CN102094086A

CN102094086A - Method for detecting Solanum rostratum Dunal by PCR (Polymerase Chain Reaction) primer

Info

Publication number: CN102094086A
Application number: CN2010105670590A
Authority: CN
Inventors: 刘勇; 廖芳; 杨秀丽; 牛春敬; 刘鹏
Original assignee: Tianjin Entry Exit Inspection and Quarantine Bureau of Animals Plants and Food Inspection Center
Current assignee: Tianjin Entry Exit Inspection and Quarantine Bureau of Animals Plants and Food Inspection Center
Priority date: 2010-12-01
Filing date: 2010-12-01
Publication date: 2011-06-15
Anticipated expiration: 2030-12-01
Also published as: CN102094086B

Abstract

The invention relates to a method for detecting a Solanum rostratum Dunal in plants and plant products by the PCR (Polymerase Chain Reaction) process, belonging to the field of toxic and harmful weed detection. In the method, two sets of primers are designed and combined and comprise a Solanaceae general primer and a Solanum rostratum Dunal specific primer, wherein the general primer is used for amplifying a Solanaceae plant gene group DNA and monitors the processes for extracting and amplifying the DNA, thereby avoiding false negative for detection; and the specific primer is used for the specific detection of the Solanum rostratum Dunal gene group DNA. The invention builds a modular detection method for the Solanum rostratum Dunal, which is quick, convenient, accurate and reliable and has strong specificity, and the whole detection can be finished within one working day.

Description

A kind of method of using the PCR primer to detect thorn calyx black nightshade

Technical field

The present invention relates to use round pcr to detect the method for thorn calyx black nightshade (Solanum rostratum Dunal) in plant and the plant prod, belong to poisonous and harmful weeds detection range.

Background technology

Alien Weeds is that a class is gone beyond the species spatial distribution interference by certain approach, the phyto-group that the height of successfully growing surely, continuing in other zone is evolved.Alien Weeds invasion is that needs are paid much attention to and the urgent problem that solves, and after in a single day Alien Weeds invades new habitat, control its harm and spread and need pay great cost.

Thorn calyx black nightshade (Solanum rostratum Dunal) has another name called chrysanthemum thorn eggplant, it is Solanaceae (Solanaceae) Solanum (Solanum) plant, originate from North America, in more than 10 countries and regions such as the U.S., Canada, Mexico, Russia, South Africa, Australia (Gao Fang takes place at present, Xu Chi, Zhou Yunlong. exotic plant thorn calyx black nightshade potentially dangerous assessment and Preventing Countermeasures thereof. Beijing Normal University's journal (natural science edition), 2005,41 (4): 420-424).China found in Liaoning early than 1981, existing main ease be born in areas such as Liaoning, Jilin, Shanxi, Xinjiang, Hebei and Beijing (Yunnan, car Shanxi, Liu Quanru, Hu Bin. exotic invasive weed thorn calyx black nightshade. weeds science, 2006, (3): 58-60).Farm crop such as thorn calyx black nightshade main harm wheat, corn, cotton and soybean, the growth competitive capacity is very strong, and the growth of severe inhibition other plant destroys local species diversity.The suitable natural disposition of thorn calyx black nightshade is extremely strong, barren-resistant, arid, often grows in the pasture of wasteland, grassland, river shoal and overgraze, also can invade farmland, orchard, melon ground, roadside and garden harm.Thorn calyx black nightshade has stronger fecundity, and the general solid amount of plant reaches 10,000～20,000, and whole plant produced seed promptly forms large stretch of single excellent population next year.Seed has dormancy, can resist bad environment, make its under harsh conditions long-term keep vitality (Zhu Dianmin is etc. the investigation of injurious weed thorn calyx black nightshade for Wang Weisheng, Zheng Hongqi. Plant Quarantine, 2005,19 (4): 247-248).Thorn calyx black nightshade still is the host of colorado potato bug (Leptinotarsa decemlineata) and corium solani important disease and pests such as (PLRV), wherein colorado potato bug is as the dangerous harmful organism of a Chinese class, it is the most important and destructive quarantine pest insect of tool of world harm potatoes and other crops, it had brought huge disaster (Thomas P E once for human agriculture production, Hassan S.First report of twenty-two new hosts of potato leafroll virus.Plant Disease, 2002,86 (5): 561).Thorn calyx black nightshade is a poisonous plants, containing solanine in its leaf, carpel, berry and the root is a kind of neurotoxin, after eating by mistake, livestock can cause poisoning even dead (Bah M, Gutierrez D M, Escobedo C, et a1.Methylprotodioscin from the Mexican medical plant Solanum rostratum (Solanaceae) .Biochemical Systematics and Ecology, 2004,32:197-202).Thorn calyx black nightshade complete stool except that corolla is close by seta, can injure livestock, and influence is herded and mankind's activity.It also belongs to Solanaceae together with tomato simultaneously, sibship is nearer, these species are by the diffusion of gene flows such as pollen, seed and the heritable variation of itself, in case with nearly edge crop hybrid such as tomato, potato, will cause immeasurable potential impact to the invasion ground eubiosis.

Wei Shouhui etc. are according to the characteristics such as difficulty of bioecology characteristic, potentially dangerous and management control, thorn calyx black nightshade has been carried out risk assessment, drawing its value-at-risk is 86, belong to highly dangerous (Wei Shouhui, Zhang Chaoxian, Liu Yan, etc. Alien Weeds thorn calyx black nightshade and risk assessment thereof. Chinese agronomy circular, 2007,23 (3): 347-351).In view of the danger of thorn calyx black nightshade, it is listed in the quarantine weeds in the U.S., Canada, Russia, Ukraine and China.Therefore, should pay much attention to sting the invasion of calyx black nightshade, actively take measures to control its harm and spread, strengthen the Plant Quarantine dynamics, resist great quarantine harmful organisms invasion, protection China's agriculture production safety and Agricultural Products Trade safety.

In recent years, development along with China's economy and foreign trade, and the demand of husbandry production, overseas plants and plant product enters China in a large number, thorn calyx black nightshade is that China provides against inward quarantine weeds, be the important quarantine item of inward plant and plant prod, its risk of importing China into by the port is increasing.Thorn calyx black nightshade is mainly derived from the U.S., though China as yet not big area spread harm, it possesses the ability in Chinese wide geographic area growth and breeding fully.The port for the detection of thorn calyx black nightshade mainly according to the morphological specificity of its seed; but the seed morphology feature of Solanaceae numerous species is closely similar with thorn calyx black nightshade; and the morphological specificity of seed is subjected to environmental influence easily and changes; therefore be necessary to set up thorn calyx black nightshade molecular detecting method fast and accurately, protect China's agriculture production safety and Agricultural Products Trade safety at the port.

Summary of the invention

The present invention collects domestic 4 kinds of thorn calyx black nightshades and 5 kinds of equal allied specieses thereof of having invaded China at present and amounts to 11 experiment materials (seeing Table 1), set up fast and convenient, high specificity, stung calyx black nightshade molecular detecting method accurately and reliably, can not come in thorn calyx black nightshade and equal floral regions such as silver hair black nightshade, black nightshade, bittersweet, tomato, eggplant.This method detects fast, and method is reliable, and whole process was finished in a working days, can effectively be applicable in the port is detected.

Concrete technical scheme is as follows:

The object of the invention provides a kind of PCR method that detects thorn calyx black nightshade genomic dna, and it may further comprise the steps:

(1) extraction of vegetable material genomic dna;

(2) design Solanaceae universal primer, primer sequence is as follows:

SRF2：CCAATGATTGGCACACAG

SRR2：GTCAACAGGCAAGCCAAC

This primer is used for the pcr amplification of plant of Solanaceae genomic dna, and as the quality monitoring to amplification procedure, amplified fragments is about 750bp.

(3) design thorn calyx black nightshade is different from the special primer of equal plants such as silver hair black nightshade, black nightshade, bittersweet, tomato, eggplant, and primer sequence is as follows:

SRSF1：CACACAGCTCTCATTCCA

SRSR1：GGCCTTCATCCAGTTGAG

This primer is used to sting the PCR specific amplified of calyx black nightshade genomic dna, the about 500bp of amplified fragments.

Another object of the present invention is to detect thorn calyx black nightshade with PCR method and 2 cover primers.

The present invention designs and has synthesized 2 cover primers, comprises Solanaceae universal primer and thorn calyx black nightshade special primer.Universal primer is used for the pcr amplification of plant of Solanaceae genomic dna, and amplified fragments is about 750bp, and DNA extraction and amplification procedure are implemented monitoring, avoids the false negative that detects.Designed and stung the special primer that the calyx black nightshade is different from equal plants such as silver hair black nightshade, black nightshade, bittersweet, tomato, eggplant, be used for the specific amplified of genomic dna, the about 500bp of amplified fragments.

Compared with prior art, the invention has the beneficial effects as follows: the present invention has set up fast and convenient, high specificity, has stung calyx black nightshade molecular detecting method accurately and reliably, thorn calyx black nightshade and equal allied species differences such as silver hair black nightshade, black nightshade, bittersweet, tomato, eggplant can be come, adopt the monitoring of universal primer realization, avoided false negative experimentation.This method detects fast, and method is reliable, and whole process was finished in a working days, can effectively be applicable in the port is detected.

Below in conjunction with accompanying drawing and embodiment the present invention is described in further detail.

Description of drawings

Fig. 1: the amplification of genomic dna universal primer;

Fig. 2: the amplification of thorn calyx black nightshade genomic dna special primer.

Embodiment

Embodiment 1: the extraction of vegetable material genomic dna

The material that experiment relates to comprises that 6 kinds of allied specieses of Solanaceae amount to 11 materials, be stored in Animal-Plant and food Detecting Center, Tianjin Exit-Entery Inspection ﹠ Quarant plant quarantine laboratory, because the strong rich tomato of gingko and red holy girl's cherry tomato do not belong to together with thorn calyx black nightshade is equal, but their seed morphologies are closely similar, so the present invention introduces this two kinds of experiment materials, other is congener, and relevant information sees Table 1.

Table 1 is for the examination material

Use sterilization tweezers picking plant seed, perhaps use the sterilization scissors that plant leaf, stem are shredded, plant tissue is put into 0.5mL Eppendorf centrifuge tube, adopt Shanghai to give birth to worker's genomic dna purification kit (numbering SK1252) and extract plant genome DNA, the genomic dna of extraction is dissolved among 70 μ L, 1 * TE.

Embodiment 2: the design of universal primer and special primer

1. universal primer

Synthetic Solanaceae universal primer, primer sequence is as follows:

SRF2：CCAATGATTGGCACACAG

SRR2：GTCAACAGGCAAGCCAAC

2. synthetic thorn calyx black nightshade is different from the special primer of equal plants such as silver hair black nightshade, black nightshade, bittersweet, tomato, eggplant, and primer sequence is as follows:

SRSF1：CACACAGCTCTCATTCCA

SRSR1：GGCCTTCATCCAGTTGAG

Embodiment 3: the pcr amplification of universal primer

The pcr amplification related reagent is precious biological (TaKaRa) engineering corporation in Dalian product.

1. the preparation of reaction mixture

Reaction system is that 20 μ L:10 * Buffer, 2.0 μ L (wherein contain the MgCl that final concentration is 1.5mmol/L ₂), concentration respectively is four kinds of dNTPs totally 0.4 μ L of 2.5mmol/L, concentration is that the primer (SRF2/SRR2) of 20umol/L respectively is 0.2 μ L, 0.2 μ L Taq archaeal dna polymerase (5U/ μ L), 0.5 μ L template DNA (being about 40ng), adding sterilized water to 20 μ L, is that template is as negative control to add sterilized water.

2.PCR response procedures is

94 ℃ of pre-sex change 4min;

94 ℃ of sex change 30s;

56 ℃ of renaturation 20s;

72 ℃ are extended 45s, 35 circulations;

72 ℃ are extended 7min.

3. interpretation of result

The genomic dna of 11 experiment materials is carried out the amplification of universal primer SRF2/SRR2, is that template is as negative control to add sterilized water.Amplified production is through 1.5% agarose gel electrophoresis, EB dyeing all can be observed specific purpose size band, the fragment of universal primer amplification should be about 750bp, all genomic dnas all can obtain the purpose fragment of specific size, negative control does not have corresponding product and (sees the swimming lane 1～12 of Fig. 1, wherein 1～11 be respectively SR1, SR2, SR3, SR4, SE, SN, SL, LE1, LE2, SM1, SM2,12 negative contrasts), M is 2000bp DNA marker.

Embodiment 4: the pcr amplification of thorn calyx black nightshade special primer

1. the preparation of reaction mixture

The amplification system of special primer is with among the embodiment 31, and wherein primer changes SRSF1/SRSR1 into.

2.PCR response procedures

94 ℃ of pre-sex change 4min;

94 ℃ of sex change 30s;

58 ℃ of renaturation 20s;

72 ℃ are extended 30s, 35 circulations;

72 ℃ are extended 7min.

2.3 interpretation of result

The genomic dna of 11 experiment materials is carried out the amplification of universal primer SRSF1/SRSR1, is that template is as negative control to add sterilized water.Amplified production is through 1.5% agarose gel electrophoresis, EB dyeing all can be observed specific purpose size band, the fragment of universal primer amplification should be about 500bp, has only SR1, SR2, SR3,4 strains such as SR4 thorns calyx black nightshade shows that the purpose band of specific size (sees 1～4 swimming lane among Fig. 2,1～4 swimming lane is respectively SR1, SR2, SR3, SR4), SE, SN, SL, LE1, LE2, SM1, SM2 and negative control do not have corresponding product and (see 6～12 swimming lanes among Fig. 2,6～12 swimming lanes are respectively SE, SN, SL, LE1, LE2, SM1, SM2 and negative control), M is 2000bpDNA marker.

Claims

1. one kind is used the PCR primer to detect the method for stinging the calyx black nightshade, it is characterized in that designed primer and purposes are as follows:

(1) Solanaceae universal primer, sequence is as follows:

SRF2：CCAATGATTGGCACACAG

SRR2：GTCAACAGGCAAGCCAAC

Amplified fragments is about 750bp, is used for the amplification of plant of Solanaceae genomic dna, and DNA extraction and amplification procedure are implemented monitoring, avoids the false negative in the testing process;

(2) thorn calyx black nightshade is different from the special primer of equal plants such as silver hair black nightshade, black nightshade, bittersweet, tomato, eggplant, and sequence is as follows:

SRSF1：CACACAGCTCTCATTCCA

SRSR1：GGCCTTCATCCAGTTGAG

Amplified fragments is about 500bp, is used to sting calyx black nightshade genomic dna specific detection.

2. a method of using the PCR primer to detect thorn calyx black nightshade is characterized in that it comprises the steps:

(1) extraction of plant genome DNA;

(2) the PCR detection method of setting up Solanaceae and stinging calyx black nightshade genomic dna,

Solanaceae universal primer sequence is as follows:

SRF2：CCAATGATTGGCACACAG

SRR2：GTCAACAGGCAAGCCAAC

Thorn calyx black nightshade special primer sequence is as follows:

SRSF1：CACACAGCTCTCATTCCA

SRSR1：GGCCTTCATCCAGTTGAG。

3. a kind of method of using the PCR primer to detect thorn calyx black nightshade according to claim 1 and 2 is characterized in that, this method and the designed application of 2 cover primers aspect detection thorn calyx black nightshade.