CN107385086A - Application and its kit and detection method of the dextran in ring mediated isothermal amplification - Google Patents
Application and its kit and detection method of the dextran in ring mediated isothermal amplification Download PDFInfo
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- CN107385086A CN107385086A CN201710787459.4A CN201710787459A CN107385086A CN 107385086 A CN107385086 A CN 107385086A CN 201710787459 A CN201710787459 A CN 201710787459A CN 107385086 A CN107385086 A CN 107385086A
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- isothermal amplification
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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 present invention relates to a kind of application of dextran in ring mediated isothermal amplification and its kit and detection method, by adding dextran in ring mediated isothermal amplification system to final concentration of 0.1~0.5mol/L (with glucose meter), improve the stability of polymerase, " polymerase " and " glycine betaine " expensive in common LAMP kit can partly be substituted, the amplification of high content GC fragments can be promoted well, the detection reaction time can be reduced to 20 30 minutes, testing cost can be reduced while shorten detection cycle, ring mediated isothermal amplification is detected in pathogenic microorganism significant.
Description
Technical field
The invention belongs to biological technical field, is related to application of the dextran in ring mediated isothermal amplification, further relates to contain
There are the kit and detection method of dextran.
Background technology
At present, the detection method of pathogenic microorganism is mainly separately cultured identification method, PCR (PCR) method and glimmering
Fluorescent Quantitative PCR (FQ-PCR) method, but it is separately cultured that identification method is cumbersome, time-consuming, PCR methods and FQ-PCR methods need expensive instrument
Device and reagent, testing cost is high, is not suitable for middle-size and small-size unit and Site Detection application.
Ring mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) technology with it is other
Nucleic acid amplification technologies are compared, and can fast, efficiently, specifically expand target sequence under isothermal conditions, and easy to operate, it is not necessary to
Expensive instrument and reagent, cost is low, shows vast potential for future development in the pathogenic microorganism examination field.But amplification procedure
In still need the higher archaeal dna polymerase of price, if can reduce archaeal dna polymerase usage amount will to further reduce the micro- life of cause of disease
Analyte detection cost is significant.
Klebsiella (Klebsiella) is gram-Negative bacillus.Mainly there is Friedlander's bacillus
(K.peneumoniae), Klebsiella ozaenae (K.ozaenae) and nose scleroma Klebsiella
(k.rhinoscleromatis).Wherein Klebsiella ozaenae is pathogenic to poultry relatively strong, be important conditioned pathogen and
One of hospital-acquired infection bacterium.The chicken rhinitis type klebsiellosis Pnemoniae being induced by it is to endanger the poultry such as chicken, turkey and pheasant and open country
A kind of contagious disease of fowl.The sick morbidity and mortality are generally 10~30%, but the chicken house infected chicken rhinitis type having
The diseased chicken death rate of Klebsiella is up to 75%.Once the chicken mass-sending live chickens rhinitis type Klebsiella infection in somewhere, should
Disease will turn into endemic illness, it is difficult to thoroughly eradicate, and frequent repeated explosion, bring huge economic loss.Chicken rhinitis
Type Klebsiella is infected to cause atrophic rhinitis, foul smelling, and septicemia, urinary infection etc. for main pathology
Feature, it is difficult to be distinguished with the infection of haemophilus paragallinarum rhinitis on pathology.It is therefore desirable to establish a kind of low cost
Detection chicken rhinitis type klebsiellosis Pnemoniae method.
The content of the invention
In view of this, an object of the present invention is improving enzyme stabilization in offer dextran in ring mediated isothermal amplification
Property shorten application in the reaction time simultaneously；The second object of the present invention is to provide the ring mediated isothermal containing dextran and expanded
The detection kit of increasing, high sensitivity, specificity is good, and easy to operate quick, as a result accurately and reliably, testing cost is low, in being adapted to
Small unit and Site Detection application；The third object of the present invention is that provide described kit is based on ring in pathogenic microorganism
Application in mediated isothermality amplification detection；The fourth object of the present invention is to provide detects chicken rhinitis based on ring mediated isothermal amplification
The method of type Klebsiella.
For achieving the above object, the present invention provides following detection scheme：
1st, dextran improves enzyme stability or/and the application in the shortening reaction time in ring mediated isothermal amplification.
2nd, the detection kit of the ring mediated isothermal amplification containing dextran, including following component：Ring mediated isothermal expands
Increase inner primer and outer primer, thermophilic lactic bacillus subtilis archaeal dna polymerase, 10 × heat polymerization buffer solution, triphosphoric acid alkali
Base deoxynucleotide mixture, magnesium sulfate, glycine betaine, dextran and fluorescent dye match rich green I；10 × the heat polymerization
Buffer solution by concentration be 150-250mmol/L, pH be 8.5-8.8 trishydroxymethylaminomethane-hydrochloric acid, concentration 50-
The magnesium sulfate and quality that ammonium sulfate that 100mmol/L potassium chloride, concentration are 50-100mmol/L, concentration are 15-20mmol/L
The triton x-100 that fraction is 0.5-1% forms；The triphosphoric acid base deoxynucleotide mixture is taken off by Adenosine triphosphate purine
Oxygen nucleotides, triphosphoric acid guanine deoxyribonucleoside acid, triphosphoric acid deoxycytidylic acid and triphosphoric acid thymidine deoxidation core
Thuja acid forms.
Mentioned reagent is remaining equal conventional reagent in addition to inner primer and outer primer need to synthesize as requested, can be direct
Bought from commercial channel, be generally standing reagent in laboratory, therefore above-mentioned examination can not be put into kit of the present invention
Agent, or the one or more being simply placed in mentioned reagent, also can be all put into certainly, there is provided multiple choices are purchased with convenient use person
Buy.
Preferably, the ring mediated isothermal amplification inner primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2；Institute
Outer primer sequence is stated as shown in SEQ ID NO.3 and SEQ ID NO.4.
Preferably, the ring mediated isothermal amplification inner primer concentration is respectively 5 μm of ol/L；The outer primer concentration is respectively
20μmol/L。
Preferably, the thermophilic lactic bacillus subtilis archaeal dna polymerase concentration is that 8U/ μ l, the triphosphoric acid base take off
In oxygen mixture of ribonucleotides each component concentration be 10mmol/L, magnesium sulfate concentration 100mmol/L, beet alkali concn be 2mol/L
It is 10% to be matched with fluorescent dye and win green I mass fraction, and dextran concentration is 4mol/L.
Preferably, 10 × heat polymerization buffer solution is Tris-HCl, dense that 250mmol/L, pH are 8.8 by concentration
Spend the magnesium sulfate and quality point that the potassium chloride for 100mmol/L, the ammonium sulfate that concentration is 100mmol/L, concentration are 20mmol/L
Number forms for 1% Triton X-100.
3rd, application of the described kit in pathogenic microorganism is detected based on ring mediated isothermal amplification.
Preferably, the pathogenic microorganism is chicken rhinitis type Klebsiella.
4th, the method based on ring mediated isothermal amplification detection chicken rhinitis type Klebsiella, comprises the following steps：
A, the DNA of bacteria of measuring samples is extracted as template DNA, the OD of Control architecture aqueous dna260/OD280It is worth and is
1.6~2.0, concentration is 10~100ng/ μ l；
B, the ring mediated isothermal amplification of chicken rhinitis type Klebsiella：Loop-mediated isothermal amplification is prepared in reaction tube
System, each component final concentration are as follows：Concentration is respectively 0.1-0.2 μm of ol/L inner primer, and concentration is respectively the outer of 0.5-0.8 μm of ol/L
Primer, 5-8U thermophilic lactic bacillus subtilis archaeal dna polymerases, 1 × heat polymerization buffer solution, concentration are respectively 0.5-1mmol/
L triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid guanine deoxyribonucleoside acid, triphosphoric acid deoxycytidylic acid and three phosphorus
Sour thymidylic acid, concentration be 2-3mmol/L magnesium sulfate, concentration be 0.25-0.5mol/L glycine betaine, concentration
For 0.1~0.5mol/L dextran, template DNA aqueous solution 0.5-1 μ l to be checked, surplus is water；By reaction tube in 60~65
Insulation reaction 20~30 minutes, is incubated 3~5 minutes terminating reactions in 80~95 DEG C of water-baths in DEG C water-bath；The inner primer
Sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2；The outer primer sequence such as SEQ ID NO.3 and SEQ ID NO.4 institutes
Show；
C, color developing detection：The fluorescent dye match that mass fraction is 10% is added in reaction tube and wins green I aqueous solution 0.5-1 μ
L, detect by an unaided eye color change, if color is yellow, illustrates not containing chicken rhinitis type Klebsiella in measuring samples；Such as
Fruit color is changed into green, illustrates to contain chicken rhinitis type Klebsiella in measuring samples.
The beneficial effects of the present invention are：Dextran is used for ring mediated isothermal amplification by the present invention first, can be notable
The heat endurance of enzyme is improved, (with glucose meter) when concentration is not more than 0.5M, can partly substitute common LAMP kit
In (half or so) expensive " polymerase " and " glycine betaine ", the amplification of high content GC fragments can be promoted well, can be with
The detection reaction time is reduced to 20-30 minutes, it is not only cost-effective, also shorten detection cycle, have to ring mediated isothermal amplification
It is significant.Invention additionally discloses the kit containing dextran, kit chicken rhinitis type Klebsiella 16S
Six specific regions of rRNA (Genbank Accession No.EU376364) conserved region devise two specific inner primers
With two specific outer primers, so as to ensure that the reliability of testing result；The present invention is examined based on loop-mediated isothermal amplification technique
Chicken rhinitis type Klebsiella is surveyed, is identified by six specific regions of four primer pair target sequences, high specificity；In isothermal
Under the conditions of expand, will not because temperature change and caused by the loss of time, take it is short, can be by target sequence amplification to 10 in 1 hour9
Times, and influenceing by non-target sequences small, sensitivity is higher compared with PCR methods, and test limit is only several copies；In addition, the skill
Art need not be special, expensive instrument and reagent, amplified production need not carry out gel electrophoresis, directly be with fluorescent dye colour developing
Can be with naked eyes judged result, easy to operate quick, testing cost is low.The kit and method of the present invention is particularly suitable for middle-size and small-size list
Position and Site Detection application.
Brief description of the drawings
In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below and carried out
Explanation：
Fig. 1 is that loop-mediated isothermal amplification technique detects chicken rhinitis type Klebsiella result (1：Blank control；2：Contrast inspection
Survey 1；3：Experimental group；4：Contrasting detection 2).
Fig. 2 is specificity experiments result (1：Pseudomonas aeruginosa；2：Staphylococcus aureus；3：Salmonella；4：Chicken rhinitis type
Klebsiella).
Fig. 3 is sensitivity experiments result (P：Negative control；1：10-1；2：10-2；3：10-3；4：10-4；5：10-5；6：10-6；
7：10-7；8：10-8；9：10-9；10：10-10)。
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.It is unreceipted specific in embodiment
The experimental method of condition, generally according to normal condition or according to the condition proposed by manufacturer.
Embodiment 1, the method based on loop-mediated isothermal amplification technique detection pathogenic microorganism
Based on the method for loop-mediated isothermal amplification technique detection pathogenic microorganism, the present embodiment is with chicken rhinitis type citric acid
Exemplified by bacterium, it is as follows using reagent：
A, concentration is respectively respectively for the 5 μm of ol/L upstream inner primer FIP aqueous solution and the downstream inner primer BIP aqueous solution, concentration
The 20 μm of ol/L upstream outer primer F3 aqueous solution and the downstream outer primer B3 aqueous solution：Primer sequence is as follows：
Upstream inner primer FIP：5’-ggctggtcatcctctcagacca-agtaggtggggtaatggctc-3’(SEQ
ID NO.1)；
Downstream inner primer BIP：5’-ggcagcagtggggaatattgca-ggccttcttcatacacgcg-3’(SEQ ID
NO.2)；
Upstream outer primer F3：5’-tgcccagatgggattagct-3’(SEQ ID NO.3)；
Downstream outer primer B3：5’-ccttcctcctcgctgaaag-3’(SEQ ID NO.4)；
B, concentration is the 8U/ μ l Bst archaeal dna polymerase aqueous solution；
C, 10 × heat polymerization buffer solution：By concentration be 250mmol/L, pH be 8.8 Tris-HCl, concentration be
The magnesium sulfate and mass fraction that ammonium sulfate that 100mmol/L potassium chloride, concentration are 100mmol/L, concentration are 20mmol/L is
1% Triton X-100 compositions；
D, concentration is the 10mmol/L dNTPs aqueous solution：By concentration respectively for 10mmol/L dATP, dGTP, dCTP and
DTTP is formed；
E, concentration is 100mmol/L magnesium sulfate solution；
F, concentration is 2mol/L aqueous solutions of betaine；
G, concentration is 4mol/L dextran；
H, mass fraction is the 10% fluorescent dye SYBR Green I aqueous solution.
Loop-mediated isothermal amplification technique detection chicken rhinitis type Klebsiella, including following step are utilized using mentioned reagent
Suddenly：
(1) DNA of bacteria of measuring samples is extracted as template DNA：The present embodiment sets experimental group and blank control simultaneously
Group, wherein experimental group are chicken rhinitis type Klebsiella, from Chongqing Academy of Animal Sciences's veterinary institute；Carried using DNA
Take kit (Tiangeng biochemical technology Co., Ltd) to extract each group DNA of bacteria, operated according to kit specification, obtained by experimental group
The OD of the DNA of bacteria aqueous solution260/OD280It is worth for 1.8, concentration is 20ng/ μ l.
(2) ring mediated isothermal amplification of chicken rhinitis type Klebsiella：The ring that cumulative volume is 25 μ l is prepared in reaction tube
Mediated isothermal amplification system：Add the upstream inner primer FIP aqueous solution and downstream inner primer BIP that concentration is 5 μm of ol/L
Each 1 μ l of the aqueous solution, concentration is the 20 μm of ol/L upstream outer primer F3 aqueous solution and downstream each 1 μ l of the outer primer B3 aqueous solution, dense
The μ l of the Bst archaeal dna polymerases aqueous solution 1 for 8U/ μ l, the μ l of 10 × heat polymerization buffer solution 2.5 are spent, concentration is 10mmol/L's
The μ l of the dNTPs aqueous solution 2.5, concentration are the 100mmol/L μ l of magnesium sulfate solution 0.75, and concentration is that 2mol/L glycine betaine is water-soluble
The μ l of liquid 6.5, concentration are the 4mol/L μ l of dextran 2.5, are diluted to 24 μ l with the water without DNA enzymatic, it is water-soluble to add template DNA
The μ l of liquid 1, it is well mixed, produces；By reaction tube in 60~65 DEG C of water-baths insulation reaction 30 minutes, be incubated in 80 DEG C of water-baths
5 minutes terminating reactions；
(3) color developing detection：The fluorescent dye match that mass fraction is 10% is added in reaction tube and wins the green μ l of I aqueous solution 1, is shaken
Shake uniformly, detect by an unaided eye color change.
As a result as shown in figure 1, result is shown, the color of blank control group is yellow, illustrates not containing chicken rhinitis type Cray
Primary Salmonella；The color of experimental group is changed into green, illustrates containing chicken rhinitis type Klebsiella, consistent with expected results.
Contrasting detection 1：According to method same as described above, distinguish and dextran is not being added in reaction, as a result such as Fig. 1
It is shown.As a result show, color is yellow under conditions of dextran is not added, it is impossible to detect containing chicken rhinitis type Cray primary
Salmonella.
Contrasting detection 2：According to method same as described above, distinguish and dextran is not being added in reaction, then increase
Aqueous solutions of betaine addition is to 12.5 μ l, insulation reaction time lengthening to 60min, as a result as shown in Figure 1.As a result show,
Under conditions of not adding dextran, the usage amount and extension insulation reaction time that increase glycine betaine are capable of detecting when chicken rhinitis type
Klebsiella.
In order to prove accuracy that above-mentioned detection reagent and method detect to chicken rhinitis type Klebsiella, specificity is carried out
Experiment and sensitivity experiment, it is specific as follows：
A, LAMP specificity experiments
Non- Friedlander's bacillus (Pseudomonas aeruginosa, the Staphylococcus aureus that Friedlander's bacillus and experimental mouse are often sent out
Bacterium, salmonella) genomic DNA and big mouse gene group DNA according to above-mentioned reaction system and condition establish LAMP detect
Method, carry out specific test.It is positive control to set Friedlander's bacillus genomic DNA, and ultra-pure water is negative control, knot
Fruit is as shown in Figure 2.As a result show, positive reaction occurs in only Friedlander's bacillus genome, and remaining is negative.
B, LAMP sensitivity tests
By Friedlander's bacillus genomic DNA carry out 10 times of gradient dilutions, respectively 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9With 10-10(2 copy), while negative control (ultra-pure water) is set, according to above-mentioned reaction system and
Condition establishes LAMP detection method, to determine the sensitiveness of detection method, as a result as shown in Figure 3.As a result show：The mouse lung of foundation
Scorching Klebsiella LAMP detection method can examine the e coil k 1 pneumonia DNA of 5.6 copy numbers/reaction in the sample of side.
It can be seen from the results above that the more conventional PCR of LAMP amplification methods and fluorescence PCR method have：
High specificity：Only by whether amplification can determine that the presence or absence of target gene, so as to complete thin rice seedling and virus
Qualitative detection.
High sensitivity：The sensitivity of Standard PCR detection method is that 100pg/ reacts (l00 copy numbers/reaction) order of magnitude, is used
Detection method, Monitoring lower-cut are that 13.5fg/ reacts (5.6 copy numbers/reaction).
Operate and identify and be simple and efficient：Standard PCR whole process can just go out result, quantitative fluorescent PCR in 2-4 hour
1-1.5 hour is needed, detection method provided by the invention may occur in which positive findings at 20~30 minutes, and operate letter
Just, it is low to instrument requirements, it is only necessary to common water-bath, and can be by the direct observed result of dyestuff, the step of eliminating electrophoresis,
Have wide practical use in the practice of basic unit's detection chicken rhinitis type Klebsiella.
Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical
Cross above preferred embodiment the present invention is described in detail, it is to be understood by those skilled in the art that can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Sequence table
<110>Chongqing Academy of Animal Sciences
<120>Application and its kit and detection method of the dextran in ring mediated isothermal amplification
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 42
<212> DNA
<213>Artificial sequence (Artificial)
<400> 1
ggctggtcat cctctcagac caagtaggtg gggtaatggc tc 42
<210> 2
<211> 41
<212> DNA
<213>Artificial sequence (Artificial)
<400> 2
ggcagcagtg gggaatattg caggccttct tcatacacgc g 41
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence (Artificial)
<400> 3
tgcccagatg ggattagct 19
<210> 4
<211> 19
<212> DNA
<213>Artificial sequence (Artificial)
<400> 4
ccttcctcct cgctgaaag 19
Claims (9)
1. dextran improves enzyme stability or/and the application in the shortening reaction time in ring mediated isothermal amplification.
2. the detection kit of the ring mediated isothermal amplification containing dextran, it is characterised in that including following component：Ring mediates
Isothermal duplication inner primer and outer primer, thermophilic lactic bacillus subtilis archaeal dna polymerase, 10 × heat polymerization buffer solution, three
Phosphoric acid base deoxynucleotide mixture, magnesium sulfate, glycine betaine, dextran and fluorescent dye match rich green I；10 × the hot polymerization
Close reaction buffer by concentration be 150-250mmol/L, pH be 8.5-8.8 trishydroxymethylaminomethane-hydrochloric acid, concentration be
The magnesium sulfate and matter that ammonium sulfate that 50-100mmol/L potassium chloride, concentration are 50-100mmol/L, concentration are 15-20mmol/L
The triton x-100 that fraction is 0.5-1% is measured to form；The triphosphoric acid base deoxynucleotide mixture is by Adenosine triphosphate purine
Deoxynucleotide, triphosphoric acid guanine deoxyribonucleoside acid, triphosphoric acid deoxycytidylic acid and triphosphoric acid thymidine deoxidation
Nucleotides forms.
3. kit according to claim 2, it is characterised in that：The ring mediated isothermal amplification inner primer sequence such as SEQ
Shown in ID NO.1 and SEQ ID NO.2；The outer primer sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4.
4. the kit according to Claims 2 or 3, it is characterised in that：The ring mediated isothermal amplification inner primer concentration point
Wei not 5 μm of ol/L；The outer primer concentration is respectively 20 μm of ol/L.
5. the kit according to Claims 2 or 3, it is characterised in that：The thermophilic lactic bacillus subtilis DNA polymerizations
Enzyme concentration is 8U/ μ l, the triphosphoric acid base deoxynucleotide mixture each component concentration is 10mmol/L, magnesium sulfate concentration is
100mmol/L, beet alkali concn are 2mol/L, dextran concentration is 4mol/L and rich green I mass fraction of fluorescent dye match is
10%.
6. the kit according to Claims 2 or 3, it is characterised in that：10 × heat polymerization buffer solution is by concentration
The sulfuric acid that potassium chloride that the Tris-HCl for being 8.8 for 250mmol/L, pH, concentration are 100mmol/L, concentration are 100mmol/L
The Triton X-100 compositions that the magnesium sulfate and mass fraction that ammonium, concentration are 20mmol/L are 1%.
7. kit the answering in pathogenic microorganism is detected based on ring mediated isothermal amplification described in any one of claim 2~6
With.
8. application according to claim 7, it is characterised in that：The pathogenic microorganism is chicken rhinitis type Klebsiella.
9. the method based on ring mediated isothermal amplification detection chicken rhinitis type Klebsiella, it is characterised in that：Comprise the following steps：
A, the DNA of bacteria of measuring samples is extracted as template DNA, the OD of Control architecture aqueous dna260/OD280Be worth for 1.6~
2.0, concentration is 10~100ng/ μ l；
B, the ring mediated isothermal amplification of chicken rhinitis type Klebsiella：Loop-mediated isothermal amplification body is prepared in reaction tube
System, each component final concentration are as follows：Concentration is respectively 0.1-0.2 μm of ol/L inner primer, and concentration is respectively drawn for 0.5-0.8 μm of the outer of ol/L
Thing, 5-8U thermophilic lactic bacillus subtilis archaeal dna polymerases, 1 × heat polymerization buffer solution, concentration are respectively 0.5-1mmol/L
Triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid guanine deoxyribonucleoside acid, triphosphoric acid deoxycytidylic acid and three phosphorus
Sour thymidylic acid, concentration be 2-3mmol/L magnesium sulfate, concentration be 0.25-0.5mol/L glycine betaine, concentration
For 0.1~0.5mol/L dextran, template DNA aqueous solution 0.5-1 μ l to be checked, surplus is water；By reaction tube in 60~65
Insulation reaction 20~30 minutes, is incubated 3~5 minutes terminating reactions in 80~95 DEG C of water-baths in DEG C water-bath；The inner primer
Sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2；The outer primer sequence such as SEQ ID NO.3 and SEQ ID NO.4 institutes
Show；
C, color developing detection：The fluorescent dye match that mass fraction is 10% is added in reaction tube and wins green I aqueous solution 0.5-1 μ l, is used
Visual color changes, if color is yellow, illustrates not containing chicken rhinitis type Klebsiella in measuring samples；If face
Discoloration is green, illustrates to contain chicken rhinitis type Klebsiella in measuring samples.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710787459.4A CN107385086A (en) | 2017-09-04 | 2017-09-04 | Application and its kit and detection method of the dextran in ring mediated isothermal amplification |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710787459.4A CN107385086A (en) | 2017-09-04 | 2017-09-04 | Application and its kit and detection method of the dextran in ring mediated isothermal amplification |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009086608A2 (en) * | 2007-12-26 | 2009-07-16 | Eppendorf Array Technologies S.A. | Method and kit to perform a pcr amplification and micro- array detection in the same medium and/or same chamber |
| CN101871005A (en) * | 2009-12-29 | 2010-10-27 | 重庆市畜牧科学院 | Riemerella anatipestifer detection kit and method based on loop-mediated isothermal amplification technology |
-
2017
- 2017-09-04 CN CN201710787459.4A patent/CN107385086A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009086608A2 (en) * | 2007-12-26 | 2009-07-16 | Eppendorf Array Technologies S.A. | Method and kit to perform a pcr amplification and micro- array detection in the same medium and/or same chamber |
| CN101871005A (en) * | 2009-12-29 | 2010-10-27 | 重庆市畜牧科学院 | Riemerella anatipestifer detection kit and method based on loop-mediated isothermal amplification technology |
Non-Patent Citations (2)
| Title |
|---|
| NELSON ENRIQUE ARENAS 等: "Design of a molecular method for subspecies specific identification of Klebsiella pneumoniae by using the 16S ribosomal subunit gene", 《COLOMBIA MEDICA》 * |
| PIERO CARNINCI 等: "Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA", 《PROC. NATL. ACAD. SCI.》 * |
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