CN105969853A - Composition capable of rapidly detecting aquatic pathogenic bacteria and preparation method thereof - Google Patents
Composition capable of rapidly detecting aquatic pathogenic bacteria and preparation method thereof Download PDFInfo
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- CN105969853A CN105969853A CN201610317558.1A CN201610317558A CN105969853A CN 105969853 A CN105969853 A CN 105969853A CN 201610317558 A CN201610317558 A CN 201610317558A CN 105969853 A CN105969853 A CN 105969853A
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- 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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- C12Q1/6888—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
The invention relates to a composition capable of detecting aquatic pathogenic bacteria. The composition particularly has specificity on aeromonas salmonicida salmonicida. The composition is capable of rapidly detecting the aeromonas salmonicida salmonicida. Primers are designed according to aeromonas salmonicida salmonicida specific conservative genes vaPA, spsM and spsE; the aeromonas salmonicida salmonicida can be detected in about 40 minutes; the sensitivity reaches 20cfu/reaction. When the composition is applied to detection, reaction results are convenient and quick to detect; the composition is capable of determining by directly observing the color change of a reaction system; if the reaction system is changed from light brown to fluorescent green, the results are positive results; meanwhile, the equipment requirement is simple; the operation is easy; the reaction speed is more rapid.
Description
Technical field
The present invention relates to compositions of a kind of quick detection aquatic pathogenic bacterium and preparation method thereof, more specifically, this
The bright one that relates to has specific compositions and preparation method thereof to aeromonas salmonicida.
Background technology
Aquaculture is one of industry with fastest developing speed in China's multi-form agriculture, but along with cultivation popularization, disease problem
Have become as the principal element that restriction industry develops in a healthy way.The most bacillary class principal disease being to endanger aquaculture of causing a disease,
Huge economic loss is caused every year to culture fishery.The quickly detection of pathogen can be that the daily production of fish culture provides
Cause of disease change information, provides foundation for disease early warning, it is to avoid the massive losses that disease is likely to result in.
Aeromonas salmonicida (Aeromonas salmonicida) is gram negative bacteria, widely distributed, it is possible to initiation is many
Plant the disease of aquatic animal, be the aquatic pathogenic bacterium that a kind of pathogenicity is stronger.It is possible not only to cause multiple fish skin ulcer
Disease can also infect echinoderm Hemicentrotus seu Strongylocentrotus and Radix Morinae Bulleyanae, and diseases induced, causes huge economic loss.
At present, the domestic research to fish bacterial disease quick diagnosis technology is scarcely out of swaddling-clothes, conventional qualification
Method wastes time and energy, and have is the most inaccurate, even needs professional and technical personnel or professional technique equipment, it is difficult in breeding production
Application, can not meet far away the needs in production, and be difficult to causal organism monitoring in early days, periodic detection, on-the-spot quick diagnosis,
Just cause concern after having broken out extensive epidemic disease the most often and pay attention to.There is no both at home and abroad for aeromonas salmonicida
The detection method being prone to execute-in-place fast and effectively.
Summary of the invention
It is an object of the invention to provide a kind of target sequence amplification specificity height, reaction result is easy to detect and detects efficiently
The compositions of aquatic pathogenic bacterium.
It is a further object to provide a kind of equipment requirements simple and easily operation, response speed soon, the most effective
And it is prone to the detection method of the detection aeromonas salmonicida of execute-in-place.
It is an object of the invention to: the selection of aeromonas salmonicida specific gene and the design of primer and optimize after isothermal
The proportioning of amplification reaction system and the setting of reaction condition.
The present invention is based on ring mediated isothermal amplification (loop-mediated isothermal amplification, letter
It is referred to as LAMP) a kind of method that technology sets up quick detection aeromonas salmonicida, the core content of the method is to kill salmon gas unit cell
The selection of bacterium specific gene and the design of primer and the proportioning of the isothermal amplification system after optimizing and the setting of reaction condition.
Ring mediated isothermal amplification (loop-mediated isothermal amplification, referred to as LAMP) technology
Depend on and be capable of identify that 4 primers of 6 specific regions on target DNA and a kind of archaeal dna polymerase with strand-displacement activity, in perseverance
Efficient amplification nucleic acid under the conditions of temperature, can complete for 1 hour, and product is by stem-cyclic DNA and the Brassica oleracea L. var. botrytis L. shape repeating target sequence more
The mixture that DNA is formed, has high specific, rapid sensitive, the feature such as easy and simple to handle.The ring mediated isothermal that the present invention sets up
Amplification system comes Indicator Reaction result, naked eyes i.e. observable by the color change of dyestuff calcein.
LAMP is the highest to the requirement of design of primers, and 2 pairs of primers of LAMP are called FIP and BIP, F3 and B3, can know
6 different regions of other target gene.The F3c regional complementarity of F3: forward outer primer, F3 district and target gene；B3: draw outside reversely
The B3c regional complementarity of thing, B3 region and target gene；FIP: forward inner primer, is made up of F2 district and F1c region, F2 district and target base
Because of the F2c regional complementarity of 3' end, F1c district is identical with the F1c regional sequence of target gene 5' end；BIP: reversely inner primer, by B1c and
B2 region forms, and B2 district and the B2c regional complementarity of target gene 3' end, B1c region is identical with target gene 5' end B1c regional sequence.
In LAMP reacts, add ring primer (forward ring primer LF, reverse ring primer LB) can substantially accelerate the speed of isothermal duplication,
Substantially increase detection efficiency.
LAMP primer designs
Follow the principle of " conservative in planting, special between kind ", kill salmon gas unit cell by consulting literatures with in ncbi database
Bacterium genome and this laboratory have checked order the comparison analysis of aeromonas salmonicida genome, screen vapA, spsM, spsE
Drawing of isothermal duplication is designed as target gene in the conserved region (about 300bp) of three special virulence genes of aeromonas salmonicida
Thing.Use online isothermal duplication primer-design software PrimerExplorer V4oftware to design primer.Primer sequence is such as
Under: (remarks: due to the primers F 1 and F2 of design on target gene close together, it is not necessary to add forward ring primer LF again, only need
Add reverse ring primer LB)
vapA
spsM
spsE
LAMP reaction system Evaluation on specificity: include vibrio alginolyticus, Atlantic Ocean arc with common bacteria in culture environment of aquatic products
Bacterium, west watt Salmonella, Staphylococcus equorum, Psychrobacter, bacillus firmus, Bacillus cereus and 3 strain aeromonas salmonicidas
DNA be template, evaluation response system specificity.3 strain aeromonas salmonicida detection of nucleic acids all occurred in that positive expansion in 1 hour
Increasing, all there is not positive amplification in 7 strain non-aeromonas salmonicida detection of nucleic acids, shows that this LAMP reaction system specificity is preferable.Sun
Property result is for be light brown from the light brown fluorescence green that becomes, negative findings and negative control.
The optimization of LAMP reaction system: the LAMP reaction system of 25 μ L includes 2 inner primer FIP and BIP, 2 outer primers
F3 and B3,1 ring primer LB, 10 × Thermpol buffer, glycine betaine (Betaine), deoxyribonucleotide (dNTP),
Magnesium sulfate, manganese chloride, calcein, Bst archaeal dna polymerase, DNA profiling, aquesterilisa supply system.Select in LAMP system
Key factor be optimized, including outer primer and inner primer concentration ratio (1: 4,1: 6,1: 8,1: 10,1:12), magnesium ion concentration
(3,4,5,6,7,8,9mmol/L), the ratio (1:5,1:10,1:15,1:20,1:25,1:30) of calcein and manganese chloride, anti-
Answer temperature (61,62,63,64,65 DEG C).The amplification system finally giving optimum is as follows: the LAMP reaction system of 25 μ L, and 63 degree permanent
Temperature amplification.
LAMP reaction system sensitivity evaluation: after 10 times of serial dilutions of aeromonas salmonicida culture fluid, the company of choosing
Continuous 7 dilution factors blood counting chamber counts, and each dilution factor takes 3 sample count respectively, obtain each dilution averagely
Value.With the DNA of bacteria of above-mentioned 10 times of gradient dilutions as template, the LAMP reaction system after optimizing is used to carry out amplification test.?
LAMP reaction system sensitivity after being optimized eventually is 20cfu/ reaction, and reaction condition is 63 degree of constant-temperature amplifications 45 minutes.
The present invention achieves following effect: 1) LAMP is high to the specificity of target sequence amplification.Owing to LAMP is by 4 primers
6 specific regions on target sequence are accurately combined and produce amplified reaction, therefore can obtain more higher specificity than PCR.
2) reaction result is easy to detect, quick.Can directly observing response system color change judge, reaction system is by light brown change
It is positive findings for fluorescence green.3) equipment requirements is simple, easily operates.LAMP has only to a common water-bath or constant temperature
Device, simple to operate, easy to carry, technical threshold is low.4) response speed is faster.LAMP is isothermal amplification reactions, does not has PCR anti-
In should, variations in temperature be time-consuming, typically can complete in 30min-60min, than quantitative fluorescent PCR response speed more faster, and can be more
The requirement of the good quickly detection meeting production scene.5) generally, the detection method based on molecular level is such as
PCR and quantitative fluorescent PCR all can be affected by mortifier in clinical samples, the Bst DNA that some researcheres report LAMP uses
The tolerance of mortifier in sample is eager to excel by polymerase than the Taq enzyme in quantitative fluorescent PCR.Therefore LAMP compares fluorescent quantitation
PCR has broader practice prospect.
Accompanying drawing explanation
Fig. 1 primer spsM specificity experiments compares
Fig. 2 primer spsE specificity experiments compares
Fig. 3 primer vapA specificity experiments compares
Fig. 4 LAMP primer designs
1 Bacillus cereus；2 vibrio alginolyticus；3 Staphylococcus equorums；
4 west watt Salmonellas；5 Atlantic Ocean vibrios；6 bacillus firmuses；
7 Psychrobacters；8 aeromonas salmonicida S7；9 aeromonas salmonicida S53；
10 aeromonas salmonicida S121；11 negative controls.
Detailed description of the invention
Embodiment 1
Said composition includes by volume:
The time of detection aeromonas salmonicida is 65 minutes (reaction temperature is 63 degree, and DNA profiling concentration is 50ng/ μ L).
Embodiment 2
Said composition includes by volume:
The time of detection aeromonas salmonicida is 58 minutes (reaction temperature is 63 degree, and DNA profiling concentration is 50ng/ μ L).
Embodiment 3
Said composition includes by volume:
The time of detection aeromonas salmonicida is 40 minutes, positive findings and the relatively low (reaction of negative findings color discrimination
Temperature is 63 degree, and DNA profiling concentration is 50ng/ μ L).
Embodiment 4
Said composition includes by volume:
The time of detection aeromonas salmonicida is 40 minutes, positive findings and the relatively low (reaction of negative findings color discrimination
Temperature is 63 degree, and DNA profiling concentration is 50ng/ μ L).
Embodiment 5
Said composition includes by volume:
The time of detection aeromonas salmonicida is 55 minutes (reaction temperature is 63 degree, and DNA profiling concentration is 50ng/ μ L).
Embodiment 6
Said composition includes by volume:
The time of detection aeromonas salmonicida is 50 minutes (reaction temperature is 63 degree, and DNA profiling concentration is 50ng/ μ L).
Embodiment 7
Said composition includes by volume:
The time of detection aeromonas salmonicida is 45 minutes (reaction temperature is 63 degree, and DNA profiling concentration is 50ng/ μ L).
Embodiment 8
Said composition includes by volume:
The time of detection aeromonas salmonicida is 45 minutes (reaction temperature is 63 degree, and DNA profiling concentration is 50ng/ μ L).
Embodiment 9
Said composition includes by volume:
The time of detection aeromonas salmonicida is 40 minutes, positive findings and the relatively low (reaction of negative findings color discrimination
Temperature is 63 degree, and DNA profiling concentration is 50ng/ μ L).
Embodiment 10
Said composition includes by volume:
The time of detection aeromonas salmonicida is 40 minutes, positive findings and the relatively low (reaction of negative findings color discrimination
Temperature is 63 degree, and DNA profiling concentration is 50ng/ μ L).
Embodiment 11
Said composition includes by volume:
The time of this reaction system detection aeromonas salmonicida is 40 minutes, positive findings and negative findings color discrimination
Higher (reaction temperature is 63 degree, and DNA profiling concentration is 50ng/ μ L).
Embodiment 12
LAMP primer designs
Follow the principle of " conservative in planting, special between kind ", kill salmon gas unit cell by consulting literatures with in ncbi database
Bacterium genome and this laboratory have checked order the comparison analysis of aeromonas salmonicida genome, screen vapA, spsM, spsE tri-
The primer of isothermal duplication is designed in the conserved region (about 300bp) of the special virulence gene of individual aeromonas salmonicida as target gene.
Use online isothermal duplication primer-design software PrimerExplorer V4software to design primer.Primer sequence is as follows:
(remarks: due to the primers F 1 and F2 of design on target gene close together, it is not necessary to add forward ring primer LF again, only need to add
Add reverse ring primer LB)
vapA
spsM
spsE
LAMP reaction system Evaluation on specificity: include vibrio alginolyticus, Atlantic Ocean arc with common bacteria in culture environment of aquatic products
Bacterium, west watt Salmonella, Staphylococcus equorum, Psychrobacter, bacillus firmus, Bacillus cereus and 3 strain aeromonas salmonicidas
DNA be template, evaluation response system specificity.3 strain aeromonas salmonicida detection of nucleic acids all occurred in that positive expansion in 1 hour
Increasing, all there is not positive amplification in 7 strain non-aeromonas salmonicida detection of nucleic acids, shows that this LAMP reaction system specificity is preferable.Sun
Property result is for be light brown from the light brown fluorescence green that becomes, negative findings and negative control.
The optimization of LAMP reaction system: the LAMP reaction system of 25 μ L includes 2 inner primer FIP and BIP, 2 outer primers
F3 and B3,1 ring primer LB, 10 × Thermpol buffer, glycine betaine (Betaine), deoxyribonucleotide (dNTP),
Magnesium sulfate, manganese chloride, calcein, Bst archaeal dna polymerase, DNA profiling, aquesterilisa supply system.Select in LAMP system
Key factor be optimized, including outer primer and inner primer concentration ratio (1: 4,1: 6,1: 8,1: 10,1:12), magnesium ion concentration
(3,4,5,6,7,8,9mmol/L), the ratio (1:5,1:10,1:15,1:20,1:25,1:30) of calcein and manganese chloride, anti-
Answer temperature (61,62,63,64,65 DEG C).The amplification system finally giving optimum is as follows: the LAMP reaction system of 25 μ L, 63 degree
Constant-temperature amplification
LAMP reaction system sensitivity evaluation: after 10 times of serial dilutions of aeromonas salmonicida culture fluid, the company of choosing
Continuous 7 dilution factors blood counting chamber counts, and each dilution factor takes 3 sample count respectively, obtain each dilution averagely
Value.With the DNA of bacteria of above-mentioned 10 times of gradient dilutions as template, the LAMP reaction system after optimizing is used to carry out amplification test.?
LAMP reaction system sensitivity after being optimized eventually is 20cfu/ reaction, and reaction condition is 63 degree of constant-temperature amplifications 45 minutes.
Claims (9)
1. a compositions for quick detection aquatic pathogenic bacterium, said composition includes by volume:
Compositions the most according to claim 1, wherein the volume ratio content of 10 × Thermpol buffer is 10%.
Compositions the most according to claim 1, wherein the volume ratio content of 5M glycine betaine is 16%.
Compositions the most according to claim 1, wherein the volume ratio content of 100mM magnesium sulfate is 6%.
Compositions the most according to claim 1, wherein the volume ratio content of 12.5mM manganese chloride is 4%.
Compositions the most according to claim 1, wherein the volume ratio content of 0.625mM calcein is 4%.
Compositions the most according to claim 1, said composition also includes by volume:
Compositions the most according to claim 7,100 μMs of wherein said primer mixtures are included by its volume ratio:
9. the application of the compositions of a quick detection aquatic pathogenic bacterium, it is characterised in that aeromonas salmonicida is had specificity.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103290099A (en) * | 2012-02-24 | 2013-09-11 | 中国科学院微生物研究所 | Quick molecular detection kit for colletotrichum kahawae and application thereof |
| CN104212885A (en) * | 2014-06-26 | 2014-12-17 | 舟山出入境检验检疫局综合技术服务中心 | LAMP kit for Vibrio cholera in aquatic product |
| CN104357570A (en) * | 2014-11-11 | 2015-02-18 | 中国水产科学研究院淡水渔业研究中心 | Rapid detection primer for aeromonas salmonicida subsp.salmonicida and application of rapid detection primer |
-
2016
- 2016-05-12 CN CN201610317558.1A patent/CN105969853B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103290099A (en) * | 2012-02-24 | 2013-09-11 | 中国科学院微生物研究所 | Quick molecular detection kit for colletotrichum kahawae and application thereof |
| CN104212885A (en) * | 2014-06-26 | 2014-12-17 | 舟山出入境检验检疫局综合技术服务中心 | LAMP kit for Vibrio cholera in aquatic product |
| CN104357570A (en) * | 2014-11-11 | 2015-02-18 | 中国水产科学研究院淡水渔业研究中心 | Rapid detection primer for aeromonas salmonicida subsp.salmonicida and application of rapid detection primer |
Non-Patent Citations (1)
| Title |
|---|
| 刘宗晓等: "杀鲑气单胞菌的实时定量PCR检测方法的建立和应用", 《海洋水产研究》 * |
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