CN101900729A - Test paper for detecting streptococcus suis, and preparation method and application thereof - Google Patents
Test paper for detecting streptococcus suis, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a test paper for detecting streptococcus suis serotype 2(SS2), a method for preparing the test paper and application of the test paper. The test paper comprises a sample pad, a colloidal gold pad, an NC membrane and an absorbent pad, and is prepared by a gold immunochromatographic assay (GICA). The method for preparing the test paper comprises the steps of SS2 polyclonal antibody preparation, colloidal gold preparation, colloidal gold probe preparation, membrane praying, test paper assembly, and the like. In case of using the test paper to detect the streptococcus suis serotype 2(SS2), the detection results can be observed in 10 minutes, and at least 106CFU/ml streptococcus suis serotype 2(SS2) can be detected. The colloidal gold test paper has strong specificity, only reacts with the streptococcus suis serotype 2 and the streptococcus suis serotype 1/2, and has no cross reaction with other serotype streptococcus suises and other streptococcuses. The test paper has the characteristics of simpleness, convenience, speediness, and the like, can serve as a diagnostic method for preliminary screening the streptococcus suis serotype 2, and is suitable for clinical and field use.
Description
Technical field
The present invention relates to a kind of test paper that detects Streptococcus suis, be specifically related to a kind of immune chromatography test paper that detects streptococcus suis 2-type, also relate to the Preparation method and use of this test paper.
Background technology
People's Streptococcus suis belongs to two class animal epidemics of national regulation, is a kind of infectious diseases common to human beings and animals.People's infection with streptococcus suis is human infection's streptococcus suis 2-type (Streptococcus suis serotype 2, SS2) a kind of infecting both domestic animals and human disease due to the grade.Streptococcus suis belongs to the R group of Lan Shi classification, and 35 serotypes are arranged, and is popular the widest with streptococcus suis 2-type, pathogenic the strongest.Streptococcus suis not only can cause pig meningitis, endocarditis, septicemia, arthritis, chronic hyperplastic perihepatitis, pneumonia even die by visitation of God, can also invade human body through damaged skin or mucous membrane, cause people's infection morbidity, to farm and meatworker's formation threat.The streptococcus clinical manifestation of people infected pigs is septicemia, TSS (TSS) and meningitis.The main infection sources of people's infection with streptococcus suis is the pig that dies of illness, and main route of transmission is cut, cleaned the pork etc. of dying of illness for slaughtering the pig that dies of illness, and hand skin has the outstanding easy infection of the personnel of damage.Feed is not boiled, the well-done pork of dying of illness also can cause infection.China is faced with the threat that the streptococcus suis infection outbreak of epidemic increases the weight of in recent years, 1998 with Streptococcus suis in 2005 in twice eruption and prevalence of China, cause 229 people to infect altogether, wherein dead 53 examples.Cause people, the morbidity of pig coinfection China Streptococcus suis, not only cause serious economic loss, brought threat also for public health and food security to pig industry.This disease has caused the great attention in worldwide public health field, and more and more researchers begins Streptococcus suis is carried out more deep research.
Traditional isolation identification to the Streptococcus suis cause of disease mainly relies on traditional separation and Culture and biochemical identification, carries out the agglutination test somatotype with the Streptococcus suis hyper-immune serum then.Though biochemical identification can identify streptococcus suis 2-type than reliable detection, it must be based on isolated pure bacterium, but these two kinds of method complex operations are unfavorable for carrying out on-the-spot quick diagnosis.Be to improve China's security monitoring ability relevant, press for and set up that China has efficiently, the detection technique of responsive, quick, high specific Emergent Public Events.
Colloid gold immune quick diagnosis technology (fast diagnose test paper bar) is a novel vitro diagnostic techniques that has grown up on monoclonal antibody technique, colloidal gold immunochromatographimethod technology and new material technology basis since the nineties in 20th century.This technology mainly is that specific antigen or antibody are fixed on the nitrocellulose filter (NC) with ribbon, colloid gold label reagent is adsorbed on the pad, be added on the sample pad of test strips one end when testing sample after, move forward by capillary action, react to each other behind the colloid gold label reagent on the dissolving pad, when moving to fixing antigen or antibody regional again, the specificity combination takes place again with it and is trapped in the compound of determinand and gold marked reagent, accumulate in to detect and be with, by the colloid gold label thing that can the estimate result that developed the color intuitively.The label of wandering about as a refugee is then crossed and is detected band, reaches the purpose of separating automatically with binding label.Colloidal gold immunochromatographimethod quick diagnosis technology can provide the result at short notice, and this detection method is quick and easy, accurate, reliable, cheap and operation is easy.
Up to the present, do not see the report that adopts colloidal gold immunochromatographimethod technology for detection streptococcus suis 2-type as yet.
Summary of the invention
The invention discloses a kind of immune chromatography test paper that detects streptococcus suis 2-type, also disclose the preparation method of this test paper and the purposes of this test paper.
Immune chromatography test paper of the present invention is a colloidal gold immune chromatography test, comprises sample pad, glue gold pad, NC film and absorption pad four parts.On glue gold pad, colloidal gold probe is arranged, the band of detection is arranged on the NC film and accuse band, detect band and accuse that band is coated with streptococcus suis 2-type antibody and anti-streptococcus suis 2 type antiantibodys respectively.Above-mentioned streptococcus suis 2-type antibody is streptococcus suis 2-type.Streptococcus suis 2-type antibody can be the antibody of streptococcus suis 2-type immunity different animals acquisition, preferred rabbit anti-streptococcus suis 2 type antibody; Preparation streptococcus suis 2-type antibody can select the streptococcus suis 2-type antigen immune of hydroformylation to form, and as formaldehyde and glutaraldehyde, preferred 1% formaldehyde promptly can be used as immunogene in 37 ℃ of deactivation 7d, can guarantee the specificity that antibody is good.Anti-streptococcus suis 2 type antiantibodys can obtain by immune different animals, and preferred goat-anti rabbit immune serum can be bought commercialization antibody.Above-mentioned two kinds detect band and accuse with on the antibody of bag quilt can adopt variable concentrations, it is 2.0mg/ml that preferred concentration is respectively rabbit anti-streptococcus suis 2 type antibody concentration, goat-anti rabbit immune serum concentration is 0.5mg/ml.
The preparation method of the immune chromatography test paper of detection streptococcus suis 2-type of the present invention comprises preparation streptococcus suis 2-type antibody; The preparation of collaurum; The preparation of colloidal gold probe; Streptococcus suis 2-type antibody and anti-streptococcus suis 2 type antiantibodys are wrapped quilt on the NC film; Golden pad of glue and the stack of NC film with absorption pad, sample pad and after handling adhere on the PVC plate, are assembled into complete steps such as test paper.
Prepare the employing of streptococcus suis 2-type antibody and directly carry out with the method for streptococcus suis 2-type immune animal, the antibody of acquisition is polyclonal antibody.At first need to prepare streptococcus suis 2-type antigen, earlier streptococcus suis 2-type is cultivated, formalin-inactivated promptly can be used as antigen behind the collection bacterium liquid.Use antigen-immunized animal then, immunity is carried out according to a conventional method, passes through the ELISA method with each immunity back blood sampling before the immunity and measures the animal's antibody titre.Reaching general immune response level through 6 immunity back antibody titers, is 10
7, the antibody that is obtained can be used for the preparation of colloidal gold immuno-chromatography test paper strip.The streptococcus suis 2-type that obtains will pass through purifying earlier, can adopt albumin A-agarose affinity chromatography method to carry out.Antibody dialysis-70 ℃ of preservations in back that purifying is good are standby.
The preparation of collaurum adopts the sodium citrate reducing process to be prepared, and obtains the collaurum of the about 25nm of diameter.
The preparation of colloidal gold probe: the streptococcus suis 2-type antibody of preparation is dialysed with pure water, and at the dimer that uses antibody to want centrifugal removal earlier easily to form as last.With the pH value that solution of potassium carbonate is regulated colloidal gold solution, mix with the streptococcus suis 2-type antibody of preparation, the centrifugal PBS dissolution precipitation that goes usefulness behind the supernatant to contain 1%BSA, centrifugal after resuspended once more precipitation, the centrifuging and taking supernatant is the good colloidal gold probe of mark.Wherein the pH value of colloidal gold solution is 〉=8.5, preferred 8.5.The suitableeest antibody protective number of streptococcus suis 2-type antibody is 22 μ g/mL.
Then be that streptococcus suis 2-type antibody and the many antiantibodys of anti-streptococcus suis 2 types are wrapped quilt on the NC film.At first streptococcus suis 2-type antibody is dialysed with the PBS working fluid, will dilute good streptococcus suis 2-type antibody and goat anti-rabbit antibody then, quilt is wrapped in line on the NC film respectively, becomes T band and C band, sees Fig. 1.The antibody concentration of bag quilt is 〉=2.0mg/ml, is best with 2.0mg/ml, seals with BSA solution, and 1.5% is optium concentration.
The invention also discloses the purposes of the immune chromatography test paper that detects Streptococcus suis.This purposes is verified by following test.
At first verifying the susceptibility of test paper of the present invention, the Streptococcus suis of cultivating is diluted to the bacterium liquid of variable concentrations, add the sample dilution and detect, is blank with the sample liquid that does not contain streptococcus suis 2-type.The sensitivity that colloidal gold immune chromatography test of the present invention detects Streptococcus suis is 1.0 * 10
6CFU/ml.
Verify the specificity of test paper of the present invention then.Streptococcus suis, other streptococcus and gold-coloured staphylococci, Listeria monocytogenes, Salmonella paratyphi A, Escherichia coli, the C.perfringens cultivated are cultivated respectively, be diluted to concentration＞10
8The bacteria suspension of CFU/ml, the immune chromatography test paper that detects Streptococcus suis with the present invention detects.Detect domestic and external epidemic strain and non-epidemic strain and pathogenic strain and the non-pathogenic strain of streptococcus suis 2-type with this test strips, the result is all positive; Especially surprisingly,, have good serological specificity although this antibody adopts the Streptococcus suis crude antigen preparation of hydroformylation, except that Streptococcus suis 1/2 type, the Streptococcus suis no cross reaction of this test strips and other serotype.Illustrate that this test strips sensing range is wide, specificity is good.15 groups of this immune test paper and common pathogenic bacteria and streptococcus are equal no cross reactions also.Show that according to above result this test strips has good specificity.
Then be the stability of checking test paper of the present invention.Respectively 4 ℃ and room temperature preservation 3 months, 37 ℃ deposit 15 days after, testing result does not all change, and the test paper good stability is described.
Test paper of the present invention has also passed through the detection of simulated samples.By detecting, the sensitivity result of test paper of the present invention can reach 10
6CFU/ml, the testing result of dummy is negative.
The immune chromatography test paper of detection of the present invention Streptococcus suis is a polyclonal antibody of using streptococcus suis 2-type, develops according to the principle of double antibody sandwich method.Overcome traditional bacterial immune and learned that the full bacterium antigen preparation of employing antibody, the specificity of diagnostic reagent is relatively poor, normal to exist defectives such as cross reaction, practicality be relatively poor with near edge bacterium.Utilize detection paper streptococcus suis 2-type of the present invention, sample disposal is simple, gets final product observations in 10 minutes, minimumly detects 10
6CFU/ml.By the detection to analog sample, it is highly sensitive, specificity good.Especially surprisingly,, have good serological specificity although this antibody adopts the Streptococcus suis crude antigen preparation of hydroformylation, except that Streptococcus suis 1/2 type, the Streptococcus suis no cross reaction of this test strips and other serotype.That is to say that though how anti-the antibody that the present invention uses be, the colloidal gold immune chromatography test of preparation has higher specificity, this be those skilled in the art can not expect having beyond thought effect.Test paper of the present invention has characteristics such as susceptibility, specificity, simplicity and rapidity, be suitable in clinical and on-the-spot use, can be used as a kind of diagnostic method of people Streptococcus suis primary dcreening operation, the application of this test paper helps our understanding and the control to the Streptococcus suis epidemic situation.
Description of drawings
Fig. 1 is a testing result interpretation synoptic diagram
Fig. 2 is a streptococcus suis 2-type serum titer measurement result
Fig. 3 is the sensitivity Detection of test strips.Wherein 1 is blank 2,3, and 4,5,6 is streptococcus suis 2-type, and wherein 2 is 1.0 * 10
8CFU/ml, 3 is 1.0 * 10
7CFU/ml, 4 is 1.0 * 10
6CFU/ml, 5 is 1.0 * 10
5CFU/ml, 6 is 1.0 * 10
4CFU/ml.
Fig. 4 is the Detection of Stability of test strips.Wherein 1 is gold-coloured staphylococci 10
8CFU/ml, 2 is streptococcus suis 2-type 10
8CFU/ml, 3 is S.suis 149 10
6CFU/ml, 4 is blank
Embodiment
Embodiment one streptococcus suis 2-type Antibody Preparation
One, material:
Bacterial strain: streptococcus suis 2-type (Streptococcus suis S.suis 149 or 98012, also can adopt phase homologous serotype streptococcus suis 2-type bacterial strain), gold-coloured staphylococci, Salmonella paratyphi A, Escherichia coli, streptococcus suis 2-type, C.perfringens is provided by microorganism of military medical sciences academy, the Streptococcus suis of 15 kinds of serotypes is provided by the Harbin veterinary institute, and all the other bacterial strains are purchased in Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
Animal used as test: 2 of 2kg left and right sides New Zealand large ear rabbits, female.Provide by Military Medical Science Institute's Experimental Animal Center.
Nutrient culture media: LB nutrient culture media, THB nutrient culture media, blood plate be preparation according to a conventional method all
Major equipment and instrument: Biohazard Safety Equipment (U.S. NUAIR company), the full-automatic protein nucleic acid enzyme connection of Spectra Max Plus detector (U.S. Molecular Devices company), the miniature half-dried electrophoretic blotting instrument of ST-I (Dalian is competed to step biotechnology company and produced), AKTA FPLC protein purification analyser (Sweden AmershamPharmacia Biotech company), every stream metal spraying pen machine (USA I AGEN ARISTA company), ND-1000 ultramicron protein quantification instrument (U.S. NanoDrop company), enzyme linked immunological absorption instrument, 868 type pH meters (U.S. Thermo company).
Two, methods and results:
1. the preparation of streptococcus suis 2-type antigen
Pathogen is a comlete antigen, can obtain antibody by the direct immunization animal, so the S.suis 149 of numeration and deactivation is the antigen of preparation streptococcus suis 2-type polyclonal antibody.The Streptococcus suis S.suis149 that preserves is seeded in the THB fluid nutrient medium, leaves standstill in 37 ℃ and cultivate 7h, sterilized water is collected bacterium liquid, the blood plate counting.1% formaldehyde promptly can be used as immunogene in 37 ℃ of deactivation 7d.
2. streptococcus suis 2-type Polyclonal Antibody Preparation and evaluation
2.1 streptococcus suis 2-type Polyclonal Antibody Preparation
The streptococcus suis 2-type polyclonal antibody obtains by the new zealand white rabbit about immune 2kg.Ear vein is got blood ELISA and is measured immune animal and whether have natural antibody before immunity for the first time, abandons as if having then.Do not get blood if having then from the each immunity of immunity beginning for the second time back 1 all ear veins, carry out indirect ELISA and survey antibody titer.Immunization protocol sees Table 1.
Table 1 streptococcus suis 2-type immunization protocol
2.2 the streptococcus suis 2-type antibody titer is measured
Immunity back ear vein is got blood 500 μ L, places 1h for 37 ℃, the centrifugal 10min of 3000rpm then, and the centrifugal 3min of 10000rpm again, supernatant is immune serum.With tiring of antibody in the indirect ELISA mensuration serum.Method is as follows:
1) be 10 with wrapping the bacterium that is cushioned the liquid dilution
9CFU/mL, 100 μ L/ holes, 4 ℃ of bags spent the night or 37 ℃ 1～2 hour,
2) use the PBST hole flushing, 100 μ L/ holes are washed 3 times, each 3min,
3) with the PB sealing that contains 3%BSA, 200 μ L/ holes place in 37 ℃ of incubators and sealed 2 hours,
4) PBST washes 3 times, 100 μ L/ holes, and each 3min,
5) anti-with the PBST gradient series dilution one that contains 3%BSA, every hole adds 100 μ L, 37 ℃ of effect 1h
6) PBST washes 3 times, 100 μ L/ holes, each 3min
7) with the PBST dilution enzyme labelled antibody (1: 2000) that contains 3%BSA, every hole 100 μ L, 37 ℃, 30～45min
8) PBST washes 4 times, 100 μ L/ holes, 3min/ time
9) add A, B colour developing liquid, each 1 in every hole, the lucifuge colour developing adds stop buffer (2NH when blueness appears in negative control
2SO
4) stop, 450nm records OD, with the blank well zeroing, if treat that gaging hole OD450 value more than or equal to 2.1 times of negative control holes, promptly is judged to be the positive, thereby draws serum titer.
After 6 immunity of 7 weeks, antibody titer can reach 10
7, reached general immunoreactive degree, therefore, the streptococcus suis 2-type antibody that this test obtains can be used for the preparation of colloidal gold immuno-chromatography test paper strip.Concrete outcome is seen Fig. 2.
2.3 the purifying of polyclonal antibody
Adopt albumin A-agarose affinity chromatography method antibody purification, with MabTrapTMG affinity column antibody purification, sample is through level pad (PB, 5mmol/L, pH7.0) after the suitable dilution, be splined on the good MabTrapTMG affinity column of level pad balance, behind the thorough flush away foreign protein of A liquid, changing eluent B liquid washes antibody, the antibody that collection washes is collected test tube and has been added 10% 1mol/L, the Tris-HCI of pH 9.0 in advance, pH is returned to about 7.0, in order to avoid lose activity.The good antibody of purifying was with 4 ℃ of PBS working fluid dialysis two days, and the centrifugal 30min of 10000r/min collects supernatant again, and-70 ℃ of preservations are standby.
The preparation of embodiment two streptococcus suis 2-type colloidal gold test paper
One, material: adopt commercial chemical reagent, with embodiment one.
Two, methods and results:
1. the preparation of collaurum
Adopt the sodium citrate reducing process to prepare the collaurum of the about 25nm of diameter.HAuCl4 is made into 1% aqueous solution earlier, gets the 99ml pure water and be heated to boiling. stir the HAuCl4 that adds 1mL1% down, add the 1.5mL trisodium citrate aqueous solution more simultaneously.Continue about heated and boiled 15min, observe and add behind the liquid color and become blackly earlier, slowly become claret subsequently, continue to stop heating behind heating 3～5min after seeing claret, deionized water is mended to volume 100mL.What obtain is the collaurum that diameter is 25nm, cools off back 4 ℃ and keeps in Dark Place.
2. the preparation of streptococcus suis 2-type colloidal gold test strip
2.1 the processing before the standard gold of streptococcus suis 2-type antibody
Because streptococcus suis 2-type antibody should be used for standard gold, also will be used to spray film, so antibody processing before use also is different.Streptococcus suis 2-type antibody respectively with PBS working fluid and pure water dialysis, is determined to be suitable for the optimum antibody disposal route of standard gold and spray film with this.
The result: the condition of groping according to inventor's actual tests shows that different application targets has different requirements: the streptococcus suis 2-type antibody that is used to spray film will make it that certain ionic strength be arranged, and be beneficial to the bag quilt with the dialysis of PBS working fluid.The streptococcus suis 2-type antibody that is used for standard gold requires not contain salt ion, must dialyse with pure water; Antibody generally places-20 ℃ of preservations before use, is easy to form dimer and influences standard gold, when particularly antibody concentration is higher, so using antibody to want 4 ℃ of centrifugal 30min of 10000rpm earlier as last.
2.2 the determination test of the optimal pH of colloid gold label streptococcus suis 2-type antibody
With the solution of potassium carbonate of 0.1mol/L the pH value of colloidal gold solution is adjusted to 6.0,6.5,7.0,7.5,8.0,8.5,9.0 respectively, respectively getting the 1mL adding uses PBS (pH7.2) dilution of 0.01mol/L to be the rabbit anti-streptococcus suis 2 type antibody 100 μ L of 0.2mg/ml, mix, room temperature reaction 10 minutes, left standstill 2 hours, and observed solution colour and change.12000rpm is centrifugal 10 minutes then, removes supernatant, adds the PBS dissolution precipitation that contains 1%BSA, and dissolving the pH value that is uniform transparent violet red solution pipe fully with precipitation is the best.
Test findings is shown in table 2-4, solution colour is blueness in various degree in each pipe of pH≤7.5, pH 〉=7.5, solution colour no change in each pipe, precipitate and to dissolve fully in the mark pipe of centrifugal back pH≤8.0, precipitation dissolving fully in the mark pipe of pH 〉=8.5, solution is transparent claret, shown in table 2.4.Therefore, the optimal pH of colloid gold label is 8.5.
The mensuration of table 2.4 streptococcus suis 2-type antibody colloidal gold optimal pH
2.3 the determination test (test tube observation) of the best standard gold amount of streptococcus suis 2-type antibody
Collaurum is a kind of very unsettled colloid, particularly regulate the pH value after, can make behind the suitable albumen collaurum stable but add.So must determine the optimum mark amount of colloid gold label streptococcus suis 2-type antibody.Measuring its optimum mark amount must carry out under optimal pH and ion concentration situation.Collaurum is transferred to best pH.PBS (pH7.2) with 0.01mol/L dilutes rabbit anti-streptococcus suis 2 type antibody to 0.2mg/mL.Press table 2 operation.
Table 2 test tube observation is measured stable colloid gold minimum mark amount
Leave standstill the back and observe, contain the few pipe of antibody amount and present coagulation phenomenon, add the solution that antibody meets or exceeds in the quantitative test tube of minimum steady and then keep red constant by red stain indigo plant.The antibody amount of the test tube that the antibody content that redness is remained unchanged is minimum is exactly the suitableeest protective number of antibody, and increasing by 20% on this basis is the optimum mark amount of the antibody of stable colloid gold.
Collaurum is transferred to pH8.5, with PBS (pH7.2) the dilution rabbit anti-streptococcus suis antibody 0.2mg/mL of 0.01mol/L.After pressing table 2 and adding all ingredients, observe the solution casting situation, show, contain the few pipe of antibody amount and present coagulation phenomenon, add the solution that antibody meets or exceeds in the quantitative test tube of minimum steady and then keep red constant by red stain indigo plant as table 3 result.Therefore, the suitableeest antibody protective number of streptococcus suis 2-type colloidal gold probe is 22 μ g/mL.
The mensuration of table 3 streptococcus suis 2-type antibody colloidal gold optimum mark amount
2.4 the preparation of colloidal gold probe
K with 0.1mol/L
2CO
3Adjust collaurum pH to 8.5, get 1ml and place the 1.5mlEP pipe, the streptococcus suis 2-type antibody 110 μ l that add 0.2mg/ml behind the mixing 30min, add 10%BSA 100 μ l, (or 4 ℃ spend the night) is under 4 ℃ behind the mixing 30min, behind the centrifugal 30min of 12000r/min, and the resuspended liquid of usefulness 1ml (0.01mol/L Tris-HCl, pH 8.2,1%BSA, 5% sucrose) resuspended precipitation; Behind 4 ℃ of centrifugal 30min of following 12000r/min, with 4 ℃ of centrifugal 4min of following 1000r/min of the resuspended precipitation of the resuspended liquid of 500 μ l, supernatant is colloidal gold probe, 4 ℃ of preservations again.
2.5 definite test of best spray film concentration and best sealing concentration
(dilution is 3.5,2.5,2.0,1.5,1.0,0.5mg/mL concentration respectively streptococcus suis 2-type antibody and goat anti-rabbit antibody with the PB (pH7.2) of 0.01mol/L, and the antibody of each concentration all contains 2.0%, 1.5%, 1.0%, 0.75%, each pipe of 0.5%BSA, is used for sealing.Get dilution good streptococcus suis 2-type antibody (T band) and goat anti-rabbit antibody (C band), see Fig. 1, use every stream metal spraying pen machine and on the NC film, rule, the NC film of having drawn line is placed 37 ℃ of dry 2h.Detect simultaneously to determine best antibody sandwich concentration and sealing concentration with the bacteria suspension of streptococcus suis 2-type and the blank that do not contain streptococcus suis 2-type.
Test findings shows that along with the increase of antibody sandwich concentration, the color and luster of index line is also deepened gradually, and color and luster is no longer deepened after arriving more than the 2.0mg/ml, determines that therefore 2.0mg/ml is best rabbit anti-streptococcus suis 2 type IgG antibody sandwich point sample concentration.BSA concentration when false positive not occurring with blank is best sealing concentration, determines that therefore 1.5% is best sealing concentration.
2.6 the assembling of test strips
Colloidal gold test strip is made up of sample pad, glue gold pad, NC film and absorption pad four parts.Sample pad and glue gold pad adopt glass fibre, and absorption pad adopts absorbent filter.Add colloidal gold probe, 37 ℃ of dry 2h on the glue gold pad.Detect band on the NC film and accuse that band wraps respectively that (be dissolved in 10mmol/L PB, pH 7.2,1.5%BSA) and goat-anti rabbit immune serum 0.5mg/ml (be dissolved in 10mmol/L PBS, pH 7.2) by rabbit anti-streptococcus suis 2 type antibody 2.0mg/ml.37 ℃ of dry 1.5h behind the film have been sprayed.Glue gold pad with absorption pad, sample pad and after handling, the stack of NC film adhere on the PVC plate, are assembled into complete test strips, cut into the 4mm/ bar then, and drying keeps in Dark Place.
2.7 testing result interpretation
(1%Tween-20,10mmol PBS pH8.5) are added on the test strips sample pad for preparing, and observe testing result behind the 15min to get the testing sample of 50 μ l and 50 μ l sample preparation liquid.Judgement to the result: positive (+): detect band (T) and quality control band (C) and red stripes all occurs; Negative findings (-): only a red stripes appears in quality control band (C), is detecting the appearance of band (T) redfree band; Invalid: red stripes does not appear in quality control band (C).As shown in Figure 1:
The evaluation of embodiment three streptococcus suis 2-type colloidal gold test paper
One, material: with embodiment one
Two, methods and results:
1. the sensitivity tests of streptococcus suis 2-type test strips
With bacterial strain S.suis 149 streak inoculations in containing on the inferior agar blood of the 5% sheep blood taxi driver brother rival plate, 37 ℃, 5%CO2 cultivates 24h, picking colony, in the switching THB fluid nutrient medium, 37 ℃, 5%CO2 cultivates 6h, 4 ℃ of centrifugal 10min of 8000r/min are diluted to the bacteria suspension of varying number level again with PBS, the coating blood plate carries out colony count.Getting the different dilution bacterium liquid of 100 μ l adds the equal-volume sample diluting liquid (1%Tween 20, and 10mmol/L PBS pH8.0) is used for detecting.
From 1.0 * 10
810 times of serial dilutions of CFU/ml streptococcus suis 2-type sample, the sample liquid that does not contain streptococcus suis 2-type is a blank, and testing result is seen Fig. 3, and immunochromatography bar detection sensitivity is 1.0 * 10
6CFU/ml.
2. the specificity of streptococcus suis 2-type test strips test
Get the frozen streptococcus suis 2-type bacterial classification of glycerine and recover according to a conventional method, be inoculated in and contain on the inferior agar blood of the 5% sheep blood taxi driver brother rival plate, the single bacterium colony switching of picking Todd-Hewitt broth (THB) nutrient culture media during cultivation, 37 ℃, 5%CO2 cultivates; Gold-coloured staphylococci, Listeria monocytogenes, Salmonella paratyphi A, Escherichia coli, C.perfringens are with turbidimetry preparation 10
8CFU/ml bacterium liquid is got 100 μ l bacterium liquid and is added the equal-volume sample diluting liquid and be used for detecting.
Get fresh bacteria suspension (concentration＞10 of different strains
8CFU/ml), detect with immune test paper.Use this method and detect 15 serotypes of Streptococcus suis, only cross reaction is arranged with SS1/2.SS1/2 contains 1 type and 2 type antigen bacterial strains simultaneously, so test strips can not be distinguished SS2 and SS1/2.Detect domestic and external epidemic strain and non-epidemic strain and pathogenic strain and the non-pathogenic strain of streptococcus suis 2-type with this test strips, the result is all positive, illustrates that this test strips sensing range is wide, and specificity is good.This immune test paper and common pathogenic bacteria and 15 equal no cross reactions of group of streptococcus.Show that according to above result this test strips has good specificity.
3. the stability test of streptococcus suis 2-type test strips
Colloidal gold immuno-chromatography test paper strip respectively at 4 ℃ and room temperature preservation 3 months, is deposited for 37 ℃ and got the streptococcus suis 2-type bacteria suspension behind the 15d (concentration is 10
8CFU/ml and 10
6CFU/ml) and staphylococcus aureus (concentration is 10
9CFU/ml) detect, and contrast as sample blank with same PBS solution.
Test findings shows: colloidal gold immuno-chromatography test paper strip respectively at 4 ℃ and room temperature preservation 3 months, is deposited to take out behind the 15d for 37 ℃ and detected, the results are shown in Figure 4, testing result is not seen change.
4. simulated samples detects
The lean meat 50mg and the serum 100 μ l that rub join 1ml respectively and contain in the sample diluting liquid of variable concentrations streptococcus suis 2-type, and mixing leaves standstill 10min or of short duration centrifugal (the centrifugal 15s of 8000rpm), and sample thief detects.
Testing result shows, still can reach 10 to lean meat and serum simulated samples detection sensitivity result
6CFU/ml.The testing result of dummy is negative.Serum is owing to its component and viscosity etc., and the chromatography speed on the NC film is slow, also can reach 10 but detect detection sensitivity
6CFU/ml.
Claims (9)
1. immune chromatography test paper that detects streptococcus suis 2-type, comprise sample pad, glue gold pad, NC film and absorption pad, it is characterized in that on the glue gold pad colloidal gold probe being arranged, this probe mark has streptococcus suis 2-type antibody, the band of detection and quality control band are arranged on the NC film, detect band and be coated with streptococcus suis 2-type antibody, wherein streptococcus suis 2-type antibody is the polyclonal antibody at streptococcus suis 2-type antigen.
2. according to the immune chromatography test paper of the described detection streptococcus suis 2-type of claim 1, wherein streptococcus suis 2-type antibody is the rabbit anti-streptococcus suis 2 type antibody of the streptococcus suis 2-type antigen immune preparation of employing hydroformylation deactivation.
3. according to the immune chromatography test paper of the described detection streptococcus suis 2-type of claim 2, wherein rabbit anti-streptococcus suis 2 type antibody concentration are 2.0mg/ml.
4. the preparation method of the immune chromatography test paper of claim 1 or 2 described detection streptococcus suis 2-types comprises the steps:
(1) preparation streptococcus suis 2-type antibody;
(2) collaurum and be marked with the preparation of the colloidal gold probe of streptococcus suis 2-type antibody;
(3) streptococcus suis 2-type antibody and the many antiantibodys of anti-streptococcus suis 2 types are sprayed on the NC film wrap quilt;
(4) golden pad of glue and the stack of NC film with absorption pad, sample pad and after handling adheres on the PVC plate, is assembled into complete test paper.
5. according to the described method of claim 4, wherein prepare streptococcus suis 2-type antibody and adopt direct streptococcus suis 2-type antigen-immunized animal with the hydroformylation deactivation to carry out, the antibody of acquisition is polyclonal antibody.
6. according to claim 4 or 5 described methods, wherein prepare streptococcus suis 2-type antigen and adopt formalin-inactivated, streptococcus suis 2-type antibody and anti-streptococcus suis 2 type antiantibodys are respectively rabbit anti-streptococcus suis 2 type antibody and goat-anti rabbit immune serum, and the two concentration is respectively 2.0mg/ml and 0.5mg/ml.
7. according to claim 4 or 5 described methods, wherein sample pad and glue gold pad adopts glass fibre, and absorption pad adopts absorbent filter.
8. claim 1 or the 2 described immune chromatography test papers purposes in detecting streptococcus suis 2-type.
9. according to the described purposes of claim 7, detect streptococcus suis 2-type sensitivity and reach 10
6CFU/ml.
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