CN101900729B - 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.Human Streptococcus suis infection disease is a kind of zoonosis due to human infection's streptococcus suis 2-type (Streptococcus suis serotype 2, SS2) etc.Streptococcus suis belongs to the R group of Lan Shi classification, has 35 serotypes, popular the widest with streptococcus suis 2-type, pathogenic the strongest.Streptococcus suis not only can cause the even die by visitation of God of pig meningitis, endocarditis, septicemia, arthritis, chronic hyperplastic perihepatitis, pneumonia, can also invade human body through damaged skin or mucous membrane, cause people's infection morbidity, the formation to farm and meatworker threat.Human Streptococcus suis infection clinical manifestation is septicemia, TSS (TSS) and meningitis.The main infection sources of Human Streptococcus suis infection disease is the pig that dies of illness, and main route of transmission, for slaughtering the pig that dies of illness, is cut, cleaned Pork form diseased or dead pigs etc., and hand skin has the outstanding easy infection of the personnel of damage.Feed is not boiled, well-done Pork form diseased or dead pigs also can cause infection.China is faced with the threat that 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 altogether 229 people infect, wherein dead 53 examples.Cause people, the morbidity of pig coinfection China Streptococcus suis, not only cause serious economic loss to pig industry, brought threat also to public health and food security.This disease has caused the great attention of worldwide field of public health, and increasing researcher starts Streptococcus suis to carry out more deep research.
Traditional traditional separation cultivation and biochemical identification of the main dependence of the qualification of the separation to Streptococcus suis cause of disease, then carries out agglutination test somatotype with Streptococcus suis hyper-immune serum.Although biochemical identification can detect more reliably and identify streptococcus suis 2-type, its must taking isolated pure bacterium as basis, but these two kinds of method complex operations are unfavorable for carrying out on-the-spot quick diagnosis.For improving China security monitoring ability relevant to Emergent Public Events, have efficiently in the urgent need to setting up China, the detection technique of responsive, quick, high specific.
Colloid gold immune quick diagnosis technology (fast diagnose test paper bar) is the novel vitro diagnostic techniques having grown up on monoclonal antibody technique, colloidal gold immunochromatographimethod technology and new material technology basis since the nineties in 20th century.This technology is mainly that specific antigen or antibody are fixed on nitrocellulose filter (NC) with ribbon, colloid gold label reagent is adsorbed on pad, after testing sample is added in the sample pad of test strips one end, move forward by capillary action, after dissolving the colloid gold label reagent on pad, react to each other, while moving to again the region of fixing antigen or antibody, there is specific binding with it again and be trapped in the compound of determinand and gold marked reagent, be gathered in to detect and bring, 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 crossed and is detected band, reaches and the automatic object separating of binding label.Colloidal gold immunochromatographimethod quick diagnosis technology can provide result at short notice, and this detection method is quick and easy, accurate, reliable, cheap and operation is easy.
Up to the present, there is not yet the report that adopts colloidal gold immunochromatographimethod technology for detection streptococcus suis 2-type.
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 colloidal gold immune chromatography test, comprises sample pad, glue gold pad, NC film and absorption pad four parts.On glue gold pad, there is colloidal gold probe, on NC film, have the band of detection and accuse band, detect band and accuse that band is coated with respectively streptococcus suis 2-type antibody and anti-streptococcus suis 2 type antiantibodys.Above-mentioned streptococcus suis 2-type antibody is streptococcus suis 2-type.Streptococcus suis 2-type antibody can be the antibody that streptococcus suis 2-type immunity different animals obtains, preferably rabbit anti-streptococcus suis 2 type antibody; Prepare streptococcus suis 2-type antibody, can select the streptococcus suis 2-type antigen immune of hydroformylation to form, as formaldehyde and glutaraldehyde, preferably 1% formaldehyde can be used as immunogene in 37 DEG C of deactivation 7d, can ensure the specificity that antibody is good.Anti-streptococcus suis 2 type antiantibodys can obtain by immune different animals, and preferably goat-anti rabbit immune serum, can buy commercialization antibody.Above-mentioned two kinds are being detected band and are accusing and bring coated antibody can adopt variable concentrations, and it is 2.0mg/ml that preferred concentration is respectively rabbit anti-streptococcus suis 2 type antibody concentration, and 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 and prepares 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 coated with on NC film; Absorption pad, sample pad and glue after treatment gold pad and the stack of NC film are adhered on PVC plate, be assembled into the steps such as complete test paper.
Prepare the employing of streptococcus suis 2-type antibody and directly undertaken by the method for streptococcus suis 2-type immune animal, the antibody of acquisition is polyclonal antibody.First need to prepare streptococcus suis 2-type antigen, first streptococcus suis 2-type is cultivated, after collection bacterium liquid, formalin-inactivated can be used as antigen.Then use antigen-immunized animal, immunity is carried out according to a conventional method, and blood sampling is measured animal's antibody titre by ELISA method before immunity and after each immunity.After 6 immunity, antibody titer reaches general immune response level, is 10
7, the antibody obtaining can be used for the preparation of colloidal gold immuno-chromatography test paper strip.The streptococcus suis 2-type obtaining first will pass through purifying, can adopt albumin A-agarose affinity chromatography method to carry out.The antibody that purifying is good is dialysed latter-70 DEG C and is saved backup.
The preparation of collaurum adopts sodium citrate reducing process to be prepared, and obtains the collaurum of diameter 25nm.
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 first centrifugal removal easily to form as last.Regulate the pH value of colloidal gold solution with solution of potassium carbonate, mix with the streptococcus suis 2-type antibody of preparation, the centrifugal PBS dissolution precipitation that goes use after supernatant to contain 1%BSA, centrifugal after resuspended precipitation again, centrifuging and taking supernatant is the colloidal gold probe that mark is good.Wherein the pH value of colloidal gold solution is >=8.5, preferably 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 type are coated with on NC film.First by PBS working fluid dialysis for streptococcus suis 2-type antibody, then, by the streptococcus suis 2-type antibody and the goat anti-rabbit antibody that have diluted, on NC film, rule and be coated with respectively, become T band and C band, see Fig. 1.Coated antibody concentration is >=2.0mg/ml, taking 2.0mg/ml as best, 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.
First verify the susceptibility of test paper of the present invention, the Streptococcus suis of cultivation be diluted to the bacterium liquid of variable concentrations, add sample diluting liquid and detect, taking not containing the sample liquid of streptococcus suis 2-type as blank.The sensitivity that colloidal gold immune chromatography test of the present invention detects Streptococcus suis is 1.0 × 10
6cFU/ml.
Then verify the specificity of test paper of the present invention.The Streptococcus suis of cultivation, other streptococcus and gold-coloured staphylococci, Listeria monocytogenes, Salmonella paratyphi A, Escherichia coli, C.perfringens 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.With the domestic and external epidemic strain of this ELISA test strip streptococcus suis 2-type and non-epidemic strain and pathogenic strain and non-pathogenic strain, result is all positive; Especially surprisingly, although this antibody adopts the Streptococcus suis crude antigen preparation of hydroformylation, there is good serological specificity, except Streptococcus suis 1/2 type, the Streptococcus suis no cross reaction of this test strips and other serotype.Illustrate that this ELISA test strip scope is wide, specificity is good.Also equal no cross reaction of 15 groups of this immune test paper and common pathogenic bacteria and streptococcus.Show according to above result, this test strips has good specificity.
Then be the stability of checking test paper of the present invention.4 DEG C and room temperature preservation 3 months, deposit after 15 days at 37 DEG C respectively, testing result does not all change, and 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 Streptococcus suis of the present invention is the polyclonal antibody of application 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 of employing Dispersal risk, the specificity of diagnostic reagent is poor, normal exists the defects such as cross reaction, practicality be poor with near edge bacterium.Utilize detection paper streptococcus suis 2-type of the present invention, sample disposal is simple, within 10 minutes, gets final product observations, minimumly detects 10
6cFU/ml.By the detection to analog sample, it is highly sensitive, specificity good.Especially surprisingly, although this antibody adopts the Streptococcus suis crude antigen preparation of hydroformylation, there is good serological specificity, except Streptococcus suis 1/2 type, the Streptococcus suis no cross reaction of this test strips and other serotype.That is to say, although the antibody that the present invention uses is to resist more, the colloidal gold immune chromatography test of preparation has higher specificity, and this is that those skilled in the art institute is not thinkable, and tool has an unexpected effect.Test paper of the present invention has the features 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 is conducive to our understanding and the control to Streptococcus suis epidemic situation.
Brief description of the drawings
Fig. 1 is testing result interpretation schematic diagram
Fig. 2 is 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 mono-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 Harbin veterinary institute, and all the other bacterial strains are purchased from Nat'l Pharmaceutical & Biological Products Control Institute.
Animal used as test: 2 of 2kg left and right New Zealand large ear rabbits, female.Provided by Military Medical Science Institute's Experimental Animal Center.
Nutrient culture media: LB nutrient culture media, THB nutrient culture media, blood plate is preparation according to a conventional method all
Major equipment and instrument: Biohazard Safety Equipment (NUAIR company of the U.S.), the full-automatic protein nucleic acid enzyme connection detector of Spectra Max Plus (Molecular Devices company of the U.S.), the miniature half-dried electrophoretic blotting instrument of ST-I (production of Dalian Jing Mai biotechnology company), AKTA FPLC protein purification analyser (AmershamPharmacia Biotech company of Sweden), every stream metal spraying pen machine (USA I/M AGEN ARISTA company), ND-1000 ultramicron protein quantification instrument (NanoDrop company of the U.S.), Enzyme-linked Immunosorbent Assay instrument, 868 type pH meters (Thermo company of the U.S.).
Two, methods and results:
1. the preparation of streptococcus suis 2-type antigen
Pathogen is comlete antigen, can obtain antibody by direct immunization animal, so the S.suis 149 of numeration deactivation is the antigen of preparing streptococcus suis 2-type polyclonal antibody.The Streptococcus suis S.suis149 of preservation is seeded in THB fluid nutrient medium, leaves standstill and cultivate 7h in 37 DEG C, sterilized water is collected bacterium liquid, blood plate counting.1% formaldehyde can be used as immunogene in 37 DEG C of deactivation 7d.
2. the Preparation and identification of streptococcus suis 2-type polyclonal antibody
The preparation of 2.1 streptococcus suis 2-type polyclonal antibodies
Streptococcus suis 2-type polyclonal antibody obtains by the new zealand white rabbit of immune 2kg left and right.Measure immune animal at immune vestibule venous blood sampling ELISA for the first time and whether there is natural antibody, abandon if having.If without from immunity for the second time starts each immunity 1 week ear vein get blood, carry out indirect ELISA survey antibody titer.Immunization protocol is in table 1.
Table 1 streptococcus suis 2-type immunization protocol
2.2 streptococcus suis 2-type antibody titers are measured
After immunity, ear vein is got blood 500 μ L, places 1h for 37 DEG C, the centrifugal 3min of the then centrifugal 10min of 3000rpm, then 10000rpm, and supernatant is immune serum.Measure tiring of Serum Antibody with indirect ELISA.Method is as follows:
1) be 10 with the bacterium of coated damping fluid dilution
9cFU/mL, 100u L/ hole, 4 DEG C coated spend the night or 37 DEG C 1～2 hour,
2) use PBST hole flushing, 100 μ L/ holes, wash 3 times, each 3min,
3) with the PB sealing containing 3%BSA, 200 μ L/ holes, are placed in 37 DEG C of incubators and seal 2 hours,
4) PBST washes 3 times, 100 μ L/ holes, and each 3min,
5) by the PBST gradient series dilution primary antibodie containing 3%BSA, every hole adds 100 μ L, 37 DEG C of effect 1h
6) PBST washes 3 times, 100 μ L/ holes, each 3min
7) with the PBST dilution enzyme labelled antibody (1: 2000) containing 3%BSA, every hole 100 μ L, 37 DEG C, 30～45min
8) PBST washes 4 times, 100 μ L/ holes, 3min/ time
9) add A, B nitrite ion, each 1 of every hole, lucifuge colour developing adds stop buffer (2NH in the time that blueness appears in negative control
2sO
4) stop, 450nm records OD, and with blank well zeroing, if treat, gaging hole OD450 value is more than or equal to 2.1 times of negative control holes, is judged to be the positive, thereby draws serum titer.
After 6 immunity in 7 weeks, antibody titer can reach 10
7, reach 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 shown in Fig. 2.
The purifying of 2.3 polyclonal antibodies
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 suitably diluting, be splined on the good MabTrapTMG affinity column of level pad balance, thoroughly wash away after foreign protein with A liquid, changing eluent B liquid washes down antibody, the antibody of collection under washing, collects test tube and has added in advance 10% 1mol/L, the Tris-HCI of pH 9.0, make pH return to 7.0 left and right, in order to avoid lose activity.4 DEG C of PBS working fluid dialysis two days for the good antibody of purifying, the more centrifugal 30min of 10000r/min, collect supernatant, and-70 DEG C save backup.
The preparation of embodiment bis-streptococcus suis 2-type colloidal gold test paper
One, material: adopt commercial chemical reagent, with embodiment mono-.
Two, methods and results:
1. the preparation of collaurum
Adopt sodium citrate reducing process to prepare the collaurum of diameter 25nm.HAuCl4 is first made into 1% aqueous solution, gets 99ml pure water and be heated to boiling. under stirring, add the HAuCl4 of 1mL1%, then add 1.5mL trisodium citrate aqueous solution simultaneously.Continue heating and boil about 15min, observe that to add after liquid color first to become black, slowly become subsequently claret, continue to stop heating after heating 3～5min after seeing claret, deionized water is mended to volume 100mL.What obtain is the collaurum that diameter is 25nm, and cooling latter 4 DEG C keep in Dark Place.
2. the preparation of streptococcus suis 2-type colloidal gold test strip
Processing before 2.1 streptococcus suis 2-type antibody standard gold
Because streptococcus suis 2-type antibody should be used for standard gold, also to be used for spraying film, so antibody processing is before use also different.Streptococcus suis 2-type antibody, respectively with PBS working fluid and pure water dialysis, is determined to the optimum antibody disposal route that is suitable for standard gold and spray film with this.
Result: the condition demonstration of groping according to inventor's actual tests, different application targets has different requirements: for spraying the streptococcus suis 2-type antibody PBS working fluid dialysis of film, make it have certain ionic strength, and be beneficial to coated.Require not contain salt ion for the streptococcus suis 2-type antibody of standard gold, must dialyse with pure water; Antibody is generally placed in-20 DEG C of preservations before use, is easy to form dimer and affects standard gold, when particularly antibody concentration is higher, so using antibody to want first 4 DEG C of centrifugal 30min of 10000rpm as last.
The determination test of the optimal pH of 2.2 colloid gold label streptococcus suis 2-type antibody
The pH value of colloidal gold solution is adjusted to respectively to 6.0,6.5,7.0,7.5,8.0,8.5,9.0 with the solution of potassium carbonate of 0.1mol/L, respectively get 1mL and add the rabbit anti-streptococcus suis 2 type antibody 100 μ L that PBS (pH7.2) dilution of use 0.01mol/L is 0.2mg/ml, mix, room temperature reaction 10 minutes, leave standstill 2 hours, observe solution colour and change.Then centrifugal 10 minutes of 12000rpm, removes supernatant, adds the PBS dissolution precipitation containing 1%BSA, dissolves the pH value that is uniform transparent violet red solution pipe completely as best to precipitate.
Test findings as shown in Table 2-4, in the each pipe in pH≤7.5, solution colour is blueness in various degree, pH >=7.5, in each pipe, solution colour is unchanged, in the mark pipe of centrifugal rear pH≤8.0, precipitation can not be dissolved completely, in the mark pipe of pH >=8.5, precipitation is dissolved completely, and solution is transparent wine redness, as 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
The determination test (test tube observing method) of the best standard gold amount of 2.3 streptococcus suis 2-type antibody
Collaurum is the very unsettled colloid of one, particularly regulates after pH value, but can make collaurum stable after adding suitable albumen.So must determine the optimum mark amount of colloid gold label streptococcus suis 2-type antibody.Measuring its optimum mark amount must carry out in optimal pH and ion concentration situation.Collaurum is adjusted to best pH.With PBS (pH7.2) the dilution rabbit anti-streptococcus suis 2 type antibody of 0.01mol/L to 0.2mg/mL.Operate by table 2.
Table 2 test tube observing method is measured stable colloid gold minimum mark amount
After leaving standstill, observe, present the coagulation phenomenon by red stain indigo plant containing the few pipe of antibody amount, the solution that adds antibody to meet or exceed in the quantitative test tube of minimum steady keeps red constant.The antibody amount of the test tube that antibody content that redness is remained unchanged is minimum is exactly the suitableeest protective number of antibody, increases on this basis the optimum mark amount of 20% antibody that is stable colloid gold.
Collaurum is adjusted to pH8.5, with PBS (pH7.2) the dilution rabbit anti-streptococcus suis antibody 0.2mg/mL of 0.01mol/L.Add after various reagent by table 2, observe solution casting situation, as table 3 result shows, present the coagulation phenomenon by red stain indigo plant containing the few pipe of antibody amount, the solution that adds antibody to meet or exceed in the quantitative test tube of minimum steady keeps red constant.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
The preparation of 2.4 colloidal gold probes
With the K of 0.1mol/L
2cO
3adjust collaurum pH to 8.5, get 1ml and be placed in 1.5mlEP pipe, the streptococcus suis 2-type antibody 110 μ l that add 0.2mg/ml, mix after 30min, add 10%BSA 100 μ l, mix after 30min (or 4 DEG C spend the night) at 4 DEG C, after the centrifugal 30min of 12000r/min, with the resuspended liquid of 1ml (0.01mol/L Tris-HCl, pH 8.2,1%BSA, 5% sucrose) resuspended precipitation; Again at 4 DEG C after the centrifugal 30min of 12000r/min, with the centrifugal 4min of 1000r/min at 4 DEG C of resuspended precipitations of the resuspended liquid of 500 μ l, supernatant is colloidal gold probe, 4 DEG C of preservations.
Definite test of 2.5 best spray film concentration and best sealing concentration
With the PB (pH7.2) of 0.01mol/L, streptococcus suis 2-type antibody and goat anti-rabbit antibody, (dilution is 3.5,2.5,2.0,1.5,1.0,0.5mg/mL concentration respectively, and the antibody of each concentration all contains 2.0%, 1.5%, 1.0%, 0.75%, each pipe of 0.5%BSA, for sealing.Get the streptococcus suis 2-type antibody (T band) and the goat anti-rabbit antibody (C band) that have diluted, see Fig. 1, use every stream metal spraying pen machine and rule on NC film, the NC film of having drawn line is placed in to 37 DEG C of dry 2h.With the bacteria suspension of streptococcus suis 2-type with do not detect to determine the coated concentration of best antibody and sealing concentration containing the blank of streptococcus suis 2-type simultaneously.
Test findings demonstration, along with the increase of the coated concentration of antibody, the color and luster of index line is also deepened gradually, and after arriving more than 2.0mg/ml, color and luster is no longer deepened, and therefore definite 2.0mg/ml is the coated point sample concentration of best rabbit anti-streptococcus suis 2 type IgG antibody.BSA concentration while there is not false positive taking blank is best sealing concentration, therefore determines that 1.5% is best sealing concentration.
The assembling of 2.6 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.On glue gold pad, add colloidal gold probe, 37 DEG C of dry 2h.On NC film, detect band and accuse that band is coated with respectively rabbit anti-streptococcus suis 2 type antibody 2.0mg/ml and (is dissolved in 10mmol/L PB, pH 7.2,1.5%BSA) and goat-anti rabbit immune serum 0.5mg/ml (being dissolved in 10mmol/L PBS, pH 7.2).37 DEG C of dry 1.5h after film are sprayed.Absorption pad, sample pad and glue after treatment gold pad, the stack of NC film are adhered on PVC plate, be assembled into complete test strips, then cut into 4mm/ bar, dry keeping in Dark Place.
2.7 testing result interpretations
Testing sample and the 50 μ l sample preparation liquid (1%Tween-20,10mmol PBS, pH8.5) of getting 50 μ l are added in the test strips sample pad preparing, and observe testing result after 15min.Judgement to result: positive (+): detect band (T) and quality control band (C) and all occur red stripes; Negative findings (-) a: only 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 tri-streptococcus suis 2-type colloidal gold test paper
One, material: with embodiment mono-
Two, methods and results:
1. the sensitivity tests of streptococcus suis 2-type test strips
By bacterial strain S.suis 149 streak inoculations in containing on Colombia's agar blood plate of 5% sheep blood, 37 DEG C, 5%CO2 cultivates 24h, picking colony, in switching THB fluid nutrient medium, 37 DEG C, 5%CO2 cultivates 6h, 4 DEG C of centrifugal 10min of 8000r/min, then be diluted to the bacteria suspension of varying number level with PBS, coating blood plate carries out colony count.Get the different dilution bacterium liquid of 100 μ l add equal-volume sample diluting liquid (1%Tween 20,10mmol/L PBS, pH8.0) for detection of.
From 1.0 × 10
810 times of serial dilutions of CFU/ml streptococcus suis 2-type sample, are not blank containing the sample liquid of streptococcus suis 2-type, and testing result is shown in Fig. 3, and immunochromatography bar detection sensitivity is 1.0 × 10
6cFU/ml.
2. the specific test of streptococcus suis 2-type test strips
Get the frozen streptococcus suis 2-type bacterial classification of glycerine and recover according to a conventional method, be inoculated in containing on Colombia's agar blood plate of 5% sheep blood, the single bacterium colony switching of picking Todd-Hewitt broth (THB) nutrient culture media when cultivation, 37 DEG C, 5%CO2 cultivates; Gold-coloured staphylococci, Listeria monocytogenes, Salmonella paratyphi A, Escherichia coli, C.perfringens, prepare 10 by turbidimetry
8cFU/ml bacterium liquid, get 100 μ l bacterium liquid add equal-volume sample diluting liquid for detection of.
Get fresh bacteria suspension (the concentration > 10 of different strains
8cFU/ml), detect with immune test paper.Apply this method and detect 15 serotypes of Streptococcus suis, only have cross reaction with SS1/2.SS1/2 contains 1 type and 2 type antigen bacterial strains simultaneously, and therefore test strips can not be distinguished SS2 and SS1/2.With the domestic and external epidemic strain of this ELISA test strip streptococcus suis 2-type and non-epidemic strain and pathogenic strain and non-pathogenic strain, result is all positive, illustrates that this ELISA test strip scope 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 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 DEG C and room temperature preservation 3 months, is deposited for 37 DEG C and got streptococcus suis 2-type bacteria suspension after 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 DEG C and room temperature preservation 3 months, is deposited after 15d for 37 DEG C and taken out and detect, the results are shown in Figure 4, testing result has no change.
4. simulated samples detects
The lean meat 50mg and the serum 100 μ l that rub, join respectively 1ml containing in the sample diluting liquid of variable concentrations streptococcus suis 2-type, mixes, and 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 due to its component and viscosity etc., and the chromatography speed on NC film is slow, also can reach 10 but detect detection sensitivity
6cFU/ml.
Claims (9)
1. one kind is detected the immune chromatography test paper of streptococcus suis 2-type, comprise sample pad, collaurum pad, NC film and absorption pad, it is characterized in that there is colloidal gold probe on collaurum pad, this probe mark has streptococcus suis 2-type antibody, on NC film, there are the band of detection and quality control band, detect band and be coated with streptococcus suis 2-type antibody, wherein streptococcus suis 2-type antibody is the polyclonal antibody for streptococcus suis 2-type antigen, and described streptococcus suis 2-type antigen is the full bacterium antigen of Streptococcus suis S.suis149.
2. detect according to claim 1 the immune chromatography test paper of streptococcus suis 2-type, wherein streptococcus suis 2-type antibody is the rabbit anti-streptococcus suis 2 type antibody that adopt the streptococcus suis 2-type antigen immune of hydroformylation deactivation to prepare.
3. detect according to claim 2 the immune chromatography test paper of streptococcus suis 2-type, wherein detecting the coated rabbit anti-streptococcus suis 2 type antibody concentration of band is 2.0mg/ml.
4. the preparation method who detects the immune chromatography test paper of streptococcus suis 2-type described in claim 1 or 2, comprises the steps:
(1) prepare 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 type are sprayed on NC film and are coated with;
(4) absorption pad, sample pad and collaurum pad after treatment and the stack of NC film are adhered on PVC plate, be assembled into complete test paper.
5. method according to claim 4, wherein prepares streptococcus suis 2-type antibody and adopts directly and carry out with the streptococcus suis 2-type antigen-immunized animal of hydroformylation deactivation, and the antibody of acquisition is polyclonal antibody.
6. according to method described in claim 4 or 5, wherein on NC film, detect that band, quality control band are coated is respectively rabbit anti-streptococcus suis 2 type antibody and goat-anti rabbit immune serum with streptococcus suis 2-type antibody and anti-streptococcus suis 2 type antiantibodys, the two concentration is respectively 2.0mg/ml and 0.5mg/ml.
7. according to method described in claim 4 or 5, wherein sample pad and collaurum pad adopt glass fibre, and absorption pad adopts absorbent filter.
8. described in claim 1 or 2, immune chromatography test paper detects the purposes in reagent preparing streptococcus suis 2-type.
9. purposes according to claim 8, detects streptococcus suis 2-type sensitivity and reaches 10
6cFU/ml.
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