CN108048462A - LAMP primer group, detection kit and its application of bovine - Google Patents
LAMP primer group, detection kit and its application of bovine Download PDFInfo
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- CN108048462A CN108048462A CN201810144742.XA CN201810144742A CN108048462A CN 108048462 A CN108048462 A CN 108048462A CN 201810144742 A CN201810144742 A CN 201810144742A CN 108048462 A CN108048462 A CN 108048462A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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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Abstract
The present invention relates to identification calf-derived Cyclospora detection technique research field, in particular to LAMP primer group, detection kit and its application of a kind of bovine.The kit includes LAMP primer group, indicator, archaeal dna polymerase and reaction buffer；The LAMP primer group includes the inner primer pair of F3 and B3 composition outer primers pair and FIP and BIP compositions.Using the detection kit can effectively, quick, high differentiation specifically is detected to the tissue of ox and other animals.
Description
Technical field
The present invention relates to identification calf-derived Cyclospora detection technique research field, in particular to a kind of bovine
LAMP primer group, detection kit and its application.
Background technology
Dairy produce is the main source of China's current Major Nutrient intake, in milk containing abundant protein, fat and
The nutritional ingredients such as amino acid are the fluid products for easily being digested and being absorbed by human body.Milk with its is sweet and dilitious, full of nutrition,
The advantages that being easy to digest and assimilate and is suitable for people of all ages comes into rapidly ten million.It, may be only but for most consumer
It is a kind of good nutriment to know milk, does not know the harm for having drunk adulterated milk to human body but, let alone how to differentiate
Adulterated milk.Therefore, some illegal retailers take this opportunity to make adulterated milk, at present, although enterprise has done largely
Work, sternly hits adulterated, Milk quality is made to have apparent improvement, but fresh milk adulteration is still one very universal
Problem, these behaviors have not only seriously affected the nutritional quality of fresh milk, also compromise the legitimate rights and interests of consumer.At present milk into
The discriminating divided is mainly based on sense organ method and chemical detection method.Organoleptic detection method mainly passes through the inspections such as vision, the sense of taste
Method tests to food, has many advantages, such as time saving and energy saving, efficient, but there are certain error, accuracy is poor, by subjectivity
It is affected, it is necessary to combine other detection techniques.Round pcr is used for the advantages that its specificity is good, high sensitivity at present
The detection of adulterations of dairy produce.This research pretends to be milk constituents to make without milk ingredient primarily directed to what China occurred with non-milk ingredient
Adulterated behavior establish detection dairy produce in calf-derived Cyclospora qualitative checking method.
With the improvement of the development of molecular biology and people constantly to traditional nucleic acid amplification technologies, in 2000, Japan
Doctor Notomi has invented a kind of novel, quick, nucleic acid amplification method-ring mediated isothermal amplification (Loop- of high sensitivity
mediated isothermal amplification,LAMP).Its basic principle is 4-6 special primers of design, this
Primer can be with the archaeal dna polymerase (Bst DNA polymerase) with strand-displacement activity under constant temperature (65 DEG C or so)
Keep the temperature 50min, you can realize the amplification to target sequence.LAMP technology have high specificity, be swift in response, high sensitivity is set simultaneously
Standby to require the features such as low, simultaneous reactions result can carry out naked eyes by carrying out the formation of byproduct of reaction magnesium pyrophosphate white precipitate
Visual observation is observed by fluorescent dye, and the Aerosol Pollution as caused by uncapping electrophoresis can be controlled from source, because
This is more applicable for field quick detection.
In view of this, it is special to propose the present invention.
The content of the invention
Present invention is primarily intended to obtain to differentiate the gene of calf-derived Cyclospora by bioinformatics means, and it is directed to
The a set of loop-mediated isothermal amplification (LAMP) primer group quickly differentiated for calf-derived Cyclospora of gene design, available for detection ox source property into
The loop-mediated isothermal amplification divided.
The second object of the present invention be to provide it is a kind of using ring mediated isothermal amplification differentiate calf-derived Cyclospora kit with
Detection method.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
The present invention relates to a kind of LAMP primer group of bovine, the LAMP primer group includes F3 and B3 composition outer primers
Pair and FIP and BIP composition inner primer pair；
The nucleotide sequence of described F3, B3, FIP and BIP are successively such as SEQ ID NO:Shown in 1~4.
According to an aspect of the present invention, the invention further relates to a kind of detection kit of bovine, the kit bags
Include LAMP primer group described in claim 1, indicator, archaeal dna polymerase and reaction buffer.
Detection kit of the present invention have the characteristics that with sample is few, high specificity, sensibility are high, quick and precisely, to distinguish ox
Simple and direct, effective quick diagnosis approach is provided with other animals.
According to an aspect of the present invention, ox and the method for the animal specimen of other species are distinguished the invention further relates to a kind of,
Including：
The genomic DNA of sample to be detected is extracted, the reagent added in kit as described above carries out amplified reaction；
If reaction solution is in the reaction or the color change after reaction or fluorescence signal intensity variation and the sun of the indicator
Property reaction it is consistent, then sample to be detected is the sample of ox.
The present invention searches out the specific gene of calf-derived Cyclospora using bioinformatics, and establishes a set of discriminating Niu Yuan
The loop-mediated isothermal amplification kit and detection method of property ingredient, a kind of letter is provided for the discriminating and detection of calf-derived Cyclospora
Just, fast and accurately approach.
Description of the drawings
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution of the prior art
Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in describing below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, can also be obtained according to these attached drawings other attached drawings.
Fig. 1 is species specificity result in one embodiment of the invention；
Fig. 2 is one embodiment of the invention medium sensitivity laboratory test results；
Fig. 3 is the testing result of actually detected 7 beef samples in one embodiment of the invention.
Specific embodiment
The present invention relates to a kind of LAMP primer group of bovine, the LAMP primer group includes F3 and B3 composition outer primers
Pair and FIP and BIP composition inner primer pair；
The nucleotide sequence of described F3, B3, FIP and BIP are successively such as SEQ ID NO:Shown in 1~4.
According to an aspect of the present invention, the invention further relates to a kind of detection kit of bovine, the kit bags
Include LAMP primer group described in claim 1, indicator, archaeal dna polymerase and reaction buffer.
Preferably, the detection kit of bovine as described above, the indicator be selected from SYBR Green I,
One kind in EvaGreen, PicoGreen, Peko Green, propidium iodide, calcein or hydroxynaphthol blue.
Preferably, the detection kit of bovine as described above, the archaeal dna polymerase are Bst archaeal dna polymerases.
Preferably, the detection kit of bovine as described above, the reaction buffer include following ingredient：
PH be 8.6~9.0 25mM~55mM Tris-HCl, 2.4mM~3.2mM dNTPs, 16mM~24mM KCl,
8mM~12mM (NH4)2SO4, 12mM~20mM MgSO4And 1.2M~2.0M glycine betaines；
It is furthermore preferred that the reaction buffer includes following ingredient：
PH is 8.8 40mM Tris-HCl, 2.8mM dNTPs, 20mM KCl, 10mM (NH4)2SO4、16mM MgSO4With
And 1.6M glycine betaines.
Preferably, the detection kit of bovine as described above, the kit further include sample pretreatment liquid, sun
Property control and water in one or more.
According to an aspect of the present invention, ox and the method for the animal specimen of other species are distinguished the invention further relates to a kind of,
Including：
The genomic DNA of sample to be detected is extracted, the reagent added in kit as described above carries out amplified reaction；
If reaction solution is in the reaction or the color change after reaction or fluorescence signal intensity variation and the sun of the indicator
Property reaction it is consistent, then sample to be detected is the sample of ox.
Preferably, ox and the method for the animal specimen of other species are distinguished as described above, when carrying out amplified reaction, instead
The primer concentration for answering described F3, B3, FIP and BIP in system be respectively 0.013mol/L~0.019mol/L,
0.013mol/L~0.019mol/L, 1.24mol/L~1.32mol/L, 1.24mol/L~1.32mol/L；
It is furthermore preferred that when carrying out amplified reaction, the primer concentration of described F3, B3, FIP and BIP in reaction system
Respectively 0.016mol/L, 0.016mol/L, 1.28mol/L, 1.28mol/L.
Preferably, ox and the method for the animal specimen of other species, the condition of the amplified reaction are distinguished as described above
For：
62 DEG C~68 DEG C reactions 35min~55min, 80 DEG C~90 DEG C reaction 5min~15min；
It is furthermore preferred that the condition of the amplified reaction is：
65 DEG C of reactions 45min, 85 DEG C of reaction 10min；
Preferably, ox and the method for the animal specimen of other species, the animal of other species are distinguished as described above
Including any one of pig, chicken, duck；
It is furthermore preferred that the sample to be detected includes tissue, cell, blood, saliva, sperm, dairy produce, bone or hair
Hair；
It is furthermore preferred that described be organized as musculature.
Unless otherwise defined, all technical and scientific terms for using of the present invention have with belonging to disclosed embodiment
The normally understood identical meaning of those of ordinary skill in field.It is although similar or equivalent with method of the present invention and material
Method and material can be used in the practice or test of present embodiment, but hereafter still describe suitable method and material.
All publications, patent application, patent and other bibliography that the present invention refers to are incorporated into this by quoting full content
Wen Zhong.In the case of a conflict, this specification (including definition) will play dominating role.In addition, material, method and embodiment are only
It is merely illustrative, and is not intended to be limited to.Other feature and advantage of embodiment will be from following detailed description of book and right
Become in it is required that apparent.
In order to promote to understand implementations described herein this purpose, certain embodiments will be referred to, and will be used
Language-specific describes these embodiments.Term as used herein is only used for description specific embodiment purpose, without purport
In limitation the scope of the present disclosure.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person, the condition suggested according to normal condition or manufacturer carry out.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
Embodiment 1
Test material and method
1.1 test material
Unless otherwise specified, all reagents are to analyze pure or biochemical reagents.Experimental water：It should meet one in GB/T6682
The specification of grade water.DNA extraction kit：TaKaRa.Bst archaeal dna polymerases:8U/μl.2x reaction buffers：40mM Tris-
HCl (pH 8.8), 20mM potassium chloride, 16mM magnesium sulfate, 1.6M glycine betaines, dNTPs 2.8mM each.Developing solution：Calcein
Visualize dyestuff：The bright reports of Loopamp.
1.2 design of primers
The chondriogen of calf-derived Cyclospora is compared with NCBI softwares, selects this gene order as target sequence
Row, with online software PrimerExplorer V5 (http：//primerexplorer.jp/e/e) design ring mediated isothermal
Amplimer group.Its primer sets includes a pair of of outer primer (F3, B3) and is formed with a pair of of inner primer (FIP, BIP), and primer sequence is such as
Under：
1.3 loop-mediated isothermal amplification
The total volume of loop-mediated isothermal amplification system is 25ul, including loop-mediated isothermal amplification (LAMP) primer group solution
7.2ul, loop-mediated isothermal amplification buffer solution 14.8ul, Bst archaeal dna polymerase 1ul, sample to be tested, the positive or feminine gender refer to
Each 2ul of control product, developing solution calcein 2ul；
1.4 ring mediated isothermal amplification programs are：1. 65 DEG C, 45min, 2. 85 DEG C, 10min.React on PCR instrument carry out or
Water-bath is completed on constant-temperature metal bath.In reaction process, if there is target DNA amplification just has chromogenic reaction, at any time may be used
To observe color change, orange for negative reaction, green is positive reaction.
2 result of the tests
The specificity experiments of 2.1 primer mixed liquors
It respectively using ox, pig, chicken, duck gene as DNA profiling, is tested, is examined with loop-mediated isothermal amplification (LAMP) primer mixed liquor
The specificity of primer is surveyed, the results are shown in Figure 1.1 is negative charging, and 2 accuse to be positive, and 3 be pig gene, and 4 be chicken gene, and 5 are
Duck gene.
2.2 sensitivity technique
After positive DNA profiling dilution, often the final concentration of 10ng-10fg of pipe, amplification are shown in Fig. 2, show this method spirit
Sensitivity is 1pg.1 is negative charging, 2-8 10ng, 1ng, 0.1ng, 10pg, 1pg, 100fg, 1fg.
2.3 actual samples detect
It is tested using different samples, is respectively 1,2,3,4,5,6, No. 7 sample, is tested with this primer sets,
The results are shown in Figure 3.The results show that there is a kind of product inspection to be caused not measure without calf-derived Cyclospora or content are too low.1 refers to for feminine gender
Control, 2-8 are 7 kinds of different products.
Embodiment 2
Test material and method
1.1 test material
Unless otherwise specified, all reagents are to analyze pure or biochemical reagents.Experimental water：It should meet one in GB/T6682
The specification of grade water.DNA extraction kit：TaKaRa.Bst archaeal dna polymerases:8U/μl.2x reaction buffers：30mM Tris-
HCl (pH8.6), 24mM potassium chloride, 12mM magnesium sulfate, 1.4M glycine betaines, dNTPs 2.6mM each.Developing solution：240 μM of hydroxyls
Naphthol blue.
1.2 the same as embodiment 1；
1.3 loop-mediated isothermal amplification
The total volume of loop-mediated isothermal amplification system is 25ul, including loop-mediated isothermal amplification (LAMP) primer group solution
7.2ul, loop-mediated isothermal amplification buffer solution 14.8ul, Bst archaeal dna polymerase 1ul, sample to be tested, the positive or feminine gender refer to
Each 2ul of control product, developing solution hydroxynaphthol blue 2ul；
1.4 ring mediated isothermal amplification programs are：1. 60 DEG C, 55min, 2. 90 DEG C, 5min.React on PCR instrument carry out or
Water-bath is completed on constant-temperature metal bath.In reaction process, if there is target DNA amplification just has chromogenic reaction, at any time may be used
To observe color change, lilac is negative reaction, and sky blue is positive reaction.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe is described in detail the present invention with reference to foregoing embodiments, but it will be understood by those of ordinary skill in the art that：Its
It can still modify to the technical solution recorded in foregoing embodiments either to which part or all technical characteristic
Carry out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is not made to depart from various embodiments of the present invention skill
The scope of art scheme.
SEQUENCE LISTING
<110>Institute of Quality Standards and Testing Technology for Agri-Products, Chinese
<120>LAMP primer group, detection kit and its application of bovine
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
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ctcacaggcc tattcctag 19
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<211> 23
<212> DNA
<213>Artificial sequence
<400> 2
gtgtaagacc cataatataa gcc 23
<210> 3
<211> 42
<212> DNA
<213>Artificial sequence
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tctcggcaga tatgggtaac acaatacact acacatccga ca 42
<210> 4
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<212> DNA
<213>Artificial sequence
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cgtgaactac ggctgaatca tctcctacgt gcatatataa gcag 44
Claims (10)
1. the LAMP primer group of a kind of bovine, which is characterized in that the LAMP primer group includes F3 and B3 composition outer primers
Pair and FIP and BIP composition inner primer pair；
The nucleotide sequence of described F3, B3, FIP and BIP are successively such as SEQ ID NO:Shown in 1~4.
2. a kind of detection kit of bovine, which is characterized in that the kit is drawn including LAMP described in claim 1
Object group, indicator, archaeal dna polymerase and reaction buffer.
3. kit according to claim 2, which is characterized in that the indicator be selected from SYBR Green I,
One kind in EvaGreen, PicoGreen, Peko Green, propidium iodide, calcein or hydroxynaphthol blue.
4. the detection kit of bovine according to claim 2, which is characterized in that the archaeal dna polymerase is Bst
Archaeal dna polymerase.
5. the detection kit of bovine according to claim 2, which is characterized in that the reaction buffer is included such as
Lower ingredient：
PH be 8.6~9.0 25mM~55mM Tris-HCl, 2.4mM~3.2mM dNTPs, 16mM~24mM KCl, 8mM~
12mM(NH4)2SO4, 12mM~20mM MgSO4And 1.2M~2.0M glycine betaines.
6. the detection kit of bovine according to claim 2, which is characterized in that the kit further includes sample
One or more in pretreatment fluid, positive control and water.
7. a kind of distinguish ox and the method for the animal specimen of other species, which is characterized in that including：
The genomic DNA of sample to be detected is extracted, the reagent added in claim 2~6 any one of them kit carries out
Amplified reaction；
If reaction solution in the reaction or the color change after reaction or fluorescence signal intensity variation and the positive of the indicator it is anti-
Should be consistent, then sample to be detected is the sample of ox.
8. according to claim 7 distinguish ox and the method for the animal specimen of other species, which is characterized in that is being expanded
When increasing reaction, the primer concentration of described F3, B3, FIP and BIP in reaction system be respectively 0.013mol/L~
0.019mol/L, 0.013mol/L~0.019mol/L, 1.24mol/L~1.32mol/L, 1.24mol/L~1.32mol/L.
9. according to claim 7 distinguish ox and the method for the animal specimen of other species, which is characterized in that the amplification
The condition of reaction is：
62 DEG C~68 DEG C reactions 35min~55min, 80 DEG C~90 DEG C reaction 5min~15min.
10. the method for the animal specimen according to claim 7 for distinguishing ox and other species, which is characterized in that it is described its
The animal of his species includes any one of pig, chicken, duck；
Preferably, the sample to be detected includes tissue, cell, blood, saliva, sperm, dairy produce, bone or hair；It is more excellent
Choosing, it is described to be organized as musculature.
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| CN201810144742.XA CN108048462A (en) | 2018-02-12 | 2018-02-12 | LAMP primer group, detection kit and its application of bovine |
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| CN201810144742.XA CN108048462A (en) | 2018-02-12 | 2018-02-12 | LAMP primer group, detection kit and its application of bovine |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116024353A (en) * | 2022-09-20 | 2023-04-28 | 西藏自治区食品药品检验研究院（西藏自治区医疗器械检测中心） | Identification method of yak meat and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586436A (en) * | 2012-02-21 | 2012-07-18 | 山东省农业科学院畜牧兽医研究所 | Loop-mediated isothermal amplification (LAMP) detection method for identifying beef and mutton |
| CN105838808A (en) * | 2016-05-18 | 2016-08-10 | 珠海出入境检验检疫局检验检疫技术中心 | Primer for LAMP detection method of bovine-derived ingredients, detection method and kit |
| CN106048062A (en) * | 2016-08-10 | 2016-10-26 | 厦门出入境检验检疫局检验检疫技术中心 | LAMP primer used for detecting bovine-derived material, and designing method thereof |
-
2018
- 2018-02-12 CN CN201810144742.XA patent/CN108048462A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586436A (en) * | 2012-02-21 | 2012-07-18 | 山东省农业科学院畜牧兽医研究所 | Loop-mediated isothermal amplification (LAMP) detection method for identifying beef and mutton |
| CN105838808A (en) * | 2016-05-18 | 2016-08-10 | 珠海出入境检验检疫局检验检疫技术中心 | Primer for LAMP detection method of bovine-derived ingredients, detection method and kit |
| CN106048062A (en) * | 2016-08-10 | 2016-10-26 | 厦门出入境检验检疫局检验检疫技术中心 | LAMP primer used for detecting bovine-derived material, and designing method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116024353A (en) * | 2022-09-20 | 2023-04-28 | 西藏自治区食品药品检验研究院（西藏自治区医疗器械检测中心） | Identification method of yak meat and application thereof |
| CN116024353B (en) * | 2022-09-20 | 2023-10-24 | 西藏自治区食品药品检验研究院（西藏自治区医疗器械检测中心） | Identification method of yak meat and application thereof |
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Application publication date: 20180518 |