CN108676842A - A kind of method of quick detection Listeria monocytogenes and staphylococcus aureus - Google Patents
A kind of method of quick detection Listeria monocytogenes and staphylococcus aureus Download PDFInfo
- Publication number
- CN108676842A CN108676842A CN201810789480.2A CN201810789480A CN108676842A CN 108676842 A CN108676842 A CN 108676842A CN 201810789480 A CN201810789480 A CN 201810789480A CN 108676842 A CN108676842 A CN 108676842A
- Authority
- CN
- China
- Prior art keywords
- sample
- listeria monocytogenes
- staphylococcus aureus
- detection
- added
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/14—Streptococcus; Staphylococcus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
- G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of methods of quick detection Listeria monocytogenes and staphylococcus aureus, belong to field of biological detection.The step of this method includes：（1）Sample is taken under aseptic condition, homogeneous in the phosphate buffer of the fifth component containing bovine serum albumin(BSA) is added to and obtains the equal liquid of sample；（2）The equal liquid of low-temperature centrifugation sample collects thalline, growth-promoting media oscillation mixing is added into the thalline of collection and forms bacteria suspension, immunomagnetic beads are taken uniformly to be mixed with bacteria suspension, after water-bath heat treatment, in stratification on magnetic frame, abandon supernatant, add eluent washing lower layer sample, obtains sample prepare liquid, be applied on tablet and cultivate, observe detection.Detection method provided by the invention can effectively shorten detection time, reduce time cost, find in time and cut off the infection sources.
Description
Technical field
The invention belongs to technical field of microbial detection, and in particular to a kind of quickly detection Listeria Monocytogenes In Food and
The method of staphylococcus aureus.
Background technology
Listeria monocytogenes are a kind of infecting both domestic animals and human food-borne pathogens being distributed widely in nature, it can cause people and animals
Listeriosis, be mainly shown as septicemia, meningitis and monocytosis after infection, especially to pregnant woman, newborn,
The elderly and immune deficiency patient harm are serious.The bacterium can be widely present in numerous food, such as meat products, dairy produce, aquatic products
Product, vegetables etc., the bacterium once led to multiple extensive food poisoning in American-European countries, therefore to Listeria Monocytogenes In Food
Quickly detection is very necessary, effectively controls the Listeria monocytogenes in food, is one of important topic of food security.
Staphylococcus aureus is food-borne and iatrogenic pathogenic bacteria, has stronger resistance to various chemical factors,
And the food such as meat easy to pollute, dairy products, it is widely present in natural environment, the enterotoxin that it is generated is the master for causing food poisoning
Virulence factor, caused food poisoning is wanted to account for larger proportion in food posioning.Research staphylococcus aureus exists
Pollution situation in food, to prevent the breaking out of food origin disease being induced by it, popular and tracking pollution sources provide foundation.
The detection method of Listeria monocytogenes and staphylococcus aureus at present also depends on traditional microbiology
Detection method.Traditional national standard detection method（GB 4789.10 and GB 4789.30）Can just go out at least 2-5 days as a result, it is desirable to by
Increase bacterium, medium culture, separation, detection etc., operation and complex steps, time-consuming, separation rate is low, and can only detect in sample and deposit
Thalline living, recall rate are low compared with the content in actual sample, false negative result easily occur, endanger consumers in general's safety.Cause
This, proposes a kind of ways and means quickly detecting both bacteriums, is carried out to staphylococcus aureus and Listeria monocytogenes
It quickly detects very necessary, to find the infection sources early, cuts off route of transmission.
Invention content
The purpose of the present invention is to propose to a kind of methods of quick detection Listeria monocytogenes and staphylococcus aureus, and
It is early to find the infection sources, cut off route of transmission.
To achieve the goals above, the present invention uses following technical scheme：
A kind of method of quick detection Listeria monocytogenes and staphylococcus aureus, includes the following steps：
（1）Sample is taken under aseptic condition, is added to fifth component containing bovine serum albumin(BSA)（BSAV）Phosphate buffer in
Matter obtains the equal liquid of sample；
（2）The equal liquid of low-temperature centrifugation sample collects thalline, and growth-promoting media is added into the thalline of collection, and oscillation mixing makes growth-promoting media and bacterium
Liquid fully combines, formed bacteria suspension, take immunomagnetic beads uniformly to be mixed with bacteria suspension, water-bath be heat-treated kill non-targeted bacterium after, in
Stratification on magnetic frame, abandons supernatant, adds eluent washing lower layer sample, obtains sample prepare liquid, be applied on tablet and cultivate, observe
Detection.
Preferably, step（1）In, the amount ratio of the sample and phosphate buffer is 1:9, the phosphate buffer
The mass concentration of middle bovine serum albumin(BSA) fifth component is 1%~10%.
Preferably, the growth-promoting media by 95wt% defatted milks, 65wt% tetramethyls glutaric acid, 40wt% Nafusakus and
0.15wt% ammonium chlorides are according to volume ratio 4:3:3:2 blend together；The growth-promoting media is 1 with the equal liquid volume ratio of sample：（1~50）.
Preferably, step（2）In, the temperature of the low-temperature centrifugation is 4 DEG C, rotating speed 8000rmp.
Preferably, step（2）In, the duration of oscillation is 1min~10min, and mode of oscillation is supersonic oscillations；It is described
Water-bath heat treatment temperature is 36 DEG C~60 DEG C, and water bath time is 10min~60min.
Preferably, the immunomagnetic beads are that the single of couple biotin label increases Li Si after modifying magnetic bead by Streptavidin
Special bacterium and Staphylococcus aureus antibody are made, and include the following steps：
a）Contain the phosphate buffer of 0.05% Tween-20 with 0.01mol/L（PBST buffer solutions, pH 7.4, Tween-
20）Listeria monocytogenes and Staphylococcus aureus antibody are diluted to 1mg/ml；10mg/ml biotins are prepared with anhydrous DMSO
N-hydroxysuccinimide eater solution（BNHS solution）, BNHS solution is added in diluted antibody and mixed, BNHS solution with
Antibody diluent volume ratio is 4:1, at room temperature, it is incubated the Listeria monocytogenes and Staphylococcus aureus to get biotin labeling
Bacteria antibody；
b）Then be added 1mg active dissolution Streptavidin modification magnetic bead mixed, be placed at room temperature for；
c）Add 9.6 μ L 1mol/L NH4Cl solution, incubation at room temperature；The molecular sieve column of upper 1ml, is eluted with PBS buffer solution,
1ml/ pipes are collected, the mixed liquor of PBS buffer solution and 0.01% Sodium azide is added, sets -20 DEG C and is kept in dark place.
Since the BNHS solution of high concentration can cause multiple biotin molecules to be incorporated on antibody, all antibody may be made
All labeled, lower concentration can then make biotinylation be maintained at bottom line, therefore when immunomagnetic beads preparation, select with anhydrous
DMSO prepares 10mg/ml BNHS；It may cause biotinylation insufficient since the amino of antibody is not accessible, can be added at this time
Antibody-solutions PBST buffer solutions are eluted, remove unbonded biotin, prevent in reaction mixture by dirty agent such as Tween 20
In with the presence of Sodium azide or free amine group, inhibit label reaction.
Preferably, step（1）In, a concentration of 0.02mol/L of phosphate buffer；Step（2）In, the eluent
For 0.02mol/L phosphate buffers, washing steps are 2~3 times.
Compared with prior art, the device have the advantages that it is as follows：
（1）The present invention is by BSAV（Biotechnology grade）It is added in buffer solution, prevents the macromolecular chaff interferences such as albumen, fat, make
The more stable growth of object bacteria in sample, and eluting rate is reduced in subsequent experiment, impurity is reduced, can effectively be selected
Property protection thalline.
（2）The growth-promoting media of the present invention is by 95% defatted milk, 65% tetramethyl glutaric acid, 40% Nafusaku, 0.15% ammonium chloride
Composition, wherein 95% defatted milk can protected protein, play the role of protectant, 0.15% ammonium chloride can remove in bacteria suspension
Impurity, 65% tetramethyl glutaric acid is root-inducing hormone, can promote preferably to be combined with sample, and Nafusaku, which has, to be promoted carefully
Growth-promoting media is added after collecting thalline in the function that born of the same parents quickly expand, low-temperature centrifugation, can promote object bacteria fast-growth, shortens culture
Time, and solve the test problems of low-level pathogenic microorganism, increase the recall rate of object bacteria.
（3）The present invention can specifically capture Listeria monocytogenes and staphylococcus aureus simultaneously using immunomagnetic beads,
By object bacteria, quick separating effectively avoids or reduces false positive phenomenon to reduce the interference to detection accuracy from sample
Occur, and realize two kinds of object bacterias of synchronous detection, reduces detection time.
To sum up, the verification and measurement ratio of detection method is high in the present invention and sensitivity is all higher, can be present in complete detection sample
Object bacteria avoids the occurrence of the generation of false negative phenomenon, reduces the harm to consumers in general.Meanwhile detection provided by the invention
Method is easy to operate, precise and high efficiency, using growth-promoting media and immunomagnetic beads, can effectively reduce increasing compared with tradition is separately cultured
Bacterium solution incubation time and tablet incubation time, shorten entire detection time, 12 are foreshortened to by 48h~96h of conventional method~
48h。
Specific implementation mode
With reference to specific embodiment, the present invention will be further described.
Prepare immunomagnetic beads
Immunomagnetic beads are that the single of couple biotin label increases Liszt and golden yellow grape after modifying magnetic bead by Streptavidin
Coccus antibody is made, and includes the following steps：
a）With 0.01mol/L PBST buffer solutions（PH 7.4, Tween-20）The single increasing Liszt of dilution and staphylococcus aureus are anti-
Body is to 1mg/ml；10mg/ml biotins N-hydroxysuccinimide eater solution is prepared with anhydrous DMSO（BNHS solution）, BNHS
Solution is added in diluted antibody and is mixed, and BNHS solution is 4 with antibody diluent volume ratio:1, at room temperature, be incubated to get
The single of biotin labeling increases Liszt and Staphylococcus aureus antibody；
b）Then be added 1mg active dissolution Streptavidin modification magnetic bead mixed, be placed at room temperature for；
c）Add 9.6 μ L 1mol/L NH4Cl solution, incubation at room temperature；The molecular sieve column of upper 1ml, is eluted with PBS buffer solution,
1ml/ pipes are collected, the mixed liquor of PBS buffer solution and 0.01% Sodium azide is added, sets -20 DEG C and is kept in dark place.
The screening of 1 BASV concentration of embodiment
1.1 Listeria monocytogenes
The PBS buffer solution of 6 parts of BASV for taking 225mL to contain various concentration respectively（A concentration of 0.02mol/L of PBS）In homogenizing bag
In, the sample homogeneous 30s that 25g contains Listeria monocytogenes is added in each homogenizing bag, the concentration for adding BASV is respectively 0%（Not
Add BASV）, 0.5%, 1%, 5%, 8% and 10%.
In addition, 225mL Li Shi is taken to increase bacterial context soup LB1 liquid as a comparison again, the sample that 25g contains Listeria monocytogenes is added
Sample liquid is made as a comparison case in homogeneous 30s.
According to traditional detection method, the operations such as increasing bacterium, scribing line separation, identification are carried out to above-mentioned sample liquid respectively, obtain examination
It tests as a result, shown in result table 1.
Staphylococcus aureus
With reference to the above-mentioned experimentation of Listeria monocytogenes, with above-mentioned experimentation the difference is that：It selects containing golden yellow
Staphylococcic sample experiments；7.5% sodium chloride broth liquid as a comparison is used in comparative example, remaining operation is identical, the results are shown in Table
1。
Table 1 selects the test result of different buffer concentrations
The clump count highest that can detect after the PBS buffer solution culture containing 1%BSAV as can be known from Table 1, and continue to increase
Its concentration, clump count is added also not to further increase, therefore the concentration of BSAV is preferably 1%.
The optimal addn of 2 growth-promoting media of embodiment
It is taken respectively containing Listeria monocytogenes and S. aureus-positive sample 25g, is added to the 225mL containing 1%BASV
In the homogenizing bag of PBS buffer solution, homogeneous 30s.Take 6 parts of 5mL sample bacterium solutions, 4 DEG C, 8000rpm thalline is collected after centrifugation, respectively plus
Enter growth-promoting media 0.1mL, 0.5mL, 1mL, 2mL, 4mL, 5mL, mixing.Then it is applied on the corresponding tablet of two kinds of bacteriums, presses respectively
The operations such as scribing line separation, biochemical test are carried out according to GB4789, obtain test result, as shown in table 2, GB4789 are selected simultaneously in table
Whole process detection method as a comparison.
The bacterium colony testing result of not same amount growth-promoting media is added in table 2
As seen from table, with the increase of growth-promoting media, bacterial population is consequently increased, when growth-promoting media is 1mL, Bacteria cold shock
At most, the positive clump count identified is also most；If continuing growing growth-promoting media quantity, bacterial population will not increase, and cannot
It is apparent to distinguish positive bacterium colony；And growth-promoting media additive amount it is low when, clump count is less, therefore the addition of growth-promoting media is preferably 1mL.
3 sonic oscillation mixing Best Times of embodiment
It takes containing Listeria monocytogenes and S. aureus-positive sample 25g, is added separately to the 225mL containing 1%BASV
In the homogenizing bag of PBS buffer solution, homogeneous 30s.Take 5 parts of 5mL sample bacterium solutions, 4 DEG C, 8000rpm thalline is collected after centrifugation, be added
1mL growth-promoting medias mix growth-promoting media with bacterium solution, and respectively sonic oscillation, 5 sample duration of oscillation be followed successively by 1min, 3min,
5min, 8min and 10min are applied on the corresponding tablet of two kinds of bacteriums after oscillation and carry out scribing line and operations, the detection knot such as be separately cultured
Fruit is as shown in table 3.
The clump count of 3 sonic oscillation different time of table detection
The results show that when oscillation incorporation time is 5min, the clump count detected is most, increases the time, clump count is not at any time
Growth and increase；If duration of oscillation is long, it can make to have been coupled to mycoprotein or compound on immunomagnetic beads and occur to break
It splits, is unfavorable for the rapid aggregation of later stage immunomagnetic beads；And it is too short be not completely separated, waste bacterium solution, the final result that reduces
Accuracy.Therefore, the preferred 5min of oscillation incorporation time.
Embodiment 4 sterilizes optimal bath temperature
First, it takes respectively containing Listeria monocytogenes and S. aureus-positive sample 25g, addition fills 225mL 1%
In the sterile homogenizing bags of PBS of BSAV, homogeneous 30s.
Then, take 4 parts of 5mL sample bacterium solutions, 4 DEG C, 8000rpm thalline is collected after centrifugation, 1mL growth-promoting medias, oscillation is added
5min mixings form bacteria suspension.
Take 4 part of 200 μ L immunomagnetic beads uniformly to be mixed with 4 parts of bacteria suspensions respectively again, bath temperature be set to 36 DEG C, 45
DEG C, 60 DEG C and 70 DEG C, kill non-targeted bacterium.
2min on magnetic frame is stood, waits for that magnetic bead fully by magnetic aggregation, abandons supernatant, is separately added into 1mL 0.02mol/L
PBS eluents, mix well, and after above step is repeated 3 times, 100 μ L 0.02mol/L PBS concussions is added, magnetic bead is resuspended, obtain
Sample prepare liquid takes 100 μ L to be applied on the corresponding tablet of two kinds of bacteriums respectively, and the operations such as scribing line separation are carried out according to GB4789,
Detect that clump count is as shown in table 4 on different colour developing tablets.
The detection clump count of the different bath temperatures of table 4
As shown in Table 4, bacterial population is not to increase with the increase of bath temperature, and bath temperature is more than that bacterium is not after 60 DEG C
Increase, is while to be easy immunomagnetic beads is made to be combined with bacteria suspension upper because of the excessively high progress not only bad for reaction of bath temperature
Protein cleavage；And bath temperature is too low that the purpose for removing non-targeted bacterium is not achieved.When bath temperature is 60 DEG C, Li Si is detected
The clump count of special bacterium and staphylococcus aureus is most, therefore bath temperature is preferably 60 DEG C when sterilizing.
Embodiment 5 sterilizes optimal water bath time
The quickly single method for increasing Liszt and staphylococcus aureus of detection, steps are as follows：
（1）It takes out respectively and contains Listeria monocytogenes and S. aureus-positive sample 25g, addition fills 225mL 1%
In the sterile homogenizing bags of PBS of BSAV, homogeneous 30s.
（2）Take 5 parts of 5mL sample bacterium solutions, 4 DEG C, 8000rpm thalline is collected after centrifugation, growth-promoting media 1mL is added, vibrates 5min
Mixing forms bacteria suspension.It is then respectively adding 200 μ L immunomagnetic beads uniformly to mix, 5 parts of samples are respectively at 60 DEG C of heat treatments of water-bath
10min, 20min, 30min, 45min and 60min kill non-targeted bacterium.2min on magnetic frame is stood, waits for magnetic bead fully by magnetic force
Supernatant is abandoned in aggregation, and 1mL 0.02mol/L PBS eluents are added, mix well, and after above step is repeated 3 times, 100 μ L are added
Magnetic bead is resuspended in 0.02mol/L PBS concussions, obtains sample prepare liquid, 100 μ L is taken to be applied on the corresponding tablet of two kinds of bacteriums respectively.
（3）The operations such as scribing line separation are carried out according to GB4789, are then detected, the results are shown in Table 5.
The detection clump count of the different water bath times of table 5
The result shows that with the increase of water bath time, bacterial population is consequently increased, when water bath time is 20min, bacterial growth shape
State is most；If continuing growing water bath time, the increase of nonspecific reaction can be caused；And water bath time is too short fully to combine
Object bacteria, therefore water-bath heat treatment time is preferably 20min when sterilizing.
6 immunomagnetic beads sensitivity tests of embodiment
By Listeria monocytogenes and Staphylococcus aureus bacterium solution, doubling dilution is carried out by certain gradient.Each gradient is passed
Tablet culture of uniting counts as a control group.
Separately take Listeria monocytogenes and staphylococcus aureus immunity magnetic bead（5mg/mL）100 μ L, are added separately to 1mL's
In Listeria monocytogenes and staphylococcus aureus dilution, 60 DEG C of heat treatment 20min, stand on magnetic frame after mixing well
2min waits for that magnetic bead fully by magnetic aggregation, abandons supernatant, 1mL0.02mol/L PBS eluents is added, mix well, stand magnetic force
2min on frame waits for that magnetic bead is fully adsorbed, and abandons supernatant, washes repeatedly 3 times, and 100 μ L 0.02mol/L PBS concussions are added and are resuspended
Magnetic bead draws 100 μ L and is applied to bacterium and corresponds to and develops the color on tablet, and 36 DEG C are cultivated for 24 hours~48h observation bacterium colonies and counted as experiment
Group, the results are shown in Table 6.
The sensitivity tests of 6 immunomagnetic bead technique of table
As shown in Table 6, it is 10 in dilution when Listeria monocytogenes and staphylococcus aureus culture solution-8When, control group is
Object bacteria can not be detected, and test group can still detect object bacteria, compared with the control group, testing result is higher than control group, sensitive
Property it is high.
Claims (7)
1. a kind of method of quick detection Listeria monocytogenes and staphylococcus aureus, which is characterized in that include the following steps：
（1）Sample is taken under aseptic condition, homogeneous in the phosphate buffer of the fifth component containing bovine serum albumin(BSA) is added to and obtains sample
The equal liquid of product；
（2）The equal liquid of low-temperature centrifugation sample collects thalline, and growth-promoting media oscillation mixing is added into the thalline of collection and forms bacteria suspension, takes
Immunomagnetic beads are uniformly mixed with bacteria suspension, after water-bath heat treatment, in stratification on magnetic frame, abandon supernatant, eluent is added to wash
Lower layer's sample obtains sample prepare liquid, is applied on tablet and cultivates, observes detection.
2. the method for quickly detecting Listeria monocytogenes and staphylococcus aureus as described in claim 1, which is characterized in that step
Suddenly（1）In, the amount ratio of the sample and phosphate buffer is 1:9, bovine serum albumin(BSA) in the phosphate buffer
The mass concentration of five components is 1%~10%.
3. the method for quickly detecting Listeria monocytogenes and staphylococcus aureus as described in claim 1, which is characterized in that institute
Growth-promoting media is stated by 95wt% defatted milks, 65wt% tetramethyls glutaric acid, 40wt% Nafusakus and 0.15wt% ammonium chlorides according to volume
Than 4:3:3:2 blend together；The growth-promoting media is 1 with the equal liquid volume ratio of sample：（1~50）.
4. the method for quickly detecting Listeria monocytogenes and staphylococcus aureus as claimed in claim 3, which is characterized in that step
Suddenly（2）In, the temperature of the low-temperature centrifugation is 4 DEG C, rotating speed 8000rmp.
5. quickly the method for detection Listeria monocytogenes and staphylococcus aureus, feature exist as described in claim 3 or 4
In step（2）In, the oscillation uses supersonic oscillations 1min~10min；The water-bath heat treatment temperature is 36 DEG C~60
DEG C, water bath time is 10min~60min.
6. the method for quickly detecting Listeria monocytogenes and staphylococcus aureus as described in claim 1, which is characterized in that institute
It is the Listeria monocytogenes and golden yellow grape that couple biotin label after magnetic bead is modified by Streptavidin to state immunomagnetic beads
Coccus antibody is made.
7. the method for quickly detecting Listeria monocytogenes and staphylococcus aureus as described in claim 1, which is characterized in that step
Suddenly（1）In, a concentration of 0.02mol/L of phosphate buffer；Step（2）In, the eluent is slow for 0.02mol/L phosphate
Fliud flushing, washing steps are 2~3 times.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810789480.2A CN108676842B (en) | 2018-07-18 | 2018-07-18 | Method for rapidly detecting Listeria monocytogenes and staphylococcus aureus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810789480.2A CN108676842B (en) | 2018-07-18 | 2018-07-18 | Method for rapidly detecting Listeria monocytogenes and staphylococcus aureus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108676842A true CN108676842A (en) | 2018-10-19 |
| CN108676842B CN108676842B (en) | 2022-04-12 |
Family
ID=63814115
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810789480.2A Active CN108676842B (en) | 2018-07-18 | 2018-07-18 | Method for rapidly detecting Listeria monocytogenes and staphylococcus aureus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108676842B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109541205A (en) * | 2018-12-07 | 2019-03-29 | 河南省商业科学研究所有限责任公司 | The detection method of lactic acid bacteria in a kind of probiotics |
| CN109557308A (en) * | 2019-01-18 | 2019-04-02 | 河南省商业科学研究所有限责任公司 | A kind of method of Listeria Monocytogenes in quick detection dairy products |
| CN109679878A (en) * | 2019-01-18 | 2019-04-26 | 河南省商业科学研究所有限责任公司 | A method of Listeria monocytogenes in enrichment meat and meat products |
| CN109696546A (en) * | 2019-02-25 | 2019-04-30 | 河南省商业科学研究所有限责任公司 | A method of detection yersinia enterocolitica |
| CN109735599A (en) * | 2019-01-18 | 2019-05-10 | 河南省商业科学研究所有限责任公司 | A method of improving staphylococcus aureus detection limit in food liquid easy to foaming |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468823A (en) * | 2013-09-30 | 2013-12-25 | 山东出入境检验检疫局检验检疫技术中心 | Listeria monocytogenes LAMP detection primer and detection method, kit and preparation method |
| CN104894219A (en) * | 2015-06-29 | 2015-09-09 | 河南省商业科学研究所有限责任公司 | Method for detecting salmonella and staphylococcus aureus in food simultaneously |
-
2018
- 2018-07-18 CN CN201810789480.2A patent/CN108676842B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468823A (en) * | 2013-09-30 | 2013-12-25 | 山东出入境检验检疫局检验检疫技术中心 | Listeria monocytogenes LAMP detection primer and detection method, kit and preparation method |
| CN104894219A (en) * | 2015-06-29 | 2015-09-09 | 河南省商业科学研究所有限责任公司 | Method for detecting salmonella and staphylococcus aureus in food simultaneously |
Non-Patent Citations (3)
| Title |
|---|
| XIANGYU MENG等: "Sensitive Detection of Staphylococcus aureus with Vancomycin-Conjugated Magnetic Beads as Enrichment Carriers Combined with Flow Cytometry", 《ACS APPL MATER INTERFACES》 * |
| 周莉等: "免疫磁珠检测食品中单增李斯特菌的研究", 《中国食品添加剂》 * |
| 周莉等: "免疫磁珠检测食品中金黄色葡萄球菌的研究", 《河南科学》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109541205A (en) * | 2018-12-07 | 2019-03-29 | 河南省商业科学研究所有限责任公司 | The detection method of lactic acid bacteria in a kind of probiotics |
| CN109541205B (en) * | 2018-12-07 | 2022-01-28 | 河南省商业科学研究所有限责任公司 | Method for detecting lactic acid bacteria in probiotics |
| CN109557308A (en) * | 2019-01-18 | 2019-04-02 | 河南省商业科学研究所有限责任公司 | A kind of method of Listeria Monocytogenes in quick detection dairy products |
| CN109679878A (en) * | 2019-01-18 | 2019-04-26 | 河南省商业科学研究所有限责任公司 | A method of Listeria monocytogenes in enrichment meat and meat products |
| CN109735599A (en) * | 2019-01-18 | 2019-05-10 | 河南省商业科学研究所有限责任公司 | A method of improving staphylococcus aureus detection limit in food liquid easy to foaming |
| CN109696546A (en) * | 2019-02-25 | 2019-04-30 | 河南省商业科学研究所有限责任公司 | A method of detection yersinia enterocolitica |
| CN109696546B (en) * | 2019-02-25 | 2022-04-12 | 河南省商业科学研究所有限责任公司 | Method for detecting yersinia enterocolitica |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108676842B (en) | 2022-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108676842A (en) | A kind of method of quick detection Listeria monocytogenes and staphylococcus aureus | |
| Do Carmo et al. | Food poisoning due to enterotoxigenic strains of Staphylococcus present in Minas cheese and raw milk in Brazil | |
| Khambaty et al. | Application of pulsed-field gel electrophoresis to the epidemiological characterization of Staphylococcus intermedius implicated in a food-related outbreak | |
| Allerberger et al. | Hemolytic-uremic syndrome associated with enterohemorrhagic Escherichia coli O26: H infection and consumption of unpasteurized cow's milk | |
| Loncarevic et al. | Occurrence of Listeria monocytogenes in soft and semi-soft cheeses in retail outlets in Sweden | |
| CN101412978B (en) | Culture medium for composite enrichment of salmonella, Listeria monocytogenes and Staphylococcus aureus, and preparation thereof | |
| CN101412977B (en) | Culture medium for composite enrichment of salmonella, Vibrio parahaemolyticus and Vibrio cholerae, and preparation thereof | |
| CN110229918A (en) | A kind of method and its kit of quick detection Staphylococcus aureus in food | |
| Day et al. | A comparison of ELISA and RPLA for detection of Bacillus cereus diarrhoeal enterotoxin | |
| Rowan et al. | Maltodextrin stimulates growth of Bacillus cereus and synthesis of diarrheal enterotoxin in infant milk formulae | |
| Lindqvist et al. | The prevalence of verocytotoxin-producingEscherichia coli (VTEC) andE. coliO157: H7 in beef in Sweden determined by PCR assays and an immuno-magnetic separation (IMS) method | |
| CN106947756A (en) | The method of quick Magneto separate bacillus cereus | |
| Batchoun et al. | Molecular characterization of Bacillus cereus toxigenic strains isolated from different food matrices in Jordan | |
| CN104726534B (en) | Method for fast detecting salmonella in fresh milk | |
| CN109696546A (en) | A method of detection yersinia enterocolitica | |
| WU et al. | RAPID PROTOCOL (5.25 H) FOR THE DETECTION OF ESCHERICHIA COLI O157: H7 IN RAW GROUND BEEF BY AN IMMUNO‐CAPTURE SYSTEM (PATHATRIX) IN COMBINATION WITH COLORTRIX AND CT‐SMAC 1 | |
| CN109541205A (en) | The detection method of lactic acid bacteria in a kind of probiotics | |
| Ueda et al. | Detection of Listeria monocytogenes from food samples by PCR after IMS-plating | |
| CN104388522A (en) | Selective chromogenic culture medium of listeria monocytogenes and preparation method of selective chromogenic culture medium | |
| Ng et al. | Enterotoxigenic strains of coagulase-positive Staphylococcus aureus in drinks and ready-to-eat foods | |
| CN105203758A (en) | Monotone increasing listeria monocytogene detecting method based on colloidal gold test strip | |
| CN109557308A (en) | A kind of method of Listeria Monocytogenes in quick detection dairy products | |
| Domján Kovács et al. | Testing of a chemiluminescence enzyme immunoassay for selective detection of E. Coli O157 from ground beef samples | |
| CN104450628A (en) | Escherichia coli resistance O157:H7 monoclonal antibody and application thereof | |
| CN102286630A (en) | Listeria monocytogenes (LM) immuno-capture and polymerase chain reaction (IC-PCR) detection reagent kit and preparation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |