CN108048586A - A kind of detection method of ox kind Brucella sp attenuated vaccine strain S19 - Google Patents
A kind of detection method of ox kind Brucella sp attenuated vaccine strain S19 Download PDFInfo
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- CN108048586A CN108048586A CN201711402789.3A CN201711402789A CN108048586A CN 108048586 A CN108048586 A CN 108048586A CN 201711402789 A CN201711402789 A CN 201711402789A CN 108048586 A CN108048586 A CN 108048586A
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- brucella
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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
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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
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The detection method of present invention offer kind ox kind Brucella sp attenuated vaccine strain S19 and the primer sets of detection, wherein the sequence of used primer sets is SEQ ID NO:1‑4.The advantages of primer sets and method of the present invention, is as follows：1) efficient and sensible：Amplification can be completed in 30min, and the minimum detectability for expanding template is 1.0 × 10‑3ng/μl；2) high specificity：Discriminating detection is carried out to S19 using two pairs of PRA primers group-specifics, with other kinds, the Brucella sp of bion, Brucella vaccine strain and common non-Brucella sp strain, no cross reaction；3) it is easy to operate：The RPA reaction systems configured are placed under 39 DEG C of constant temperatures and complete reaction, need not use PCR instrument；4) it is high-throughput：A large amount of sample detections can be disposably carried out in water-bath or constant incubator, from the detection hole number limitation of PCR instrument equipment.
Description
Technical field
The invention belongs to technical field of microbial detection, and in particular to a kind of weight of ox kind Brucella sp attenuated vaccine strain S19
Group enzymatic polymerization enzymatic amplification technology (recombinase polymerase amplification, RPA) method for quick identification.
Background technology
Brucellosis (abbreviation cloth disease) is the pandemic Arbo infectious disease in the whole world as caused by Brucella sp, to herding
Industry and public health security cause serious threat.The situation is tense for China's cloth disease Prevention and cure of epidemic situation in recent years, presents and is herded from pastoral area to non-
Area shifts, the trend spread from professional population to non-professional crowd.Carrying out attenuated vaccine immunity to domestic animal can contain cloth disease to people
Between propagate, but the immune of attenuated vaccine can interfere the diagnosis and monitoring of cloth disease.Realize Brucella sp attenuated vaccine strain and open country
The discriminating of raw type bacterial strain, is of great significance for cloth disease prevention and control.
S19 plants of Brucella sp is one of widely applied Brucella sp attenuated vaccine strain and world animal health group in the world
Knit the attenuated vaccine strain of (OIE) recommendation.S19 plants can continue that body is stimulated to generate antibody, be widely used in the immunity inoculation of ox.
S19 plants belong to smooth type Brucella sp, and containing LPS lipopolysaccharides, the antibody generated after animal immune can not mutually be distinguished with natural infection,
Difficulty is brought to cloth disease diagnosis.And Brucella sp separation identification needs to carry out in P3 laboratory, and time-consuming, to people
Member and equipment requirement are higher.
The development of Protocols in Molecular Biology provides means for S19 plants of quick differentiate, successively establishes be based in the world
The method that PCR and fluorescence quantifying PCR method differentiate S19 plants.RPA is in recent years a kind of constant temperature nucleic acid amplification technology newly developed, should
Method can be completed to detect under 36~40 DEG C of constant temperatures in a large amount of amplifying target genes in 20min, have high specificity,
The advantages that high sensitivity, reaction speed are fast, easy to operate.Compared with conventional PCR and fluorescence quantifying PCR method, PRA reaction items
Part is constant temperature, need not be denatured, annealed, extending Xun Huan, and experimental instrument and equipment is relied on less and reaction speed is fast, especially suitable
For Site Detection.The technology is applied in the quick context of detection of some cause of diseases at present, but without based on RPA technologies
Differentiate S19 plants of detection method.
The content of the invention
The object of the present invention is to provide a kind of RPA of ox kind Brucella sp attenuated vaccine strain S19 quickly to differentiate detection method, from
And make up the deficiency of existing Brucella sp detection technique.
Present invention firstly provides one group for differentiating S19 plants of RPA primer sets, primer information is as follows：
Omp2-F:CGTGCGGCATGTAACCGTCGATCGTGTAATC(SEQ ID NO:1)、
Omp2-R:TGCATATATCACGCTTGGTGGTTTCAAGGTT(SEQ ID NO:2)、
S19E-F:CGCTAACCCGGACGACTACGATCCTTACGCAT(SEQ ID NO:3)、
S19E-R:GGCAGATTTCATTATCCACCGCGCCGCCTTC(SEQ ID NO:4)；
S19 plants of method is detected using above-mentioned RPA primer sets the present invention also provides a kind of, including the steps：
1) extraction of genomic DNA to be detected：It is thin with bacterial genomes DNA extraction kit (QIAGEN companies) extraction
Bacterium DNA；
2) RPA steps：Present invention design RPA primer sets and each reactive component are separately added into reaction system, 39 DEG C anti-
Answer 30min.
3) result detects：Recovery purifying RPA reaction products, 2% agarose gel electrophoresis detection, judge testing result.
On the other hand, RPA primer sets of the invention can be used for preparing detection kit.
The advantages of primer sets and method of the present invention, is as follows：1) efficient and sensible：Amplification can be completed in 30min, expands template
Minimum detectability is 1.0 × 10-3ng/μl；2) high specificity：Discriminating inspection is carried out to S19 using two pairs of PRA primers group-specifics
It surveys, with other kinds, the Brucella sp of bion, Brucella vaccine strain and common non-Brucella sp strain, no cross reaction；3) operation letter
Just：The RPA reaction systems configured are placed under 39 DEG C of constant temperatures and complete reaction, need not use PCR instrument；4) it is high-throughput：It can
A large amount of sample detections are disposably carried out in water-bath or constant incubator, from the detection hole number limitation of PCR instrument equipment.
Description of the drawings
Fig. 1：RPA methods detect the gel electrophoresis figure of S19 plants and non-S19 Brucella sps strain
Band 1 is DL1000DNA Marker；Band 2 is Omp2 primer detection S19 pnca gene group DNA masterplates；Band 3 is
S19E primer detection S19 pnca gene group DNA masterplates；Band 4 is the non-S19 Brucella sps representative strains M5-90 genes of Omp2 primer detections
Group DNA masterplates；Band 5 is S19E primer detection M5-90 pnca gene group DNA masterplates；Band 6 is right for Omp2 primer detection feminine genders
According to；Band 7 is S19E primer detection negative controls.
Fig. 2:RPA method sensitivity techniques gel electrophoresis figure (a containing Fig. 2 and 2b)
Fig. 2 a. using the diluted S19 pnca genes group DNA masterplates of RPA methods detection series, masterplate concentration for 10ng/ μ l~
1.0×10-4ng/μl.Wherein, band 2~7 is Omp2 primer detections as a result, band 9~14 is S19E primer detection results.Tool
Body is as follows：Band 1 and 8 is DL1000Marker；Band 2 and 9 is 10ng/ μ l；Band 3 and 10 is 1ng/ μ l；Band 4 and 11 is
1.0×10-1ng/μl；Band 5 and 12 is 1.0 × 10-2ng/μl；Band 6 and 13 is 1.0 × 10-3ng/μl；Band 7 and 14 is
1.0×10-4ng/μl。
Fig. 2 b. are serially diluted non-S19 Brucella sps representative strains M5-90 genomic DNA masterplates, masterplate using the detection of RPA methods
Concentration is 10ng/ μ l~1.0 × 10-4ng/μl.Wherein, band 2~7 is Omp2 primer detections as a result, band 9~14 is S19E
Primer detection result.It is specific as follows：Band 1 and 8 is DL1000Marker；Band 2 and 9 is 10ng/ μ l；Band 3 and 10 is
1ng/μl；Band 4 and 11 is 1.0 × 10-1ng/μl；Band 5 and 12 is 1.0 × 10-2ng/μl；Band 6 and 13 for 1.0 ×
10-3ng/μl；Band 7 and 14 is 1.0 × 10-4ng/μl。
Fig. 3:RPA method specific detections gel electrophoresis figure (a containing Fig. 3,3b and 3c)
Fig. 3 a and 3b. are using PRA methods detection Brucella sp Common Species, bion Reference Strains and the gene of Brucella vaccine strain
Group DNA masterplates.Wherein, Fig. 3 a are Omp2 primer detections as a result, Fig. 3 b are S19E primer detection results.It is specific as follows：Band 1,
DL1000DNA Marker；Band 2, Brucella suis biovar 1S1330；Band 3, B.suis biovar
2Thomsen；Band 4, B.suis biovar 3 686；Band 5, B.suis biovar 4 40；Band 6, B.abortus
biovar 1A544；Band 7, B. abortus biovar 2 86/8/59；Band 8, B.abortus biovar 3Tulya；
Band 9, B.abortus biovar 4 292；Band 10, B.abortus biovar 5B3196；Band 11, B.abortus
biovar 6 870；Band 12, B.abortus biovar 7 63/75；Band 13, B.abortus biovar 9C68；Item
Band 14,1 16M of B.melitensis biovar；Band 15, B.melitensis biovar 2 63/9；Band 16,
B.melitensis biovar 3Ether；Band 17, B.ovis 63/290；Band 18, B.canis RM6/66；Band 19,
B.neotomae 5K33；Band 20, S19；Band 21, M5-90；Band 22, S2；Band 23, A19；Band 24, it is negative right
According to；Band 25, positive control.
Fig. 3 c. detect four kinds of common non-Brucella sp pnca gene group DNA masterplates using PRA methods.Wherein, band 1~7 is
Omp2 primer detections are as a result, band 8-14 is S19E primer detection results.It is specific as follows：Band 1 and 8, DL1000DNA
Marker；Band 2 and 9, Escherichia coli K99；Band 3 and 10, Pasteurella multocida C48-1；Item
Band 4 and 11, Streptococcus suis ST171；Band 5 and 12, Pseudomonas aeruginosa DI-1；Band 6
With 13, negative control；Band 7 and 14, positive control.
Specific embodiment
The method of the present invention is described in detail with reference to embodiment.
Embodiment 1, the design for detecting S19 plants of RPA primer sets
S19 plants of ox kind Brucella sp is separated by the U.S. from milk in nineteen twenty-three, after passing on attenuation naturally through laboratory, is become
The less-virulent strain that one plant of virulence is stablized.S19 plants are antierythrite sensitive strains, with other Brucella sp genome comparisons, S19 plants of presence
Significant EryC gene delections.Omp2 genes are the highly conserved genes of Brucella sp, are present in all kind type Brucella sp genes
In group.The present invention uses OligoSoftware devises Brucella sp strain universal detector primer Omp2 according to Omp2 genes first, then
S19 plants of diagnostic primers S19E are devised according to EryC genes, S19 plants of effective discriminating can be realized using above-mentioned two groups of primers.If
It is the key that carry out RPA reactions that meter, which screens suitable primer, needs to consider base composition, G/C content, two level knot when designing primer
The factors such as formation, the Tm values of structure, then carry out many experiments, therefrom the best primer pair of selective mechanisms effect.
During using above-mentioned primer detection, the amplifiable all kinds of types Brucella sp strain Omp2 genetic fragments of Omp2 primers, S19E draws
EryC genetic fragments in the amplifiable all Brucella sp strains in addition to S19 plants of object.Recovery product after the completion of reaction carries out agarose and coagulates
Gel electrophoresis, Omp2 primer detections are positive, and S19E primer detections are negative, can determine whether the template source in S19 plants；Omp2 primer detections
The positive, S19E primer detections are positive, can determine whether the template source in other Brucella sp bacterial strains of non-S19.Primer is farsighted rich by Beijing
Xing Ke Bioisystech Co., Ltd synthesizes, and primer is as shown in table 1：
Table 1：The sequence information of RPA primers
The effect detection of embodiment 2, ox kind Brucella sp attenuated vaccine strain S19RPA
1.1 test material
Bacterial strain：Brucella sp Common Species, bion Reference Strains and the Brucella vaccine strain used, is shown in Table 2；Four non-Brucella sps
Reference strain：E. coli k99, Pasteurella C48-1, Streptococcus suis ST171, Pseudomonas aeruginosa DI-1.
Table 2：The Brucella sp strain used and accession number
1.2 DNA of bacteria extract
Above-mentioned bacterial strains strain is inoculated with tryptose agar medium, 37 DEG C of 24~72h of culture are not added or added as needed
Add 5~10%CO2.Bacterium colony is cultivated with after the brine containing 0.5% formaldehyde, 37 DEG C of inactivations for 24 hours, use bacterial genomes
DNA extraction kit (QIAGEN companies) extracts DNA of bacteria, and trace dna protein assay measures bacterial genomes DNA concentration,
It freezes in -20 DEG C, it is spare, expand template as RPA.
1.3RPA reaction systems and condition
Reaction condition：39℃30min.
S19E primer reaction systems are：
Reaction condition：39℃30min.
The detection of 1.4RPA reaction products
After reaction, using DNA fragmentation QIAquick Gel Extraction Kit (Beijing Bioisystech Co., Ltd of polymerization U.S.) recovery purifying
RPA reaction products, the detection of 2% agarose gel electrophoresis.
The sensitivity evaluation of 1.5RPA methods
Sensitivity evaluation：It is measured through ultramicron nucleic acid-protein analyzer, S19 plants of Brucella sp and non-S19 Brucella sps representative strains
The concentration of M5-90 genomic DNAs is respectively 12.0ng/ μ l and 30.0ng/ μ l.First by S19 plants and M5-90 pnca gene groups DNA
10.0ng/ μ l are quantitatively diluted to, then carry out 10 times of serial dilutions.The diluted Bu Shi of series is detected respectively with RPA methods
S19 plants of bacterium and M5-90 pnca gene group DNA masterplates, each reaction add in 1 μ l as template.Recovery product after reaction,
2% agarose gel electrophoresis carries out imaging analysis after electrophoresis.Assess the Brucella sp that PRA methods detect S19 plants and non-S19
The sensitivity of strain.
The Evaluation on specificity of 1.6RPA methods
Evaluation on specificity：Brucella sp Common Species, bion Reference Strains and Brucella vaccine strain is extracted respectively (to be shown in Table 2, compile
Number 1~23) and e. coli k99, Pasteurella C48-1, Streptococcus suis ST171, the genomic DNA of Pseudomonas aeruginosa DI-1.
DNA of bacteria template is replaced as negative control using sterile water.Assess the specificity that PRA methods differentiate S19 plants.
2 results and analysis
2.1RPA sensitivity technique results
Fig. 2 a show that the sensitivity of RPA methods detection S19 pnca gene groups DNA is 1.0 × 10-3ng/μl；Fig. 2 b show,
The sensitivity that RPA methods detect non-S19 Brucella sps pnca gene group DNA is 1.0 × 10-3ng/μl。
2.2RPA specific detection results
Fig. 3 a are shown, in RPA methods, Omp2 primers can detect Common Species in sample disc, bion Brucella sp and vaccine strain
Genomic DNA；3b is shown, in RPA methods, S19E primers can detect in sample disc except S19 plants of outer Common Species, bion Brucella sp
With vaccine strain genomic DNA；Fig. 3 c show, two groups of primer detection Escherichia coli of PRA methods, Pasteurella, Streptococcus suis, green
The equal no cross reaction of purulence vaccae genomic dna.As a result illustrate, the RPA methods specificity that this research is established is good, passes through two groups of primers
Common detection is, it can be achieved that effective discriminating of S19 plants of Brucella sp.
The above results show that S19 plants of RPA primers of Brucella sp of the invention and detection method high sensitivity, specificity are good, lead to
Crossing detection can quickly and efficiently differentiate whether detection sample is S19 plants of Brucella sp.Meanwhile it is easy to operate, in water bath with thermostatic control or training
It can be reacted, be detected from the limitation of PCR instrument and equipments detection hole number under the conditions of foster case, high-volume can be carried out simultaneously
Detection.
Sequence table
<110>China Animal Health and Epidemiology Center
<120>A kind of detection method of ox kind Brucella sp attenuated vaccine strain S19
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 31
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
cgtgcggcat gtaaccgtcg atcgtgtaat c 31
<210> 2
<211> 31
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
tgcatatatc acgcttggtg gtttcaaggt t 31
<210> 3
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
cgctaacccg gacgactacg atccttacgc at 32
<210> 4
<211> 31
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ggcagatttc attatccacc gcgccgcctt c 31
Claims (8)
- A kind of 1. primer sets for being used to differentiate S19 plants of ox kind Brucella sp attenuated vaccine strain, which is characterized in that the primer sets bag Contain two pairs of primers；Wherein the sequence of the sense primer of the first primer pair is SEQ ID NO:1；The sequence of anti-sense primer is SEQ ID NO:2；The sequence of the sense primer of second primer pair is SEQ ID NO:3；The sequence of anti-sense primer is SEQ ID NO:4.
- 2. application of the primer sets described in claim 1 in the product for preparing S19 plants of detection ox kind Brucella sp attenuated vaccine strain.
- 3. application as claimed in claim 2, which is characterized in that the product is detection kit.
- 4. a kind of detection kit, which is characterized in that include primer sets described in claim 1 in the kit.
- 5. application of the kit in S19 plants of ox kind Brucella sp attenuated vaccine strain is detected described in claim 4.
- A kind of 6. method for detecting S19 plants of ox kind Brucella sp attenuated vaccine strain, which is characterized in that the method includes following step Suddenly：1) extraction of genomic DNA to be detected：With the DNA of bacteria of bacterial genomes DNA extraction kit extraction detected sample；2) RPA steps：Primer sets described in claim 1 and each reactive component are separately added into reaction system, 39 DEG C of reaction 30min；3) result detects：Recovery purifying RPA reaction products, 2% agarose gel electrophoresis detection, judge testing result.
- 7. method as claimed in claim 6, which is characterized in that testing result is judged in the method, if the first primer To amplified production for the positive, and the amplified production of the second primer pair for feminine gender；Then bacterial strain to be detected is the weak poison of ox kind Brucella sp S19 plants of vaccine strain；If the amplified production of the first primer pair is the positive, the amplified production of the second primer pair is the positive, then to be checked Survey other Brucella sp bacterial strains that bacterial strain is non-ox kind Brucella sp attenuated vaccine strain S19.
- 8. a kind of examination criteria for judging ox kind Brucella sp, which is characterized in that the standard is for Brucella sp to be detected Bacterial strain, the amplified production of the first primer pair described in claim 1 is the positive, and the amplified production of the second primer pair is the moon Property；Then bacterial strain to be detected is S19 plants of ox kind Brucella sp attenuated vaccine strain；If the amplified production of the first primer pair is the positive, the The amplified production of two primer pairs is the positive, then bacterial strain to be detected is other Bu Shi of non-S19 plants of ox kind Brucella sp attenuated vaccine strain Bacteria strain.
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| CN110734993A (en) * | 2019-12-04 | 2020-01-31 | 军事科学院军事医学研究院军事兽医研究所 | nucleic acid detection test paper strip for distinguishing Brucella vaccine strain S19 from naturally infected strain |
| CN114058719A (en) * | 2021-07-28 | 2022-02-18 | 内蒙古农业大学 | Primer pair and kit for identifying Brucella and application of primer pair and kit |
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Cited By (3)
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| CN110734993A (en) * | 2019-12-04 | 2020-01-31 | 军事科学院军事医学研究院军事兽医研究所 | nucleic acid detection test paper strip for distinguishing Brucella vaccine strain S19 from naturally infected strain |
| CN114058719A (en) * | 2021-07-28 | 2022-02-18 | 内蒙古农业大学 | Primer pair and kit for identifying Brucella and application of primer pair and kit |
| CN114058719B (en) * | 2021-07-28 | 2023-09-29 | 内蒙古农业大学 | Primer pair and kit for identifying brucella and application of primer pair and kit |
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