PCR (polymerase chain reaction) detection primers for fusarium verticillioide, kit containing primers and application

A technology for detecting primers and kits, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve the problems that there are no reports of specific primers for the detection of Verticillium pseudomonas, and achieve specificity. Good performance, easy operation and high sensitivity

Inactive Publication Date: 2016-07-27
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

[0006] The translation elongation factor (TEF-1α) gene widely exists in eukaryotes, and there is ...

Method used

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  • PCR (polymerase chain reaction) detection primers for fusarium verticillioide, kit containing primers and application
  • PCR (polymerase chain reaction) detection primers for fusarium verticillioide, kit containing primers and application
  • PCR (polymerase chain reaction) detection primers for fusarium verticillioide, kit containing primers and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 is used for detecting the synthesis of the specific PCR primer of Fusarium pseudoverticillium

[0025] Download the translation elongation factor (TEF-1α) gene sequence (KC599245.1) of F.verticillioides from the FusariumCenter'sdatabase at PennState database, and use DNAMAN and other biological software to analyze the F.verticillioides and other Fusarium The TEF-1α gene sequences of spore species were compared and analyzed, and several highly specific sites were found that only existed in Verticillium spp., and a series of primers for detecting F. spp. were designed by using the software Primer5. , from which the most specific and sensitive primers Pr40 / Pf40 (Table 1) were screened out, and on this basis a PCR system for detecting Fusarium pseudoverticillium was established.

[0026] Table 1 Information of Pr40 / Pf40 primers for specific PCR detection of Verticillium pseudomonas

[0027]

[0028] The primers were synthesized by Shanghai Yingjun Biotechno...

Embodiment 2

[0031] Example 2 Utilize PCR primers Pr40 / Pf40 to detect the specificity and sensitivity analysis of Fusarium pseudoverticillium

[0032] 1.1 Sample source:

[0033] Fusarium used in this example includes Fusarium pseudoverticillium, Fusarium incognita, Fusarium asiatica, Fusarium oxysporum complex, Fusarium fujikura, Fusarium laminarum, Fusarium equisetum, Fusarium yellow and Solanum Fusarium, etc., provided by the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

[0034] 1.2 Fusarium genomic DNA extraction:

[0035]The isolates of different Fusarium species isolated from single spores were transferred to different PDA medium respectively, 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-VISspectrophotometer Q5000 ultraviolet-visible s...

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Abstract

The invention provides specific PCR (polymerase chain reaction) detection primers for fusarium verticillioide. The primers comprise Pr40 and Pf40 (represented as Seq ID No.1 and 2). On the basis of differences between the fusarium verticillioide and other fusarium strains on a TEF-1alpha gene sequence, a series of primers for detecting the fusarium verticillioide are designed, the primers Pr40/Pf40 with the highest specificity and the highest sensitivity are screened out, a PCR detection system is built on that basis, and the fusarium verticillioide can be detected rapidly and accurately from pathogenic plant tissue. A detection kit prepared with the method is simple and convenient to operate, good in specificity and high in sensitivity, can be used for detecting various forms of propagules such as mycelia, conidia and the like of the fusarium verticillioide and has great significance in the aspects of early warning of the fusarium verticillioide epidemic situation, pathogen surveillance in an epidemic area and the like.

Description

technical field [0001] The invention relates to molecular biology detection technology, in particular to PCR detection primers for Fusarium pseudoverticillium, kits containing the primers and applications. Background technique [0002] Corn (Zeamays L.) is not only an important food crop, but also an important raw material for feed and industrial alcohol. At present, my country's corn planting area and total output have ranked first in the production of cereal crops, about 10% of the total output is used as staple food, and 70% is used as feed. Therefore, the production safety and sustainable development of corn directly affect the production stability of my country's grain crops and the development safety of extended industries. [0003] Corn ear rot is a kind of fungal disease that commonly occurs in corn production areas in the world. In addition to causing direct yield loss caused by grain rot, the pathogenic bacteria in the grain can also produce a variety of mycotoxin...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6895
Inventor 段灿星周丹妮王晓鸣杨洋朱振东孙素丽武小菲
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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