Primer for multi-PCR (Polymerase Chain Reaction) detection aiming at fusaria and application of primer

A fusarium, multiple technology, applied in the field of molecular biology, can solve time-consuming and troublesome problems, and achieve the effect of accurate distinction

Active Publication Date: 2014-04-23
南京微测生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the different types of mycotoxins produced by different Fusarium species, and the traditional morphological detection method is time-consuming and labor-intensive, it is urgent to establish a quick and easy way to accurately distinguish these three different mycotoxins through multiplex PCR detection primers. The detection method of Fusarium lays a theoretical foundation for the population distribution and dynamic analysis of corn ear rot, and provides a scientific basis for the comprehensive management of the disease

Method used

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  • Primer for multi-PCR (Polymerase Chain Reaction) detection aiming at fusaria and application of primer
  • Primer for multi-PCR (Polymerase Chain Reaction) detection aiming at fusaria and application of primer
  • Primer for multi-PCR (Polymerase Chain Reaction) detection aiming at fusaria and application of primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The extraction of embodiment 1 DNA

[0024] Reagents involved in the examples: CTAB extract: containing 20 mM EDTA, 100 mM Tris-HCl, 1.4 M NaCl and 2% CTAB by volume.

[0025] The bacterial strain involved in the embodiment: 3 strains of Fusarium graminearum ( F. graminearum ), 3 strains of Fusarium verticillium ( F. verticillioides ), 3 strains of Fusarium laminarum ( F. proliferatum ), 1 strain of Fusarium asiatica ( F. asiaticum ), 1 rice bakanae strain ( F. fujikuroi ), 1 strain of Fusarium oxysporum ( F. oxysproum ), 1 Magnaporthe grisea strain ( Magnaporthe oryzea ), 1 Botrytis cinerea ( Botrytis cinerea ) and 1 strain of Sclerotinia sclerotiorum ( Sclerotinia sclerotiorium ). The above-mentioned strains are common plant pathogenic fungi, which can be obtained by conventional separation and purification methods, that is: take the disease-healthy junction of plant-susceptible samples, disinfect them with 70% ethanol for 5 seconds under aseptic co...

Embodiment 2

[0028] Example 2 Primer Design and Synthesis

[0029] According to the ketoacid-reductoisomerase encoded by different Fusarium ILV5 Genes and genes encoding sterol 24-C reductase ERG24B For the base difference of the gene, three sets of allele-specific PCR primers Fg-F + Fg-R, Fv-F + Fv-R and Fp-F + Fp-R were designed. The primer sequences are shown in Table 1.

[0030]

[0031] The above primers were synthesized by Shanghai Biological Engineering Co., Ltd.

Embodiment 3

[0032] Example 3 Analysis of Primer Specificity

[0033] With the DNA of 9 kinds of fungi described in embodiment 1 as template, with three groups of primers described in embodiment 2 to SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.1 and SEQ ID NO.2 and SEQ ID NO.2 and SEQ ID NO.1 ID NO.1 and SEQ ID NO.2 were amplified by PCR respectively, and amplified according to the following PCR reaction system and reaction conditions, and a negative control was set for each reaction (sterilized double-distilled water was used to replace the DNA template), and the PCR reaction The products were detected by gel electrophoresis and photographed by EB staining.

[0034] PCR reaction system: 10×PCR Buffer 2.5 μl, 1.5U Taq enzyme, dNTPs 0.2 μM, MgCl 2 2 mM, 2 μl of DNA template and 0.2 μM each of primers Fg-F and Fg-R described in Example 2 or 0.2 μM each of Fv-F and Fv-R or 0.2 μM each of Fp-F and Fp-R, plus sterilization Double distilled water to 25 μl.

[0035] The Taq enzyme and its correspo...

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Abstract

The invention provides a primer for multi-PCR (Polymerase Chain Reaction) detection aiming at fusaria and an application of the primer. The primer is formed by three groups of primer pairs, and can be used for rapidly and accurately identifying three types of fusaria comprising fusarium graminearum, fusarium verticillioides and fusarium proliferatum resulted in maize kernel rots through a multi-PCR amplification system by one step. The detection primer has the good specificity, namely the primer only can be used for detecting stripes with specific sizes in an amplifying manner from the target fusaria but cannot be used for detecting the stripes from proximal species or other pathogenic fungi; only the time of 2 hours is consumed in the whole PCR and electrophoresis process. Thus, the primer provided by the invention is used for rapidly, simply, conveniently and accurately distinguishing the three various types of fusaria resulted in the maize kernel rots.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a primer sequence for identifying Fusarium and its application. Background technique [0002] Corn is an important food crop and feed source. Ear rot is one of the most common diseases in the late growth period of corn. It occurs in all corn producing areas and is caused by a variety of pathogenic bacteria. Fusarium is the main pathogen causing corn ear rot, which not only causes huge economic losses to corn production, but also reduces the quality of corn due to the mycotoxins accumulated by Fusarium during the pathogenic process, posing a serious threat to food safety and feed safety. [0003] The population structure of Fusarium fungi that cause corn ear rot is different in corn producing areas in different climatic zones. In central-eastern Europe, the main causative agent of corn ear rot is Fusarium graminearum Fusarium graminearum , Fusarium verticillium F. verticilli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/77
CPCC12Q1/04C12Q1/686C12Q2537/143
Inventor 刘馨史建荣徐剑宏仇剑波王健
Owner 南京微测生物科技有限公司
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