Primer and detection method for real-time fluorescent quantitative PCR detection of colletotrichum gloeosporioides of hevea brasiliensis

A technology for real-time fluorescence quantification and glomus anthracnose, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of lack of monitoring means for rubber tree anthracnose pathogens, and achieve fast and quantitative Accurate and repeatable results

Pending Publication Date: 2020-08-14
HAINAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently a lack of monitoring methods for rubber tree anthracnose pathogens, and pathogen live detection technology needs to be established and applied urgently

Method used

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  • Primer and detection method for real-time fluorescent quantitative PCR detection of colletotrichum gloeosporioides of hevea brasiliensis
  • Primer and detection method for real-time fluorescent quantitative PCR detection of colletotrichum gloeosporioides of hevea brasiliensis
  • Primer and detection method for real-time fluorescent quantitative PCR detection of colletotrichum gloeosporioides of hevea brasiliensis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Cloning and sequencing of the ITS sequence of Hevea anthracnose

[0043] (1) Preparation of Hevea anthracnose DNA:

[0044] Put Hevea anthracnose bacteria on PDA medium, and culture them at 28° C. in the dark for 5-6 days. The hyphae on the surface of the plate were scraped off with a sterilized glass slide, quickly ground to powder with liquid nitrogen, and the Omega kit was used to extract DNA.

[0045] (2) Amplification of Hevea anthracnose ITS sequence:

[0046] The general primer ITS1 / ITS4 is used to amplify the ITS sequence of Hevea anthracnose by using the ITS sequence amplification of pathogenic fungi. The primer sequences are as shown in SEQ ID No.1 and SEQ ID No.2, specifically:

[0047] ITS1: 5'-TCGTAGGTGAACCTGCGG-3'

[0048] ITS4: 5'-TCCTCCGCTTATTGATATGC-3'

[0049] PCR amplification system: 50ng / μL DNA 1μL, 2× Max Master Mix 25, 2 μL each of 10 μM ITS1 / ITS4, plus sterilized ddH 2 0 to 50 μL. The PCR amplification program was pre-denaturat...

Embodiment 2

[0051] Embodiment 2: Specific detection of Hevea anthracnose primers

[0052] The characteristic of the detection of the specificity of the primers of Hevea anthracnose is that it is pre-detected by conventional PCR and then detected by fluorescent quantitative PCR. This method can reduce the material loss in the screening process of specific primers.

[0053] Materials: DNA from 5 strains of Glucospora anthracnose as positive control, 1 strain of C. acutatum, 1 strain of Oidium heveae, 1 strain of Corynesporacasslicola, The DNA of the rubber tree (Hevea brasiliensis) was subjected to conventional PCR, and the specific information of the strains is shown in Table 1.

[0054] Table 1: Strain specific information

[0055]

[0056] Note: The microorganisms used in the present invention have been recorded in the existing literature, and are also preserved in this laboratory, which can be distributed to the public for verification tests.

[0057] (1) Sample DNA preparation:

...

Embodiment 3

[0076] Example 3: Establishment of Fluorescent Quantitative PCR Standard Curve and Detection of Primer Sensitivity

[0077] (1) Construction of plasmid standards

[0078] The connection system using the ITS fragment of Hevea anthracis amplified in Example 1 and the PMD18-T vector is as follows, and the mixed connection system was incubated at 16°C for 4-6 hours.

[0079] The connection system of ITS fragment of Hevea anthracnose and PMD18-T vector: 1 μL of PMD18-T vector, 3 μL of ITS sequence, 5 μL of Solution Ⅰ, plus sterilized dd H 2 0 to 10 μL.

[0080] Colony PCR detection system: ExTaq 10 μL, 10 μM ITS1 / ITS4 1 μL each, sterilized ddH 2 O 9 μL. Colony PCR detection program: pre-denaturation at 94°C for 4 minutes, denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 42s, a total of 35 cycles, and finally extension at 72°C for 5 minutes, and storage at 12°C.

[0081] The transformation and extraction methods of recombinant plasmids refer to "Mol...

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Abstract

The invention discloses a primer and a detection method for real-time fluorescent quantitative PCR detection of colletotrichum gloeosporioides of a hevea brasiliensis. A specific primer is designed according to a ribosome ITS sequence of colletotrichum gloeosporioides of the hevea brasiliensis, quantitative analysis is performed on the colletotrichum gloeosporioides through real-time fluorescent quantitative PCR, and the change rule of DNA of the colletotrichum gloeosporioides in tissue after inoculation of leaves of the hevea brasiliensis along with time is determined. The hevea brasiliensiscolletotrichum gloeosporioides quantitative detection technology provided by the invention is high in specificity, high in sensitivity, high in repeatability, accurate in quantification, high in speedand capable of achieving a full-closed reaction, colletotrichum gloeosporioides is identified and can be accurately quantified, the thallus content of hevea brasiliensis leaves at different time points after thallus infection is obtained, and therefore, early warning and preventive measures are carried out when the disease does not appear, the loss caused by the disease is reduced to the minimum,and the method is of great significance to early diagnosis, disease prediction and comprehensive prevention and treatment of the disease.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection of plant fungi, and in particular relates to primers and a detection method for real-time fluorescent quantitative PCR detection of Hevea anthracnose. Background technique [0002] Rubber tree is a tropical forest tree with important economic value. More than 98% of the world's natural rubber comes from rubber tree. my country is the largest consumer and importer of natural rubber, and rubber tree planting is limited to hot-spot provinces such as Hainan, Yunnan, and Guangxi. Pests and diseases have always been the main reason for the decline in the rubber production capacity of rubber trees, restricting the smooth development of the rubber planting industry, and its effective prevention and control has always been a difficult problem at home and abroad. Among them, rubber tree anthracnose is one of the main reasons for the decline of rubber production in my country. Infection ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/06C12N15/11C12R1/01
CPCC12Q1/689C12Q1/686
Inventor 张宇梁晓宇杜艳楠王萌王立丰
Owner HAINAN UNIVERSITY
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