CN108226496A - A kind of method for detecting brucella - Google Patents
A kind of method for detecting brucella Download PDFInfo
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- CN108226496A CN108226496A CN201810084667.2A CN201810084667A CN108226496A CN 108226496 A CN108226496 A CN 108226496A CN 201810084667 A CN201810084667 A CN 201810084667A CN 108226496 A CN108226496 A CN 108226496A
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- brucella
- outer membrane
- membrane protein
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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
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/23—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Brucella (G)
Abstract
The invention discloses a kind of method of quick detection Infected with Brucella, this method is solved difficult present in current brucella detection using brucella thalline in the antibody test sample of specific recognition brucella outer surface epitope.Contribute to diagnosis, the prevention and treatment effect assessment of brucellosis.
Description
Technical field
The present invention relates to biotechnologies, and in particular to a kind of method for detecting brucella.
Background technology
Brucellosis（Brucellosis）It is one of current most wide infectious diseases common to human beings and animals popular in the world, according to the world
Health organization statistics shows that 500,000 people's brucellosis occurs every year in the whole world, it is annual caused by economic loss up to 3,000,000,000 dollars
.The popular mixed infection based on sheep kind Brucella of China's cloth disease, sheep kind account for the 80% of epidemic strain.
Brucellosis clinical symptoms are complicated, and the pathological lesions such as caused fever, miscarriage are easily obscured with certain common diseases；Doctor
It is difficult to diagnose Brucella infection only according to ill domestic animal contact history is whether there is to give birth to, and brings certain difficulty to diagnosis, is easy to cause mistake
It examines, causes chronic infection.On the other hand lacking effective treatment means, a large amount of be used for a long time of antibiotic remains difficult to take effect, and
Easily cause drug resistance problems.Therefore, the vaccine prevention of Brucella and Rapid＆Early diagnosis have the prevention and control of cloth bacterium great
Realistic meaning；The wherein immune effect of vaccine and the accuracy of cloth bacterium diagnosis is just particularly important.
Although biochemical culture is the goldstandard of brucellosis detection, but this method, in the presence of time-consuming, bio-safety etc. is square
The many limitations in face, therefore be still the serological method that mainly uses with the high Serological testing of specificity, as China is main
Primary dcreening operation and the diagnosis of brucellosis are carried out by the method that RBPT and SAT are combined, judges therapeutic effect.But both are detected
Method all there are problems that, first, cannot distinguish between the problem of natural infection and vaccine immunization generation antibody, seriously affect
The detection of brucellosis and vaccine immunity preventative strategies.Second is that RBPT method false positive rates are higher, the operation of SAT methods is relatively complicated,
The consuming time is long, is susceptible to false negative result.M5-90 is attenuation sheep kind Brucella vaccine strain, and OMP31 albumen is fenestra
Albumen, it is related to the weak toadstool seedling strain virulence, and contain protective epitope, gene order is between different kinds
Homology is up to more than 98%.Therefore, using the monoclonal antibody for OMP31 albumen, can establish Brucella pathogen and
Brucellosis diagnostic method and its diagnostic reagent, and then solve the side such as brucellosis antidiastole, prevention, treatment and Index for diagnosis
The technical barrier in face.
Invention content
The defects of in order to make up prior art, the present invention provide a kind of method for detecting brucella.
The technical problems to be solved by the invention are achieved by the following technical programs：
A kind of method for detecting brucella, which is characterized in that using immunofluorescence technique or Flow Cytometry to cloth Lu Shi
Bacterium is detected.
Further, antibody specificity identification brucella outer membrane protein used is detected to brucella or fat is more
Sugar.
Further, antibody specificity identification brucella lipopolysaccharides used, cloth Lu Shi are detected to brucella
Bacterial outer membrane protein OMP10, brucella outer membrane protein OMP16, brucella outer membrane protein OMP19, brucella outer membrane protein
One in OMP21, brucella outer membrane protein OMP28, brucella outer membrane protein OMP31, brucella outer membrane protein OMP35
Kind is several.
Further, one or more be detected to brucella in the following epitope of antibody specificity identification used
It is a：
（1）The 27aa-42aa of brucella outer membrane protein omp10；
（2）The 37aa-52aa of brucella outer membrane protein OMP16；
（3）36aa-50aa, 67aa-80aa of brucella outer membrane protein OMP19；
（4）The 57aa-71aa of brucella outer membrane protein OMP21；
（5）The 33aa-49aa of brucella outer membrane protein OMP25；
（6）22aa-35aa, 47aa-62aa, 102aa-121aa of brucella outer membrane protein OMP28；
（7）42aa-57aa, 77aa-91aa, 95aa-102aa of brucella outer membrane protein OMP31.
Further, it is specific recognition brucella outer membrane protein that antibody used is detected to brucella
The mixture or specific recognition brucella outer membrane egg OMP25 and cloth of OMP31 and brucella outer membrane protein OMP28
The mixture of Shandong Salmonella outer membrane egg OMP28.
Further, brucella is detected in antibody incubation system used and adds polyethylene glycol.
Further, the polyethylene glycol is PEG1500.
The present invention has the advantages that：
The detection of brucella never has a kind of not only simple and efficient, but also accurate method.Present invention utilizes antibody identification is anti-
Former high specific and immunofluorescence technique, Flow Cytometry it is easy, quick the features such as have devised and a kind of conscientiously may be used
Capable rapid detection method.This method does not need to that brucella sample is carried out to operate up to the culture as long as two weeks, detection
Sensitivity has also reached molecule rank, solves the problems, such as that existing biochemical culture method omission factor is high, has fabulous application
Prospect.
Specific embodiment
With reference to embodiment, the present invention will be described in detail, and embodiment is only the preferred embodiment of the present invention,
It is not limitation of the invention.
1 immunofluorescence technique of embodiment detects human peripheral sample
(1) peripheral blood mononuclear cells is isolated from human peripheral（PBMCs）, and with the cell culture medium equal with blood volume
It is resuspended；
(2) according to 100 μ L/ holes, by PBMCs plating cells in 96 porocyte culture plates, it is placed in 37 DEG C of 5% carbon dioxide environment
Middle culture is for 24 hours；
(3) supernatant is discarded, the methanol of 100 μ L, 4 DEG C of precoolings is added in per hole, in -20 DEG C of fixed 20min；
(4) liquid is abandoned, 100 μ L IF Buffer are added in per hole（1 × PBS, 1%BSA, 2.5 mM EDTA, pH7.2）, room temperature incubates
Educate 1 h；
(5) liquid is discarded, brucella detection antibody is diluted to 0.2 μ g-2 μ g with IF Buffer, 100 μ L, room are added in per hole
Temperature is incubated 1h；
(6) liquid is discarded, with 1 × PBST（20 contents 0.05% of Tween, pH7.2）Wash micropore three times；
(7) with IF Buffer by sheep anti-mouse igg-FITC（Fluorescein isothiocynate）3000 times of dilution adds in 100 μ L, room per hole
Temperature is incubated 1h；
(8) liquid is discarded, with 1 × PBST（20 contents 0.05% of Tween, pH7.2）Wash micropore three times；
(9) sentence read result under fluorescence microscope：Yellow green luminous point prompts for brucella thalline, is otherwise negative findings.
(10) experimental result is referring to table 1.
Table 1 detects brucella in 187 parts of human blood samples using Flow Cytometry
* it is performed with reference to " WS268-2007 brucellosis diagnostic criteria "
Brucella in 2 Flow Cytometry of embodiment detection human peripheral blood cell
(1) peripheral blood mononuclear cells is isolated from peripheral blood（PBMCs）, with 1 × PBS（pH7.2）PBMCs cells are resuspended, and
It is 1 × 10 to adjust cell concentration6A/ml；
(2) 100 μ L are taken in EP pipes, 1ml is added in and fixes/rupture of membranes working solution（Such as：The fix＆Perm ruptures of membranes of Invitrogen
Agent）, it is incubated at room temperature 40min；
(3) 1000rpm centrifuges 10min, discards supernatant；
(4) with 1 × PBST of 1mL（20 contents 0.05% of Tween, pH7.2）Cell is resuspended, 1000rpm centrifugation 10min are discarded
Supernatant；It is primary to repeat the operation of this step；
(5) the brucella detection antibody of 200 μ L FITC (fluorescein isothiocynate) labels is added in, cell is resuspended, is incubated in 37 DEG C
Educate 1h；
(6) with 1 × PBST of 1mL（20 contents 0.05% of Tween, pH7.2）Cell is resuspended, 1000rpm centrifugation 10min are discarded
Supernatant；It is primary to repeat the operation of this step；
(7) with 200 1 × PBS of μ L（pH7.2）Cell is resuspended；
(8) cell is detected with flow cytometer.
(9) experimental result is referring to table 2.
Table 2 detects brucella in 187 parts of human blood samples using Flow Cytometry
* it is performed with reference to WS268-2007 brucella and diagnostic criteria
3 immunofluorescence technique of embodiment detects sheep aborted fetus sample
(1) prepared by sample
Smear preparation method：First by glass slide by flame 3 times, after cooling, with oese（Or platinum ear）Select tested material
It is uniformly coated into the round smear of diameter about 1cm.If material is too dense, then it should add appropriate sterile saline in advance in another slide
On, with the mixed dilution of oese picking material, physiological saline can also be added in tested material, mixed by the smear after uniformly
Smear after closing uniformly.After coating, naturally dry.
Imprint sample method：Tissue specimen to be checked is cut off with sterile scissors, with sterile cotton balls or the filter paper of cleaning by section
Blood blot, then with the light forming dough into noodles of glass slide, be allowed to be stained with 1-2 confluent monolayer cells, then be applied again at another place of slide with section
It wipes, obtains a thicker smear, by sample naturally dry.
(2) sample is fixed：3-10min is fixed with 10% formaldehyde room temperature.
(3) slide sample fixed is placed in wet disk, 0.01mol/L is added dropwise, the PBS of pH7.4 is in sample slice to be checked
On, it is discarded after 10min, sample is made to keep certain humidity.
(4) antibody-solutions of appropriate diluted fluorescent marker are added dropwise, make it that sample area be completely covered.
(5) slide is placed in light tight container with cover（Bottom pad is put glass frame on paper, suitable quantity of water is added to make with filter paper
Filter paper soaks, and plate, the built-in 2-3 cotton balls soaked can be used when slide quantity is few）, Mi Gai is positioned over the dyeing of 37 DEG C of incubators
30min。
(6) slide is taken out, is put in glass frame, unbonded labelled antibody liquid is first washed away with PBS, is then put in a large amount of PBS
It rinses 15min or slowly rinses slide using deionized water or distilled water and repeatedly dry afterwards, be then soaked in 1min in PBS.
(7) slide is taken out, excessive moisture is sucked with filter paper, sample moistening is kept, a drop is added to buffer glycerine, is covered with coverslip
Lid.
(8) fluorescence microscope is used immediately.
(9) experimental result is referring to table 3.
3 immunofluorescence technique of table detects 130 parts of sheep aborted fetus samples
* it is performed with reference to " GB/T 18646-2002 animal brucellosis diagnostic techniques "
Brucella pollutes in 4 Flow Cytometry of embodiment detection milk
(1) 200 μ L milk are taken in EP pipes, add in 20 μ g Proteinase Ks, 5 μ L Triton X-100,56 DEG C of processing 15min；
(2) 13000rpm centrifuges 5min；
(3) it inhales and abandons supernatant, retain precipitation；
(4) precipitation is placed in boiling water bath 10min；
(5) 200 1 × PBS of μ L are added in（pH7.4）Precipitation is resuspended；
(6) the brucella detection antibody of 200 μ L fluorescent markers is added in, mixing is incubated 0.5h in 37 DEG C；
(7) 1 × PBST of 1mL are added in（20 contents 0.05% of Tween, pH7.2）, mixing, 13000rpm centrifugation 5min, discard
Supernatant；It is primary to repeat the operation of this step；
(8) with 200 1 × PBS of μ L（pH7.2）Thalline is resuspended；
(9) thalline is detected with flow cytometer.
(10) experimental result is referring to table 4.
4 Flow Cytometry of table detects brucella in 104 portions of milk
* it is performed with reference to " NY/T 1467-2007 milk Bovine brucellosis PCR diagnostic techniques "
Influence of the 5 different disposal liquid of embodiment for recall rate
We have found that positive detection can be significantly improved in brucella cohesive process, adding polyethylene glycol in detection antibody
Rate.It is explained below with embodiment 5.
(1) 200 μ L milk are taken in EP pipes, add in 20 μ g Proteinase Ks, 5 μ L Triton X-100,56 DEG C of processing 15min；
(2) 13000rpm centrifuges 5min；
(3) it inhales and abandons supernatant, retain precipitation；
(4) precipitation is placed in boiling water bath 10min；
(5) 200 1 × PBS of μ L are added in（pH7.4）Precipitation is resuspended；
(6) the brucella detection antibody of 200 μ L fluorescent markers is added in（Contain 0,0.5%, 1%, 3%, 5%, 7%, 10%
PEG1500）, mixing, in 37 DEG C of incubation 0.5h；
(7) 1 × PBST of 1mL are added in（20 contents 0.05% of Tween, pH7.2）, mixing, 13000rpm centrifugation 5min, discard
Supernatant；It is primary to repeat the operation of this step；
(8) with 200 1 × PBS of μ L（pH7.2）Thalline is resuspended；
(9) thalline is detected with flow cytometer.
(10) experimental result is referring to table 5.
Influences of the PEG for testing result is added in 5 antibody incubation system of table
Embodiment described above only expresses embodiments of the present invention, therefore description is more specific and detailed, but can not be
And the limitation to the scope of the claims of the present invention is interpreted as, as long as the technical side obtained using the form of equivalent substitution or equivalent transformation
Case should all be fallen within the scope and spirit of the invention.
Claims (7)
- A kind of 1. method for detecting brucella, which is characterized in that using immunofluorescence technique or Flow Cytometry to cloth Shandong Salmonella is detected.
- 2. the method for detection brucella as described in claim 1, which is characterized in that used in brucella is detected Antibody specificity identifies brucella outer membrane protein or lipopolysaccharides.
- 3. the method for detection brucella as claimed in claim 2, which is characterized in that used in brucella is detected Antibody specificity identification brucella lipopolysaccharides, brucella outer membrane protein omp10, brucella outer membrane protein OMP16, Bu Lu Salmonella outer membrane protein OMP19, brucella outer membrane protein OMP21, brucella outer membrane protein OMP28, brucella outer membrane egg One or more of white OMP31, brucella outer membrane protein OMP35.
- 4. the method for detection brucella as claimed in claim 3, which is characterized in that used in brucella is detected Antibody specificity identifies one or more of following epitope：（1）The 27aa-42aa of brucella outer membrane protein omp10；（2）The 37aa-52aa of brucella outer membrane protein OMP16；（3）36aa-50aa, 67aa-80aa of brucella outer membrane protein OMP19；（4）The 57aa-71aa of brucella outer membrane protein OMP21；（5）The 33aa-49aa of brucella outer membrane protein OMP25；（6）22aa-35aa, 47aa-62aa, 102aa-121aa of brucella outer membrane protein OMP28；（7）42aa-57aa, 77aa-91aa, 95aa-102aa of brucella outer membrane protein OMP31.
- 5. the method for detection brucella as claimed in claim 3, which is characterized in that used in brucella is detected Antibody is mixture or the spy of specific recognition brucella outer membrane protein OMP31 and brucella outer membrane protein OMP28 The mixture of opposite sex identification brucella outer membrane egg OMP25 and brucella outer membrane egg OMP28.
- 6. the method for detection brucella as described in claim 1, which is characterized in that used in brucella is detected Polyethylene glycol is added in antibody incubation system.
- 7. the method for detection brucella as claimed in claim 6, which is characterized in that the polyethylene glycol is PEG1500.
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Cited By (3)
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| CN110218763A (en) * | 2019-05-27 | 2019-09-10 | 厦门大学 | A method of in edible raw egg pathogenic bacteria and total bacteria count carry out quantitative detection |
| CN110592184A (en) * | 2019-09-30 | 2019-12-20 | 通辽市地方病防治站 | Method for extracting and identifying Brucella genome DNA |
| CN117603366A (en) * | 2024-01-19 | 2024-02-27 | 北京纳百生物科技有限公司 | Brucella specific fusion antigen and application thereof |
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