CN105886619A - Kit and method for detecting claviceps purpurea - Google Patents
Kit and method for detecting claviceps purpurea Download PDFInfo
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- CN105886619A CN105886619A CN201610268387.8A CN201610268387A CN105886619A CN 105886619 A CN105886619 A CN 105886619A CN 201610268387 A CN201610268387 A CN 201610268387A CN 105886619 A CN105886619 A CN 105886619A
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- 241000221751 Claviceps purpurea Species 0.000 title abstract description 5
- 239000011535 reaction buffer Substances 0.000 claims abstract description 11
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 8
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 8
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 8
- 239000000980 acid dye Substances 0.000 claims abstract description 7
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims abstract description 5
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims abstract description 5
- 238000007397 LAMP assay Methods 0.000 claims abstract description 4
- 241001480006 Clavicipitaceae Species 0.000 claims description 28
- 238000012360 testing method Methods 0.000 claims description 14
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 9
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- 239000011536 extraction buffer Substances 0.000 claims description 7
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- 241000209140 Triticum Species 0.000 claims description 6
- 235000021307 Triticum Nutrition 0.000 claims description 6
- 235000013305 food Nutrition 0.000 claims description 6
- 239000013641 positive control Substances 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 5
- 239000000376 reactant Substances 0.000 claims description 5
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 3
- 229960003237 betaine Drugs 0.000 claims description 3
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 101150092075 FIP1 gene Proteins 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000006116 polymerization reaction Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 12
- 230000035945 sensitivity Effects 0.000 abstract description 4
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- 235000013339 cereals Nutrition 0.000 description 3
- 244000025254 Cannabis sativa Species 0.000 description 2
- 241000221760 Claviceps Species 0.000 description 2
- 241000221785 Erysiphales Species 0.000 description 2
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
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- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 240000008067 Cucumis sativus Species 0.000 description 1
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 1
- 108050009160 DNA polymerase 1 Proteins 0.000 description 1
- 206010072289 Eye colour change Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
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- 239000004459 forage Substances 0.000 description 1
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- 238000007689 inspection Methods 0.000 description 1
- 238000011901 isothermal amplification Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
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- 239000011780 sodium chloride Substances 0.000 description 1
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- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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- 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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- 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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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Abstract
The invention provides a kit for detecting claviceps purpurea. The kit comprises a primer, Bst DNA polymerase, a reaction buffer solution and nucleic acid dye. The invention further provides a LAMP method for detecting the claviceps purpurea. By the LAMP kit or method, the claviceps purpurea can be authenticated and detected fast and accurately. The kit is high in specificity, short in detection time, high in sensitivity, simple in authentication, applicable to laboratory or field detection, and the like.
Description
Technical field
The present invention relates to microorganism detection field, specifically, the present invention relates to a kind of for examining
Survey test kit and the detection method of Clavicipitaceae.
Background technology
Clavicipitaceae (Claviceps purpurea) genus claviceps section, Claviceps, be a class master
Will with grassy weed and grain cereal the fungus as parasitic host.It parasitizes the son of host
In room, the ovary infected is formed without seed but forms hard sclerotium.Sclerotium structure comparison
Densification, and outside is surrounded by one layer of horn shape shell, and thus gain the name " Ergota " (Ergots)
(Alderman,1999).Clavicipitaceae grows vigorous under the conditions of high humility, high-moisture, easily
Infected plant.The Ergota of major part Ergota pathogenic bacteria is 1-4 times of its host plant seed.By wheat
The disease that angle pathogenic bacteria and the caused host plant of sclerotium thereof are occurred is commonly referred to as " ergot " (Ergot
diseases).Ergot is the important disease of wheat and barley and grass herbage, not only makes wheat and barley
The significantly underproduction, and the alkaloid in Ergota is poisonous to people, animal, in history because eating by mistake containing wheat
The corn at angle and cause poisoning to happen occasionally, as domestic animal eats the forage grass death by poisoning of band Ergota by mistake
Dying, people takes in too much cereal foods containing Ergota and also can miscarry the even phenomena of mortality.Therefore,
Exploitation is needed to be suitable to test kit or the method that Clavicipitaceae is used for quickly detecting by field or laboratory.
The feature that molecular Biological Detection is quick with it, sensitive at present, in microorganism detection field
Gradually replace traditional Morphological Identification, become popular research direction.Ring mediated isothermal amplification skill
Art (loop-mediated isothermal amplification of DNA, LAMP) is Japanology
A kind of novel nucleic acid amplification technologies that person Notomi set up in 2000.This technology is for target
Gene 4 primers of 6 region designs, utilize the archaeal dna polymerase with strand-displacement activity to exist
Expanding target gene under constant temperature quickly, with high specificity, product is that two ends are with loop-stem structure
Dumbbell shaped DNA molecular.Compared with the PCR method of laboratory conventional sense, LAMP
Technology has the advantages such as easy and simple to handle, highly sensitive, high specificity, in food, animals and plants inspection
Test and quarantine is widely used in various Pathogen test.
Summary of the invention
It is an object of the invention to provide a kind of LAMP kit for detecting Clavicipitaceae and
LAMP method.
In order to realize the purpose of the present invention, in an aspect, the present invention provides a kind of for examining
Surveying the LAMP kit of Clavicipitaceae, it includes that primer, reaction buffer, Bst DNA are poly-
Synthase and nucleic acid dye, wherein said primer is as follows:
Outer primer F3:CGGACACTCAAGATCGACC (SEQ ID NO:1)
Outer primer B3:CGATATCGGGCCTTGTGAAT (SEQ ID NO:2)
Inner primer FIP:TGGGGTTCGTTTCGGCTTGAA-GACAAGCGTG
CTTGACCAAT(SEQ ID NO:3)
Inner primer BIP:GAGAATCTGAGGCCGGCTACTG-TCCTTCCTAT
GCCCTGCT(SEQ ID NO:4)。
Preferably, described reaction buffer is slow by 2mM dNTP, 10 × ThermoPol reaction
Rush liquid, 0.6mM glycine betaine and 6mM Mg2+Composition.
Preferably, described nucleic acid dye is 1000 × SYBR Green I.
Preferably, the test kit of the present invention farther includes DNA extraction reagent and the positive is right
According to and negative control.
Preferably, described DNA extraction reagent such as CTAB extraction buffer.
In one aspect of the method, the invention provides primer in preparation for detecting Clavicipitaceae
Application in LAMP kit.Described primer is as shown in sequence SEQ ID NOs:1-4.
In a further aspect, present invention also offers a kind of LAMP side detecting Clavicipitaceae
Method, wherein uses the test kit of the present invention, said method comprising the steps of:
(1) in testing sample, add CTAB extraction buffer, carry according to conventional CTAB method
Take DNA；
(2) in PCR pipe, LAMP reaction system is prepared, including the outer primer of 0.2 μm ol/ μ l
F3 1 μ l, the outer primer B3 1 μ l of 0.2 μm ol/ μ l, the inner primer FIP 1 μ l of 1.2 μm ol/ μ l,
The inner primer BIP 1 μ l of 1.2 μm ol/ μ l, reaction buffer 2.5 μ l, the Bst DNA polymerization of 8U/ μ l
Enzyme 1 μ l, template DNA 2 μ l, add water and be supplemented to 25 μ l, and using Clavicipitaceae genomic DNA as
Positive control, using 100mM Tris-HCl pH 8.0 and 50mM EDTA as negative control；
(3) by the PCR pipe in step (2) in 63 DEG C of isothermal reaction 60min；
(4) analysis judges product result: add 1 μ l in (3) in gained product
Nucleic acid dye SYBR Green I, if reactant liquor color is orange, represents that result is negative, food
Without Clavicipitaceae in product, if reactant liquor color is green, represent that result is positive.
The present inventor designs 4 primers for 6 regions of Clavicipitaceae sequence, sensitive
Degree is high, specificity is good.The present invention can for the LAMP kit and method detecting Clavicipitaceae
Precise Identification food, corn, the Clavicipitaceae in field, false positive rate is low, quick, efficient, and
Observe by the naked eye color change and get final product result of determination, it is not necessary to the step such as electrophoresis and ultraviolet visualization,
Low cost, simple to operate, qualification simplicity, be suitable to Basic Laboratory and Site Detection, be worth pushing away
Wide application.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.Ability
Field technique personnel it is intended that, can carry out many in the case of without departing from the spirit of the present invention
Planting amendment, these amendments will be contained in the scope of the present invention.
The preparation of the test kit of embodiment 1 present invention
1.1 reagent
Primer is synthesized by TIANGEN Biotech (Beijing) Co., Ltd.；Bst archaeal dna polymerase and
10 × ThermoPol reaction buffer is purchased from Takara；SYBR Green I is purchased from Invitrogen；
Reagent needed for remaining PCR reagent and preparation CTAB extraction buffer is purchased from Sigma.
The preparation of 1.2 test kits:
Obtain following reagent, to prepare the test kit of the present invention:
CTAB extraction buffer: prepare according to formula as below: 100mM Tris-HCl pH 8.0,
50mM EDTA, 1M NaCl, 1% (v/v) beta-mercaptoethanol, 2%CTAB, adjust pH
Value is to 7.2；
Reaction buffer: prepare according to formula as below: 2mM dNTP, 10 × ThermoPol
Reaction buffer, 0.6mM glycine betaine, 6mM MgSO4；
Primer: outer primer F3, its nucleotide sequence is as shown in SEQ ID NO:1；Outer primer B3,
Its nucleotide sequence is as shown in SEQ ID NO:2；Inner primer FIP, its nucleotide sequence such as SEQ
Shown in ID NO:3, inner primer BIP, its nucleotide sequence is as shown in SEQ ID NO:4.Its
The concentration of primers F 3 and B3 is 0.2 μm ol/ μ l at home and abroad, and the concentration of inner primer FIP and BIP is
1.2μmol/μl。
Concentration is the Bst archaeal dna polymerase of 8U/ μ l；
Nucleic acid dye: 1000 × SYBR Green I；
Positive control: Clavicipitaceae genomic DNA；
Negative control: 100mM Tris-HCl (pH 8.0) and 50mM EDTA.
Embodiment 2 Clavicipitaceae specific detection
2.1LAMP specific detection
Plant the most to be measured
Plant to be measured includes (including black from ergot plant 8 strain of Hebei Prov. Academy of Agricultural &. Forest Sciences
Wheat 3 strain, Fructus Hordei Vulgaris 5 strain), anthrax Fructus Cucumidis sativi 2 strain, powdery mildew Semen Pisi sativi 2 strain and the Fructus Hordei Vulgaris 2 of health
Strain.On ergot plant fringe, the most visible Ergota is formed.
2.1.2 sample pretreatment:
Ergot plant is cut off from basal part of stem, after the aseptic water washing of top, cuts with sterilization
Cutter is cut into segment, is placed in PDA culture medium, 25 degree of constant temperature culture, until forming bacterium colony.
With the mould layer of white on inoculating loop scraping powdery mildew Pea Plants blade, it is inoculated in PDA and puts down
On plate, streak culture.Cut anthrax cucumber leaves with sterilization shears appropriate, cultivate at PDA
Separation and Culture anthrax bacteria on base.
The mycelia of picking above-mentioned pathogenic bacteria bacterium colony is a small amount of, observes its morphology under an optical microscope special
Levy.Observe display, cultivate gained bacterium colony and all meet the colony characteristics of corresponding pathogenic bacteria.
2.1.3LAMP detection
The test kit using embodiment 1 preparation follows the steps below detection:
(1) collect the bacterium colony on PDA plate and be centrifuged, adding CTAB extraction buffer,
DNA is extracted according to conventional CTAB method；
(2) in PCR pipe, LAMP reaction system is prepared, wherein four kinds of each 1 μ l of primer, anti-
Answer buffer 2.5 μ l, the Bst archaeal dna polymerase 1 μ l of 8U/ μ l, step (1) gained template DNA
2 μ l, add water and are supplemented to 25 μ l, and using Clavicipitaceae genomic DNA as positive control, with
100mM Tris-HCl pH 8.0 and 50mM EDTA is as negative control；
(3) PCR pipe in step (2) is placed in 63 DEG C of isothermal reaction 60min；
(4) analysis judges product result: add 1 μ l in (3) in gained product
1000 × SYBR Green I, if reactant liquor color is orange, represents that result is negative, as instead
Answer liquid color for green, represent that result is positive.
2.2 testing result
The PCR pipe of 8 strain ergot plant and positive control all presents green, and Semen Pisi sativi,
The colour developing result of Fructus Cucumidis sativi, healthy Fructus Hordei Vulgaris and negative control is orange, shows that primer can be accurate
Really identify the disease plant carrying Clavicipitaceae, there is the strongest specificity.
Embodiment 3 Clavicipitaceae sensitivity technique
3.1LAMP sensitivity technique
Extracting Clavicipitaceae DNA according to the step (1) of embodiment 2, ultraviolet spectrophotometer is surveyed
Measure its OD value, and with 10 times of concentration series dilution methods be diluted to 10ng, 1ng, 100pg, 10pg,
1pg, 100fg totally 6 gradients.
LAMP reaction system and reaction condition and interpretation of result save with embodiment 2 2.1.3
Step (2)-(4).
3.2 testing result
Clavicipitaceae DNA concentration be 10ng, 1ng, 100pg, 10pg, 1pg PCR pipe in all
Present green, show that the lowest detection limit of the detection method of the present invention reaches 1pg DNA, spirit
Sensitivity is the highest.
Claims (5)
1. the LAMP kit being used for detecting Clavicipitaceae, it is characterised in thatPermissibleBag
Include specific primer group, reaction buffer, Bst archaeal dna polymerase and nucleic acid dye, Qi Zhongsuo
State primer as follows:
Outer primer F3:CGGACACTCAAGATCGACC (SEQ ID NO:1)
Outer primer B3:CGATATCGGGCCTTGTGAAT (SEQ ID NO:2)
Inner primer FIP:TGGGGTTCGTTTCGGCTTGAA-GACAAGCGTG
CTTGACCAAT(SEQ ID NO:3)
Inner primer BIP:GAGAATCTGAGGCCGGCTACTG-TCCTTCCTAT
GCCCTGCT(SEQ ID NO:4)。
LAMP kit for detecting Clavicipitaceae the most according to claim 1, its
Described in reaction buffer by 2mM dNTP, 10 × ThermoPol reaction buffer, 0.6mM
Glycine betaine and 6mM Mg2+Composition.
LAMP kit for detecting Clavicipitaceae the most according to claim 1 and 2,
It farther includes DNA extraction reagent and positive control and negative control.
LAMP kit for detecting Clavicipitaceae the most according to claim 3, its
Described in DNA extraction reagent be CTAB extraction buffer.
5. detecting a LAMP method for Clavicipitaceae, described method is for the wheat in food
Bacterium is identified at angle, and it comprises the following steps:
(1) in testing sample, add CTAB extraction buffer, carry according to conventional CTAB method
Take DNA；
(2) preparing LAMP reaction system in PCR pipe, it includes outer the drawing of 0.2 μm ol/ μ l
Thing F3 1 μ l, the outer primer B3 1 μ l of 0.2 μm ol/ μ l, the inner primer FIP 1 μ l of 1.2 μm ol/ μ l,
The inner primer BIP 1 μ l of 1.2 μm ol/ μ l, reaction buffer 2.5 μ l, the Bst DNA polymerization of 8U/ μ l
Enzyme 1 μ l, template DNA 2 μ l, add water and be supplemented to 25 μ l, and using Clavicipitaceae genomic DNA as
Positive control, using 100mM Tris-HCl pH 8.0 and 50mM EDTA as negative control,
Wherein said primer is as follows:
Outer primer F3:CGGACACTCAAGATCGACC (SEQ ID NO:1)
Outer primer B3:CGATATCGGGCCTTGTGAAT (SEQ ID NO:2)
Inner primer FIP:TGGGGTTCGTTTCGGCTTGAA-GACAAGCGTG
CTTGACCAAT(SEQ ID NO:3)
Inner primer BIP:GAGAATCTGAGGCCGGCTACTG-TCCTTCCTAT
GCCCTGCT(SEQ ID NO:4)；
(3) by the PCR pipe in step (2) in 63 DEG C of isothermal reaction 60min；
(4) analysis judges product result: add 1 μ l in (3) in gained product
Nucleic acid dye SYBR Green I, if reactant liquor color is orange, represents that result is negative, food
Without Clavicipitaceae in product, if reactant liquor color is green, represent that result is positive.
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| CN201710261299.XA CN106916896B (en) | 2016-04-27 | 2016-04-27 | A kind of kit and detection method for being used to detect ergot |
| CN201610268387.8A CN105886619B (en) | 2016-04-27 | 2016-04-27 | A kind of method for being identified the ergot in food |
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| CN110257544A (en) * | 2019-06-14 | 2019-09-20 | 宁波检验检疫科学技术研究院 | Ergot germ fluorescence quantitative PCR detection reagent and detection kit and application |
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| CN104561358A (en) * | 2015-02-02 | 2015-04-29 | 邓继红 | LAMP detection kit and detection method for dendrobium officinale-phytophthora nicotianae |
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| CN104419761A (en) * | 2013-09-10 | 2015-03-18 | 保龄宝生物股份有限公司 | Detection kit for buckwheat allergen ingredients by employing loop-mediated isothermal amplification and application method of kit |
| EP2871243B1 (en) * | 2013-11-06 | 2018-03-21 | Omya International AG | Nucleic acids and methods for detecting pathogens and beneficial microorganisms |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561358A (en) * | 2015-02-02 | 2015-04-29 | 邓继红 | LAMP detection kit and detection method for dendrobium officinale-phytophthora nicotianae |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257544A (en) * | 2019-06-14 | 2019-09-20 | 宁波检验检疫科学技术研究院 | Ergot germ fluorescence quantitative PCR detection reagent and detection kit and application |
| CN110257544B (en) * | 2019-06-14 | 2023-01-06 | 宁波检验检疫科学技术研究院 | Ergota germ fluorescent quantitative PCR detection reagent, detection kit and application |
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| CN106916896B (en) | 2018-04-13 |
| CN105886619B (en) | 2017-10-27 |
| CN106916896A (en) | 2017-07-04 |
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