CN101561436A - Colloidal gold test paper for listeria monocytogenes, preparation method thereof and application thereof - Google Patents
Colloidal gold test paper for listeria monocytogenes, preparation method thereof and application thereof Download PDFInfo
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- CN101561436A CN101561436A CNA2009100849703A CN200910084970A CN101561436A CN 101561436 A CN101561436 A CN 101561436A CN A2009100849703 A CNA2009100849703 A CN A2009100849703A CN 200910084970 A CN200910084970 A CN 200910084970A CN 101561436 A CN101561436 A CN 101561436A
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Abstract
The invention discloses a detection test paper. The test paper comprises (1) a reaction support, (2) a water absorption pad, (3) a nitrocellulose membrane, (4) a gold-labeled antibody adsorption membrane and (5) a sample pad, wherein the nitrocellulose membrane is coated with a detection strip and a quality control strip of a listeria monocytogenes P60 antibody and a quality control antibody; and the gold-labeled antibody adsorption membrane contains the listeria monocytogenes P60 antibody labeled by colloidal gold. The test paper can be matched with a small instrument to carry out semi-quantitative detection, does not need professional training, has clear and identifiable result, can objectively store data, is simple to operate, easy to popularize, suitable for a basic level, suitable for field detection of emergency and suitable for epidemiological investigation, and plays an auxiliary role in diagnosis of listeria monocytogenes infection.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to a kind of detection test paper and preparation method thereof and application in Listeria monocytogenes of Listeria monocytogenes.
Background technology
Listeria monocytogenes (Listeria monocytogenes, Listeria monocytogenes), it is most important human foodborne bacterial pathogens in the listeria (Listeria), also be a kind of pathogenic bacteria of infecting both domestic animals and human, belong to listeria, it can cause humans and animals trouble meningitis, encephalitis, septicemia, endocarditis, miscarriage, stillborn foetus and abscess etc., sends out patient's mortality ratio and can reach 30%-40%.Existing in recent years many countries have reported owing to pollute the food poisoning that Listeria monocytogenes takes place.Therefore, countries in the world government department more and more payes attention to the food poisoning that Listeria monocytogenes causes, has formulated some new food security rules one after another, and Listeria monocytogenes is included in legal strong inspection project.The detection method of Listeria monocytogenes mainly relies on traditional separation to support and biochemical identification at present, and this method wastes time and energy.Collaurum fast diagnose test paper bar technology is a novel vitro diagnostic techniques that has grown up since the nineties in 20th century.This method development has in recent years particularly obtained widespread use in the medical test at biomedical sector rapidly, but it is less to be used for the product that detects in the food hygiene field.The immune colloidal gold detection test paper strip of food pollution Listeria monocytogenes has been developed in this research at Listeria monocytogenes.The collaurum detection method sensitivity that this research is set up, accurate, high specificity can further be applied in the detection real work of inspection and quarantine department to Listeria monocytogenes in the import and export food.After the method is set up, can with classical conventional method complementation, expectation will have wider application prospect in inspection and quarantine, food industry department and sanitation monitoring department, and certain economic and social benefit are arranged.Simultaneously, can be food microorganisms checks the revision of international method or augments scientific basis is provided.
Quantitative fluorescent PCR and traditional microbe growth, these two kinds of methods are all longer detection time, still need 8 hours the soonest, and need the experimental situation and the expensive instrument of appointment, and PCR also is prone to false positive, is not suitable for field quick detection, and can not carry out detection by quantitative.
Summary of the invention
The purpose of this invention is to provide a kind of test paper that detects Listeria monocytogenes quickly and easily, the semi-quantitative detection method that can utilize the gold-marking immunity analyser to carry out simultaneously.The principle of work of this test paper is to utilize the combination of specific antigen-antibody, uses colloid gold label antibody, after antigen to be checked combines, make with test paper on the capture antibody colour developing that combines.The invention still further relates to the preparation method of the above-mentioned detection test paper of preparation.
The present invention can detect the Listeria monocytogenes in the sample quickly and easily based on a sample and a test paper, has saved a large amount of manpower and materials, easily and fast, simple and direct.
The present invention relates to a kind of detection test paper that detects Listeria monocytogenes quickly and easily, it comprises:
(1) reaction holder;
(2) adsorptive pads;
(3) nitrocellulose membrane, this film are coated with the Quality Control band of Listeria monocytogenes P60 polyclonal antibody (for example can from Jilin Province Entry-Exit Inspection and Quarantine Bureau) test strip and Quality Control goat anti-rabbit igg (for example available from ancient cooking vessel state Bioisystech Co., Ltd).(Xiong Guohua, in jasmine, Cao Jijuan etc., Listeria monocytogenes immunity colloidal gold test paper strip fast detecting [J] Chinese public health magazine the 24th the 2nd phase of volume of February in 2008)
(4) golden labeling antibody adsorption film, how anti-the Listeria monocytogenes P60 that wherein contains colloid gold label is.
The detection test paper of the above-mentioned Listeria monocytogenes that the present invention relates to, it also comprises sample pad.
The detection test paper of the above-mentioned Listeria monocytogenes that the present invention relates to wherein reacts holder and selects the PVC plate for use.
The detection test paper of the above-mentioned Listeria monocytogenes type that the present invention relates to, wherein adsorptive pads is selected filter paper for use.
The detection test paper of the above-mentioned Listeria monocytogenes that the present invention relates to, wherein golden labeling antibody adsorption film is selected polyester film, glass fibre or filter paper fibre for use.
On the other hand, the detection test paper of the above-mentioned Listeria monocytogenes that the present invention relates to, wherein adsorptive pads, nitrocellulose membrane, golden labeling antibody adsorption film, sample pad and reaction holder constitute according to mode shown in the accompanying drawing 1; Reaction holder 5 is positioned at bottom, and nitrocellulose membrane 2 is positioned at the middle part on the reaction holder 5, and the T place of this film is the test strip of Listeria monocytogenes P60 polyclonal antibody bag quilt, and the C place is the Quality Control band of goat anti-rabbit igg bag quilt; Gold labeling antibody adsorption film 3 is positioned at a side on nitrocellulose membrane top and overlaps with it, and this film contains the Listeria monocytogenes P60 polyclonal antibody of colloid gold label; Adsorptive pads 1 be positioned at nitrocellulose membrane 2 tops the opposite side for golden labeling antibody absorption 3 and with 2 overlap; Sample pad 4 be positioned at a side opposite on 2 with 1 and with 3 overlap.
Another aspect, the detection test paper of the above-mentioned Listeria monocytogenes that the present invention relates to is an initiating terminal with adsorptive pads one side, and sample pad one side is terminal, the test strip of Listeria monocytogenes P60 polyclonal antibody is positioned near terminal, and the Quality Control band of goat anti-rabbit igg bag quilt approaches initiating terminal.
Further object of the present invention provides the method for the test paper of the described detection Listeria monocytogenes of preparation, and this method may further comprise the steps: the preparation of colloidal gold probe, the preparation of golden labeling antibody adsorption film, the spraying bag quilt of nitrocellulose membrane.
1, the preparation of colloidal gold probe
(1) HAuCl4 is formulated as 0.01% aqueous solution earlier.The HAuCl4 that accurately adds 1ml 1% under the magnetic agitation adds the trisodium citrate aqueous solution of 1.5-3ml 1% simultaneously.Continue heating behind the colour stable, be cooled to and add pure water after the room temperature and complement to 100ml, 4 ℃ keep in Dark Place.
(2) transferring pH value with K2CO3 is about 7-9, is preferably about 7.8-8.9, more preferably from about 8.2-8.5.
(3) collaurum is transferred to pH 8.2-8.5,, dilute antibody to 0.2mg/ml with 5mM PB (pH 7.2).
(4) keep collaurum stable.
The present invention also is to provide a kind of acquisition to keep the method for the stable antibody optimum mark dosage of collaurum, and this method comprises:
A. get the 1.5ml centrifuge tube of 10 cleanings, add colloidal gold solution 1ml respectively.
B. the PBS that in the 1-10 pipe, adds 10ml, 20ml, 30ml, 40ml, 50ml, 60ml, 70ml, 80ml, 90ml, 100ml respectively, the antibody 90ml to be marked, 80ml, 70ml, 60ml, 50ml, 40ml, 30ml, 20ml, 10ml, the 0ml that add the good 0.2mg/ml of dilution more successively, mixing left standstill 2 minutes.
C. add 10%NaCl solution 100ml in 10 pipes respectively, room temperature left standstill 2 hours behind the mixing, observed change color.
Do not add liquid color in the test tube that antibody and addition be not enough to the stable colloid gold and present variation, add the test tube that the antibody amount meets or exceeds minimum consistent dose and then keep red constant by red stain indigo plant.Compare with control tube (No. 1 pipe), color is the most approaching, contain the contained antibody dosage of the minimum test tube of antibody dosage, is the necessary antibody consistent dose of 1ml collaurum, adds 20% antibody on this basis again, is antibody optimum mark dosage.The present invention is through repetition test and analysis, and the result who draws shows: keeping the suitable antibody amount of colloidal gold solution is 2-10ug, preferred antibody amount 5-9ug, and more preferably 6.2-8.2ug most preferably is 7.2ug.
2, the preparation of golden labeling antibody adsorption film
The collaurum of getting above-mentioned pH is sub-packed in the centrifuge tube of 1.5ml with the 1ml/ pipe.Add 3-8ug mark dosage in every arm, preferred antibody amount 4-6ug, more preferably 4.5-5.5ug most preferably is the antibody of 5ug, places behind the jog mixing.The BSA100 of adding 10% in every pipe? 1, mixing is placed.The top colloidal gold probe that tentatively makes 12000rpm, 4 ℃ centrifugal 30 minutes down, is abandoned supernatant.It is the same centrifugal to add the resuspended liquid suspension of 1ml post precipitation in every centrifuge tube.Abandon supernatant, the pipe end, obtain bolarious loose shape precipitation, add 500? 1 resuspended liquid is resuspended back in 4 ℃, 1000rpm, centrifugal 10 minutes; Get supernatant, evenly drip on the wide glass fibre of 1cm 37 ℃ of dryings.
3, the spraying bag quilt of nitrocellulose membrane
(1) utilizes the nitrocellulose membrane that increases Listeria P60 polyclonal antibody and two bands of Quality Control goat anti-rabbit igg every stream metal spraying pen machine with the spray film speed Sheet of 50mm/s;
In the step of this spraying coated film, spray film speed is 10-100mm/s preferably, is more preferably 45-55mm/s, most preferably is 50mm/s; Described Listeria monocytogenes P60 polyclonal antibody, its addition is 1-5mg/ml, the dilution of this polyclonal antibody can be PBS; The Quality Control goat anti-rabbit igg can be available from ancient cooking vessel state biotech company, and its concentration is that 0.1-5mg/ml is more preferably 0.5-2.5mg/ml, most preferably is 1mg/ml;
(2) a kind of many anti-glass fibre membranes of Listeria monocytogenes P60 that contain colloid gold label of preparation are with many anti-being uniformly coated on the glass fibre membrane of Listeria monocytogenes P60 of colloid gold label.Antibody dilutes with PBS, and pH is 7-9, preferred 7.5-9, and optimum PH is 8.5.
The invention also discloses the application of described test paper in detecting the Listeria monocytogenes type, referring to accompanying drawing 2.
Detect in the method for Listeria monocytogenes in the present invention, earlier sample to be measured is put into the bottle that dilution is housed, with pipettor sample mix liquid is added test paper " 4 " again and locate (promptly terminal), the liquid in the sample relies on syphonic effect up, generally needs 10-15 minute sentence read result:
As containing Listeria monocytogenes in the sample, then it will form corresponding compound with the Listeria monocytogenes P60 polyclonal antibody of colloid gold label on the test paper, developing liquid is up and combine with Listeria monocytogenes P60 polyclonal antibody on the nitrocellulose membrane, form red lines, promptly locate to form red stripes at " T ".
No matter whether contain corresponding antigen, the colloid gold label polyclonal antibody continues to continue up and form the red precipitate line with goat anti-rabbit igg on this film with liquid, promptly locates to form red stripes at " C ".This line is a nature controlling line, loses efficacy as collaurum, and this line just can not occur, and illustrates that test paper lost efficacy.
2 red precipitate lines can appear in positive findings, and 1 red precipitate line appears in negative findings, lose efficacy as lines explanation test paper not occurring.If naked eyes can't be distinguished T place red stripes, can utilize the gold-marking immunity analyser to carry out half-quantitative detection.
Technical scheme of the present invention is: the Listeria monocytogenes P60 polyclonal antibody of employing purifying and goat anti-rabbit igg difference solid phase are on nitrocellulose membrane, how anti-the Listeria monocytogenes P60 of association colloid gold mark is, uses rete and analyse the principle of double antibody sandwich method and detect Listeria monocytogenes in the sample.
Description of drawings
The front schematic view of Figure 1A test paper of the present invention.
The side schematic view of Figure 1B test paper of the present invention.
Wherein, Figure 1A and 1B:1: adsorptive pads; 2: (T: Sheet increases Listeria P60 polyclonal antibody test strip to nitrocellulose membrane; C: bag is by the Quality Control band of goat anti-rabbit igg); 3: the glass fibre membrane that contains colloid gold label Listeria monocytogenes P60 polyclonal antibody; 4: sample pad; 5: the reaction holder.
Fig. 2: testing result synoptic diagram; Two line positives of T, C; Line feminine gender of C; Invalid.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
The preparation of the colloid gold test paper of embodiment 1, detection Listeria monocytogenes
1, the preparation of colloidal gold probe
(1) HAuCl4 is mixed with 0.01% aqueous solution earlier, gets the 100ml pure water and be heated to boiling.The HAuCl4 that accurately adds 1ml 1% under the magnetic agitation adds the 1.5ml massfraction and is 1% trisodium citrate aqueous solution simultaneously.Continued heated and boiled behind the colour stable 15 minutes, and be cooled to and add pure water after the room temperature and complement to 100ml, 4 ℃ keep in Dark Place.
(2) K2CO3 accent pH value is about 8.2-8.5.
(3) collaurum is transferred to suitable pH, preferred 8.0-8.5, more preferably 8.2, with 5mM PB (pH7.2) dilution antibody to 0.2mg/ml.
2, the preparation of golden labeling antibody adsorption film
The collaurum of getting pH 8.2 is sub-packed in the centrifuge tube of 1.5ml with the 1ml/ pipe.Add the antibody of optimum mark dosage in every arm, placed 5 minutes or long slightly behind the jog mixing, this step combines with colloid gold particle for antibody.The BSA100ml of adding 10% in every pipe, mixing was placed 15 minutes or long slightly, and this step is sealing.The top colloidal gold probe that tentatively makes with 12000rpm, centrifugal 30 minutes, 4 ℃, is abandoned supernatant.It is the same centrifugal to add the resuspended liquid suspension of 1ml post precipitation in every centrifuge tube.Abandon supernatant, stay the loose shape precipitation of pipe end kermesinus, add the resuspended back of the resuspended liquid of 500ml in 4 ℃, 1000rpm, centrifugal 10 minutes; Get supernatant, drip uniformly on the wide glass fibre of 1cm, 37 ° dry 2.5-3 hour.
3, the spraying bag quilt of nitrocellulose membrane
(1) utilize every stream metal spraying pen machine with the spray film speed Sheet of 50mm/s increase Listeria P60 polyclonal antibody (1.5mg/ml+2%BSA) and Quality Control goat anti-rabbit igg (available from ancient cooking vessel state biotech company, the 1mg/ml) nitrocellulose membrane of two bands;
(2) contain the glass fibre membrane of colloid gold label Listeria monocytogenes P607.2ug, the PB dilution; Optimum PH is 8.5.
Referring to Fig. 1, the reaction holder is 6.2cm * 0.4cm PCV plate; Adsorptive pads is the filter paper for oil of 2cm * 0.4cm; 1.8cm the nitrocellulose membrane of * 0.4cm wraps by goat anti-rabbit igg successively (available from ancient cooking vessel state biotech company, 1mg/ml), Listeria monocytogenes P60 polyclonal antibody 2mg/ml (containing the 1%BSA sealer) contains Listeria monocytogenes P60 polyclonal antibody (the standard gold concentration: 7.2ug PB dilution of 0.4cm * 0.4cm colloid gold label; Optimum PH is 8.5) glass fibre membrane; Sample pad is the glass fibre membrane of 2.7cm * 0.4cm; Promptly form Listeria monocytogenes and detected test paper.
Embodiment 3, detection method
Referring to accompanying drawing 2, with sample to be measured and sample diluting liquid mixing, with pipettor sample mix liquid is added test paper sample well place again, the liquid in the sample relies on syphonic effect up, 10-15 minute sentence read result:
As contain Listeria monocytogenes, then with the Listeria monocytogenes P60 of colloid gold label is how anti-forms corresponding compound on the test paper, up and the many resistive connections of stream Listeria monocytogenes P60 that be coated on the nitrocellulose membrane close, and form red lines, promptly locate to form red stripes at " T ".
No matter whether contain corresponding antigen, the how anti-continuation of colloid gold label is upwards creeped and the goat anti-rabbit igg that is coated on the film forms the red precipitate line, promptly locates to form red stripes at " C ".This line is a nature controlling line, loses efficacy as collaurum, and this line just can not occur, and illustrates that test paper lost efficacy.
2 red precipitate lines can appear in positive findings, and 1 red precipitate line appears in negative findings, lose efficacy as lines explanation test paper not occurring.
The quantitative gold-marking immunity analyser of embodiment 4, DJM-3 half-quantitative detection
1, can detect 20 negative values of Listeria monocytogenes test strips with detection method earlier, the average of relatives that utilizes gold mark instrument to detect its T/C of examination calculates the threshold value of deciding of golden staphylococcal enterotoxin Type B test strips.
Secondly 2, utilize and decide threshold value and come to mark the least concentration that instrument machine detection Listeria monocytogenes test strips can reach with gold.
3, positive more than or equal to deciding threshold value during sentence read result, negative less than deciding threshold value.
Detecting the variable concentrations Listeria monocytogenes, is 0.017 through computational discrimination value (CUT-OFF), and concentration is that the value that average T/C ratio is of the Listeria monocytogenes of 101cfu/ml, 102cfu/ml is 0.00, is lower than 0.017, and is negative; It is positive that the value that the average T of the Listeria monocytogenes of 103cfu/ml～108cfu/ml/C ratio is respectively all is higher than 0.017 result; When Listeria monocytogenes concentration during greater than 103cfu/ml, T/C average of relatives value is greater than 0.017, so detection by quantitative sensitivity is 103cfu/ml.
Each concentration testing result of table 1 Listeria monocytogenes colloidal gold immuno-chromatography test paper strip
Advantage of the present invention:
Test paper of the present invention can cooperate miniature instrument to carry out half-quantitative detection, does not need the specialty training Instruction, the result is clear easily to be distinguished and the objective save data of energy, simple to operate, is easy to promote, and is fit to basic unit, The Site Detection that is suitable for accident is fit to epidemiology survey, and to Listeria monocytogenes Infect And Diagnose plays booster action.
Claims (11)
1, a kind of detection test paper is characterized in that it comprises:
(1) reaction holder;
(2) adsorptive pads;
(3) nitrocellulose membrane, this film are coated with Listeria monocytogenes antibody and Quality Control detection of antibodies band and Quality Control band;
(4) golden labeling antibody adsorption film wherein contains the Listeria monocytogenes P60 antibody of colloid gold label;
(5) sample pad.
2, test paper according to claim 1, wherein, adsorptive pads is selected filter paper for use, and the reaction holder is selected the PVC plate for use, the material of gold labeling antibody adsorption film is selected from polyester film, glass fibre or filter paper fibre, and the material of sample pad is selected from polyester film, glass fibre or filter paper fibre.
3, according to each described test paper of claim 1-2, wherein, reaction holder (5) is positioned at bottom, nitrocellulose membrane (2) is positioned at the middle part on the reaction holder (5), the T place of this film is the test strip of Listeria monocytogenes P60 polyclonal antibody bag quilt, and the C place is the Quality Control band of goat anti-rabbit igg bag quilt; Gold labeling antibody adsorption film (3) is positioned at a side on nitrocellulose membrane top and overlaps with it, and this film contains the Listeria monocytogenes P60 polyclonal antibody of colloid gold label; Adsorptive pads (1) is positioned at the opposite side for golden labeling antibody adsorption film (3) on nitrocellulose membrane (2) top and overlaps with nitrocellulose membrane (2); Sample pad (4) is positioned at upward opposite with adsorptive pads (1) side of nitrocellulose membrane (2) and overlaps with golden labeling antibody adsorption film (3).
4, according to each described test paper of claim 1-3, wherein, adsorptive pads one side is an initiating terminal, and sample pad one side is terminal, and the band that detects antibody is positioned near terminal, and the Quality Control band approaches initiating terminal.
5, according to each described test paper of claim 1-4, wherein, how anti-the antibody of described anti-Listeria monocytogenes can be, also monoclonal antibody; Quality Control antibody can be selected the IgG of goat-anti rabbit, mouse-anti rabbit, the anti-rabbit of people, the anti-sheep of rabbit, mouse-anti sheep, the anti-sheep of people, sheep anti mouse, the anti-mouse of people, the anti-mouse of rabbit etc. according to the immunogenic of golden labeling antibody.
6, according to each described test paper of claim 1-5, wherein, the concentration of described anti-Listeria monocytogenes P60 polyclonal antibody is 0.5-5mg/ml.
7, according to each described test paper of claim 1-6, wherein, the Quality Control antibody concentration is 0.1-2mg/ml.
8, according to each described test paper of claim 1-7, wherein, the amount of described anti-Listeria monocytogenes P60 antibody labeling 1ml collaurum is 5-20ug.
9, a kind of preparation method who prepares the detection test paper of each described Listeria monocytogenes of claim 1-8, this method comprises:
(1) use every stream metal spraying pen machine with certain spray film speed spray anti-Listeria monocytogenes antibody and
Quality ControlThe nitrocellulose membrane of two bands of antibody;
(2) a kind of glass fibre membrane that contains the anti-Listeria monocytogenes antibody of colloid gold label of preparation is uniformly coated on the anti-Listeria monocytogenes antibody of colloid gold label on the glass fibre membrane, and oven dry or freeze drying.
10, the application of each described test paper of claim 1-8 in detecting Listeria monocytogenes, comprising with sample to be measured and sample diluting liquid mixing, sample mix liquid is added test paper sample well place, the liquid in the sample relies on syphonic effect up, 10-15 minute sentence read result again.
11, by the test paper of the detection Listeria monocytogenes of the described method of claim 10 preparation.
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| CN102507948B (en) * | 2011-11-15 | 2013-12-04 | 吉林出入境检验检疫局检验检疫技术中心 | Method using liquid phase chip to detect mononuclear cell proliferation listeria |
| CN102507948A (en) * | 2011-11-15 | 2012-06-20 | 吉林出入境检验检疫局检验检疫技术中心 | Method using liquid phase chip to detect mononuclear cell proliferation listeria |
| CN102539770A (en) * | 2011-12-29 | 2012-07-04 | 何鑫 | Reagent kit for detecting antiuninuclear cell proliferation Listeria bacteria by colloidal gold Hly gene monoclonal antibodies |
| CN103941012A (en) * | 2014-04-29 | 2014-07-23 | 上海理工大学 | Quick detection test strip for listeria immune colloidal gold |
| CN103941012B (en) * | 2014-04-29 | 2015-12-02 | 上海理工大学 | Listeria immune colloid gold Rapid detection test strip |
| CN104483459A (en) * | 2014-12-23 | 2015-04-01 | 北京出入境检验检疫局检验检疫技术中心 | Low-noise excitation type fluorescent marker based chromatography test strip for listeria monocytogenes |
| CN107462718A (en) * | 2017-08-06 | 2017-12-12 | 潘荣兰 | A kind of food-borne pathogens quick detection kit |
| CN110308274A (en) * | 2019-07-24 | 2019-10-08 | 南京黎明生物制品有限公司 | A kind of kit and preparation method thereof detecting listeria monocytogenes |
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