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

A technology of Solanum thorns and universal primers, which is applied in the direction of biochemical equipment and methods, and the determination/inspection of microorganisms, can solve the problems of changes in the impact of seed morphological characteristics and environmental conditions, achieve rapid detection, avoid false negatives, and methods reliable effect

Inactive Publication Date: 2011-06-15
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The detection of Solanum nigrum at the port is mainly based on the morphological characteristics of its seeds. However, the morphological characteristics of the seeds of many species of Solanaceae are very similar to Solanum nigrum, and the morphological characteristics of the seeds are easily affected by environmental conditions. Therefore, it is necessary to The port establishes a fast and accurate molecular detection method for Solanum solanum to protect my country's agricultural production safety and agricultural product trade safety

Method used

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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: Extraction of plant material genomic DNA

[0025] The materials involved in the experiment include 6 similar species of Solanaceae, a total of 11 materials, which are stored in the Plant Inspection Laboratory of the Animal, Plant and Food Testing Center of Tianjin Entry-Exit Inspection and Quarantine Bureau. Solanum nigrum belongs to the same family but has different genus, but their seed forms are very similar, so the present invention introduces these two experimental materials, and the others are plants of the same genus. See Table 1 for relevant information.

[0026] Table 1 Test materials

[0027]

[0028] Use sterilized tweezers to pick plant seeds, or use sterilized scissors to cut plant leaves and stems into pieces, put the plant tissue into a 0.5mL Eppendorf centrifuge tube, and use Shanghai Sangon Genomic DNA Purification Kit (No. SK1252) to extract plant genomic DNA. The extracted genomic DNA was dissolved in 70 μL of 1×TE.

Embodiment 2

[0029] Embodiment 2: the design of universal primer and specific primer

[0030] 1. Universal primers

[0031] Artificially synthesized Solanaceae universal primers, the primer sequences are as follows:

[0032] SRF2:CCAATGATTGGCACACAG

[0033] SRR2: GTCAACAGGCAAGCCAAC

[0034] 2. Artificially synthesized specific primers for the artificially synthesized Solanum solanum and other plants of the same family such as Solanum nigrum, Solanum nigrum, Baiying, tomato, and eggplant. The primer sequences are as follows:

[0035] SRSF1: CACACAGCTCTCATTCCA

[0036] SRSR1: GGCCTTCATCCAGTTGAG

Embodiment 3

[0037] Embodiment 3: the PCR amplification of universal primer

[0038] Reagents related to PCR amplification were products of TaKaRa Engineering Company.

[0039] 1. Preparation of reaction mixture

[0040] The reaction system is 20 μL: 10×Buffer 2.0 μL (which contains MgCl with a final concentration of 1.5 mmol / L 2 ), a total of 0.4 μL of four dNTPs at a concentration of 2.5 mmol / L, 0.2 μL of each primer (SRF2 / SRR2) at a concentration of 20 μmol / L, 0.2 μL Taq DNA polymerase (5 U / μL), 0.5 μL template DNA (about 40ng), add sterile water to 20 μL, and use sterile water as a template as a negative control.

[0041] 2. The PCR reaction procedure is

[0042] Pre-denaturation at 94°C for 4 minutes;

[0043] Denaturation at 94°C for 30s;

[0044] Refolding at 56°C for 20s;

[0045] 72°C extension for 45s, 35 cycles;

[0046] Extend at 72°C for 7 minutes.

[0047] 3. Result Analysis

[0048] The genomic DNA of 11 experimental materials was amplified with universal primers SRF2...

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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

technical field [0001] The invention relates to a method for detecting Solanum rostratum Dunal in plants and plant products by using PCR technology, and belongs to the field of detection of toxic and harmful weeds. Background technique [0002] Alien weeds are a class of highly evolved plant groups that have successfully colonized and continued in other regions by overcoming the spatial distribution barriers of species in certain ways. The invasion of alien weeds is a problem that needs to be highly valued and urgently resolved. Once alien weeds invade new habitats, it will cost a lot to control their spread. [0003] Solanum rostratum Dunal, also known as Solanum rostratum Dunal, is a plant of the Solanaceae (Solanaceae) genus Solanum. It originated in North America and has been grown in more than 10 countries in the United States, Canada, Mexico, Russia, South Africa, Australia, etc. Occurred in several countries and regions (Gao Fang, Xu Chi, Zhou Yunlong. Potential risk...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 刘勇廖芳杨秀丽牛春敬刘鹏
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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