CN104911269A - Primers, probe and kit for identifying Brucella A19 vaccine strain in aerosol - Google Patents
Primers, probe and kit for identifying Brucella A19 vaccine strain in aerosol Download PDFInfo
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- CN104911269A CN104911269A CN201510355509.2A CN201510355509A CN104911269A CN 104911269 A CN104911269 A CN 104911269A CN 201510355509 A CN201510355509 A CN 201510355509A CN 104911269 A CN104911269 A CN 104911269A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Abstract
The invention discloses primers, a probe and a kit for identifying a Brucella A19 vaccine strain in aerosol. RPA-LFD (Recombinase Polymerase Amplification and Lateral Flow Dipstick) primers, a probe combination and the kit which are applied to differential diagnosis of Brucella bacteria and the A19 vaccine strain have high sensitivity and strong specificity and can be used for minimally detecting 3*10<0>cfu level Brucella bacteria or Brucella A19 vaccine bacteria. The kit provided by the invention can be used for identifying the Brucella A19 vaccine strain and an aerosol sample and can be further applied to the differential detection of other clinical samples such as blood and milk samples.
Description
Technical field
The invention belongs to biological technical field, be specifically related to apply the primer of brucella A19 vaccine strain in the clinical samples such as recombinase polymeric enzymatic amplification-Sidestream chromatography ELISA test strip technology (Recombinase Polymerase Amplification and Lateral Flow Dipstick, RPA-LFD) rapid differential diagnosis aerosol and probe and test kit.
Background technology
Brucellosis (Brucellosis) is the transmissible disease that the people that caused by Brucella bacterium (being mainly ox kind, sheep kind, pig kind and Br. cants etc.) and multiple domestic animal suffer from altogether, the important pathogen of the legal quarantine of Ye Shi China and purification.Sickness rate presents ascendant trend year by year in recent years, especially very harmful to dairy stock, and abortion ratio is up to 50%-80%, and breast, meat production reduce 10%-20%.According to " animal doctor's publication " statistics, 2013 there is brucellosis 3620 times in the whole nation altogether, 42720 cattle and sheep morbidities.Therefore, brucellosis constitutes a serious threat safely etc. to socio-economic development and stable, National Public Health.
Contagium mainly infected animal and the carrier of brucellosis, brucella is excreted by secretory product, milk, body fluid etc. by they, contaminate environment, and brucella sticks on koniology suspended particles simultaneously, form aerosol and then propagation, infect other animal and humans.The aerocolloidal distribution of brucella in plant's environment can cause the infection of brucellosis and popular, bring serious harm to Animal husbandry production and human health, under especially current intensive, high-density breeding environment, more easily form the popular of brucella aerosol.Therefore, by brucella aerosol in monitoring plant environment, can the breaking out with popular of early-warning and predicting brucellosis effectively.
The main policies of China's animal brucellosis prevention and control is quarantine and purification.The conventional B. abortus A19 living vaccine strain of current China cows immunity is understood in the diagnosis of antigen with antibody cause interference to brucellosis wild virus infection.This is because the brucellosis diagnostic method that China uses at present can not carry out differential diagnosis to natural infection and artificial vaccines's immunity, the ill domestic animal evading quarantine inspection of some natural infections must be caused, make infected animal long-term existence, the safety of cows and the mankind is constituted a threat to for a long time, therefore, China is difficult to the quarantine carrying out brucellosis, the prevention and control code of slaughtering, purifying.
Summary of the invention
The object of this invention is to provide a kind of primer, probe and the test kit of differentiating brucella A19 vaccine strain in aerosol, this test kit can special, sensitive, simple and easy, brucella A19 vaccine strain in on-the-spot differential diagnosis aerosol fast.
For realizing the object of the invention, adopt following technical scheme:
Differentiate primer pair and the probe combinations of brucella A19 vaccine strain in aerosol, wherein Brucella-RPA-LFD forward primer sequence is as shown in SEQIDNo.1, and reverse primer sequences is as shown in SEQIDNo.2, and probe sequence is as shown in SEQIDNo.3; A19-RPA-LFD forward primer sequence is as shown in SEQIDNo.4, and reverse primer sequences is as shown in SEQIDNo.5, and probe sequence is as shown in SEQIDNo.6.
Present invention also offers a kind of test kit differentiating brucella A19 vaccine strain in aerosol, this test kit comprises above-mentioned primer pair and probe combinations.
Invention further provides a kind of method for brucella A19 vaccine strain in rapid differential diagnosis aerosol sample, comprise the steps:
(1) detect the extraction of sample DNA or detect sample cracking process;
(2) with the sample of process in step (1) for template, carry out RPA amplification;
(3) with the above-mentioned RPA amplified production of Sidestream chromatography ELISA test strip, whether judge in sample containing brucella A19 vaccine strain according to detected result.
Preferably, described in step (2), RPA amplification system is: RPA reaction system is 50 μ L: comprising the forward primer of 2.1 μ L10 μMs, the reverse primer of 2.1 μ L 10 μMs, the probe of 0.6 μ L 10 μMs, rehydration damping fluid 29.5 μ L, sample to be tested DNA or thick split product 2 μ L, ddH
2o 11.2 μ L; Above-mentioned 47.5 μ L mixtures are added RPA-nfo reaction tubes, and fully mixing, dissolving, finally add the magnesium acetate solution 2.5 μ L of 280mM, in water bath with thermostatic control 38 DEG C reaction 20-30 minute after mixing of turning upside down.
Preferably, the concrete steps of step (3) are: each RPA of getting reaction product is got 2 μ L and mixed with 98 μ L PBST ELISA test strips damping fluid (1 × PBS+0.1% polysorbas20), a flow measurement chromatograph test strip (LFD) is immersed, observations within 5min, if there is test strip and contrast band during Brucella-RPA-LFD and A19-RPA-LFD two test set contracts, then in this sample, brucella A19 vaccine strain is positive; There is test strip and contrast band in Brucella-RPA-LFD test set, and band only appears contrasting in A19-RPA-LFD test set, then illustrate in this sample containing other brucella beyond brucella A19 vaccine strain; All there is not test strip and occur contrasting band simultaneously in Brucella-RPA-LFD and A19-RPA-LFD two test set, then illustrates and detect in sample not containing Brucella bacterium.Occur that other situations then illustrate that RPA-LFD brucella A19 differentiates that detection is false.
The present invention also provides the test kit for differential diagnosis brucella A19 vaccine strain containing above-mentioned two groups of primers and probe combinations.The recA recombinase of Escherichia coli, strand displacement archaeal dna polymerase, single-stranded DNA binding protein (SSB), rehydration damping fluid, 280mM magnesium acetate (MgAc) solution, Sidestream chromatography test strip (specific detection vitamin H and FAM marker gene amplified production), PBST ELISA test strip damping fluid (1 × PBS+0.1% polysorbas20), sterilizing deionization distilled water (ddH is also comprised in this test kit
2o), TE damping fluid, 10% (w/v) SDS, 2% (w/v) Proteinase K, brucella A19 vaccine strain standard positive template.
With brucella A19 vaccine strain and other brucella genome difference DNA sequence dnas for differentiating target sequence, application RPA-LFD technology, can realize brucella A19 vaccine strain and other brucella strains scene rapid differential diagnosis.RPA technology is by the mixture of three kinds of enzymes, can in conjunction with the recombinase of single-chain nucleic acid (Oligonucleolide primers), single-stranded DNA binding protein (SSB) and strand displacement archaeal dna polymerase, also activity is had at normal temperatures, optimal reaction temperature is at 37-39 DEG C, and whole process generally can obtain the amplified production that can detect level within ten minutes.The sensitivity that RPA detects is very high, by nucleic acid (especially DNA) template amplification of trace level (trace levels) to the level that can detect, can obtain about 10 from single template molecule
12amplified production, is more suitable for the clinical detection template of lower concentration.And RPA reaction is not high to template purity requirement, is applicable to the detection on the spot cannot extracting nucleic acid.At present, RPA reaction can pass through agarose gel electrophoresis, and amplified fluorescence curve and LFD test strip three kinds of different modes detected results, the primer of these three kinds of modes is different from probe reaction principle.Gel electrophoresis and amplified fluorescence curve method still rely on laboratory and special instruments and equipment, and LFD is a kind of method that can be used for scene without the need to specific apparatus.
The primer of RPA-nfo reaction and the design of probe do not have concrete rule at present, can only detect, could screen the primer and the probe combinations that obtain and can be used for clinical detection through RPA reaction Sidestream chromatography test strip (LFD).The specific probe that a band FAM marks is designed in the amplified target sequence of RPA-nfo forward primer, reverse primer biotin labeling simultaneously, after the specific probe that the specific probe hybridization that biotin labeled RPA product and FAM mark, FAM mark is combined with the gold mark thing of anti-FAM antibody, this immunocomplex is added drop-wise in test strip, immunocomplex is spread by chromatographic film, when being diffused into detection line, biotin labeled amplified production is caught by biotin ligand, form the coloured detection line of tool, i.e. test strip.Not captured immunocomplex continue to be diffused into nature controlling line catch by specific antibody, form the coloured nature controlling line of tool, namely contrast band.Such RPA technology and Sidestream chromatography combine with technique, i.e. RPA-LFD technology, result can be read by Sidestream chromatography test strip (LFD), present method neither requires special instruments and equipment, also without the need to the sample preparation of complexity, common heating unit such as water-bath and Sidestream chromatography test strip is only used just can to pass through visual results.Therefore, RPA-LFD technology has broad application prospects in the field diagnostics of clinical sample such as brucella aerosol and the discriminating of vaccine strain A19.
Compared with prior art, the invention has the advantages that:
(1) RPA technology provided by the invention is that emerging one is all better than the novel method of PCR and ring mediated isothermal amplification (LAMP) technology in rate of amplification and output in recent years, and its reaction just can obtain about 10 from single template molecule in 30min
12amplified production.
(2) Brucella bacterium provided by the invention and the differential diagnosis of A19 vaccine strain RPA-LFD primer and probe combinations and test kit is highly sensitive, high specificity, minimumly all can detect 3 × 10
0the Brucella bacterium of cfu or brucella A19 vaccine strain bacterium, the equal no cross reaction of bacterium such as Brucella-RPA-LFD test set and Escherichia coli O 157: H7, Yersinia enterocolitica O:9, Salmonellas, streptococcus aureus.The equal no cross reactions of bacterium such as A19-RPA-LFD test set and other brucella strain and Escherichia coli O 157: H7, Yersinia enterocolitica O:9, Salmonellas, streptococcus aureus.
(3) brucella A19 vaccine strain differential diagnosis RPA-LFD primer provided by the invention and probe combinations and test kit not only can be used for the detection of brucella A19 vaccine strain bacterial strain and aerosol sample, also can be used for the detection of other clinical samples as blood, milk sample etc.
(4) brucella A19 vaccine strain differential diagnosis RPA-LFD detection method provided by the invention is easy to use, without the need to special instruments and equipment, only at 38 DEG C, 30min just can to the thick lysate of sample to be tested carry out brucella A19 vaccine strain sensitive, special, rapidly differentiate detect, be applicable to on-the-spot or basic unit brucellosis A19 vaccine strain and street strain differential diagnosis work, also can be used for the detection of Brucella bacterium.
(5) use test kit of the present invention can the natural infection of effectively differential diagnosis brucellosis and artificial vaccines's immunity, avoid the ill domestic animal evading quarantine inspection of natural infection, find the existence of infected animal timely.
Accompanying drawing explanation
Fig. 1 brucella A19 vaccine strain differential diagnosis RPA-LFD detects the primer of combination and the screening of probe, the RPA amplification of primer, probe and template that ControlPrimer/Probe Mix amplification group provides for TwistAmp nfo kit; The amplification that the primer of the specific detection Brucella bacterium that Brucella-RPA-LFD group filters out for the present invention and probe are template with brucella A19 vaccine strain genomic dna; The amplification that the primer of the specific detection brucella A19 vaccine strain that A19-RPA-LFD group filters out for the present invention and probe are template with brucella A19 vaccine strain genomic dna; NC is with ddH
2o replaces the negative control of template.
Fig. 2 brucella A19 vaccine strain differential diagnosis RPA-LFD sensitivity technique, template is respectively 3 × 10
8cfu – 3 × 10
-1cfu B. abortus A19 vaccine strain genomic dna, NC is with ddH
2o replaces the negative control group of template.
Fig. 3 brucella A19 vaccine strain differential diagnosis RPA-LFD specific detection, 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, the template of M.bovis is respectively B. abortus vaccine strain A19 strain, pig kind brucella vaccine strain S2 strain, brucella melitensis vaccine strain M5-90 strain, Br.ovis 63/290 strain, dog brucella RM6/66 strain, Escherichia coli O 157: H7, Yersinia enterocolitica O:9, Salmonellas, streptococcus aureus, cow mycobacteria genomic dna, NC is with ddH
2o replaces the negative control group of template.
The brucella A19 vaccine strain RPA-LFD differential diagnosis of the clinical samples such as Fig. 4 aerosol ,+: template is B. abortus A19 vaccine strain genomic dna, NC: with ddH
2o replaces the negative control group of template, 1-10: the positive aerosol sample of brucella nucleic acid, wherein 1-3 sample is the positive aerosol sample of A19 vaccine strain nucleic acid, 11-13: brucella nucleic acid positive blood sample, wherein 11 and No. 12 samples are A19 vaccine strain nucleic acid positive blood sample, 14-16: the positive milk sample sample of brucella nucleic acid, wherein 14 and No. 15 samples are the positive milk sample sample of A19 vaccine strain nucleic acid, 17-20: be respectively brucella negative aerosol, blood, milk sample and aborted fetus clinical sample.
Embodiment
Following embodiment is used for further illustrating the present invention, but is not used for limiting the scope of the invention.
Following examples all conveniently test conditions and method are carried out, or according to the test conditions that manufacturer advises.
1. test materials
B. abortus vaccine strain A19 strain (B.abortus A19), pig kind brucella vaccine strain S2 strain (B.suis S2), brucella melitensis vaccine strain M5-90 strain (B.melitensis M5-90), Br.ovis 63/290 strain (B.ovis63/290), dog brucella RM6/66 strain (B.canis RM6/66), Escherichia coli O 157: H7, Yersinia enterocolitica O:9, Salmonellas, streptococcus aureus (CMCC26003), cow mycobacteria (M.bovis ATCC 19210), be diagnosed as the aerosol of the brucella positive clinically, blood and milk sample DNA sample etc. are preserved by Cow Research Center, Shandong Academy of Agricultural Sciences's disease research room.
Primer and probe are synthesized by the Shanghai biological company limited of raw work.TwistAmp DNA Amplification nfo Kits is purchased from TwistDX company, and Sidestream chromatography test strip (HybriDetect Dipsticks) is purchased from Milenia company, and other biochemical reagents are import packing or domestic analytical pure.
2. laboratory apparatus
Thermostat water bath, whizzer, palm whizzer, vortex instrument, MS-I type multifunctional microbial sampling thief (Qingdao Zhongrui Intelligent Instrument Co., Ltd.) etc.
The design of embodiment one, primer and probe and screening
The VirB gene (GengBank No.AF226278) that the present invention is conservative according to Brucella bacterium designs 2 specific probes of the universal Brucella-RPA-LFD of Brucella bacterium, devises 4 forward primers and 2 reverse primers in the both sides of probe.Lack 7 nucleotide sequences (AGATTTC) according to A19 vaccine strain in genome 22840-22846 site and design the A19-RPA-LFD reverse primer 4 of specific detection A19 vaccine strain, at this deletion segment upstream design 2 forward primers and 4 specific probes simultaneously.RPA-nfo reverse primer is positioned this can carry out differential diagnosis by A19 vaccine strain and other Brucella bacteriums.
The primer length of RPA-nfo is generally 30-35nt, and the too short meeting of primer has a strong impact on the activity of recombinase.Long primer might not improve amplification capability, also can increase the possibility forming secondary structure on the contrary.LF probe length generally, about 46-52nt, nfo Ribozyme cleavage site distance probes 5 ' end 30 bases, is held about 16-22 base apart from 3 '.At present, the design of RPA-LFD primer and probe does not have concrete working rule, has to pass through RPA reaction Sidestream chromatography test strip (LFD) and detects, by experiment sieving, could obtain the primer and probe that can be used for clinical detection.
Need in this test to design multipair primer from target sequence two ends and probe is optimized, screens, replacement or the increase and decrease of Individual base all can produce material impact to test-results.When primer and probe design, we also contemplate three lime lights: (1) 5 ' 3-5 a held Nucleotide should avoid poly-guanine (G), and cytosine(Cyt) (C) can urge the combination of primer and amplified target gene like this.For 3 Nucleotide of 3 ' end, guanine and cytosine(Cyt) contribute to the stable bond of polysaccharase, can promote the amplification capability of primer.(2) special sequence had better not be there is in primer, the poly-purine of such as long string or poly-pyrimidine.GC too high levels (>70%) or too low (<30%) are unfavorable for that RPA increases.(3) in addition, should avoid easily forming secondary structure when primer and probe design as far as possible, primer-primer is done mutually, primer-probe is done mutually, the sequence of hairpin structure, reduces dimeric formation.
With B. abortus A19 vaccine strain DNA for template, the various combination of above-mentioned Brucella primer and probe and A19 diagnostic primers and probe is screened, finally select one group of amplification efficiency respectively high, the primer of high specificity and probe combinations, differentiate to detect for brucella A19 vaccine strain RPA-LFD, primer and probe sequence are 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 is nfo nuclease cleavage site; C3-Spacer: polymerase extension blocker.
The foundation of embodiment two, brucella A19 vaccine strain differential diagnosis RPA-LFD detection method
1. experimental procedure
(1) extraction of bacterial genomes DNA
Get 1ml bacterium liquid, carry out the extraction of bacteria total DNA according to TIANGEN Biotech's bacterial genomes DNA extraction kit specification sheets.
(2) foundation of brucella A19 vaccine strain differential diagnosis RPA-LFD reaction system
RPA reaction system is 50 μ L:
Above-mentioned 47.5 μ L mixtures are added RPA-nfo reaction tubes, fully mixing, dissolving.Finally add 2.5 μ L 280mM magnesium acetate solution, mixing of turning upside down, after centrifugal gently, reaction tubes is placed in 38 DEG C of water bath processing 20-30min.Get 2 μ L amplified productions after each RPA reaction terminates to detect damping fluid with 98 μ L LFD and mix, the corresponding LFD of each reaction, LFD is immersed corresponding reaction mixture, observations within 5min, if there is test strip and contrast band during Brucella-RPA-LFD and A19-RPA-LFD two test set contracts, then in this sample, brucella A19 vaccine strain is positive; There is test strip and contrast band in Brucella-RPA-LFD test set, and band only appears contrasting in A19-RPA-LFD test set, then illustrate in this sample containing other brucella beyond brucella A19 vaccine strain; All there is not test strip and occur contrasting band simultaneously in Brucella-RPA-LFD and A19-RPA-LFD two test set, then illustrates and detect in sample not containing Brucella bacterium.Occur that other situations then illustrate that RPA-LFD brucella A19 differentiates that detection is false.
(3) brucella A19 vaccine strain differential diagnosis RPA-FLD sensitivity technique
Utilize colony counting method to calculate B. abortus A19 bacterial concentration, become 3 × 10 with 10 times of doubling dilutions respectively
8– 3 × 10
-1cfu/mL bacterium liquid, gets each 1mL of bacterium liquid of corresponding extension rate, adopts bacterial genomes DNA extraction kit to extract the bacterium liquid genomic dna of each extension rate, dissolve with 100 μ L TE, getting 2 μ L genomic dnas is template, carries out RPA amplification according to above-mentioned steps (2), simultaneously with ddH
2o is negative control, the susceptibility of inspection present method.
(4) brucella A19 vaccine strain differential diagnosis RPA-FLD specific detection
With the RPA-FLD test kit of differential diagnosis A19 vaccine strain of the present invention to B. abortus A19 vaccine strain, pig kind brucella S2 vaccine strain, brucella melitensis M5-90 vaccine strain, Br.ovis, Br. cants, Escherichia coli O 157: H7, Yersinia enterocolitica O:9, Salmonellas, streptococcus aureus, cow mycobacteria genomic dna increase, simultaneously with ddH
2o is negative control, the specificity of checking present method.
2. test-results
Carry out RPA-LFD respectively to two groups of RPA-LFD primers and probe combinations to detect and screen, the primer that optimization 2 kinds is optimum and probe combinations, result as shown in Figure 1.With the B. abortus A19 vaccine strain genomic dna of 10 of 10 times of doubling dilutions gradients for template detects, as shown in Figure 2, two groups of RPA-LFD detectabilities of 50 μ L systems are 3 × 10 to result
0cfu.To brucella and other bacteriums of 5 strains bacterial strain in contrast of 5 strain different generas, carry out two kinds of RPA-LFD respectively to detect, as shown in Figure 3, all there is band at the detection line place of Sidestream chromatography test strip in 5 strain brucella Brucella-RPA-LFD reaction tubess to result, is positive findings; And other 5 strain bacterial isolates and ddH
2all there is not test strip in O contrast, all occurs contrast band.A19-RPA-LFD amplification group only has B. abortus A19 vaccine strain genome to occur test strip, and test strip appears in other amplifications Guan Junwei, is feminine gender.Illustrate that two groups of RPA-LFD primers and probe combinations respectively can specific detection Brucella bacterium and brucella A19 vaccine strains.
The differential diagnosis 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 adopted international standards, be called for short AGI), with the phosphate buffered saline buffer of 10mL pH value 7.0 (PBS) for sampling media, gather 20min according to the sampling flow of 12.5L/min, collect the aerosol sample of different zones in plant's environment.At room temperature the centrifugal 10min of 12000r/min, abandons supernatant liquor, and precipitation is used for By Direct Pyrolysis process.
(2) in situ preparation of the clinical sample such as aerosol
By the clinical samples such as aerosol 200 μ L TE damping fluid (1.0M Tris-HCl (pH8.0) 10mL, 0.5MNa
2eDTA2H
2o (pH8.0) 2mL, adding distil water is to l000mL) resuspended, liquid sample directly gets 200 μ L, then adds 30 μ L 10% (w/v) SDS and 3 μ L 2% (w/v) Proteinase Ks, mixes rear 37 DEG C of incubation 1h.
(3) discriminating of the clinical sample brucella A19 vaccine strain such as aerosol detects
According to the method for the on-the-spot cracking process of clinical sample in step (2), respectively to this laboratory preserve with checking order 10 parts of positive aerosol samples of brucella after pcr amplification correct, wherein 3 parts of brucella A19 vaccine strains are positive, 3 parts of milk samples and 3 parts of blood brucella positive sample, and 2 parts of aerosols, 1 part of milk sample and 1 part of blood brucella negative sample process, simultaneously with B. abortus A19 vaccine strain DNA and ddH
2o is respectively positive, negative control, carries out two groups of RPA-LFD augmentation detection respectively.
2. test-results
To 12 parts of aerosol samples of this Laboratory Diagnosed, 4 parts of blood samples and 4 parts of milk sample samples, carry out Brucella-RPA-LFD and A19-RPA-LFD respectively to detect, wherein negative 2 parts, the aerosol sample of brucella, brucella negative blood and each 1 part of milk sample sample, result is as shown in Fig. 4 and table 2.Above result absolutely proves and can be used for differential diagnosis Brucella bacterium and A19 vaccine strain with two groups of RPA-LFD primers, probe combinations and test kits provided by the invention, the method has higher sensitivity and specificity, the most important thing is that can directly carry out scene to clinical aerosol equal samples diagnoses fast.
The discriminating detected result of the clinical sample brucella A19 vaccine strains such as table 2 aerosol
By reference to the accompanying drawings the specific embodiment of the present invention is described although above-mentioned; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.
Claims (10)
1. differentiate primer pair and the probe combinations of brucella A19 vaccine strain in aerosol for one kind, it is characterized in that, Brucella-RPA-LFD forward primer sequence is as shown in SEQIDNo.1, and reverse primer sequences is as shown in SEQIDNo.2, and probe sequence is as shown in SEQIDNo.3; A19-RPA-LFD forward primer sequence is as shown in SEQIDNo.4, and reverse primer sequences is as shown in SEQIDNo.5, and probe sequence is as shown in SEQIDNo.6.
2. primer pair as claimed in claim 1 and probe combinations, is characterized in that, the in situ preparation method of the clinical samples such as described aerosol is as follows:
Clinical sample is resuspended with 200 μ L TE damping fluids, and liquid sample directly gets 200 μ L, then adds 30 μ L 10% (w/v) SDS and 3 μ L 2% (w/v) Proteinase Ks, mixes rear 37 DEG C of incubation 1h; Described TE damping fluid is the Tris-HCl 10mL of 1.0M pH8.0, the Na of 0.5M pH8.0
2eDTA2H
2o 2mL, adding distil water is to 1000mL.
3. differentiate a test kit for brucella A19 vaccine strain in aerosol, it is characterized in that, this test kit comprises primer pair according to claim 1 and probe combinations.
4. test kit as claimed in claim 3, it is characterized in that, comprise the recA recombinase of Escherichia coli, strand displacement archaeal dna polymerase, single-stranded DNA binding protein, rehydration damping fluid, 280mM magnesium acetate solution, Sidestream chromatography test strip, PBST ELISA test strip damping fluid (1 × PBS+0.1% polysorbas20), sterilizing deionization distilled water, TE damping fluid, 10% (w/v) SDS, 2% (w/v) Proteinase K, brucella A19 vaccine strain standard positive template.
5., for a method for brucella A19 vaccine strain in rapid differential diagnosis aerosol sample, it is characterized in that, comprise the steps:
(1) detect the extraction of sample DNA or detect sample cracking process;
(2) with the sample of process in step (1) for template, carry out RPA amplification;
(3) with the above-mentioned RPA amplified production of Sidestream chromatography ELISA test strip, whether judge in sample containing brucella A19 vaccine strain according to detected result.
6. method as claimed in claim 5, it is characterized in that, in described step (2), the amplification system of RPA is: RPA reaction system is 50 μ L: comprising the forward primer of 2.1 μ L 10 μMs, the reverse primer of 2.1 μ L 10 μMs, the probe of 0.6 μ L 10 μMs, rehydration damping fluid 29.5 μ L, sample to be tested DNA or thick split product 2 μ L, ddH
2o 11.2 μ L.
7. method as claimed in claim 5, it is characterized in that, in described step (2), the amplification program of RPA is: amplification system mixed solution 47.5 μ L is added RPA-nfo reaction tubes, mixing, dissolving, finally add the magnesium acetate solution of 2.5 μ L 280mM, in 37-39 DEG C of reaction 20-30 minute after mixing of turning upside down.
8. method as claimed in claim 5, it is characterized in that, the concrete steps of described step (3) are: get 2 μ L amplified productions respectively for each RPA reaction tubes and mix with 98 μ L PBST ELISA test strip damping fluids, a LFD is immersed corresponding reaction mixture, observations within 5min.
9. the method for brucella A19 vaccine strain in discriminating aerosol according to claim 5, is characterized in that: in step (3), and the decision method whether containing brucella A19 vaccine strain in sample is:
If there is test strip and contrast band, then brucella A19 vaccine strain in this sample during Brucella-RPA-LFD and A19-RPA-LFD two test set contracts;
There is test strip and contrast band in Brucella-RPA-LFD test set, and band only appears contrasting in A19-RPA-LFD test set, then illustrate in this sample containing other brucella beyond brucella A19 vaccine strain;
All there is not test strip and occur contrasting band in Brucella-RPA-LFD and A19-RPA-LFD two test set, then illustrates and detect in sample not containing Brucella bacterium.
10. method as claimed in claim 7, is characterized in that, in 38 DEG C of reactions 25 minutes after mixing of turning upside down.
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| CN108048584A (en) * | 2017-12-08 | 2018-05-18 | 吴斌 | The brucellar probe of RAA Fluorometric assays and kit |
| CN108220461A (en) * | 2018-01-29 | 2018-06-29 | 中国农业科学院兰州兽医研究所 | A kind of primer, probe and kit based on recombinase polymeric enzymatic amplification method detection brucella |
| 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 |
| CN115948588A (en) * | 2023-02-11 | 2023-04-11 | 昆明理工大学 | Multiple fluorescent quantitative PCR (polymerase chain reaction) seed separating detection reagent for four Brucella species |
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| CN108220461A (en) * | 2018-01-29 | 2018-06-29 | 中国农业科学院兰州兽医研究所 | A kind of primer, probe and kit based on recombinase polymeric enzymatic amplification method detection brucella |
| 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 |
| CN115948588A (en) * | 2023-02-11 | 2023-04-11 | 昆明理工大学 | Multiple fluorescent quantitative PCR (polymerase chain reaction) seed separating detection reagent for four Brucella species |
| CN115948588B (en) * | 2023-02-11 | 2024-04-19 | 昆明理工大学 | Multiple fluorescent quantitative PCR (polymerase chain reaction) seed separation detection reagent for four species of Brucella |
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