CN106086206A - Green Wei Si Salmonella loop-mediated isothermal gene amplification fast detecting kit and detection method - Google Patents
Green Wei Si Salmonella loop-mediated isothermal gene amplification fast detecting kit and detection method Download PDFInfo
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- CN106086206A CN106086206A CN201610593823.9A CN201610593823A CN106086206A CN 106086206 A CN106086206 A CN 106086206A CN 201610593823 A CN201610593823 A CN 201610593823A CN 106086206 A CN106086206 A CN 106086206A
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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
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- C12Q1/6844—Nucleic acid amplification reactions
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Abstract
The invention discloses a kind of green Wei Si Salmonella loop-mediated isothermal gene amplification fast detecting kit and detection method, wherein test kit carries out extensive genome analysis by Bioinformatics Platform, according to green Wei Si SalmonellarecNFour specific primers of gene design, in conjunction with LAMP technology, establish rapid sensitive detection method accurately for green Wei Si Salmonella, and build the quick detection kit for the method.Use quick detection kit and the detection method of the present invention, qualification can be observed by the naked eye after the reaction, without other any analytical procedures such as electrophoresis, have that the detection time is short, the advantage such as high specificity, instrument and equipment requirement are low and easy and simple to handle, can be used for the detection safely and fast of the samples such as food, environment and clinic.
Description
Technical field
The present invention relates to one and utilize loop-mediated isothermal gene amplification (loop-mediated isothermal
Amplification of DNA, LAMP) technology carries out the biological detection reagent that bacterium sample quickly detects, and belongs to green Wei Si Salmonella
Rapid gene molecular diagnosis agents technical field, be specifically related to a kind of green Wei Si Salmonella loop-mediated isothermal gene amplification quick
Detection kit and detection method.
Background technology
Green Wei Si Salmonella (Weissella viridescens) belong to lactobacillus mesh (Lactobacillales) bright string
Pearl Cordycepps (Leuconostocaceae) Wei Si Bordetella (Weissella).This bacterium is in terms of microcosmic and macroscopic form and lactic acid
Other representative strain of bacterium is similar, particularly easily and leukonid (Leuconostoc) and lactobacillus (Lactobacillus)
Obscure mutually.Green Wei Si Salmonella is gram positive bacteria, and in irregular rod-short, two ends are rounded or the most tiny, in pairs or
Short chain arranges；MRS agar culture medium forms transparent petite, in micro-protuberance circular contour.
Green Wei Si Salmonella has heterofermentation metabolism, it is possible to participate in the corrupt change of food particularly meat products
Matter.When exposed to oxygen, some meat productss, as sausage, vacuum packaging meat, bacon etc. there will be the phenomenon turing green.Grind
Studying carefully and think, green Wei Si Salmonella is to cause meat products to show main cause mucus greening occur.Mucus is originally single bacterium
Fall upper appearance, the most just gradually forms the mucus of a piece of green, and from the surface of meat products toward internal penetration.It is present in meat products
In some antibacterial can produce hydrogen peroxide by its normal physiological activity, and green Wei Si Salmonella has relatively low oxidation also
Former current potential, it is possible to cause hydrogen peroxide accumulation in meat products.Hydrogen peroxide, by oxidation nitrosomyochromogen, ultimately results in
Meat products greening.Green Wei Si Salmonella has become as the important putrefaction bacteria threatening meat products quality and safety, to meat packing row
Industry causes significant economic loss.
But, the detection method currently for green Wei Si Salmonella is extremely limited.Traditional authentication method needs through front
Increase bacterium, selective medium and again increase bacterium, culture at the flat lining out containing one or more tested bacteria growing preparations of suppression
Cultivate, macroscopic characteristic bacterium colony is confirmed and this bacterium colony is carried out a series of biochemistry and serotype detection make
Identify, the longest, complex operation, and result reliability is low；Amplification rDNA restriction analysis (amplified
Ribosomal DNA restriction analysis, ARDRA) Wei Si Salmonella can be distinguished in the level planted, but grasp
Make complex steps (PCR expands, enzyme action and cluster analysis), need special instrument (PCR instrument) and multiple restricted enzyme, become
This is higher；Real-Time Fluorescent Quantitative PCR Technique is successfully used for the Molecular Identification of green Wei Si Salmonella, although the method has accurately
Property the advantage such as good, highly sensitive, high specificity, but the necessary real-time fluorescence quantitative PCR instrument of experiment is expensive, it is difficult to wide
General application and Site Detection.
DNA circle mediated constant temperature nucleic acid amplification technology (LAMP) is a kind of novel constant temperature nucleic acid amplification method, this technology gram
Take the deficiency of conventional gene amplification method, it is possible under constant temperature, carry out the amplification of nucleic acid, there is simple, quick, cost
The advantages such as low and high specificity, are suitable to Site Detection, have preferable application.
Summary of the invention
Present invention solves the technical problem that and there is provided a kind of rapid sensitive green Wei Si Salmonella loop-mediated isothermal accurately
Gene amplification fast detecting kit and detection method.
The present invention solves that above-mentioned technical problem adopts the following technical scheme that, green Wei Si Salmonella loop-mediated isothermal gene expands
Increase quick detection kit, it is characterised in that including:
(1) reactant liquor 1: by 10mmol/L deoxynucleoside triphosphate (dNTP), 10 × ThermoPol Buffer reaction buffer,
150mmol/L magnesium sulfate (MgSO4), 5mol/L glycine betaine and sterilizing distilled water (ddH2O) composition；
(2) reactant liquor 2: by 10 μm ol/L outer primer 1(F3), 10 μm ol/L outer primer 2(B3), 40 μm ol/L inner primer 1(FIP)
With 40 μm ol/L inner primer 2(BIP) composition, primer sequence is as follows:
Outer primer 1:CTTTGCTCAACGCAACAG,
Outer primer 2:ATTTTCGCGTCGCTCATG,
Inner primer 1:ACCTGCATTAACGCTTGGTG-GAAGTGGGCAATCATTTGG,
Inner primer 2:TAGATGAATACGCATCAAACGCT-CTAACTGTCGATACTCCGC；
(3) 8U/ μ L Bst archaeal dna polymerase；
(4) developer: 10wt% SYBR Green I fluorescent dye；
In above-mentioned loop-mediated isothermal gene amplification fast detecting kit, reactant liquor 1 converts into every sample 19 μ L, consisting of:
10mmol/L deoxynucleoside triphosphate (dNTP) 4 μ L, 10 × ThermoPol Buffer reaction buffer 2.5 μ L, 150mmol/L
Magnesium sulfate (MgSO4) 1 μ L, 5mol/L glycine betaine 5 μ L and sterilizing distilled water (ddH2O) 6.5 μ L；Reactant liquor 2 converts into every sample 3 μ L,
Consisting of: 10 μm ol/L outer primer 1(F3) 0.5 μ L, 10 μm ol/L outer primer 2(B3) 0.5 μ L, 40 μm ol/L inner primers 1
(FIP) 1 μ L and 40 μm ol/L inner primer 2(BIP) 1 μ L.
The detection method of green Wei Si Salmonella loop-mediated isothermal gene amplification fast detecting kit of the present invention, its
It is characterised by concretely comprising the following steps:
Step (1), extracts the genomic DNA of measuring samples
Isolation kit method is used to prepare measuring samples genomic DNA；
Step (2), loop-mediated isothermal amplification reacts
First take 2 μ L measuring samples Genomic DNA solution, add 19 μ L reactant liquor 1,3 μ L reactant liquor 2 and 1 μ L Bst DNA and gather
Synthase, then the reaction system configured is taken out to be checked after 65 DEG C of amplified reaction 50-70min；
Step (3), analyzes and judges reaction result
Add 1 μ L developer in the reaction product, mixing, stand 2min, if reactant liquor shows green is the positive, orange, be
Negative.
The method have the advantages thatrecNGene is the restructuring of Wei Si Salmonella and repair protein gene, at Si Shi Wei
There are differences between bacterium is the most of the same race.The present invention is based on green Wei Si SalmonellarecNFour specific primers of gene design, should
Gene order is that green Wei Si Salmonella is common, to ensure the reliability of the green Wei Si Salmonella of detection separate sources.The present invention
Use LAMP technology, establish rapid sensitive detection method accurately for green Wei Si Salmonella, and build for the method
Quick detection kit.Use quick detection kit and the detection method of the present invention, mirror can be observed by the naked eye after the reaction
Fixed, it is not necessary to other any analytical procedures such as electrophoresis, have that the detection time is short, high specificity, instrument and equipment requirement be low and operation letter
The advantage such as just, can be used for the detection safely and fast of the samples such as food, environment and clinic.
Accompanying drawing explanation
Fig. 1 is the positive amplification result comparison diagram of different strains, and wherein in No. 1 reaction tube, sample is green, No. 2-8 reaction
In pipe, sample is orange.
Detailed description of the invention
By the following examples the foregoing of the present invention is described in further details, but this should be interpreted as this
The scope inventing above-mentioned theme is only limitted to below example, and all technology realized based on foregoing of the present invention belong to this
Bright scope.
Embodiment 1
Green Wei Si Salmonella loop-mediated isothermal gene amplification fast detecting kit and the foundation of detection method
Step one, the design of primer, the assembling of synthetic agent box:
The present embodiment determines that the primer sequence for detection is as follows:
Outer primer 1:CTTTGCTCAACGCAACAG,
Outer primer 2:ATTTTCGCGTCGCTCATG,
Inner primer 1:ACCTGCATTAACGCTTGGTG-GAAGTGGGCAATCATTTGG,
Inner primer 2:TAGATGAATACGCATCAAACGCT-CTAACTGTCGATACTCCGC.
The green Wei Si Salmonella loop-mediated isothermal gene amplification fast detecting kit of design, this test kit bag on this basis
Include:
(1) reactant liquor 1: by 10mmol/L deoxynucleoside triphosphate (dNTP), 10 × ThermoPol Buffer reaction buffer,
150mmol/L magnesium sulfate (MgSO4), 5 mol/L glycine betaines and sterilizing distilled water (ddH2O) composition；
(2) reactant liquor 2: by 10 μm ol/L outer primer 1(F3), 10 μm ol/L outer primer 2(B3), 40 μm ol/L inner primer 1(FIP)
With 40 μm ol/L inner primer 2(BIP) composition；
(3) 8U/ μ L Bst archaeal dna polymerase；
(4) developer: 10wt% SYBR Green I fluorescent dye；
In above-mentioned loop-mediated isothermal gene amplification fast detecting kit, reactant liquor 1 converts into every sample 19 μ L, consisting of:
10mmol/L deoxynucleoside triphosphate (dNTP) 4 μ L, 10 × ThermoPol Buffer reaction buffer 2.5 μ L, 150mmol/L
Magnesium sulfate (MgSO4) 1 μ L, 5mol/L glycine betaine 5 μ L and sterilizing distilled water (ddH2O) 6.5 μ L；Reactant liquor 2 converts into every sample 3 μ L,
Consisting of: 10 μm ol/L outer primer 1(F3) 0.5 μ L, 10 μm ol/L outer primer 2(B3) 0.5 μ L, 40 μm ol/L inner primers 1
(FIP) 1 μ L and 40 μm ol/L inner primer 2(BIP) 1 μ L.
Step 2, the preparation of measuring samples: use isolation kit method to prepare measuring samples genomic DNA:
Detection strain green Wei Si Salmonella (Weissella viridescens) preserved by this laboratory, numbering N08.Use
The bacterial genomes that Beijing Tian Gen bio-engineering corporation produces is extracted test kit and is extracted sample gene group DNA.
Step 3, carries out loop-mediated isothermal amplification reaction:
First take 2 μ L measuring samples Genomic DNA solution, add 19 μ L reactant liquor 1,3 μ L reactant liquor 2 and 1 μ L Bst DNA and gather
Synthase, then the reaction system configured is taken out to be checked after 65 DEG C of amplified reaction 50-70 min.
Step 4, analyzes and judges reaction result:
Add 1 μ L developer in the reaction product, mixing, stand 2min, visual color changes, and reactant liquor color becomes green
Color, illustrates that bacterial strain to be checked is for green Wei Si Salmonella.
Embodiment 2
Negative control
Step one, the design of primer, the assembling of synthetic agent box:
The present embodiment determines for the primer sequence detected with embodiment 1.
The green Wei Si Salmonella constant temperature gene amplification fast detecting kit of design on this basis, this test kit includes:
(1) reactant liquor 1: by 10mmol/L deoxynucleoside triphosphate (dNTP), 10 × ThermoPol Buffer reaction buffer,
150mmol/L magnesium sulfate (MgSO4), 5mol/L glycine betaine and sterilizing distilled water (ddH2O) composition；
(2) reactant liquor 2: by 10 μm ol/L outer primer 1(F3), 10 μm ol/L outer primer 2(B3), 40 μm ol/L inner primer 1(FIP)
With 40 μm ol/L inner primer 2(BIP)；
(3) 8U/ μ L Bst archaeal dna polymerase；
(4) developer: 10wt% SYBR Green I fluorescent dye；
In above-mentioned loop-mediated isothermal gene amplification fast detecting kit, reactant liquor 1 converts into every sample 19 μ L, consisting of:
10mmol/L deoxynucleoside triphosphate (dNTP) 4 μ L, 10 × ThermoPol Buffer reaction buffer 2.5 μ L, 150mmol/L
Magnesium sulfate (MgSO4) 1 μ L, 5mol/L glycine betaine 5 μ L and sterilizing distilled water (ddH2O) 6.5 μ L；Reactant liquor 2 converts into every sample 3 μ L,
Consisting of: 10 μm ol/L outer primer 1(F3) 0.5 μ L, 10 μm ol/L outer primer 2(B3) 0.5 μ L, 40 μm ol/L inner primers 1
(FIP) 1 μ L and 40 μm ol/L inner primer 2(BIP) 1 μ L.
Step 2, the preparation of measuring samples: use isolation kit method to prepare measuring samples genomic DNA:
The bacterial genomes using Beijing Tian Gen bio-engineering corporation to produce is extracted test kit and is extracted enterococcus faecalis, plant breast bar
Bacterium, Lactobacillus paracasei, breast bright string coccus, cibarium Wei Si Salmonella, fusion Wei Si Salmonella and the base of Greece 7 kinds of antibacterials of Wei Si Salmonella
Because of group DNA.
Step 3, carries out loop-mediated isothermal amplification reaction:
First take 2 μ L measuring samples Genomic DNA solution, add 19 μ L reactant liquor 1,3 μ L reactant liquor 2 and 1 μ L Bst DNA and gather
Synthase；Again the reaction system configured is taken out to be checked after 65 DEG C of amplified reaction 50-70min.
Step 4, analyzes and judges reaction result:
Add 1 μ L developer in the reaction product, mixing, stand 2min, visual color changes, if reactant liquor shows green
Being the positive, orange is then negative.
As it is shown in figure 1, the reaction tube of numbered 1-8 respectively corresponding green Wei Si Salmonella, enterococcus faecalis, Lactobacillus plantarum,
Lactobacillus paracasei, breast bright string coccus, cibarium Wei Si Salmonella, fusion Wei Si Salmonella and Greece Wei Si Salmonella.
Described embodiment 1 compares with each negative control group in embodiment 2, produces positive amplification result, such as figure in embodiment 1
No. 1 reaction tube in 1, and embodiment 2 negative control bacterial strain does not produce positive amplification result, such as 2-8 reaction tube in Fig. 1, from
And prove that this test kit and detection method have stronger specificity, other bacterial strain beyond green Wei Si Salmonella will not be produced
False positive results.
SEQUENCE LISTING
<110>He'nan Normal University
<120>green Wei Si Salmonella loop-mediated isothermal gene amplification fast detecting kit and detection method
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>artificial sequence
<400> 1
ctttgctcaa cgcaacag 18
<210> 2
<211> 18
<212> DNA
<213>artificial sequence
<400> 2
attttcgcgt cgctcatg 18
<210> 3
<211> 39
<212> DNA
<213>artificial sequence
<400> 3
acctgcatta acgcttggtg gaagtgggca atcatttgg 39
<210> 4
<211> 42
<212> DNA
<213>artificial sequence
<400> 4
tagatgaata cgcatcaaac gctctaactg tcgatactcc gc 42
The ultimate principle of the present invention, principal character and advantage are more than shown and described, without departing from spirit and scope of the invention
On the premise of, the present invention also has various changes and modifications, and these changes and improvements both fall within the scope of claimed invention.
SEQUENCE LISTING
<110>He'nan Normal University
<120>green Wei Si Salmonella loop-mediated isothermal gene amplification fast detecting kit and detection method
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>artificial sequence
<400> 1
ctttgctcaa cgcaacag 18
<210> 2
<211> 18
<212> DNA
<213>artificial sequence
<400> 2
attttcgcgt cgctcatg 18
<210> 3
<211> 39
<212> DNA
<213>artificial sequence
<400> 3
acctgcatta acgcttggtg gaagtgggca atcatttgg 39
<210> 4
<211> 42
<212> DNA
<213>artificial sequence
<400> 4
tagatgaata cgcatcaaac gctctaactg tcgatactcc gc 42
Claims (2)
1. green Wei Si Salmonella loop-mediated isothermal gene amplification fast detecting kit, it is characterised in that including:
(1) reactant liquor 1: by 10mmol/L deoxynucleoside triphosphate, 10 × ThermoPol Buffer reaction buffer,
150mmol/L magnesium sulfate, 5mol/L glycine betaine and sterilizing distilled water composition；
(2) reactant liquor 2: by 10 μm ol/L outer primer 1,10 μm ol/L outer primer 2,40 μm ol/L inner primer 1 and 40 μm ol/L
Primer 2 forms, and primer sequence is as follows:
Outer primer 1:CTTTGCTCAACGCAACAG,
Outer primer 2:ATTTTCGCGTCGCTCATG,
Inner primer 1:ACCTGCATTAACGCTTGGTG-GAAGTGGGCAATCATTTGG,
Inner primer 2:TAGATGAATACGCATCAAACGCT-CTAACTGTCGATACTCCGC；
(3) 8U/ μ L Bst archaeal dna polymerase；
(4) developer: 10wt% SYBR Green I fluorescent dye；
In above-mentioned loop-mediated isothermal gene amplification fast detecting kit, reactant liquor 1 converts into every sample 19 μ L, consisting of:
10mmol/L deoxynucleoside triphosphate 4 μ L, 10 × ThermoPol Buffer reaction buffer 2.5 μ L, 150mmol/L magnesium sulfate
1 μ L, 5mol/L glycine betaine 5 μ L and sterilizing distilled water 6.5 μ L；Reactant liquor 2 converts into every sample 3 μ L, consisting of: outside 10 μm ol/L
Primer 1 0.5 μ L, 10 μm ol/L outer primer 2 0.5 μ L, 40 μm ol/L inner primer 11 μ L and 40 μm ol/L inner primer 21 μ L.
2. the detection side of the green Wei Si Salmonella loop-mediated isothermal gene amplification fast detecting kit described in a claim 1
Method, it is characterised in that concretely comprise the following steps:
Step (1), extracts the genomic DNA of measuring samples
Isolation kit method is used to prepare measuring samples genomic DNA；
Step (2), loop-mediated isothermal amplification reacts
First take 2 μ L measuring samples Genomic DNA solution, add 19 μ L reactant liquor 1,3 μ L reactant liquor 2 and 1 μ L Bst DNA and gather
Synthase, then the reaction system configured is taken out to be checked after 65 DEG C of amplified reaction 50-70min；
Step (3), analyzes and judges reaction result
Add 1 μ L developer in the reaction product, mixing, stand 2min, if reactant liquor shows green is the positive, orange, be
Negative.
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| CN201610593823.9A CN106086206A (en) | 2016-07-26 | 2016-07-26 | Green Wei Si Salmonella loop-mediated isothermal gene amplification fast detecting kit and detection method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755540A (en) * | 2017-03-06 | 2017-05-31 | 河南师范大学 | The molten bacillus loop-mediated isothermal gene amplification fast detecting kit of antibiotic and detection method |
| CN106906296A (en) * | 2017-04-05 | 2017-06-30 | 江南大学 | A kind of label of high frequency zone Wei Si Salmonellas and its application |
| CN108504754A (en) * | 2018-04-03 | 2018-09-07 | 河南师范大学 | Green Wei Si Salmonellas real-time fluorescence quantitative PCR quick detection kit and its detection method |
-
2016
- 2016-07-26 CN CN201610593823.9A patent/CN106086206A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| ERICA M. GÓMEZ-ROJO等: "A novel real-time PCR assay for the specific identification and quantification of Weissella viridescens in blood sausages", 《INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY》 * |
| JOHN SAMELIS等: "Usefulness of Rapid GC Analysis of Cellular Fatty Acids for Distinguishing Weissella viridescens, Weissella paramesenteroides, Arginine-negative Weissella Strains of Meat Origin", 《SYSTEM. APPL. MICROBIOL. 》 * |
| 邵秀玲等主编: "《植物病原生物现代检测技术及应用》", 30 November 2015, 中国标准出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755540A (en) * | 2017-03-06 | 2017-05-31 | 河南师范大学 | The molten bacillus loop-mediated isothermal gene amplification fast detecting kit of antibiotic and detection method |
| CN106906296A (en) * | 2017-04-05 | 2017-06-30 | 江南大学 | A kind of label of high frequency zone Wei Si Salmonellas and its application |
| CN108504754A (en) * | 2018-04-03 | 2018-09-07 | 河南师范大学 | Green Wei Si Salmonellas real-time fluorescence quantitative PCR quick detection kit and its detection method |
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Application publication date: 20161109 |