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CN106636348A - Zymomonas mobilis loop-mediated isothermal amplification of DNA rapid detection kit, and detection method - Google Patents

Zymomonas mobilis loop-mediated isothermal amplification of DNA rapid detection kit, and detection method Download PDF

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CN106636348A
CN106636348A CN201610979995.XA CN201610979995A CN106636348A CN 106636348 A CN106636348 A CN 106636348A CN 201610979995 A CN201610979995 A CN 201610979995A CN 106636348 A CN106636348 A CN 106636348A
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primer
zymomonas mobilis
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徐雅梦
姜晓冰
姜晓杰
于涛
张艺鸽
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Henan Normal University
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
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    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • 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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Abstract

本发明公开了一种运动发酵单胞菌环介导恒温基因扩增快速检测试剂盒及检测方法，其中试剂盒借助生物信息平台进行大规模基因组分析，根据运动发酵单胞菌的甘油醛‑3‑磷酸脱氢酶基因gap设计了四条特异性引物，结合LAMP技术，针对运动发酵单胞菌建立了快速灵敏准确的检测方法，并构建用于该方法的快速检测试剂盒。使用本发明的快速检测试剂盒和检测方法，在反应后可通过肉眼观察鉴定，无需电泳等其他任何分析步骤，具有检测时间短、特异性强、仪器设备要求低、操作简便等优点，可用于运动发酵单胞菌的快速检测。

The invention discloses a Zymomonas mobilis ring-mediated constant temperature gene amplification rapid detection kit and detection method, wherein the kit performs large-scale genome analysis by means of a biological information platform, and according to the glyceraldehyde-3 of Zymomonas mobilis ‑Phosphate dehydrogenase gene gap designed four specific primers, combined with LAMP technology, established a rapid, sensitive and accurate detection method for Zymomonas mobilis, and constructed a rapid detection kit for this method. Using the rapid detection kit and detection method of the present invention, it can be identified by naked eyes after the reaction, without any other analysis steps such as electrophoresis, and has the advantages of short detection time, strong specificity, low requirements for equipment and easy operation, etc., and can be used in Rapid detection of Zymomonas mobilis.

Description

Zymomonas mobilis loop-mediated isothermal gene amplification fast detecting kit and detection Method
Technical field
The present invention relates to a kind of utilize loop-mediated isothermal gene magnification（loop-mediated isothermal Amplification of DNA, LAMP）Technology carries out the kit of bacterium sample quick detection, and in particular to a kind of motion fermentation list Born of the same parents' collarium mediated constant temperature gene amplification fast detecting kit and detection method.
Background technology
In recent years as the quick continuous growth of population and industry has greatly accelerated energy resource consumption, the whole world is caused to face potential Energy crisis.The continuous improvement of simultaneous fuel price and the drastically reduction of fossil fuel, this promotes the whole world and seek Look for alternative regenerative resource.Wherein bio-fuel is considered as the topmost substitute of conventional fossil fuel.Bio-ethanol It is a kind of regenerative resource, its process of consumption not only solves the problem of environmental pollution of traditional fuel, solves agriculture and forestry product The huge pollution that brings to environment of leftover bits and pieces, and be also greatly reduced the cost for obtaining the mineral matter energy.Biological fermentation process Production ethanol is widely used in some countries and regions, and Brazil produces 11,000,000 tons of fuel second every year using sugarcane as raw material Alcohol；The U.S. then about produces every year more than 5,500,000 tons of alcohol fuel；China's bio-ethanol annual production at present is more than 300 ten thousand tons, It is only second to Brazil, U.S. row third place in the world.
Zymomonas mobilis（Zymomonas mobilis,Zm）For the gramnegative bacterium of amphimicrobian, mainly lead to Cross Entner-Doudoroff（ED）Approach metabolizable glucose, fructose and sucrose etc., its unique metabolic pathway and efficient second Alcohol fermentation characteristic causes the bacterial strain to there is huge application potential in fields such as food, medicine, chemical industry and bioenergies, thus right The research of zymomonas mobilis is also deepening continuously.What zymomonas mobilis had a high-yield quick converts glucose into second Alcohol, can to tolerate high temperature, concentration of alcohol and sugared concentration, the non-growth for coupling and fermentation i.e. alcohol production process uncorrelated to growth etc. Feature so as to which the application in terms of alcohol is produced especially is paid attention to.
There are various detection methods currently for zymomonas mobilis.Traditional Zengjing Granule method generally require 10 days with On, the cycle is longer, and needs to use animal used as test, relatively costly；Immunology detection（Immunoassay, IA）It is a kind of root According to antigen, the principle of antibody response, unknown antibody is detected or using known antibody test unknown antigen using known antigen Technology, mainly including Enzyme-linked Immunosorbent Assay（ELISA）, radio-immunity（RIA）, reverse indirect blood coagulation（RPHA）With reverse breast Gelling collection experiment（RPLA）Deng, such method cycle is short, great amount of samples is once can detect, and be not required to animal used as test, but it lacks Point is the antibody that must have high-affinity；Real time fluorescence quantifying PCR method can be used in the detection of pathogen, but to instrument and equipment It is higher with the requirement of the level professional technology of personnel, and complex operation, it is time-consuming longer, limit the popularization of the technology.
DNA circle mediated constant temperature nucleic acid amplification technology（LAMP）It is a kind of new constant temperature nucleic acid amplification method, this technology gram The deficiency of conventional gene amplification method is taken, the amplification of nucleic acid can have been carried out under constant temperature, with simple, quick, cost The advantages of low and high specificity, Site Detection is suitable to, there is preferable application.At present, motion fermentation list also it is not specifically designed for The loop-mediated isothermal gene amplification fast detecting kit of born of the same parents bacterium and the related record of detection method.
The content of the invention
It is an object of the invention to provide a kind of accurate zymomonas mobilis loop-mediated isothermal gene of rapid sensitive expands Increase quick detection kit and detection method.
The present invention is adopted the following technical scheme that for achieving the above object：Zymomonas mobilis loop-mediated isothermal gene magnification Quick detection kit, it is characterised in that include：
（1）Reactant liquor 1：By 10mmol/L deoxynucleoside triphosphates, 10 × ThermoPol Buffer reaction buffers, 150mmol/L magnesium sulfate, 5mol/L glycine betaines and sterilizing distilled water composition；
（2）Reactant liquor 2：By in 10 μm of 1,10 μm of ol/L outer primers, 2,40 μm of ol/L outer primers ol/L inner primers 1 and 40 μm of ol/L Primer 2 is constituted, and primer sequence is as follows：
Outer primer 1：GTGCAAGATGCGTTGGAAAC,
Outer primer 2：GGCGTTGATATCGTGATGGA,
Inner primer 1：GCGATGTTGATCGCACCGTTGT-CTTGTCATCTGCGGTCAGG,
Inner primer 2：AGCCGGAGCAGAAATCAGAACC-CGGGTATCTTCACCAACACC；
（3）8U/ μ L Bst archaeal dna polymerases；
（4）Developer：Mass concentration is 10% SYBR Green I fluorescent dyes；
Reactant liquor 1 is equivalent per the μ L of sample 19 in above-mentioned loop-mediated isothermal gene amplification fast detecting kit, consisting of： The μ L of 10mmol/L deoxynucleoside triphosphates 4, μ L, 150mmol/L magnesium sulfate of 10 × ThermoPol Buffer reaction buffers 2.5 The μ L of 1 μ L, 5mol/L glycine betaine 5 and sterilizing μ L of distilled water 6.5；Reactant liquor 2 is equivalent per the μ L of sample 3, consisting of：Outside 10 μm of ol/L The μ L of primer 1 0.5,10 μm of the μ L of ol/L outer primers 2 0.5,40 μm of μ L of ol/L inner primers 11 and 40 μm of μ L of ol/L inner primers 21.
The detection method of zymomonas mobilis loop-mediated isothermal gene amplification fast detecting kit of the present invention, It is characterized in that concretely comprising the following steps：
Step（1）, extract the genomic DNA of measuring samples；
Step（2）, loop-mediated isothermal amplification reaction takes 2 μ L measuring samples DNA solutions, adds 19 μ L reactant liquors 1,3 μ L reactant liquors 2 and 1 μ L 8U/ μ L Bst archaeal dna polymerases, the reaction system for having configured are taken out after 65 DEG C of amplified reaction 50-70min and are treated Inspection；
Step（3）, analyze and judge reaction result, 1 μ L developers are added in the reaction product, mix, 2min is stood, if reactant liquor Shows green is the positive, and orange is then feminine gender.
Glyceraldehyde-3-phosphate dehydrogenase gene of the present invention according to zymomonas mobilisgapDevise four specificity Primer, the gene order be zymomonas mobilis it is common, with ensure detect separate sources zymomonas mobilis can By property.The present invention adopts LAMP technology, and for zymomonas mobilis the accurate detection method of rapid sensitive is established, and builds For the quick detection kit of the method.Using the quick detection kit and detection method of the present invention, can lead to after the reaction Cross and visually observe identification, without the need for other any analytical equipments such as electrophoresis, with detection time is short, high specificity, instrument and equipment will The advantages of asking low and easy to operate, can be used for the quick detection of zymomonas mobilis.
Description of the drawings
Fig. 1 is the positive amplification result comparison diagram of different strains.
Specific embodiment
With reference to specific embodiment, the foundation and application to kit in the present invention and detection method is made furtherly It is bright, but present disclosure is not limited in any form.
Embodiment 1
The foundation of zymomonas mobilis loop-mediated isothermal gene amplification fast detecting kit and detection method
Step（1）, the design of primer, the assembling of synthetic agent box
The present embodiment determines as follows for the primer sequence of detection：
Outer primer 1：GTGCAAGATGCGTTGGAAAC,
Outer primer 2：GGCGTTGATATCGTGATGGA,
Inner primer 1：GCGATGTTGATCGCACCGTTGT-CTTGTCATCTGCGGTCAGG,
Inner primer 2：AGCCGGAGCAGAAATCAGAACC-CGGGTATCTTCACCAACACC；
Zymomonas mobilis loop-mediated isothermal gene amplification fast detecting kit, the kit are designed on this basis Including following reagent：
（1）Reactant liquor 1：By 10mmol/L deoxynucleoside triphosphates（dNTP）, 10 × ThermoPol Buffer reaction buffers, 150mmol/L magnesium sulfate（MgSO4）, 5 mol/L glycine betaines and sterilizing distilled water（ddH2O）Composition；
（2）Reactant liquor 2：By 10 μm of ol/L outer primers 1（F3）, 10 μm of ol/L outer primers 2（B3）, 40 μm of ol/L inner primers 1（FIP） With 40 μm of ol/L inner primers 2（BIP）Composition；
（3）8U/ μ L Bst archaeal dna polymerases；
（4）Developer：Mass concentration is 10% SYBR Green I fluorescent dyes；
Reactant liquor 1 is equivalent per the μ L of sample 19 in above-mentioned loop-mediated isothermal gene amplification fast detecting kit, consisting of： 10mmol/L deoxynucleoside triphosphates（dNTP）4 μ L, 10 × ThermoPol Buffer reaction buffers 2.5 μ L, 150mmol/L Magnesium sulfate（MgSO4）The μ L of 1 μ L, 5mol/L glycine betaine 5 and sterilizing distilled water（ddH2O）6.5μL；Reactant liquor 2 is equivalent per the μ L of sample 3, Consisting of：10 μm of ol/L outer primers 1（F3）0.5 μ L, 10 μm of ol/L outer primers 2（B3）0.5 μ L, 40 μm of ol/L inner primers 1 （FIP）1 μ L and 40 μm of ol/L inner primers 2（BIP）1μL.
Step（2）, the preparation of measuring samples
Measuring samples genomic DNA is prepared using isolation kit method
Detection bacterial classification zymomonas mobilis（Zymomonas mobilis, Zm）From He'nan Normal University's microbe The bacterial classification of Laboratories Accession.The bacterial genomes extracts kit produced using Beijing Tiangeng bio-engineering corporation extracts sample base Because of a group DNA.
Step（3）, carry out loop-mediated isothermal amplification reaction
（1）2 μ L measuring samples DNA solutions are taken, 19 μ L reactant liquors 1,3 μ L reactant liquors 2 and 1 μ L 8U/ μ L Bst DNA polymerization is added Enzyme；
（2）The reaction system for preparing is taken out into be checked after 65 DEG C of amplified reaction 50min.
Step（4）, analyze and judge reaction result
1 μ L developers being added in the reaction product, being mixed, stand 2min, visual color change, reactant liquor color is changed into green Color, illustrates that bacterial strain to be checked is zymomonas mobilis.
Embodiment 2
Negative control
Step（1）, the design of primer, the assembling of synthetic agent box
The present embodiment determine for detection primer sequence with embodiment 1.
Zymomonas mobilis loop-mediated isothermal gene amplification fast detecting kit, the examination are designed on this basis Agent box includes following reagent：
（1）Reactant liquor 1：By 10mmol/L deoxynucleoside triphosphates（dNTP）, 10 × ThermoPol Buffer reaction buffers, 150mmol/L magnesium sulfate（MgSO4）, 5mol/L glycine betaines and sterilizing distilled water（ddH2O）Composition；
（2）Reactant liquor 2：By 10 μm of ol/L outer primers 1（F3）, 10 μm of ol/L outer primers 2（B3）, 40 μm of ol/L inner primers 1（FIP） With 40 μm of ol/L inner primers 2（BIP）Composition；
（3）8U/ μ L Bst archaeal dna polymerases；
（4）Developer：Mass concentration is 10% SYBR Green I fluorescent dyes；
Reactant liquor 1 is equivalent per the μ L of sample 19 in above-mentioned loop-mediated isothermal gene amplification fast detecting kit, consisting of： 10mmol/L deoxynucleoside triphosphates（dNTP）4 μ L, 10 × ThermoPol Buffer reaction buffers 2.5 μ L, 150mmol/L Magnesium sulfate（MgSO4）The μ L of 1 μ L, 5mol/L glycine betaine 5 and sterilizing distilled water（ddH2O）6.5μL；Reactant liquor 2 is equivalent per the μ L of sample 3, Consisting of：10 μm of ol/L outer primers 1（F3）0.5 μ L, 10 μm of ol/L outer primers 2（B3）0.5 μ L, 40 μm of ol/L inner primers 1 （FIP）1 μ L and 40 μm of ol/L inner primers 2（BIP）1μL.
Step（2）, the preparation of measuring samples
Measuring samples genomic DNA is prepared using isolation kit method
The bacterial genomes extracts kit produced using Beijing Tiangeng bio-engineering corporation extracts pseudomonas aeruginosa, green Wei This Salmonella, Listeria monocytogenes, staphylococcus aureus, Escherichia coli, Klebsiella Pneumoniae and Salmonella The genomic DNA of 7 kinds of bacteriums such as bacterium.
Step（3）, carry out loop-mediated isothermal amplification reaction
（1）2 μ L measuring samples DNA solutions are taken, 19 μ L reactant liquors 1,3 μ L reactant liquors 2,1 μ L 8U/ μ L Bst DNA polymerizations is added Enzyme；
（2）The reaction system for preparing is taken out into be checked after 65 DEG C of amplified reaction 50min.
Step（4）, analyze and judge reaction result
1 μ L developers are added in the reaction product, is mixed, stand 2min, visual color change, if reactant liquor shows green As positive, orange is then feminine gender.
As shown in figure 1, the reaction tube that numbering is 1-8 corresponds to respectively pseudomonas aeruginosa, zymomonas mobilis, green Wei This Salmonella, Listeria monocytogenes, staphylococcus aureus, Escherichia coli, Klebsiella Pneumoniae and Salmonella Bacterium.
Described embodiment 1 compares with each negative control group in embodiment 2, positive amplification result is produced in embodiment 1, such as No. 2 reaction tubes in Fig. 1, and do not carrygapThe negative control bacterial strain of embodiment 2 of gene does not produce positive amplification result, such as Fig. 1 In No. 1,3-8 reaction tubes, so as to prove that this kit and detection method have stronger specificity, to not carryinggapGene Other bacterial strains will not produce false positive results.
Have been shown and described above the general principle of the present invention, principal character and advantage, without departing from spirit of the invention and On the premise of scope, the present invention also has various changes and modifications, and these changes and improvements both fall within claimed invention Scope.
SEQUENCE LISTING
<110>He'nan Normal University
<120>Zymomonas mobilis loop-mediated isothermal gene amplification fast detecting kit and detection method
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
gtgcaagatg cgttggaaac 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
ggcgttgata tcgtgatgga 20
<210> 3
<211> 41
<212> DNA
<213>Artificial sequence
<400> 3
gcgatgttga tcgcaccgtt gtcttgtcat ctgcggtcagg 41
<210> 4
<211> 42
<212> DNA
<213>Artificial sequence
<400> 4
agccggagca gaaatcagaa cccgggtatc ttcaccaacacc 42

Claims (2)

1.运动发酵单胞菌环介导恒温基因扩增快速检测试剂盒，其特征在于包括：1. Zymomonas mobilis ring-mediated constant temperature gene amplification rapid detection kit, characterized in that it comprises: （1）反应液1：由10mmol/L脱氧核苷三磷酸、10×ThermoPol Buffer反应缓冲液、150mmol/L硫酸镁、5mol/L甜菜碱和灭菌双蒸水组成；(1) Reaction solution 1: composed of 10mmol/L deoxynucleoside triphosphate, 10×ThermoPol Buffer reaction buffer, 150mmol/L magnesium sulfate, 5mol/L betaine and sterilized double distilled water; （2）反应液2：由10μmol/L外引物1、10μmol/L外引物2、40μmol/L内引物1和40μmol/L内引物2组成，引物序列如下：(2) Reaction solution 2: composed of 10 μmol/L outer primer 1, 10 μmol/L outer primer 2, 40 μmol/L inner primer 1 and 40 μmol/L inner primer 2, the primer sequences are as follows: 外引物1：GTGCAAGATGCGTTGGAAAC，Outer primer 1: GTGCAAGATGCGTTGGAAAC, 外引物2：GGCGTTGATATCGTGATGGA，Outer primer 2: GGCGTTGATATCGTGATGGA, 内引物1：GCGATGTTGATCGCACCGTTGT-CTTGTCATCTGCGGTCAGG，Inner primer 1: GCGATGTTGATCGCACCGTTGT-CTTGTCATCTGCGGTCAGG, 内引物2：AGCCGGAGCAGAAATCAGAACC-CGGGTATCTTCACCAACACC；Inner primer 2: AGCCGGAGCAGAAATCAGAACC-CGGGTATCTTCACCAACACC; （3）8U/μL Bst DNA聚合酶；(3) 8U/μL Bst DNA polymerase; （4）显色剂：质量浓度为10%的SYBR Green I荧光染料；(4) Chromogen: SYBR Green I fluorescent dye with a mass concentration of 10%; 上述环介导恒温基因扩增快速检测试剂盒中反应液1折合每样品19μL，其组成为：10mmol/L脱氧核苷三磷酸4μL、10×ThermoPol Buffer反应缓冲液2.5μL、150mmol/L硫酸镁1μL、5mol/L甜菜碱5μL和灭菌双蒸水6.5μL；反应液2折合每样品3μL，其组成为：10μmol/L外引物1 0.5μL、10μmol/L外引物2 0.5μL、40μmol/L内引物1 1μL和40μmol/L内引物2 1μL。The reaction solution 1 in the above-mentioned loop-mediated constant temperature gene amplification rapid detection kit is equivalent to 19 μL per sample, and its composition is: 4 μL of 10 mmol/L deoxynucleoside triphosphate, 2.5 μL of 10×ThermoPol Buffer reaction buffer, 150 mmol/L magnesium sulfate 1 μL, 5 mol/L betaine 5 μL and sterilized double distilled water 6.5 μL; reaction solution 2 is equivalent to 3 μL per sample, and its composition is: 10 μmol/L outer primer 1 0.5 μL, 10 μmol/L outer primer 2 0.5 μL, 40 μmol/L 1 μL of internal primer 1 and 1 μL of 40 μmol/L internal primer 2. 2.一种权利要求1所述的运动发酵单胞菌环介导恒温基因扩增快速检测试剂盒的检测方法，其特征在于具体步骤为：2. the detection method of a Zymomonas mobilis ring-mediated constant temperature gene amplification rapid detection kit according to claim 1, is characterized in that concrete steps are: 步骤（1），提取待检样品的基因组DNA；Step (1), extracting the genomic DNA of the sample to be tested; 步骤（2），环介导恒温扩增反应，取2μL待检样品DNA溶液，加入19μL反应液1、3μL反应液2和1μL 8U/μL Bst DNA聚合酶，将配置好的反应体系在65℃扩增反应50-70min后取出待检；Step (2), loop-mediated constant temperature amplification reaction, take 2 μL of the DNA solution of the sample to be tested, add 19 μL of reaction solution 1, 3 μL of reaction solution 2 and 1 μL of 8U/μL Bst DNA polymerase, and put the prepared reaction system at 65 °C After 50-70 minutes of amplification reaction, take it out for inspection; 步骤（3），分析判断反应结果，在反应产物中加入1μL显色剂，混匀，静置2min，若反应液显现绿色即为阳性，橙色则为阴性。Step (3), analyze and judge the reaction result, add 1 μL of chromogen to the reaction product, mix well, and let it stand for 2 minutes. If the reaction solution appears green, it is positive, and if it is orange, it is negative.
CN201610979995.XA 2016-11-08 2016-11-08 Zymomonas mobilis loop-mediated isothermal amplification of DNA rapid detection kit, and detection method Pending CN106636348A (en)

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Publication number Priority date Publication date Assignee Title
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Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1633500A (en) * 2002-02-20 2005-06-29 希森美康株式会社 Nucleic acid amplification primer for detection of housekeeping gene mRNA and inspection method using same
CN104561263A (en) * 2014-11-24 2015-04-29 河南师范大学 Rapid detection kit and detection method for clostridium botulinum B employing loop-mediated isothermal gene amplification

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Title
T. CONWAY ET AL: "Glyceraldehyde-3-Phosphate Dehydrogenase Gene from Zymomonas mobilis: Cloning, Sequencing, and Identification of Promoter Region", 《JOURNAL OF BACTERIOLOGY》 *
TSUGUNORI NOTOMI ET AL: "Loop-mediated isothermal amplification of DNA", 《NUCLEIC ACIDS RESEARCH》 *
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Application publication date: 20170510