CN110456049A - A kind of detection method of platelet product germ contamination - Google Patents

A kind of detection method of platelet product germ contamination Download PDF

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Publication number
CN110456049A
CN110456049A CN201910589644.1A CN201910589644A CN110456049A CN 110456049 A CN110456049 A CN 110456049A CN 201910589644 A CN201910589644 A CN 201910589644A CN 110456049 A CN110456049 A CN 110456049A
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China
Prior art keywords
magnetic nanoparticle
tested
reaction
buffer
detection
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Pending
Application number
CN201910589644.1A
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Chinese (zh)
Inventor
王明元
严伟斌
吴建香
陈炜
汤龙海
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Suzhou central blood station
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Suzhou central blood station
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Priority to CN201910589644.1A priority Critical patent/CN110456049A/en
Publication of CN110456049A publication Critical patent/CN110456049A/en
Pending legal-status Critical Current

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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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria

Abstract

The present invention relates to technical field of medical examination, more particularly to a kind of method of platelet product germ contamination detection, the following steps are included: concentration and separation goes out tested bacteria from sample to be tested using magnetic nanoparticle, the magnetic nanoparticle surface coupling has and the monoclonal antibody of tested bacteria specific binding or polyclonal antibody or aptamers；There is the magnetic nanoparticle of tested bacteria to be resuspended with buffer capture；Chromatographic test paper is inserted into buffer and is reacted；The chromatographic test paper that reaction is completed, which is taken out, and is immersed to magnetic nanoparticle to carry out secondary color reaction in the substrate of catalyzed coloration；Paper slip, color development stopping reaction are taken out, observation detects the result at band and quality control band.The processing reacted by setting secondary color, can enhance the significant degree of color change at detection line, to improve the sensibility of detection, allow testing result more acurrate.

Description

A kind of detection method of platelet product germ contamination
Technical field
The present invention relates to technical field of medical examination, and in particular to a kind of detection method of platelet product germ contamination.
Background technique
Blood product is the important component of clinical disease treatment and first aid, is directly related to the life security of patient.But At present blood product clinically especially Bacteria Contamination of Platelets the problem of it is increasingly serious.Studies have shown that single every about 3000 Just there is 1 unit that germ contamination occurs in the platelet product of position, pollution main source has blood donor's skin flora, blood donor The pollution occurred there are asymptomatic bacteremia and during the preparation process.And since the patient of clinically platelet transfusion is mostly Patient with severe symptoms, physical basis condition is poor, hypoimmunity, the blood platelet that not can effectively clear the bacterium of input, therefore pollute If product is infused into patient's body, almost the transfusion reaction without exception that light and heavy degree will be caused not wait, even fatal Death can occur for bacteremia, septicemia and pyemia, serious person, therefore, carry out before infusion to platelet product necessary thin Bacterium pollution detection, to reducing patients with transfusion septicemia as far as possible and pyemic be of great significance.
Chromatograph test strip detection is a kind of detection method for capableing of quick bacterium examination, by by antibody or aptamers It is coupled on the carrier particles, such as gold nano grain, magnetic nanoparticle, there is the carrier granular of antibody or aptamers by being coupled The bacterium in identification sample to be tested is gone, and the detection on chromatograph test strip takes general scribing line and has another monoclonal is/mostly anti-/ to fit Then ligand again has carefully capture by detecting band by identifying the bacterium captured in sample to be tested first with carrier granular The carrier granular of bacterium carries out secondary capture, so that carrier granular takes aggregation in detection, has color by itself at this time Carrier granular aggregation so that naked eyes are it can be observed that the color change that takes of detection, thus to the bacterium in sample to be tested Pollution is determined.
But in this way, when bacterial concentration is lower in sample to be tested, carrier granular is detecting the aggregation taken It is less, so that the color change that detection takes is weaker, at this point, being easy to cause naked eyes that can not really judge that detection takes Whether there is color change, mistake occurs so as to cause testing result, i.e. detection sensitivity is not high.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is that the aggregation in the prior art by carrier granular is overcome to develop the color The not high defect of germ contamination situation sensibility in interpretation sample to be tested, to provide a kind of platelet product germ contamination detection Method.
In order to solve the above technical problems, the technical solution adopted by the present invention are as follows:
A kind of method of platelet product germ contamination detection, comprising the following steps:
Using magnetic nanoparticle, concentration and separation goes out tested bacteria from sample to be tested, and the magnetic nanoparticle surface is even Be associated with can specific recognition tested bacteria monoclonal antibody or polyclonal antibody or aptamers, the magnetic nanoparticle have Enzymatic activity；
There is the magnetic nanoparticle of bacteria to be tested to be resuspended with buffer capture；
Chromatographic test paper is inserted into re-suspension liquid and is reacted；
The chromatographic test paper that reaction is completed is taken out and immersed to magnetic nanoparticle and can carry out two in the substrate of catalyzed coloration Secondary chromogenic reaction；
Paper slip, color development stopping reaction are taken out, observation detects the result at band and quality control band.
Further, the magnetic nanoparticle is Fe3O4、Fe3O4@Pt、γ-Fe2O3And Fe3O4In@carbon at least It is a kind of.
Further, the substrate be sedimentation type TMB (tetramethyl benzidine), in DAB (diaminobenzidine) at least It is a kind of.
Further, the buffer is phosphate buffered saline solution (PBS), in Tris buffered saline (TBS), TE buffer At least one.
Technical solution of the present invention has the advantages that
1. the method for platelet product Bacteria Detection provided by the invention, by setting magnetic Nano for carrier granular Grain, and magnetic nanoparticle has the property of enzyme, chromogenic reaction can occur with catalyzed coloration substrate, complete in chromatographic test paper to be measured After strain captures, using the catalytic activity of magnetic nanoparticle, substrate is allowed to react shape at the detection line of chromatographic test paper It develops the color at precipitability particle, the processing reacted by setting secondary color can enhance the obvious of color change at detection line Degree, so that testing result can be more acurrate, improves the sensibility of detection, simultaneously as carrier granular is that have magnetism Nano enzyme, therefore tested bacteria can be separated with sample to be tested by way of magnetic, with centrifugation in the prior art Formula separation is compared, and tested bacteria is enriched with and is separated by the way of magnetic, during the separation process, platelet component will not It is separated from sample to be tested, so as to reduce blood platelet to influence caused by testing result.
Specific embodiment
There is provided following embodiments is to preferably further understand the present invention, it is not limited to the best embodiment party Formula is not construed as limiting the contents of the present invention and protection scope, anyone under the inspiration of the present invention or by the present invention and its The feature of his prior art is combined and any and identical or similar product of the present invention for obtaining, all falls within of the invention Within protection scope.
Specific experiment step or condition person are not specified in embodiment, according to the literature in the art described routine experiment The operation of step or condition can carry out.Reagents or instruments used without specified manufacturer, being can be by commercially available acquisition Conventional reagent product.
Embodiment 1
This programme is related to a kind of detection method of the germ contamination in platelet product, specifically with gram-positive bacteria or yin For property bacterium, comprising the following steps:
The preparation of S1, reaction film: it chooses nitrocellulose filter (NC film) and is used as reaction film, it is big to be cut into 30 × 1.8cm Small, two spare.The concentration of anti-mouse IgG is adjusted to 0.25mg/mL with the phosphate buffer that the pH of 0.02M is 7.2, is added The Tween-20 that volume fraction is 1% uses a stroke film instrument to be sprayed on film surface as nature controlling line, and drawing film amount is 1 μ L/cm.It will Specificity uses the pH of 0.02M for the adjusting of 7.2 phosphate buffers respectively for the monoclonal antibody of lipopolysaccharides and phosphatide Teichaic acid Its concentration be 0.2mg/mL, be added volume fraction be 1% Tween-20, use respectively draw a film instrument be sprayed on film surface as Detection line.5mm is divided between every line.It is immediately placed in 37 DEG C of vacuum ovens and is dried overnight after drawing film, room temperature preservation is standby With.
The assembling of S2, chromatograph test strip
Nitrocellulose filter is chosen as sample cushion material, it is spare to be cut into 30 × 3cm size.Water absorption pad is chosen, and It is cut into 30 × 1.8cm size, two spare.Two reaction films are first pasted on the PVC bottom plate that specification is 30 × 9cm, Reaction film interval 2.4cm；Sample pad two sides are taken that overlapped again, and method sticks in reaction film, remaining is sticked on bottom plate, is taken The length connect is 0.3cm；Water absorption pad side is overlapped on reaction mould respectively and sticks on bottom plate, and length that overlapped is 0.3cm.Group After installing, the test strips of 0.5cm wide are cut into, are put in aluminium foil bag, is sealed at 4 DEG C.
The preparation of S3, magnetic nanoparticle: 1.35g FeCl is weighed3·6H2O, 1.0g urea and 1.5gPEG4000 are added Into 40mL ethylene glycol solution, 20min is stirred, is allowed to be completely dissolved；Mixed liquor is added to poly- the four of hydrothermal synthesis reaction kettle In vinyl fluoride liner, then reaction kettle closing is placed in electric drying oven with forced convection, 200 DEG C, reacts 10-16h；After natural cooling Reactant is taken out, cleans 3 times with dehydrated alcohol repeated washing 5 times, then with deionized water, finally with deionized water by resulting production Object disperses again, room temperature preservation.
S4, magnetic nanoparticle coupled antibody: lipoteichoicacid monoclonal antibody or lipopolysaccharides monoclonal antibody are used respectively It is 5mg/mL that 0.1M phosphate buffer (pH=7.2), which is diluted to concentration, and isometric 10mg/mL succinimide -4- ring is added Hexane -1- carbonic ester, 4 DEG C of reaction 3h, then dialysed with the phosphate buffer that pH is 7.2, remove unreacted succinyl Imines -4- thiacyclohexane -1- carbonic ester, obtains antibody-solutions.Then use 0.1M phosphate buffer (pH=7.2) by magnetic Nano Particle is diluted to 5% (w/v), the antibody-solutions after isometric dialysis is added, and is placed in blending instrument room temperature reaction 4-6h.Reaction is completed Afterwards, Magnetic Isolation removes supernatant, is washed 3 times with phosphate buffer, and being diluted to volume fraction is 10% stand-by, obtains being coupled The magnetic nanoparticle of lipopolysaccharides antibody or phosphatide antiteichoic acid antibody.
S5, detection: in 1.5mL centrifuge tube, the magnetism that coupling respectively has lipopolysaccharides antibody and phosphatide antiteichoic acid antibody is added Each 50 μ L of nano particle and 200 μ L sample to be examined are uniformly mixed, after reacting at room temperature 10min, magnetic-adsorption, and after removing supernatant, It is resuspended in 100 μ L phosphate buffers, then test strips is perpendicularly inserted into re-suspension liquid and react 15min.Take out test strips, It immerses in the EP pipe containing sedimentation type substrate TMB (tetramethyl benzidine), reacts 10min.Test strips are taken out, are rushed with deionized water Wash color development stopping reaction, the result at visual results detection band and quality control band.
The processing that the present embodiment is reacted by setting secondary color, can enhance the significant degree of color change at detection line, So that testing result can be more acurrate, the sensibility of detection is improved.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or It changes still within the protection scope of the invention.

Claims (4)

1. a kind of method of platelet product germ contamination detection, which comprises the following steps:
Using magnetic nanoparticle, concentration and separation goes out bacteria to be tested from sample to be tested, and the magnetic nanoparticle surface coupling has The monoclonal antibody or polyclonal antibody or aptamers of energy specific recognition tested bacteria, the magnetic nanoparticle have enzyme activity Property；
There is the magnetic nanoparticle of tested bacteria to be resuspended with buffer capture；
Chromatographic test paper is inserted into re-suspension liquid and is reacted；
The chromatographic test paper that reaction is completed, which is taken out, and is immersed to magnetic nanoparticle to carry out secondary show in the substrate of catalyzed coloration Colour response；
Paper slip, color development stopping reaction are taken out, observation detects the result at band and quality control band.
2. the method according to claim 1, wherein the magnetic nanoparticle is Fe3O4、Fe3O4@Pt、γ- Fe2O3And Fe3O4At least one of@carbon.
3. according to the method described in claim 2, it is characterized in that, the substrate be sedimentation type TMB (tetramethyl benzidine), At least one of DAB (diaminobenzidine).
4. method according to any one of claim 1-3, which is characterized in that the buffer is phosphate buffered saline solution (PBS), at least one of Tris buffered saline (TBS), TE buffer.
CN201910589644.1A 2019-07-02 2019-07-02 A kind of detection method of platelet product germ contamination Pending CN110456049A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104407131A (en) * 2014-10-22 2015-03-11 中国检验检疫科学研究院 Immunomagnetic bead substrate color development test paper and preparation method thereof
CN104502589A (en) * 2014-12-17 2015-04-08 中国科学院苏州生物医学工程技术研究所 Chromatographic test strip for detecting platelet product bacterial pollution and detection method
US20160334397A1 (en) * 2014-01-14 2016-11-17 Gill Biotechnology (Tianjin) Co., Ltd. Nanozyme immunochromatographic detection method
CN108982834A (en) * 2018-04-28 2018-12-11 公安部物证鉴定中心 The method of nano enzyme immuno-sandwich new technology detection biomolecule

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160334397A1 (en) * 2014-01-14 2016-11-17 Gill Biotechnology (Tianjin) Co., Ltd. Nanozyme immunochromatographic detection method
CN104407131A (en) * 2014-10-22 2015-03-11 中国检验检疫科学研究院 Immunomagnetic bead substrate color development test paper and preparation method thereof
CN104502589A (en) * 2014-12-17 2015-04-08 中国科学院苏州生物医学工程技术研究所 Chromatographic test strip for detecting platelet product bacterial pollution and detection method
CN108982834A (en) * 2018-04-28 2018-12-11 公安部物证鉴定中心 The method of nano enzyme immuno-sandwich new technology detection biomolecule

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