PCR primer and quantitative detection method for rapidly detecting strawberry colletotrichum gloeosporioides

A technology for the detection of glyospora anthracnose bacteria, applied in biochemical equipment and methods, microbe measurement/inspection, DNA/RNA fragments, etc., can solve problems such as high information error rate, difficulty in detection, and decline in real quality, and achieve High amplification efficiency, good specificity, time-saving and cost-saving effects

Inactive Publication Date: 2020-11-06
SHANGHAI ACAD OF AGRI SCI
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Problems solved by technology

Therefore, seedling quarantine is essential in the process of strawberry planting. However, due to the characteristics of latent infection of strawberry anthracnose, the infected seedlings do not show symptoms, which brings difficulties to detection.
[0005] The traditional identification method of anthrax requires the isolation and cultivation of pathogenic bacteria, and further judgment based on its culture characteristics and micro-morphological structure, which is time-consuming and labor-intensive; and, based on the marker genes ITS, β-tubulin, ACT, CAL, CHS, GAPDH The PCR-specific detection such as ect. has the characteristics of high information error rate and the inability to distinguish the relationship within the species. Although the phylogenetic analysis based on this multi-gene joint tree building is recognized as the standard operation for the identification of anthrax bacteria, due to its complexity and consumption It takes a long time and cannot be applied to rapid detection in the field
[0006] At present, the most important means of prevention and control of the disease is still the spraying of chemical pesticides. With the increase of the amount of pesticides, a series of problems such as the decline of fruit quality, environmental pollution, and the emergence of pathogen resistance have appeared.

Method used

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  • PCR primer and quantitative detection method for rapidly detecting strawberry colletotrichum gloeosporioides
  • PCR primer and quantitative detection method for rapidly detecting strawberry colletotrichum gloeosporioides
  • PCR primer and quantitative detection method for rapidly detecting strawberry colletotrichum gloeosporioides

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1 A kind of PCR rapid detection method of G. fragariae anthracnose

[0033] 1. Primer design

[0034]In order to detect the compound species of G. glyospora anthracnose on strawberries in the field in my country as far as possible, the cutinase gene sequences of 35 strains of G. anthracis gloeosporum collected from different regions of the country were respectively amplified and sequenced. After sequence comparison, the selected It has a common nucleic acid sequence at the 3' end of the gene, as shown in SEQ ID NO.1.

[0035] The selected conserved sequence is located at the 3' end of the cutinase gene, which is unique to the physiological race of G. fragariae. The cutinase encoded by this gene is involved in assisting the invasion process of anthracnose. It is speculated that it may be related to the high prevalence of G. It is related to pathogenicity, and the expansion and identification of specialized physiological races in different regions of my country ...

Embodiment 2

[0056] Example 2 A method for absolute quantification of the biomass of strawberry anthracnose pathogenic bacteria Gleospora anthracnose

[0057] 1. Establish a standard curve

[0058] The standard plasmid pGEM-T-cut series after 10-fold isocratic dilution was used as a template, and the concentrations were 3.28×10 1 copies / μL, 3.28×10 2 copies / μL, 3.28×10 3 copies / μL, 3.28×10 4 copies / μL, 3.28×10 5 copies / μL, use primers Cfcut1 and Cfcut2 to carry out real-time fluorescent quantitative PCR amplification, obtain the cycle number Cq value of the corresponding standard, use the obtained cycle number Cq value and the corresponding plasmid template copy number to plot, and obtain the standard curve equation Y= -3.3348 (log 10 X)+34.726, Y is the cycle number Cq value, X is the initial copy number, correlation coefficient R 2 The value is 0.9992, and the primer amplification efficiency is 99.47%.

[0059] Among them, the real-time fluorescent quantitative PCR reaction syste...

Embodiment 3

[0065] Four physiological races of G. spp. C.aenigma, C. fructicola, C. siamense and C. gloeosporioide, C. nymphaeae and other three common pathogenic fungi in the field, Botrytiscinerea, Sphaerotheca macularis and Pestalotiopsis clavispora Genomic DNA of healthy strawberries and healthy strawberries were used as samples for detection.

[0066] 1. Extraction of fungal genomic DNA:

[0067] (1) Take 100 mg of fresh mycelia into a centrifuge tube, quickly add 600 μL of FG1 buffer solution, shake vigorously to fully disperse the mycelia, incubate at 65°C for 10 minutes, and mix by inverting twice during the period;

[0068] (2) Add 140 μL of FG2 buffer, shake and mix well, centrifuge at ≥10,000 g for 10 minutes, carefully absorb about 600 μL of the supernatant and transfer it to a new centrifuge tube, add 0.7 times the volume of isopropanol and mix well;

[0069] (3) Discard the supernatant after centrifugation at 10,000 g for 2 minutes, add 300 μL sterile deionized water prehea...

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Abstract

The invention relates to a PCR primer and quantitative detection method for rapidly detecting strawberry colletotrichum gloeosporioides. The PCR primer is designed according to a cutinase gene consensus sequence of colletotrichum gloeosporioides physiological races and comprises two primers Cfcut1 and Cfcut2 and an amplification product 115bp; by utilizing the PCR primer and the quantitative detection method, fungal genome DNA or field sample DNA can be subjected to PCR detection without separation and culture of pathogenic bacteria, the amplification efficiency is high, the specificity is good, the amplification efficiency of the primer is 99.47%, and colletotrichum gloeosporioides compound species causing strawberry anthracnose can be rapidly detected and identified; and through real-time fluorescent quantitative PCR, absolute quantitative detection can be carried out on the biomass of the colletotrichum gloeosporioides in a sample, the occurrence period and the prevalence intensityof the anthracnose are predicted timely and accurately, the anthracnose is prevented and treated and even avoided, negative influences caused by use of pesticide can be reduced, and a basis is provided for comprehensive prevention and treatment of the anthracnose.

Description

technical field [0001] The invention belongs to the field of detection of plant pathogens, in particular to a PCR primer and a quantitative detection method for rapid detection of Gleosporium fragariae anthracnose. Background technique [0002] Strawberry belongs to Rosaceae, Fragaria genus, and is a perennial herb plant. It is one of the traditional excellent fruits in my country. As a characteristic and advantageous industry among horticultural crops in Shanghai, strawberry has very significant economic benefits. The suburban area is maintained at about 40,000 mu all year round. However, strawberry diseases have always restricted the development of strawberry industry. Strawberry anthracnose, as one of the main diseases in strawberry production, has an increasing trend in recent years. [0003] "Strawberry anthracnose" refers to all strawberry diseases caused by fungi of the genus Anthracnose. The reported pathogens of strawberry anthracnose mainly include Colletotrichum g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6851C12Q1/686C12Q1/06C12N15/11
CPCC12Q1/6895C12Q1/6851C12Q1/686C12Q2600/166C12Q2565/125C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 杨静高清华段可
Owner SHANGHAI ACAD OF AGRI SCI
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