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Method for establishing number of Fusarium sp. copies in rhizosphere soil in growth period of transgenic rice by fluorescence real-time quantitative PCR (polymerase chain reaction)

A transgenic wheat, real-time quantitative technology, applied in biochemical equipment and methods, fluorescence/phosphorescence, microbial measurement/inspection, etc., can solve problems that have not been reported, and achieve simple identification methods, high amplification efficiency, and good specificity sexual effect

Inactive Publication Date: 2013-07-10
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, researchers have selected fluorescent dyes such as SYBR GreenI and Eva Green for absolute quantitative analysis of Fusarium in environmental samples. research has not yet been reported

Method used

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  • Method for establishing number of Fusarium sp. copies in rhizosphere soil in growth period of transgenic rice by fluorescence real-time quantitative PCR (polymerase chain reaction)
  • Method for establishing number of Fusarium sp. copies in rhizosphere soil in growth period of transgenic rice by fluorescence real-time quantitative PCR (polymerase chain reaction)
  • Method for establishing number of Fusarium sp. copies in rhizosphere soil in growth period of transgenic rice by fluorescence real-time quantitative PCR (polymerase chain reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Select the specific primer of Fusarium, and this primer sequence comes from Kamel A. Abd-Elsalam etc. (PCR identification of Fusarium genus based on nuclear ribosomal-DNA sequence data, African Journal of Biotechnology Vol. 2 (4), pp. 82-85 , April 2003)

[0028] SEQ ID No.2: ITS-Fu-F: 5'-CAACTCCCAAACCCCTGTGA-3';

[0029] SEQ ID No. 3: ITS-Fu-R: 5'-GCGACGATTACCAGTAACGA-3'.

[0030] In the transgenic wheat evaluation base of Jiangsu Academy of Agricultural Sciences, 64㎡ was selected as the experimental base, and 5 sampling points on the diagonal line were established in the field, and positioning marks were set during the experiment, and the soil samples collected at each point before sowing were blank. Control each sampling 0.5g at each point, sow the transgenic wheat line N12-1 in the field, and collect roots at the above-mentioned sampling soil points during the growth period of wheat at the seedling stage, turning green stage, jointing stage, filling stage, and matu...

Embodiment 2

[0038] Primer Specificity Detection

[0039] Select five kinds of pathogenic fungi commonly found in soil, Fusarium graminearum, Fusarium nivale, Fusarium axysporum, Rhizoctonia cerealis and Rhizoctonia solani Bacteria (Rhizoctonia solani) to verify the primers, the specific steps are:

[0040] (1) Extract the total DNA of the pure culture of these bacteria as a template

[0041] (2) Using the constructed plasmid DNA, the specific fragment with a size of about 400 bp obtained in Example 1 was recovered from agarose gel and connected to the pMD-19T vector (TaKaRa, Dalian) for cloning and transformation.

[0042] (3) Using wheat rhizosphere soil DNA as a positive control, Real-Time QPCR amplification was performed referring to the conditions of Example 1. The results showed that only in Fusarium graminearum, Fusarium nivale, and Fusarium oxysporum had DNA amplification products, and the other two bacteria had no corresponding amplification products. The results showed that th...

Embodiment 3

[0046] Establishment and detection analysis of fluorescent quantitative PCR system

[0047] Specific primer pair is identical with embodiment 1

[0048] (1) Preparation of Fusarium DNA template required for making standard curve

[0049] The product obtained in Example 1 was recovered by agarose gel and connected to the pMD-19T vector (TaKaRa, Dalian). After clone screening, sequencing analysis was performed. The sequencing result (SEQ ID No.1) showed that it was consistent with multiple Fusarium species in GenBank. The sequence is 100% homologous, indicating that the obtained sequence is correct, and the positive plasmid can be used to make plasmid standards. Use ultraviolet spectrophotometer to measure that this standard plasmid concentration is 4.30 * 10 10 copies / μl, the initial standard plasmid solution was serially diluted 10 times.

[0050] (2) Calculation of standard plasmid concentration

[0051] Extract the plasmids of the positive clones verified by sequencing,...

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Abstract

The invention relates to a method for establishing number of Fusarium sp. copies in rhizosphere soil in growth period of transgenic rice by fluorescence real-time quantitative PCR (polymerase chain reaction), which comprises the following steps: establishing five soil sampling points on a diagonal line and marking and locating, and collecting rhizosphere soil samples at the above soil sampling points before seeding and at the growing period; extracting total DNA from the soil samples collected at the soil sampling points, dissolving the total DNA of the samples in double distilled water, and carrying out PCR amplification of the sample DNA at each period with a Fusarium-specific universal primer pair SEQ ID No.2 and SEQ ID No.3 to obtain a specific fragment of size 418bp (base pairs); directly measuring the number of copies of the corresponding Fusarium sp. with a fluorescence quantitative PCR instrument according to the variation of fluorescence signals and a standard curve; and summarizing the number of Fusarium sp. copies in the soil samples at different periods to obtain the number of Fusarium sp. copies in rhizosphere soil in the growth period of transgenic rice.

Description

technical field [0001] The invention relates to molecular diagnosis technology of crop disease spreading bacteria and belongs to the field of biotechnology. Background technique [0002] Since transgenic crops are introduced with specific traits that people need, such as making crops resistant to insects and diseases, and improving crop yield and quality, genetically modified crops are of great significance to food production. Whether genetically modified crops pose a safety threat to environmental organisms, such as the impact of genetically modified crops on soil microorganisms, has always been concerned. However, there are few reports in this area at home and abroad. [0003] Fusarium (Fusarium. sp) is the main group of soil fungi. There are more than 50 species of fungi. Fusarium can infect the vascular system and organs of the host plant, causing symptoms such as plant wilting and root, stem, leaf and fruit rot. The main pathogen of rot. Among them, head blight in c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/06G01N21/64
Inventor 史建荣王秀宇林凡云祭芳董飞曹欢
Owner JIANGSU ACAD OF AGRI SCI
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