CN107326097A - Dextran combines application and its kit and detection method of the astragalus polyose in ring mediated isothermal amplification - Google Patents
Dextran combines application and its kit and detection method of the astragalus polyose in ring mediated isothermal amplification Download PDFInfo
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- CN107326097A CN107326097A CN201710787561.4A CN201710787561A CN107326097A CN 107326097 A CN107326097 A CN 107326097A CN 201710787561 A CN201710787561 A CN 201710787561A CN 107326097 A CN107326097 A CN 107326097A
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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
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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
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
Abstract
The present invention relates to application and its kit and detection method of a kind of dextran joint astragalus polyose in ring mediated isothermal amplification, it is 0.05~0.25mol/L by adding dextran and astragalus polyose to final concentration in ring mediated isothermal amplification system (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 shortening detection cycle, significant is detected in pathogenic microorganism to ring mediated isothermal amplification.
Description
Technical field
The invention belongs to biological technical field, it is related to dextran joint astragalus polyose answering in ring mediated isothermal amplification
With, further relate to containing dextran combine astragalus polyose kit and detection method.
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 are high, are 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 target sequence can be fast, efficiently, specifically expanded 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 reduction 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 poultry and the open countries such as harm chicken, turkey and pheasant
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 often repeated explosion, bring huge economic loss.Chicken rhinitis
Type Klebsiella infects to cause atrophic rhinitis, foul smelling, and septicemia, urinary infection etc. for main pathology
Feature, is difficult to be distinguished with the infection of haemophilus paragallinarum rhinitis on pathology.It is therefore desirable to set up 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 that providing dextran combines astragalus polyose in ring mediated isothermal amplification
The middle application improved in the enzyme stability shortening reaction time simultaneously；The second object of the present invention be to provide containing dextran and
The detection kit of the ring mediated isothermal amplification of astragalus polyose, sensitivity is high, and specificity is good, easy to operate quick, as a result accurately
Reliably, testing cost is low, is adapted to middle-size and small-size unit and Site Detection application；The third object of the present invention is the examination described in offer
Application of the agent box in pathogenic microorganism is detected based on ring mediated isothermal amplification；The fourth object of the present invention, which is to provide, is based on ring
The method that mediated isothermality amplification detects chicken rhinitis type Klebsiella.
For achieving the above object, the present invention provides following detection scheme：
1st, dextran joint astragalus polyose improves enzyme stability in ring mediated isothermal amplification and shortened in the reaction time
Application.
2nd, the detection kit of the ring mediated isothermal amplification containing dextran and astragalus polyose, including following component：Ring
Mediated isothermality amplification inner primer and outer primer, thermophilic lactic bacillus subtilis archaeal dna polymerase, 10 × heat polymerization buffering
Liquid, triphosphoric acid base deoxynucleotide mixture, magnesium sulfate, glycine betaine, dextran, astragalus polyose and fluorescent dye match are rich green
Ⅰ；10 × heat polymerization buffer solution is by trihydroxy methyl amino first that concentration is that 150-250mmol/L, pH are 8.5-8.8
Ammonium sulfate that potassium chloride that alkane-hydrochloric acid, concentration are 50-100mmol/L, concentration are 50-100mmol/L, concentration are 15-
20mmol/L magnesium sulfate and mass fraction constitutes for 0.5-1% triton x-100；The triphosphoric acid base deoxynucleotide
Mixture is by triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid guanine deoxyribonucleoside acid, triphosphoric acid deoxycytidylic acid
With triphosphoric acid thymidylic acid composition.
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 and be purchased there is provided multiple choices with convenient use person certainly
Buy.
It is preferred that, 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.
It is preferred that, the ring mediated isothermal amplification inner primer concentration is respectively 5 μm of ol/L；The outer primer concentration is respectively
20μmol/L。
It is preferred that, the thermophilic lactic bacillus subtilis archaeal dna polymerase concentration is 8U/ μ l, the triphosphoric acid base takes off
Each component concentration is that 10mmol/L, magnesium sulfate concentration are that 100mmol/L, beet alkali concn are 2mol/L in oxygen mixture of ribonucleotides
It is that 10%, dextran concentration is that 4mol/L, astragalus polyose concentration are 4mol/L to win green I mass fraction with fluorescent dye match.
It is preferred that, 10 × heat polymerization buffer solution is by Tris-HCl, dense that concentration is that 250mmol/L, pH are 8.8
Magnesium sulfate and quality point that ammonium sulfate that potassium chloride that degree is 100mmol/L, concentration are 100mmol/L, concentration are 20mmol/L
Number constitutes for 1% Triton X-100.
3rd, application of the described kit in pathogenic microorganism is detected based on ring mediated isothermal amplification is detected.
It is preferred that, the pathogenic microorganism is chicken rhinitis type Klebsiella.
4th, the method that chicken rhinitis type Klebsiella is detected based on ring mediated isothermal amplification, is comprised the following steps：
A, the DNA of bacteria of extraction measuring samples are used 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, chicken rhinitis type Klebsiella ring mediated isothermal amplification：Loop-mediated isothermal amplification is prepared in reaction tube
System, each component final concentration is as follows：The inner primer of concentration respectively for 0.1-0.2 μm of ol/L, 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 is 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 is 2-3mmol/L magnesium sulfate, and concentration is 0.25-0.5mol/L glycine betaine, concentration
For 0.05~0.25mol/L dextran, concentration is 0.05~0.25mol/L astragalus polyose, the template DNA aqueous solution to be checked
0.5-1 μ l, surplus is water；By reaction tube in 60~65 DEG C of water-baths insulation reaction 20~30 minutes, then at 80~95 DEG C of water-baths
3~5 minutes terminating reactions of middle insulation；The inner primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2；Draw outside described
Thing sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4；
C, color developing detection：Mass fraction is added in reaction tube and wins green I aqueous solution 0.5-1 μ for 10% fluorescent dye match
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：The present invention first expands dextran and astragalus polyose for ring mediated isothermal
Increase, can significantly improve the heat endurance of enzyme, (with glucose meter) when concentration is not more than 0.5M, can partly substitute common
(half or so) expensive " polymerase " and " glycine betaine ", can promote high content GC fragments well in LAMP kit
In the detection reaction time, can be reduced to 20-30 minutes by amplification, not only cost-effective, also shorten detection cycle, to ring mediation etc.
Temperature amplification is significant.Invention additionally discloses the kit for combining astragalus polyose containing dextran, the kit chicken nose
Six specific regions design of scorching type Klebsiella 16S rRNA (Genbank Accession No.EU376364) conserved region
Two specific inner primers and two specific outer primers, so as to ensure that the reliability of testing result；The present invention is based on ring
Mediated isothermal amplification technology detects chicken rhinitis type Klebsiella, is known by six specific regions of four primer pair target sequences
Not, high specificity；Expand under isothermal conditions, will not because temperature change and caused by the loss of time, take it is short, in 1 hour i.e.
Can be by target sequence amplification to 109Times, and influenceing small by non-target sequences, sensitivity is higher compared with PCR methods, and test limit is only
Several copies；In addition, the technology need not be special, expensive instrument and reagent, amplified production need not carry out gel electrophoresis, directly
Connecing can be with naked eyes judged result with fluorescent dye colour developing, and easy to operate quick, testing cost is low.The kit of the present invention and side
Method is particularly suitable for middle-size and small-size unit 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 loop-mediated isothermal amplification technique detection 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
The method that pathogenic microorganism is detected based on loop-mediated isothermal amplification technique, the present embodiment is with chicken rhinitis type citric acid
Exemplified by bacterium, it is as follows using reagent：
A, concentration are respectively the 5 μm of ol/L upstream inner primer FIP aqueous solution and the downstream inner primer BIP aqueous solution, and concentration is respectively
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’-gttccagtgtggctggtcatcc-taatggctcacctaggcgac-3’(SEQ
ID NO.1)；
Downstream inner primer BIP：5’-ggaggcagcagtggggaatatt-aaggccttcttcatacacgc-3’(SEQ
ID NO.2)；
Upstream outer primer F3：5’-tgcccagatgggattagct-3’(SEQ ID NO.3)；
Downstream outer primer B3：5’-cttaatgccttcctcctcgc-3’(SEQ ID NO.4)；
B, concentration are 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 are the 10mmol/L dNTPs aqueous solution：By concentration respectively for 10mmol/L dATP, dGTP, dCTP and
DTTP is constituted；
E, concentration are 100mmol/L magnesium sulfate solution；
F, concentration are 2mol/L aqueous solutions of betaine；
G, concentration are 4mol/L dextran；
H, concentration are 4mol/L astragalus polyose；
I, mass fraction are the 10% fluorescent dye SYBR Green I aqueous solution.
Using mentioned reagent chicken rhinitis type Klebsiella, including following step are detected using loop-mediated isothermal amplification technique
Suddenly：
(1) DNA of bacteria for extracting measuring samples is used 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 is the 100mmol/L μ l of magnesium sulfate solution 0.75, and concentration is water-soluble for 2mol/L glycine betaine
The μ l of liquid 6.5, concentration is the 4mol/L μ l of dextran 1.25, and concentration is the 4mol/L μ l of astragalus polyose 1.25, with without DNA enzymatic
Water is diluted to 24 μ l, adds the μ l of the template DNA aqueous solution 1, is well mixed, produces；Reaction tube is protected in 60~65 DEG C of water-baths
Temperature reaction 20 minutes, is incubated 5 minutes terminating reactions in 80 DEG C of water-baths；
(3) color developing detection：Mass fraction is added in reaction tube and wins the green μ l of I aqueous solution 1 for 10% fluorescent dye match, is shaken
Shake uniform, 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, difference is many without dextran and the Radix Astragali in reacting
Sugar, as a result as shown in Figure 1.As a result show, color is yellow under conditions of without dextran and astragalus polyose, it is impossible to examined
Measure containing chicken rhinitis type Klebsiella.
Contrasting detection 2：According to method same as described above, difference is many without dextran and the Radix Astragali in reacting
Sugar, then increases aqueous solutions of betaine addition 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 without dextran and astragalus polyose, increase the usage amount and extension insulation reaction of glycine betaine
Time is capable of detecting when chicken rhinitis type Klebsiella.
In order to prove accuracy that above-mentioned detection reagent and method are detected 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, 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 set up LAMP detect
Method, carries 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
Friedlander's bacillus genomic DNA is subjected to 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 set negative control (ultra-pure water), according to above-mentioned reaction system and
Condition sets up 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.
Sensitivity is high：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 is 13.5fg/ reactions (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, the detection method that the present invention is provided 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, it is possible to by the direct observed result of dyestuff, the step of eliminating electrophoresis,
Have wide practical use in the practice that chicken rhinitis type Klebsiella detects in basic unit.
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>Dextran combines application and its kit and detection method of the astragalus polyose in ring mediated isothermal amplification
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 42
<212> DNA
<213>Artificial sequence (Artificial)
<400> 1
gttccagtgt ggctggtcat cctaatggct cacctaggcg ac 42
<210> 2
<211> 42
<212> DNA
<213>Artificial sequence (Artificial)
<400> 2
ggaggcagca gtggggaata ttaaggcctt cttcatacac gc 42
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence (Artificial)
<400> 3
tgcccagatg ggattagct 19
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 4
cttaatgcct tcctcctcgc 20
Claims (9)
1. dextran joint astragalus polyose improves enzyme stability in ring mediated isothermal amplification and shortens answering in the reaction time
With.
2. the detection kit of the ring mediated isothermal amplification containing dextran and astragalus polyose, it is characterised in that including as follows
Component：Ring mediated isothermal amplification inner primer and outer primer, thermophilic lactic bacillus subtilis archaeal dna polymerase, 10 × thermal polymerization are anti-
Answer buffer solution, triphosphoric acid base deoxynucleotide mixture, magnesium sulfate, glycine betaine, dextran, astragalus polyose and fluorescent dye
Match rich green I；10 × heat polymerization buffer solution is by trihydroxy methyl that concentration is that 150-250mmol/L, pH are 8.5-8.8
Ammonium sulfate that potassium chloride that aminomethane-hydrochloric acid, concentration are 50-100mmol/L, concentration are 50-100mmol/L, concentration are 15-
20mmol/L magnesium sulfate and mass fraction constitutes for 0.5-1% triton x-100；The triphosphoric acid base deoxynucleotide
Mixture is by triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid guanine deoxyribonucleoside acid, triphosphoric acid deoxycytidylic acid
With triphosphoric acid thymidylic acid composition.
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 that 8U/ μ l, the triphosphoric acid base deoxynucleotide mixture each component concentration are that 10mmol/L, magnesium sulfate concentration are
100mmol/L, beet alkali concn are that 2mol/L, dextran concentration are 4mol/L, and astragalus polyose concentration is 4mol/L and fluorescence
It is 10% that green I mass fraction is won in dyestuff match.
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 for chicken rhinitis type Klebsiella is detected based on ring mediated isothermal amplification, it is characterised in that：Comprise the following steps：
A, the DNA of bacteria of extraction measuring samples are used as template DNA, the OD of Control architecture aqueous dna260/OD280Be worth for 1.6~
2.0, concentration is 10~100ng/ μ l；
B, chicken rhinitis type Klebsiella ring mediated isothermal amplification：Loop-mediated isothermal amplification body is prepared in reaction tube
System, each component final concentration is as follows：The inner primer of concentration respectively for 0.1-0.2 μm of ol/L, 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 is 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 is 2-3mmol/L magnesium sulfate, and concentration is 0.25-0.5mol/L glycine betaine, concentration
For 0.05~0.25mol/L dextran, concentration is 0.05~0.25mol/L astragalus polyose, the template DNA aqueous solution to be checked
0.5-1 μ l, surplus is water；By reaction tube in 60~65 DEG C of water-baths insulation reaction 20~30 minutes, then at 80~95 DEG C of water-baths
3~5 minutes terminating reactions of middle insulation；The inner primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2；Draw outside described
Thing sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4；
C, color developing detection：Mass fraction is added in reaction tube and wins green I aqueous solution 0.5-1 μ l for 10% fluorescent dye match, 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.
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| 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 |
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2017
- 2017-09-04 CN CN201710787561.4A patent/CN107326097A/en active Pending
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| 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 |
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