CN103134927A - Method used for detecting shigella and reagent box - Google Patents
Method used for detecting shigella and reagent box Download PDFInfo
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Abstract
The invention provides a reagent box used for detecting shigella. The reagent box comprises an immune magnetic bead and a detection reagent strip. The immune magnetic bead is used for gathering the shigella in a sample. The detection reagent strip is a shigella double resistant clamping core detection card and used for detecting the shigella in the sample. The immune magnetic bead is adopted and the shigella is gathered by the immune magnetic bead, thus steps of increasing bacteria which are time-consuming in the pathogenic bacterium detection can be effectively shortened.
Description
Technical field
The present invention relates to biological technical field, more specifically, the present invention relates to method and kit for detection of Shigella.
Background technology
Shigella (Shigella spp.) common name shigella dysenteriae is the common pathogenic entero becteria with hyperinfection of a class.According to the difference of biochemical reaction and O antigenic structure, Shigella can be divided into shigella dysenteriae (S.dysenteriae), shigella flexneri (S.flexneri), Shigella bogdii (S.boydii) and bacillus ceylonensis A (S.sonnei).Infect Shigella and can cause serious gastrointestinal reaction such as pus and blood stool, dehydration, a few peoples' initiating system complication such as convulsions, hemolytic uremic syndrome, hyponatremia, hypoglycemia and enterobrosis, even dead.
, often have because water source or food are subject to Shigella and pollute the report that causes the disease popularities such as dysentery, diarrhoea because sanitary condition is poor in the vast rural area of China.Therefore, fast, effective, economic, method that detect easily Shigella seems particularly important.
But at present, for the detection of Shigella, separately unsatisfactory part is arranged still: although traditional propagation cultivation, biochemical test, serological test check accuracy are good, required time is long; Molecular biology method, mainly contain conventional PCR, multiplex PCR, real-time PCR method, although sensitive, high specificity fast,, but need well-trained professional and technical personnel, and need to be by the checkout equipment (as the quantitative PCR instrument) of costliness and than the hi-tech level, be unfavorable for promoting in common laboratory, therefore only limit at present national high mechanism and department's employings such as customs, commodity inspection; VITEK (automatic bacterial Biochemical Analyzer), VDAS (automatic lmunoassays analyzer) although etc. fast, accurately, detection limit is large, expense is quite high, is not suitable for individual samples to detect; Genetic chip, protein-chip is accurate, detection limit is large, but manufacturing cost is too high, also is not suitable for China present stage for detection method fast, economical, easy an urgent demand accurately.
Summary of the invention
The present invention one of is intended to solve the problems of the technologies described above at least to a certain extent or provides at least a kind of useful business to select.For this reason, one object of the present invention is to propose a kind of kit and method that can effectively detect Shigella.
The kit for detection of Shigella according to the embodiment of the present invention comprises: immunomagnetic beads, described immunomagnetic beads are used for the Shigella of sample is carried out enrichment; And the detection reagent strip, described detection reagent strip is Shigella double antibodies sandwich test card, is used for the Shigella of sample is detected.Thus, can carry out enrichment by adopting immunomagnetic beads, can effectively shorten the bacterium step that increases the most consuming time in pathogenic microbes detect.According to embodiments of the invention, can adopt and detect reagent strip is that Shigella double antibodies sandwich test card has higher specificity and sensitivity, and easy and simple to handle, need not specialized facilities and equipment.The Shigella that is particularly suitable for food, feed, environment and clinical sample detects and examination.In addition, the preparation method of Shigella detection kit of the present invention is easy, good stability, good reproducibility.
With reference to figure 1, detect that Shigella double antibodies sandwich test card is got stuck by plastics and detector bar forms, detector bar is arranged within plastics get stuck.The Shigella detector bar is by pasting successively polyester film 1 on adhesive base 6, sample pad 2, glass fibre membrane 3, nitrocellulose filter 4 and adsorptive pads 5, be coated with the anti-Shigella monoclonal antibody (or polyclonal antibody) of colloid gold label on glass fibre membrane, be coated with anti-Shigella polyclonal antibody (or monoclonal antibody) on nitrocellulose filter as detection line 6 and be coated with anti-mouse antibody as nature controlling line 7.
According to embodiments of the invention, described immunomagnetic beads is nanometer or sub-micron magnetic bead, and on described nanometer or sub-micron magnetic bead, coupling has anti-Shigella polyclone or monoclonal antibody.Thus, can further improve detection efficiency.
According to embodiments of the invention, described magnetic bead and anti-Shigella antibody are the coupling of affinity single-point.Thus, can further improve detection efficiency.
According to embodiments of the invention, described magnetic bead is the streptavidin magnetic bead, and described anti-Shigella antibody is the anti-Shigella antibody of biotinylation.Thus, can further improve detection efficiency.
According to embodiments of the invention, the anti-Shigella antibody of biotinylation is that long-chain biotin and anti-Shigella antibody pass through the covalent coupling gained, and wherein, the mol ratio of biotin and anti-Shigella antibody is lower than 1:1.Thus, can further improve detection efficiency.
According to embodiments of the invention, anti-Shigella polyclone or monoclonal antibody all are positive at least a Shigella serotype of shigella dysenteriae (S.dysenteriae), shigella flexneri (S.flxneri), Shigella bogdii (S.boydii) and bacillus ceylonensis A (S.sonnei), to the reaction that is negative of ovobiocin tolerant bacteria.Thus, can further improve detection efficiency.
In still another aspect of the invention, the present invention proposes a kind of method of utilizing foregoing kit to detect Shigella, it is characterized in that, comprise the following steps: utilize described immunomagnetic beads to carry out enrichment to the Shigella in sample to be detected, in order to obtain Shigella through the sample of enrichment; And utilize described detection reagent strip that described Shigella is detected.Thus, can carry out enrichment by adopting immunomagnetic beads, can effectively shorten the bacterium step that increases the most consuming time in pathogenic microbes detect.According to embodiments of the invention, can adopt and detect reagent strip is that Shigella double antibodies sandwich test card has higher specificity and sensitivity, and easy and simple to handle, need not specialized facilities and equipment.The Shigella that is particularly suitable for food, feed, environment and clinical sample detects and examination.In addition, the preparation method of Shigella detection kit of the present invention is easy, good stability, good reproducibility.
According to embodiments of the invention, before carrying out enrichment, in advance described sample is increased bacterium and process.Thus, can further improve detection efficiency.
According to embodiments of the invention, comprise the following steps:
1) sample increases bacterium in advance: get sample 25g or liquid sample 25mL with sterile working, add the homogeneous cup that sterilization 225mL Shigella increases bacterial context soup-ovobiocin is housed, use rotating knife chip homogenizer with 8000r/min ~ 10000r/min homogeneous; Or add and be equipped with in the homogenizing bag that the 225mL Shigella increases bacterial context soup-ovobiocin, with slap type homogenizer continuous homogenizing 1 ~ 2min, and with the sample after homogeneous in 40 ℃ ± 1 ℃, anaerobism is cultivated 10h;
2) right to use requires 1 ~ 6 described kit of any one, carry out the magnetic separation and concentration by described immunomagnetic beads: get the pre-germ-increasing liquid of 1mL and join the 2mL centrifuge tube, adding wherein 50 μ L concentration is the described immunomagnetic beads of 2mg/mL, rotating 10rpm reaction 30min on mixed instrument under room temperature, after take off, magnetic frame separates, supernatant discarded.Use at last the resuspended magnetic bead of PBS of 100 μ L pH=7.2;
3) detect front deactivation and processing: with the resuspended liquid of resulting magnetic bead water-bath 5min in 90 ℃ of water-baths, gained liquid carries out magnetic in magnetic field separates, and getting clear liquid is sample liquid to be checked;
4) step 3) gained liquid to be checked is got 80 μ L and dripped in described Shigella double antibodies sandwich test card, sentence read result in 5 ~ 10min.
Thus, can further improve detection efficiency.
Additional aspect of the present invention and advantage part in the following description provide, and part will become obviously from the following description, or recognize by practice of the present invention.
Description of drawings
Above-mentioned and/or additional aspect of the present invention and advantage are from obviously and easily understanding becoming the description of embodiment in conjunction with following accompanying drawing, wherein:
Fig. 1 is the structural representation of test-strips according to an embodiment of the invention.
Embodiment
The below describes embodiments of the invention in detail, and these embodiment are exemplary, is intended to for explanation the present invention, and can not be interpreted as limitation of the present invention.
1.1 full bacterium antigen preparation
respectively with shigella dysenteriae (S.dysenteriae), shigella flexneri (S.flexneri), Shigella bogdii (S.boydii) and bacillus ceylonensis A (S.sonnei) type strain line LB are dull and stereotyped, picking list bacterium colony after 37 ℃ of cultivation 20h, 37 ℃ of activation 10h in 10mL LB test tube, after activating, bacterium liquid is pressed 1% inoculum concentration in large volume culture 18h, counting, 3 ‰ (V/V) formalin deactivation 12h, remove nutrient culture media after the centrifugal 5min of 7000r/min, and clean for several times with aseptic PBS (pH=7.2), after four strain bacterium are pressed thalline and count mixed in equal amounts, be resuspended in stroke-physiological saline solution standby.
1.2 broken antigen preparation
1.1 gained antigens are adjusted after concentration 200w on Ultrasonic Cell Disruptor, broken 9s, interval 9s, 25 circulations and so forth add a small amount of DNA and RNA enzyme to process, and then gained antigen is the broken antigen of Shigella.
2.1 how anti-preparation
Choose 2 large ear rabbits of the female Japan of body weight 1.8 ~ 2.5kg, immunity 1.1 prepared full bacterium antigens.Volume injected is 500 μ L/, immunizing dose ~ 2 * 10
9Individual cell/only, every immunity in 7 days once, hypodermic injection first, auricular vein injection afterwards, the blood monitoring that takes a morsel before each immunity is tired, and carries out the last immunity of impacting when antiserum titre no longer raises, and immunizing dose doubles, and after 7 days, femoral artery is got blood.After 37 ℃ of temperature bath 1h of blood, 4 ℃ are spent the night, and sucking-off serum adopts caprylic acid-ammonium to carry out must resisting the Shigella polyclonal antibody after purifying and dialysis.
2.2 monoclonal antibody preparation
Take the 1.2 broken antigens of gained Shigellas as immunogene, 46 of immunity BALB/c mouse in age in week, immunizing dose is 2 * 10
8Individual cell/only, injection system is: hypodermic injection, impacting immunity at last is tail vein injection.Immunity interval 20 days, first immunisation is aided with Freund's complete adjuvant (adjuvant antigen volume ratio is 1:1), is aided with afterwards incomplete Freunds adjuvant.Tire no longer increase or meet the demands after, impact immunity, put to death mouse, get spleen cell under aseptic condition and induce with PEG-4000, conventional method merges, and cell cultivates, indirect ELISA method detects cells and supernatant.Concrete steps are as follows:
(1) antigen coated: coated shigella dysenteriae, shigella flexneri, Shigella bogdii and bacillus ceylonensis A respectively, 100 μ L/ holes, 4 ℃ are spent the night or 37 ℃ of incubation 1h;
(2) washing: abandon coating buffer, wash 3 times with PBST, 320 μ L/ holes, button is done the PBST cleaning fluid;
(3) sealing: add the gelatin of confining liquid 10mg/mL, 320 μ L/ holes, 37 ℃ of incubation 1h;
(4) washing: with step 2;
(5) antibody sandwich: add cells and supernatant, and establish blank, feminine gender, positive control, 100 μ L/ holes, 37 ℃ of incubation 30min;
(6) washing: abandon coating buffer, wash 4 times with PBST, 320 μ L/ holes, button is done the PBST cleaning fluid;
(7) add enzyme labelled antibody: 5000 times of dilution sheep anti-mouse igg+M two are anti-, and 30min are placed for 37 ℃ in 100 μ L/ holes;
(8) washing: abandon coating buffer, wash 5 times with PBST, 320 μ L/ holes, button is done the PBST cleaning fluid;
(9) colour developing: add the TMB chromophoric solution, 100 μ L/ holes, 37 ℃ of reaction 15min;
(10) cessation reaction: every hole adds 50 μ L10%(V/V) H
2SO
4, color development stopping;
(11) in the OD value of measuring on microplate reader under 450nm.
With to each strain bacterium all positive cell conditioned medium calculate positive, strong positive hole inner cell is cloned cultivation with limiting dilution assay, and track record, to take turns above clone through 3 and cultivate and detect, the hole inner cell that all is positive is the hybridoma of secrete monoclonal antibody.This hybridoma is cultivated through enlarging, selected 4 male BALB/c mouse, lumbar injection whiteruss 0.5mL/, 7 days pneumoretroperitoneum injection hybridomas 0.5 ~ 1 * 10
6/ only, when obviously expanding, collects mouse web portion ascites.After adopting sad-ammonium sulfate method purifying and dialysis, the albumen that the judgement of monoclonal antibody subclass kit obtains is IgG albumen.
3.1 carboxylated magnetic bead preparation
3.1.1 preparation oleic acid modified magnetic microspheres
1) add the 150mL high purity water in there-necked flask, logical nitrogen drove oxygen in 15 minutes, and 500rpm stirs, and 50 ℃ of water-baths add 8mM FeCl
36H
2O and 16mM FeSO
47H
2O
2) add 12.5mL ammoniacal liquor simultaneously in there-necked flask, 500rpm, logical nitrogen, 50 ℃ of water-baths were reacted 30 minutes.
3) the ultrapure washing of deoxygenation is 5 times, washes away ammoniacal liquor.
4) nano particle that obtains is resuspended in 100mL deoxygenation ultrapure water 500rpm, logical nitrogen, 70 ℃ of water-baths.
5) slowly drip the potpourri of 1.2mL oleic acid and 0.4mL undecenoic acid, keep each reaction conditions, reaction 3h.
6) after 3h, magnetic is attached, and the supernatant that inclines after washing 5 times with absolute ethyl alcohol, dries up under nitrogen protection, is resuspended in octane.
3.1.2 synthetic carboxyl-functional magnetic microsphere
1) take the modified nanoparticles that 2g has dried up, add 2.53g styrene and 0.51g divinylbenzene, ultrasonic dispersion.
2) add 0.44g SDS and 0.48g NaHCO in 200mL deoxygenation ultrapure water
3, oil phase and water mix, and ultrasonic emulsification is after 20 minutes, broken 20min under cell crushing instrument 300w.
3) above-mentioned reaction system is transferred in there-necked flask, put into water-bath, logical nitrogen, rotating speed is adjusted to 300rpm, when temperature rises to 70 ℃, dropwise adds the 0.06M potassium persulfate, the acrylic acid of 150 μ L, reaction 24h.
4) magnetic is attached, and the transfer reaction product is used the ultrapure water cyclic washing, then uses 95% ethanol water washing, does not have foam to produce until rock.
5) after the ultrapure water washing, be stored in the 50mL centrifuge tube stored refrigerated.
The gained magnetic bead is surperficial carboxylated magnetic bead.
3.2 streptavidin magnetic bead preparation
Streptavidin embedding magnetic bead (EDC/NHSS method):
1) get the carboxylated magnetic bead of 20mg step 3.1 gained in four 5mL centrifuge tubes, use successively the 3mL absolute ethyl alcohol, 3mL1M NaOH, the HCL of 3mL1M washs magnetic bead respectively once, and 3mL PB (0.02M, pH=6.0) washes three times, use 3mL0.05M after washing, the pH6.0MES dissolving is resuspended.
2) add NHSS0.8mg, EDC0.7mg (using 0.05M, the pH6.0MES dissolving), 37 ℃ of activation 2h.
3) magnetic separates, and sucks supernatant, and 3mL PB (0.02M, pH=8.0) is resuspended, and magnetic bead is disperseed, and then 400 μ g Streptavidins is joined in magnetic bead again 37 ℃ of coupling 2h.
4) get 240 μ g monoethanolamine sealings, with a small amount of PB (0.02M, pH=8.0) dissolving (solution is adjusted to neutrality), directly add in reactant liquor, rotation mixes the suspended state that keeps magnetic bead, room temperature sealing 2h.
5) magnetic separates, and draws supernatant, uses ELISA to survey not the coupling Streptavidin and tires or measure protein concentration monitoring coupling efficiency in supernatant, with PB (0.02M contains 0.05%Tween20, pH=8.0) washing magnetic bead 3 times.
6) (contain 0.05%NaN with pH8.0PB
3, 0.5%BSA) 3mL is resuspended, 4 ℃ of preservations.
3.3 polyclonal antibody biotinylation
Taking 1.8mg long-chain biotin is dissolved in 324 μ L ultrapure waters, be mixed with 10mM biotin mother liquor, according to biotin: Shigella polyclonal antibody mol ratio 0.8:1 adds the biotin mother liquor, incubated at room 120min, solution is put into bag filter, dialysed 3 days for 4 ℃, to remove unconjugated biotin, 1:1 (V/V) adds glycerine to be stored in-20 ℃.
3.4 Shigella immunomagnetic beads preparation
Ratio in every mg magnetic bead 80 μ g antibody adds the described biotinylation Shigella of step 3.3 how anti-, is placed in 37 ℃ of coupling 30min on blending instrument.Take off centrifuge tube and be placed in magnetic separation on magnetic frame, remove supernatant, (contain 0.05%NaN with pH7.4PBS
3, 0.5%BSA) 2mg/mL is resuspended, 4 ℃ of preservations.
4.1 the preparation of nitrocellulose filter
The preparation of detection line and nature controlling line:
To resist respectively Shigella polyclonal antibody and anti-mouse two anti-coated to nitrocellulose filter with BIODOT Dispensing System, make detection line and nature controlling line.For detection line with the PBS (phosphate buffered solution) of 0.01M pH7.2 respectively adjustment kit be 1.0 ~ 2.0mg/mL by substrate concentration, spray film amount is 0.74 μ L/cm; Nature controlling line spraying concentration is 1.0 ~ 1.5mg/mL, and spray film amount is 0.74 μ L/cm, the two lines 6mm of being separated by.The nitrocellulose filter for preparing is preserved under the environment of drying at room temperature after 37 ℃ of drying and processings spend the night.
4.2 the preparation of glue gold pad
1) preparation of collaurum: prepare 40nm particle diameter collaurum with conventional method reduction gold chloride, all once reaching with ultraviolet and visible absorption peak position and peak width monitoring collaurum particle diameter.
2) with colloid gold label Shigella monoclonal antibody, and it is sprayed on glass fibre membrane.
With collaurum by the anti-Shigella monoclonal antibody of the mode mark of static coupling: get collaurum 50mL, at the centrifugal 10 ~ 15min of 8000 * g, centrifugal rear collecting precipitation is transferred pH to 8.0 with 0.01M sal tartari with 0.1.Under stirring, colloidal gold solution is mixed with anti-Shigella monoclonal antibody, antibody and collaurum ratio are 20 ~ 40 μ g antibody/mL collaurums, stir lower reaction 1h, add final concentration and be 1% BSA sealing 1h.4 ℃, the centrifugal 30min of 4000r/min precipitate 10 times and concentrate resuspended.Be sprayed into (5 ~ 8 μ L/cm) on glass fibre membrane with BIODOT Dispensing System, 30 ℃ of vacuum drying 1 ~ 2h make glue gold pad, are put under the environment of drying at room temperature to deposit.
4.3 assembling test strips;
Following material is pasted on overlap joint ground successively on adhesive base:
(1) polyester film; (2) sample pad; (3) glue gold pad; (4) nitrocellulose filter; (5) thieving paper, cutting knife are cut into the 4mm/ bar, install additional get stuck Shigella double antibodies sandwich immuno-chromatographic test paper strip of the present invention, closed chamber is gentle in the aluminium foil bag that drying agent is arranged puts for this test strips.
In embodiment 5 actual sample, Shigella detects
Adopt institute's method for building up and national standard method to measure the pollution condition of Shigella in 60 ripe chicken meat samples of taking from the different market of farm produces.Detect according to the following steps:
Increase in advance bacterium: get sample 25g with sterile working, add the homogeneous cup that sterilization 225mL Shigella increases bacterial context soup is housed, use rotating knife chip homogenizer with 8000r/min ~ 10000r/min homogeneous; Or add and be equipped with in the homogenizing bag that the 225mL Shigella increases bacterial context soup, with slap type homogenizer continuous homogenizing 1 ~ 2min, fluid sample vibration mixing gets final product.In 40 ℃ ± 1 ℃, anaerobism is cultivated 10h.
Get the pre-germ-increasing liquid of 1mL and join the 2mL centrifuge tube, adding wherein 50 μ L concentration is that the described immunomagnetic beads of claim 1 of 2mg/mL (is that the magnetic bead amount is: 100 μ g/mL), take off after reacting 30min (10rpm) on the rotation mixed instrument under room temperature, magnetic frame separates, supernatant discarded.Use at last the 100 resuspended magnetic beads of μ L PBS (pH=7.2).
With the resuspended liquid of above-mentioned magnetic bead water-bath 5min in 90 ℃ of water-baths, gained liquid carries out magnetic in magnetic field separates, and getting clear liquid is sample liquid to be checked.
Test card being returned to room temperature, keeps flat, with 3) step gained liquid to be checked gets 80 μ L and drips in the described kit of claim 1 in the test card well, sentence read result in 5 ~ 10min.
Interpretation as a result: if red stripes appears in the test card detection line, red stripes appears in nature controlling line, increases in the bacterium sample Shigella positive; If the not aobvious red stripes of detectability, red stripes appears in nature controlling line, and in sample, Shigella is negative.If the not aobvious band of nature controlling line detects and lost efficacy.
Result shows, in 60 parts of ripe chicken products, two kinds of methods all only detect 4 parts of positive, and visible accuracy of the present invention is good.The contrast colony counting, Shigella kit of the present invention detects and is limited to 10
4Cfu/mL increases sample after bacterium, can realize sample after increasing bacterium 10h in the Shigella Rapid Detection, effectively shortened and increased in advance bacterium time and sense cycle.
In the description of this instructions, the description of reference term " embodiment ", " some embodiment ", " example ", " concrete example " or " some examples " etc. means to be contained at least one embodiment of the present invention or example in conjunction with specific features, structure, material or the characteristics of this embodiment or example description.In this manual, the schematic statement of above-mentioned term not necessarily referred to identical embodiment or example.And the specific features of description, structure, material or characteristics can be with suitable mode combinations in any one or more embodiment or example.
Although the above has illustrated and has described embodiments of the invention, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art is not in the situation that break away from principle of the present invention and aim can change above-described embodiment within the scope of the invention, modification, replacement and modification.
Claims (9)
1. the kit for detection of Shigella, is characterized in that, comprising:
Immunomagnetic beads, described immunomagnetic beads are used for the Shigella of sample is carried out enrichment; And
Detect reagent strip, described detection reagent strip is Shigella double antibodies sandwich test card, is used for the Shigella of sample is detected.
2. kit according to claim 1, is characterized in that, described immunomagnetic beads is nanometer or sub-micron magnetic bead, and on described nanometer or sub-micron magnetic bead, coupling has anti-Shigella polyclone or monoclonal antibody.
3. kit according to claim 2, is characterized in that, described magnetic bead and anti-Shigella antibody are the coupling of affinity single-point.
4. kit according to claim 2, is characterized in that, described magnetic bead is the streptavidin magnetic bead, and described anti-Shigella antibody is the anti-Shigella antibody of biotinylation.
5. kit according to claim 4, is characterized in that, the anti-Shigella antibody of biotinylation is that long-chain biotin and anti-Shigella antibody pass through the covalent coupling gained, and wherein, the mol ratio of biotin and anti-Shigella antibody is lower than 1:1.
6. kit according to claim 2, it is characterized in that, anti-Shigella polyclone or monoclonal antibody all are positive at least a Shigella serotype of shigella dysenteriae (S.dysenteriae), shigella flexneri (S.ftexneri), Shigella bogdii (S.boydii) and bacillus ceylonensis A (S.sonnei), to the reaction that is negative of ovobiocin tolerant bacteria.
7. a method of utilizing the described kit of claim 1 ~ 6 any one to detect Shigella, is characterized in that, comprises the following steps:
Utilize described immunomagnetic beads to carry out enrichment to the Shigella in sample to be detected, in order to obtain Shigella through the sample of enrichment; And
Utilize described detection reagent strip that described Shigella is detected.
8. method according to claim 7, is characterized in that, before carrying out enrichment, in advance described sample increased bacterium and process.
9. method according to claim 8, is characterized in that, comprises the following steps:
1) sample increases bacterium in advance: get sample 25g or liquid sample 25mL with sterile working, add the homogeneous cup that sterilization 225mL Shigella increases bacterial context soup-ovobiocin is housed, use rotating knife chip homogenizer with 8000r/min ~ 10000r/min homogeneous; Or add and be equipped with in the homogenizing bag that the 225mL Shigella increases bacterial context soup-ovobiocin, with slap type homogenizer continuous homogenizing 1 ~ 2min, and with the sample after homogeneous in 40 ℃ ± 1 ℃, anaerobism is cultivated 10h;
2) right to use requires 1 ~ 6 described kit of any one, carry out the magnetic separation and concentration by described immunomagnetic beads: get the pre-germ-increasing liquid of 1mL and join the 2mL centrifuge tube, adding wherein 50 μ L concentration is the described immunomagnetic beads of 2mg/mL, rotating 10rpm reaction 30min on mixed instrument under room temperature, after take off, magnetic frame separates, supernatant discarded.Use at last the resuspended magnetic bead of PBS of 100 μ L pH=7.2;
3) detect front deactivation and processing: with the resuspended liquid of resulting magnetic bead water-bath 5min in 90 ℃ of water-baths, gained liquid carries out magnetic in magnetic field separates, and getting clear liquid is sample liquid to be checked;
4) with step 3) gained liquid to be checked gets 80 μ L and drips in described Shigella double antibodies sandwich test card, sentence read result in 5 ~ 10min.
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Cited By (5)
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| CN107478834A (en) * | 2017-09-05 | 2017-12-15 | 杨蕾 | A kind of preparation method for detecting salmonella magnetic resonance imaging kit |
| CN110658338A (en) * | 2019-09-12 | 2020-01-07 | 武汉大学 | Portable mastitis pathogen MRSA detection method in lactation period |
| CN113109566A (en) * | 2021-05-24 | 2021-07-13 | 上海理工大学 | Fluorescence immunochromatography joint inspection method for food-borne pathogenic bacteria based on antigen colorization |
| CN113552346A (en) * | 2021-07-15 | 2021-10-26 | 上海理工大学 | Shigella boydii immune colloidal gold detection test strip and detection method thereof |
| CN115856296A (en) * | 2022-12-16 | 2023-03-28 | 华北理工大学 | Anti-shigella monoclonal antibody and application thereof in detection |
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| CN115856296A (en) * | 2022-12-16 | 2023-03-28 | 华北理工大学 | Anti-shigella monoclonal antibody and application thereof in detection |
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