CN104911269B - Differentiate primer, probe and the kit of brucella A19 vaccine strains in aerosol - Google Patents
Differentiate primer, probe and the kit of brucella A19 vaccine strains in aerosol Download PDFInfo
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Abstract
The invention discloses a kind of primer, probe and kit for differentiating brucella A19 vaccine strains in aerosol, Brucella bacterium provided by the invention and the RPA LFD primers and probe combinations and kit high sensitivity, high specificity of A19 vaccine strain antidiastoles are minimum to detect 3 × 100Cfu Brucella bacterium or brucella A19 vaccine strain bacteriums, kit provided by the invention not only can be used for differentiating brucella A19 vaccine strains bacterial strain and aerosol sample, it may also be used for the discriminating detection of other clinical samples such as blood, milk sample etc..
Description
Technical field
The invention belongs to biological technical field, and in particular to be examined using recombinase polymeric enzymatic amplification-Sidestream chromatography test strips
Survey technology (Recombinase Polymerase Amplification and Lateral Flow Dipstick, RPA-
LFD) primer of brucella A19 vaccine strains and probe and kit in the clinical sample such as rapid differential diagnosis aerosol.
Background technology
Brucellosis (Brucellosis) is by Brucella bacterium (predominantly ox kind, sheep kind, pig kind and kind of dog
Brucella etc.) caused by the infectious disease suffered from altogether of people and a variety of domestic animals, and the important pathogen of the legal quarantine in China and purification.Closely
Ascendant trend year by year is presented in the incidence of disease over year, especially very harmful to dairy stock, and abortion ratio is up to 50%-80%, breast, meat production
Reduce 10%-20%.According to《Animal doctor's publication》Brucellosis occurs altogether for statistics, the whole nation in 2013 3620 times, 42720 cattle and sheep hairs
Disease.Therefore, brucellosis to socio-economic development with stably, National Public Health safety etc. constitute a serious threat.
The infection sources of brucellosis is mainly infected animal and carrier, and they will by secretion, milk, body fluid etc.
Brucella is excreted, and pollutes environment, while brucella is adhered on aerial dust suspended particles, forms aerosol
And then propagate, infect other animals and people.The distribution of brucella aerosol in plant's environment can cause brucellosis
Infection with it is popular, bring seriouss harm to Animal husbandry production and human health, especially current intensive, high-density breeding ring
The prevalence of brucella aerosol is more readily formed under border.Therefore, by monitoring brucella aerosol in plant's environment,
Can effectively early-warning and predicting brucellosis break out with it is popular.
The main policies of China's animal brucellosis prevention and control are quarantine and purification.Conventional ox is immunized in China cows at present
Kind brucella A19 live vaccines strain can interfere in the diagnosis of antigen and antibody to brucellosis wild virus infection.This is
Because the brucellosis diagnostic method that China uses at present immune to natural infection and artificial vaccines can not carry out antidiastole,
Necessarily cause the ill domestic animal evading quarantine inspection of some natural infections, make infected animal long-term existence, it is long-term to the safety of cows and the mankind
Constitute a threat to, therefore, China is difficult the quarantine for carrying out brucellosis, the prevention and control code slaughtered, purified.
The content of the invention
It is an object of the invention to provide a kind of primer, probe and reagent for differentiating brucella A19 vaccine strains in aerosol
Box, the kit being capable of brucella A19 vaccine strains in special, sensitive, simple, quick live antidiastole aerosol.
To realize the object of the invention, adopt the following technical scheme that：
The primer pair and probe combinations of brucella A19 vaccine strains, wherein Brucella-RPA- in a kind of discriminating aerosol
LFD forward primer sequences are as shown in SEQIDNo.1, and reverse primer sequences are as shown in SEQIDNo.2, and probe sequence is such as
Shown in SEQIDNo.3；A19-RPA-LFD forward primer sequences are as shown in SEQIDNo.4, reverse primer sequences such as SEQIDNo.5
Shown, probe sequence is as shown in SEQIDNo.6.
Present invention also offers a kind of kit for differentiating brucella A19 vaccine strains in aerosol, the kit includes
Above-mentioned primer pair and probe combinations.
It is used for brucella A19 vaccine strains in rapid differential diagnosis aerosol sample invention further provides a kind of
Method, comprise the following steps：
(1) extraction or the cracking processing of detection sample of sample DNA are detected；
(2) using the sample of processing in step (1) as template, RPA amplifications are carried out；
(3) the above-mentioned RPA amplified productions of Sidestream chromatography ELISA test strip are used, judge whether contain in sample according to testing result
Brucella A19 vaccine strains.
Preferably, RPA amplification systems are described in step (2)：RPA reaction systems are 50 μ L：Including 2.1 μ L
10 μM of forward primer, 2.1 10 μM of μ L reverse primer, 0.6 10 μM of μ L probe, the μ L of rehydration buffer solutions 29.5,
Sample to be tested DNA or thick pyrolysis products 2 μ L, ddH2O 11.2μL；Above-mentioned 47.5 μ L mixtures are added into RPA-nfo reaction tubes,
Fully mix, dissolving, be eventually adding the 280mM μ L of magnesium acetate solution 2.5, mixing of turning upside down is after 38 DEG C of reactions of water bath with thermostatic control
20-30 minutes.
Preferably, step (3) concretely comprise the following steps：Each RPA reaction products are taken to take 2 μ L and 98 μ L PBST test strips
Buffer solution (1 × PBS+0.1% polysorbas20s) mixing is detected, a flow measurement chromatograph test strip (LFD) is immersed, is observed within 5min
As a result, if Brucella-RPA-LFD test strip and control stripes band occurs simultaneously with two detection combinations of A19-RPA-LFD,
Brucella A19 vaccine strains are positive in the sample；There is test strip and control stripes band in Brucella-RPA-LFD detection groups, and
Only there is control stripes band in A19-RPA-LFD detections group, then illustrates to contain other beyond brucella A19 vaccine strains in the sample
Brucella；There is not test strip and occurred simultaneously pair in Brucella-RPA-LFD and two detection groups of A19-RPA-LFD
According to band, then Brucella bacterium is not contained in explanation detection sample.There are other situations and then illustrate RPA-LFD brucella
A19 differentiates that detection is invalid.
The present invention also provides is used for antidiastole brucella A19 vaccine strains containing above-mentioned two groups of primers and probe combinations
Kit.Also the recA recombinases including Escherichia coli, strand displacement archaeal dna polymerase, single stranded DNA combine in the kit
(specific detection is given birth to for albumen (SSB), rehydration buffer solutions, 280mM magnesium acetates (MgAc) solution, Sidestream chromatography test strips
Thing element with FAM marker gene amplified production), PBST ELISA test strips buffer solution (1 × PBS+0.1% polysorbas20s), sterilizing go from
Sub- distilled water (ddH2O), TE buffer solutions, 10% (w/v) SDS, 2% (w/v) Proteinase K, brucella A19 vaccine strain standard males
Property template.
Using brucella A19 vaccine strains and other brucella genome difference DNA sequence dnas as target sequence is differentiated, apply
RPA-LFD technologies, it is possible to achieve brucella A19 vaccine strains and other brucella strain scene rapid differential diagnosis.RPA skills
Art, can be with reference to recombinase, the single-stranded DNA binding protein of single-chain nucleic acid (Oligonucleolide primers) by the mixture of three kinds of enzymes
(SSB) and strand displacement archaeal dna polymerase, also active at normal temperatures, optimal reaction temperature at 37-39 DEG C, typically may be used by whole process
Horizontal amplified production can be detected by being obtained within ten minutes.The sensitivity of RPA detections is very high, can be by trace level (trace
Levels nucleic acid (especially DNA) template amplification) obtains about 10 to the level that can be detected from single template molecule12Expand
Increase production thing, be more suitable for the clinical detection template of low concentration.And RPA reactions are not high to template purity requirement, suitable for that can not carry
Take the detection on the spot of nucleic acid.At present, RPA reactions can pass through agarose gel electrophoresis, fluorescent amplification curve and LFD test strips three
Kind of different modes testing result, the primers of these three modes are different from probe reaction principle.Gel electrophoresis and amplified fluorescence
Curve method still relies on laboratory and special instruments and equipment, and LFD is a kind of method that can be used for scene without specific apparatus.
The design of the primer and probe of RPA-nfo reactions can only pass through RPA reaction effluents currently without specific rule
Chromatograph test strip (LFD) detects, and could screen the primer and probe combinations obtained available for clinical detection.It is positive in RPA-nfo
The specific probe of band FAM marks, while reverse primer biotin labeling, biotin are designed in the amplification target sequence of primer
Specific probe hybridization that the RPA products of mark mark with FAM, the specific probe of FAM marks are combined with the gold mark thing of anti-FAM antibody
Afterwards, the immune complex being added drop-wise in test strips, immune complex is by chromatographing membrane diffusion, when being diffused into detection line, biology
The amplified production of element mark is captured by biotin ligand, is formed and has coloured detection line, i.e. test strip.Not captured exempts from
Epidemic disease compound continues to diffuse into nature controlling line and captured by specific antibody, is formed and has coloured nature controlling line, i.e. control stripes band.This
Sample RPA technologies are combined with Sidestream chromatography technology, i.e. RPA-LFD technologies, can be read and tied by Sidestream chromatography test strips (LFD)
Fruit, this method neither require special instruments and equipment, without the sample treatment of complexity, only using only common heater such as water
Bath and Sidestream chromatography test strips can observe by the naked eye result.Therefore, RPA-LFD technologies are in brucella aerosol etc.
Had broad application prospects in terms of the discriminating of the field diagnostic and vaccine strain A19 of clinical sample.
Compared with prior art, the advantage of the invention is that：
(1) RPA technologies provided by the invention are that emerging one kind is superior to PCR in rate of amplification and yield in recent years
With the new method of ring mediated isothermal amplification (LAMP) technology, its reaction can just obtain about in 30min from single template molecule
1012Amplified production.
(2) Brucella bacterium provided by the invention and the RPA-LFD primers and probe groups of A19 vaccine strain antidiastoles
Conjunction and kit high sensitivity, high specificity are minimum to detect 3 × 100Cfu Brucella bacterium or cloth Shandong
Salmonella A19 vaccine strain bacteriums, Brucella-RPA-LFD detections group and Escherichia coli O 157:H7, YE
O:9th, the equal no cross reaction of the bacterium such as salmonella, staphylococcus aureus.A19-RPA-LFD detections group and other brucella
Strain and Escherichia coli O 157:H7, YE O:9th, the equal nothing of the bacterium such as salmonella, staphylococcus aureus
Cross reaction.
(3) brucella A19 vaccine strains antidiastole RPA-LFD primers provided by the invention and probe combinations and reagent
Box not only can be used for the detection of brucella A19 vaccine strains bacterial strain and aerosol sample, it may also be used for other clinical samples are such as
The detection of blood, milk sample etc..
(4) brucella A19 vaccine strains antidiastole RPA-LFD detection methods provided by the invention are easy to use, without
Special instruments and equipment, only at 38 DEG C, 30min just can carry out brucella A19 vaccine strains to the thick lysate of sample to be tested
It is sensitive, special, rapidly differentiate detection, be adapted to scene or the discriminating of basic unit's brucellosis A19 vaccine strains and street strain to examine
Disconnected work, it can also be used to the detection of Brucella bacterium.
(5) effectively the natural infection of antidiastole brucellosis and artificial vaccines can be exempted from using the kit of the present invention
Epidemic disease, the ill domestic animal evading quarantine inspection of natural infection is avoided, timely find the presence of infected animal.
Brief description of the drawings
The screening of the primer and probe of Fig. 1 brucella A19 vaccine strain antidiastole RPA-LFD detection combinations, Control
Primer/Probe Mix amplifications group is the RPA amplifications of primer, probe and template that TwistAmp nfo kit are provided；
The primer for the specific detection Brucella bacterium that Brucella-RPA-LFD groups filter out for the present invention is with probe with cloth Shandong
Salmonella A19 vaccine strains genomic DNA is the amplification of template；The specificity inspection that A19-RPA-LFD groups filter out for the present invention
Survey the primer and amplification of the probe using brucella A19 vaccine strains genomic DNA as template of brucella A19 vaccine strains；
NC is with ddH2O replaces the negative control of template.
Fig. 2 brucella A19 vaccine strain antidiastole RPA-LFD sensitivity techniques, template is respectively 3 × 108cfu–3×
10-1Cfu B. abortus A19 vaccine strain genomic DNAs, NC is with ddH2O replaces the negative control group of template.
Fig. 3 brucella A19 vaccine strain antidiastole RPA-LFD specific detections, B.abortus A19, B.suis
S2、B.melitensis M5-90、B.ovis、B.canis、E.coli O157:H7、Y.enterocolitica O:9、
Salmonella, S.aureus, M.bovis template are respectively B. abortus vaccine strain A19 strains, pig kind brucella epidemic disease
Seedling strain S2 strains, brucella melitensis vaccine strain M5-90 strains, 63/290 plant of Br.ovis, Brucellacanis RM6/66 strains,
Escherichia coli O 157:H7, YE O:9th, salmonella, staphylococcus aureus, cow mycobacteria gene
Group DNA；NC is with ddH2O replaces the negative control group of template.
The brucella A19 vaccine strain RPA-LFD antidiastoles of the clinical samples such as Fig. 4 aerosols ,+：Template is ox kind cloth
Shandong Salmonella A19 vaccine strain genomic DNAs, NC：With ddH2O replaces the negative control group of template, 1-10：Brucella nucleic acid is positive
Aerosol sample, wherein 1-3 samples are A19 vaccine strain nucleic acid positive aerosol samples, 11-13：Brucella nucleic acid is positive
Blood sample, wherein 11 and No. 12 samples are A19 vaccine strain nucleic acid positive blood samples, 14-16：Brucella nucleic acid positive milk
All, wherein 14 and No. 15 samples are A19 vaccine strain nucleic acid positive milk sample samples, 17-20：Respectively brucella feminine gender gas
Colloidal sol, blood, milk sample and aborted fetus clinical sample.
Embodiment
Following embodiments are used to further illustrate the present invention, but are not limited to the scope of the present invention.
Following examples are carried out according to normal test conditions and method, or according to the test bar proposed by manufacturer
Part.
1. test material
B. abortus vaccine strain A19 strains (B.abortus A19), pig kind brucella vaccine strain S2 strains (B.suis
S2), brucella melitensis vaccine strain M5-90 strains (B.melitensis M5-90), 63/290 plant of Br.ovis
(B.ovis63/290), Brucellacanis RM6/66 strains (B.canis RM6/66), Escherichia coli O 157:H7, enterocolitis
Yersinia O:9th, salmonella, staphylococcus aureus (CMCC26003), cow mycobacteria (M.bovis ATCC
19210) brucella positive aerosol, blood and milk sample DNA sample etc., are clinically diagnosed as by Agriculture in Shandong Province science
Institute milk cow research center disease research room preserves.
Primer is given birth to work biology Co., Ltd by Shanghai with probe and synthesized.TwistAmp DNA Amplification nfo
Kits is purchased from TwistDX companies, and Sidestream chromatography test strips (HybriDetect Dipsticks) are purchased from Milenia companies, other
Biochemical reagents are that import packing or domestic analysis are pure.
2. laboratory apparatus
Thermostat water bath, centrifuge, palm centrifuge, vortex instrument, (Qingdao crowd is auspicious for MS-I type multifunctional microbials sampler
Intelligence instrument Co., Ltd) etc..
The design and screening of embodiment one, primer and probe
The VirB genes (GengBank No.AF226278) that the present invention guards according to Brucella bacterium design cloth Shandong
The universal Brucella-RPA-LFD of Bordetella bacterium 2 specific probes, 4 forward primers are devised in the both sides of probe
With 2 reverse primers.7 nucleotide sequences are lacked at genome 22840-22846 sites according to A19 vaccine strains simultaneously
(AGATTTC) the A19-RPA-LFD reverse primers 4 of specific detection A19 vaccine strains are designed, are set in the deletion segment upstream
Count 2 forward primers and 4 specific probes.RPA-nfo reverse primers are positioned at this can be by A19 vaccine strains and other cloth Shandongs
Bordetella bacterium carries out antidiastole.
RPA-nfo primer length is generally 30-35nt, the too short activity that can have a strong impact on recombinase of primer.Long primer
Amplification capability might not can be improved, can also increase the possibility to form secondary structure on the contrary.LF probe lengths are typically in 46-
52nt, nfo Ribozyme cleavage site distance probes 5 ' hold 30 bases or so, and distance 3 ' holds 16-22 base or so.At present,
RPA-LFD primers and probe are designed without specific operation rules, it is necessary to by RPA reactions Sidestream chromatography test strips
(LFD) detect, by experiment sieving, the primer and probe available for clinical detection could be obtained.
Need to optimize with probe from the target sequence both ends multipair primer of design in this experiment, screen, Individual base
Replace or increase and decrease all can produce material impact to result of the test.When primer and probe design, we also contemplate three notes
Meaning point：The 3-5 nucleotides at (1) 5 ' end should avoid poly- guanine (G), cytimidine (C), can so promote primer and amplification target base
The combination of cause.For 3 nucleotides of 3 ' ends, guanine and cytimidine contribute to the stable bond of polymerase, can be with
Lift the amplification capability of primer.(2) had better not occur special sequence, such as long string of poly- purine or poly- pyrimidine in primer.GC
Too high levels (>70%) or it is too low (<30%) all it is unfavorable for RPA amplifications.(3) in addition, should be tried one's best when primer designs with probe
Avoid easily forming secondary structure, primer-primer interaction, primer-probe interaction, the sequence of hairpin structure, reduce dimer
Formed.
Using B. abortus A19 vaccine strains DNA as template, above-mentioned Brucella primer and probe and A19 are reflected
The various combination of other primer and probe is screened, and finally selects one group of amplification efficiency height, the primer of high specificity and spy respectively
Pin combines, and differentiates for brucella A19 vaccine strains RPA-LFD and detects, and primer and probe sequence is shown in SEQ ID NO.1 respectively,
SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 (table 1).
Table 1
Note：Biotin：Biotin labeling；FAM：Fluoresceincarboxylic acid marks；dSpacer：Nucleobase analog, it is
Nfo nucleic acid cleavage sites；C3-Spacer：Polymerase extends blocker.
The foundation of embodiment two, brucella A19 vaccine strain antidiastole RPA-LFD detection methods
1. experimental procedure
(1) bacterial genomes DNA extraction
1ml bacterium solutions are taken, according to TIANGEN Biotech's bacterial genomes DNA extraction kit specification
Carry out the extraction of bacteria total DNA.
(2) foundation of brucella A19 vaccine strains antidiastole RPA-LFD reaction systems
RPA reaction systems are 50 μ L：
Above-mentioned 47.5 μ L mixtures are added into RPA-nfo reaction tubes, fully mixed, dissolving.It is eventually adding 2.5 μ L 280mM
Magnesium acetate solution, mixing of turning upside down, reaction tube is placed in 38 DEG C of water bath processing 20-30min after gently centrifuging.Each RPA is anti-
Take 2 μ L amplified productions to detect buffer solution with 98 μ L LFD after should terminating to mix, each corresponding LFD of reaction, LFD is immersed into phase
The reaction mixture answered, result is observed within 5min, if two detection combinations of Brucella-RPA-LFD and A19-RPA-LFD are same
When there is test strip and control stripes band, then brucella A19 vaccine strains are positive in the sample；Brucella-RPA-LFD is detected
There is test strip and control stripes band in group, and control stripes band only occurs in A19-RPA-LFD detections group, then illustrates to contain in the sample
Other brucella beyond brucella A19 vaccine strains；Two detection groups of Brucella-RPA-LFD and A19-RPA-LFD are equal
There is not test strip and occur control stripes band simultaneously, then do not contain Brucella bacterium in explanation detection sample.There is it
His situation then illustrates that RPA-LFD brucella A19 differentiates that detection is invalid.
(3) brucella A19 vaccine strains antidiastole RPA-FLD sensitivity techniques
B. abortus A19 bacterial concentrations are calculated using colony counting method, respectively with 10 times of doubling dilutions into 3 × 108–
3×10-1Cfu/mL bacterium solutions, each 1mL of bacterium solution of corresponding extension rate is taken, extracted using bacterial genomes DNA extraction kit each
The bacterium solution genomic DNA of extension rate, dissolved with 100 μ L TE, it is template to take 2 μ L genomic DNAs, is entered according to above-mentioned steps (2)
Row RPA is expanded, while with ddH2O is negative control, examines the sensitiveness of this method.
(4) brucella A19 vaccine strains antidiastole RPA-FLD specific detections
With the RPA-FLD kits of antidiastole A19 vaccine strains of the present invention to B. abortus A19 vaccine strains, pig kind
Brucella S2 vaccine strains, brucella melitensis M5-90 vaccine strains, Br.ovis, Br. cants, Escherichia coli
O157:H7, YE O:9th, salmonella, staphylococcus aureus, cow mycobacteria genomic DNA are carried out
Amplification, while with ddH2O is negative control, verifies the specificity of this method.
2. result of the test
Two groups of RPA-LFD primers and probe combinations are carried out with RPA-LFD detections respectively to screen, 2 kinds of optimization is optimal to draw
Thing and probe combinations, as a result as shown in Figure 1.With the B. abortus A19 vaccine strain bases of 10 gradients of 10 times of doubling dilutions
Because group DNA is that template is detected, as a result as shown in Fig. 2 two groups of RPA-LFD test limits of 50 μ L systems are 3 × 100cfu。
Brucella and 5 plants of other bacteriums to 5 plants of different generas carry out two kinds of RPA-LFD detections, as a result respectively as control strain
As shown in figure 3, there is bar at the detection line of Sidestream chromatography test strips in 5 plants of brucella Brucella-RPA-LFD reaction tubes
Band, it is positive findings；And other 5 plants of bacterium bacterial strains and ddH2O controls do not occur test strip, control stripes band occur.
A19-RPA-LFD amplification groups only have B. abortus A19 vaccine strain genomes test strip occur, and other amplifications Guan Junwei goes out
Existing test strip, is feminine gender.Illustrating two groups of RPA-LFD primers, energy specific detection Brucella is thin respectively with probe combinations
Bacterium and brucella A19 vaccine strains.
The antidiastole of the clinical sample brucella A19 vaccine strains such as embodiment three, aerosol
1. experimental procedure
(1) collection of aerosol sample
The full glass precursor solution impact-actuated sampler (all-glass-impinger, abbreviation AGI) adopted international standards, with
The phosphate buffer (PBS) of 10mL pH value 7.0 is sampling media, gathers 20min according to 12.5L/min sampling flow, receives
Collect the aerosol sample of different zones in plant's environment.12000r/min centrifuges 10min at room temperature, abandons supernatant, precipitates
For Direct Pyrolysis processing.
(2) clinical sample such as aerosol is prepared in situ
By the clinical samples such as aerosol with 200 μ L TE buffer solutions (1.0M Tris-HCl (pH8.0) 10mL,
0.5MNa2EDTA·2H2O (pH8.0) 2mL, adds distilled water to l000mL) it is resuspended, liquid sample directly takes 200 μ L, then added
(w/v) SDS of 30 μ L 10% and 3 μ L 2% (w/v) Proteinase Ks, 37 DEG C of incubation 1h after mixing.
(3) the discriminating detection of the clinical sample such as aerosol brucella A19 vaccine strains
According to the method for clinical sample scene cracking processing in step (2), PCR has been used to what this laboratory preserved respectively
Correct 10 parts of brucella positive aerosol sample is sequenced after amplification, wherein 3 parts of brucella A19 vaccine strains are positive, 3 portions of milk
Sample and 3 parts of blood brucella positive samples, and 2 parts of aerosols, 1 part of milk sample and 1 part of blood brucella negative sample are entered
Row processing, while with B. abortus A19 vaccine strains DNA and ddH2O is respectively positive, negative control, carries out two groups respectively
RPA-LFD augmentation detections.
2. result of the test
To 12 parts of aerosol samples, 4 parts of blood samples and 4 parts of milk sample samples of this Laboratory Diagnosed, carry out respectively
Brucella-RPA-LFD and A19-RPA-LFD detections, wherein 2 parts of brucella feminine gender aerosol sample, brucella are negative
Blood and each 1 part of milk sample sample, as a result as shown in Fig. 4 and table 2.Result above is absolutely proved with two groups of RPA- provided by the invention
LFD primers, probe combinations and kit can be used for antidiastole Brucella bacterium and A19 vaccine strains, this method to have
Higher sensitivity and specificity, it is most important that live quickly diagnosis can be directly carried out to clinical aerosol equal samples.
The discriminating testing result of the clinical sample brucella A19 vaccine strains such as aerosol of table 2
Although above-mentioned the embodiment of the present invention is described with reference to accompanying drawing, model not is protected to the present invention
The limitation enclosed, one of ordinary skill in the art should be understood that on the basis of technical scheme those skilled in the art are not
Need to pay various modifications or deformation that creative work can make still within protection scope of the present invention.
Claims (6)
1. a kind of primer pair and probe for differentiating brucella A19 vaccine strains in aerosol, it is characterized in that, Brucella-RPA-
LFD forward primers sequence is as shown in SEQ ID No.1, and reverse primer sequences are as shown in SEQ ID No.2, and probe sequence is such as
Shown in SEQ ID No.3；A19-RPA-LFD forward primers sequence is as shown in SEQ ID No.4, reverse primer sequences such as SEQ
Shown in ID No.5, probe sequence is as shown in SEQ ID No.6.
2. a kind of kit for differentiating brucella A19 vaccine strains in aerosol, it is characterized in that, the kit will comprising right
Seek the primer pair and probe described in 1.
3. the kit as described in claim 2, it is characterized in that, including rehydration buffer solutions, 280mM magnesium acetates be molten
Liquid, Sidestream chromatography test strips, PBST ELISA test strips buffer solution, sterilizing deionization distilled water, TE buffer solutions, 10% (w/
V) SDS, 2% (w/v) Proteinase K, brucella A19 vaccine strain standard positive templates；
Wherein, the recA recombinases containing Escherichia coli, strand displacement DNA polymerizations in the rehydration buffer solutions
Enzyme and single stranded DNA associated proteins.
4. a kind of method for being used for brucella A19 vaccine strains in quick discriminating aerosol sample, methods described are not used in disease
The diagnosis of disease, it is characterized in that, comprise the following steps：
(1) extraction or the cracking processing of detection sample of sample DNA are detected；
(2) using the sample of processing in step (1) as template, RPA amplifications are carried out；
RPA amplification system is in the step (2)：RPA reaction systems are 50 μ L：Including 10 μM of 2.1 μ L's
Forward primer, as shown in SEQ ID No.1 or 4,2.1 10 μM of μ L reverse primer, as shown in SEQ ID No.2 or 5,
0.6 10 μM of μ L probe, as shown in SEQ ID No.3 or 6, rehydration buffer solutions 29.5 μ L, sample to be tested DNA or
Thick pyrolysis product 2 μ L, ddH2The μ L of O 11.2, the μ L of amplification system mixed liquor 47.5 are added into RPA-nfo reaction tubes, mixes, is molten
Solution, 2.5 μ L 280mM magnesium acetate solution is eventually adding, mixing of turning upside down reacts 20-30 minutes after 37-39 DEG C；
(3) the above-mentioned RPA amplified productions of Sidestream chromatography ELISA test strip are used, judge whether contain in sample according to testing result
Brucella A19 vaccine strains；
In step (3), whether the decision method containing brucella A19 vaccine strains is in sample：
If there is test strip and control stripes band simultaneously with two detection combinations of A19-RPA-LFD in Brucella-RPA-LFD,
Then brucella A19 vaccine strains in the sample；
There is test strip and control stripes band in Brucella-RPA-LFD detection groups, and A19-RPA-LFD detection groups only occur
Control stripes band, then illustrate in the sample containing other brucella beyond brucella A19 vaccine strains； Brucella-
There is not test strip and control stripes band occurs with two detection groups of A19-RPA-LFD in RPA-LFD, then explanation detection sample
In do not contain Brucella bacterium.
5. the method as described in claim 4, it is characterized in that, the step (3) is：Distinguish for each RPA reaction tubes
Take 2 μ L amplified productions to be mixed with 98 μ L PBST ELISA test strip buffer solutions, a Sidestream chromatography test strips are immersed corresponding
Reaction mixture, result is observed within 5min.
6. the method as described in claim 4, it is characterized in that, mixing of turning upside down is reacted 25 minutes after 38 DEG C.
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| CN108315476A (en) * | 2018-01-29 | 2018-07-24 | 杭州德同生物技术有限公司 | A kind of RPA fluorescent quantitations primer pair, probe, kit and method for detecting HPV18 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102146466A (en) * | 2011-02-15 | 2011-08-10 | 浙江国际旅行卫生保健中心 | Reagent for detecting brucella and complex probe fluorescence quantitative PCR (polymerase chain reaction) brucella detection method |
| CN102776220A (en) * | 2012-06-11 | 2012-11-14 | 新疆维吾尔自治区畜牧科学院兽医研究所 | Construction of brucellosis A19 molecular marker vaccine strain and determination of virulence and immunogenicity |
| CN103981261A (en) * | 2014-05-06 | 2014-08-13 | 山东省农业科学院奶牛研究中心 | Method for detection and differential diagnosis of Brucella in aerosol |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102146466A (en) * | 2011-02-15 | 2011-08-10 | 浙江国际旅行卫生保健中心 | Reagent for detecting brucella and complex probe fluorescence quantitative PCR (polymerase chain reaction) brucella detection method |
| CN102776220A (en) * | 2012-06-11 | 2012-11-14 | 新疆维吾尔自治区畜牧科学院兽医研究所 | Construction of brucellosis A19 molecular marker vaccine strain and determination of virulence and immunogenicity |
| CN103981261A (en) * | 2014-05-06 | 2014-08-13 | 山东省农业科学院奶牛研究中心 | Method for detection and differential diagnosis of Brucella in aerosol |
Non-Patent Citations (3)
| Title |
|---|
| Recombinase Polymerase Amplification Assay for Rapid Detection of Francisella tularensis;Milena Euler,et al;《Journal of Clinical Microbiology》;20120731;第50卷(第7期);2234–2238 * |
| Recombinase polymerase amplification combined with a lateral flow dipstick for discriminating between infectious Penaeus stylirostris densovirus and virus-related sequences in shrimp genome;Wansadaj Jaroenram, Leigh Owens;《Journal of Virological Methods》;20140822;第208卷;144－151 * |
| 应用PCR法鉴定布鲁氏菌种型的方法研究;易新萍等;《第三届全国人畜共患病学术研讨会》;20111231;278－284 * |
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