CN101949927A - Francisella tularensis detection test paper, preparation method thereof, detection method using detection test paper and quantitative detection system - Google Patents
Francisella tularensis detection test paper, preparation method thereof, detection method using detection test paper and quantitative detection system Download PDFInfo
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Abstract
The invention discloses Francisella tularensis detection test paper, a preparation method thereof, a detection method using the detection test paper and a quantitative detection system. By using the colloidal gold labeling and double-antibody sandwich immunochromatography technologies, the invention establishes a method for the fast detection of Francisella tularensis. The established colloidal gold immunochromatographic method for the detection of the Francisella tularensis can fast, sensitively, specifically and accurately detect the Francisella tularensis in a sample and can realize quantitative detection, thereby being applicable to on-site fast detection.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to fast qualitative and quantitative detecting method and the colloid gold immune test strip of Francisella tularensis.
Background technology
Francisella tularensis (Francisella tularensis, Tula) be called for short soil and draw bacterium, this bacterium can cause the zoonosis of " soil draws heat ", and exanthemv, lymph gland enlargement, pulmonary infection and typhosis can appear in the infected, is in a bad way even death.This bacterium has widely distributed, and the route of transmission is various, low, the pathogenic characteristics such as strong of infective dose.Inject 10cfu, contact 10-50cfu with skin or take in 10
2-10
8This bacterium of cfu can both cause tularemia to infect.Therefore this bacterium is listed in and the terrified source of the category-A of same columns such as the plague, anthrax, has caused global great attention.Chemical Weapons Convention also draws soil heat also to be listed in the strictest controlling object.
The bio-safety problem has caused whole world concern, and U.S. disease prevention and control center draws bacterium to classify the pathogen that most possibly is used for biological weapons as in soil.From national bio-safety strategic height, China is domestic, and especially the research of the anti-bio-terrorism of bacterium should be strengthened soil is drawn in the entry and exit port.The top priority of anti-bio-terrorism is to make diagnosis rapidly, and spreading of epidemic situation controlled and prevented and treated to clear and definite cause of disease so that in time correct curing patient reduces Secondary cases and propagates, quickly and effectively.That the immune chromatography method of colloid gold label has is quick, easy, do not need to rely on important equipment, can realize characteristics such as on-the-spot detection, becomes the focus of studying in the present infectious disease quick diagnosis.
Summary of the invention
The object of the present invention is to provide a kind of Francisella tularensis to detect test paper and preparation method thereof, utilize above-mentioned detection test paper fast to detect the method for Francisella tularensis and utilize the detection system of above-mentioned detection test paper detection by quantitative Francisella tularensis.
For achieving the above object; a kind of Francisella tularensis of the present invention detects test paper; comprise the reaction stilt; closely be connected in the nitrocellulose membrane of reaction stilt upper surface middle part; the upper surface of nitrocellulose membrane initiating terminal is overlapped with adsorptive pads and is connected; the upper surface of nitrocellulose membrane end is overlapped with the golden labeling antibody diaphragm of the FopA-S polyclonal antibody that contains colloid gold label and is connected; the overlapping sample pad that is connected with of upper surface portion of gold labeling antibody diaphragm; wherein, be provided with the Quality Control band of Quality Control antibody sandwich on the nitrocellulose membrane near its initiating terminal place; be provided with the anti-reorganization of rabbit FopA-L polyclonal antibody test strip near its end.
Further, described reaction holder is the PCV plate, and adsorptive pads is a filter paper for oil.
Further, described golden labeling antibody diaphragm is glass fibre membrane, polyester film or filter paper fibre.
Further, described sample pad is glass fibre membrane, polyester film or filter paper fibre, and it adopts the phosphate buffer that contains polysorbas20 to carry out pre-service.
Further, described Quality Control antibody is chosen as anti-rabbit igg or SPA according to the immunogenic of golden labeling antibody, and it is preferably goat anti-rabbit igg.
Further, the external packets of described test paper integral body is wrapped with shell, is provided with sample well corresponding to described sample pad place on this shell, is provided with view window corresponding to described Quality Control band, test strip place on the shell.
A kind of above-mentioned Francisella tularensis detects the preparation method of test paper, is specially:
1) preparation collaurum: compound concentration is 0.01% HAuCl4 aqueous solution, and adjusts its pH value for 6-8, and diluting antibody to concentration with PBS is 0.2mg/ml;
2) the golden labeling antibody diaphragm of preparation: get the collaurum for preparing in the step 1) and be sub-packed in the 1ml/ pipe in the centrifuge tube of several 1.5ml, the antibody amount is 2-10ug in every arm, places behind the jog mixing; The BSA100ul of adding 10% in every pipe, mixing is placed; The top colloidal gold probe that tentatively makes is carried out first step centrifugal treating, abandon supernatant; Add the resuspended liquid suspension of 1ml post precipitation in every centrifuge tube and carry out the second step centrifugal treating; Abandon supernatant, the pipe end, obtain bolarious loose shape precipitation, carries out the 3rd step centrifugal treating after the resuspended liquid of adding 500ul is resuspended, gets supernatant, evenly drip on the film body of golden labeling antibody diaphragm, and dried;
3) spraying nitrocellulose membrane: the Quality Control band of spraying goat anti-rabbit igg bag quilt, the anti-reorganization of rabbit FopA-L polyclonal antibody test strip on film body, the FopA-S polyclonal antibody with colloid gold label is uniformly coated on the film body again;
4) will react stilt, nitrocellulose membrane, adsorptive pads, golden labeling antibody diaphragm and sample pad assembles according to test paper version and prepares the detection test paper.
Further, concrete steps in the described step 1) are: accurately add the HAuCl4 of 1ml 1% under the magnetic agitation, add the trisodium citrate aqueous solution of 1.5-3ml 1% simultaneously, continue heating behind the colour stable, add pure water to 100ml after being cooled to room temperature, 4 ℃ keep in Dark Place; Use K
2CO
3After transferring the pH value optimum to be 7.0-7.5, dilute antibody to 0.2mg/ml with PBS, and keep collaurum stable.
Further, described step 2) described in the first step centrifugal treating 12000rpm, 4 ℃ centrifugal 30 minutes down, the described second step centrifugal treating is 12000rpm, 4 ℃ centrifugal 30 minutes down, the 3rd step centrifugal treating was 1000rpm, 4 ℃ centrifugal 10 minutes down.
Further, utilize in the described step 3) every flowing the spray film speed of metal spraying pen machine with 10-100mm/s and spray described Quality Control band and test strip, the anti-reorganization of described rabbit FopA-L polyclonal antibody concentration is 1-5mg/ml, adopts the PB diluted; Described goat anti-rabbit igg concentration is 0.1-5mg/ml.
Further, the amount of described FopA-S polyclonal antibody mark 1ml collaurum is 3～5ug, adopts the PBS dilution, and its pH is 6-8.
A kind ofly utilize above-mentioned detection test paper fast to detect the method for Francisella tularensis, be specially: with sample to be measured and sample diluting liquid mixing, again sample mix liquid is added test paper sample well place, liquid relies on syphonic effect to expand to the test paper other end, the Francisella tularensis that contains in the sample to be measured will form corresponding compound with the FopA-S polyclonal antibody of colloid gold label, the anti-reorganization of this compound and rabbit FopA-L polyclonal antibody combines, and forms red lines at the test strip place and points out and detect corresponding mushroom; Compound and goat anti-rabbit igg form the red precipitate line, with the validity of check test paper.
A kind of detection system of utilizing above-mentioned detection test paper detection by quantitative Francisella tularensis, comprise and detect test paper and colloidal gold test strip interpretoscope, wherein detect the golden labeling antibody diaphragm that test paper comprises nitrocellulose membrane and contains the FopA-S polyclonal antibody of colloid gold label, close its initiating terminal place is provided with the Quality Control band of Quality Control antibody sandwich on the nitrocellulose membrane, close its end is provided with the anti-reorganization of rabbit FopA-L polyclonal antibody test strip; The colloidal gold test strip interpretoscope can this test strips of interpretation to the testing result of Francisella tularensis, detect realizing.
Francisella tularensis of the present invention detects test paper, based on the how anti-mark collaurum of the FopA of purifying, makes up double antibody sandwich method and detects antigen, has simple to operately, and detection time is short, need not the professional, is fit to advantages such as scene detection fast.
Description of drawings
Fig. 1 is the range estimation sensitivity maps of Francisella tularensis colloidal gold immuno-chromatography test paper strip;
Fig. 2 is the specific detection Test Drawing;
Fig. 3 is that Francisella tularensis colloidal gold immuno-chromatography test paper strip analog sample detects figure;
Fig. 4 detects the fitting a straight line figure of test paper for the present invention;
Fig. 5 A detects the front schematic view of test paper for the present invention;
Fig. 5 B detects the side schematic view of test paper for the present invention;
Fig. 6 A detects the positive test symbol synoptic diagram of test paper for the present invention;
Fig. 6 B detects the negative result synoptic diagram of test paper for the present invention;
Fig. 6 C detects the invalid detection result schematic diagram of test paper for the present invention.
Embodiment
As Fig. 1 to Fig. 4, shown in Fig. 5 A, Fig. 5 B, Fig. 6 A, Fig. 6 B, Fig. 6 C, Fig. 1 is the range estimation sensitivity maps of Francisella tularensis colloidal gold immuno-chromatography test paper strip, and 1-5 represents that respectively the sample concentration that detects is followed successively by blank, 75ug/mL, 7.5ug/mL, 750ng/mL, 375ng/mL.As shown in Figure 1,75ug/mL, 7.5ug/mL, the positive reaction of 750ng/mL; 375ng/mL does not have positive reaction; Be that the observable least concentration of naked eyes is 750ng/mL.
Fig. 2 is the specific detection Test Drawing, and 1 expression detects the horse beautiful eyes GP2 albumen of 750ng/mL, 2 expression smallpox M1R albumen, 3 expression bird flu NP albumen, 4 expression dengue virus Deng-E-D3 albumen, 5 expression Rickettsia prowazeki 120N albumen.
Fig. 3 is that Francisella tularensis colloidal gold immuno-chromatography test paper strip analog sample detects; Test-strips 1: negative control, 2～5: be respectively and contain FopA protein 75 0ng/mL pear juice, flour, biscuit, jelly.
Among Fig. 5 A, Fig. 5 B, 1: adsorptive pads; 2: nitrocellulose membrane, T: bag is by the anti-reorganization of rabbit FopA-L polyclonal antibody test strip; C: bag is by the Quality Control band of goat anti-rabbit igg; 3: the glass fibre membrane that contains colloid gold label FopA-S polyclonal antibody; 4: sample pad; 5: the reaction holder.
Fig. 6 A, Fig. 6 B, Fig. 6 C are testing result synoptic diagram of the present invention; Two lines of T, C occur and represent the positive; Line of C only occurs and represent feminine gender; It is invalid that representing does not all appear in two lines of T, C.
The present invention adopts colloid gold label, and essence is that biomacromolecules such as antibody protein are adsorbed to the bag on colloid gold particle surface by process.Collaurum depends primarily on the minimum steady concentration of pH value and protein to absorption of proteins, and ion concentration and protein molecular weight etc. also influences the absorption between the two.The present invention finds that pH value and ion concentration condition are keys in the preparation process of colloidal gold colloidal gold detection test paper strip, collaurum is a kind of very unsettled colloid, it has been generally acknowledged that, adsorption effect is best when the pH value equals isoelectric points of proteins or is higher than isoelectric point, and easily forms firm compound and come the stable colloid gold.This moment, protein molecule mainly was electric neutrality, less with the mutual electrostatic attraction of colloid gold particle, and the surface tension of protein molecule is maximum, be in a kind of faint hydrated state, therefore the surface that more easily is adsorbed in gold grain forms a protein layer, stoped being in contact with one another of colloid gold particle, made collaurum be in a metastable state.When the pH value was lower than the isoelectric point of protein, protein was mainly positively charged, and collaurum is electronegative, and the two very easily loses the ability of conjugated protein in conjunction with forming big polymkeric substance.When the pH value was higher than isoelectric points of proteins, the protein belt negative charge repelled mutually with the negative charge of gold grain and can not interosculate.Bovine serum albumin(BSA) therefore commonly used, ovalbumin, polyglycol or gelatin prevent the polymerization and the precipitation of protein and collaurum as stabilizing agent.Through experimental results show that originally native system adopts bovine serum albumin(BSA) as stabilizing agent, its specificity, susceptibility are all fine.This experiment gropes to draw through condition repeatedly: K
+Influence to whole collaurum detection system is bigger, so selecting antibody diluent, sample preparation liquid etc. all will avoid introducing K
+, also can adopt dialysis to remove K
+
The invention provides a kind of Francisella tularensis detection test paper, semi-quantitative detection method that can utilize the gold-marking immunity analyser to carry out simultaneously of detecting quickly and easily.The principle of work of this detection 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 Francisella tularensis 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.
A kind of method that detects Francisella tularensis quickly and easily comprising with sample to be measured and sample diluting liquid mixing, adds sample mix liquid test paper sample well place again, and the liquid in the sample relies on syphonic effect up, 10-15 minute sentence read result; Described detection test paper comprises:
(1) reaction holder;
(2) adsorptive pads;
(3) nitrocellulose membrane, this film are coated with rabbit anti-reorganization FopA-L polyclonal antibody and Quality Control detection of antibodies band and Quality Control band;
(4) golden labeling antibody diaphragm wherein contains the FopA-S polyclonal antibody of colloid gold label;
(5) sample pad adopts the phosphate buffer that contains polysorbas20 that the plain film of glass fibre is carried out pre-service;
Wherein reacting holder is the PCV plate; adsorptive pads is a filter paper for oil; nitrocellulose membrane wraps successively by goat anti-rabbit igg, the anti-reorganization of rabbit FopA-L polyclonal antibody 1mg/ml; gold labeling antibody diaphragm is glass fibre membrane or polyester film or the filter paper fibre that contains the FopA-S polyclonal antibody of colloid gold label, and sample pad is selected glass fibre membrane or polyester film or filter paper fibre for use.
Francisella tularensis of the present invention detects test paper, and wherein adsorptive pads, nitrocellulose membrane, golden labeling antibody diaphragm, sample pad and reaction holder constitute according to mode shown in the accompanying drawing 5A; 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 anti-reorganization of a rabbit FopA-L polyclonal antibody test strip, and the C place is the Quality Control band of goat anti-rabbit igg bag quilt; Glass fibre membrane 3 is positioned at a side on nitrocellulose membrane top and overlaps with it, and this film contains the FopA-S polyclonal antibody of colloid gold label; Adsorptive pads 1 be positioned at nitrocellulose membrane 2 tops the opposite side for glass fibre membrane 3 and with 2 overlap.Sample pad 4 be positioned at a side opposite on 2 with 1 and with 3 overlap.
Francisella tularensis of the present invention detects test paper, is end with adsorptive pads one side, and sample pad one side is terminal, and the anti-reorganization of rabbit FopA-L polyclonal antibody test strip is positioned near initiating terminal, and the Quality Control band of goat-anti rabbit I gG bag quilt approaches initiating terminal.
Francisella tularensis of the present invention detects the preparation method of test paper, and this preparation method may further comprise the steps: the preparation of colloidal gold probe, the preparation of gold mark pad, the spraying bag quilt of nitrocellulose membrane, the pre-service of sample pad.
1, the preparation of colloidal gold probe
(1) HAuCl 4 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) use K
2CO
3Transferring pH value is about 6-8, is preferably about 7.0-8.0, more preferably from about behind the 7.0-7.5.
(3) collaurum is transferred to pH 7.0-7.5, dilute antibody to 0.2mg/ml with PBS.
(4) keep collaurum stable.
The present invention also provides 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 PB 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 2-8ug, and more preferably 3-6ug most preferably is 3-5ug.
2, the preparation of gold mark pad
The collaurum of getting above-mentioned pH is sub-packed in the centrifuge tube of 1.5ml with the 1ml/ pipe.In every arm 2-10ug, preferred antibody amount 2-8ug, more preferably 3-6ug most preferably is 3-5ug, places behind the jog mixing.The BSA100ul of adding 10% in every pipe, 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, adds the resuspended back of the resuspended liquid of 500ul in 4 ℃, 1000rpm, centrifugal 10 minutes; Get supernatant, evenly drip on the wide glass fibre of 1cm 37 ℃ of dryings.
3, the spraying of nitrocellulose membrane
(1) utilizes every stream metal spraying pen machine with the detected rabbit of the spray film speed bag anti-reorganization FopA-L polyclonal antibody of 50mm/s and the nitrocellulose membrane of two bands of Quality Control goat anti-rabbit igg;
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; The anti-reorganization of described rabbit FopA-L polyclonal antibody, its addition is 1-5mg/ml, the dilution of this polyclonal antibody can be PB; 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 glass fibre membrane that contains the FopA-S polyclonal antibody of colloid gold label of preparation is uniformly coated on the FopA-S polyclonal antibody of colloid gold label on the glass fibre membrane.Antibody dilutes with PBS, and pH is 6-8, preferred 7-8, and optimum PH is 7.0-7.5.
The invention also discloses the application of described test paper in detecting Francisella tularensis, referring to accompanying drawing 3.
Detect in the method for Francisella tularensis in the present invention, earlier sample to be measured is put into the bottle that dilution is housed, with sample pad 4 places (promptly terminal) of pipettor with sample mix liquid adding test paper, the liquid in the sample relies on syphonic effect up, generally needs 10-15 minute sentence read result again:
As containing Francisella tularensis in the sample, then it will form corresponding compound with the FopA-S polyclonal antibody of colloid gold label on the test paper, developing liquid is up and combine with the anti-reorganization of rabbit FopA-L 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.As shown in Figure 6.
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: adopt rabbit anti-reorganization FopA-L polyclonal antibody and goat anti-rabbit igg difference solid phase on nitrocellulose membrane, the FopA-S polyclonal antibody of association colloid gold mark is used rete and is analysed the principle of double antibody sandwich method and detect Francisella tularensis in the sample.
The present invention also provides the detection system of a kind of detection by quantitative Francisella tularensis with colloidal gold test strip and collaurum biology sensor organic combination.The advantage of utilization colloidal gold test strip interpretoscope is that interpretoscope can can't correctly be discerned suspicious item at human eye and detect whether there is under the positive situation correct this test strips testing result of interpretation.Reduce the error rate of bringing because of artificial subjective interpretation, increased objectivity, accuracy; And can realize detection by quantitative.In addition, the collaurum interpretoscope is easy to carry, use is simple, judge accurate, objective, the easy reservation of result, for the check work for inspection has brought convenience.
The above-mentioned Francisella tularensis that the present invention relates to detects test paper, and it also comprises golden labeling antibody diaphragm.
The above-mentioned Francisella tularensis that the present invention relates to detects test paper, wherein reacts holder and selects the PVC plate for use.
The above-mentioned Francisella tularensis that the present invention relates to detects test paper, and wherein adsorptive pads is selected filter paper for use.
The above-mentioned Francisella tularensis that the present invention relates to detects test paper, and wherein golden labeling antibody diaphragm is selected polyester film, glass fibre or filter paper fibre for use.
Embodiment 1: the preparation of colloidal gold immuno-chromatography test paper strip
Material and method
1, antigen and antibody
The anti-reorganization of rabbit FopA-L polyclonal antibody, FopA-S polyclonal antibody are provided by the Health Office of institute of China inspection section.Goat anti-rabbit igg (available from ancient cooking vessel state biotech company)
2, chromatography test-strips consumptive material
Pad (glass fibre), nitrocellulose filter (NC film, SHF 1350225), sample pad and adsorptive pads, filter paper are available from Minipore company.
3, experimental apparatus
The gold-marking immunity analyser can be available from the analyser of Shanghai Optics and Precision Mechanics institute, Chinese Academy of Sciences, China Inst. of Quarantine Inspection Sciences's joint research and development.
4, the preparation of colloidal gold immuno-chromatography test paper strip
4.1 collaurum pad
The FopA-S polyclonal antibody of pH value 7.0～7.5, concentration 0.2mg/ml is marked on the colloid gold particle that the sodium citrate legal system is equipped with the colloid gold particle of 25nm, 37 ℃ of dryings.
4.2 nitrocellulose filter
Detect band: the anti-reorganization of rabbit FopA-L polyclonal antibody 1mg/ml; Quality control band: goat anti-rabbit igg: 1mg/ml, 37 ℃ of dryings.
4.3 assembling
Sample pad, pad, adsorptive pads are attached to the end liner card that has the mixture that slits successively, are cut into the bar of 0.4cm, drying, the dress shell, room temperature storage is standby.
Embodiment 2: the judgement of the qualitative and detection by quantitative of sample
1, the processing of sample
(1) liquid--pear juice at first dilutes mixing respectively in the proper ratio with damping fluid; (2) solid/semisolid--take by weighing white powder: after (flour, biscuit) and semisolid jelly add the damping fluid dissolving, get supernatant solution after the dissolving and will recombinate and natively draw bacterium FopA albumen to add respectively wherein to make the final concentration of FopA albumen reach 750ng/mL.
Table 1 soil draws bacterium sample detection and sensitivity
2 qualitative detection
Sample and sample preparation liquid (as negative sample) 100ul after handling are added to the chromatography strip sample pad end for preparing, leave standstill 15min, observations.Detection band and quality control band redness all occurs and are judged to the positive, and it is negative that only redness appears in quality control band, and detection is with and quality control band does not all develop the color, and then are that test strips lost efficacy.
3, detection by quantitative
3.1 determining of decision content (CUT-OFF)
Press 1.6.1 negative sample is detected 20 times, read signal T/C ratio with the scanning of gold-marking immunity analyser.The mean value (AVERAGE) of 20 sample T/C ratios is the CUT-OFF value with 3 times of standard deviations (STDEVA) sum.
3.2 detection by quantitative is judged
Colloidal-gold strip after the colour developing is put into the scanning of gold-marking immunity analyser, read signal T/C ratio, positive greater than decision content.
Embodiment 3: sensitivity test
1, the sensitivity of detection by quantitative
Detecting the variable concentrations Francisella tularensis, is 0.003 through computational discrimination value (CUT-0FF), and it is positive that concentration is that the value that average T/C ratio is respectively of the Francisella tularensis of 750ng/ml all is higher than 0.003 result; When Francisella tularensis concentration during greater than 750ng/ml, T/C average of relatives value is greater than 0.003, so detection by quantitative sensitivity is 750ng/ml.
Table 2 soil draws each concentration testing result of bacterium colloidal gold immuno-chromatography test paper strip
2, visual inspection sensitivity is estimated
Prepare colloidal gold immuno-chromatography test paper strip by the optimum reaction condition of determining, detect the Francisella tularensis of variable concentrations.Francisella tularensis is diluted to concentration with sample preparation liquid to be followed successively by concentration and to be followed successively by 75ug/mL, 7.5ug/mL, 750ng/mL, 375ng/mL.As shown in Figure 1,75ug/mL, 7.5ug/mL, 750ng/mL detect simultaneously.
The result as shown in Figure 1, directly the sensitivity 750ng/mL colour developing of range estimation is more clear.
3, the sample practicality detects
Having chosen pear juice, flour, biscuit, jelly respectively is that analog sample detects, the processing mode of the various samples of its disposal route difference (table 1) of different materials, and the detection sensitivity of handling the back sample is 750ng/mL.
Embodiment 4: the specificity test
Prepare colloidal gold immuno-chromatography test paper strip by the optimum reaction condition of determining, detect horse beautiful eyes GP2 albumen, smallpox M1R albumen, bird flu NP albumen, dengue virus Deng-E-D3 albumen and the Rickettsia prowazeki 120N albumen of 750ng/mL with sample diluting liquid equally.Detect with the colloidal gold immuno-chromatography test paper strip for preparing, and draw the contrast of bacterium FopA protein sample, detect blank simultaneously with the soil of same concentration.The result as shown in Figure 2, this test strip for these albumen and toxin all nothing but specific phenomenon occur, respond well to the Francisella tularensis specific detection.
Embodiment 5: the typical curve match
Drawing the concentration of bacterium FopA albumen with soil is horizontal ordinate (X), and the gold mark analyser value of reading T/C ratio (Y) is set up collaurum examination criteria curve for the ordinate mapping.(referring to Fig. 4).
Detecting under each concentration of Francisella tularensis, is T/C value of reading of 0.003 through computational discrimination value (CUT-OFF).As seen concentration is that the soil of 375ng/ml draws the average T/C ratio of bacterium FopA albumen to be 0.002, be lower than 0.003, negative, when soil draws bacterium FopA protein concentration to be 750～24000ng/ml, be good linear relationship between the concentration of albumen and its gold mark analyser value of the reading T/C ratio, Fig. 4 is seen in matched curve.Linear equation is: y=0.003x+0.037; R
2=0.9861.
Embodiment 6: recovery test
In linear detection range, detect the Francisella tularensis of concentration known, calculate detectable concentration according to the gold mark analyser value of reading T/C ratio and typical curve, the ratio percent of detectable concentration and theoretical concentration is the recovery.By table 2 as seen, etection theory concentration is 750,1500,3000, and when the soil of 6000ng/ml drew bacterium FopA albumen, its recovery was between 56.7%～89.2%.
The recovery that table 2 soil draws the bacterium colloidal gold immuno-chromatography test paper strip to detect
Embodiment 7: stability test
Referring to Fig. 3, the Francisella tularensis colloidal gold colloidal gold detection test paper strip that is taken at 37 ℃ of placements detects, having good stability of the visible Francisella tularensis test-strips of the testing result of 7d still can specificly detect Francisella tularensis behind 37 ℃ of placement 7d, and susceptibility descends also.Compare with freshly prepd test strip, sensitivity does not obviously descend, and specificity is good.
Claims (10)
1. a Francisella tularensis detects test paper; it is characterized in that; this detection test paper comprises the reaction stilt; closely be connected in the nitrocellulose membrane of reaction stilt upper surface middle part; the upper surface of nitrocellulose membrane initiating terminal is overlapped with adsorptive pads and is connected; the upper surface of nitrocellulose membrane end is overlapped with the golden labeling antibody diaphragm of the FopA-S polyclonal antibody that contains colloid gold label and is connected; the overlapping sample pad that is connected with of upper surface portion of gold labeling antibody diaphragm; wherein, be provided with the Quality Control band of Quality Control antibody sandwich on the nitrocellulose membrane near its initiating terminal place; be provided with the anti-reorganization of rabbit FopA-L polyclonal antibody test strip near its end.
2. Francisella tularensis as claimed in claim 1 detects test paper, it is characterized in that described reaction holder is the PCV plate, and adsorptive pads is a filter paper for oil; Described golden labeling antibody diaphragm is glass fibre membrane, polyester film or filter paper fibre.
3. Francisella tularensis as claimed in claim 1 detects test paper, it is characterized in that described sample pad is glass fibre membrane, polyester film or filter paper fibre, and it adopts the phosphate buffer that contains polysorbas20 to carry out pre-service; Described Quality Control antibody is chosen as anti-rabbit igg or SPA according to the immunogenic of golden labeling antibody, and it is preferably goat anti-rabbit igg.
4. Francisella tularensis as claimed in claim 1 detects test paper, it is characterized in that, the external packets of described test paper integral body is wrapped with shell, is provided with sample well corresponding to described sample pad place on this shell, is provided with view window corresponding to described Quality Control band, test strip place on the shell.
5. an above-mentioned Francisella tularensis detects the preparation method of test paper, it is characterized in that this preparation method is specially:
1) preparation collaurum: compound concentration is 0.01% HAuCl4 aqueous solution, and adjusts its pH value for 6-8, and diluting antibody to concentration with PBS is 0.2mg/ml;
2) the golden labeling antibody diaphragm of preparation: get the collaurum for preparing in the step 1) and be sub-packed in several centrifuge tubes, the antibody amount is 2-10ug in every centrifuge tube, places behind the jog mixing; The BSA100ul of adding 10% in every pipe, mixing is placed; The top colloidal gold probe that tentatively makes is carried out first step centrifugal treating, abandon supernatant; Add the resuspended liquid suspension of 1ml post precipitation in every centrifuge tube and carry out the second step centrifugal treating; Abandon supernatant, the pipe end, obtain bolarious loose shape precipitation, carries out the 3rd step centrifugal treating after the resuspended liquid of adding 500ul is resuspended, gets supernatant, evenly drip on the film body of golden labeling antibody diaphragm, and dried;
3) spraying nitrocellulose membrane: the Quality Control band of spraying goat anti-rabbit igg bag quilt, the anti-reorganization of rabbit FopA-L polyclonal antibody test strip on film body, the FopA-S polyclonal antibody with colloid gold label is uniformly coated on the film body again;
4) will react stilt, nitrocellulose membrane, adsorptive pads, golden labeling antibody diaphragm and sample pad assembles according to test paper version and prepares the detection test paper.
6. preparation method as claimed in claim 7, it is characterized in that, concrete steps in the described step 1) are: the HAuCl4 that accurately adds 1ml 1% under the magnetic agitation, the trisodium citrate aqueous solution that adds 1.5-3ml 1% simultaneously, continue heating behind the colour stable, add pure water to 100ml after being cooled to room temperature, 4 ℃ keep in Dark Place; Use K
2CO
3After the adjust pH optimum is 7.0-7.5, dilute antibody to 0.2mg/ml, and keep collaurum stable with PBS.
7. preparation method as claimed in claim 7, it is characterized in that, described step 2) described in the first step centrifugal treating 12000rpm, 4 ℃ centrifugal 30 minutes down, the described second step centrifugal treating is 12000rpm, 4 ℃ centrifugal 30 minutes down, and the 3rd step centrifugal treating was 1000rpm, 4 ℃ centrifugal 10 minutes down.
8. preparation method as claimed in claim 7, it is characterized in that, utilize in the described step 3) every flowing the spray film speed of metal spraying pen machine with 10-100mm/s and spray described Quality Control band and test strip, the anti-reorganization of described rabbit FopA-L polyclonal antibody concentration is 1-5mg/ml, adopts the PB diluted; Described goat anti-rabbit igg concentration is 0.1-5mg/ml.
9. one kind is utilized above-mentioned detection test paper fast to detect the method for Francisella tularensis, it is characterized in that, this method is specially: with sample to be measured and sample diluting liquid mixing, again sample mix liquid is added test paper sample well place, liquid relies on syphonic effect to expand to the test paper other end, the Francisella tularensis that contains in the sample to be measured will form corresponding compound with the FopA-S polyclonal antibody of colloid gold label, the anti-reorganization of this compound and rabbit FopA-L polyclonal antibody combines, and forms red lines at the test strip place and points out and detect corresponding mushroom; Compound and goat anti-rabbit igg form the red precipitate line, with the validity of check test paper.
10. detection system of utilizing above-mentioned detection test paper detection by quantitative Francisella tularensis, it is characterized in that, this detection system comprises detection test paper and colloidal gold test strip interpretoscope, wherein detect the golden labeling antibody diaphragm that test paper comprises nitrocellulose membrane and contains the FopA-S polyclonal antibody of colloid gold label, close its initiating terminal place is provided with the Quality Control band of Quality Control antibody sandwich on the nitrocellulose membrane, close its end is provided with the anti-reorganization of rabbit FopA-L polyclonal antibody test strip; The colloidal gold test strip interpretoscope can this test strips of interpretation to the testing result of Francisella tularensis, detect realizing.
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| CN2010102496003A CN101949927A (en) | 2010-08-10 | 2010-08-10 | Francisella tularensis detection test paper, preparation method thereof, detection method using detection test paper and quantitative detection system |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103941003A (en) * | 2013-03-15 | 2014-07-23 | 河南省农业科学院 | Swine listeriosis, swine necrobacillosis and swine francisella tularensis tri-combination detection test paper strip |
| CN111024661A (en) * | 2019-12-12 | 2020-04-17 | 潍坊科技学院 | Fluorescent detection card and method for environmental pollutants |
-
2010
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103941003A (en) * | 2013-03-15 | 2014-07-23 | 河南省农业科学院 | Swine listeriosis, swine necrobacillosis and swine francisella tularensis tri-combination detection test paper strip |
| CN103941003B (en) * | 2013-03-15 | 2016-07-06 | 河南省农业科学院 | Pig Listeria monocytogenes, pig necrobacillus and pig Bacillus tularensis three test strip |
| CN111024661A (en) * | 2019-12-12 | 2020-04-17 | 潍坊科技学院 | Fluorescent detection card and method for environmental pollutants |
| CN111024661B (en) * | 2019-12-12 | 2022-06-24 | 潍坊科技学院 | Fluorescence detection card and detection method for environmental pollutants |
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