by digestion products PMD19-T-OmpC with pET-32a(+) carrier is connected, transforms afterwards, from transformation plate, random picking colony, carries out double digestion checking.
(3) abduction delivering of OmpC
Will containing the streak inoculation of positive plasmid BL21-pET32a-OmpC recombinant bacterium in being 100 μ g/mL containing Amp(concentration) LB solid medium, in 37 DEG C of constant incubators, cultivate 12-24 h; The fresh single bacterium colony of picking respectively, aseptic inoculation is in 5 mL Amp LB fluid nutrient mediums, and 37 DEG C of shaken cultivation 12-16 h, are then inoculated in LB/Amp fluid nutrient medium with the bacterium liquid measure of 1%, 37 DEG C of shaken cultivation 4 h (bacterium liquid OD
450reach 0.6-1.0) time, get the bacterium liquid that 1mL do not induce and cultivate separately, all the other bacterium liquid add IPTG to final concentration 1.0 mmol/L, and 25 DEG C are continued thermal agitations and cultivate 4 h.Do empty vector control simultaneously, observe expression;
Get the rear bacterium liquid 5500rpm 15min of IPTG induction, collecting cell after cell washing.Add 20mmol/L Tris-Cl(pH8.0) suspension sedimentation cell, ultrasonic disruption, then the centrifugal 20min of 12000 rpm, preserve upper cleer and peaceful precipitation respectively, add 100 μ L 1 × sds gel sample loading buffers, centrifugal 1 min of boiling water bath 5min, 12000 rpm after mixing, gets the expression of the SDS-PAGE electrophoresis detection recombinant protein of supernatant in 12%.
(4) purifying of restructuring destination protein: adopt the imidazoles of 200mM to carry out eluting to restructuring destination protein, get eluent 10 μ l afterwards and carry out electrophoresis checking, observe the purity of albumen.
(5) immunogenic preparation: get the albumen after purifying and isopyknic Freund's complete adjuvant and incomplete Freund's adjuvant and grind, after fully emulsified, then immunization experiment rabbit.
(6) immunity of new zealand rabbit: choose test with rabbit, from rabbit ear edge vein exploitating blood, carry out aggegation experiment with S. pullonum, selects not occur that the rabbit that coagulation sedimentation reacts is tested;
Divide and carry out immunity four times: Jia Fushi Freund's complete adjuvant during first time immunity, immunizing dose is 1ml/, and immunogenic form is foot pad and subcutaneous multi-point injection; Interval is after 14 days, and carry out second time immunity, Jia Fushi Freund's incomplete adjuvant, immunizing dose is 0.5ml/, and immunogenic form is subcutaneous multi-point injection; Interval is after 22 days, and carry out third time immunity, Jia Fushi Freund's incomplete adjuvant, immunizing dose is 0.5ml/, and immunogenic form is subcutaneous multi-point injection; Interval, after 25 days, is carried out the 4th immunity, is not added adjuvant, and immunizing dose is 0.2ml/, and immunogenic form is auricular vein injection.To take a blood sample after 4th immunity extraction antibody.
(7) serum antibody titer measures
Rabbit anteserum to be measured is pressed 1:2; 1:2
2; 1:2
3... 1:2
15gradient dilution, adopts slide agglutination to measure antibody titer.
(8) saturated ammonium sulfate grading purification antibody
First carry out pre-service to serum, low-temperature and high-speed centrifugal (4 DEG C, 12000rpm/min, 15-20min), can remove cell residue and finely ground particle substance.The antibody of antibody isolation and purification method to preparation of reference antibody Theory and technology carries out purifying;
Gained antibody is salmonella specific antibody.
For an application process for the specific immunity magnetic bead of enrichment salmonella, be by salmonella specific immunity magnetic bead is added in various measuring samples, reach separation or enrichment under certain condition and detect the object of salmonella;
Concrete steps are:
(1) testing sample phosphate buffer (0.01M ~ 0.1M, PH6.0 ~ 7.4) is made the even liquid of a certain proportion of testing sample, get 100 ~ 200 μ l, join in 150 ~ 300 μ l magnetic beads, 37 DEG C of reaction 30 ~ 60min.
(2) be placed on magnet and act on 2 ~ 3min, after magnet is fully adsorbed, discard suspending liquid, add the PBS repeated washing 3 times of 0.01 ~ 0.1M PH6.0 ~ 7.4 of 1ml.
(3) by the 37 DEG C of cultivation 24h on XLT4 agar medium of the salmonella after enrichment, counting, obtains the quantity of salmonella in sample.
Described testing sample comprises egg, RP-HPLC, feed, water, animal dung sample in step (1).
RP-HPLC described is in step (1) 1:9 with the W:V ratio of phosphate buffer; Feed is 1:9 with the W:V ratio of phosphate buffer; Animal dung sample is 1:9 with the W:V ratio of phosphate buffer; The volume ratio of water and phosphate buffer is 1:9; The volume ratio of the yellow and white potpourri and phosphate buffer is 1:9; Eggshell joins in 50 ~ 100ml phosphate buffer.
The invention has the beneficial effects as follows:
(1) detection sensitivity and accuracy is improved: utilize salmonella specific immunity magnetic bead to carry out enrichment to salmonella, can to catch in sample while micro-salmonella, other compositions in sample except object bacteria can also be removed, to prevent from, to the interference of detection accuracy, effectively avoiding or reducing false positive.
(2) detection time is shortened: compared with salmonella tradition isolated culture, utilize immunomagnetic beads enrichment salmonella, effectively can replace the increasing bacterium step in testing process, detection time foreshortens to 25h by 72h.
(3) easy and simple to handle, be widely used: the preparation method of the specific immunity magnetic bead for enrichment salmonella in the present invention is easy and simple to handle, do not need the professional of large-scale instrument and Special Training, sample does not need special pre-treatment, can be applicable to the detection of salmonella in egg, RP-HPLC, feed, water, animal dung sample.
Embodiment
Be described in further details the present invention below by embodiment, these embodiments are only used for the present invention is described, do not limit the scope of the invention.
One, the preparation of salmonella specific immunity magnetic bead:
Embodiment 1,
Get the magnetic carrier of 500 μ l, be placed in centrifuge tube, discard conserving liquid, add the PBS damping fluid of 0.01M PH 7.4; Then add 500 μ l salmonella specific antibodies, under room temperature, after reacting 6h on the oscillator, be placed in magnetic field, separation of supernatant.
Embodiment 2
Get the magnetic carrier of 500 μ l, be placed in centrifuge tube, discard conserving liquid, add the PBS damping fluid of 0.1M PH6.0; Then add 500 μ l salmonella specific antibodies, under room temperature, after reacting 6 h on the oscillator, be placed in magnetic field, separation of supernatant.
Two, salmonella specific antibody is to the application of salmonella enrichment in sample:
Embodiment 1
Egg is opened, egg white, yolk is poured in sterile chamber, fully mixes, draw 25mL potpourri in the aseptic conical flask filling 225mL 0.01M PH 7.4 PBS damping fluid, fully mix, make the even liquid of sample of 1:10.Get the even liquid of 200 μ l sample, join in 300 μ l magnetic beads, 37 DEG C of reaction 30min.Be placed on magnet and act on 2 ~ 3min, after magnet is fully adsorbed, discard suspending liquid, add the PBS repeated washing 3 times of the 0.01M PH7.4 of 1ml.By the 37 DEG C of cultivation 24h on XLT4 agar medium of the salmonella after enrichment, counting determines the quantity of salmonella in sample.
Embodiment 2
Egg is opened, remove egg white, yolk, with aseptic 0.01M PH 7.4 PBS wash buffer eggshell inner membrance 2 ~ 3 times, crumb after egg shell gently, moved into fill 50mL 0.01M PH 7.4 PBS damping fluid aseptic homogeneous cup in, 10000r/min homogeneous 1 ~ 2 minute, makes the even liquid of sample.Get the even liquid of 200 μ l sample, join in 300 μ l magnetic beads, 37 DEG C of reaction 30min.Be placed on magnet and act on 2 ~ 3min, after magnet is fully adsorbed, discard suspending liquid, add the PBS repeated washing 3 times of the 0.01M PH7.4 of 1ml.By the 37 DEG C of cultivation 24h on XLT4 agar medium of the salmonella after enrichment, counting determines the quantity of salmonella in sample.
Embodiment 3
Egg is opened, remove egg white, yolk, with aseptic 0.01M PH 7.4 PBS wash buffer eggshell inner membrance 2 ~ 3 times, crumb after egg shell gently, moved into fill 100mL 0.01M PH 7.4 PBS damping fluid aseptic homogeneous cup in, 10000r/min homogeneous 1 ~ 2 minute, makes the even liquid of sample.Get the even liquid of 200 μ l sample, join in 300 μ l magnetic beads, 37 DEG C of reaction 30min.Be placed on magnet and act on 2 ~ 3min, after magnet is fully adsorbed, discard suspending liquid, add the PBS repeated washing 3 times of the 0.01M PH7.4 of 1ml.By the 37 DEG C of cultivation 24h on XLT4 agar medium of the salmonella after enrichment, counting determines the quantity of salmonella in sample.
Embodiment 4
Take 25g RP-HPLC class sample in the aseptic homogeneous cup filling 225mL 0.01M PH 7.4 PBS damping fluid, 10000r/min homogeneous 1 ~ 2 minute, makes the even liquid of sample of 1:10.Get the even liquid of 200 μ l sample, join in 300 μ l magnetic beads, 37 DEG C of reaction 30min.Be placed on magnet and act on 2 ~ 3min, after magnet is fully adsorbed, discard suspending liquid, add the PBS repeated washing 3 times of the 0.01M PH7.4 of 1ml.By the 37 DEG C of cultivation 24h on XLT4 agar medium of the salmonella after enrichment, counting determines the quantity of salmonella in sample.
Embodiment 5
Draw 25mL water sample to be measured in the aseptic conical flask filling 225mL 0.01M PH 7.4 PBS damping fluid, fully mix, make the even liquid of sample of 1:10.Get the even liquid of 200 μ l sample, join in 300 μ l magnetic beads, 37 DEG C of reaction 30min.Be placed on magnet and act on 2 ~ 3min, after magnet is fully adsorbed, discard suspending liquid, add the PBS repeated washing 3 times of the 0.01M PH7.4 of 1ml.By the 37 DEG C of cultivation 24h on XLT4 agar medium of the salmonella after enrichment, counting, obtains the quantity of salmonella in sample.
Embodiment 6
Take 25g feed (mash feed, particulate material) in the aseptic homogeneous cup filling 225mL 0.01M PH 7.4 PBS damping fluid, 10000r/min homogeneous 1 ~ 2 minute, makes the even liquid of sample of 1:10.Get the even liquid of 200 μ l sample, join in 300 μ l magnetic beads, 37 DEG C of reaction 30min.Be placed on magnet and act on 2 ~ 3min, after magnet is fully adsorbed, discard suspending liquid, add the PBS repeated washing 3 times of the 0.01M PH7.4 of 1ml.By the 37 DEG C of cultivation 24h on XLT4 agar medium of the salmonella after enrichment, counting, obtains the quantity of salmonella in sample.
Embodiment 7
Take 25g animal dung sample in the aseptic homogeneous cup filling 225mL 0.01M PH 7.4 PBS damping fluid, 10000r/min homogeneous 1 ~ 2 minute, makes the even liquid of sample of 1:10.Get the even liquid of 200 μ l sample, join in 300 μ l magnetic beads, 37 DEG C of reaction 30min.Be placed on magnet and act on 2 ~ 3min, after magnet is fully adsorbed, discard suspending liquid, add the PBS repeated washing 3 times of the 0.01M PH7.4 of 1ml.By the 37 DEG C of cultivation 24h on XLT4 agar medium of the salmonella after enrichment, counting, obtains the quantity of salmonella in sample.
Claims (3)
1. based on a method for immunomagnetic beads enrichment salmonella, it is characterized in that: the specific antibody for the preparation of described immunomagnetic beads is that salmonella outer membrane protein OmpC immunize New Zealand rabbit obtains.
2., based on a method for immunomagnetic beads enrichment salmonella, it is characterized in that carrying out as follows:
(1) testing sample phosphate buffer (0.01M ~ 0.1M, PH6.0 ~ 7.4) is made the even liquid of a certain proportion of testing sample, draw 100 ~ 200 μ l, join in 150 ~ 300 μ l magnetic beads, 37 DEG C of reaction 30 ~ 60min;
(2) be placed on magnet and act on 2 ~ 3min, after magnet is fully adsorbed, discard suspending liquid, add the PBS(0.01M ~ 0.1M of 1ml, PH6.0 ~ 7.4) repeated washing 2 ~ 3 times;
(3) by the 37 DEG C of cultivation 24h on XLT4 agar medium of the salmonella after enrichment, counting determines the quantity of salmonella in sample;
RP-HPLC described is in step (1) 1:9 with the W:V ratio of phosphate buffer; Feed is 1:9 with the W:V ratio of phosphate buffer; Animal dung sample is 1:9 with the W:V ratio of phosphate buffer; The volume ratio of water and phosphate buffer is 1:9; The volume ratio of the yellow and white potpourri and phosphate buffer is 1:9; Eggshell joins in 50 ~ 100ml phosphate buffer.
3. a kind of method based on immunomagnetic beads enrichment salmonella according to claims 2, is characterized in that: to detect sample be egg, RP-HPLC, feed, water, animal dung sample.
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| CN104726534A (en) * | 2015-04-23 | 2015-06-24 | 河南省商业科学研究所有限责任公司 | Method for fast detecting salmonella in fresh milk |
| CN104726534B (en) * | 2015-04-23 | 2017-04-19 | 河南省商业科学研究所有限责任公司 | Method for fast detecting salmonella in fresh milk |
| CN108950060A (en) * | 2017-09-06 | 2018-12-07 | 武汉中科志康生物科技有限公司 | A kind of microorganism detection method |
| CN108085377A (en) * | 2017-12-29 | 2018-05-29 | 北京和益源生物技术有限公司 | The detection method of salmonella under a kind of high background |
| CN110819686A (en) * | 2019-10-24 | 2020-02-21 | 广东环凯生物科技有限公司 | Salmonella immunomagnetic bead washing liquor |
| CN111366726A (en) * | 2020-03-12 | 2020-07-03 | 天津海关动植物与食品检测中心 | Method for detecting salmonella in food by combining immune enrichment with MALDI-TOF MS and application |
| WO2022048350A1 (en) * | 2020-09-03 | 2022-03-10 | 南昌大学 | Immunomagnetic adsorbent based on phenylboronic acid directional coupling antibody and preparation method therefor |
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