Method and special kit for detecting Ustilago maydis

A maize black powder fungus and kit technology are applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., and can solve the problems that cannot be used for rapid and quantitative analysis of pathogens, time-consuming, complicated operations, etc. achieve the effect of preventing the further occurrence of the disease

Active Publication Date: 2018-03-23
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have disadvantages such as complicated operation and time-consuming to a certain extent.
Furthermore, it cannot be used as a tool for rapid and quantitative analysis of pathogens

Method used

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  • Method and special kit for detecting Ustilago maydis
  • Method and special kit for detecting Ustilago maydis
  • Method and special kit for detecting Ustilago maydis

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0047] Preparation of individual samples used in the following examples:

[0048] U. maydis SG200: Activated on PDA plates, U. maydis colonies grown on PDA medium were transferred to YEPS L In liquid medium [0.4% (w / v) yeast extracts (BD, Franklin Lakes, NJ, USA), 0.4% (w / v) peptone (BD) 2% (w / v) sucrose (Fisher Sci., Pittsburgh , PA, USA)] at 28 ℃ shaking culture for propagation.

[0049] Plant sample preparation: According to the previously described method, the plants of different maize inbred lines were artificially inoculated with the mating-type compatible strain or the haploid pathogenic strain SG200, and the maize plants were collected before the typical symptoms of maize smut appeared sample.

[0050] Soil sample preparation: Teliospores were isolated from tuberculous fungal galls collected in the field. Hemocytometer adjusts the number of prepared teliospores to 10 9 spores / ml. Inoculate 1ml teliospore suspension into sterilized soil (10g), incubate at 25°C for ...

Embodiment 1

[0056] Embodiment 1, the design screening of the LAMP primer that detects Ustilago maize and the preparation of kit

[0057] 1. LAMP primer design

[0058] UmPep1 (XM_753041), UmPit2 (XM_752429) and UmSee1 (XM_011390277) genes of Ustilago zeae, using PrimerExplorer V4 software (http: / / primerexplorer.jp / e / ; EikenChemical Co., Ltd., Tokyo, Japan) Design 4 sets of primers (synthesized by Shanghai Sangon Bioengineering Co., Ltd., purified by HPLC).

[0059] The primer sequences are shown in Table 1:

[0060] Table 1

[0061]

[0062]

[0063]

[0064] 2. Screening of LAMP primer screening

[0065] U. maydis SG200 strain DNA group was used as a template, and each set of primers shown in Table 1 was used for LAMP amplification. Curvularia was used as a negative control (Negative Control, NC).

[0066] The 25 μL reaction system includes: 12.5 μL LAMP reaction buffer (Guangzhou Diao Technology Co., Ltd.) [【The final concentration of each component in the buffer is 1.6 mM...

Embodiment 2

[0076] Embodiment 2, the LAMP system optimization and application of detection maize smut

[0077] 1. Optimum LAMP system optimization and establishment of detection method

[0078] In order to explore the possibility of optimizing the LAMP reaction. After determining the primers, the concentration of Bst DNA polymerase in the system (U) (2.0, 4.0, 6.0, 8.0), the concentration ratio of internal and external primers (μM)

[0079] (0.4:0.2,0.8:0.2,1.2:0.2,1.6:0.2) and Mg 2+ Concentration (mmol / L) (5.0, 6.0, 7.0, 8.0) was carried out single-factor test, and then three-factor four-level L16 (45) orthogonal experiment (Table 2) was carried out on the basis of single-factor test to explore the relationship between each factor Interaction.

[0080] Table 2 is the LAMP reaction system optimization [L 16 (4 5 )]Orthogonal design

[0081]

[0082] LAMP was performed on a real-time fluorescent quantitative PCR instrument CFX96TM real-time PCR (Bio-Rad, Hercules, CA, USA).

[00...

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Abstract

The invention discloses a method and a special kit for detecting Ustilago maydis. The invention also provides a set of ring-mediated isothermal amplification primers for detecting the Ustilago maydis.The set of primers comprise a primer Pep-F3-2, a primer Pep-B3-2, a primer Pep-FIP(F1c+F2)-2, a primer Pep-BIP(B1c+B2)-2, a primer Pep-LoopF-2 and a primer Pep-LoopB-2. The method can be used for field sample detection, and provide a basis for the prevention and control of common diseases of corn and the resistance breeding. An LAMP detection system developed in the invention can provide effective guidance for the comprehensive prevention and control of the Ustilago maydis, avoids the further occurrence of diseases, and is also an important auxiliary means for the research work of plant-pathogen molecular interaction and disease resistance breeding.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting smut and a special kit. Background technique [0002] Maize tumor smut, caused by Ustilago maydis infection, is one of the major diseases of maize. In the main corn producing areas of my country, the average incidence of the disease is between 5% and 10%, and it can reach 20% when it occurs seriously. Control measures including crop rotation, seed coating treatment, fungicide foliar spraying and biological control cannot effectively control the occurrence of the disease. Planting resistant varieties is considered the only way to effectively prevent the disease from occurring, however, there are currently no resistant varieties or inbred lines available. Therefore, it is particularly important to establish an effective and convenient rapid detection system for Ustilago maizee for the prevention and control of the disease and for disease-resistant breeding. In...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12Q1/06C12N15/11
CPCC12Q1/6844C12Q1/6851C12Q1/6895C12Q2537/1376C12Q2531/119C12Q2563/107C12Q2545/114
Inventor 曹言勇
Owner HENAN ACAD OF AGRI SCI
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