CN104911269A - Primers, probe and kit for identifying Brucella A19 vaccine strain in aerosol - Google Patents

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

Primers, probes and kits for identifying Brucella A19 vaccine strains in aerosol

technical field

The invention belongs to the field of biotechnology, in particular to the application of Recombinase Polymerase Amplification and Lateral Flow Dipstick (RPA-LFD) rapid differential diagnosis of brucellosis in aerosol and other clinical samples. Primers, probes and kits for Bacillus A19 vaccine strain.

Background technique

Brucellosis is an infectious disease caused by bacteria of the genus Brucella (mainly Brucella in cattle, sheep, pigs and dogs, etc.) Important pathogens for statutory quarantine and purification. In recent years, the incidence rate has shown an increasing trend year by year, especially for dairy animals. The abortion rate is as high as 50%-80%, and the output of milk and meat is reduced by 10%-20%. According to the "Veterinary Bulletin" statistics, in 2013, there were 3,620 cases of brucellosis nationwide, involving 42,720 cattle and sheep. Therefore, brucellosis poses a serious threat to social and economic development and stability, and national public health security.

The source of infection of brucellosis is mainly sick animals and carriers. They excrete Brucella through secretions, milk, body fluids, etc., polluting the environment, while Brucella adheres to the dust in the air and suspends particles, form aerosols and spread, infecting other animals and humans. The distribution of Brucella aerosol in the farm environment can cause the infection and prevalence of brucellosis, and bring serious harm to animal husbandry production and human health, especially in the current intensive and high-density breeding environment. The formation of Brucella aerosol epidemic. Therefore, by monitoring Brucella aerosol in the farm environment, the outbreak and prevalence of Brucellosis can be effectively forecasted.

The main strategies for the prevention and control of animal brucellosis in my country are quarantine and purification. At present, the commonly used live vaccine strain of Brucella bovis A19 in herd immunity in my country will interfere with the diagnosis of brucellosis wild virus in the diagnosis of antigens and antibodies. This is because the diagnostic methods for brucellosis currently used in my country cannot differentiate between natural infection and artificial vaccine immunity, which will inevitably lead to some naturally infected sick animals escaping quarantine, making sick animals exist for a long time, and affecting cattle and humans. Security has long been a threat. Therefore, it is difficult for my country to implement the prevention and control procedures for brucellosis quarantine, culling, and purification.

Contents of the invention

The purpose of the present invention is to provide a kind of primer, probe and test kit for distinguishing Brucella A19 vaccine strain in aerosol, and this test kit can be specific, sensitive, simple and fast on-the-spot differential diagnosis Brucella in aerosol A19 vaccine strain.

For realizing the object of the present invention, adopt following technical scheme:

A primer pair and probe combination for identifying Brucella A19 vaccine strains in aerosol, wherein the Brucella-RPA-LFD forward primer sequence is shown in SEQIDNo.1, and the reverse primer sequence is shown in SEQIDNo.2. The needle sequence is shown in SEQIDNo.3; the A19-RPA-LFD forward primer sequence is shown in SEQIDNo.4, the reverse primer sequence is shown in SEQIDNo.5, and the probe sequence is shown in SEQIDNo.6.

The present invention also provides a kit for identifying the Brucella A19 vaccine strain in the aerosol, the kit comprising the above-mentioned primer pair and probe combination.

The present invention further provides a kind of method for rapid differential diagnosis Brucella A19 vaccine strain in aerosol sample, comprises the steps:

(1) Extraction of test sample DNA or test sample lysis treatment;

(2) using the sample processed in step (1) as a template to perform RPA amplification;

(3) Use a lateral flow chromatography test strip to detect the above-mentioned RPA amplification product, and determine whether the sample contains the Brucella A19 vaccine strain according to the detection result.

Preferably, the RPA amplification system described in step (2) is: RPA reaction system is 50 μL: including 2.1 μL 10 μM forward primer, 2.1 μL 10 μM reverse primer, 0.6 μL 10 μM probe, rehydration buffer 29.5 μL, 2 μL of DNA or crude lysate of the sample to be tested, 11.2 μL of ddH ₂ O; add the above 47.5 μL mixture into the RPA-nfo reaction tube, mix well and dissolve, and finally add 2.5 μL of 280 mM magnesium acetate solution, mix up and down After mixing, react in a constant temperature water bath at 38°C for 20-30 minutes.

Preferably, the specific steps of step (3) are as follows: 2 μL of each RPA reaction product is mixed with 98 μL PBST test strip detection buffer (1×PBS+0.1% Tween 20), and a flow chromatography test Immerse the paper strip (LFD) and observe the results within 5 minutes. If the detection band and the control band appear in the two detection combinations of Brucella-RPA-LFD and A19-RPA-LFD at the same time, it means that the Brucella A19 vaccine strain in the sample Positive; Brucella-RPA-LFD detection group appeared detection bands and control bands, while only control bands appeared in A19-RPA-LFD detection group, indicating that the sample contained other Brucella strains other than Brucella A19 vaccine strains. Bacteria; Brucella-RPA-LFD and A19-RPA-LFD two test groups do not appear detection bands and control bands appear at the same time, indicating that the test samples do not contain Brucella bacteria. The occurrence of other conditions indicates that the differential detection of RPA-LFD Brucella A19 is not established.

The present invention also provides a kit for differential diagnosis of Brucella A19 vaccine strain containing the above two sets of primers and probe combinations. The kit also includes recA recombinase derived from Escherichia coli, strand-displacing DNA polymerase, single-strand DNA binding protein (SSB), rehydration buffer, 280mM magnesium acetate (MgAc) solution, lateral flow chromatography test strips (specific Biotin and FAM marker gene amplification product), PBST test strip detection buffer (1×PBS+0.1% Tween 20), sterilized deionized double distilled water (ddH ₂ O), TE buffer, 10 % (w/v) SDS, 2% (w/v) proteinase K, Brucella A19 vaccine strain standard positive template.

Using the genome differential DNA sequence of Brucella A19 vaccine strain and other Brucella strains as the identification target sequence, the application of RPA-LFD technology can realize rapid on-site differential diagnosis of Brucella A19 vaccine strain and other Brucella strains . RPA technology uses a mixture of three enzymes, namely, recombinase capable of binding single-stranded nucleic acid (oligonucleotide primer), single-stranded DNA binding protein (SSB) and strand-displacing DNA polymerase, which is also active at room temperature and has the best response The temperature is 37-39°C, and the whole process can generally obtain a detectable level of amplification product within ten minutes. The sensitivity of RPA detection is very high, and it can amplify trace levels (trace levels) of nucleic acid (especially DNA) templates to a detectable level, and obtain about 10 ¹² amplification products from a single template molecule, which is more suitable for low-concentration Clinical testing template. Moreover, the RPA reaction does not require high template purity, and is suitable for on-site detection where nucleic acid cannot be extracted. At present, the RPA reaction can be detected by three different methods: agarose gel electrophoresis, fluorescence amplification curve and LFD test strip. The principles of primer and probe reactions in these three methods are different. Gel electrophoresis and fluorescence amplification curve methods still rely on laboratories and special instruments and equipment, while LFD is a method that can be used on-site without special instruments.

There are currently no specific rules for the design of primers and probes for the RPA-nfo reaction. Only through lateral flow chromatography test strips (LFD) for RPA reactions can screening primers and probe combinations that can be used for clinical testing be obtained. A FAM-labeled specific probe was designed in the amplified target sequence of the RPA-nfo forward primer, and the reverse primer was labeled with biotin. The biotin-labeled RPA product hybridized with the FAM-labeled specific probe, and the FAM-labeled After the specific probe is combined with the gold standard of the anti-FAM antibody, the immune complex is dropped onto the test strip, and the immune complex diffuses through the chromatographic membrane. When it diffuses to the detection line, the biotin-labeled amplification product is detected. The biotin ligand is captured to form a colored detection line, the detection band. The uncaptured immune complex continues to diffuse to the quality control line and is captured by the specific antibody, forming a colored quality control line, that is, the control strip. In this way, the combination of RPA technology and lateral flow chromatography technology, that is, RPA-LFD technology, can read the results through lateral flow chromatography test strips (LFD). This method neither requires special equipment nor complicated sample processing. Results can be observed visually using common heating devices such as water baths and lateral flow test strips. Therefore, RPA-LFD technology has broad application prospects in the on-site diagnosis of clinical samples such as Brucella aerosol and the identification of vaccine strain A19.

Compared with the prior art, the present invention has the advantages of:

(1) The RPA technology provided by the present invention is a new method emerging in recent years that is superior to PCR and loop-mediated isothermal amplification (LAMP) technology in terms of amplification rate and yield, and its reaction can be completed within 30 minutes. Approximately 10 ¹² amplification products were obtained from a single template molecule.

(2) The combination of RPA-LFD primers and probes and the kit for the differential diagnosis of Brucella bacteria and A19 vaccine strains provided by the present invention have high sensitivity and strong specificity, and a minimum of 3×10 ⁰ cfu can be detected. Rutella bacteria or Brucella A19 vaccine strain bacteria, Brucella-RPA-LFD detection group and Escherichia coli O157:H7, Yersinia enterocolitica O:9, Salmonella, Staphylococcus aureus and other bacteria had no Cross reaction. The A19-RPA-LFD test group had no cross-reaction with other Brucella strains, Escherichia coli O157:H7, Yersinia enterocolitica O:9, Salmonella, Staphylococcus aureus and other bacteria.

(3) The Brucella A19 vaccine strain differential diagnosis RPA-LFD primer and probe combination provided by the present invention and the test kit can not only be used for the detection of Brucella A19 vaccine strain bacterial strain and aerosol sample, also can be used for other Detection of clinical samples such as blood, milk samples, etc.

(4) The Brucella A19 vaccine strain differential diagnosis RPA-LFD detection method provided by the present invention is easy to use, does not need special instrument and equipment, only at 38 ℃, the crude lysate of 30min just can be tested Brucella The sensitive, specific and rapid differential detection of the A19 vaccine strain is suitable for the differential diagnosis of the A19 vaccine strain and the wild strain of brucellosis in the field or at the grassroots level, and can also be used for the detection of bacteria belonging to the genus Brucella.

(5) Use the test kit of the present invention to effectively differentially diagnose the natural infection and artificial vaccine immunity of brucellosis, avoid the sick animals of natural infection from evading quarantine, and find the existence of sick animals in time.

Description of drawings

Figure 1 Screening of primers and probes for differential diagnosis of Brucella A19 vaccine strain RPA-LFD detection combination, RPA amplification results of primers, probes and templates provided by the ControlPrimer/Probe Mix amplification group for TwistAmp nfo kit; Brucella -RPA-LFD group is the primer and probe of the specific detection Brucella bacterium that the present invention screens out and takes Brucella A19 vaccine strain genome DNA as the amplification result of template; A19-RPA-LFD group is based on The primers and probes screened by the invention to specifically detect the Brucella A19 vaccine strain use the genomic DNA of the Brucella A19 vaccine strain as a template for the amplification results; NC is a negative control in which ddH ₂ O replaces the template.

Figure 2 Sensitivity detection of RPA-LFD for the differential diagnosis of Brucella A19 vaccine strain, the templates are 3×10 ⁸ cfu–3×10 ^-1 cfu genomic DNA of Brucella bovis A19 vaccine strain, NC is based on ddH ₂ O Negative control group in place of template.

Figure 3 Differential diagnosis of Brucella A19 vaccine strain RPA-LFD specific detection, B.abortus A19, B.suis S2, B.melitensis M5-90, B.ovis, B.canis, E.coli O157:H7, The templates of Y.enterocolitica O:9, Salmonella, S.aureus, and M.bovis are Brucella bovis vaccine strain A19, Brucella suis vaccine strain S2 strain, and Brucella melis vaccine strain M5-90 strain, Brucella ovis 63/290 strain, Brucella canis RM6/66 strain, Escherichia coli O157:H7, Yersinia enterocolitica O:9, Salmonella, Staphylococcus aureus, Genomic DNA of Mycobacterium bovis; NC is a negative control group in which ddH ₂ O was used instead of template.

Figure 4 Differential diagnosis of Brucella A19 vaccine strain RPA-LFD in aerosol and other clinical samples, +: template is genomic DNA of Brucella bovis A19 vaccine strain, NC: negative control group with ddH ₂ O instead of template, 1-10: Brucella nucleic acid-positive aerosol samples, of which samples 1-3 are nucleic acid-positive aerosol samples of the A19 vaccine strain, 11-13: Brucella nucleic acid-positive blood samples, of which samples 11 and 12 are A19 vaccine strain nucleic acid positive blood samples, 14-16: Brucella nucleic acid positive milk samples, of which samples 14 and 15 are A19 vaccine strain nucleic acid positive milk samples, 17-20: Brucella negative gas samples respectively Sol, blood, milk samples and clinical samples of aborted fetuses.

Detailed ways

The following examples are used to further illustrate the present invention, but are not intended to limit the scope of the present invention.

The following examples are all carried out according to conventional test conditions and methods, or according to the test conditions suggested by the manufacturer.

1. Test material

Brucella bovis vaccine strain A19 (B.abortus A19), Brucella suis vaccine strain S2 strain (B.suis S2), Brucella sheep species vaccine strain M5-90 strain (B.melitensis M5-90), Brucella ovis 63/290 strain (B.ovis63/290), Brucella canis RM6/66 strain (B.canis RM6/66), Escherichia coli O157:H7, enterocolitis Yersinia O:9, Salmonella, Staphylococcus aureus (CMCC26003), Mycobacterium bovis (M.bovis ATCC 19210), clinically confirmed Brucella positive aerosol, blood and milk DNA samples, etc. Preserved by the Disease Research Office of Dairy Cow Research Center, Shandong Academy of Agricultural Sciences.

Primers and probes were synthesized by Shanghai Sangon Biotechnology Co., Ltd. TwistAmp DNA Amplification nfo Kits were purchased from TwistDX Company, lateral flow chromatography test strips (HybriDetect Dipsticks) were purchased from Milenia Company, and other biochemical reagents were imported or domestically produced analytically pure.

2. Experimental equipment

Constant temperature water bath, centrifuge, handheld centrifuge, vortex instrument, MS-I multifunctional microbial sampler (Qingdao Zhongrui Intelligent Instrument Co., Ltd.), etc.

Embodiment 1. Design and screening of primers and probes

The present invention designs 2 specific probes of Brucella-RPA-LFD according to the conserved VirB gene (GengBank No.AF226278) of Brucella bacteria, and designs 4 probes on both sides of the probe. 1 forward primer and 2 reverse primers. At the same time, 4 A19-RPA-LFD reverse primers for specific detection of the A19 vaccine strain were designed according to the deletion of 7 nucleotide sequences (AGATTTC) at the 22840-22846 position in the genome of the A19 vaccine strain, and the upstream of the deletion site was designed 2 forward primers and 4 specific probes. The RPA-nfo reverse primer is positioned here to differentiate the A19 vaccine strain from other Brucella bacteria.

The primer length of RPA-nfo is generally 30-35nt, and if the primer is too short, it will seriously affect the activity of the recombinase. Longer primers do not necessarily improve amplification performance, but rather increase the likelihood of secondary structure formation. The length of the LF probe is generally 46-52nt, and the nfo ribozyme cleavage site is about 30 bases away from the 5' end of the probe, and about 16-22 bases away from the 3' end. At present, there are no specific operating rules for the design of RPA-LFD primers and probes. The primers and probes that can be used in clinical testing can only be obtained through the detection of lateral flow chromatography test strips (LFD) for RPA reactions and the screening of tests.

In this test, multiple pairs of primers and probes need to be designed from both ends of the target sequence for optimization and screening, and the substitution or increase or decrease of individual bases will have an important impact on the test results. When designing primers and probes, we also considered three points for attention: (1) 3-5 nucleotides at the 5' end should avoid polyguanine (G) and cytosine (C), which can promote primers and probes. Amplify the binding of the target gene. For the three nucleotides at the 3' end, guanine and cytosine contribute to the stable binding of the polymerase, which can improve the amplification performance of the primer. (2) It is best not to have special sequences in the primers, such as long strings of polypurine or polypyrimidine. Too high (>70%) or too low (<30%) GC content is not conducive to RPA amplification. (3) In addition, when designing primers and probes, try to avoid sequences that are easy to form secondary structures, primer-primer interactions, primer-probe interactions, and hairpin structures, so as to reduce the formation of dimers.

Using the DNA of Brucella bovis A19 vaccine strain as a template, different combinations of primers and probes for the above-mentioned Brucella genus and A19 identification primers and probes were screened, and finally a group with high amplification efficiency and specificity was selected. A strong combination of primers and probes for the identification and detection of Brucella A19 vaccine strain RPA-LFD, the primers and probe sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, 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: carboxyfluorescein labeling; dSpacer: nucleotide base analogue, which is the nfo nuclease cleavage site; C3-Spacer: polymerase extension blocker.

Embodiment two, the establishment of the differential diagnosis RPA-LFD detection method of Brucella A19 vaccine strain

1. Experimental steps

(1) Extraction of bacterial genomic DNA

Take 1ml of the bacterial liquid, and extract the total bacterial DNA according to the instructions of the Genomic DNA Extraction Kit of Tiangen Biochemical Technology (Beijing) Co., Ltd.

(2) Establishment of RPA-LFD reaction system for differential diagnosis of Brucella A19 vaccine strains

The RPA reaction system is 50 μL:

Add 47.5 μL of the above mixture into the RPA-nfo reaction tube, mix well and dissolve. Finally, 2.5 μL of 280 mM magnesium acetate solution was added, mixed upside down, gently centrifuged, and the reaction tube was placed in a 38°C water bath for 20-30 minutes. After each RPA reaction, take 2 μL of the amplification product and mix it with 98 μL of LFD detection buffer. Each reaction corresponds to one LFD. Immerse the LFD in the corresponding reaction mixture and observe the results within 5 minutes. If Brucella-RPA-LFD and A19- The detection band and the control band appear in the two detection combinations of RPA-LFD at the same time, and the Brucella A19 vaccine strain in the sample is positive; the detection band and the control band appear in the Brucella-RPA-LFD detection group, and the A19-RPA - Only the control band appears in the LFD detection group, which means that the sample contains other Brucella bacteria other than the Brucella A19 vaccine strain; Brucella-RPA-LFD and A19-RPA-LFD detection groups are not detected If the band appears at the same time as the control band, it means that the test sample does not contain bacteria of the genus Brucella. The occurrence of other conditions indicates that the differential detection of RPA-LFD Brucella A19 is not established.

(3) Differential diagnosis of Brucella A19 vaccine strain RPA-FLD sensitivity detection

Calculate the concentration of Brucella bovis A19 bacterial solution by plate counting method, and dilute it into 3×10 ⁸ -3×10 ^-1 cfu/mL bacterial solution by 10 times, and take 1 mL of the bacterial solution corresponding to the dilution multiple, Use the bacterial genomic DNA extraction kit to extract the bacterial liquid genomic DNA of each dilution factor, dissolve it in 100 μL TE, take 2 μL genomic DNA as a template, and perform RPA amplification according to the above step (2), and use ddH ₂ O as a negative control to test sensitivity of this method.

(4) Differential diagnosis of Brucella A19 vaccine strain RPA-FLD specific detection

With the RPA-FLD kit of differential diagnosis A19 vaccine strain of the present invention, to Brucella bovis A19 vaccine strain, Brucella suis S2 vaccine strain, Brucella melis M5-90 vaccine strain, sheep seed cloth Genomic DNA of Rubyella, Brucella canis, Escherichia coli O157:H7, Yersinia enterocolitica O:9, Salmonella, Staphylococcus aureus, and Mycobacterium bovis were amplified with ddH ₂ O as Negative control to verify the specificity of this method.

2. Test results

The two sets of RPA-LFD primer and probe combinations were respectively screened for RPA-LFD detection, and two optimal primer and probe combinations were optimized. The results are shown in Figure 1. The genomic DNA of the 10-fold diluted Brucella bovis A19 vaccine strain in 10 gradients was used as a template for detection, and the results are shown in Figure 2. The detection limits of the two groups of RPA-LFD in the 50 μL system were both 3×10 ⁰ cfu. 5 strains of different species of Brucella and 5 strains of other bacteria were used as control strains, and two RPA-LFD detections were carried out respectively. The results are shown in Figure 3. Five strains of Brucella-RPA-LFD reaction tubes were There were bands on the detection line of the lateral flow chromatography test strips, all of which were positive results; while the other five bacterial strains and the ddH ₂ O control did not have detection bands, and all had control bands. In the A19-RPA-LFD amplification group, only the genome of the Brucella bovis A19 vaccine strain showed detection bands, and no detection bands appeared in other amplification tubes, all of which were negative. It shows that the two sets of RPA-LFD primers and probe combinations can specifically detect the bacteria of the genus Brucella and the vaccine strain of Brucella A19 respectively.

Embodiment three, the differential diagnosis of clinical samples such as aerosol Brucella A19 vaccine strain

1. Experimental steps

(1) Collection of aerosol samples

The international standard all-glass-impinger (AGI) was used, and 10 mL of phosphate buffer saline (PBS) with a pH value of 7.0 was used as the sampling medium, and the sampling flow rate was 12.5 L/min for 20 minutes. Collect aerosol samples from different areas of the farm environment. Centrifuge at 12000r/min for 10min at room temperature, discard the supernatant, and precipitate for direct lysis.

(2) On-site preparation of clinical samples such as aerosol

Resuspend clinical samples such as aerosols with 200 μL TE buffer (1.0M Tris-HCl (pH8.0) 10mL, 0.5MNa ₂ EDTA·2H ₂ O (pH8.0) 2mL, add distilled water to 1000mL), liquid samples directly Take 200 μL, then add 30 μL 10% (w/v) SDS and 3 μL 2% (w/v) proteinase K, mix and incubate at 37° C. for 1 h.

(3) Identification and detection of Brucella A19 vaccine strains in aerosol and other clinical samples

According to the method of on-site cracking treatment of clinical samples in step (2), the 10 Brucella positive aerosol samples that have been sequenced correctly after PCR amplification and 3 Brucella A19 vaccine strains stored in our laboratory were respectively analyzed. Positive, 3 milk samples and 3 blood samples positive for Brucella, and 2 aerosol samples, 1 milk sample and 1 blood sample negative for Brucella were treated with Brucella bovis A19 vaccine Strain DNA and ddH ₂ O were used as positive and negative controls, respectively, and two groups of RPA-LFD amplification tests were carried out.

2. Test results

The 12 aerosol samples, 4 blood samples and 4 milk samples confirmed by our laboratory were tested for Brucella-RPA-LFD and A19-RPA-LFD, of which 2 Brucella negative aerosol samples, Brucella-negative blood and milk samples were 1 each, and the results are shown in Figure 4 and Table 2. The above results fully illustrate that two groups of RPA-LFD primers, probe combinations and test kits provided by the present invention can be used for differential diagnosis of Brucella bacteria and A19 vaccine strains, the method has higher sensitivity and specificity, the most important The most important thing is that samples such as clinical aerosol can be directly diagnosed on-site and quickly.

Table 2 The identification and detection results of Brucella A19 vaccine strains in aerosol and other clinical samples

Although the specific implementation of the present invention has been described above in conjunction with the accompanying drawings, it does not limit the protection scope of the present invention. Those skilled in the art should understand that on the basis of the technical solution of the present invention, those skilled in the art do not need to pay creative work Various modifications or variations that can be made are still within the protection scope of the present invention.

Claims (10)

1. a kind of primer pair and probe combination of distinguishing Brucella A19 vaccine strain in aerosol, it is characterized in that, Brucella-RPA-LFD forward primer sequence is as shown in SEQIDNo.1, and reverse primer sequence is as shown in SEQIDNo. 2, the probe sequence is shown in SEQIDNo.3; the A19-RPA-LFD forward primer sequence is shown in SEQIDNo.4, the reverse primer sequence is shown in SEQIDNo.5, and the probe sequence is shown in SEQIDNo.6 . 2. primer pair and probe combination as claimed in claim 1, is characterized in that, the on-the-spot preparation method of clinical samples such as described aerosol is as follows: Resuspend the clinical sample with 200 μL TE buffer, take 200 μL of the liquid sample directly, then add 30 μL 10% (w/v) SDS and 3 μL 2% (w/v) proteinase K, mix and incubate at 37°C for 1 h; TE buffer was 10 mL of 1.0M Tris-HCl at pH 8.0, 2 mL of 0.5 M Na ₂ EDTA·2H ₂ O at pH 8.0, and added distilled water to 1000 mL. 3. a test kit for distinguishing Brucella A19 vaccine strain in aerosol, it is characterized in that, this test kit comprises primer pair and probe combination described in claim 1. 4. kit as claimed in claim 3 is characterized in that, comprises the recA recombinase of Escherichia coli source, strand displacement DNA polymerase, single-stranded DNA binding protein, rehydration buffer, 280mM magnesium acetate solution, lateral flow chromatography Test strips, PBST test strip detection buffer (1×PBS+0.1% Tween 20), sterilized deionized double distilled water, TE buffer, 10% (w/v) SDS, 2% (w/v ) proteinase K, Brucella A19 vaccine strain standard positive template. 5. a method for rapid differential diagnosis of Brucella A19 vaccine strain in the aerosol sample, is characterized in that, comprises the steps: (1) Extraction of test sample DNA or test sample lysis treatment; (2) using the sample processed in step (1) as a template to perform RPA amplification; (3) Use a lateral flow chromatography test strip to detect the above-mentioned RPA amplification product, and determine whether the sample contains the Brucella A19 vaccine strain according to the detection result. 6. The method according to claim 5, wherein the amplification system of RPA in the step (2) is: the RPA reaction system is 50 μL: including 2.1 μL of 10 μM forward primer, 2.1 μL of 10 μM reverse primer For primers, 0.6 μL of 10 μM probe, 29.5 μL of rehydration buffer, 2 μL of sample DNA or crude lysate, and 11.2 μL of ddH ₂ O. 7. The method according to claim 5, characterized in that, the amplification program of RPA in the described step (2) is: 47.5 μ L of the amplification system mixed solution is added to the RPA-nfo reaction tube, mixed, dissolved, and finally Add 2.5μL of 280mM magnesium acetate solution, mix upside down and react at 37-39℃ for 20-30 minutes. 8. the method as claimed in claim 5, is characterized in that, the concrete step of described step (3) is: get 2 μ L amplified product and 98 μ L PBST test strip detection buffer respectively for each RPA reaction tube and mix, put One LFD is immersed in the corresponding reaction mixture, and the results can be observed within 5 minutes. 9. the method for distinguishing Brucella A19 vaccine strain in the aerosol according to claim 5 is characterized in that: in step (3), whether contain the judging method of Brucella A19 vaccine strain in the sample is: If the detection band and the control band appear in the two detection combinations of Brucella-RPA-LFD and A19-RPA-LFD at the same time, the Brucella A19 vaccine strain is contained in the sample; The detection band and control band appear in the Brucella-RPA-LFD detection group, but only the control band appears in the A19-RPA-LFD detection group, indicating that the sample contains other Brucella bacteria other than the Brucella A19 vaccine strain ; Both the Brucella-RPA-LFD and A19-RPA-LFD test groups did not appear detection bands but appeared control bands, indicating that the test samples did not contain Brucella bacteria. 10. The method according to claim 7, characterized in that, after mixing upside down, react at 38° C. for 25 minutes.