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Specific PCR amplification primers and specific PCR detection system of fusarium graminearum and applications

A technology of Fusarium graminearum and amplification primers, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve the effects of high sensitivity, simple operation and good specificity

Pending Publication Date: 2017-06-20
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] β-tubulin (β-tubulin) gene widely exists in eukaryotes, so far there is no report on specific detection primers for Fusarium graminearum designed for this target gene

Method used

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  • Specific PCR amplification primers and specific PCR detection system of fusarium graminearum and applications
  • Specific PCR amplification primers and specific PCR detection system of fusarium graminearum and applications
  • Specific PCR amplification primers and specific PCR detection system of fusarium graminearum and applications

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1 is used for detecting the synthesis of the specific PCR primer of Fusarium graminearum

[0034]Download the β-tubulin (β-tubulin) gene sequence (KX087134.1) of Fusarium graminearum from the Fusarium Center's database at Penn State database, and use biological software such as DNAMAN to analyze the β-tubulin gene sequence (KX087134.1) of Fusarium graminearum and other Fusarium species. -tubulin gene sequences were compared and analyzed, and several highly specific sites that only existed in Fusarium graminearum were found. A series of primers for detecting Fusarium graminearum were designed by using the software Primer 5, and specificity, The most sensitive primers are Fg18TF / Fg18TR (Table 1), nested outer primers HW18TF / HW18TR (Table 2), and on this basis, a PCR reaction system for detecting Fusarium graminearum was established.

[0035] Table 1 Information of Fg18TF / Fg18TR primers for detection of Fusarium graminearum-specific PCR

[0036]

[0037] Tab...

Embodiment 2

[0046] Example 2 Utilize PCR primers Fg18TF / Fg18TR and HW18TF / HW18TR to detect the specificity and sensitivity analysis of Fusarium graminearum

[0047] 1.1 Sample source:

[0048] The pathogens used in this example include Fusarium oxysporum, Fusarium graminearum, Fusarium yellow, Fusarium solani, Fusarium laminarum, Fusarium verticillium, Fusarium rubrum, Fusarium fujikura, Pythium, Mold, Aspergillus flavus, and Trichoderma were all provided by the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

[0049] 1.2 Fusarium genomic DNA extraction:

[0050] Different Fusarium isolates isolated from single spores were transferred to different PDA medium, cultured at 25°C for 7 days, the mycelia were scraped and dried and placed in 2.0mL centrifuge tubes. DNA was extracted with the Fungal Genomic DNA Rapid Extraction Kit (Shanghai Sangong). The content and purity were double detected by agarose gel electrophoresis and UV-VIS spectrophotometer Q5000 ultraviolet-...

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Abstract

The invention provides specific PCR amplification primers and a specific PCR detection system of fusarium graminearum. The primers comprise Fg18TF and Fg18TR (Seq ID NO:1 and 2) and a pair of nested outer primers HW18TF / HW18TR (Seq ID No:3 and 4). Based on the differences of fusarium graminearum and other fusaria on the aspect of beta-tubulin gene sequences, a series of primers for detecting fusarium graminearum are designed, the primers Fg18TF and Fg18TR with the strongest specificity are selected, meanwhile, the detection sensitivity is improved by designing the pair of nested outer primers HW18TF / HW18TR, on the basis, the PCR detection system is established, and fusarium graminearum can be rapidly and accurately detected from the tissue of diseased plants and the complicated pathogenic bacteria environment in the soil. A detection kit constructed according to the method is easy and convenient to operate, good in specificity, and high in sensitivity, can detect propagules in various forms of fusarium graminearum, such as hyphae and conidia, and has the great significance in the aspects of early warning for fusarium graminearum epidemic situation and pathogen surveillance of epidemic areas.

Description

technical field [0001] The invention relates to molecular biology detection technology, in particular to a specific PCR amplification primer and detection system for Fusarium graminearum, a test kit containing the primer and application thereof. Background technique [0002] Corn is one of the most important food crops in my country. With the development of food industry and feed industry, the status of corn as the main food and feed crop is also increasing. Ear rot is an important disease in corn production. It is caused by a variety of pathogenic fungi. It not only directly causes the loss of corn yield, but also the pathogenic bacteria in the grain can also produce a variety of mycotoxins, such as trichothecenes and fumonisins. and zearalenone, etc., leading to a decline in the quality of corn, bringing major safety hazards to food and feed, and threatening the health of humans and animals. [0003] Fusarium spp. is one of the common pathogenic fungi in corn production, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6895C12Q1/6848C12Q2549/119
Inventor 段灿星葛波王晓鸣陈国康周丹妮孙素丽朱振东
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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