CN110468186A - Whether a kind of 11 type Riemerellosis Anatipestifers of identification are to the drug resistant method of fluoroquinolones - Google Patents
Whether a kind of 11 type Riemerellosis Anatipestifers of identification are to the drug resistant method of fluoroquinolones Download PDFInfo
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- CN110468186A CN110468186A CN201910860174.8A CN201910860174A CN110468186A CN 110468186 A CN110468186 A CN 110468186A CN 201910860174 A CN201910860174 A CN 201910860174A CN 110468186 A CN110468186 A CN 110468186A
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- 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
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- C12Q1/686—Polymerase chain reaction [PCR]
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- 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
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Abstract
Whether the present invention provides a kind of 11 type Riemerellosis Anatipestifers of identification to the drug resistant method and its application of fluoroquinolones, belong to disease of poultry research field, using primer and restriction endonuclease (EcoRV enzyme), whether foundation can identify 11 type Riemerellosis Anatipestifers to the drug resistant method of fluoroquinolones, the sensibility and PCR of this method are suitable, it only needs to carry out conventional agarose gel electrophoresis again after carrying out the digestion of EcoRV enzyme to PCR product, 11 type Riemerellosis Anatipestifers can be identified whether to fluoroquinolones drug resistance according to electrophoretic band, the method simple practical of foundation, it is convenient and efficient, without carrying out drug sensitive test, to carry out whether 11 type Riemerellosis Anatipestifers provide rapid detection method to fluoroquinolones drug resistance in poultry, it will instruct clinical science medication, it is ground with highly important Study carefully meaning.
Description
Technical field
The invention belongs to disease of poultry research fields, and in particular to whether a kind of 11 type Riemerellosis Anatipestifers of identification are to fluorine quinoline promise
The method and its application of ketone Drug-resistant.
Background technique
11 type Riemerellosis Anatipestifers can encroach on duckling, young goose, poult, and caused characteristic lesion is perihepatitis, pericardium
Scorching and peritonitis etc., is commonly called as infectious serositis.It finds within 1932, currently still in whole world prevalence, and endangers China's water
One of the common bacteria disease of fowl cultivation.Fluoroquinolones (Norfloxacin, Ciprofloxacin, Enrofloxacin etc.) belongs to third generation quinoline
Promise ketone, due to its has a broad antifungal spectrum, antibacterial activity is high, penetration into tissue is strong the features such as, be widely used in treating the whole body of poultry
Sexuality dye, the usage amount of veterinary clinic is only second to beta-lactam class antibiotic at home.In recent years, 11 type Riemerellosis Anatipestifer pair
The drug resistance of fluoroquinolones has been reported that more, how quickly to measure the bacterium to the sensibility of fluoroquinolones, refer to
The selection for leading drug seems very urgent.Bacterium is to there are many drug resistant mechanism of fluoroquinolones, wherein DNA gyrase
The mutation of (action target spot of fluoroquinolones) is one of the major reasons.
Early-stage study discovery, the quinolone drugs drug resistance of the DNA gyrase A subunit gene of 11 type Riemerellosis Anatipestifers are determined
Area (quinolone resistance-determining region, QRDR) 247-249 bit base is determined, if by AGC
ATC is sported, then the bacterium affirms drug resistance to fluoroquinolones.Based on this feature, the present invention design pair of primers across
The otherness site of QRDR, establishes PCR method, expands to it, two class bacteriums (refer to fluoroquinolones drug resistance or
Not drug resistant 11 type Riemerellosis Anatipestifer) pcr amplification product be 533 bp.Since the amplification region inner nucleotide sequence is deposited
In EcoRV enzyme Restriction Fragment Length difference, sporting ATC by AGC will lead to one EcoRV enzyme site of increase in the region
(GAT ↓ ATC).That is, the bacterium (herein can for GATAGC) PCR product to the not drug resistant bacterial strain of fluoroquinolones
Using constant (being still 533 bp) as size by the digestion of EcoRV enzyme；And the bacterium is to drug resistant bacterial strain (this of fluoroquinolones
Place is GAT ↓ ATC), PCR product PCR product is different two sections (353 bp and 180 bp) by EcoRV enzyme digestion.This method
Sensibility and PCR it is suitable, only need to carry out PCR product additional EcoRV enzyme digestion (without carrying out glue recycling to product)
Carry out electrophoresis again afterwards, it is simple and practical, without carrying out drug sensitive test, convenient and efficient, currently there is not yet the identification based on similar principles
Whether to the report of the drug resistant method of fluoroquinolones, this research can fill up Related Research Domain to 11 type Riemerellosis Anatipestifers
Blank.
Summary of the invention
Present invention employs primer and restriction endonuclease (EcoRV enzyme), foundation can identify whether 11 type Riemerellosis Anatipestifers are right
The drug resistant method of fluoroquinolones, the sensibility and PCR of this method are suitable, only need to carry out EcoRV enzyme to PCR product
Conventional agarose gel electrophoresis is carried out after digestion again, 11 type Riemerellosis Anatipestifers can be identified whether to fluorine quinoline according to electrophoretic band
Promise ketone Drug-resistant, the method simple practical of foundation, it is convenient and efficient, without carrying out drug sensitive test.
To achieve the above object, the invention adopts the following technical scheme:
Whether a kind of 11 type Riemerellosis Anatipestifers of identification are to the drug resistant primer of fluoroquinolones, and the primer sequence is such as
Under:
R11F:5 '-TCGATTATGCCATGTCCGTCA -3 ',
R11R:5 '-TTCGATATACGCTAAGCAACC -3 '.
Whether a kind of 11 type Riemerellosis Anatipestifers of identification are to the drug resistant method of fluoroquinolones, comprising the following steps:
(1) strain gene group is extracted；
(2) PCR amplification genome；
(3) PCR product digestion identification is carried out using EcoRV enzyme.
The beneficial effects of the present invention are: the sensibility and PCR of the method for the present invention are suitable, only need to PCR product into
Carry out electrophoresis after the additional EcoRV enzyme digestion of row (without carrying out glue recycling to product) again, it is simple and practical, without carrying out susceptibility examination
It tests, is convenient and efficient, it is current there is not yet whether the 11 type Riemerellosis Anatipestifer of identification based on similar principles is to fluoroquinolones
The report of drug resistant method, this research can fill up Related Research Domain blank.Detailed description of the invention
Fig. 1 is the electrophoretogram of PCR amplification；Wherein M:DL2000 molecular weight standard；1:R315 plants；2:R267 plants；3:R315 plants of enzymes
It cuts；4:R267 plants of digestions；5:DEV；6:MDPV；7:DuCV；8:GPV；9: sterile deionized water.
Fig. 2 is paper disk method drug sensitivity test.Specific embodiment
Embodiment 1
1, material
1.1 strains and bacterial strain
Test is with 11 type Riemerellosis Anatipestifer R267(bacterial strain of bacterial strain to fluoroquinolones not drug resistance), Riemerellosis Anatipestifer
The R315(bacterial strain is to fluoroquinolones drug resistance) it is saved by Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute.
Test control strain duck enteritis virus (DEV), Muscovy duck parvovirus (MDPV), duck circovirus (DuCV), duck
Source goose parvovirus (GPV) is saved by Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute.
1.2 design of primers
According to National Center for Biotechnology Information (National Center of Biotechnology
Information, NCBI) database 1 type Riemerellosis Anatipestifer DNA gyrase A subunit (DNA gyrase subunit A)
Gene is carried out nucleotide series analyses and comparison using Lasergene DNAStar, is set using primer-design software Oligo 7.0
A set of primer is counted, as follows:
R11F:5 '-TCGATTATGCCATGTCCGTCA -3 ',
R11R:5 '-TTCGATATACGCTAAGCAACC -3 '.
Above-mentioned primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
It should be pointed out that when being expanded using R11F/ R11R to R267 plants and R315 plants, R267 plants and R315
The PCR product purpose band size of strain is that 533 bp(conventional agarose electrophoresis can not be distinguished effectively).But in the amplification region
EcoRV enzyme (GAT ↓ ATC) restriction enzyme site difference of R267 plants and R315 plants of existing characteristics in domain, wherein R315 plants in the amplification
There are GAT ↓ ATC sequences (can be identified by EcoRV enzyme enzyme) in region, and R267 plants do not have that (usual drug resistant sequence is GATAGC, is not had
There is EcoRV enzyme digestion recognition site).
1.3 main agents
2×TransTaq-T PCR SuperMix、EasyPure Viral DNA/RNA Kit、EasyPure Bacteria
Genomic DNA Kit, DNA molecular amount standard DL2000, FlyCut EcoRV enzyme is purchased from Beijing Quan Shijin biotechnology has
Limit company.
The foundation of 2 test methods
The extraction of 2.1 genomic DNAs
Experimental control strain DEV, MDPV, DuCV, GPV is mentioned according to the method for EasyPure Viral DNA/RNA Kit kit
Take corresponding genomic DNA, -80 DEG C freeze it is spare.
The R267 plants and R315 plants methods according to EasyPure Bacteria Genomic DNA Kit kit are extracted
Corresponding genomic DNA, -80 DEG C freeze it is spare.
The configuration of 2.2 reaction solutions and the optimization of annealing temperature
It is expanded according to the 50 μ L systems that 2 × TransTaq-T PCR SuperMix kit is recommended, wherein 2 × PCR
25 μ L of Master Mix amplification reaction solution, primer R11F/ R11R(10 μM) it is respectively that 1.0 μ L, the nucleic acid-templated of extraction are
1.0 μ L, supplement sterile deionized water carry out PCR amplification after mixing to 50 μ L of final volume.
Amplification condition enters circulation, 94 DEG C of denaturation 30 s, △ T(52 DEG C -62 DEG C after being 94 DEG C of 5 min of initial denaturation) it moves back
30 s of fire, 72 DEG C of extension 45s, 30 after circulation terminates, and 72 DEG C extend 10 min eventually, after reaction according to conventional agarose
Gel electrophoresis identification.- 62 DEG C of △ T(52 DEG C) indicate annealing temperature optimized in this section, it is optimized after best annealing
Temperature is 56 DEG C.
As a result there is the band (the 2nd swimming lane) that size is 533 bp when template is only added R267 plants in visible (Fig. 1)；When
When template is only added R315 plants, there is the band (the 1st swimming lane) that size is 533 bp, conventional agarose electrophoresis naked eyes can not area
Point.
2.3 specific test
By the PCR system and amplification condition after optimization, DEV, MDPV, DuCV, GPV and sterile deionized water control are expanded
Increase, be showed no amplified band, the result is shown in Figure 1, the 5th swimming lane of DEV(), the 6th swimming lane of MDPV(), the 7th swimming lane of DuCV(), GPV(the 8th
Swimming lane) and sterile deionized water (the 9th swimming lane), show the method high specificity established, it is anti-without intersecting to common aquatic bird cause of disease
It answers.
2.4 digestions identification
PCR product is subjected to digestion identification using FlyCut EcoRV enzyme, digestion system is 20 μ L systems: wherein 10 ×
2 μ L of FlyCut Buffer, 2 μ L of FlyCut EcoRV enzyme, 10 μ L of PCR product, supplement sterile deionized water to final volume 20
μL.Mix gently rear brief centrifugation, 37 DEG C of 15 min of water-bath.The result is shown in Figure 1, R315 plants of products have two, size difference
For the 3rd swimming lane of 353 bp and 180 bp()；R267 plants of digestion products sizes are constant, are still the 4th swimming lane of 533 bp().Prove duck
Salmonella R315 is write from memory in epidemic disease to fluoroquinolones drug resistance, 11 type Riemerellosis Anatipestifer R267 to fluoroquinolones intolerant to
Medicine.
2.5 bacteria drug sensitivity test
Disk diffusion method carries out the drug sensitive test of bacterium, and Riemerellosis Anatipestifer R267 is 19 to the antibacterial circle diameter of Norfloxacin
Mm is determined as sensitivity；Riemerellosis Anatipestifer R315 is 11 mm to the antibacterial circle diameter of Norfloxacin, is determined as drug resistance.See Fig. 2.
3 clinical applications
11 type Riemerellosis Anatipestifers being clinically separated to 28 parts, are extracted corresponding nucleic acid DNA, are reflected using the method after optimization
Whether determine to fluoroquinolones drug resistance.EcoRV enzyme digestion mirror is carried out after it is carried out PCR amplification using R11F/ R11R
Fixed, as a result visible product size is that 533 bp have 9 plants, show this 9 plants to fluoroquinolones not drug resistance, accounting 32.1%；
Visible product size is that two segments of 353 bp and 180 bp have 19 plants, show this 19 plants to fluoroquinolones drug resistance,
Accounting 67.9%.It is compareed with the drug sensitive test in 2.5, as a result visible 19 plants through method identification of the invention are to fluorine quinoline
The bacterial strain of promise ketone Drug-resistant, through drug sensitive test to fluoroquinolones drug resistance, coincidence rate 100%；Through of the invention
Method identification, 9 plants to the not drug resistant bacterial strain of fluoroquinolones, through drug sensitive test to fluoroquinolones intolerant to
Medicine, coincidence rate 100%.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
SEQUENCE LISTING
<110>Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<120>whether a kind of 11 type Riemerellosis Anatipestifers of identification are to the drug resistant method of fluoroquinolones
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
<400> 1
tcgattatgc catgtccgtc a 21
<210> 2
<211> 21
<212> DNA
<213>artificial sequence
<400> 2
ttcgatatac gctaagcaac c 21
Claims (2)
1. whether a kind of 11 type Riemerellosis Anatipestifers of identification are to the drug resistant primer of fluoroquinolones, which is characterized in that
The primer sequence is as follows:
R11F:5 '-TCGATTATGCCATGTCCGTCA -3 ',
R11R:5 '-TTCGATATACGCTAAGCAACC -3 '.
2. whether a kind of 11 type Riemerellosis Anatipestifers of identification are to the drug resistant method of fluoroquinolones, it is characterised in that: including
Following steps:
(1) strain gene group is extracted；
(2) PCR amplification genome；
(3) PCR product digestion identification is carried out using EcoRV enzyme.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU3372399A (en) * | 1998-04-01 | 1999-10-18 | Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The | Oligonucleotide probes for detecting Enterobacteriaceae and quinolone-resistant Enterobacteriaceae |
| US20030049668A1 (en) * | 2001-09-04 | 2003-03-13 | Yasuhiko Suzuki | Method for determining quinolon resistance of tubercle bacilli |
| JP2012085582A (en) * | 2010-10-20 | 2012-05-10 | Kanagawa Acad Of Sci & Technol | Method for detecting bacteria resistant to fluoroquinolones, and primer for the same |
| CN104611422A (en) * | 2015-01-09 | 2015-05-13 | 四川大学 | Method for detecting escherichia coli fluoroquinolone-resisting gyrA/parC gene point mutation |
| CN104774932A (en) * | 2015-03-25 | 2015-07-15 | 扬州大学 | Molecular detection kit for determining resistance level of Escherichia coli to quinolone drugs based on high resolution melting curve |
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| CN108938661A (en) * | 2018-07-27 | 2018-12-07 | 四川农业大学 | The application in the drug for inhibiting Riemerellosis Anatipestifer is being prepared in conjunction with the gyrA gene PNA joint antibiotic of cell-penetrating peptide |
-
2019
- 2019-09-11 CN CN201910860174.8A patent/CN110468186A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU3372399A (en) * | 1998-04-01 | 1999-10-18 | Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The | Oligonucleotide probes for detecting Enterobacteriaceae and quinolone-resistant Enterobacteriaceae |
| US20030049668A1 (en) * | 2001-09-04 | 2003-03-13 | Yasuhiko Suzuki | Method for determining quinolon resistance of tubercle bacilli |
| JP2012085582A (en) * | 2010-10-20 | 2012-05-10 | Kanagawa Acad Of Sci & Technol | Method for detecting bacteria resistant to fluoroquinolones, and primer for the same |
| CN104611422A (en) * | 2015-01-09 | 2015-05-13 | 四川大学 | Method for detecting escherichia coli fluoroquinolone-resisting gyrA/parC gene point mutation |
| CN104878082A (en) * | 2015-02-26 | 2015-09-02 | 闽南师范大学 | Aeromonas hydrophila fluoroquinolone drug-resistance gene detection method |
| CN104774932A (en) * | 2015-03-25 | 2015-07-15 | 扬州大学 | Molecular detection kit for determining resistance level of Escherichia coli to quinolone drugs based on high resolution melting curve |
| CN108938661A (en) * | 2018-07-27 | 2018-12-07 | 四川农业大学 | The application in the drug for inhibiting Riemerellosis Anatipestifer is being prepared in conjunction with the gyrA gene PNA joint antibiotic of cell-penetrating peptide |
Non-Patent Citations (2)
| Title |
|---|
| 谭炳乾: ""耐氟喹诺酮类药物猪源致病性沙门氏菌gyrA基因PCR-RFLP及序列分析"", 《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》 * |
| 金龙翔: ""鸭疫里默氏菌对氟喹诺酮类药物耐药机制的研究"", 《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》 * |
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Application publication date: 20191119 |