CN104502589A - Chromatographic test strip for detecting platelet product bacterial pollution and detection method - Google Patents
Chromatographic test strip for detecting platelet product bacterial pollution and detection method Download PDFInfo
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- CN104502589A CN104502589A CN201410781842.5A CN201410781842A CN104502589A CN 104502589 A CN104502589 A CN 104502589A CN 201410781842 A CN201410781842 A CN 201410781842A CN 104502589 A CN104502589 A CN 104502589A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The invention discloses a chromatographic test strip for detecting platelet product bacterial pollution and a detection method. The test strip consists of a sample pad, reaction films and an absorption pad which are mutually lapped on a bottom plate, wherein two ends of the sample pad are respectively lapped with two reaction films; each reaction film is fixedly provided with an inner quality control band and at least one detection band; the two reaction films are capable of respectively detecting the gram positive bacterium and negative bacterium in the platelet pollution; the sample in the sample pad can simultaneously diffuse toward the reaction films at two sides. Through the manner, the chromatographic test strip is capable of solving the technical problem that the traditional detection technology of the platelet product bacterial pollution is long in time, tedious to operate and low in sensitivity, so that the detection of conventional platelet product bacterial pollution before blood transfusion can be carried out clinically so as to avoid the blood transfusion diseases caused by bacterial pollution, improve the life quality of the patients and guarantee the clinically safe, effective and scientific blood transfusion.
Description
Technical field
The present invention relates to microorganism detection field, particularly relate to a kind of chromatograph test strip and the detection method that detect platelet product bacterial contamination.
Background technology
Blood product is the important component of clinical disease treatment and first aid, is directly connected to the life security of patient.But at present the problem of blood product especially Bacteria Contamination of Platelets is increasingly serious clinically.Research shows, just have 1 unit generation bacterial contamination in the platelet product of approximately every 3000 units, main source has blood donor's skin flora, and blood donor exists asymptomatic bacteremia and pollutes in preparation process.If the platelet product that bacterial contamination occurs is infused in patient body, the almost transfusion reaction that light and heavy degree all will be caused not wait without exception, or even fatal bacteremia, septicemia and pyemia.Its reason has two aspects: (1), for keeping biologically active pdgf, platelet product need be preserved 22 DEG C of concussions, and this temperature is conducive to the growth and breeding of bacterium.And containing plasma fraction in platelet product, be the nutrient culture media of bacterium near ideal.Therefore, to compare other component bloods much higher for the incidence of Bacteria Contamination of Platelets.(2) patient of platelet transfusion mostly is patient with severe symptoms clinically, and physical basis condition is poor, hypoimmunity, effectively can not remove the bacterium of input, is easy to bacteremia, septicemia and pyemia occur, and severe patient even death occurs.Because China's aging population and major disease such as the patient populations such as cancer, leukaemia, again barrier increase, blood platelet demand is day by day soaring, the annual platelet transfusion amount of current China reaches 1,000 ten thousand person-portions/year, and rise with the speed of 10% every year, there is the absolute value cumulative year after year of bacterial contamination in it, therefore need to keep the vigilant of height to platelet product bacterial contamination, carry out necessary bacterial contamination before infusion and detect, reduce patients with transfusion septicemia and pyemic generation as far as possible.
American association of blood banks (AABB) specifies that all platelet product all should be done bacterial contamination and detect before infusion for 2004.In Europe, Belgium carried out blood platelet Sterility testing with regard to forcing from 1998, and Holland also starts pressure from calendar year 2001 and carries out bacterial screening.
In the research of platelet product Bacteria Detection technology, the multiple detection technique of external research and development is mainly based on the indirect indexes of bacterial growth breeding, as abnormal in pH value change, concentration of glucose etc.; Bacterium consumes O
2with release CO
2principle; Antigen-antibody immunology principle; Molecular biology detection of nucleic acids principle etc.Wherein by FDA certification and be applied to platelet product bacterial contamination detect have Bact/ALERT microbe growth monitoring system, PalleBDS Bacteria Detection system, Scansystem system and blood platelet PGD detection system.Current detection method mainly contains three problems: length consuming time, and complex operation, susceptibility are low, well can not adapt to the demand of clinical quick, convenient, the safety blood of China.
The Ministry of Public Health of China discussed the bacterium safety problem of blood product specially in 2004, but also put into effect, also without quick, practical domestic detection reagent and instrument less than the relevant laws and regulations detected about blood and blood constituent bacterial contamination, code so far.
Summary of the invention
The technical matters that the present invention mainly solves is to provide a kind of chromatograph test strip and the detection method that detect platelet product bacterial contamination, and the method detects quick, convenient, accurate, reliable.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is: provide a kind of chromatograph test strip detecting platelet product bacterial contamination, comprise sample pad, reaction film, absorption pad and base plate, described reaction film is positioned on described base plate, described sample pad is positioned on described base plate and two ends and is overlapped on one end of described reaction film respectively, described absorption pad is positioned on described base plate and one end and is overlapped on the other end of described reaction film, described reaction film is fixed with detection zone and inner quality control band, described detection zone is positioned at the side near described sample pad, described inner quality control band is positioned at the side near described absorption pad.
In a preferred embodiment of the present invention, each described reaction film is fixed with detection zone described in a described inner quality control band and at least one, the length of described overlap joint is 0.3cm.
In a preferred embodiment of the present invention, described detection zone is fixed with the part with the albumen specific bond on bacteria cell wall or cell membrane, described quality control band is fixed with against murine IgG, and the material of described reaction film is nitrocellulose membrane, cellulose acetate film, nylon membrane or poly tetrafluoroethylene.
In a preferred embodiment of the present invention, described part is specificity for one or more antibody in lipopolysaccharides, phosphatide Teichaic acid, peptide glycan or is aptamer.
In a preferred embodiment of the present invention, described chromatograph test strip is for detecting the gram-positive bacteria and negative bacterium that pollute in blood platelet simultaneously.
A kind of chromatograph test strip is provided to detect the method for platelet product bacterial contamination, comprising step is: mixed with the Nano microsphere of labelled antibody by platelet contamination sample and hatch, magnetic resolution also removes supernatant, dilution is obtained with phosphate buffer dilution, joined by described dilution in the sample pad of described chromatograph test strip, the colour developing according to detection line obtains result.
In a preferred embodiment of the present invention, described Nano microsphere is magnetic microsphere or the color magnetic microballoon of nm of gold parcel, and the size of described magnetic microsphere is 5nm-50nm, and described antibody is lipoteichoicacid antibody and lipopolysaccharides antibody.
There is provided a kind of chromatograph test strip to detect the method for platelet product bacterial contamination, comprising step is: mixed with the Nano microsphere marking polyclonal antibody by platelet contamination sample and hatch, and magnetic resolution is also removed supernatant and is precipitated; The fluorescently-labeled monoclonal antibody phosphate buffer washing of described precipitation hatched, magnetic resolution supernatant, removes precipitation; The separation supernatant obtained is added in the sample pad of described chromatograph test strip, described chromatograph test strip is placed in fluorescence detector and detects.
In a preferred embodiment of the present invention, described Nano microsphere is magnetic microsphere or the color magnetic microballoon of nm of gold parcel, and the size of described magnetic microsphere is 5nm-50nm, and described polyclonal antibody is lipopolysaccharides polyclonal antibody and phosphatide Teichaic acid polyclonal antibody.
In a preferred embodiment of the present invention, described fluorescence labeling adopts fluorescein, and described fluorescein is anthocyanidin Cy line fluorescent element or Alexa Fluor line fluorescent element, and described monoclonal antibody is lipopolysaccharides monoclonal antibody and phosphatide Teichaic acid monoclonal antibody.
The invention has the beneficial effects as follows: the chromatograph test strip of detection platelet product of the present invention bacterial contamination and detection method, the operation of this immunochromatography technique is fast and convenient, can visual observation or realize sxemiquantitative in conjunction with detecting instrument or quantitatively detect, testing process is simple, efficiently, accurately, inexpensive and practical, be not easy undetected, not high to operating personnel's technical requirement, make the clinical detection can carrying out the front conventional platelet product bacterial contamination of blood transfusion, avoid the bacteremia that bacterial contamination causes, septicemia and pyemic generation, improve the quality of life of patient, alleviate the financial burden of patient, ensure clinical safety, effectively, the platelet transfusion of science, also can save valuable blood platelet resource simultaneously.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing used required in describing embodiment is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings, wherein:
Fig. 1 is the structural representation of chromatograph test strip one preferred embodiment of detection platelet product of the present invention bacterial contamination;
In accompanying drawing, the mark of each parts is as follows: 1, sample pad, and 2, reaction film, 3, absorption pad, 4, base plate, 5, detection zone, 6, inner quality control band.
Embodiment
Be clearly and completely described to the technical scheme in the embodiment of the present invention below, obviously, described embodiment is only a part of embodiment of the present invention, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making other embodiments all obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment one:
Refer to Fig. 1, a kind of chromatograph test strip detecting platelet product bacterial contamination is provided, comprise sample pad 1, two reaction films 2, two absorption pads 3 and base plate 4.Two described reaction films 2 lay respectively on described base plate 4.Described sample pad 1 is positioned on the centre position of described base plate 4, and the two ends of described sample pad 1 overlap respectively and partially overlap on one end of described reaction film 2.Two described absorption pads 3 are positioned on the two ends, left and right of described base plate 4, and described absorption pad 3 one end overlaps and partially overlaps on overlapping with described sample pad 1 other end of described reaction film 2.Described reaction film 2 is fixed with detection zone 5 and inner quality control band 6, general described each reaction film 2 is fixed with an inner quality control band 6 and at least one detection zone 5, described detection zone 5 is positioned at the side near described sample pad 1, and described inner quality control band 6 is positioned at the side near described absorption pad 3.Described two reaction films 2 can detect the gram-positive bacteria and negative bacterium that pollute in blood platelet respectively, and in described sample pad 1, sample can spread to both sides reaction film 2 simultaneously.
Embodiment two:
There is provided a kind of chromatograph test strip to detect the method for platelet product bacterial contamination, the method is visual assessment result:
One, the preparation of test strips
(1) preparation of reaction film
Choose Whatman Immunopore RP as reaction film, be cut into 30 × 1.8cm size, two for subsequent use.Be 0.25mg/ml with the phosphate buffer that the pH of 0.015M is 7.2 by the concentration adjustment of against murine IgG, add the Tween-20 that volume fraction is 1%, use and draw a film instrument and be sprayed on film surface as nature controlling line, drawing a film amount is 1 μ l/cm.With the pH of 0.015M be 7.2 phosphate buffers regulate its concentration be 0.25mg/ml respectively by specificity for the monoclonal antibody of lipopolysaccharides and phosphatide Teichaic acid, add the Tween-20 that volume fraction is 1%, use a stroke film instrument to be sprayed on two kinds of films surfaces as detection line respectively.Every bar line is spaced apart 5mm.Draw after film terminates and put into 37 DEG C of positive empty drying box dried overnight immediately, room temperature preservation is for subsequent use.
(2) assembling of test strips
Choose Whatman Rapid 27 as sample pad material, be cut into 30 × 3 cm sizes for subsequent use.Choose Whatman adsorptive pads CF6, and be cut into 30 × 1.8cm size, two for subsequent use.In specification be 30 × 9cm PVC base plate on first paste two reaction films, reaction film interval 2.4cm; Taked sample pad both sides the method overlapped to stick in reaction film again, all the other stick on base plate, and the length of overlap joint is 0.3cm; Adsorptive pads side is overlapped on reaction mould respectively and sticks on base plate, and the length of overlap joint is 0.3cm.After assembling, be cut into the test strips that 0.5cm is wide, be put in aluminium foil bag, 4 DEG C of lower seals are preserved.
Two, the preparation of the Nano microsphere of tagged ligand
(1) lipoteichoicacid antibody or lipopolysaccharides antibody 0.0.1M phosphate buffer (pH=7.2) being diluted to concentration is 5mg/ml, add equal-volume 10mg/ml succinimide-4-thiacyclohexane-1-carbonic ester, 4 DEG C of reaction 3h, dialyse with the phosphate buffer that pH is 7.2 again, remove unreacted succinimide-4-thiacyclohexane-1-carbonic ester, obtain antibody-solutions.
(2) with 0.0.1M phosphate buffer (pH=7.2), gold nano magnetic microsphere being diluted to volume fraction is 5%, adds the antibody-solutions after equal-volume dialysis, is placed in blending instrument room temperature reaction 4h.
(3) after having reacted, magnetic resolution removes supernatant, washs 3 times with phosphate buffer, and it is 10% stand-by for being diluted to volume fraction, obtains the gold nano magnetic microsphere of the good lipopolysaccharides antibody of coupling or phosphatide antiteichoic acid antibody.
Three, detecting step
(1) each for the gold nano magnetic microsphere of coupling two kinds of different antibodies 50 μ l are mixed;
(2) Nano microsphere is joined in 1ml blood platelet sample, mix, hatch 10min for 37 DEG C.
(3) magnetic resolution, removes supernatant, precipitation phosphate buffer (pH=7.2) is diluted to 200 μ l.
(4) above-mentioned dilution is joined in sample pad;
(5) colour developing is assembled according to gold nano magnetic microsphere at detection line place, sentence read result.
Embodiment three:
There is provided a kind of chromatograph test strip to detect the method for platelet product bacterial contamination, the method is Fluorescence Identification result:
One, the preparation of test strips
(1) preparation of reaction film
Choose Whatman PRIMA60 as reaction film, be cut into 30 × 1.8cm size, two for subsequent use.Be 0.25mg/ml with the phosphate buffer that the pH of 0.015M is 7.2 by the concentration adjustment of against murine IgG, add the Tween-20 that volume fraction is 1%, use and draw a film instrument and be sprayed on film surface as nature controlling line, drawing a film amount is 1 μ l/cm.Its concentration is regulated to be 0.25mg/ml with the phosphate buffer that the pH of 0.015M is 7.2 respectively for the monoclonal antibody of lipopolysaccharides and phosphatide Teichaic acid specificity, add the Tween-20 that volume fraction is 1%, use a stroke film instrument to be sprayed on two kinds of films surfaces as detection line respectively.Every bar line is spaced apart 5mm.Draw after film terminates and put into 37 DEG C of positive empty drying box dried overnight immediately, room temperature preservation is for subsequent use.
(2) assembling of test strips
Choose Whatman VF2 as sample pad material, be cut into 30 × 3 cm sizes for subsequent use.Choose Whatman adsorptive pads CF6, and be cut into 30 × 1.8cm size, two for subsequent use.In specification be 30 × 9cm PVC base plate on first paste two reaction films, reaction film interval 2.4cm; Taked sample pad both sides the method overlapped to stick in reaction film again, all the other stick on base plate, and the length of overlap joint is 0.3cm; Adsorptive pads side is overlapped on reaction mould respectively and sticks on base plate, and the length of overlap joint is 0.3cm.After assembling, be cut into the test strips that 0.5cm is wide, be put in aluminium foil bag, 4 DEG C of lower seals are preserved.
Two, the preparation of nano-magnetic microsphere coupled antibody
(1) with 0.01M phosphate buffer (pH=7.2), nano-magnetic microsphere being diluted to volume fraction is 5%, add lipoteichoicacid polyclonal antibody or lipopolysaccharides polyclonal antibody that isopyknic concentration is 5mg/ml, gently after mixing, add 50mg phosphinylidyne diimmonium salt immediately, be placed in blending instrument room temperature reaction 6h.
(2) after having reacted, magnetic resolution removes supernatant, washs 3 times with phosphate buffer, and it is 10% stand-by for being diluted to volume fraction.Obtain the nano-magnetic microsphere of the good lipopolysaccharides polyclonal antibody of coupling or phosphatide Teichaic acid polyclonal antibody.
Three, detecting step
(1) each for the nano-magnetic microsphere of coupling two kinds of different antibodies 50 μ l are mixed.
(2) microballoon is joined in 1ml blood platelet sample, mix, hatch 10min for 37 DEG C.
(3) magnetic resolution, removes supernatant, adds lipopolysaccharides monoclonal antibody and the phosphatide Teichaic acid monoclonal antibody of 200ul Cy5 dye marker respectively, 50 DEG C, hatch 10min, magnetic resolution supernatant in precipitation, removes precipitation.
(4) separation of supernatant is joined in sample pad.
(5) test strips is placed in fluorescence signal and reads instrument, read fluorescence signal and measure.
The invention discloses a kind of chromatograph test strip and the detection method that detect platelet product bacterial contamination, this test strips is made up of the sample pad mutually overlapped on base plate, reaction film and absorption pad, described sample pad two ends overlap two kinds of reaction films respectively, described reaction film is fixed with an inner quality control band and at least one detection zone, described two kinds of reaction films can detect the gram-positive bacteria and negative bacterium that pollute in blood platelet respectively, and in described sample pad, sample can spread to both sides reaction film simultaneously.
The present invention can will solve the technical barriers such as the traditional sensing techniques of platelet product bacterial contamination for a long time length consuming time, complex operation, susceptibility be low, make the clinical detection can carrying out the front conventional platelet product bacterial contamination of blood transfusion, the blood transfusion disease avoiding bacterial contamination to cause, improve the quality of life of patient, ensure clinical safety, effective, science blood transfusion.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize description of the present invention to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other relevant technical field, be all in like manner included in scope of patent protection of the present invention.
Claims (10)
1. one kind is detected the chromatograph test strip of platelet product bacterial contamination, it is characterized in that, comprise sample pad, reaction film, absorption pad and base plate, described reaction film is positioned on described base plate, described sample pad is positioned on described base plate and two ends and is overlapped on one end of described reaction film respectively, described absorption pad is positioned on described base plate and one end and is overlapped on the other end of described reaction film, described reaction film is fixed with detection zone and inner quality control band, described detection zone is positioned at the side near described sample pad, and described inner quality control band is positioned at the side near described absorption pad.
2. chromatograph test strip according to claim 1, is characterized in that, each described reaction film is fixed with detection zone described in a described inner quality control band and at least one, and the length of described overlap joint is 0.3cm.
3. chromatograph test strip according to claim 1, it is characterized in that, described detection zone is fixed with the part with the albumen specific bond on bacteria cell wall or cell membrane, described quality control band is fixed with against murine IgG, and the material of described reaction film is nitrocellulose membrane, cellulose acetate film, nylon membrane or poly tetrafluoroethylene.
4. chromatograph test strip according to claim 3, is characterized in that, described part is specificity for one or more antibody in lipopolysaccharides, phosphatide Teichaic acid, peptide glycan or is aptamer.
5. chromatograph test strip according to claim 1, is characterized in that, described chromatograph test strip is for detecting the gram-positive bacteria and negative bacterium that pollute in blood platelet simultaneously.
6. provide a kind of chromatograph test strip to detect the method for platelet product bacterial contamination, it is characterized in that, comprising step is: mixed with the Nano microsphere of labelled antibody by platelet contamination sample and hatch, magnetic resolution also removes supernatant, dilution is obtained with phosphate buffer dilution, joined by described dilution in the sample pad of described chromatograph test strip, the colour developing according to detection line obtains result.
7. detection method according to claim 6, is characterized in that, described Nano microsphere is magnetic microsphere or the color magnetic microballoon of nm of gold parcel, and the size of described magnetic microsphere is 5nm-50nm, and described antibody is lipoteichoicacid antibody and lipopolysaccharides antibody.
8. provide a kind of chromatograph test strip to detect the method for platelet product bacterial contamination, it is characterized in that, comprising step is: mixed with the Nano microsphere marking polyclonal antibody by platelet contamination sample and hatch, and magnetic resolution is also removed supernatant and is precipitated; The fluorescently-labeled monoclonal antibody phosphate buffer washing of described precipitation hatched, magnetic resolution supernatant, removes precipitation; The separation supernatant obtained is added in the sample pad of described chromatograph test strip, described chromatograph test strip is placed in fluorescence detector and detects.
9. detection method according to claim 8, it is characterized in that, described Nano microsphere is magnetic microsphere or the color magnetic microballoon of nm of gold parcel, and the size of described magnetic microsphere is 5nm-50nm, and described polyclonal antibody is lipopolysaccharides polyclonal antibody and phosphatide Teichaic acid polyclonal antibody.
10. detection method according to claim 8, it is characterized in that, described fluorescence labeling adopts fluorescein, and described fluorescein is anthocyanidin Cy line fluorescent element or Alexa Fluor line fluorescent element, and described monoclonal antibody is lipopolysaccharides monoclonal antibody and phosphatide Teichaic acid monoclonal antibody.
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| CN110095600A (en) * | 2019-05-16 | 2019-08-06 | 无锡市人民医院 | A kind of Test paper and kit of bacterial endotoxin |
| CN110456049A (en) * | 2019-07-02 | 2019-11-15 | 苏州市中心血站 | A kind of detection method of platelet product germ contamination |
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Application publication date: 20150408 |