Ustilago esculenta typing identification method and application thereof

A type identification, smut technology, applied in biochemical equipment and methods, microbe measurement/inspection, DNA/RNA fragments, etc., can solve problems affecting sales, unsuitable for quantitative analysis, economic losses, etc., to achieve The effect of quantitative detection

Pending Publication Date: 2022-07-05
CHINA JILIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After T-type fungus Ustilago infects Zizania plants, a large number of gray-brown teliospores accumulated on the enlarged stems seriously affect the quality of Zizania, which in turn affects sales, causing certain economic losses
[0004] The traditional typing and identification of Ustilago smut is mainly based on the phenotype of the plant. However, the time when the plant phenotype can be used for identification is often in the later stage of cultivation, and economic losses cannot be recovered at this time.
The patent application with the publication number CN109680090A discloses a molecular marker for identifying the phenotypic characteristics of Zizania zizania and its application and acquisition method. It is analyzed by the method of high-resolution melting curve, and the phenotypic identification and analysis of Zizania zizania can be carried out at the seedling stage. But this method is only suitable for qualitative analysis of Ustilago smut, not for quantitative analysis
The latest research shows that a single plant may contain both T-type and MT-type Ustilago sativa, neither the method based on plant phenotype nor high-resolution melting curve can quantitatively analyze the two kinds of Ustilago sativa

Method used

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  • Ustilago esculenta typing identification method and application thereof
  • Ustilago esculenta typing identification method and application thereof
  • Ustilago esculenta typing identification method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Genome resequencing analysis and quantitative PCR of T-type wild black powder.

[0032] (1) Obtaining materials.

[0033] The upstream and downstream primers and probes were synthesized by Nanjing GenScript Biotechnology Co., Ltd.; sterile nuclease-free water, PerfectStart II Probe qPCR SuperMix were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; plant genome extraction kit was purchased from Tiangen Biotechnology Technology Co., Ltd.; wild black powder fungus strain samples were isolated, identified and preserved by Zhejiang Key Laboratory of Biometrics and Inspection and Quarantine Technology, China Jiliang University.

[0034] (2) DNA extraction, sequencing and sequence analysis

[0035] Use a plant genomic DNA extraction kit (Tiangen, DP305), and operate according to the instructions in the instructions, and finally dissolve the DNA in 50 μL of sterile water.

[0036] A total of 121 T-type and MT-type wild black powder bacteria were collected in Zhejia...

Embodiment 2

[0056] Digital PCR detection.

[0057] (1) Obtaining materials.

[0058] The upstream and downstream primers and probes were synthesized by Nanjing GenScript Biotechnology Co., Ltd.; sterile nuclease-free water, ddPCRSupermix for Probes (dUTP-free) were purchased from Bole Bioengineering Co., Ltd.; plant genome extraction kit was purchased from Tiangen Biotechnology Co., Ltd.; wild black powder fungus strain samples were isolated, identified and preserved by Zhejiang Key Laboratory of Biometrics and Inspection and Quarantine Technology of China Jiliang University.

[0059] (2) Digital PCR detection.

[0060] Take out the primers, probes and other main reagents required for digital PCR, and then return to room temperature (15-25°C), mix gently, and then centrifuge briefly for later use; Mix well and centrifuge briefly.

[0061] table 5

[0062] reagent Usage amount Final concentration ddPCR Supermix for Probes (no dUTP) 10μL 1x Forward primer (10μ...

Embodiment 3

[0076] Actual sample testing.

[0077] (1) Acquisition of materials.

[0078] The upstream and downstream primers and probes were synthesized by Nanjing GenScript Biotechnology Co., Ltd.; sterile nuclease-free water, PerfectStart II Probe qPCR SuperMix were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; plant genome extraction kit was purchased from Tiangen Biotechnology Technology Co., Ltd.; plant samples were provided by Jinhua Agricultural Science Research Institute, Hangzhou Agricultural Technology Promotion Center and other units.

[0079] (2) DNA extraction and fluorescence quantitative PCR detection.

[0080] Use a plant genomic DNA extraction kit (Tiangen, DP305), and operate according to the instructions in the instructions, and finally dissolve the DNA in 50 uL of sterile water.

[0081] Take out the main reagents such as primers and probes (sequences are shown in Table 1) required for fluorescence quantitative PCR, return to room temperature (15-25°C),...

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PUM

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Abstract

The invention discloses an Ustilago esculenta typing identification method and application thereof, and belongs to the technical field of molecular biology. The invention provides a target for typing identification of Ustilago esculenta. The target is derived from T-type Ustilago esculenta, and the nucleotide sequence of the target is as shown in SEQ ID No. 1. According to the Ustilago esculenta typing identification method provided by the invention, T-type Ustilago esculenta in a complex sample can be quantitatively detected, the detection limit is as low as 0.0088 ng / [mu] L (22.36 copies / [mu] L), and meanwhile, the quantitative detection of the T-type Ustilago esculenta in different tissues in different stages of plants is realized.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a type identification method of wild black powder and its application. Background technique [0002] Ustilago esculenta (Ustilago esculenta), belonging to the order Ustilaginales (Ustilaginales), the family Ustilaginaceae, the genus Ustilago (Pers.) Roussel, together with Ustilago Hordei, corn smut Bacteria (Ustilago maydis) have a relatively close relationship. Wild black powder fungus is a biotrophic fungus obligately parasitized on the plants of wild water. The mycelium of this fungus is perennial and exists in the underground stems of wild wild mushroom (Zizania caduciflora), which forms water bamboo in summer and autumn. In addition to being a vegetable, water jasmine can also be used medicinally, with the effects of diuretic and thirst-quenching, heat-clearing and detoxifying. [0003] The infection of wild black powder fungus is the key to the expansion of wat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2563/107C12Q2545/114C12Q2563/159
Inventor 李士玉夏文强赵丽张雅芬汤近天崔海峰叶子弘俞晓平
Owner CHINA JILIANG UNIV
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