CN103571950B - Rapid detection kit for aeromonas schubertii and detection method - Google Patents
Rapid detection kit for aeromonas schubertii and detection method Download PDFInfo
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- CN103571950B CN103571950B CN201310482323.4A CN201310482323A CN103571950B CN 103571950 B CN103571950 B CN 103571950B CN 201310482323 A CN201310482323 A CN 201310482323A CN 103571950 B CN103571950 B CN 103571950B
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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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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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
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a rapid detection kit for aeromonas schubertii. The rapid detection kit comprises a DNA (Deoxyribonucleic Acid) extraction reagent (1) and a PCR (Polymerase Chain Reaction) reagent (2), wherein the DNA extraction reagent comprises a TE (Tris-EDTA) buffer solution with 5-15mM of Tris and 0.5-1.5mM of EDTA (Ethylene Diamine Tetraacetic Acid), proteinase K with the concentration of 15-20mg/mL, 100% chloroform, 100% isopropanol, ethanol with the concentration of 70% and double distilled water; each PCR tube contains 2.5muL of 10*PCR buffer solution, 0.5muL of dNTPs with the concentration of 10muM, 0.3muL of Taq DNA polymerase with the concentration of 5U/muL, 0.5muL of specific oligonucleotide primers A with the concentration of 10muM, 0.5muL of specific oligonucleotide primers B with the concentration of 10muM, 19.2muL of sterilized deionized water, a primer A (5'-CAGCGAGGAGGAAAGGTTGGTGGT-3') and a primer B (5'-AAGCCACGCCTCAAGGGCACAA-3'). The rapid detection kit is used for detecting aeromonas schubertii.
Description
Technical field
The present invention relates to the detection technique of target DNA segment, be specifically related to a kind of Aeromonas schubertii quick detection kit and the method with this test kit detection Aeromonas schubertii.
Background technology
Aeromonas schubertii (Aeromonas schubertii) belongs to Aeromonas section (Aeromonadaceae) Aeromonas (Aeromonas), is gram negative bacillus, one pole flagellum, moves very active.This bacterium is extensively present in fresh water, seawater, soil, fish and vertebrates enteron aisle, can cause infection after human contact, is the important pathogenic bacteria of acute diarrhea.Though but about the detection kit of this bacterium and the existing patent application (application number: 201210467910.1 of detection technique, title: " double PCR of a kind of pathogenic murrel source Aeromonas schubertii detects primer sets, test kit and method ") disclose, but the test kit of this application and detection technique are only applicable to " murrel source Aeromonas schubertii ", can accurately not detect the Aeromonas schubertii of other Hosts, analysis according to detecting primer " gyrB primer pair " to the core of this application finds, this primer pair gene corresponding to the Aeromonas schubertii that part aquatic animal is originated can not well mate, as the HQ731455 in GeneBank database, HQ731454, HQ731457, JQ319030.And core primers group of the present invention is special and make up completely to the corresponding gene order of all Aeromonas schubertiis of having announced sequence.Therefore the present invention is applicable to according to conservative region design special in Aeromonas schubertii genome the primer assembling test kit, and conjunctive tissue and bacterial genomes extraction reagent establish Aeromonas schubertii detection kit, compensate for this technical field and can not solve the defect that all sources Aeromonas schubertii is detected.The quick diagnosis being established as aquatic animal Aeromonas schubertii and the Etiology survey of test kit of the present invention and detection method provide technical support.
Summary of the invention
For solving traditional problem that Aeromonas schubertii authentication method is time-consuming, accuracy rate is low, the invention provides a kind of Aeromonas schubertii quick detection kit and detection method thereof.According to Aeromonas schubertii genome conserved regions design Auele Specific Primer, use two-step approach round pcr development Aeromonas schubertii quick detection kit and detection method.The method not only has detection efficiency, has more the specificity of detection, to other Aeromonas no cross reaction belonged to together, amplification object clip size is moderate, in detection technique PCR program annealing and amplification carry out simultaneously, greatly reduce the reaction times, and add atopic.This test kit can detect for initiator with the DNA carried at the beginning of tissue sample, is beneficial to the application of clinical reagent box.This also compensate for the blank of the Aeromonas schubertii detection kit lacking universality now.
For solving the problem, the present invention includes a kind of Aeromonas schubertii quick detection kit and detecting the method for Aeromonas schubertii with this test kit.
A kind of Aeromonas schubertii quick detection kit of the present invention, be made up of DNA extraction reagent and PCR reaction reagent, its special character is that described DNA extraction reagent and PCR reaction reagent are respectively:
(1) DNA extraction agent formulations is the TE damping fluid of pH8.0 of 5-15mM Tris, 0.5-1.5mM EDTA; Proteinase K, concentration 15-20mg/mL; The chloroform of 100%; The Virahol of 100%; Ethanol, concentration 70%; Distilled water;
(2) PCR reaction reagent is sub-packed in some reaction tubess, containing 10 × PCR reaction buffer 2.5 μ L in each PCR reaction tubes, concentration is the dNTPs0.5 μ L of 10 μMs, concentration is the Taq archaeal dna polymerase 0.3 μ L of 5U/ μ L, concentration is the specific oligonucleotide primer A0.5 μ L of 10 μMs, the specific oligonucleotide primer B0.5 μ L of 10 μMs, sterilizing deionized water 19.2 μ L, wherein---
Primer A:5 '-CAGCGAGGAGGAAAGGTTGGTGGT-3 ',
Primer B:5 '-AAGCCACGCCTCAAGGGCACAA-3 '.
The described test kit of the present invention detects the method for Aeromonas schubertii, and step is: (1) extracts the DNA in sample with described extraction reagent; (2) with described reaction reagent, pcr amplification is done to described DNA; (3) described amplification product does gel electrophoresis; Its special character is described pcr amplification process is 95 DEG C of denaturation 3min; 98 DEG C of sex change 10s, 68 DEG C of renaturation extend 30s, circulate 35 times; Last 72 DEG C extend 5 ~ 10min; 413bp amplified production is observed after described gel electrophoresis.
Aeromonas schubertii specific detection primer designed in the present invention, it is the corresponding genome sequence according to 25 kinds of Aeromonas in GenBank, and all 18 strain Aeromonas schubertiis of having announced genome sequence, carry out the comparison of sequence similarity district and arrangement, to find for Aeromonas schubertii nucleotide sequence conservative region and site as design of primers district, design the primer (primer A and primer B) special to Aeromonas schubertii.There is not yet the research report to all sources Aeromonas schubertii specific detection design of primers both at home and abroad.
Special for Aeromonas schubertii for ensureing, and obtain amplification fast, the present invention carries out local optimization design to the primer chosen, annealing temperature during PCR is reacted is close to elongating temperature, thus 2 step PCR methods can be utilized to increase to object district, this not only increases the specificity of detection, more greatly reduces detection required time.Test kit of the present invention, compensate for this technical field and can not solve the defect detected all sources Aeromonas schubertii.The quick diagnosis being established as aquatic animal Aeromonas schubertii and the Etiology survey of test kit of the present invention and detection method provide technical support.
Accompanying drawing explanation
Fig. 1 is the specificity figure that the present invention detects Aeromonas schubertii.
M:DL1000DNA Marker in Fig. 1; 1:A.veronii; 2:A.sobria; 3:A.hydrophila; 4:A.caviae; 5:A.bestiarum; 6:A.allosaccharophila; 7:A.media; 8:A.eucrenophila; 9:A.ichthiosmia; 10:A.caviae; 11:Edwardsiella tarda; 12:Escherichia coli; 13:Pseudomonas aeruginosa; 14:Staphylococcus aureus; 15:Enterobacter cloacae; 16:Streptococcusagalactiae; 17:Vibrio harveyi; 18:V.parahaemolyticus; 19:V.alginolyticus; 20:Aeromonas schubertii; 21:Aeromonas schubertii; 22: illing tissue.
Embodiment
The present invention is further illustrated below in conjunction with embodiment.
Embodiment one
Aeromonas schubertii quick detection kit, and with this test kit, specific detection is done to different bacterium genomic dna.
Test kit consists of:
DNA extraction reagent its consist of the TE damping fluid of pH8.0 of 5mM Tris, 0.5mM EDTA; Proteinase K, concentration 15mg/mL; The chloroform of 100%; The Virahol of 100%; Ethanol, concentration 70%; Distilled water;
PCR reaction reagent is sub-packed in some reaction tubess, containing 10 × PCR reaction buffer 2.5 μ L in each PCR reaction tubes, concentration is the dNTPs0.5 μ L of 10 μMs, concentration is the Taq archaeal dna polymerase 0.3 μ L of 5U/ μ L, concentration is the specific oligonucleotide primer A0.5 μ L of 10 μMs, the specific oligonucleotide primer B0.5 μ L of 10 μMs, sterilizing deionized water 19.2 μ L.
Test kit provided by the present invention is to the detection method of Aeromonas schubertii in sample, first with DNA extraction reagent, DNA extraction is carried out to sample, with PCR reaction reagent, pcr amplification is carried out to DNA again, get 5 μ L PCR reaction product after amplified reaction terminates and carry out 1.5% agarose gel electrophoresis, whether can find in sample containing Aeromonas schubertii.Whole testing process can complete in 3 hours, was specially:
1, the DNA in sample is extracted
(1). take disease sample 0.1g, fully add the TE damping fluid consisting of the pH8.0 of 5mM Tris and 0.5mM EDTA described in 500 μ L after grinding, and add 20 μ L Proteinase K Solution, after mixing, place 1hr in 56 DEG C; Add 100 μ L chloroforms mixings, the centrifugal 15min of 12000rpm, gets upper liquid and moves to another centrifuge tube, adds 400 μ L Virahols, the centrifugal 10min of precipitation at room temperature 10min, 12000rpm, taking precipitate, by 70% washing with alcohol 2 times;
(2). 21 kinds of different strains of liquid culture, comprise Aeromonas schubertii, respectively get 500 μ L inoculums centrifugal, taking precipitate, add the TE damping fluid consisting of the pH8.0 of 5mM Tris and 0.5mMEDTA described in 500 μ L, and add 20 μ L Proteinase K Solution, place 1hr in 56 DEG C after mixing; Add 100 μ L chloroforms mixings, the centrifugal 15min of 12000rpm, gets upper liquid and moves to another centrifuge tube, adds 400 μ L Virahols, the centrifugal 10min of precipitation at room temperature 10min, 12000rpm, taking precipitate, by 70% washing with alcohol 2 times;
The throw out of step (1) and the throw out of step (2) are respectively at drying at room temperature, and after dry, sample is dissolved in distilled water respectively, obtains the Genomic DNA solution of 22 samples.
2, PCR reaction
Get the reaction tubes 22 in described test kit, with the Genomic DNA solution one_to_one corresponding of described 22 samples, a kind of described Genomic DNA solution 1 μ L is added respectively in each reaction tubes, and mark respectively, after DNA solution and PCR reaction reagent fully mix in reaction tubes, put into PCR amplification instrument reaction.Reaction process is 95 DEG C of denaturation 3min; 98 DEG C of sex change 10s, 68 DEG C of renaturation extend 30s, circulate 35 times; Last 72 DEG C extend 5min.
3, agarose gel electrophoresis
Get 5 μ L pcr amplification products containing 5 smelling of μ g/ml second pyridine (EB) 1.5% sepharose on electrophoresis, after 5V/cm, 30min on ultraviolet transmission reflectometer observations, object amplified fragments is 413bp.
4, the detection specificity of test kit
Result shows the nucleic acid amplification product of the tissue only having Aeromonas schubertii and infected Aeromonas schubertii through agarose gel electrophoresis, at 413bp place appearance specific nucleic acid band; And other bacterial species has no the specific nucleic acid band (see figure 1) that clip size is 413bp.Result shows that this test kit detects special to Aeromonas schubertii.
Embodiment two
Aeromonas schubertii quick detection kit, and with this test kit, specific detection is done to different bacterium genomic dna.
Test kit consists of:
DNA extraction reagent its consist of the TE damping fluid of pH8.0 of 15mM Tris, 1.5mM EDTA; Proteinase K, concentration 18mg/mL; The chloroform of 100%; The Virahol of 100%; Ethanol, concentration 70%; Distilled water;
PCR reaction reagent is sub-packed in some reaction tubess, containing 10 × PCR reaction buffer 2.5 μ L in each PCR reaction tubes, concentration is the dNTPs0.5 μ L of 10 μMs, concentration is the Taq archaeal dna polymerase 0.3 μ L of 5U/ μ L, concentration is the specific oligonucleotide primer A0.5 μ L of 10 μMs, the specific oligonucleotide primer B0.5 μ L of 10 μMs, sterilizing deionized water 19.2 μ L.
Test kit provided by the present invention is to the detection method of Aeromonas schubertii in sample, first with DNA extraction reagent, DNA extraction is carried out to sample, with PCR reaction reagent, pcr amplification is carried out to DNA again, get 5 μ L PCR reaction product after amplified reaction terminates and carry out 1.5% agarose gel electrophoresis, whether can find in sample containing Aeromonas schubertii.Whole testing process can complete in 3 hours, was specially:
1, the DNA in sample is extracted
(1). take disease sample 0.1g, fully add the TE damping fluid consisting of the pH8.0 of 15mM Tris and 1.5mM EDTA described in 500 μ L after grinding, and add 20 μ L Proteinase K Solution, after mixing, place 1hr in 56 DEG C; Add 100 μ L chloroforms mixings, the centrifugal 15min of 12000rpm, gets upper liquid and moves to another centrifuge tube, adds 400 μ L Virahols, the centrifugal 10min of precipitation at room temperature 10min, 12000rpm, taking precipitate, by 70% washing with alcohol 2 times;
(2). 21 kinds of different strains of liquid culture, comprise Aeromonas schubertii, respectively get 500 μ L inoculums centrifugal, taking precipitate, add the TE damping fluid consisting of the pH8.0 of 15mM Tris and 1.5mMEDTA described in 500 μ L, and add 20 μ L Proteinase K Solution, place 1hr in 56 DEG C after mixing; Add 100 μ L chloroforms mixings, the centrifugal 15min of 12000rpm, gets upper liquid and moves to another centrifuge tube, adds 400 μ L Virahols, the centrifugal 10min of precipitation at room temperature 10min, 12000rpm, taking precipitate, by 70% washing with alcohol 2 times;
The throw out of step (1) and the throw out of step (2) drying at room temperature respectively, after dry, sample is dissolved in distilled water, obtains sample gene group DNA solution.
Following PCR reaction, agarose gel electrophoresis and observations and the detection specificity of test kit and the identical of upper example, repeat no more.
Claims (1)
1. an Aeromonas schubertii quick detection kit, is made up of DNA extraction reagent and PCR reaction reagent, it is characterized in that described DNA extraction reagent and PCR reaction reagent are respectively:
(1) DNA extraction agent formulations is the TE damping fluid of pH8.0 of 5-15mM Tris, 0.5-1.5mM EDTA; Proteinase K, concentration 15-20mg/mL; The chloroform of 100%; The Virahol of 100%; Ethanol, concentration 70%; Distilled water;
(2) PCR reaction reagent is sub-packed in some reaction tubess, containing 10 × PCR reaction buffer 2.5 μ L in each PCR reaction tubes, concentration is the dNTPs 0.5 μ L of 10 μMs, concentration is the Taq archaeal dna polymerase 0.3 μ L of 5U/ μ L, concentration is the specific oligonucleotide primer A 0.5 μ L of 10 μMs, the specific oligonucleotide primer B 0.5 μ L of 10 μMs, sterilizing deionized water 19.2 μ L, wherein---
Primer A:5 '-CAGCGAGGAGGAAAGGTTGGTGGT-3 ',
Primer B:5 '-AAGCCACGCCTCAAGGGCACAA-3 '.
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| CN105734165A (en) * | 2016-05-11 | 2016-07-06 | 辽宁大学 | Aeromonas schubertii specific primer and application thereof in turbot farming process |
| CN106148379A (en) * | 2016-07-26 | 2016-11-23 | 中国水产科学研究院珠江水产研究所 | A kind of method of labeled with green fluorescent protein gene murrel source Aeromonas schubertii |
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| CN108148815A (en) * | 2017-12-11 | 2018-06-12 | 中国水产科学研究院珠江水产研究所 | The bacteriophage of one plant of fish bacteria and its application |
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