CN106893773A - Identification alfalfa seed carries the kit and detection method of Semen Cuscutae seed - Google Patents
Identification alfalfa seed carries the kit and detection method of Semen Cuscutae seed Download PDFInfo
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- CN106893773A CN106893773A CN201710025422.8A CN201710025422A CN106893773A CN 106893773 A CN106893773 A CN 106893773A CN 201710025422 A CN201710025422 A CN 201710025422A CN 106893773 A CN106893773 A CN 106893773A
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- 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
Whether the method and its primer of alfalfa dodder seed are carried in alfalfa seed the invention mainly relates to identify.Specifically related to alfalfa dodder gene detecting kit and its detection method.It is a kind of to identify the primer kit that alfalfa seed carries alfalfa dodder seed, it is characterised in that：Including 2 × EcoTaq PCR SuperMix, primer C_apF (5 ' 3 ')：CGAAGATCTTCGAATACCTCCA 0.5, primer C_apR (5 ' 3 ')：ATCAATAACTGCATGCATTGCG, ddH2O.The present invention have sensitivity is high, accuracy is strong, it is fast and convenient the features such as.Prior to seeding, alfalfa dodder parasitism alfalfa has been prevented from technological layer, has drawn plant nutrient, reduces the harm of forage yield, the method has had broad application prospects in Seed Inspection, there is significant economic benefit in careless animal husbandry production.
Description
Technical field
The invention mainly relates to a kind of alfalfa seed whether carry alfalfa dodder rapid molecular detection method and
Its primer special.Kit and its detection method that specifically related to alfalfa dodder genomic DNA is detected.
Background technology
Alfalfa (Medicago sativa L.) is a kind of highly important leguminous forage, because its Herbage harvest is high, battalion
Support the stress such as abundant, quality better, drought-resistant and saline and alkaline, be not required to nitrogen fertilizer application and can fixed nitrogen improve the soil, the good characteristic such as strong adaptability, from
And plant extensively and apply in the whole world, have the good reputation of " King of Pasture ".The grown worldwide area of alfalfa is about 3500
Ten thousand hectares, up to 2,000,000 hectares of the Alfalfa Growing area of current China occupies the 5th, the world (Wang Yanhua etc., 2009).
The seed of high-quality is the important leverage of high quality forage, but alfalfa dodder is usually mixed with alfalfa seed
Seed.Alfalfa dodder belongs to the malignant weed of worldwide alfalfa grasslands, after it is colonized on clover, can draw clover and support
Point, so that the growth retardation of clover, weak, short and small by parasitic plant growing way, solid difficulty is developed, it is dead in flakes ahead of time,
The Yield and quality and its economic benefit of final influence clover.In Xinjiang of China Tacheng Prefecture, Semen Cuscutae kind in alfalfa seed
The content of son is up to 13.4%, seed production (Jiang Haiyan etc., 2005) of alfalfa dodder serious harm locality alfalfa.
And alfalfa is similar with the particle size of alfalfa dodder seed, color is close, once alfalfa dodder silk is mixed with alfalfa
Sub- seed, conventional method is difficult to identify and remove (see Fig. 1, Fig. 2), will bring irremediable damage to the production of alfalfa
Lose.
The content of the invention
A kind of identification alfalfa seed is provided and is carried it is an object of the invention to avoid the deficiencies in the prior art part
The primer of alfalfa dodder seed.Fill up whether existing detection means is difficult to carry alfalfa dodder in identification alfalfa seed
The primer that quick detection alfalfa seed carries alfalfa dodder is capable of in the blank of seed, proposition for a pair.
The purpose of the present invention two is to provide a kind of kit for identifying alfalfa seed carrying alfalfa dodder seed.
The purpose of the present invention three is to disclose a kind of detection method for identifying alfalfa seed carrying alfalfa dodder seed.
The method is time saving and energy saving, easy to operate, being capable of short period interior completion in laboratory conditions.This research has very
Strong application value, contributes to whether Seed Inspection personnel carry lucerne in quickly, accurately and efficiently detecting alfalfa
Mu Semen Cuscutae.In the popularizing planting of China's alfalfa, the technology has boundless application prospect, can be China
Huge contribution is made in the development of grass industry and animal husbandry.
To achieve the above object, the technical scheme taken of the present invention is：One kind identification alfalfa seed carries alfalfa dodder
The primer of the sub- seed of silk, it is mainly characterized by, and the primer of alfalfa dodder is：
C_apF(5′-3′)：CGAAGATCTTCGAATACCTCCA；
C_apR(5′-3′)：ATCAATAACTGCATGCATTGCG.
A kind of to identify the kit that alfalfa seed carries alfalfa dodder seed, it is mainly characterized by and includes：
Alfalfa dodder PCR detection architectures, including 2 × EcoTaq PCR SuperMix,
Primer C_apF (5 ' -3 ')：CGAAGATCTTCGAATACCTCCA；
Primer C_apR (5 ' -3 ')：ATCAATAACTGCATGCATTGCG and ddH2O。
A kind of to identify the detection method that alfalfa seed carries alfalfa dodder seed, it is mainly characterized by including such as
Lower step：
A. seed is collected, it is standby to extract seed cdna group DNA to be measured using SDS cracking process；
B. alfalfa dodder primer is utilized, performing PCR detection is entered to seed cdna group DNA to be measured.The totality of PCR detection architectures
Product is 10 μ l, and (work, production code member are given birth in Shanghai including 2 × EcoTaq PCR SuperMix：SK2082) 5 μ l, primer C_
apF(5′-3′)：CGAAGATCTTCGAATACCTCCA 0.5 μ l, primer C_apR (5 ' -3 ')：
ATCAATAACTGCATGCATTGCG 0.5 μ l, ddH2The μ l of the O 3.5 and μ l of seed DNA profiling 50ng/ μ l 0.5 to be measured.Amplification bar
Part is：(1) 94 DEG C of predegeneration 3min；(2) 94 DEG C of denaturation 30s；(3) 57 DEG C of annealing 30s；(4) 72 DEG C of extension 50s；(5) 2-4 steps
Rapid circulation 33 times；(6) 72 DEG C of extension 7min；
C. by PCR primer in the Ago-Gel of 1-2% electrophoresis, electrophoretic voltage is 135-130V, and the time is 14-
18min, takes pictures in gel imaging system：
D. if there is obvious band, then illustrate to carry alfalfa dodder in alfalfa seed；If without obvious bar
Band, then illustrate not carry alfalfa dodder in alfalfa seed.
The beneficial effects of the invention are as follows whether can fast and effectively detect carry in alfalfa seed alfalfa dodder silk
Sub- seed, can also be 1: 10000 and 1: 5000 (the secondary expansion of PCR primer in alfalfa dodder and alfalfa DNA concentration ratio
Increase) level on, detect the micro alfalfa dodder seed being mixed with alfalfa.The method has low cost, stability
By force, repetitive rate is high and the features such as simple and fast, and whether can identify carry alfalfa dodder in large batch of alfalfa seed,
For the high-quality and safety for ensureing alfalfa germplasm provides feasible foundation.
Brief description of the drawings
Fig. 1 alfalfa seeds and alfalfa dodder seed particle size similitude；
In figure：A is alfalfa seed, and B is alfalfa dodder seed, and engineer's scale is 1mm.
The photo of Fig. 2 alfalfa dodders parasitism alfalfa；
In figure：Point out alfalfa and alfalfa dodder in figure respectively with arrow.
The PCR qualification results of Fig. 3 alfalfa dodders and alfalfa DNA；
In figure：The 1-3 numberings of alfalfa dodder (C.approximata) represent 3 kinds of alfalfa dodder kinds of separate sources
Son, the 1-8 numberings of alfalfa (M.sativa) represent 8 different cultivars of alfalfa, and " M " represents the DNA of DL2000
Marker, " H2O " is represented with ddH2O is the negative control group of template.
The PCR qualification results of different proportion alfalfa dodder are mixed with Fig. 4 (a) alfalfa seeds；
The secondary PCR product qualification result of 1: 5000 ratio alfalfa dodder is mixed in Fig. 4 (b) alfalfa seeds；
The secondary PCR product qualification result of 1: 10000 ratio alfalfa dodder is mixed with Fig. 4 (c) alfalfa seeds.
In figure：1: 1,1: 5,1: 25,1: 100,1: 200,1: 500,1: 1000,1: 2000,1: 5000,1: 10000 represents
Alfalfa dodder seed and the mass ratio of alfalfa seed genomic DNA template mixing, " M " represents the DNA of DL2000
Marker, " H2O " is represented with ddH2O is the negative control group of template.
Specific embodiment
Principle of the invention and feature are described below in conjunction with embodiment, example is served only for explaining this hair
It is bright, it is not intended to limit the scope of the present invention.
Embodiment 1：It is a kind of to identify the primer that alfalfa seed carries alfalfa dodder seed, alfalfa dodder primer
For：
C_apF(5′-3′)：CGAAGATCTTCGAATACCTCCA
C_apR(5′-3′)：ATCAATAACTGCATGCATTGCG.
Embodiment 2：A kind of to identify the kit that alfalfa seed carries alfalfa dodder seed, its main feature includes
Have：
Alfalfa dodder PCR detection architectures, including 2 × EcoTaq PCR SuperMix,
Primer C_apF (5 ' -3 ')：CGAAGATCTTCGAATACCTCCA
Primer C_apR (5 ' -3 ')：ATCAATAACTGCATGCATTGCG and ddH2O。
Embodiment 3：A kind of to identify the method that alfalfa seed carries alfalfa dodder seed, its main feature is included such as
Lower step：
A. following seeds are collected, it is standby to extract seed cdna group DNA to be measured using SDS cracking process；
The alfalfa dodder of table 1. and alfalfa are participated in the experiment information
B. alfalfa dodder primer special is utilized, performing PCR detection is entered to above-mentioned seed cdna group DNA.PCR detection architectures
Cumulative volume is 10 μ l, and (work, production code member are given birth in Shanghai including 2 × EcoTaq PCR SuperMix：SK2082) 5 μ l, primer
C_apF(5′-3′)：CGAAGATCTTCGAATACCTCCA 0.5 μ l, primer C_apR (5 ' -3 ')：
ATCAATAACTGCATGCATTGCG 0.5 μ l, ddH2The μ l of the O 3.5 and μ l of above-mentioned seed DNA profiling (50ng/ μ l) 0.5.Amplification
Condition is：(1) 94 DEG C of predegeneration 3min；(2) 94 DEG C of denaturation 30s；(3) 57 DEG C of annealing 30s；(4) 72 DEG C of extension 50s；(5)2-4
Step cycle 33 times；(6) 72 DEG C of extension 7min；
C. by PCR primer in the Ago-Gel of 1-2% electrophoresis, electrophoretic voltage is 135-130V, and the time is 14-
18min, takes pictures in gel imaging system：
D. if there is obvious band, then illustrate to carry alfalfa dodder in alfalfa seed；If without band,
Illustrate not carry alfalfa dodder in alfalfa seed.
PCR primer detects that its effect is through 1% agarose gel electrophoresis：
Fig. 3 shows three kinds of different alfalfa dodder genomic DNAs of originating for the electrophoretic band of template is limpid in sight, and with
Alfalfa seed genome for template control in without electrophoretic band, with ddH2O is the negative control group of template also without electrophoresis
Band.
Embodiment 4：Whether the method for alfalfa dodder is carried in checking quick detection alfalfa seed includes following step
Suddenly：
Alfalfa dodder and alfalfa seed DNA are respectively with 1: 1,1: 5,1: 25,1: 100,1: 200,1: 500,1:
1000th, 1: 2000,1: 5000,1: 10000 ratio mixing, extracts after mixing the genomic DNA of seed and as template, with
ddH2O is compared for template, using C_apF (5 ' -3 ')：CGAAGATCTTCGAATACCTCCA, C_apR (5 ' -3 ')：
ATCAATAACTGCATGCATTGCG primers enter performing PCR amplification.PCR system and method are with embodiment 3.
PCR primer detects that its effect is through 1% agarose gel electrophoresis：
Fig. 4 display DNA profiling mixing after, alfalfa dodder than alfalfa DNA concentration be 1: 1,1: 5,1: 25,1:
100th, under 1: 200,1: 500,1: 1000,1: 2000,1: 5000 level, alfalfa dodder can go out band with ddH2O is template
Negative control group also without electrophoretic band.Alfalfa dodder than alfalfa DNA concentration be 1：10000 and 1；5000 water
Under flat, first PCR primer is detected through 1% Ago-Gel, and without band, secondary PCR product has obvious band to occur.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all it is of the invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.
Claims (3)
1. it is a kind of to identify the primer that alfalfa seed carries alfalfa dodder seed, it is characterised in that primer is：
C_apF(5′-3′)：CGAAGATCTTCGAATACCTCCA；
C_apR(5′-3′)：ATCAATAACTGCATGCATTGCG.
2. it is a kind of to identify the kit that alfalfa seed carries alfalfa dodder seed, it is characterised in that to include：
Alfalfa dodder PCR detection architectures, including 2 × EcoTaq PCR SuperMix,
Primer C_apF (5 ' -3 ')：CGAAGATCTTCGAATACCTCCA、
Primer C_apR (5 ' -3 ')：ATCAATAACTGCATGCATTGCG and ddH2O。
3. it is a kind of to identify the detection method that alfalfa seed carries alfalfa dodder seed, it is characterised in that including following step
Suddenly：
A. seed is collected, it is standby to extract seed cdna group DNA to be measured using SDS cracking process；
B. alfalfa dodder primer is utilized, performing PCR detection is entered to seed cdna group DNA to be measured；The cumulative volume of PCR detection architectures is
10 μ l, including 2 × EcoTaq PCR SuperMix 5 μ l, primer C_apF (5 ' -3 ')：
CGAAGATCTTCGAATACCTCCA 0.5 μ l, primer C_apR (5 ' -3 ')：The μ l of ATCAATAACTGCATGCATTGCG 0.5,
ddH2The μ l of O 3.5 and seed DNA profiling 50ng/ μ l0.5 μ l to be measured；Amplification condition is：(1) 94 DEG C of predegeneration 3min；(2)94℃
Denaturation 30s；(3) 57 DEG C of annealing 30s；(4) 72 DEG C of extension 50s；(5) 2-4 step cycles 33 times；(6) 72 DEG C of extension 7min；
C. by PCR primer in the Ago-Gel of 1-2% electrophoresis, electrophoretic voltage is 135-130V, and the time is 14-18min, coagulate
Taken pictures in glue imaging system：
D. if there is obvious band, then illustrate to carry alfalfa dodder in alfalfa seed；If without obvious band,
Illustrate not carry alfalfa dodder in alfalfa seed.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710025422.8A CN106893773A (en) | 2017-01-13 | 2017-01-13 | Identification alfalfa seed carries the kit and detection method of Semen Cuscutae seed |
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| CN201710025422.8A CN106893773A (en) | 2017-01-13 | 2017-01-13 | Identification alfalfa seed carries the kit and detection method of Semen Cuscutae seed |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110229929A (en) * | 2019-07-22 | 2019-09-13 | 兰州大学 | Identify specific molecular marker and its application of the alfalfa seed true and false |
| CN114231659A (en) * | 2022-01-05 | 2022-03-25 | 兰州大学 | Primer pair for detecting thistle plants in alfalfa, application and detection method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110229929A (en) * | 2019-07-22 | 2019-09-13 | 兰州大学 | Identify specific molecular marker and its application of the alfalfa seed true and false |
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| CN114231659A (en) * | 2022-01-05 | 2022-03-25 | 兰州大学 | Primer pair for detecting thistle plants in alfalfa, application and detection method thereof |
| CN114231659B (en) * | 2022-01-05 | 2023-06-23 | 兰州大学 | Primer pair for detecting thistle plants in alfalfa, application of primer pair and detection method |
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