CN106353496A - Brucella fluorescence polarization (FPA) detection kit - Google Patents
Brucella fluorescence polarization (FPA) detection kit Download PDFInfo
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- CN106353496A CN106353496A CN201610987091.1A CN201610987091A CN106353496A CN 106353496 A CN106353496 A CN 106353496A CN 201610987091 A CN201610987091 A CN 201610987091A CN 106353496 A CN106353496 A CN 106353496A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The invention relates to a brucella fluorescence polarization (FPA) detection kit. In the kit, fluorescein isothiocyanate (FITC) is adopted for marking smooth brucella O-chain lipopolysaccharide micromolecule segment (OPS) and serves as an antigen. On the basis of FPA experimental principle, the kit can be used for detecting brucellosis of various animals and simultaneously detecting IgG and IgM antigens in serum, so that the kit has more obvious advantages at sensibility aspect. According to the invention, the main reagent formula is improved and optimized, especially, the titer of positive serum is accurately marked, so that the sensitivity and specificity of the kit are effectively promoted, the problems of sensibility, repeatability, storage period, operation convenience, and the like, are solved and the time for high throughput test is greatly shortened. The invention provides a novel efficient detection method for brucellosis diagnosis in China.
Description
Technical field
The present invention relates to a kind of diagnostic method brucella fluorescence polarization (fpa) the detection examination of animal brucellosis
Agent box, belongs to biological product detection technique field.
Background technology
Brucellosis (brucellosis) abbreviation brucellosis, are the people based on infection domestic animal being caused by brucella
Poultry suffers from infectious disease altogether.Primary disease not only breeding and production performance to animal there is serious harm it is often more important that, people infect cloth Shandong
It tends to be difficult to cure, thus causing serious public health problem after Salmonella.The main of this disease is eliminated at present in worldwide
Method is to slaughter to combine with immunity, so it is very necessary to preventing and treating and removing brucellosis to set up fast and accurately diagnostic method.
The conventional serological method of Brucella antibody detection has rose bengal precipitation test (rbt), tube agglutination test
(sat), complement fixation test (cft), elisa (elisa) and fluorescence polarization assay (fpa).Wherein sat because
Specificity is not high to be detected it is generally recognized that being not suitable for international trade；Rbt, cft and elisa specify test for international trade；Fpa is
International trade Selection experiment.Fpa achieves the quick detection to disease using biophysical techniques, and its ultimate principle is fluorescence
After the blue polarized light (485nm) through single plane for the material irradiates, absorb luminous energy and leap to excited state, subsequently return back to ground state, concurrently
Go out the polarized fluorescence (525nm) of single plane.The degree of strength of polarized fluorescence is proportionate with the size of fluorescence molecule, is subject to it
The speed rotating when exciting is in inverse correlation.In the present invention, we are in the upper labelling fluorescein molecule of brucella o chain polysaccharide (ops)
(fitc), after brucella specific antibody forms antigen/antibody complex with antigen binding, molecule quantitative change is big, now examines
The fluorescence polarization degree measuring is also higher, when the polarization intensity value detecting is more than cutoff value, that is, is judged to brucellosis positive, instead
For feminine gender.Then with respect to elisa method, fpa spy has higher detection efficiency, and has good specificity and sensitivity
Property；In addition, its test operation is also simple than elisa, it is particularly suitable for the detection to a large amount of samples, in brucella serology inspection
There is specific advantage in survey.
The applicant is successfully developing cattle brucellosis indirect elisa test kit, animal brucellosis competition elisa test kit and cloth
On the basis of the Salmonella antibody test test strips of Shandong, succeeded in developing brucella fluorescence polarization detection kit again.
Content of the invention
It is an object of the invention to establishing a kind of Brucella antibody high throughput testing technology.Its core is by purification
Fluorescein isothiocyanate on smooth type brucella o chain lipopolysaccharide (ops) labelling, is used as detection antigen after accurate quantification.With
Artificial immunity health cattle prepares positive serum, makees negative serum with healthy nonimmune Ox blood serum, and the group such as 25 × sample diluting liquid
Divide and assemble, for detecting the smooth type Brucella antibody in many animals serum.
Technical scheme
1. a kind of brucella fluorescence polarization detection kit is it is characterised in that this test kit is by brucella fluorescent labeling
Antigen ops, brucella positive control serum, negative control sera and sample diluting liquid composition.
2. as claimed in claim 1 a kind of brucella fluorescence polarization detection kit it is characterised in that cloth Shandong therein
Salmonella fluorescent labeled antigen ops is little point of the fluorescent labeling smooth type brucella s2 strain lipopolysaccharide of detection Brucella antibody
Sub-piece, this ops is respectively provided with good specificity and sensitivity to common brucella pathogenic autoantibody.
3. the application of the brucella fluorescence polarization detection kit described in claim 1, this can detect many animals blood
Brucella specific antibody in clear, that is, this test kit the brucellosis of pig, cattle, sheep can be carried out high flux and quickly examine
Survey.
The specific embodiment of the invention
Brucella fluorescence polarization detection kit.By multi-party to labelled antigen, reaction condition, result criterion etc.
The optimization of noodles part, has particularly carried out Accurate Calibration to the potency of positive serum, has solved brucella glimmering more satisfactoryly
In light polarization test diagnosis method the problems such as specificity, sensitivity, repeatability, storage life, operation convenience.Use in this patent
Fluorescein isothiocyanate (fitc) labelling smooth type brucella o chain lipopolysaccharide small molecule segment (ops), as antigen.Base
In the experimental principle of fpa, this test kit can carry out brucellosis detection to many animals, can detect igg the and igm class in serum simultaneously
Antibody, therefore this test kit have more obvious advantage in terms of sensitivity.
In implementation process of the present invention, for specificity and the sensitivity of objective evaluation test kit, collect within nearly 6 years
343 parts of field cattle, serum samples of sheep, after brave red agglutination test primary dcreening operation, then with tube agglutination test and complement fixation test
Detection, eliminate the inconsistent sample of partial detection, determine altogether 148 parts of brucellosis positive serums (wherein 70 parts of Ox blood serum,
78 parts of sheep blood serum), 155 parts of brucellosis negative serums (wherein 82 parts of Ox blood serum, 73 parts of sheep blood serum).The test kit of present invention exploitation,
Coincidence rate with confirmed 148 parts of brucellosis positive serum samples is 98.0% (145/148), with confirmed 155 parts of feminine genders
The coincidence rate of sample is 98.7% (153/155), thereby determines that test kit is 98.0% to the sensitivity of field sample, specificity
For 98.7% it is shown that the reliability of test kit diagnostic result.
Brucella fluorescence polarization detection kit of the present invention, chooses pig kind brucella s2 strain and carries as ops
The production strain taking, on the basis of traditional extraction ops method, increased Proteinase K digestion and concentration step, improves ops
Concentration and purity.Concrete technical scheme is as follows:
1.ops extracts
(1) bacteria suspension will be inactivated 20min will be centrifuged with 10000g, collect precipitation.Weigh and calculate thalline weight in wet base, thalline is wet
Add in 2% (v/v) acetic acid in 1:8 (w/v) ratio, 121 DEG C of autoclaving 15min again.4 DEG C are gone with 10000g centrifugation 20min
Except bacterial chip.
(2) add final concentration of 5% trichloroacetic acid in supernatant, after 15min is stirred at room temperature, 4 DEG C of 10000g centrifugations
10min, discards precipitation.Add a certain amount of protease k so as to final concentration reaches 50 μ g/ml, put 56 DEG C of water-baths and digest 2 hours.
(3) supernatant puts 100 times of distilled water dialysed overnight, concentrates 40 times with peg8000, is again placed in distilled water and dialysed
At night, collect bag filter content, be distributed into 2ml/ bottle lyophilizing and preserve, this ops purifying.
2. establish fitc-ops molecule labelling method
(1) 4mg ops is added in the triethylamine solution of 2ml 0.5% (v/v), puts supersound process 15min on ice, surpass
Add the edta solution of 200 μ l 100mmol/l after sound, and with the hydrochloric acid of 10 μ l 1mol/l, solution ph is adjusted to 5.0.
(2) (isomer i) is dissolved in 800 μ l 0.25mol/l boric acid solution (ph10.5), Ran Houquan to take 20mg fitc
Portion is added in ops solution, continues supersound process 1min.Add 1ml 1.6% sodium deoxycholate solution, 37 DEG C of stirring incubations 18
Hour.
(3) 4 DEG C of 10000g are centrifuged 30min, supernatant is transferred in bag filter and is concentrated with peg6000, subsequently saturating with pbs
Analysis 1 hour, dialysate pd-10 post desalination is collected all fitc-ops labels, is used peg6000 reconcentration, after merging
Dialysed 1 hour with pbs more afterwards, collect 4 DEG C of preservations of product after dialysis, as labelled antigen stock solution.
The potency of 3.fpa test kit positive serum is demarcated
(1) use brucella rose bengal precipitation test, tube agglutination test and complement fixation test that clinical manifestation is good for
2 years old of health about Adult Bovine detected, screen brucellosis negative antibody cattle.
(2) by brucella with reference to virulent strain m28 inactivation antigen with 5 × 1010Cfu/ cattle cervical region subcutaneous inoculation, after 3 months
Doubling dosage booster immunization 1 time.Blood sampling in 2 weeks after booster immunization.
(3) measure agglutination titer with tube agglutination test, when potency is more than 800iu/ml, venesection, separate serum.
According to the potency of practical measurement, with cattle brucellosis negative serum, its potency is diluted to 320iu/ml, is crossed with 0.45 μm of filter and filter
Bacterium, as brucellosis positive serum in test kit.
The specificity of 4.pfa test kit and sensitivity assessment
(1) by the serum sample of 343 parts of field cattle of 2009～2015 Nian Jian seminars collection, sheep, red flat with tiger respectively
Plate agglutination test and tube agglutination test detection, be detected as negative by the serum sample of equal for two methods test positive with all
Sample is confirmed with complement fixation test again, selects three kinds of methods to be all the serum of the positive as positive reference serum, three kinds of methods
The serum being all feminine gender is as negative reference serum.
(2) 148 parts with filtering out be defined as the positive serum (wherein 70 parts of Ox blood serum, 78 parts of sheep blood serum) of brucellosis and
155 parts of brucellosis negative serums (wherein 82 parts of Ox blood serum, 73 parts of sheep blood serum) as serum to be checked, the sensitivity of kits for evaluation and
Specificity.
Brief description
The pcr identification qualification figure in figure 1:dna marker of Fig. 1 pig kind brucella s2；2: escherichia coli negative control；
The pcr amplification of 3:s2 bacterial strain.
Microbial resources information of the present invention
Microorganism involved in the present invention is: pig kind brucella (brucella suis) s2 strain (cvcc70502), comes
From National Veterinary Culture Collection, it is pig kind brucella biology 1 type, is brucellosis attenuated live vaccines strain.
Brucella melitensis (brucella melitensis) m28 strain (cvcc70003), from National Veterinary Microbiological Culture Collection
Center, is brucella melitensis biology 1 type, is brucella disease live-vaccine effect evaluation inspection malicious Reference Strains by force.Two bacterium
Strain is by ZhongGuanCun south Street, Haidian District, BeiJing City 8 China of China Veterinery Drug Inspection Office veterinary microorganism culture presevation management
Center identification, keeping and supply (ask for an interview China Veterinery Drug Inspection Office, Chinese veterinary microorganism culture presevation administrative center compiles
Write, Chinese veterinary's strain catalogue (second edition), Scientia Agricultura Sinica technology publishing house, 2002, p35, p32).
Advantages of the present invention
The present invention relates to brucella fluorescence polarization (fpa) detection kit.Present invention uses Fluorescein isothiocyanate
(fitc) labelling smooth type brucella o chain lipopolysaccharide small molecule segment (ops), as antigen.Based on the experimental principle of fpa,
This test kit can carry out brucellosis detection to many animals, can detect igg the and igm antibody-like in serum, therefore this reagent simultaneously
Box has more obvious advantage in terms of sensitivity；The present invention is by the improvement of main agents formula and optimization, particularly right
The potency of positive serum has carried out Accurate Calibration, effectively increases the Sensitivity and Specificity of test kit, sensitivity, repeatability,
The problems such as storage life, operation convenience, substantially reduce the time needed for high throughput testing, the present invention is China's brucellosis
Diagnosis provides new and effective detection method.
Embodiment
Following examples are the bright present invention further, do not constitute the restriction of the technical scheme that the present invention is claimed.
Embodiment 1
The antigen preparation quality standard of strain (pig kind brucella s2 strain, b.suiss2).
1. colonial morphology colony edge is neat, mellow and full, reveals drop-wise, and skew ray irradiates, and backlight observes micro-strip blue-opalescent.
2. dyeing form be the coccobacilluss, single be dispersed in, do not form spore and pod membrane.Size is between 0.6～2.5 μm.Leather
Blue Albert'stain Albert is feminine gender, and Ke's Albert'stain Albert is redness.
3. purely inspection by existing " Chinese veterinary pharmacopoeia " (Chinese veterinary pharmacopoeia committee, Republic of China Veterinary Pharmacopoeia, two
1 year version three, Chinese agriculture publishing house, 2011, hereinafter referred to as " Chinese veterinary pharmacopoeia ") annex is carried out, should be purely.
4. specific assay
(1) serological specificity makes antigen with culture, coagulation should with smooth type brucella positive serum, with
Rough type serum occurs without coagulation.
(2) pcr specificity synthesizes following 4 primers, is made into primer mixing storing liquid, and each primer concentration is 25 μ
m.
Feri:5 '-gcgccgcgaa gaacttatca a-3 ' 21 (sequence 1)
Reri:5 '-cgccatgtta gcggcggtga-3 ' 20 (sequence 2)
fsuis: 5 '-gcgcggtttt ctgaaggttc agg-3 ' 23 (sequence 3)
ris711: 5 '-tgccgatcac ttaagggcct tcat-3 ' 24 (sequence 4)
Template s2 cultivates the s2 genome dna that bacterium colony or test kit extract.
In the reaction system of pcr reaction system 50 μ l, containing 5 μ l 10 × buffer, 8 μ l2.5mmdntps, mix primer is stored up
Liquid storage 2 μ l, taq enzyme 2u, template dna 1 μ l (or picking colony is a little).
Pcr response procedures: after 95 DEG C of 5min, carry out 28 circulations by 94 DEG C of 1min, 60 DEG C of 1.5min, 72 DEG C of 1.5min,
Last 72 DEG C of 10min.
, should 2 specificity pcr bands, greatly in result: amplified production carries out electroresis appraisal with 1.5% agarose gel
Little respectively 178bp and 285bp (see accompanying drawing 1).
Embodiment 2
The application detects purification and labeling method with antigen ops
1. qualified b.suiss2 strain secondary seed is inoculated in the flat bottle of pancreas agar for bacteria suspension preparation, 37 DEG C
After culture 48 hours, every bottle adds the normal saline 20ml containing 0.5% phenol to soak more than phage surface 10min, washes lower training
Foster thing, is collected in sterile glass vials.
2. the bacteria suspension gathering is heated to 80 DEG C by bacterium solution inactivation and inactivation inspection, maintains 120min, treats that it is naturally cold
But, after, it is stored in 4 DEG C.Inactivated bacterial liquid should be carried out with inactivation inspection.Take inactivated bacterial liquid 0.1ml coating pancreas agar or tsa flat board, 37
DEG C culture 7 days, do not answer asepsis growth.
Bacteria suspension qualified for inactivation inspection is centrifuged 20min with 10000g by the purification of 3.ops, collects precipitation.Weigh and count
Calculate thalline weight in wet base, thalline weight in wet base is added in 2% (v/v) acetic acid in 1:8 (w/v) ratio, after fully mixing, 121 DEG C of high pressure go out
Bacterium 15min.Cool down rear 4 DEG C of 10000g centrifugation 20min and remove bacterial chip.Final concentration of 5% trichlorine is added in supernatant
Acetic acid, after 15min is stirred at room temperature, 4 DEG C of 10000g are centrifuged 10min, discard precipitation.Add a certain amount of protease k so as to end is dense
Degree reaches 50 μ g/ml, puts 56 DEG C of water-baths and digests 2 hours.Supernatant is put dialysed overnight in 100 times of distilled water, is concentrated with peg8000
After 40 times, it is again placed in distilled water dialysed overnight, collects bag filter content, be distributed into 2ml/ bottle lyophilizing and preserve, this purifies
Ops.
The labelling of 4.ops takes 4mg ops to be added in the triethylamine solution of 2ml 0.5% (v/v), puts supersound process on ice
15min, the ultrasonic rear edta solution adding 200 μ l 100mmol/l, and with the hydrochloric acid of 10 μ l 1mol/l, solution ph is adjusted to
5.0.(isomer i) is dissolved in 800 μ l 0.25mol/l boric acid solution (ph10.5), then all adds to take 20mg fitc
To in ops solution, continue supersound process 1min.Add 1ml 1.6% sodium deoxycholate solution, 37 DEG C of stirrings are incubated 18 hours.4
DEG C 10000g centrifugation 30min, supernatant is transferred in bag filter and is concentrated with peg6000, is subsequently dialysed 1 hour with pbs, will be thoroughly
Analysis thing pd-10 post desalination, collects all fitc-ops labels, uses peg6000 reconcentration after merging, finally saturating with pbs again
Analysis 1 hour, collects 4 DEG C of preservations of product after dialysis, as labelled antigen stock solution.
Embodiment 3
The preparation of negative control sera and positive control serum and touchstone in the application test kit
1. the preparation of negative control sera
(1) animal rose bengal precipitation test, tube agglutination test and complement fixation test are all detected as brucella
About 2 one full year of life of negative antibody, sexually matured health cattle.
(2) serum preparation jugular vein blood collection, separates serum, with 0.45 μm of filter filtration sterilization, the serum addition of collection
0.05%proclin300 is anti-corrosion, aseptic subpackaged, and after the assay was approved, -20 DEG C save backup.
2. the inspection of negative control sera
(1) character is orange or faint yellow supernatant liquid.
(2) steriling test is tested by existing " Chinese veterinary pharmacopoeia " annex, answers asepsis growth.
(3) specific assay should be through rose bengal precipitation test, tube agglutination test and complement fixation test inspection
Negative.Using the negative control sera of preparation as serum to be checked, carry out fpa detection, it 70 < mp < 95.
3. the preparation of positive control serum
(1) animal screening brucella rose bengal precipitation test, tube agglutination test and complement fixation test are to facing
Bed shows about 2 one full year of life of health, sexually matured health cattle is detected, screens brucellosis negative antibody cattle.
(2) brucella is made bacteria suspension with reference to virulent strain m28 inactivation antigen, with 5 × 10 by serum preparation10Cfu/ cattle neck
Portion's subcutaneous inoculation, doubling dosage booster immunization 1 time after 3 months.Blood sampling in 2 weeks after booster immunization.Measured solidifying with tube agglutination test
Collection potency, when potency is more than 800iu/ml, venesection, separate serum.According to the potency of practical measurement, negative with cattle brucellosis
Its potency is diluted to 320iu/ml by serum, with 0.45 μm of filter filtration sterilization, adds proclin 300 to final concentration of
0.05%, -20 DEG C save backup, as brucellosis positive serum in test kit.
4. the inspection of positive control serum
(1) character is orange or faint yellow supernatant liquid.
(2) steriling test is tested by " Chinese veterinary pharmacopoeia " annex, answers asepsis growth.
(3) titration adopts tube agglutination test to measure the potency of positive control serum, should be 1:320.By preparation
Positive control serum, as serum to be checked, carries out fpa detection, and it 120 < mp < 250.By positive control serum negative control blood
Carry out clearly 1:64 to dilute as serum to be checked, carry out fpa detection, calculate △ mp, 20 should be not less than.
Embodiment 4
The preparation of test kit other component and touchstone
1. the preparation of labelled antigen and inspection
(1) labelled antigen prepare labelled antigen stock solution antigen protective agent (commercialization) be diluted to 250,500,1000,
2000 times, with the reduction of antigen concentration, the sensitivity of fpa detection steps up, when labelled antigen is made 1:1000 times and is diluted,
The sensitivity of fpa detection method is 5iu/ml, consistent with elisa method and Brucella antibody test strip sensitivity, because
This determines optimal antigen diluent concentration.Dilution antigen is fpa test kit labelled antigen, keeps in Dark Place in 4 DEG C of brown bottles.
(2) check
1) character colourless transparent liquid.
2) steriling test is tested by existing " Chinese veterinary pharmacopoeia " annex, answers asepsis growth.
The preparation of 2.25 × sample diluting liquid and inspection
(1) prepare and weigh na2hpo4·12h2O 6.25g, nah2po4·2h2O 87.5g, nacl212.5g, tween-20
12.5ml, sucrose 125g, plus deionized water to 800ml, adjusts ph value 7.2～7.6, is settled to 1000ml.Add proclin 300
Solution make its final concentration of 0.05%, after filtration sterilization, aseptic subpackaged.
(2) check
1) character is colourless transparent liquid.
2) steriling test is tested by " Chinese veterinary pharmacopoeia " annex, answers asepsis growth.
3) ph value should be 7.2～7.6.
Embodiment 5
The selection of brucella fluorescence polarization test kit (fpa) diluent
Select carbonate buffer solution, pbst and the phosphate buffer containing 0.5% sucrose as sample diluting liquid respectively, carry out
Fpa tests, and positive serum reaction △ mp value soprano is as the best diluent of effect.Result by being coated the ratio of liquid to 3 kinds
Relatively, show that the phosphate buffer containing 0.5% sucrose is best (the results are shown in Table 1) as the dilution effect of diluent.
13 kinds of diluent fpa results contrast of table
Note: the meansigma methodss of the mp negative serum mp of △ mp=sample
Embodiment 6
The preparation of brucella fluorescence polarization (fpa) detection kit positive control serum
With brucella rose bengal precipitation test, tube agglutination test and complement fixation test to clinical manifestation health
About 2 one full year of life, Adult Bovine is detected, screens brucellosis negative antibody cattle.Negative cattle brucella reference virulent strain to screening
M28 inactivation antigen makes bacteria suspension, with 5 × 1010Cfu/ cattle cervical region subcutaneous inoculation, doubling dosage booster immunization 1 time after 3 months.
Blood sampling in 2 weeks after booster immunization.Measure agglutination titer with tube agglutination test, when potency is more than 800iu/ml, venesection, point
From serum.According to the potency of practical measurement, with cattle brucellosis negative serum, its potency is diluted to 320iu/ml, with 0.45 μm of filter
Filtration sterilization, adds proclin 300 to final concentration of 0.05%, -20 DEG C save backup, as brucellosis positive blood in test kit
Clearly.
1. brucellosis feminine gender sheep screening
Using rose bengal precipitation test, tube agglutination test and complement fixation test, clinical trial Niu Jinhang is detected,
The Niu Zuowei that three kinds of methods are all detected as feminine gender is selected to prepare the candidate cattle of positive serum.Have selected numbering in result test is
912nd, 936,963 three parts of Virus monitory, the results are shown in Table 2.3 parts of blood serum samples to be checked are gone out with brave red antigen all no agglutinating particles
Existing, in uniform muddiness, it is judged to brucellosis negative antibody.Comparison positive serum and brave red antigen are in obvious agglutinating particle, and liquid is almost
Fully transparent.3 parts of 4 different dilution factor (1:50,1:100,1:200,1:400) knots of blood serum sample to be checked in tube agglutination test
Fruit is feminine gender.In complement test, equal 100% haemolysis of 3 parts of blood serum samples to be checked, is judged to brucellosis negative antibody.
The testing result to serum to be checked for the 2 three kinds of distinct methods of table
Note: (1) rose bengal precipitation test result explanation: ++++: big coagulation piece or granule occur, liquid is completely saturating
Bright；+++: there is obvious agglutinating particle, liquid is almost fully transparent；++: there is obvious agglutinating particle, liquid is slightly transparent；+:
Coagulation, opaque slightly can be seen；: no coagulation, liquid is uniformly muddy.
(2) tube agglutination test result explanation: the complete coagulation of 4+: thalline and precipitation, liquid 100% is limpid；3+: thalline is several
Coagulation and precipitation completely, liquid 75% is limpid；2+: have significant coagulation and precipitation, liquid 50% is limpid；1+: have and clearly may be used
The coagulation seen and precipitation, liquid 25% is limpid；: no coagulation and precipitation, liquid bacterium solution is muddy.
(3) Evaluation of Complement Fixation Test explanation: p: positive；N: negative.
2. immunity
Immunity is general only need to be carried out twice.Just exempt from: with 2ml brucella m28 inactivation antigen (5 × 1010Cfu/ml) cervical region
The negative cattle of subcutaneous inoculation brucellosis.Booster immunization: after just exempting from 3 months, the subcutaneous booster immunization of doubling dosage cervical region 1 time.Reinforcement is exempted from
Blood sampling in 2 weeks after epidemic disease, measures agglutination titer with tube agglutination test and should be not less than 1:800.If potency does not reach standard, continue to strengthen
Immunity 1～2 time, tries blood after 15 days again, until potency is qualified.The present invention 912,936 and No. 963 cattle are adopted after carrying out secondary immunity
It is respectively 1:900,1:1000,1:1200 with the potency that tube agglutination test measures.
3. serum preparation
Two exempt from 14 after blood sampling, measure tube agglutination potency, further with negative control sera by detached positive serum
Being diluted to expected potency in proportion is 1:320, adds proclin 300 (supelco company) to final concentration of 0.05%, aseptic
Subpackage, as positive control serum to be checked.
4. check
The positive control serum that preparation is completed is tested by following project:
(1) character observation serum product color and luster is yellowish or blush clear liquid.
(2) steriling test is tested by existing " Chinese veterinary pharmacopoeia " annex, is asepsis growth.
(3) serum is made 1:140 by titration, and 1:150,1:160,1:170,1:180 dilution carries out tube agglutination test,
Measure its potency.The serum 1:64 of preparation is diluted as serum to be checked, carries out fpa detection, calculate △ mp, 20 should be not less than.
Embodiment 6
Test kit sensitivity testss
1. the sensitivity test of the Brucella antibody positive control serum of dilution
Brucella antibody positive control serum is carried out after 2 times of gradient dilutions, makes test kit by oneself with 3 batches of laboratorys and carry out
Detection, determines the sensitivity of the positive control serum to gradient dilution for this test kit, the results are shown in Table 3.From 3,3 crowdes of fpa of table
Test kit detects that the result of gradient dilution positive control serum is consistent, and serum titer all detects to 1:64 times (△ mp value >=20).
The sensitivity technique of the positive control serum to gradient dilution for the table 3
Note: the criterion of brucella fluorescence polarization (fpa) detection kit is: during sample △ mp >=20, cloth Lu Shi
The bacteria antibody positive (be designated as "+")；Sample △ mp value < when 20, is Brucella antibody feminine gender (being designated as "-").
2. the sensitivity testss of positive sample known to pair
Using 3 batches of brucella fluorescence polarization (fpa) detection kit to 148 parts of brucellosis positive serum (wherein Ox blood serums
70 parts, 78 parts of sheep blood serum) detected, the coincidence rate of brucellosis positive serum sample is 98.0% (145/148), the results are shown in Table
4 and table 5 it is shown that the reliability of the higher sensitivity of test kit and diagnostic result.
The sensitivity Detection to known positive sample for the table 4
Note: the criterion of brucella fluorescence polarization (fpa) detection kit is: during sample △ mp >=20, cloth Lu Shi
The bacteria antibody positive (be designated as "+")；Sample △ mp value < when 20, is Brucella antibody feminine gender (being designated as "-").
The sensitivity analyses to known positive serum for the table 5
Note:
Fpa method operation instruction
1 usage
1.1 sample treatment take animal's whole blood, after blood coagulation, are centrifuged 10min with 4000r/min, collect supernatant, serum
Should be limpid, no haemolysis.
The preparation of 1.2 1 × sample diluting liquid, using front, 25 × sample diluting liquid is recovered to room temperature (20～25 DEG C), and
Shake, makes crystallization dissolve (can heat 5～10min in 37 DEG C of water-baths), and then deionized water makees 1:25 dilution, fully mixed
Even.
1.3 operating procedure
1.3.1 take Sptting plate, serum to be checked, positive control serum and negative control sera are added separately to elisa plate
In, 20 μ l/ holes, wherein positive control serum add 1 hole (hole 1) and negative control sera respectively adds 3 holes (hole 2, hole 3, hole 4).Sample-adding
Process is it is noted that avoid producing bubble.
1.3.2 every hole adds 180 μ l sample diluting liquids, fully mixes；Every hole is read after incubation reaction plate 3～5min under room temperature
Blank value.
1.3.3 every hole adds 10 μ l fluorescent labeled antigens immediately, fully mixes；After incubation reaction plate 3～5min under room temperature
Read every hole polarization value.
2 result judgements
2.1 computing formula: polarization value (△ mp)=(sample mp negative control mp meansigma methodss)
2.2 test establishment conditions: the value in 3 negative control sera replication holes all should be in 70～95mp；1 positive is right
Value according to serum hole should be tested and set up in 120～250mp.
2.3 result judgements: as the △ mp >=20mp of serum sample, be brucellosis antibody positive；Work as serum sample
△ mp ＜ 20mp when, be brucellosis negative antibody.
Sequence table
<110>China Veterinery Drug Inspection Office
<120>brucella fluorescence polarization (fpa) detection kit
<130>
<160> 4
<170> patentin version 3.5
<210> 1
<211> 21
<212> dna
<213>artificial sequence
<223>description to artificial sequence: primer feri
<400> 1
Gcgccgcgaa gaacttatca a 21(sequence 1)
<210> 2
<211> 20
<212> dna
<213>artificial sequence
<223>description to artificial sequence: primer reri
<400> 2
Cgccatgtta gcggcggtga 20(sequence 2)
<210> 3
<211> 23
<212> dna
<213>artificial sequence
<223>description to artificial sequence: primer fsuis
<400> 3
Gcgcggtttt ctgaaggttc agg 23(sequence 3)
<210> 4
<211> 24
<212> dna
<213>artificial sequence
<223>description to artificial sequence: primer ris711
<400> 4
Tgccgatcac ttaagggcct tcat 24(sequence 4)
1
Claims (3)
1. a kind of brucella fluorescence polarization detection kit is it is characterised in that this test kit is by brucella fluorescent labeled antigen
Ops, brucella positive control serum, negative control sera and sample diluting liquid composition.
2. as claimed in claim 1 a kind of brucella fluorescence polarization detection kit it is characterised in that brucella therein
Fluorescent labeled antigen ops is the small molecule piece of the fluorescent labeling smooth type brucella s2 strain lipopolysaccharide of detection Brucella antibody
Section, this ops is respectively provided with good specificity and sensitivity to common brucella pathogenic autoantibody.
3. the application of the brucella fluorescence polarization detection kit described in claim 1, this can detect in many animals serum
Brucella specific antibody, that is, this test kit can carry out high flux quick detection to the brucellosis of pig, cattle, sheep.
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| CN111024947A (en) * | 2019-11-19 | 2020-04-17 | 江苏美克医学技术有限公司 | Candida albicans fluorescence immunochromatography assay kit and preparation method thereof |
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