CN101701255B - Primer for PCR identification of kidney bean and identification method - Google Patents

Abstract

The invention provides a primer for PCR identification of kidney beans and an identification method, wherein the nucleotide sequence of the kidney beans is shown as a sequence tables SEQ ID No. 1&2. The invention further provides a PCR detection method for the identification of the kidney beans, which comprises the following steps: utilizing the primer for PCR amplification by taking the total DNA of a sample as a template, and judging results according to agarose electrophoresis after reaction is finished. The primer has good specificity, high accuracy and good sensitivity, and a rapid and simple detection method is provided for identifying the species resource of the kidney beans.

Description

The PCR primers designed and the authentication method of broad bean

Technical field

The present invention relates to Measurement for Biotechnique, relate in particular to the broad bean Biological resources are detected with primer and PCR detection method.

Background technology

Vicia pulse family Vetch plant.Contain the important component calcium of regulating brain and nervous tissue, zinc, manganese, phosphatide etc. in the broad bean, and contain abundant cholelith alkali, the brain strengthening function of memory is arranged.Calcium in the broad bean helps absorption and the calcification of bone to calcium, can promote growing of skeleton.Rich in protein in the broad bean, and do not contain cholesterol, can improve food value, preventing cardiovascular disease.Vitamins C in the broad bean can delay arteriosclerosis, and the food fibre in the broad bean skin has reducing cholesterol, promotes the effect of intestinal peristalsis.In addition, the modern thinks that also broad bean also is one of anti-carcinogenic foods, and prevention intestinal cancer is had effect.Broad bean all has plantation in China various places, is important grain, dish, fertile dual-purpose type crop.Be mainly used in interplanting of paddy and wheat field and imtertilled crop and plant in the ranks, pluck blue or green tender pod and do vegetables or receive sub eating, cane turns over to press makes green manure.China is a country that the Vicia plant germplasm resource is very abundant, and many improved seeds are arranged, and main improved seeds have the blue or green lima bean in Sichuan, the white skin in Nanxiang District, Xingning, Putian etc.These kinds have become the many areas of China and have produced the kind that goes up a large amount of cultivations, have promoted the development of China's broad bean related industries widely.For preventing the loss of China's broad bean germ plasm resource, the social productive forces of China are caused loss difficult to the appraisal, should strengthen protection and research work to China's broad bean resource.

The appearance of modern biotechnology, making genetic resources more is to carry departure with the genetic material form of non-living body, make the port examination difficult more, thereby study and set up effective genetic resources departure and check authentication method and relevant capacity building just to seem very necessary.

The domestic and international at present identification for broad bean only limits on the form, does not see the research of molecular detecting method.

The present invention uses PCR method the molecular detecting method of broad bean is studied, by trnL sequences Design special primer, set up stable to broad bean, authentication method, cost are low efficiently, can judge whether have broad bean nucleic acid to exist according to the plain agar sugar electrophoresis to amplified production.

Summary of the invention

The object of the invention is to be provided for primer and the method that broad bean PCR detects.

The present invention is by on the basis of analyzing grass chloroplast(id) trnL gene orders such as broad bean and its sibling species, and the design special primer can carry out qualitative detection to broad bean quickly and accurately.

The nucleotide sequence of detection primer provided by the invention is shown in SEQ ID NO.1 and 2.

With the sample total DNA is template, and above-mentioned primer carries out pcr amplification, judges according to pcr amplification product whether sample is broad bean.Promptly detect the specific amplification that whether has 198bp in the PCR product.Specifically, can utilize agarose gel electrophoresis to detect, whether the specific band according to whether there being 198bp comes judgement sample positive.

PCR25 μ L reaction system: sample DNA 1 μ L (10-50ng), 10 * PCR buffer (Mg ²⁺Free) 2.5 μ L, 25mM MgCL ₂2 μ L, 10mM dNTPs 2 μ L, 10 μ MPrimers, 2 μ L, 5U/ μ L Taq DNA polymerase 0.1 μ L, ddH ₂O 15.4 μ L.

Reaction conditions: pre-94 ℃ of 3min of sex change, again through 94 ℃ of sex change 30sec, 66 ℃ of annealing 30sec, 72 ℃ are extended 30sec, 30 circulations, last 72 ℃ are extended 5min.

Primer of the present invention and related reagent can be assembled into test kit, use with convenient.

The present invention has set up the easy detection method that use at suitable port, promptly directly detect broad bean from Gramineae class material into and out of the border, swift to operate, the result is accurate, avoided having complied with the characteristics of current port testing with expending time in and easy defective of slipping that traditional typoiogical classification and cytology method are produced.

Description of drawings

Fig. 1 is that PCR tests the broad bean specific detection;

Among the figure, M is DNA marker DL 2,000; 1 root of Szemao crotalaria; 2 broad beans; 3 Kidney beans; 4 peas; 5 Zi Gong winter beans; 6 Jilin granules; 7 Shanxi beans 21; 8 Ji beans 12; 9 wild soybeans 236; 10 agree farming 18; 11 Japan are fine; 12 corns; 13 China spring; The contrast of CK water.

Embodiment

Following embodiment is used for further specifying of the present invention, but is not used for limiting the scope of the invention.

Embodiment 1 primer design

According to broad bean trnL sequence, utilize software and Primer 3 design primers, through multi-turns screen, the unexpected specificity of following primer of finding significantly is better than other primer, and this primer sequence is:

Forward: 5 '-TTAAATGGGCAATCCTGAGC-3 '

Oppositely: 5 '-CAATTGTGATCGAATCAAAACTAAA-3 '.

The extraction of embodiment 2 total DNA

(1) gets 0.2～0.3g plant tissue, preserve, shred and add the liquid nitrogen grind into fine powder in the rearmounted mortar, move to sterilized 1.5mL centrifuge tube, generally be added to 1/3 volume of centrifuge tube with silica gel.

(2) in centrifuge tube, add at 65 ℃ of preheatings, 500 μ L CTAB extracting solution (3% (w/v) CTAB, 100mM Tris-HCl (pH 8.0), 2M NaCl, 25mM EDTA (pH 8.0), 4%b-mercaptoethanol (v/v), and 5%PVP (w/v)) mixing places 65 ℃ of water-bath insulations 30 minutes.

(3) sample is taken out from water-bath, add and the isopyknic chloroform/primary isoamyl alcohol of extracting solution (24: 1), the abundant mixing of the centrifuge tube that turns upside down, 12000 rev/mins are centrifugal 15 minutes.

(4) draw supernatant liquor and place another sterilized 1.5mL centrifuge tube.

(5) add isopyknic chloroform/primary isoamyl alcohol (24: 1) again, repeating step (3), (4).

(6) dehydrated alcohol (-20 ℃ of precoolings) of adding equal-volume or two volumes is put upside down the centrifuge tube mixing gently, places-20 ℃ of refrigerators, places 30 minutes.

(7) 10000 rev/mins centrifugal 10 minutes, remove supernatant liquor, keep precipitation. the throw out of this moment is mainly DNA.

(8) add 400 μ L, 70% ethanol, 10000 rev/mins centrifugal 5 minutes, collecting precipitation.Place 37 ℃ or 50 ℃ of oven for drying.(9) add 100 μ L TE (perhaps aqua sterilisa) dissolving DNAs, add 2 μ L RNA enzymes (10mg/ml) again, 37 ℃ are incubated 30 minutes.

(10) 1.0% agarose electrophoresiss detect DNA concentration and quality.

The foundation of embodiment 3PCR amplification method

1, PCR reaction system

With total DNA is template, carries out the PCR reaction, sample is arranged: sample DNA 1 μ L (10-50ng), 10 * PCR buffer (Mg in the 25 μ L reaction systems ²⁺Free) 2.5 μ L, 25mMMgCL ₂2 μ L, 10mM dNTPs 2 μ L, 10 μ M Primers, 2 μ L, 5U/ μ L Taq DNApolymerase 0.1 μ L, ddH ₂O 15.4 μ L.

2, PCR reaction conditions

After sample hose put into ABI PCR instrument, following condition is set reacts: pre-94 ℃ of 3min of sex change, again through 94 ℃ of sex change 30sec, 66 ℃ of annealing 30sec, 72 ℃ are extended 30sec, 30 circulations, last 72 ℃ are extended 5min.Reaction finishes the back amplified production through the agarose gel electrophoresis result of determination.

The specificity of embodiment 4 broad bean PCR method is determined

With 13 total DNA of leguminous plants such as broad bean and its sibling specieses is template, with the water belongs with yin contrast, finishes the specific amplification band of back agarose gel electrophoresis 198bp by the PCR reaction and judges positive findings.

Test-results:

Have only the pcr amplification product of broad bean positive amplification to occur, and other nearly edge material and negative control all do not have amplified signal, the results are shown in Figure 1.

Sequence table

＜110〉China Inst. of Quarantine Inspection Sciences

＜120〉the PCR primers designed and the authentication method of broad bean

<130>KHP09113377.1

<160>2

<170>PatentIn?version?3.5

<210>1

<211>20

<212>DNA

＜213〉artificial sequence

<400>1

TTAAATGGGC?AATCCTGAGC 20

<210>2

<211>25

<212>DNA

＜213〉artificial sequence

<400>2

CAATTGTGAT?CGAATCAAAA?CTAAA 25

Claims (5)

1. a broad bean is identified and is used the PCR primer, and its nucleotides sequence is classified as:

Forward: 5 '-TTAAATGGGCAATCCTGAGC-3 '

Oppositely: 5 '-CAATTGTGATCGAATCAAAACTAAA-3 '.

2. method that detects broad bean, this method is a template with the sample total DNA, utilizes the described primer of claim 1 to carry out pcr amplification, judges according to pcr amplification product whether sample is broad bean.

3. method as claimed in claim 2 is characterized in that, utilizes agarose gel electrophoresis to detect the PCR product, and the specific amplification band that produces 198bp is then judged positive findings.

4. as claim 2 or 3 described methods, it is characterized in that the response procedures of PCR is: pre-94 ℃ of 3min of sex change, again through 94 ℃ of sex change 30sec, 54 ℃ of annealing 30sec, 72 ℃ are extended 30sec, 30 circulations, last 72 ℃ are extended 5min.

5. the test kit that contains the described primer of claim 1.

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