CN108254559A - The method of quick detection microorganism - Google Patents
The method of quick detection microorganism Download PDFInfo
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- CN108254559A CN108254559A CN201810005602.4A CN201810005602A CN108254559A CN 108254559 A CN108254559 A CN 108254559A CN 201810005602 A CN201810005602 A CN 201810005602A CN 108254559 A CN108254559 A CN 108254559A
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
Abstract
The present invention relates to microorganism detection fields, disclose a kind of method of quick detection microorganism.It the described method comprises the following steps：1) sample to be tested is subjected to pre-treatment, to obtain sample to be tested of the pH value as 6.4 7.4 liquid form；2) immunomagnetic beads are mixed with the sample to be tested of the liquid form, the condition of mixing cause when in sample to be tested there are during object bacteria, immunomagnetic beads can specifically combining target bacterium, to obtain the mixture containing immunomagnetic beads target bacteria complex；3) the mixed material for obtaining step 2) carries out the first Magneto separate, to obtain the first aggregation containing the immunomagnetic beads target bacteria complex, and removes and is not associated with the substance on immunomagnetic beads；4) the first aggregation that step 3) obtains is resuspended, to obtain re-suspension liquid；5) re-suspension liquid for obtaining step 4) carries out chemiluminescence detection.The present invention, which realizes, carries out the object bacteria in sample purpose that is quick, accurately detecting.
Description
Technical field
The present invention relates to microorganism detection fields, and in particular to a kind of method of quick detection microorganism, it is particularly (non-to examine
Disconnected destination) method of trace microorganism in detection sample.
Background technology
Food security is great livelihood issues.Food-borne microbial pathogens effectively monitoring for food quality control and
It supervises particularly important.It (is such as posted for the pathogenic microorganism (such as bacterium, fungi and virus) in food and other harmful organisms
It is infested), traditional detection method is isolated culture.Increase bacterium, biochemical identification or serological Identification before being separately cultured needs, entirely
Detection process needs the time of even 5-6 days 2-3 days that could complete, and culture medium needed for detection process prepares, tablet
Culture, bacterium colony counting, biochemical identification etc. so that Detection task labor intensity is big, take, it is impossible to micro- in timely, Fast Evaluation food
The safety of biology, is unfavorable for fast reaction of the supervision department to problem food.Traditional detection pathogenic microorganism method and step is more,
Required time is grown, and speed is slow, it is difficult to be adapted to food and quickly be circulated the needs of detection.The actually detected universal face of food-borne pathogens
Face the interference of complicated food composition, high concentration miscellaneous bacteria, it is accurate to detect that content is low and the technical bottleneck of object bacteria unevenly distributed.
Salmonella in Food, staphylococcus aureus, Listeria, the quantity of Cronobacter sakazakii are general less (being less than 10CFU/g)
And the ratio that accounts in sample whole microorganisms is smaller (is generally less than 1%, national standard allows 103-105CFU/g's is non-pathogenic micro-
Biology is present in food).However, existing detection microorganism (particularly salmonella, staphylococcus aureus, Liszt
Bacterium, Cronobacter sakazakii etc.) method detection limit it is often higher, and there are false positive or false negative testing results.For example, at present
10 are generally using the detection limit of chemiluminescence immune analysis method detection salmonella5-106CFU/mL。
It (is particularly eaten therefore, it is quite necessary to find new quick, accurate and with relatively low detection limit detection sample
Product sample) in microorganism method.
Invention content
The purpose of the invention is to overcome drawbacks described above of the existing technology, a kind of (non-diagnostic purpose) detection is provided
The method of microorganism when being detected using this method to the microorganism in sample, can be detected quickly and accurately in sample
Object bacteria, and detection limit is relatively low, so as to efficiently avoid the risk of missing inspection.
To achieve these goals, the method that microorganism is quickly detected the present invention provides a kind of (non-diagnostic destination),
This method includes the following steps：
(1) sample to be tested is subjected to pre-treatment, to obtain sample after processing of the pH value as the liquid form of 6.4-7.4；
(2) immunomagnetic beads are mixed with sample after the processing of the liquid form, the condition of mixing to work as sample to be tested
In there are during object bacteria, immunomagnetic beads can specifically combining target bacterium, to obtain containing immunomagnetic beads-target bacteria complex
Mixture；
(3) the mixed material obtained step (2) carries out the first Magneto separate, with obtain containing the immunomagnetic beads-
First aggregation of target bacteria complex, and remove and be not associated with the substance on immunomagnetic beads；
(4) the first aggregation for obtaining step (3) carries out the first resuspension, to obtain the first re-suspension liquid；Or
The first aggregation that step (3) obtains is subjected to Zengjing Granule and the second Magneto separate successively, and by the second Magneto separate
The second obtained aggregation carries out second and is resuspended, to obtain the second re-suspension liquid；
(5) the first re-suspension liquid obtained step (4) or the second re-suspension liquid carry out chemiluminescence detection, to determine to treat test sample
The presence of object bacteria in product.
The present invention is by carrying out pre-treatment to sample to be tested successively, being captured in sample to be tested using immunomagnetic beads specificity
Object bacteria (preferably carries out Zengjing Granule) to the object bacteria of immunomagnetic beads specificity capture, then carries out chemiluminescence detection, so as to
The purpose that quick (within 10 hours) are carried out to the object bacteria in sample, are accurately detected is realized, and relatively low in object bacteria concentration
(down to 10-1CFU/g (or mL)) in the case of can also detect, so as to efficiently avoid the risk of missing inspection, have it is good should
Use prospect.
Description of the drawings
Fig. 1 is to carry out the result figure of Salmeterol fluticasone propionate using the method for the present invention in embodiment 1；
Fig. 2 is to carry out the result figure of Listeria monocytogenes detection using the method for the present invention in embodiment 6；
Fig. 3 is to carry out the result figure of staphylococcus aureus detection using the method for the present invention in embodiment 9；
Fig. 4 is to carry out the result figure of Cronobacter sakazakii detection using the method for the present invention in embodiment 13.
Specific embodiment
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood to comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It between the endpoint value of a range and individual point value and can be individually combined with each other between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
The present invention provides the method for microorganism in a kind of (non-diagnostic destination) quickly detection sample, this method include with
Lower step：
(1) sample to be tested is subjected to pre-treatment, to obtain sample to be tested of the pH value as the liquid form of 6.4-7.4；
(2) immunomagnetic beads are mixed with sample after the processing of the liquid form, the condition of mixing to work as sample to be tested
In there are during object bacteria, immunomagnetic beads can specifically combining target bacterium, to obtain containing immunomagnetic beads-target bacteria complex
Mixture；
(3) the mixed material obtained step (2) carries out the first Magneto separate, with obtain containing the immunomagnetic beads-
First aggregation of target bacteria complex, and remove and be not associated with the substance on immunomagnetic beads；
(4) the first aggregation for obtaining step (3) carries out the first resuspension, to obtain the first re-suspension liquid；Or
The first aggregation that step (3) obtains is subjected to Zengjing Granule and the second Magneto separate successively, and by the second Magneto separate
The second obtained aggregation carries out second and is resuspended, to obtain the second re-suspension liquid；
(5) the first re-suspension liquid obtained step (4) or the second re-suspension liquid carry out chemiluminescence detection, to determine to treat test sample
The presence of object bacteria in product.
In the present invention, the object bacteria can be for the various common microorganisms in this field, particularly harmful microorganism (such as
Pathogenic bacteria), for example, various bacteriums, fungi or virus.Preferably, the object bacteria is salmonella (Salmonella), golden yellow
Color staphylococcus (Staphylococcus aureus), Cronobacter sakazakii (Cronobacter) and Listeria (Listeria)
At least one of；It is highly preferred that the object bacteria is salmonella typhimurium (Salmonella typhimurium), enteritis
Salmonella (Salmonella enteritidis), Salmonella paratyphi A (Salmonella paratyphi A),
Salmonella choleraesuls (Salmonella choleraesuis), Listeria monocytogenes (Listeria
Monocytogenes), staphylococcus aureus (Staphylococcus aureus) and the rugged Cronobacter sakazakii of slope
At least one of (Cronobacter sakazakii).
There is no particular limitation for form of the present invention to the sample to be tested, as long as after pre-treatment, can obtain liquid shape
Sample after the processing of formula, for example, the form of the sample to be tested can be solid (such as pork head meat, beef, dumpling and rice flour
Deng further including pasty masses, such as day cream), or liquid (such as milk, beverage, soy sauce).The present invention treats test sample to described
Also there is no particular limitation in the source of product, preferably food sources and/or medicine source and/or environment.
In the present invention, in step (1), the mode of the pre-treatment preferably mixes sample to be tested and pretreatment liquid
It closes；It is highly preferred that the pH value for using sample after the processing for causing the liquid form of the pretreatment liquid is 6.8-7.4.
Specifically, in step (1), when the sample to be tested is fluid sample (such as milk, beverage, soy sauce), institute
The mode for stating pre-treatment is：Sample to be tested (fluid sample) is mixed with pretreatment liquid, and the sample to be tested and the preceding place
The volume ratio for managing liquid is 1：0.04-0.5；When the sample to be tested is solid sample (such as meat, eggs, grain, day cream system
Product) when, the mode of the pre-treatment is：Sample to be tested (solid sample) is mixed, and patted using homogenizer with pretreatment liquid
(2-5min) is uniformly mixed it, and relative to the sample to be tested (solid sample) of 1g, the dosage of the pretreatment liquid is
0.5-2mL。
In a kind of preferred embodiment of the present invention, when the sample to be tested is solid sample, the pre-treatment
Process further include：After sample to be tested is mixed with pretreatment liquid, mixed liquor is filtered, to remove the solid in mixed liquor
Particle.The filtering preferably uses aperture as the strainer (the coarse filtration film of such as 10-30 mesh) below 30 mesh.
In the present invention, to the composition of the pretreatment liquid, there is no particular limitation, and test sample is treated as long as can be used for adjusting
The pH value of product keeps its osmotic pressure, for example, the pretreatment liquid contains phosphate buffer (namely phosphate-buffered
Liquid).In situations where it is preferred, the pretreatment liquid can also contain nutriment (such as peptone, yeast extract) and/or tween
20.Most preferably, for different types of object bacteria, with the use of different pretreatment liquid formulas.Specifically：
For salmonella and Cronobacter sakazakii (the particularly rugged Cronobacter sakazakii of slope), the pretreatment liquid (solvent is water)
The polysorbas20 of the disodium hydrogen phosphate of potassium dihydrogen phosphate, 7-14g/L, the sodium chloride of 2-6g/L, 0.2-1mL/L containing 2-7g/L and
The peptone of 5-10g/L.
For Listeria (particularly Listeria monocytogenes), the pretreatment liquid (solvent is water) contains 5-
The tryptone of 10g/L, the yeast extract of 3-6g/L, the sodium chloride of 10-20g/L, the potassium dihydrogen phosphate of 2-4g/L, 10-14g/L phosphoric acid
Disodium hydrogen, the polysorbas20 of 0.2-1mL/L, the aesculin of 0.5-1.5g/L, 0.03-0.05 volumes % nalidixic acid (a concentration of 1
Weight %) and 0.03-0.04 volumes % acridine yellow (a concentration of 1 weight %).
For staphylococcus aureus, the potassium dihydrogen phosphate of the pretreatment liquid (solvent is water) containing 2-7g/L, 7-
The disodium hydrogen phosphate of 14g/L, the sodium chloride of 50-90g/L, 0.2-1mL/L polysorbas20s and 5-10g/L peptones.
In the present invention, in step (1), the condition of the pre-treatment includes：25-37 DEG C of temperature, time 0.5-3h.
According to the present invention, in step (2), the immunomagnetic beads are anti-by what will specifically be combined with object bacteria
Body is coupled at what is be prepared on magnetic bead.There is no particular limitation for type of the present invention to the antibody, as long as it can be special
Ground is combined with object bacteria.In situations where it is preferred, for different types of object bacteria, it can be special with the use of different
The antibody that ground is combined with object bacteria.It is highly preferred that the amount for the antibody being coupled on every milligram of magnetic bead is usually 5-50 μ g.Specifically：
For salmonella, the antibody be number be LS-C103073 LSBIO monoclonal antibodies (antibody can be from
LSBIO companies are commercially available, article number LS-C103073) and/or number be 10-S05G Fitzgerald polyclonal antibodies
The KPL that (antibody can be commercially available from Fitzgerald companies, article number 10-S05G) and/or number are 019199 is more
Clonal antibody (antibody can be commercially available from KPL companies, article number 019199).Preparation method is：10mg surfaces is taken to have
The superparamagnetic ball of carboxyl modified is lived through 1- ethyls -3- (3- dimethyl aminopropyls)-carbodiimides and n-hydroxysuccinimide
Change, be dispersed in 1mL PBS buffer solution, add in the above-mentioned antibody of 50-500 μ g, vibrate (200r/min) 3h, Ran Houtong at room temperature
Magneto separate is crossed to be cleaned；Then with 10mg/mL bovine serum albumin(BSA)s (BSA) solution (being dissolved in phosphate buffer) at 37 DEG C
Closing 30min is prepared.
For Listeria (particularly Listeria monocytogenes), the antibody is that number is MC0007's
(antibody can obtain California Bioscience monoclonal antibodies commercially available from California Bioscience companies
It arrives, article number MC0007)；The antibody and carboxyl magnetic bead coupling are prepared into immunomagnetic beads using Carbodiimide condensation method, specifically
Operating process is the same as above-mentioned salmonella.
For staphylococcus aureus, the antibody is the Anti-TNF-α of Thermo Fisher that number is PA1-7246
Body (antibody can be commercially available from Thermo Fisher companies, article number PA1-7246)；Utilize Carbodiimide condensation method
The antibody and carboxyl magnetic bead coupling are prepared into immunomagnetic beads, specific operation process is the same as above-mentioned salmonella.
For Cronobacter sakazakii (the particularly rugged Cronobacter sakazakii of slope), the antibody is the polyclonal antibody of number ES-Pab
The polyclonal antibody that (being commercially available from Shanghai Huiyun Biological Technology Co., Ltd.) and/or number are 1010015 (is composed from Beijing writing brush
Medicine bioengineering research institute is commercially available).Preparation method can be sketched：Using new zealand white rabbit as immune animal, with inactivation
Cronobacter sakazakii is immunized as immunogene according to 3mg/kg/ times.Immunogene and adjuvant are mixed fully emulsified through ultrasound
Afterwards, the subcutaneous multi-point injection of nape part.It is immune for the first time by immunogene and Freund's complete adjuvant, at interval of progress second in 3 weeks and the
It is immunized three times, adjuvant is changed to incomplete Freund's adjuvant.It takes a blood sample after third time is 10 days immune, detects antibody titer, and use
Protein A affinity column antibody purifications.The antibody is coupled adaptive immune magnetic using Carbodiimide condensation method and carboxyl magnetic bead
Pearl, specific operation process is the same as above-mentioned salmonella.
In the present invention, the grain size of the immunomagnetic beads is preferably 180-2800nm, more preferably 180-700nm.
In the present invention, the immunomagnetic beads, which are preferably stored in, preserves in liquid.It is highly preferred that it is described preservation liquid be supplemented with
The phosphate buffer of the polysorbas20 of BSA, 0.5mL/L of 10mg/mL and the sodium azide of 1mg/L.
According to the present invention, in step (2), the dosage of the immunomagnetic beads is relative to object bacteria present in sample to be tested
Amount be excessive, the amount preferably with respect to object bacteria after Zengjing Granule described in step (4) is also excessive, that is, in step
(2) in, the dosages of the immunomagnetic beads can specifically combining target bacterium completely, the step of being preferably able to combine at least 80%
(4) object bacteria described in after Zengjing Granule can more preferably be completely combined the target after Zengjing Granule described in step (4)
Bacterium.In the preferred embodiments of the present invention, relative to the sample to be tested of every milliliter of liquid form, the immunomagnetic beads
Dosage is 5-30 μ g, more preferably 6-20 μ g.
In the present invention, in step (2), to the condition of the mixing, there is no particular limitation, as long as can make to be immunized
Magnetic bead specifically combining target bacterium.Preferably, the condition of the mixing includes：Temperature is 25-37 DEG C, time 0.5-
2h。
It in the present invention, can not before step (2) obtains the mixture containing immunomagnetic beads-target bacteria complex
Addition nutriment carries out increasing bacterium to object bacteria.
In the present invention, in step (3), the process of first Magneto separate can be by magnetic separating device (such as magnetic force
Frame) it carries out, to the condition of first Magneto separate, there is no particular limitation, it is preferable that in step (3), first magnetic point
From condition include：Temperature is 20-36 DEG C, time 10-30min.It is highly preferred that the process of first Magneto separate is also wrapped
It includes：The first aggregation that first Magneto separate obtains is washed, the number of the washing is 2 times or more, and the cleaning solution is
Supplemented with the phosphate buffer of 0.5mL/L polysorbas20s.
It in the present invention, can be true according to the concentration of aggregation in re-suspension liquid and the density of object bacteria in step (4)
It is fixed whether to need to carry out Zengjing Granule.Specifically, in step (4), in first re-suspension liquid, first aggregation
Concentration is preferably 100-1000 μ g/mL；It is highly preferred that in first re-suspension liquid, density >=2 of the object bacteria ×
102CFU/mL；It is further preferred that when the object bacteria is salmonella, in first re-suspension liquid, the object bacteria
Density >=3 × 102CFU/mL；It is described when the object bacteria is Listeria (particularly Listeria monocytogenes)
Density >=7 × 10 of object bacteria2CFU/mL；When the object bacteria is staphylococcus aureus, density >=2 of the object bacteria ×
102CFU/mL；When the object bacteria is Cronobacter sakazakii, density >=6 × 10 of the object bacteria2CFU/mL。
But when in the first re-suspension liquid, the density of the object bacteria is unable to reach 2 × 102It, preferably will step during CFU/mL
Suddenly the first aggregation that (3) obtain carries out Zengjing Granule and the second Magneto separate successively, and second gathers the second Magneto separate obtains
Collect object and carry out the second resuspension, to obtain the second re-suspension liquid.In step (4), in second re-suspension liquid, second aggregation
The concentration of object is preferably 100-1000 μ g/mL；It is highly preferred that in second re-suspension liquid, density >=2 of the object bacteria ×
102CFU/mL；It is further preferred that when the object bacteria is salmonella, in second re-suspension liquid, the object bacteria
Density >=3 × 102CFU/mL；It is described when the object bacteria is Listeria (particularly Listeria monocytogenes)
Density >=7 × 10 of object bacteria2CFU/mL；When the object bacteria is staphylococcus aureus, density >=2 of the object bacteria ×
102CFU/mL；When the object bacteria is Cronobacter sakazakii (particularly the rugged Cronobacter sakazakii of slope), the density of the object bacteria >=
6×102CFU/mL。
In the present invention, in step (4), the process of second Magneto separate can be by magnetic separating device (such as magnetic force
Frame) it carries out, to the condition of second Magneto separate, there is no particular limitation, it is preferable that in step (3), second magnetic point
From condition include：Temperature is 20-36 DEG C, time 5-15min.It is highly preferred that the process of second Magneto separate further includes：
The second aggregation that second Magneto separate obtains is washed, the number of the washing is 2 times or more, and the cleaning solution is the same as first
The cleaning solution that secondary Magneto separate uses.
According to the present invention, in step (4), the process of the Zengjing Granule uses enrichment liquid.
In step (4), there is no particular limitation for the condition of type and Zengjing Granule to the enrichment liquid, as long as can be with
Realize the purpose of object bacteria proliferation.Preferably, it is preferable to use microbiological culture medias for the enrichment liquid.It is highly preferred that in step
Suddenly in (4), the condition of the Zengjing Granule includes：Temperature is 30-44 DEG C, time 2-8h.It is further preferred that for difference
The object bacteria of type, with the use of the condition of different enrichment liquid and Zengjing Granule.Specifically：
If the object bacteria be salmonella, in step (4), the enrichment liquid for SC culture mediums, TTB culture mediums and
At least one of BPW culture mediums；The SC culture mediums, the TTB culture mediums and BPW culture mediums can pass through conventional quotient
Purchase means obtain, alternatively, can also voluntarily prepare.Preferably, the egg of the SC culture mediums (solvent is water) containing 5g/L
The l-cysteine of peptone, the lactose of 4g/L, the disodium hydrogen phosphate of 10g/L, the sodium hydrogen selenite of 4g/L and 0.01g/L in vain；The TTB
The peptone of culture medium (solvent is PBS buffer solution) containing 10g/L, the beef extract of 5g/L, the sodium thiosulfate of 50g/L, 4g/L
The crystalline flake of iodine, the potassium iodide of 5g/L, the brilliant green of 10mg/L and 5g/L bovine bile；The BPW culture mediums (solvent is PBS buffer solution)
The separately peptone containing 10g/L.If it is highly preferred that the object bacteria is salmonella, when the process of the Zengjing Granule makes
During with SC culture mediums, the condition of the Zengjing Granule includes：Temperature is 35-37 DEG C, time 3-6h；When the Zengjing Granule mistake
When journey uses TTB culture mediums, the condition of the Zengjing Granule includes：Temperature is 41-43 DEG C, time 4-6h；When the increasing bacterium
When incubation uses BPW culture mediums, the condition of the Zengjing Granule includes：Temperature is 35-37 DEG C, time 2-4h.
If the object bacteria is Listeria (particularly Listeria monocytogenes), described in step (4)
Enrichment liquid is LB1 culture mediums and/or LB2 culture mediums；The LB1 culture mediums and LB2 culture mediums can be by routine commercially available from
Means obtain, alternatively, can also voluntarily prepare.Preferably, the pancreas of the LB2 culture mediums (solvent is water) containing 5g/L
Peptone, the multivalence peptone of 5g/L, the yeast extract of 5g/L, the sodium chloride of 20g/L, the potassium dihydrogen phosphate of 1.4g/L, 12g/L phosphoric acid hydrogen two
Sodium, the aesculin of 1g/L, the nalidixic acid (a concentration of 1 weight %) of 0.04 volume % and the acridine yellow (concentration of 0.05 volume %
For 1 weight %)；The tryptone of the LB1 culture mediums (solvent is water) containing 5g/L, the multivalence peptone of 5g/L, 5g/L yeast extract,
The sodium chloride of 20g/L, the potassium dihydrogen phosphate of 1.4g/L, the disodium hydrogen phosphate of 12g/L, the aesculin of 1g/L, 0.05 volume %
The acridine yellow (a concentration of 1 weight %) of nalidixic acid (a concentration of 1 weight %) and 0.03 volume %.It is if it is highly preferred that described
Object bacteria is Listeria (particularly Listeria monocytogenes), and the condition of the Zengjing Granule includes：Temperature 30-32
DEG C, time 2-8h.
If the object bacteria is staphylococcus aureus, in step (4), the enrichment liquid for TSB culture mediums and/or
Broth bouillon with high salt；The TSB culture mediums and broth bouillon with high salt can be obtained by conventional commercially available means, alternatively,
It can also voluntarily prepare.Preferably, the tryptone of the TSB culture mediums (solvent is water) containing 17g/L, 3g/L it is big
Beans papain digestion object, the sodium chloride of 5g/L, the potassium dihydrogen phosphate of 2.5g/L and 2.5g/L glucose (pH for 7.3 ±
0.2)；The peptone of the broth bouillon with high salt (preferably 7.5 weight % sodium chloride broths culture mediums) containing 10g/L, 5g/
The beef extract of L and the sodium chloride of 75g/L.If it is highly preferred that the object bacteria is staphylococcus (particularly Staphylococcus aureus
Bacterium), the condition of the Zengjing Granule includes：35-37 DEG C of temperature, time 2-5h.
If the object bacteria be Cronobacter sakazakii, in step (4), the target bacterium culture medium for BPW culture mediums and/
Or mLST-Vm broth bouillons；The BPW culture mediums and mLST-Vm meat soups can be obtained by conventional commercially available means or
Person can also voluntarily prepare.Preferably, the tryptone of the mLST-Vm meat soups (solvent is water) containing 20g/L, 5g/
The lactose of L, the potassium dihydrogen phosphate of 2.75g/L, the dipotassium hydrogen phosphate of 2.75g/L and 0.1g/L lauryl sodium sulfate；It is described
The formula of BPW culture mediums is as described above, details are not described herein.If it is highly preferred that the object bacteria be Cronobacter sakazakii, it is described
The condition of Zengjing Granule includes：42-44 DEG C of temperature, time 2-6h.
In the present invention, it described first is resuspended or described second solution used is resuspended can be that conventional exempt from for suspending
The solution of epidemic disease magnetic bead-target bacteria complex, preferably cleaning solution (as previously described).
According to the present invention, in step (5), the chemiluminescence detection preferably includes following steps：
(51) the first re-suspension liquid or the second re-suspension liquid respectively obtained step (4) is mixed with detection antibody, the detection
Antibody is the antibody that can be specifically combined with object bacteria of horseradish peroxidase-labeled；
(52) mixture for obtaining step (51) carries out third Magneto separate, and the third concentrating that third Magneto separate is obtained
Object mixes, and detect luminous intensity with chemiluminescence A liquid and chemiluminescence B liquid.
According to the present invention, in step (51), relative to the first aggregation in the first re-suspension liquid described in every microgram or institute
The second aggregation in the second re-suspension liquid is stated, the dosage of the detection antibody is preferably 0.1-2ng.
In step (51), there is no particular limitation for type of the present invention to the detection antibody, as long as horseradish peroxide
Change the antibody that can be specifically combined with object bacteria that enzyme (HRP) marks.Preferably, in the detection antibody, without
The antibody that can be specifically combined with object bacteria of HRP labels and the antibody used during preparing the immunomagnetic beads
It is identical and/or different.
In situations where it is preferred, for different types of object bacteria, with the use of different detection antibody.Specifically：
For salmonella, the detection antibody is the Fitzgerald Anti-TNF-αs that the number of HRP labels is 10-S05I
Body.
For Listeria (particularly Listeria monocytogenes), the detection antibody is the number of HRP labels
For 019090 KPL polyclonal antibodies.
For staphylococcus aureus, the detection antibody is the California that the number of HRP labels is CB100401
(antibody can be commercially available Bioscience monoclonal antibodies from California Bioscience companies, and article number is
CB100401)。
For Cronobacter sakazakii (the particularly rugged Cronobacter sakazakii of slope), the detection antibody is that the number of HRP labels is ES-
The monoclonal antibody (being commercially available from Shanghai Huiyun Biological Technology Co., Ltd.) of Mab-02.
In step (51), the condition of mixing is not required particularly, as long as detection antibody and the first aggregation can be made
With reference to for example, the condition of mixing can include：Temperature is 25-37 DEG C, time 0.5-1h.
In the present invention, in step (52), the process of the third Magneto separate can be by magnetic separating device (such as magnetic force
Frame or 96 orifice plate magnetic frames) it carries out, to the condition of the third Magneto separate, there is no particular limitation, it is preferable that in step (3)
In, the condition of the third Magneto separate includes：Temperature is 20-35 DEG C, time 2-5min.It is highly preferred that the third magnetic point
From process further include：The third concentrating object that third Magneto separate obtains is washed, the number of the washing is 5 times or more,
The washing uses phosphate (PBS) buffer solution.
In step (52), the third concentrating object that is obtained relative to third Magneto separate described in every microgram, the chemiluminescence A
The total volume of liquid and the chemiluminescence B liquid is 0.5-5 μ L, preferably 2-5 μ L.
In the present invention, in step (52), the volume ratio of the chemiluminescence A liquid and the chemiluminescence B liquid is preferred
It is 1：1.
According to the present invention, in step (52), do not have to the composition of the chemiluminescence A liquid and the chemiluminescence B liquid
It is special to limit, as long as can be as the chemiluminescent substrate of horseradish peroxidase (HRP).In situations where it is preferred,
The chemiluminescence A liquid luminol of (solvent is water) containing 1-10mmol/L and 0.5-5mmol/L to imidazole radicals phenol；More
Preferably, the urea peroxide of the chemiluminescence B liquid (solvent is water) containing 0.05-0.5mmol/L, 0.01-0.3mmol/L
The citric acid of sodium tetraborate and 2-20mmol/L.
The present invention will be described in detail by way of examples below.In following embodiment,
PBS buffer solution (solvent be water, pH value 7.2-7.4)：The sodium chloride of 8g/L, the potassium chloride of 0.2g/L, 1.15g/L
Disodium hydrogen phosphate and 0.2g/L potassium dihydrogen phosphate；
Chemiluminescence A liquid (solvent is water)：The luminol of 7.5mmol/L and 1.5mmol/L to imidazole radicals phenol；
Chemiluminescence B liquid (solvent is water)：The urea peroxide of 0.4mmol/L, the sodium tetraborate of 0.2mmol/L and
The citric acid of 15mmol/L.
Unless otherwise specified, all reagents can be obtained by conventional commercially available means.
Embodiment 1
The present embodiment is used in the detection sample for illustrating (non-diagnostic purpose) provided by the invention in the method for salmonella
The sensitivity of chemiluminescence immune assay.
(1) by 10810 times of gradient dilutions of CFU/mL salmonella typhimuriums (purchased from ATCC, article No. 14028) are to 100-
105CFU/mL uses pretreatment liquid (7g potassium dihydrogen phosphates, 7g disodium hydrogen phosphates, 5g sodium chloride, 0.5mL polysorbas20s and 10g eggs
White peptone is dissolved in 1L deionized waters) adjust bacterium solution pH be 7；
(2) bacterium solution of above-mentioned gradient dilution is taken, each 1mL adds in 30 μ g immunomagnetic beads (grain size 300nm, every milligram of magnetic beads
On be coupled 25 μ g LS-C103073 monoclonal antibodies), mildly vibrate 30min at 36 DEG C；
(3) the progress Magneto separate 10min under room temperature (25 DEG C) is placed on magnetic frame, supernatant is abandoned, obtains containing immune magnetic
The aggregation of pearl-salmonella compound；
(4) above-mentioned aggregation is cleaned twice with cleaning solution (supplemented with the PBS buffer solution of 0.5mL/L polysorbas20s), magnetic point
After (5min), it is resuspended with 50 μ L PBS buffer solution, obtains re-suspension liquid (a concentration of 600 μ g/mL of aggregation)；
(5) chemiluminescence detection
50 μ L re-suspension liquids are added in microwell plate (bottom and side wall is opaque), add in the detection antibody (number of HRP labels
For the Fitzgerald polyclonal antibodies of 10-S05I, a concentration of 30ng/mL) 50 μ L, 25 DEG C of incubation 30min；
It is placed on magnetic frame under room temperature (25 DEG C) and carries out Magneto separate 3min；
5 times (about 15min) are cleaned with PBS buffer solution, after Magneto separate abandons supernatant again, chemiluminescence A liquid and chemistry are sent out
Light B liquid is according to 1：1 volume mixture adds in 100 μ L mixed liquors, after vibrating 3min, is measured and is shone by force with chemiluminescence detector
Degree.The results are shown in Figure 1.
As seen in Figure 1, when mentioned reagent box is used to carry out Salmeterol fluticasone propionate, detection is limited to 3 × 102CFU/
mL。
Embodiment 2
The method that the present embodiment is used for salmonella in the detection sample for illustrating non-diagnostic purpose provided by the invention.
(1) totally 20 commercially available pasteurized milk samples, are negative sample, randomly choose 10 additions 10-1CFU/mL
Bacterium enteritidis (purchased from ATCC, article No. 13076)；
Every sample takes 25mL, adds in 1mL pretreatment liquids (with embodiment 1), the shaken cultivation 1h at 36 DEG C, gained processing
The pH of sample is about 7 afterwards；
(2) 250 μ g immunomagnetic beads (with embodiment 1) are added in, mildly vibrate 2h at 36 DEG C；
(3) it is placed on magnetic frame at 35 DEG C of room temperature and carries out Magneto separate 30min, abandon supernatant, obtain containing immunomagnetic beads-
The aggregation of salmonella compound；
(4) BPW culture medium (purchased from Beijing overpass company, article No. GCP201) of the above-mentioned aggregation in 1mL is increased at 36 DEG C
Bacterium 3h；Bacterium solution after increasing bacterium is placed on magnetic frame after carrying out Magneto separate 10min at 35 DEG C, two are cleaned with PBS buffer solution
After secondary (about 10min), with 417 μ L PBS buffer solution be resuspended Magneto separate after aggregation, obtain re-suspension liquid (aggregation it is a concentration of
600μg/mL)；
(5) chemiluminescence detection
50 μ L re-suspension liquids are added in microwell plate, respectively according to the detection method of step (5) in embodiment 1 and according to GB
4789.4-2016 national standard method carry out Salmeterol fluticasone propionate.The results are shown in Table 1.
Table 1
| Detection method | Positive findings number | Negative findings number | Detection time | Positive rate |
| Embodiment 2 | 10 | 10 | 8h | 100% |
| National standard method | 9 | 11 | 3d | 90% |
Embodiment 3
The method that the present embodiment is used for salmonella in the detection sample for illustrating non-diagnostic purpose provided by the invention.
(1) 20 commercially available shortening pork head meat, is negative sample, randomly chooses 10 and adds in 10-1CFU/g hog cholera sramana
Salmonella (purchased from ATCC, article No. 10708)；
Sample to be tested 25g is taken, adds in 25mL pretreatment liquids (with embodiment 1), 2min is patted with homogenizer, shakes at 36 DEG C
Culture 3h is swung, the pH of sample is about 7 after gained processing；
(2) above-mentioned culture solution filters off precipitation through coarse filtration film, 200 μ g immunomagnetic beads (same embodiments is added in filtrate
1), 1h is mildly vibrated at 36 DEG C；
(3) the progress Magneto separate 30min at 35 DEG C is placed on magnetic frame, supernatant is abandoned, obtains containing immunomagnetic beads-sramana
The aggregation of Salmonella compound；
(4) above-mentioned aggregation is added in into 1mL SC culture mediums (purchased from Beijing overpass company, article No. CP202) and increases bacterium at 37 DEG C
4h；Bacterium solution after increasing bacterium is placed on magnetic frame after carrying out Magneto separate 10min at 35 DEG C, is cleaned twice with PBS buffer solution
After (about 10min), compound is resuspended with 333 μ L PBS buffer solution, obtains re-suspension liquid (a concentration of 600 μ g/mL of aggregation)；
(5) chemiluminescence detection
50 μ L re-suspension liquids are added in microwell plate, respectively according to the detection method of step (5) in embodiment 1 and according to GB
4789.4-2016 national standard method carry out Salmeterol fluticasone propionate.The results are shown in Table 2.
Table 2
| Detection method | Positive findings number | Negative findings number | Detection time | Positive rate |
| Embodiment 3 | 10 | 10 | 10h | 100% |
| National standard method | 10 | 10 | 3d | 100% |
Embodiment 4
The method that the present embodiment is used for salmonella in the detection sample for illustrating non-diagnostic purpose provided by the invention.
It is carried out according to the method for embodiment 2, unlike, chemiluminescence A liquid is the luminol of 7.5mmol/L, and chemistry is sent out
Light B liquid is the hydrogen peroxide of 0.4mmol/L.The results are shown in Table 3.
Table 3
| Detection method | Positive findings number | Negative findings number | Detection time | Positive rate |
| Embodiment 4 | 3 | 7 | 8h | 30% |
| National standard method | 10 | 10 | 3d | 100% |
Embodiment 5
(1) totally 20 commercially available pasteurized milk samples, are negative sample, randomly choose 10 additions 10-1CFU/mL
Salmonella typhimurium (with embodiment 1)；
Every sample takes 25mL, adds in 1mL pretreatment liquids (with embodiment 2), shaken cultivation 1h, gained are to be measured at 36 DEG C
The pH of sample is about 7；
(2) 25mL BPW culture mediums (purchased from Beijing overpass company, article No. GCP201) are added in and increase bacterium 3h at 36 DEG C；
(3) 250 μ g immunomagnetic beads (with embodiment 1) are added in culture solution, mildly vibrate 2h at 36 DEG C；
(4) the progress Magneto separate 30min at 35 DEG C is placed on magnetic frame, supernatant is abandoned, obtains containing immunomagnetic beads-sramana
The aggregation of Salmonella compound；Aggregation is cleaned twice with PBS buffer solution, after Magneto separate is resuspended with 417 μ L PBS buffer solution
Aggregation, obtain re-suspension liquid (a concentration of 600 μ g/mL of aggregation)；
(5) chemiluminescence detection
50 μ L re-suspension liquids are added in microwell plate, respectively according to the detection method of step (5) in embodiment 1 and according to GB
4789.4-2016 national standard method carry out Salmeterol fluticasone propionate.The results are shown in Table 4.
Table 4
| Detection method | Positive findings number | Negative findings number | Detection time | Positive rate |
| Embodiment 5 | 13 | 7 | 8h | 130% |
| National standard method | 10 | 10 | 3d | 100% |
Comparative example 1
It is carried out according to the method for embodiment 2, unlike, step (1) is added without pretreatment liquid.The results are shown in Table 5.
Comparative example 2
It is carried out according to the method for embodiment 2, unlike, in step (1), pretreatment liquid changes 0.05M citric acids into and delays
The pH of sample to be tested is adjusted to 5.5 or so by fliud flushing.The results are shown in Table 5.
Comparative example 3
It is carried out according to the method for embodiment 2, unlike, in step (1), pretreatment liquid changes 0.05M Tris-salt into
The pH of sample to be tested is adjusted to 8 or so by acid buffer.The results are shown in Table 5.
Table 5
| Detection method | Positive findings number | Negative findings number | Detection time | Positive rate |
| Comparative example 1 | 6 | 14 | 8h | 60% |
| Comparative example 2 | 8 | 12 | 8h | 80% |
| Comparative example 3 | 9 | 11 | 8h | 90% |
Embodiment 6
The present embodiment is used for monocyte hyperplasia Liszt in the detection sample for illustrating non-diagnostic purpose provided by the invention
The sensitivity of chemiluminescence immune assay in the method for bacterium.
(1) by 10710 times of gradient dilutions of CFU/mL Listeria monocytogenes (purchased from ATCC, article No. 19115)
To 100-105CFU/mL uses pretreatment liquid (10g tryptones, 5g yeast extracts, 5g sodium chloride, 4g potassium dihydrogen phosphates, 10g phosphoric acid hydrogen
Disodium, 0.5mL polysorbas20s, 1g aesculins, 0.5mL nalidixic acids (a concentration of 1 weight %) and 0.3mL acridine yellows (a concentration of 1 weight
Measure %), be dissolved in 1L deionized waters) adjust bacterium solution pH be 7；
(2) bacterium solution of above-mentioned gradient dilution is taken, each 1mL adds in 20 μ g immunomagnetic beads (grain size 600nm, every milligram of magnetic beads
On be coupled 15 μ g MC0007 monoclonal antibodies), mildly vibrate 30min at 36 DEG C；
(3) the progress Magneto separate 30min at 35 DEG C is placed on magnetic frame, supernatant is abandoned, obtains containing immunomagnetic beads-monokaryon
The aggregation of hyperplasia Listeria compound；
(4) it after above-mentioned aggregation being cleaned (about 10min) twice with PBS buffer solution, is resuspended, obtained with 50 μ L PBS buffer solution
To re-suspension liquid (a concentration of 400 μ g/mL of aggregation)；
(5) chemiluminescence detection
50 μ L re-suspension liquids are added in microwell plate (bottom and side wall is opaque), add in the detection antibody (number of HRP labels
For 019090 KPL polyclonal antibodies, a concentration of 20ng/mL) 50 μ L, 25 DEG C of incubation 30min；
It is placed on magnetic frame under room temperature (25 DEG C) and carries out Magneto separate 3min；
5 times (about 15min) are cleaned with PBS buffer solution, after Magneto separate abandons supernatant again, chemiluminescence A liquid and chemistry are sent out
Light B liquid is according to 1：1 volume mixture adds in 100 μ L mixed liquors, after vibrating 3min, is measured and is shone by force with chemiluminescence detector
Degree.The results are shown in Figure 2.
As seen in Figure 2, when mentioned reagent box is used to carry out Listeria monocytogenes detection, detection limit
It is 7 × 102CFU/mL。
Embodiment 7
The present embodiment is used for monocyte hyperplasia Liszt in the detection sample for illustrating non-diagnostic purpose provided by the invention
The method of bacterium.
(1) totally 20 commercially available fresh milk samples, are negative sample, randomly choose 10 additions 10-1CFU/mL monokaryons are thin
Born of the same parents' hyperplasia Listeria (with embodiment 6)；
Sample to be tested 25mL is taken, adds in 1mL pretreatment liquids (with embodiment 6), shaken cultivation 1h, gained are to be measured at 30 DEG C
The pH of sample is about 7；
(2) 250 μ g immunomagnetic beads (with embodiment 6) are added in, mildly vibrate 2h at 30 DEG C；
(3) the progress Magneto separate 10min at 30 DEG C is placed on magnetic frame, supernatant is abandoned, obtains containing immunomagnetic beads-monokaryon
The aggregation of hyperplasia Listeria compound；
(4) above-mentioned aggregation is added in into the LB1 culture mediums (purchased from Beijing overpass company, article No. CM505) of 1mL at 30 DEG C
Increase bacterium 4h；Bacterium solution after increasing bacterium is placed on magnetic frame after carrying out Magneto separate 10min at 30 DEG C, is cleaned with PBS buffer solution
Twice after (about 10min), it is resuspended with 625 μ L PBS buffer solution, obtains re-suspension liquid (a concentration of 400 μ g/mL of aggregation)；
(5) chemiluminescence detection
50 μ L re-suspension liquids are added in microwell plate, respectively according to the detection method of step (5) in embodiment 6 and according to GB
4789.30-2016 the national standard method of the first method carries out Listeria monocytogenes detection.The results are shown in Table 6.
Table 6
| Detection method | Positive findings number | Negative findings number | Detection time | Positive rate |
| Embodiment 7 | 10 | 10 | 9h | 100% |
| National standard method | 10 | 10 | 4d | 100% |
Embodiment 8
The present embodiment is used for monocyte hyperplasia Liszt in the detection sample for illustrating non-diagnostic purpose provided by the invention
The method of bacterium.
(1) 20 commercially available shortening beef, is negative sample, randomly chooses 10 additions 10-1CFU/g monocyte hyperplasias
Listeria (with embodiment 6), obtains sample to be tested；
Sample to be tested 25g is taken, adds in 30mL pretreatment liquids (with embodiment 6), 2min is patted with homogenizer, shakes at 30 DEG C
Culture 2h is swung, the pH of gained sample to be tested is about 7；
(2) it will be precipitated and filtered off with strainer, obtain culture solution.The culture solution 10mL after filtering is taken, adds in 200 μ g immunomagnetic beads
(with embodiment 6) mildly vibrates 1h at 36 DEG C；
(3) the progress Magneto separate 30min at 30 DEG C is placed on magnetic frame, supernatant is abandoned, obtains containing immunomagnetic beads-monokaryon
The aggregation of hyperplasia Listeria compound；
(4) the LB1 culture mediums (with embodiment 7) that 1mL is added in above-mentioned aggregation increase bacterium 4h at 30 DEG C；After bacterium is increased
Bacterium solution is placed on magnetic frame at 30 DEG C after progress Magneto separate 10min, after cleaning (about 10min) twice with PBS buffer solution, is used
500 μ L PBS buffer solution are resuspended, and obtain re-suspension liquid (a concentration of 400 μ g/mL of aggregation)；
(5) chemiluminescence detection
50 μ L re-suspension liquids are added in microwell plate, respectively according to the detection method of step (5) in embodiment 6 and according to GB
4789.30-2016 the national standard method of the first method carries out Listeria monocytogenes detection.The results are shown in Table 7.
Table 7
| Detection method | Positive findings number | Negative findings number | Detection time | Positive rate |
| Embodiment 8 | 10 | 10 | 9h | 100% |
| National standard method | 10 | 10 | 4d | 100% |
Embodiment 9
The present embodiment is used for the side for the detection Gold Samples staphylococcus aureus for illustrating non-diagnostic purpose provided by the invention
The sensitivity of chemiluminescence analysis in method.
(1) by 10810 times of gradient dilutions of CFU/mL staphylococcus aureuses (purchased from ATCC, article No. 6538) are to 100-
105CFU/mL uses pretreatment liquid (7g potassium dihydrogen phosphates, 7g disodium hydrogen phosphates, the sodium chloride of 75g, 0.5mL polysorbas20s and 10g
Peptone is dissolved in 1L deionized waters) adjust bacterium solution pH be 7；
(2) bacterium solution of above-mentioned gradient dilution is taken, each 1mL adds in 10 μ g immunomagnetic beads, and (grain size is 2.8 μm, every milligram of magnetic bead
On be coupled 12 μ g PA1-7246 polyclonal antibodies), mildly vibrate 30min at 36 DEG C；
(3) progress Magneto separate 15min is placed on magnetic frame at 35 DEG C, abandons supernatant, obtains containing immunomagnetic beads-golden yellow
The aggregation of color staphylococcus compound；
(4) it after above-mentioned aggregation being cleaned (about 15min) twice with PBS buffer solution, is resuspended, obtained with 50 μ L PBS buffer solution
To re-suspension liquid (a concentration of 200 μ g/mL of aggregation)；
(5) chemiluminescence detection
50 μ L re-suspension liquids are added in microwell plate (bottom and side wall is opaque), add in the detection antibody (number of HRP labels
For the monoclonal antibody of CB100401, a concentration of 15ng/mL) 50 μ L, 25 DEG C of incubation 30min；
It is placed on magnetic frame under room temperature (25 DEG C) and carries out Magneto separate 3min；
Cleaned 5 times, after Magneto separate abandons supernatant again with PBS buffer solution, by chemiluminescence A liquid and chemiluminescence B liquid according to
1：1 volume mixture adds in 100 μ L mixed liquors, and after vibrating 3min, luminous intensity is measured with chemiluminescence detector.As a result such as
Shown in Fig. 3.
As seen in Figure 3, when mentioned reagent box is used to carry out staphylococcus aureus detection, detection is limited to 2 ×
102CFU/mL。
Embodiment 10
The present embodiment is used for the side for the detection Gold Samples staphylococcus aureus for illustrating non-diagnostic purpose provided by the invention
Method.
(1) 20 commercially available pasteurized milk sample, is negative sample, randomly chooses 10 additions 10-1CFU/mL gold
Staphylococcus aureus (with embodiment 9), obtains sample to be tested.
Sample to be tested 25mL is taken, adds in 1mL pretreatment liquids (with embodiment 9), shaken cultivation 1h, gained are to be measured at 36 DEG C
The pH of sample is about 7；
(2) 200 μ g immunomagnetic beads (with embodiment 9) are added in, mildly vibrate 2h at 36 DEG C；
(3) progress Magneto separate 30min is placed on magnetic frame at 35 DEG C, abandons supernatant, obtains containing immunomagnetic beads-golden yellow
The aggregation of color staphylococcus compound；
(4) the TSB culture mediums that above-mentioned aggregation is added in 1mL increase bacterium 3h at 36 DEG C；Bacterium solution after increasing bacterium is placed on
After carrying out Magneto separate 10min at 35 DEG C on magnetic frame, after cleaning (about 10min) twice with PBS buffer solution, with 1000 μ L PBS
Buffer solution is resuspended, and obtains re-suspension liquid (a concentration of 200 μ g/mL of aggregation)；
(5) chemiluminescence detection
50 μ L re-suspension liquids are added in microwell plate, respectively according to the detection method of step (5) in embodiment 9 and according to GB
4789.10-2016 the national standard method of the first method carries out staphylococcus aureus detection.The results are shown in Table 8.
Table 8
| Detection method | Positive findings number | Negative findings number | Detection time | Positive rate |
| Embodiment 10 | 10 | 10 | 8h | 100% |
| National standard method | 10 | 10 | 3d | 100% |
Embodiment 11
The present embodiment is used for the side for the detection Gold Samples staphylococcus aureus for illustrating non-diagnostic purpose provided by the invention
Method.
(1) 20 commercially available day cream sample, is negative sample, randomly chooses 10 additions 10-1CFU/mL golden yellow grapes
Coccus (purchased from ATCC, article No. 25923), obtains sample to be tested；
Day cream sample 10mL is taken, adds in 15mL pretreatment liquids (with embodiment 9), 2min is patted with homogenizer, at 36 DEG C
Shaken cultivation 2h, the pH of gained sample to be tested is about 7；
(2) it will be precipitated and filtered off with strainer, obtain culture solution.The culture solution 10mL after filtering is taken, adds in 100 μ g immunomagnetic beads
(with embodiment 9) mildly vibrates 1h at 36 DEG C；
(3) the progress Magneto separate 5min under room temperature (25 DEG C) is placed on magnetic frame, supernatant is abandoned, obtains containing immune magnetic
The aggregation of pearl-staphylococcus aureus compound；
(4) the TSB culture mediums (with embodiment 10) that above-mentioned aggregation is added in 1mL increase bacterium 3h at 36 DEG C；After bacterium being increased
Bacterium solution be placed on magnetic frame and carry out Magneto separate 10min at 35 DEG C after, after cleaning (about 10min) twice with PBS buffer solution,
It is resuspended with 500 μ L PBS buffer solution, obtains re-suspension liquid (a concentration of 200 μ g/mL of aggregation)；
(5) chemiluminescence detection
50 μ L re-suspension liquids are added in microwell plate, respectively according to the detection method of step (5) in embodiment 9 and according to makeup
The method that product hygienic practice 2015 does the 5th chapter carries out staphylococcus aureus detection.The results are shown in Table 9.
Table 9
| Detection method | Positive findings number | Negative findings number | Detection time | Positive rate |
| Embodiment 11 | 10 | 10 | 8h | 100% |
| Cosmetics health specification | 10 | 10 | 4d | 100% |
Embodiment 12
The present embodiment is used for the side for the detection Gold Samples staphylococcus aureus for illustrating non-diagnostic purpose provided by the invention
Method.
(1) preparation of sample to be tested
The quick-freezing dumpling of 50 commercially available suspected infection staphylococcus aureuses.
Sample 25g is taken, adds in 30mL pretreatment liquids (with embodiment 9), homogenizer pats 2min, the shaken cultivation at 36 DEG C
2h, the pH of sample to be tested is about 7；
(2) it will be precipitated and filtered off with strainer, obtain culture solution.The culture solution 20mL after filtering is taken, adds in 200 μ g immunomagnetic beads
(with embodiment 9) mildly vibrates 1h at 36 DEG C；
(3) progress Magneto separate 30min is placed on magnetic frame at 35 DEG C, abandons supernatant, obtains containing immunomagnetic beads-golden yellow
The aggregation of color staphylococcus compound；
(4) by above-mentioned aggregation with 1000 μ L PBS buffer solution be resuspended compound, obtain re-suspension liquid (aggregation it is a concentration of
200μg/mL)；
(5) chemiluminescence detection
Re-suspension liquid is added in microwell plate, respectively according to the detection method of step (5) in embodiment 9 and according to GB
4789.10-2016 the national standard method of the second method carries out staphylococcus aureus detection.The results are shown in Table 10.
Table 10
| Detection method | Positive findings number | Negative findings number | Detection time | National standard coincidence rate |
| Embodiment 12 | 8 | 42 | 8h | 100% |
| National standard method | 8 | 42 | 3d | - |
Embodiment 13
The present embodiment is used in the detection sample for illustrating non-diagnostic purpose provided by the invention in the method for Cronobacter sakazakii
The sensitivity of chemiluminescence analysis.
(1) by 10810 times of gradient dilutions of the rugged Cronobacter sakazakii of CFU/mL slopes (purchased from ATCC, article No. 29544) are to 100-
105CFU/mL uses pretreatment liquid (7g potassium dihydrogen phosphates, 7g disodium hydrogen phosphates, 5g sodium chloride, 0.5mL polysorbas20s and 10g eggs
White peptone is dissolved in 1L deionized waters) pH of bacterium solution is adjusted to 7；
(2) bacterium solution of above-mentioned gradient dilution is taken, each 1mL adds in 20 μ g immunomagnetic beads (grain size 300nm, every milligram of magnetic beads
On be coupled 30 μ g ES-Pab polyclonal antibodies), mildly vibrate 30min at 36 DEG C；
(3) the progress Magneto separate 10min under room temperature (25 DEG C) is placed on magnetic frame, supernatant is abandoned, obtains containing immune magnetic
The aggregation of pearl-Cronobacter sakazakii compound；
(4) it after above-mentioned aggregation being cleaned (about 10min) twice with PBS buffer solution, is resuspended, obtained with 50 μ L PBS buffer solution
To re-suspension liquid (a concentration of 400 μ g/mL of aggregation)；
(5) chemiluminescence detection
Re-suspension liquid is added in microwell plate (bottom and side wall is opaque), adds in the detection antibody (ES-Mab- of HRP labels
02 monoclonal antibody, a concentration of 20ng/mL) 50 μ L, 25 DEG C of incubation 30min；
It is placed on magnetic frame under room temperature (25 DEG C) and carries out Magneto separate 3min；
5 times (about 15min) are cleaned with PBS buffer solution, after Magneto separate abandons supernatant again, chemiluminescence A liquid and chemistry are sent out
Light B liquid is according to 1：1 volume mixture adds in 100 μ L mixed liquors, after vibrating 3min, is measured and is shone by force with chemiluminescence detector
Degree.The results are shown in Figure 4.
As seen in Figure 4, when mentioned reagent box is used to carry out Cronobacter sakazakii detection, detection is limited to 6 ×
102CFU/mL。
Embodiment 14
The method that the present embodiment is used for Cronobacter sakazakii in the detection sample for illustrating non-diagnostic purpose provided by the invention.
(1) 20 commercial infant formula milk sample, is negative sample, randomly chooses 10 additions 10-1CFU/g slopes are rugged
Cronobacter sakazakii (with embodiment 13), obtains sample to be tested；
Sample to be tested 100g is taken, 150mL pretreatment liquids (with embodiment 13) is added in, 3h is vibrated at 36 DEG C, gained is to be measured
The pH of sample is about 7；
(2) it will be precipitated and filtered off with strainer, obtain culture solution.The culture solution 20mL after filtering is taken, adds in 200 μ g immunomagnetic beads
(with embodiment 13) mildly vibrates 2h at 36 DEG C；
(3) the progress Magneto separate 30min at 35 DEG C is placed on magnetic frame, supernatant is abandoned, obtains containing immunomagnetic beads-Crow
The aggregation of promise bacillus compound；
(4) above-mentioned aggregation is added in into the mLST-Vm meat soups (purchased from overpass company, article No. CM1519A-02) of 1mL 44
DEG C oscillation increases bacterium 4h；Bacterium solution after increasing bacterium is placed on magnetic frame after carrying out Magneto separate 10min at 35 DEG C, is buffered with PBS
Liquid cleaning after (about 10min), is resuspended with 500 μ L PBS buffer solution twice, obtains re-suspension liquid (a concentration of 400 μ g/ of aggregation
mL)；
(5) chemiluminescence detection
50 μ L re-suspension liquids are added in microwell plate, respectively according to the detection method of step (5) in embodiment 13 and according to GB
4789.40-2016 national standard method carry out Cronobacter sakazakii detection.As a result as shown in table 11.
Table 11
| Detection method | Positive findings number | Negative findings number | Detection time | Positive rate |
| Embodiment 14 | 10 | 10 | 10h | 100% |
| National standard method | 10 | 10 | 5d | 100% |
By by the result of above example 1-14 it is found that using method provided by the invention to the Salmonella in sample
When bacterium, Listeria, staphylococcus and Cronobacter sakazakii are detected, can quickly and accurately it obtain as a result, and detection limit
It is relatively low, it can effectively avoid false negative and false positive results.
It is first handled in addition, can be seen that the present invention according to the result of embodiment 2 and comparative example 1-3 and use with pretreatment liquid
Sample to be tested, so detection object bacteria is carried out immunocapture again the scheme of Zengjing Granule and specific pretreatment process and
Condition can be further reduced the false positive or false negative of testing result.
The preferred embodiment of the present invention has been described above in detail, and still, the present invention is not limited thereto.In the skill of the present invention
In art conception range, a variety of simple variants can be carried out to technical scheme of the present invention, including each technical characteristic with it is any its
Its suitable method is combined, and it should also be regarded as the disclosure of the present invention for these simple variants and combination, belongs to
Protection scope of the present invention.
Claims (10)
1. a kind of method that microorganism is quickly detected in non-diagnostic destination, which is characterized in that this method includes the following steps：
(1) sample to be tested is subjected to pre-treatment, to obtain sample to be tested of the pH value as the liquid form of 6.4-7.4；
(2) immunomagnetic beads are mixed with the sample to be tested of the liquid form, the condition of mixing causes exist in sample to be tested
During object bacteria, immunomagnetic beads can specifically combining target bacterium, to obtain the mixing containing immunomagnetic beads-target bacteria complex
Object；
(3) the mixed material for obtaining step (2) carries out the first Magneto separate, to obtain containing the immunomagnetic beads-target
First aggregation of bacteria complex, and remove and be not associated with the substance on immunomagnetic beads；
(4) the first aggregation for obtaining step (3) carries out the first resuspension, to obtain the first re-suspension liquid；Or
The first aggregation that step (3) obtains is subjected to Zengjing Granule and the second Magneto separate, and the second Magneto separate is obtained successively
The second aggregation carry out second be resuspended, to obtain the second re-suspension liquid；
(5) the first re-suspension liquid obtained step (4) or the second re-suspension liquid carry out chemiluminescence detection, to determine in sample to be tested
The presence of object bacteria.
2. according to the method described in claim 1, wherein, in step (1), the pH value of the sample to be tested of the liquid form is
6.8-7.4；
Preferably, in step (1), sample to be tested is is mixed by the mode of the pre-treatment with pretreatment liquid, the pre-treatment
Liquid contains phosphate buffer；
Preferably, when the sample to be tested is liquid, the sample to be tested and the volume ratio of the dosage of the pretreatment liquid are
1：0.04-0.5；
Preferably, when the sample to be tested is solid, relative to the sample to be tested of 1g, the dosage of the pretreatment liquid is
0.5-2mL；
Preferably, in step (1), the condition of the pre-treatment includes：25-37 DEG C of temperature, time 0.5-3h.
3. according to the method described in claim 1, wherein, in step (2), the immunomagnetic beads are by will be specifically
What the antibody coupling combined with object bacteria was prepared on magnetic bead；
Preferably, the grain size for preparing the magnetic bead used in the immunomagnetic beads is 180-2800nm, more preferably 180-700nm.
4. the method according to claim 1 or 3, wherein, in step (2), relative to treating for every milliliter of liquid form
Sample, the dosage of the immunomagnetic beads is 5-30 μ g, preferably 6-20 μ g；
Preferably, in step (2), the condition of the mixing includes：Temperature is 25-37 DEG C, time 0.5-2h.
5. according to the method described in claim 1, wherein, in step (4), in first re-suspension liquid, described first is poly-
Collect a concentration of 100-1000 μ g/mL of object；
Preferably, in first re-suspension liquid, density >=2 × 10 of the object bacteria2CFU/mL；
Preferably, in step (4), in second re-suspension liquid, a concentration of 100-1000 μ g/ of second aggregation
mL；
Preferably, in second re-suspension liquid, density >=2 × 10 of the object bacteria2CFU/mL。
6. method according to claim 1 or 5, wherein, in step (4), the process of the Zengjing Granule uses increasing bacterium
Liquid；
Preferably, if the object bacteria is salmonella (Salmonella), the enrichment liquid is SC culture mediums, TTB is cultivated
At least one of base and BPW culture mediums；
Preferably, if the object bacteria is Listeria (Listeria), the enrichment liquid is trained for LB1 culture mediums and/or LB2
Support base；
Preferably, if the object bacteria is staphylococcus aureus (Staphylococcus aureus), the enrichment liquid is
TSB culture mediums and/or broth bouillon with high salt；
Preferably, if the object bacteria is Cronobacter sakazakii (Cronobacter), the target bacterium culture medium is cultivated for BPW
Base and/or mLST-Vm broth bouillons；
Preferably, in step (4), the condition of the Zengjing Granule includes：Temperature is 30-44 DEG C, time 2-8h.
7. according to the method described in claim 1, wherein, in step (5), the chemiluminescence detection includes the following steps：
(51) the first re-suspension liquid or the second re-suspension liquid respectively obtained step (4) is mixed with detection antibody, the detection antibody
The antibody that can be specifically combined with object bacteria for horseradish peroxidase-labeled；
(52) mixture obtained step (51) carries out third Magneto separate, and the third concentrating object that third Magneto separate is obtained with
Chemiluminescence A liquid and the mixing of chemiluminescence B liquid, and detect luminous intensity.
8. according to the method described in claim 7, wherein, in step (51), relative in the first re-suspension liquid described in every microgram
The first aggregation or second re-suspension liquid in the second aggregation, it is described detection antibody dosage be 0.1-2ng；
Preferably, in step (52), the third concentrating object that is obtained relative to third Magneto separate described in every microgram, the chemistry hair
The total volume of light A liquid and the chemiluminescence B liquid is 0.5-5 μ L；
It is highly preferred that in step (52), the volume ratio of the chemiluminescence A liquid and the chemiluminescence B liquid is 1：1.
9. method according to claim 7 or 8, wherein, the chemiluminescence A liquid contain 1-10mmol/L luminol and
0.5-5mmol/L to imidazole radicals phenol；
Preferably, the chemiluminescence B liquid contains the tetraboric acid of the urea peroxide of 0.05-0.5mmol/L, 0.01-0.3mmol/L
The citric acid of sodium and 2-20mmol/L.
10. according to the method described in any one in claim 1-9, wherein, the object bacteria is salmonella, golden yellow Portugal
At least one of grape coccus, Cronobacter sakazakii and Listeria, preferably salmonella typhimurium (Salmonella
Typhimurium), Bacterium enteritidis (Salmonella enteritidis), Salmonella paratyphi A
(Salmonella paratyphi A), Salmonella choleraesuls (Salmonella choleraesuis), monocyte hyperplasia
Listeria (Listeria monocytogenes), staphylococcus aureus (Staphylococcus aureus) and slope are rugged
At least one of Cronobacter sakazakii (Cronobacter sakazakii).
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