CN102021241B - Primer and reagent for detecting D.ferox and application thereof - Google Patents
Primer and reagent for detecting D.ferox and application thereof Download PDFInfo
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- CN102021241B CN102021241B CN 201010293942 CN201010293942A CN102021241B CN 102021241 B CN102021241 B CN 102021241B CN 201010293942 CN201010293942 CN 201010293942 CN 201010293942 A CN201010293942 A CN 201010293942A CN 102021241 B CN102021241 B CN 102021241B
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Abstract
The invention provides a primer for detecting D.ferox. The primer comprises a nucleic acid primer DF1M shown in SEQ ID NO.1 and a nucleic acid primer DF2 shown in SEQ ID NO.2 or a complete complementary nucleic acid chain of nucleic acid primers DFIM and DF2. Nucleic acid product is obtained through augmentation by a PCR (polymerase chain reaction) technology. By utilizing the primer provided by the invention, detection can be finished within half of the working day, the detection time is shortened greatly, and the sensitivity of detection is improved obviously. A nuclear acid combination provided by the invention is used in the fields of quarantine inspection, agriculture production, plant protection, and the like, so that rapid and accurate inspection and identification can be achieved.
Description
Technical field
The present invention relates to agricultural and plant inspection Quarantine Techniques field; be specifically related to a kind of nucleic acid composition that utilizes the molecular Biological Detection technology to detect rapidly and accurately datura ferox (D.ferox); adopt the real-time fluorescence PCR detection technique, said composition is applicable in the middle of the fields such as Check and Examination of Port quarantine, agriculture production and plant protection.
Background technology
Datura (Datura L.) is plant of Solanaceae.All the daturism event can occur every year in the world, in addition, because the many kinds of Datura are widely used in pharmacology and Horticulture, therefore, the many kinds of this genus all have a large amount of cultivations all over the world with China, cause this genus worldwide extensively to distribute.In inward cargo such as soybean, the report of intercepting and capturing poisonous and injurious weed Datura seed is often arranged, and Seed Identification is obscured easily.In the present document at home and abroad there be the botanical name of the normal Datura that occurs: datura ferox (D.ferox), datura quercifolia (D.quercifolia), thorn apple (D.stramonium), flower of Maikoa (D.arborea), beautiful thorn apple (D.candida), wetland thorn apple (D.ceratocaula), heterochromatic thorn apple (D.discolor), stingless thorn apple (D.inermis), datura innoxia (D.inoxia), datura alba Nees (D.leichhardtii), Hindu Datura Flower Hairy Datura Flower (D.metel), datura (D.tatula), cypress breathe out ground thorn apple (D.bernhardii), datura sanguineas (D.sanguinea) etc., wherein the different name of datura ferox is D.quercifolia.
In the real work, the external appearance characteristic of the thorn apple weed seed that is mingled with in the agricultural-food tends to be worn and be out of shape because of transportation, simultaneously owing to environment, weather, the impact of cultivation condition, the individual difference that does not cause on an equal basis of kernel maturing, more identification of morphology has increased difficulty, and the cycle of the needs such as cell biology method is long and be difficult to precise Identification.
At present, cytology research shows that the satellite of thorn apple (D.stramonium) and datura ferox (D.ferox) and bivalent shape and number are the most approaching; Biochemically studies show that the isozyme of thorn apple (D.stramonium) and datura ferox (D.ferox) is the most approximate; At molecular level, utilize the similarity distance of AFLP scientific discovery thorn apple (D.stramonium) and datura ferox (D.ferox) nearest, be classified as same group evolutionary tree.Up to now, not yet there is the achievement in research of molecular level to can be used for identifying datura ferox (D.ferox).
Summary of the invention
One object of the present invention is to overcome in the above-mentioned prior art difficulty that exists about identification of morphology and cell biology method, propose to adopt molecular biological method, to the detection of rrna the Internal Transcribed Spacer (Intemal Transcribed Spacers, ITS) sequence difference design datura ferox (D.ferox) with primer pair.
Another object of the present invention is, sets up the PCR detection method of datura ferox (D.ferox), and nucleic acid composition in the middle of the fields such as Check and Examination of Port quarantine, agriculture production and plant protection, is identified to reach to detect fast and accurately.
The primer of detection datura ferox provided by the invention (D.ferox) is to being based on datura ferox (D.ferox) and its allied species difference design on the rDNA internal transcribed spacer sequence.The Internal Transcribed Spacer ITS of 18S～26S Nuclear ribosomal DNA (nrDNA) is divided into ITSl and ITS2 two portions.The ITS district highly repeats.All nrDNA repeating unit comprises that 40,000 copies repeat (tandemrepeats) mode with series connection and appear at (Hamby R K on one or more chromogenes site, Zimer E A.Ribosomal RNA as a phylogenetic tool in plant systematics of plants[M] .Chapman and Hall, New York, NY.1992,50～91.).In angiosperm, the ITS district had not only had the variability of nucleotide sequence but also conservative property on the length had been arranged, the sequence that these transcribed spacers are described is easy to sort between Relatives, and abundant variation can solve botanical system growth problem at (as: between genus and between planting) on the lower taxonomic category.Qu Lianggu and Chen Yueqin (1999) (biomolecules taxa key---principle and method, Zhongshan University's journal (natural science edition) [J] .1999,38 (1): 1-6) by relatively drawing ITS sequence (from the biology database) of different biological groups: the difference between species value of the ITS sequence that the most of sections of angiosperm belong to is 1.2%～10.2%, difference value is 9.6%～28.8% between genus, and this all is more suitable scope concerning phylogeny research.
A kind of primer that detects datura ferox provided by the invention pair comprises nucleic acid primer DF1M and nucleic acid primer DF2.
Nucleic acid primer DF1M, i.e. upstream primer, its 5 ' end to 3 ' terminal sequence is: TTAAGACGTGCGCGGGCT.
Nucleic acid primer DF2, i.e. upstream primer, its 5 ' end to 3 ' terminal sequence is: ATGCGTGACGCCCAGGCAGA.
Can know by inference according to the nucleotide base pairing rules, the one group of nucleic acid composition that forms with the nucleic acid chains of nucleic acid primer DF1M provided by the invention and nucleic acid primer DF2 nucleotide sequence complete complementary also is applicable in the middle of the present invention.
After extracting total DNA of target sample, utilization PCR detection technique just can be finished the amplification to target sample, and again through gel electrophoresis and dyeing, binding molecule amount mark can judge whether target sample is datura ferox (D.ferox).
The present invention is after extracting single seeded DNA, under the cooperation of the conventional reagent of various PCR, utilize nucleic acid primer DF1M/DF2 to carry out pcr amplification, again after separating (as: gel electrophoresis) and colour developing (as: ethidium bromide staining), positive can obtain the nucleic acid product of total length 357bp, as: by comparing with known molecular amount mark, on gel the 357bp electrophoretic band can appear; Negative sample behind the pcr amplification and blank do not have electrophoretic band to present in the corresponding position, can distinguish thus the sample of datura ferox (D.ferox) and other Datura.
Traditional authentication method is to utilize morphological characteristics of seeds, cell biological characteristics etc.Mode of appearance is identified as the surface that can distinguish easily different plants kindred plant is not classified and becomes one of important means of port quarantine weeds.But, owing in the agricultural-food of import because of come off, the reason such as drying, accumulating, loading and unloading, the external appearance characteristic of the weed seed that is mingled with tends to so is worn and modification, owing to environment, weather, the impact of cultivation condition, the individual difference that does not cause on an equal basis of kernel maturing also are unavoidable, so just identify to appearance and brought difficulty simultaneously.And cell biology method etc. not only need cycle of growing, and only still are difficult to identify according to chromosomal form.
The approximate kind of datura ferox (D.ferox) is more, as: thorn apple (D.stramonium), flower of Maikoa (D.arborea), beautiful thorn apple (D.candida), wetland thorn apple (D.ceratocaula), heterochromatic thorn apple (D.disColor), stingless thorn apple (D.inermis), datura innoxia (D.inoxia), datura alba Nees (D.leichhardtii), Hindu Datura Flower Hairy Datura Flower (D.metel), datura (D.tatula), cypress breathes out ground thorn apple (D.bernhardii), datura sanguinea (D.sanguinea) etc.The seed of these plants is very similar to datura ferox (D.ferox), but has nothing in common with each other in its hazardness and quarantine status.The present invention adopts the primer pair of nucleic acid primer DF1M and nucleic acid primer DF2 and complementary nucleic acid chain formation thereof; after adopting the round pcr amplification; separate the nucleic acid molecule that obtains 357bp; realization is distinguished the specificity of datura ferox (D.ferox), is conducive to the fields such as Check and Examination of Port quarantine, agriculture production and plant protection the fast and accurately detection of target sample is identified.
Description of drawings
In order to be illustrated more clearly in the technical scheme of the embodiment of the invention, the accompanying drawing of required use was done to introduce simply during the below will describe embodiment.
To be nucleic acid primer DF1M/DF2 carry out obtaining gel electrophoresis images behind the pcr amplification to the DNA of datura ferox (D.ferox) and approximate kind thereof Fig. 1; Among the figure, M represents standard molecule mark DL2000, each band represents respectively that from top to bottom length is the nucleic acid molecule of 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp, numeral 1-10 is datura ferox (D.ferox) amplified production of different sources, numeral 11-22 is the amplified production of the approximate kind of datura ferox (D.ferox), and numeral 23 is blank.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the invention, the technical scheme in the embodiment of the invention is clearly and completely described.
1. sample DNA extracts
Single seed grinds, and extracts according to following steps:
1) changes in the centrifuge tube behind the porphyrize in the liquid nitrogen, add 500～800 μ L TES[100mM Tris (pH8.0), 10mM EDTA, 2%SDS]; Add 50～100 μ g protein kinase K mixings, place 55 ℃～60 ℃ water-bath 60～90min, during mixing is several times gently;
2) regulate salt concn, add 10% cetyltriethylammonium bromide (CTAB) of 1/10 volume, place 65 ℃ of water-bath 10～20min;
3) add isopyknic SEVGA (chloroform: primary isoamyl alcohol=24: 1) mixing, hatch 30min for 0 ℃;
4) centrifugal, get supernatant, add 3～6 μ L RNase A, 37 ℃ of water-bath 30min remove RNA;
6) add pre-cold isopropanol, place 15～30min for-20 ℃;
7) centrifugal, abandon Virahol, 70% ethanol is washed 2 times.Drying at room temperature, 50～200 μ L ddH
2The O dissolving.
2.PCR amplification
Utilize nucleic acid primer DF1M (SEQ ID No 1), nucleic acid primer DF2 (SEQ ID No 2) carries out pcr amplification to the sample DNA that extracts.
PCR reaction cumulative volume is 20 μ L, comprise: ddH2O 11.4 μ L, PCR damping fluid (containing Mg2+) 2 μ L, dNTP (2.5mmol/L) 3.2 μ L, rTaq archaeal dna polymerase (2.5U/mL) 0.4 μ l, above-mentioned nucleic acid primer (20 μ mol/L) 2 μ l, template DNA l μ L (≈ 50ng).Response procedures is: 95 ℃ of denaturation 3min; 94 ℃ of sex change 30s, 68 ℃ of annealing 30s, 72 ℃ are extended 1min s, 30 circulations, then 72 ℃ are extended 6min again, last 4 ℃ of preservations.
3. gel electrophoresis
Get molecular weight marker (Marker) and amplified production 10 μ L, add 3 μ L sample-loading buffers (6 * sample-loading buffer), mixing, 1.5% agarose gel electrophoresis.Electrophoresis, imaging in the gel imaging instrument after ethidium bromide (EB) dyeing.
Adopt above-mentioned identical detecting step, the datura ferox (D.ferox) that comes from different areas and the sample (seeing Table 1) of approximate kind thereof are detected, detected result sees Table 2.
Fig. 1 is seen in the gel electrophoresis of amplified production, and digital 1-22 is corresponding one by one with the numeral in the table 1 among the figure, and the product of gel electrophoresis 1-10 is carried out nucleic acid sequencing, determines that its length is 357bp.
Table 1PCR amplification material therefor and source thereof
Table 2PCR detects light Chinese sorghum and allied species result thereof
"+" expression: the positive, the amplified production gel electrophoresis has the 357bp band;
"-" expression: feminine gender, the amplified production gel electrophoresis does not present the 357bp band.
Detected result shows, detected result conforms to fully with experiment material, the amplified production of datura ferox (D.ferox) (positive) sample, obtain specific band about 357bp in gel electrophoresis, datura ferox (D.ferox) allied species (feminine gender) sample and blank do not obtain specific band about 357bp in gel electrophoresis.
This shows that nucleic acid primer DF1M and nucleic acid primer DF2 that the present invention adopts can finish detection by round pcr within half working days, can greatly shorten detection time.The present invention just can determine that the sensitivity of detection improves greatly by single seeded detection.Traditional conventional sense method can not be distinguished form wearing and tearing and the inadequate seed of ripening degree.On nucleic acid primer DF1M of the present invention and nucleic acid primer DF2 basis, the PCR detection method that adopts various existing PCR reagent to set up can special sensitivity be identified datura ferox (D.ferox) and allied species thereof quickly and accurately.
More than by embodiment the present invention is had been described in detail, but these are not to be construed as limiting the invention.In the situation that does not break away from the principle of the invention, those skilled in the art also can make many distortion and improvement, and these changes also should be considered as protection scope of the present invention.
Claims (4)
1. a primer that detects datura ferox pair is characterized in that:
The nucleic acid primer that is respectively SEQ ID No 1 and SEQ ID No 2 by sequence forms.
2. the primer of a detection datura ferox claimed in claim 1 pair is characterized in that described primer is to obtaining the nucleic acid product of 357bp after datura ferox DNA is carried out pcr amplification.
3. the primer of a detection datura ferox claimed in claim 1 pair is characterized in that described primer is to being used for judging the datura ferox of target sample.
4. reagent that detects datura ferox is characterized in that comprising primer claimed in claim 1 pair.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101223862A (en) * | 2008-02-20 | 2008-07-23 | 西南大学 | Method of regenerative plants from hairy root of woody mandala having hyoscyamine and scopolamine |
| CN101230349A (en) * | 2008-02-20 | 2008-07-30 | 西南大学 | Method for gaining woody datura stramonium hairy root generating hyoscyamine and hyoscine |
| CN101988126A (en) * | 2010-09-28 | 2011-03-23 | 中华人民共和国上海出入境检验检疫局 | Nucleic acid composition for detecting Datura ferox and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101223862A (en) * | 2008-02-20 | 2008-07-23 | 西南大学 | Method of regenerative plants from hairy root of woody mandala having hyoscyamine and scopolamine |
| CN101230349A (en) * | 2008-02-20 | 2008-07-30 | 西南大学 | Method for gaining woody datura stramonium hairy root generating hyoscyamine and hyoscine |
| CN101988126A (en) * | 2010-09-28 | 2011-03-23 | 中华人民共和国上海出入境检验检疫局 | Nucleic acid composition for detecting Datura ferox and application thereof |
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