CN108531625A - The rapid detection method of Listeria Monocytogenes in a kind of leafy vegetable - Google Patents
The rapid detection method of Listeria Monocytogenes in a kind of leafy vegetable Download PDFInfo
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Abstract
The invention belongs to testing food pathogenic technical fields, specifically disclose a kind of rapid detection method of Listeria Monocytogenes in leafy vegetable, including collecting sample to be tested, cyclodextrin handles sample, prepare cow's milk protein, prepare cow's milk protein coating activated carbon, prepare cow's milk protein coating activated carbon processing sample, PCR detection and etc..The method whole operation step of the present invention is simple, can effectively detach pathogenic bacteria from leaf vegetables sample, is not necessarily to preceding increasing bacterium, detection time to sample is 4 hours, and the time is short and detection limit is only 10cfu/25g, and detection sensitivity is high, high specificity, it is not easy to false positive or false negative result occur.
Description
Technical field
The invention belongs to testing food pathogenic technical fields, and in particular to monocyte hyperplasia Lee in a kind of leafy vegetable
The rapid detection method of this special Salmonella.
Background technology
Food-borne pathogens are the first causes for causing food origin disease, and food-borne pathogens cause greatly human health
Harm, is the major hidden danger of food security.Food origin disease is not reduced or is disappeared with economic development and technological progress, the world
Food security malignant event occurs in succession in range, and food origin disease fails effectively to be controlled, and food origin disease incidence occupies
It is high not under, the situation is tense for food security.Although government regulator continues to increase food-safe supervision, food is pacified
Full research field science research input increases year by year, while Consciousness of food security in production process is also continuously improved in food enterprise, introduces
The advanced managements systems such as HACCP ensure food security, but food-borne pathogens contaminated food products and then cause food-borne
Outbreak of disease is increasingly frequent.At present to the control of pathogenic bacteria contaminated food products approach in, can not also find a kind of fully effective prevent
Pathogenic bacteria pollution mode, develop the highly practical pathogenic bacteria detection method of rapid sensitive just become ensure the one of food security
A important leverage.
At this stage although common rapid detection method is used widely, Standard PCR and immunological detection method, usually
Problems faced is that detection method sensitivity is not high enough, and for normal food sample after selective enrichment is handled, bacterium to be checked is dense
Degree will reach 104-105Cfu/ml can just obtain comparing accurate result, when, there are preservative, natural antibacterial is antibacterial in food samples
When ingredient, often detection limit is not achieved in pathogenic bacteria concentration to be checked, leads to false positive or false negative result occur.In addition, food
In contain a large amount of PCR response inhabitations factor, such as fat, protein, enzyme, antibiotic, organic and Inorganic chemical substance, polysaccharide
Substance etc., these inhibiting factors greatly affected the application that PCR method detects pathogenic bacteria in food.Currently, for gram
The method for taking the PCR response inhabitation factors is the means for increasing bacterium by 12-24 hours before detection, improves the sensitivity of detection, but same
When also extend detection time.
To sum up, problem of the existing technology is that conventional PCR method and enzyme-linked immunization sensitivity are not high enough, are easy out
Existing false positive or false negative result.
Invention content
The rapid detection method of Listeria Monocytogenes, solves in a kind of leafy vegetable provided by the invention
Standard PCR and enzyme-linked immunization sensitivity are not high enough, the problem of being susceptible to false positive or false negative result.
The object of the present invention is to provide a kind of rapid detection method of Listeria Monocytogenes in leafy vegetable,
Include the following steps：
S1 collects sample to be tested
S2 prepares cyclodextrin processing sample
Into leaf vegetables sample to be measured plus sterile saline, homogenization are added cyclodextrin, and make solution cyclodextrin most
Final concentration of 10g/100ml, homogenization, centrifugation removal large particulate matter, the centrifugation of gained supernatant are collected sediment and are dissolved in
In 0.01M acetic acid normal saline buffer solutions, cyclodextrin processing sample is obtained；
S3 prepares cow's milk protein
Skimmed milk powder and ultra-pure water mixing, it is ensured that skimmed milk powder thoroughly dissolves；95% ethanol solution of volume fraction is added, stirs
Mix 2min precipitation cow's milk proteins；The cow's milk protein of precipitation centrifuges under room temperature；Finally the cow's milk protein of precipitation is suspended again
In ultra-pure water, pH to 9.0 is adjusted, cow's milk protein solution is obtained；
S4 prepares cow's milk protein coating activated carbon
Activated carbon is cleaned with ultra-pure water, 55 DEG C of dryings obtain dry activated carbon；In the cow's milk protein solution prepared to S3
Dry activated carbon is added, mixing liquid after the completion of concussion, outwells supernatant liquid, bottom in 37 DEG C, 150rpm concussion coating 120min
The activated carbon ultrapure water of layer, it is dry, obtain cow's milk protein coating activated carbon；
S5 prepares cow's milk protein coating activated carbon processing sample
Cow's milk protein is cleaned with 0.85% physiological saline of mass fraction and is coated with activated carbon, by cyclodextrin processing sample and cleaning
Good cow's milk protein coating activated carbon mixing, room temperature concussion；Mixture after concussion is transferred in the container equipped with mineral wool,
It is cleaned with the 0.01M acetic acid normal saline buffer solution and collects eluent；Eluent centrifuges, and discards supernatant liquid；Precipitation is outstanding
It floats in 5mg/ml bovine serum albumin solutions, 0.05mg/ml salmon sperm dnas solution and ultra-pure water is added；Boiling water bath obtains
Cell lysate cooled on ice to room temperature, centrifugation takes supernatant, and the absolute ethyl alcohol with the 4 DEG C of precoolings of supernatant equivalent volumes is added, from
The heart discards supernatant liquid, retains the DNA of precipitation, and the DNA of sterile ultrapure water dissolution precipitation is added, obtains DNA profiling, spare；
S6, PCR are detected
PCR detects the primer sequence used：
Iap1：5’-acaagctgcagctgatgcag-3’
Iap2：5’-tgagagcgtgtgtagtagct-3’
If the PCR detection product segments obtained are 131bp, result is the positive.
Preferably, in above-mentioned leafy vegetable in the rapid detection method of Listeria Monocytogenes, S2's is specific
Step is：It takes and 0.85% physiological saline of aseptic quality score, leaf vegetables sample and aseptic quality to be measured is added in leaf vegetables sample to be measured
The ratio of 0.85% physiological saline of score is 1g：3ml, homogenization are added cyclodextrin, and make solution cyclodextrin ultimate density
For 10g/100ml, continue homogeneous 2min, 1000rpm and centrifuge 5min, remove large particulate matter, gained supernatant 10000rpm from
Heart 10min collects sediment and is dissolved in 0.01M acetic acid normal saline buffer solutions, obtains cyclodextrin processing sample.
Preferably, in above-mentioned leafy vegetable in the rapid detection method of Listeria Monocytogenes, S3's is specific
Steps are as follows：By skimmed milk powder and ultra-pure water according to 24g：The ratio mixing of 25ml, it is ensured that skimmed milk powder thoroughly dissolves；Phase is added
When in 95% ethanol solution of volume fraction of 2 times of volumes of ultra-pure water, stirring 2min precipitates cow's milk protein；The cow's milk protein room of precipitation
5000rpm centrifuges 5min under the conditions of temperature；Finally the cow's milk protein of precipitation is resuspended in and is equivalent to 3 times of volumes of ethanol solution
In ultra-pure water, with 0.1M sodium hydroxide solution tune pH to 9.0, cow's milk protein solution is obtained.
Preferably, in above-mentioned leafy vegetable in the rapid detection method of Listeria Monocytogenes, S4's is specific
Steps are as follows：The activated carbon of 0.85~2.0nm of grain size is cleaned 3 times with ultra-pure water, 55 DEG C of dryings obtain dry activated carbon；To
It is added dry activated carbon in cow's milk protein solution prepared by S3, the ratio of activated carbon and cow's milk protein solution is 44g：150ml is mixed
Liquid is closed in 37 DEG C, 150rpm concussion coating 120min, after the completion of concussion, outwells supernatant liquid, the activated carbon of bottom is with ultrapure
Water rinses 3 times, 55 DEG C of dryings, obtains cow's milk protein coating activated carbon.
Preferably, it in above-mentioned leafy vegetable in the rapid detection method of Listeria Monocytogenes, in S5, will sink
Shallow lake is suspended in 5mg/ml bovine serum albumin solutions, 0.05mg/ml salmon sperm dnas solution and ultra-pure water is added, wherein cow's milk
The ratio that albumen is coated with activated carbon, bovine serum albumin solution, salmon sperm dna solution and ultra-pure water is 4.6g:100μl:100μ
l:300μl。
Preferably, in above-mentioned leafy vegetable in the rapid detection method of Listeria Monocytogenes, in S6, PCR
Using 50 μ l PCR reaction systems：5 μ 10 × PCR of l buffer, 4 μ l dNTPs mixtures, 0.5 μ l primer Is ap1,0.5 μ l draw
Object Iap2,0.25 μ l Taq archaeal dna polymerases, template 1 μ l, ddH2O38.75μl；
PCR reaction conditions：94 DEG C of pre-degeneration 4min；94 DEG C of denaturation 1min；55 DEG C of annealing 1min；72 DEG C extension 1min, 30
A cycle；72 DEG C extend 7min eventually；4 DEG C of preservations.
Compared with prior art, in a kind of leafy vegetable Listeria Monocytogenes rapid detection method, tool
There is following advantageous effect：
(1) method of the invention is a kind of Fast Practical sensitive from leafy vegetable PCR detections without preceding increasing bacterium by force
It is (newly-built to greatly shorten the detection time of conventional Listeria Monocytogenes detection and rapid detection method at present for method
Vertical detection method is 4 hours to the detection time of sample), and it is only 10cfu/25g to detect limit, detection sensitivity is high, does not allow
Easily there is false negative result, it can be ensured that food detects in real time from farm to the food security in the dining table process of circulation, Jin Ergeng
Ensure consumer health well.
(2) due to containing a large amount of PCR response inhabitations factor in food, for example, it is fat, protein, enzyme, antibiotic, organic
And Inorganic chemical substance, polysaccharose substance etc., these inhibiting factors greatly affected PCR method and detected to pathogenic bacteria in food
Application.Currently, being the means for increasing bacterium by 12-24 hours before detection for overcoming the method for the PCR response inhabitation factors, improve
The sensitivity of detection, but detection time is also extended simultaneously.
The purpose of the present invention is establishing effective sample treatment to improve pathogenic bacteria from the rate of recovery in food, eliminate
The PCR response inhabitation factors.Since leaf vegetables surface has coating of wax matter structure, this wax structure to influence whether bentonite packet
By activated carbon to the adsorption and enrichment ability of Listeria Monocytogenes in leaf vegetables, and then influence whether the pathogenic bacteria
Testing result.We then can preferably keep the adsorption and enrichment ability to the pathogenic bacteria using cow's milk protein coating activated carbon,
Pathogenic bacteria detected in leaf vegetables that can be more sensitive and accurate, therefore for monocyte hyperplasia Listeria in leaf vegetables
Bacterium detects, and the method for the present invention then can preferably ensure detection sensitivity and accuracy using cow's milk protein coating activated carbon.In addition
Quantitative salmon sperm dna is added in sample handling processes can further remove the PCR response inhabitation factors, and it is sensitive to improve detection
Degree, so newly-established detection method only needs 4 hours to the detection time of sample.
Description of the drawings
Fig. 1 is positive and negative result electrophoretogram；
Wherein, M swimming lanes are DNA maker, 1,3-28 swimming lanes be positive test symbol, No. 2 swimming lanes are that negative detection is tied
Fruit.
Specific implementation mode
With reference to specific example, the present invention is described in detail, but should not be construed as the limitation of the present invention.It is real below
Test method without specific conditions in example is carried out according to the conventional method of this field and condition.
Embodiment 1
The rapid detection method of Listeria Monocytogenes, includes the following steps in a kind of leafy vegetable：
S1 collects sample to be tested
The sample to be tested that the present embodiment 1 is collected is artificial contaminated samples, and artificial contamination's sample is prepared as follows：It takes
(concentration range is respectively 25g leaf vegetables sample (spinach) artificial contamination's 1ml Listeria Monocytogenes bacteria suspension
100cfu/ml、101cfu/ml、102cfu/ml、103cfu/ml、104cfu/ml、105Cfu/ml), the sample of artificial contamination is put into
It is preserved overnight in 4 DEG C of refrigerators, it is ensured that pathogenic bacteria are effectively adsorbed on sample, obtain artificial contamination's sample, and detection is wanted before starting
Actual test is carried out to the pathogenic bacteria concentration to be checked in sample, test method is with reference to various pathogenic bacteria detections in existing state food
Method carries out.
S2 prepares cyclodextrin processing sample
It takes and 0.85% physiological saline of 75ml aseptic qualities score is added in 25g artificial contamination's samples, carried out using homogenizer
Cyclodextrin solution is added in homogenization, and solution cyclodextrin ultimate density is made to be 5g/100ml, continues homogenization 2min,
1000rpm low-speed centrifugal 5min, remove large particulate matter, and gained supernatant 10000rpm high speed centrifugation 10min collect sediment
It is dissolved in 30ml 0.01M acetic acid normal saline buffer solutions, obtains cyclodextrin processing sample.
Wherein, the acetic acid for the acetic acid and 0.01mol that solute is 0.01mol in every liter of 0.01M acetic acid normal saline buffer solution
Sodium, solvent are mass fraction 0.85%NaCl solution, and it is 5.0 to adjust pH with 0.1M hydrochloric acid solutions.
S3 prepares cow's milk protein
Infant's skimmed milk powder 24.0g is added in 400ml beakers, and 25ml ultra-pure waters are added, and magnetic stirring apparatus middling speed is stirred
Mix 2min, it is ensured that skimmed milk powder thoroughly dissolves；95% (volume fraction) ethanol solution 50ml is taken to be added in above-mentioned beaker, magnetic force stirs
Mix 2min precipitation cow's milk proteins；The cow's milk protein of precipitation is transferred to 250ml centrifugal bottles, under room temperature (20~25 DEG C),
5000rpm (or 8000g) centrifuges 5min；The cow's milk protein finally precipitated is resuspended in 150ml ultra-pure waters, with 0.1M hydrogen
Sodium hydroxide solution tune pH to 9.0 obtains cow's milk protein solution, spare.
S4 prepares cow's milk protein coating activated carbon
The activated carbon of 0.85~2.0nm of grain size is cleaned 3 times with ultra-pure water, 55 DEG C of dryings obtain dry activated carbon；By S3
The 150ml cow's milk protein solution of preparation is transferred in 1L beakers, 44g dry activated carbons is added, mixing liquid is in 37 DEG C, 150rpm
Concussion coating 120min, after the completion of concussion, is carefully poured off supernatant liquid, and the activated carbon of bottom is gently rinsed 3 times with ultra-pure water,
The cow's milk protein not being combined is removed, beaker is placed into drying in 55 DEG C of incubators, until the coated activated carbon of cow's milk protein is thorough
Bottom drying (dry to moisture≤2%) obtains cow's milk protein coating activated carbon, spare.
S5 prepares cow's milk protein coating activated carbon processing sample
4.6g cow's milk proteins coating activated carbon is taken to be added in 250ml beakers, with 0.85% physiology of 20ml intelligence love tinkling of pieces of jade score
Brine cleans 2 times, 30ml cyclodextrin processing sample is mixed with cleaned cow's milk protein coating activated carbon, mixture is in room temperature
Lower 160rpm rotations concussion 15min；0.2g sterilized glass wools are taken to put to 50ml aseptic plastic syringes bottom, it will be mixed after concussion
It closes object to be transferred in the syringe equipped with mineral wool, using sterile centrifugation tube as collection vessel, with the 0.01M acetic acid physiological saline
Buffer solution for cleaning in sterile centrifugation tube until be collected into 30ml eluents；30ml eluents centrifuge 10min through 10000rpm, abandon
Supernatant is gone to retain precipitation；Precipitation is suspended in 0.5ml and contains 100 μ l 5mg/ml bovine serum albumin solutions (solute is cow's serum
Albumin, solvent are sterile ultra-pure water) in, 100 μ l 0.05mg/ml salmon sperm dnas solution and the 300 sterile ultra-pure waters of μ l are added；
Centrifuge tube is put into boiling water, boiling water bath thermal cracking cell 10min, extremely by obtained cell lysate cooled on ice
Room temperature, 12000rpm centrifuge 5min, supernatant are transferred in clean centrifuge tube, are added pre- with 4 DEG C of the supernatant equivalent volumes
Cold absolute ethyl alcohol, to precipitate DNA, centrifuge tube 12000 centrifuges 15min again, discards supernatant liquid, retains precipitation, 100 μ l are added
The DNA of sterile ultrapure water dissolution precipitation, DNA sample packing, every part of 10 μ l obtain DNA profiling, spare.
S6, PCR are detected
Specific primer sequences are designed for Listeria Monocytogenes virulence gene (iap), optimize reaction condition.
The primer sequence is：
Iap1：5’-acaagctgcagctgatgcag-3’
Iap2：5’-tgagagcgtgtgtagtagct-3’
50 μ l PCR reaction systems：5 μ 10 × PCR of l buffer, 4 μ l dNTPs mixtures, 0.5 μ l primer Is ap1 (40 μ
Mol/L), 0.5 μ l primer Is ap2 (40 μm of ol/L), 0.25 μ l (5U/ μ l) Taq archaeal dna polymerases, template 1 μ l, ddH2O 38.75
μl。
PCR reaction conditions：94 DEG C of pre-degeneration 4min；94 DEG C of denaturation 1min；55 DEG C of annealing 1min；72 DEG C extension 1min, 30
A cycle；72 DEG C extend 7min eventually；4 DEG C of preservations.
If the PCR detection product segments obtained are 131bp, result is the positive, positive and negative result electrophoresis
Figure is as shown in Figure 1.Wherein, M swimming lanes are DNA maker, 1,3-28 swimming lanes be positive test symbol, No. 2 swimming lanes are negative examine
Survey result.
It should be noted that cyclodextrin used in embodiment 1 is beta-cyclodextrin.
Embodiment 2
The rapid detection method of Listeria Monocytogenes, includes the following steps in a kind of leafy vegetable：
Either Chinese cabbage prepares or directly selects spinach to artificial contamination's sample romaine lettuce, Lettuce, the rape of embodiment 2
Dish, romaine lettuce, Lettuce, rape or Chinese cabbage carry out the operation of follow-up S2 as sample to be tested.The step of remaining S2~S6 with
Embodiment 1 is identical, and it will not go into details herein.
Embodiment 3
The rapid detection method of Listeria Monocytogenes, includes the following steps in a kind of leafy vegetable：
Quality in " skimmed milk powder 24.0g " in the S3 of embodiment 1 is changed to " 12.0g, 6.0g, 3.0g, 1.5g,
Any of 0.75g or 0.38g ", remaining operation is the same as embodiment 1.
For here is the method for embodiment 1, illustrate the effect of the present invention.
One, the detection result of different primers
We carry out actual test to common microbiological in different food, are prepared using the method for the embodiment of the present invention 1
Artificial contamination's sample (leaf vegetables sample includes spinach, romaine lettuce, Lettuce, rape or Chinese cabbage) and extraction different microorganisms
DNA, and carry out PCR reactions using the primer in the embodiment of the present invention 1 and verify the specificity of detection method as experimental group.Institute
It includes extraction Listeria Monocytogenes, other listeria spps and non-listeria spp to state microorganism.
To have disclosed the Listeria Monocytogenes detection primer specificity (PrfA delivered:5’-
CCCAAGTAGCAGGACATGCTAA-3’；PrfB:5 '-GGTATCACAAAGCTCACGAG-3 ') as a contrast, remaining operation is same
Experimental group.Simultaneously as a comparison in Listeria Monocytogenes examination criteria method in existing state food, to verify
The practicability of newly-established PCR detection method, the results are shown in Table 1.
The specificity of 1 different primers detection method of table
Note："+" indicates that positive findings, "-" indicate negative findings in table 1.
Table 1 the result shows that, the primer sequence that embodiment 1 uses has preferable specificity, only to monocyte hyperplasia Lee
This special Salmonella is positive testing result, and is negative testing result to other listeria spps and non-listeria spp.And in addition
A kind of PrfA primers are negative for some monocytes monocytogenes as a result, part other listeria spps detection is in
Positive findings show that the specificity of the primer sequence is not highly desirable, and there are the defects of false negative testing result.Existing country
Test for Listeria Monocytogenes in Foods examination criteria method is identical as the result of embodiment 1.
Two, the determination of detection limit
By the Listeria Monocytogenes artificial contamination of various concentration to leaf vegetables sample (including spinach, romaine lettuce, oil
Wheat dish, rape or Chinese cabbage) in, it is detected using the detection method of embodiment 1, determines the detection sensitivity of embodiment 1,
It the results are shown in Table 2.As shown in Table 2, the detection sensitivity of the detection method of embodiment 1 is 10cfu/25g, and is had good stability.It is raw
Dish, Lettuce, rape or the testing result of Chinese cabbage are identical with the testing result of spinach, herein only with the result of spinach
As an example, i.e. when cell concentration is 1cfu/25g, testing result is feminine gender, when cell concentration is more than or equal to 10cfu/25g
When, testing result is the positive.Illustrate the present invention method for leaf vegetables sample (including spinach, romaine lettuce, Lettuce, rape or
Chinese cabbage) it is general.
The sensitivity for artificial contamination's dairy products that table 2 is prepared with spinach
Note："+" indicates that positive findings, "-" indicate negative findings in table 1.
Three, with the comparison of existing national standards method
Commercially available fresh spinach, romaine lettuce, Lettuce, rape or each 500 parts of Chinese cabbage are acquired, implementation of the present invention is respectively adopted
The rapid detection method of example 1 and existing national examination criteria are detected comparison, for verifying the practicality of the rapid detection method
Property, it the results are shown in Table 3.As shown in Table 3, recall rate, coincidence rate and the live body bacterium of the method for embodiment 1 and existing national standard method
Verification and measurement ratio all same, but the method detection time of embodiment 1 is obviously shortened, only 4 hours.
3 actual sample test result of table
Four, duration experiment is preserved
In the detection method of the present invention used main agents consumptive material have cow's milk protein, cow's milk protein coating activated carbon,
Various buffer solutions etc..Wherein, cow's milk protein, cow's milk protein coating activated carbon are prepared using the method for embodiment 1, various buffer solutions
It is referred to Ru not special, then uses existing method to prepare.
In order to test the stability of used reagent consumptive material in the method for the present invention, reagent consumptive material used in method is protected
In the presence of under -20 DEG C of freezing conditions, preserves 3 months, 6 months, 12 months respectively, then acquire commercially available spinach, romaine lettuce, oil respectively
Wheat dish, rape or each 500 parts of Chinese cabbage carry out test comparison, and are compared with existing national examination criteria, confirm present invention side
Stability and practicability of the reagent consumptive material in the case where long-time preserves used in method.
4 artificial contamination's Sample storage duration experimental results of table
Note："+" indicates that positive findings, "-" indicate negative findings in table 4.
Table 4 the results show that spinach, life after mentioned reagent consumptive material is preserved 3 months, 6 months, 12 months at -20 DEG C
As a result its detection sensitivity of the sample tests such as dish, Lettuce, rape or Chinese cabbage, detection sensitivity 10cfu/25g are demonstrate,proved
Reagent consumptive material in bright the method for the present invention can at least preserve 12 months under -20 DEG C of freezing conditions, and detection sensitivity is not
It can be affected.
In order to test the stability of used reagent consumptive material in the method for the present invention, reagent consumptive material used in method is protected
In the presence of under -20 DEG C of freezing conditions, preserves 3 months, 6 months, 12 months respectively, then acquire commercially available spinach, romaine lettuce, oil respectively
Wheat dish, rape or each 500 parts of Chinese cabbage carry out test comparison, and are compared with existing national examination criteria, confirm present invention side
Stability and practicability of the reagent consumptive material in the case where long-time preserves used in method.Table 5 is the test result of actual sample, 5 knot of table
Fruit shows that the reagent consumptive material of reagent consumptive material and national standard method preparation used in the method for the present invention preserves 3 at -20 DEG C
The moon, 6 months the samples such as spinach, romaine lettuce, Lettuce, rape or Chinese cabbage are detected under the conditions of 12 months, and use state
Family's examination criteria compares, the recall rate of the method for the present invention, and coincidence rate and national examination criteria method are consistent, live body bacterium verification and measurement ratio
Reach 100%, as a result confirm that the practicability of the method for the present invention is fabulous, agents useful for same consumptive material (live by cow's milk protein, cow's milk protein coating
Property charcoal etc.) preservation at least 12 months can be stablized under freezing conditions.
5 actual sample of table preserves duration experimental results
Five, influence of the different capsulating materials to testing result sensitivity
Comparative example：" cow's milk protein coating activated carbon " in embodiment 1 is changed to " bentonite coating activated carbon ", it is specific to walk
It is rapid as follows：The activated carbon of 200 mesh is spent into Ion Cleaning 3 times, it is drained and standby.200ml ultra-pure waters, shake are added into 4g bentonites
1min is swung, 700rpm centrifuges 1min, abandons precipitation, the 120ml bentonitic upper solutions for containing 1.52g are gone to 1 liter of beaker,
The activated carbon (weight in wet base) that 30g is drained is added, beaker is placed on 150rpm in rotary shaker, 37 DEG C of incubator overnights (>=12h), then will
Solution in beaker is placed in 55 DEG C of baking ovens and dries, until the coated activated carbon of bentonite is dried (moisture≤1%), obtains
It is coated with activated carbon to bentonite.It should be noted that bentonite needs first to be cleaned, some impurity are removed, could be used, I
Test confirm centrifugation after bentonite solution in bentonitic content be 1.52g/120ml, so cannot be directly by 1.52g
Bentonite be added in 120ml water, otherwise can influence testing result.Remaining operating procedure of comparative example is the same as embodiment 1.
According to above " two, detection limit determination " described in method measure artificial contamination's sample sensitivity, the results show that
According to Listeria Monocytogenes in the method spinach of embodiment 1, romaine lettuce, Lettuce, rape or cabbage samples
Detection be limited to 10cfu/25g；According to monokaryon in the method spinach of comparative example, romaine lettuce, Lettuce, rape or cabbage samples
The detection of Monocytogenes is limited to 100cfu/25g.
We respectively take 500 parts of commercially available spinach, romaine lettuce, rape, Lettuce and Chinese cabbage to be used as actual sample, detect practical sample
Listeria Monocytogenes in product, the results show that spinach in the cow's milk protein coating absorbent charcoal material of embodiment 1
Positive rate is 1.4%, the positive rate of romaine lettuce is 2.6%, and rape, Lettuce and the positive rate of Chinese cabbage are
0%；The positive rate of spinach is 0.6% in the bentonite coating activated carbon of comparative example, the positive rate of romaine lettuce is
1.2%, rape, Lettuce and the positive rate of Chinese cabbage are 0%.
The above results illustrate, for the leaf vegetables sample such as spinach, romaine lettuce, Lettuce, rape or Chinese cabbage, to use cow's milk egg
It is more preferable as capsulating material effect in vain.Leaf vegetables surface has coating of wax matter structure, this wax structure to influence whether swelling
Soil is coated with adsorption and enrichment ability of the activated carbon to Listeria Monocytogenes in leaf vegetables, and then influences whether that this causes a disease
The testing result of bacterium.And cow's milk protein coating activated carbon is used then can preferably to keep the adsorption and enrichment energy to the pathogenic bacteria
Power, pathogenic bacteria detected in leaf vegetables that can be more sensitive and accurate, therefore for monocyte hyperplasia Li Si in leaf vegetables
Special Salmonella detection, the method for the present invention then can preferably ensure detection sensitivity and accuracy using cow's milk protein coating activated carbon.
Six, the influence of different virulence gene pairs testing result sensitivity
The embodiment of the present invention 1 is directed to iap virulence gene design primers, in major leaf such as Hebei province, Beijing, Tianjin
472 plants of Listeria Monocytogenes for isolating identification in the leaf vegetables sample of vegetables vegetables production base acquisition
(all bacterial strains, which pass through API10300 biochemical identifications kit and the identification of BD full automatic microorganism bacterial strain assessing instruments, ensures 472 plants
Bacterial strain is Listeria Monocytogenes), wherein the iap primer sequence specificity designed by the method for the present invention reaches
100%, and the InlB primer sequences before us in oyster designed by detection Listeria Monocytogenes are detecting
It finds there are 11 plants of bacterium InlB gene delection phenomenons occur in comparison, increases in order to ensure accurately detecting monocyte in leaf vegetables
Raw listeria spp avoids the occurrence of false negative result, then uses iap primer sequences in the methods of the invention.
It should be noted that involved in the present invention when numberical range, it is thus understood that two endpoints of each numberical range with
And any one numerical value can be selected between two endpoints, since the step method of use is identical as embodiment, go to live in the household of one's in-laws on getting married in order to prevent
It stating, the present invention describes preferred embodiment 1, although preferred embodiments of the present invention have been described, but technology in the art
Personnel once know basic creative concept, then additional changes and modifications may be made to these embodiments.So appended power
Profit requires to be intended to be construed to include preferred embodiment and fall into all change and modification of the scope of the invention.
Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art
God and range.In this way, if these modifications and changes of the present invention belongs to the range of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to include these modifications and variations.
Claims (6)
1. the rapid detection method of Listeria Monocytogenes in a kind of leafy vegetable, which is characterized in that including following
Step：
S1 collects sample to be tested
S2 prepares cyclodextrin processing sample
Into leaf vegetables sample to be measured plus sterile saline, homogenization are added cyclodextrin, and keep solution cyclodextrin finally dense
Degree is 10g/100ml, and homogenization centrifuges removal large particulate matter, and the centrifugation of gained supernatant collects sediment and is dissolved in 0.01M
In acetic acid normal saline buffer solution, cyclodextrin processing sample is obtained；
S3 prepares cow's milk protein
Skimmed milk powder and ultra-pure water mixing, it is ensured that skimmed milk powder thoroughly dissolves；95% ethanol solution of volume fraction, stirring is added
2min precipitates cow's milk protein；The cow's milk protein of precipitation centrifuges under room temperature；Finally the cow's milk protein of precipitation is resuspended in
In ultra-pure water, pH to 9.0 is adjusted, cow's milk protein solution is obtained；
S4 prepares cow's milk protein coating activated carbon
Activated carbon is cleaned with ultra-pure water, 55 DEG C of dryings obtain dry activated carbon；It is added in the cow's milk protein solution prepared to S3
Dry activated carbon, mixing liquid after the completion of concussion, outwell supernatant liquid in 37 DEG C, 150rpm concussion coating 120min, bottom
Activated carbon ultrapure water, it is dry, obtain cow's milk protein coating activated carbon；
S5 prepares cow's milk protein coating activated carbon processing sample
With 0.85% physiological saline of mass fraction clean cow's milk protein be coated with activated carbon, by cyclodextrin processing sample with it is cleaned
Cow's milk protein is coated with activated carbon mixing, room temperature concussion；Mixture after concussion is transferred in the container equipped with mineral wool, institute is used
0.01M acetic acid normal saline buffer solutions are stated to clean and collect eluent；Eluent centrifuges, and discards supernatant liquid；Precipitation is suspended in
In 5mg/ml bovine serum albumin solutions, 0.05mg/ml salmon sperm dnas solution and ultra-pure water is added；Boiling water bath, obtained cell
Lysate cooled on ice to room temperature, centrifugation takes supernatant, and the absolute ethyl alcohol with the 4 DEG C of precoolings of supernatant equivalent volumes is added, centrifugation,
Liquid is discarded supernatant, the DNA of precipitation is retained, the DNA of sterile ultrapure water dissolution precipitation is added, obtains DNA profiling, it is spare；
S6, PCR are detected
PCR detects the primer sequence used：
Iap1：5’-acaagctgcagctgatgcag-3’
Iap2：5’-tgagagcgtgtgtagtagct-3’
If the PCR detection product segments obtained are 131bp, result is the positive.
2. the rapid detection method of Listeria Monocytogenes in leafy vegetable according to claim 1, special
Sign is, S2 the specific steps are：It takes and 0.85% physiological saline of aseptic quality score, leaf vegetables to be measured is added in leaf vegetables sample to be measured
The ratio of 0.85% physiological saline of sample and aseptic quality score is 1g：3ml, homogenization are added cyclodextrin, and make in solution
Cyclodextrin ultimate density is 10g/100ml, continues homogeneous 2min, and 1000rpm centrifuges 5min, removes large particulate matter, on gained
Clear liquid 10000rpm centrifuges 10min, collects sediment and is dissolved in 0.01M acetic acid normal saline buffer solutions, obtains cyclodextrin processing
Sample.
3. the rapid detection method of Listeria Monocytogenes in leafy vegetable according to claim 2, special
Sign is that S3 is as follows：By skimmed milk powder and ultra-pure water according to 24g：The ratio mixing of 25ml, it is ensured that skimmed milk powder
Thoroughly dissolving；95% ethanol solution of volume fraction for being equivalent to 2 times of volumes of ultra-pure water is added, stirring 2min precipitates cow's milk protein；
5000rpm centrifuges 5min to the cow's milk protein of precipitation under room temperature；Finally the cow's milk protein of precipitation is resuspended in and is equivalent to
In the ultra-pure water of 3 times of volumes of ethanol solution, with 0.1M sodium hydroxide solution tune pH to 9.0, cow's milk protein solution is obtained.
4. the rapid detection method of Listeria Monocytogenes in leafy vegetable according to claim 3, special
Sign is that S4 is as follows：The activated carbon of 0.85~2.0nm of grain size is cleaned 3 times with ultra-pure water, 55 DEG C of dryings obtain
To dry activated carbon；Dry activated carbon, the ratio of activated carbon and cow's milk protein solution is added in the cow's milk protein solution prepared to S3
Example is 44g：150ml, mixing liquid after the completion of concussion, outwell supernatant liquid, bottom in 37 DEG C, 150rpm concussion coating 120min
The activated carbon of layer ultrapure water 3 times, 55 DEG C of dryings obtain cow's milk protein coating activated carbon.
5. the rapid detection method of Listeria Monocytogenes in leafy vegetable according to claim 4, special
Sign is, in S5, precipitation is suspended in 5mg/ml bovine serum albumin solutions, be added 0.05mg/ml salmon sperm dnas solution and
Ultra-pure water, wherein cow's milk protein coating activated carbon, bovine serum albumin solution, salmon sperm dna solution and ultra-pure water ratio be
4.6g:100μl:100μl:300μl。
6. the rapid detection method of Listeria Monocytogenes in leafy vegetable according to claim 5, special
Sign is, in S6, PCR uses 50 μ l PCR reaction systems：5 μ 10 × PCR of l buffer, 4 μ l dNTPs mixtures, 0.5 μ l
Primer I ap1,0.5 μ l primer Is ap2,0.25 μ l Taq archaeal dna polymerases, template 1 μ l, ddH2O 38.75μl；
PCR reaction conditions：94 DEG C of pre-degeneration 4min；94 DEG C of denaturation 1min；55 DEG C of annealing 1min；72 DEG C of extension 1min, 30 are followed
Ring；72 DEG C extend 7min eventually；4 DEG C of preservations.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN203866319U (en) * | 2014-05-26 | 2014-10-08 | 上海理工大学 | Immuno-PCR (Polymerase Chain Reaction) detection kit for Listeria monocytogenes |
| CN105816414A (en) * | 2016-04-05 | 2016-08-03 | 广州市娇兰化妆品有限公司 | Yeast water and preparation method as well as application thereof to cosmetics |
| CN107164540A (en) * | 2017-07-12 | 2017-09-15 | 河北科技师范学院 | The quick determination method of Listeria Monocytogenes in one seed oyster |
| WO2020182924A1 (en) * | 2019-03-14 | 2020-09-17 | Merck Patent Gmbh | A novel sampling method for long-term monitoring of microbes |
-
2018
- 2018-04-12 CN CN201810325743.4A patent/CN108531625A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN203866319U (en) * | 2014-05-26 | 2014-10-08 | 上海理工大学 | Immuno-PCR (Polymerase Chain Reaction) detection kit for Listeria monocytogenes |
| CN105816414A (en) * | 2016-04-05 | 2016-08-03 | 广州市娇兰化妆品有限公司 | Yeast water and preparation method as well as application thereof to cosmetics |
| CN107164540A (en) * | 2017-07-12 | 2017-09-15 | 河北科技师范学院 | The quick determination method of Listeria Monocytogenes in one seed oyster |
| WO2020182924A1 (en) * | 2019-03-14 | 2020-09-17 | Merck Patent Gmbh | A novel sampling method for long-term monitoring of microbes |
Non-Patent Citations (1)
| Title |
|---|
| 杨洋: ""中国食源性单核细胞增生李斯特菌分型及毒力基因研究"", 《医药卫生科技辑》 * |
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