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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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

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

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0024] Example 1: Extraction of genomic DNA from plant materials

[0025] The materials involved in the experiment include 6 similar species of Solanaceae, a total of 11 materials, which are stored in the phytosanitary laboratory of the Animal, Plant and Food Inspection Center of Tianjin Entry-Exit Inspection and Quarantine Bureau. Ginkgo qiangfeng tomato and red sage cherry tomato and thorny calyx Solanum has different genera in the same family, but their seed morphology is very similar. Therefore, the present invention introduces these two kinds of 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 sterile tweezers to pick plant seeds, or use sterile scissors to cut plant leaves and stems, put the plant tissues in a 0.5mL Eppendorf centrifuge tube, and use Shanghai Shenggong Genomic DNA Purification Kit (No. SK1252) to extract plant genomic DNA. The extracted genomic DNA w...

Example Embodiment

[0029] Example 2: Design of universal primers and specific primers

[0030] 1. Universal primer

[0031] Artificially synthesized Solanaceae universal primer, the primer sequence is as follows:

[0032] SRF2: CCAATGATTGGCACACAG

[0033] SRR2: GTCAACAGGCAAGCCAAC

[0034] 2. Artificially synthesized special primers for Solanum vulgaris, Solanum vulgare, Solanum vulgare, Tomato, Eggplant, etc. The primer sequences are as follows:

[0035] SRSF1: CACACAGCTCTCATTCCA

[0036] SRSR1: GGCCTTCATCCAGTTGAG

Example Embodiment

[0037] Example 3: PCR amplification of universal primers

[0038] Reagents for PCR amplification are the products of TaKaRa Engineering Company.

[0039] 1. Preparation of reaction mixture

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

[0041] 2. The PCR reaction program is

[0042] 94℃ pre-denaturation for 4min;

[0043] Denaturation at 94℃ for 30s;

[0044] Refolding at 56℃ for 20s;

[0045] Extension at 72℃ 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 by the universal primer SRF2 / SRR2, and sterilized water was added as a templat...

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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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