CN107326096A - The kit and its detection method of chicken rhinitis haemophilus paragallinarum are detected based on ring mediated isothermal amplification - Google Patents
The kit and its detection method of chicken rhinitis haemophilus paragallinarum are detected based on ring mediated isothermal amplification Download PDFInfo
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- CN107326096A CN107326096A CN201710787325.2A CN201710787325A CN107326096A CN 107326096 A CN107326096 A CN 107326096A CN 201710787325 A CN201710787325 A CN 201710787325A CN 107326096 A CN107326096 A CN 107326096A
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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 a kind of kit and its detection method that chicken rhinitis haemophilus paragallinarum is detected based on ring mediated isothermal amplification, by adding dextran and astragalus polyose 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 shortening detection cycle, significant is detected in chicken rhinitis haemophilus paragallinarum to ring mediated isothermal amplification.
Description
Technical field
The invention belongs to biological technical field, it is related to and chicken rhinitis haemophilus paragallinarum is detected based on ring mediated isothermal amplification
Kit, the method for further relating to detect chicken rhinitis haemophilus paragallinarum based on ring mediated isothermal amplification.
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.
Haemophilus paragallinarum is gram-Negative bacillus, is one of important conditioned pathogen and hospital-acquired infection bacterium.By
Its caused chicken rhinitis haemophilus paragallinarum disease is to endanger a kind of contagious infection of the poultry such as chicken, turkey and pheasant and wild fowl
Disease.The sick morbidity and mortality are generally 10~30%, but the diseased chicken for the chicken house infected chicken rhinitis haemophilus paragallinarum having is dead
The rate of dying is up to 75%.Once the chicken mass-sending live chickens rhinitis haemophilus paragallinarum infection in somewhere, the disease will turn into region disease
Disease, it is difficult to thoroughly eradicate, and often repeated explosion, bring huge economic loss.Chicken rhinitis haemophilus paragallinarum infection with
It is major pathologic features to cause atrophic rhinitis, foul smelling, and septicemia, urinary infection etc., on pathology very
It is difficult to be distinguished with other bacillary rhinitis infection.It is therefore desirable to set up a kind of bloodthirsty bar of secondary chicken of inexpensive detection chicken rhinitis
The method of bacterium disease.
The content of the invention
In view of this, an object of the present invention is to provide bloodthirsty based on the secondary chicken of ring mediated isothermal amplification detection chicken rhinitis
The kit of bacillus, sensitivity is high, and specificity is good, and easy to operate quick, as a result accurately and reliably, testing cost is low, is adapted to medium and small
Type unit and Site Detection application；The second object of the present invention be provide described in kit based on ring mediated isothermal amplification
Detect the application in chicken rhinitis haemophilus paragallinarum；The third object of the present invention is to provide based on ring mediated isothermal amplification detection
The method of chicken rhinitis haemophilus paragallinarum.
For achieving the above object, the present invention provides following detection scheme：
1st, the kit of chicken rhinitis haemophilus paragallinarum, including following component are detected based on ring mediated isothermal amplification：Ring is situated between
Lead isothermal duplication inner primer and outer primer, thermophilic lactic bacillus subtilis archaeal dna polymerase, 10 × heat polymerization 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 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；The ring mediated isothermal amplification inner primer sequence such as SEQ ID NO.1 and SEQ
Shown in ID NO.2；The outer primer sequence is 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 4U/ μ l, the triphosphoric acid base takes off
Oxygen mixture of ribonucleotides each component concentration be 10mmol/L, magnesium sulfate concentration be 100mmol/L, beet alkali concn be 2mol/L,
Dextran concentration is 4mol/L, and astragalus polyose concentration is 4mol/L and rich green I mass fraction of fluorescent dye match is 10%.
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.
2nd, application of the described kit in chicken rhinitis haemophilus paragallinarum is detected based on ring mediated isothermal amplification.
3rd, the method that chicken rhinitis haemophilus paragallinarum 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 haemophilus paragallinarum 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, 3~4U 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
With triphosphoric acid thymidylic acid, concentration is 2-3mmol/L magnesium sulfate, and concentration is 0.5-1mol/L glycine betaine,
Concentration is 0.05~0.25mol/L dextran, and concentration is 0.05~0.25mol/L astragalus polyoses, and template DNA to be checked is water-soluble
Liquid 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
3~5 minutes terminating reactions are incubated in bath；The inner primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2；It is described outer
Primer 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 haemophilus paragallinarum in measuring samples；Such as
Fruit color is changed into green, illustrates to contain chicken rhinitis haemophilus paragallinarum 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 containing dextran, the bloodthirsty bar of kit chicken rhinitis pair chicken
Six specific regions of bacterium 16S rRNA (Genbank Accession No.EU376364) conserved region devise two specificity
Inner primer 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 for detection chicken rhinitis haemophilus paragallinarum, is identified, high specificity by six specific regions of four primer pair target sequences；
Expand under isothermal conditions, will not because temperature change and caused by the loss of time, take it is short, target sequence can be expanded in 1 hour
Increase to 109Times, and influenceing small by non-target sequences, sensitivity is higher compared with PCR methods, and test limit is only several copies；This
Outside, the technology need not be special, expensive instrument and reagent, amplified production need not carry out gel electrophoresis, directly be contaminated with fluorescence
Expect that 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 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 haemophilus paragallinarum 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 pair
Haemophilus gallinarum).
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 the bloodthirsty bar of the secondary chicken of chicken rhinitis
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’-gcacgtgtgtagccctactcgctggaggaaggtggggatg-3’(SEQ ID
NO.1)；
Downstream inner primer BIP：5’-ggtgcatacagagggaagcgaggttgcagactccaatccgg-3’(SEQ ID
NO.2)；
Upstream outer primer F3：5’-caaaggagactgccagtgat-3’(SEQ ID NO.3)；
Downstream outer primer B3：5’-agcgattccgacttcatgg-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 haemophilus paragallinarum, 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 haemophilus paragallinarum, 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 haemophilus paragallinarum：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 0.5 for 8U/ μ l, the μ l of 10 × heat polymerization buffer solution 2.5 are spent, concentration is 10mmol/L
The μ l of the dNTPs aqueous solution 2.5, concentration be 100mmol/L the μ l of magnesium sulfate solution 0.75, concentration be 2mol/L beet buck
The μ l of solution 12.5, 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
The water of enzyme is diluted to 24 μ l, adds the μ l of the template DNA aqueous solution 1, is well mixed, produces；By reaction tube in 60~65 DEG C of water-baths
Middle insulation reaction 25 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, illustrate not contain chicken rhinitis pair chicken thermophilic
Blood bacillus；The color of experimental group is changed into green, illustrates containing chicken rhinitis haemophilus paragallinarum, consistent with expected results.
Contrasting detection 1：According to method same as described above, difference is without dextran in reacting, as a result such as Fig. 1
Shown in.As a result show, color is yellow under conditions of without dextran, it is impossible to detect thermophilic containing the secondary chicken of chicken rhinitis
Blood bacillus.
Contrasting detection 2：According to method same as described above, difference is without dextran, polymerase to add in reacting
Dosage increases to 1 μ l, insulation reaction time lengthening to 60min, as a result as shown in Figure 1.As a result show, without dextran
Under conditions of, the usage amount and extension insulation reaction time for increasing glycine betaine are capable of detecting when chicken rhinitis haemophilus paragallinarum.
In order to prove accuracy that above-mentioned detection reagent and method are detected to chicken rhinitis haemophilus paragallinarum, 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 detect the e coil k 1 pneumonia DNA of 5.6 copy numbers/reaction in sample.
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
It need to play 1-1.5 hour, 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 haemophilus paragallinarum 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>The kit and its detection method of chicken rhinitis haemophilus paragallinarum are detected based on ring mediated isothermal amplification
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 40
<212> DNA
<213>Artificial sequence (Artificial)
<400> 1
gcacgtgtgt agccctactc gctggaggaa ggtggggatg 40
<210> 2
<211> 41
<212> DNA
<213>Artificial sequence (Artificial)
<400> 2
ggtgcataca gagggaagcg aggttgcaga ctccaatccg g 41
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 3
caaaggagac tgccagtgat 20
<210> 4
<211> 19
<212> DNA
<213>Artificial sequence (Artificial)
<400> 4
agcgattccg acttcatgg 19
Claims (6)
1. the kit of chicken rhinitis haemophilus paragallinarum is detected based on ring mediated isothermal amplification, it is characterised in that including such as the following group
Point：Ring mediated isothermal amplification inner primer and outer primer, thermophilic lactic bacillus subtilis archaeal dna polymerase, 10 × heat polymerization
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 ammonia that concentration is that 150-250mmol/L, pH are 8.5-8.8
Ammonium sulfate that potassium chloride that methylmethane-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；The ring mediated isothermal amplification inner primer sequence such as SEQ ID NO.1 and SEQ
Shown in ID NO.2；The outer primer sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4.
2. kit according to claim 1, it is characterised in that：The ring mediated isothermal amplification inner primer concentration is respectively
5μmol/L；The outer primer concentration is respectively 20 μm of ol/L.
3. kit according to claim 1, it is characterised in that：The thermophilic lactic bacillus subtilis archaeal dna polymerase
Concentration is that 4U/ 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.
4. kit according to claim 1, it is characterised in that：10 × heat polymerization buffer solution is by concentration
Ammonium sulfate that potassium chloride that Tris-HCl that 250mmol/L, pH are 8.8, concentration are 100mmol/L, concentration are 100mmol/L,
The Triton X-100 compositions that the magnesium sulfate and mass fraction that concentration is 20mmol/L are 1%.
5. the kit described in any one of Claims 1 to 4 is based on the bloodthirsty bar of the ring mediated isothermal amplification secondary chicken of detection chicken rhinitis
Application in bacterium.
6. the method for chicken rhinitis haemophilus paragallinarum 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 haemophilus paragallinarum 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, 3~4U 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 0.5-1mol/L glycine betaine, and concentration is
0.05~0.25mol/L dextran, concentration is 0.05~0.25mol/L astragalus polyoses, template DNA aqueous solution 0.5- to be checked
1 μ l, surplus is water；By reaction tube in 60~65 DEG C of water-baths insulation reaction 20~30 minutes, in 80~95 DEG C of water-baths protect
Warm 3~5 minutes terminating reactions；The inner primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2；The outer primer sequence
Row are 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 haemophilus paragallinarum in measuring samples；If face
Discoloration is green, illustrates to contain chicken rhinitis haemophilus paragallinarum in measuring samples.
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Application publication date: 20171107 |