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Primer for detecting orchid colletotrichum gloeosporioides molecules and quick detection method

A technology for molecular detection of glyospora anthracnose, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., to achieve the effects of easy operation, strong specificity and high sensitivity

Inactive Publication Date: 2012-07-04
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005]G. anthracnose is highly contagious and difficult to control once it spreads
At present, most of the detection of G. anthracnose on orchids still use traditional culture and identification methods. However, during the isolation process of the disease by conventional methods, due to the slower growth rate of G. anthracnose, it is often identified by other species including Pythium. Covered by miscellaneous bacteria, it is very difficult for the successful isolation of the bacteria and the accurate diagnosis of the disease
Therefore, the conventional disease diagnosis technology based on morphological characteristics is difficult to meet the actual needs of orchid anthracnose diagnosis due to its long time-consuming, low efficiency and sensitivity, and it is easy to miss the best period of disease control

Method used

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  • Primer for detecting orchid colletotrichum gloeosporioides molecules and quick detection method
  • Primer for detecting orchid colletotrichum gloeosporioides molecules and quick detection method
  • Primer for detecting orchid colletotrichum gloeosporioides molecules and quick detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Primer specific amplification of Colletotrichum gloeosporioides

[0040] 1. Specific detection of Colletotrichum gloeosporioides

[0041] 25μl PCR reaction system, including Taq PCR Master Mix 12.5μL, 200ng template DNA, primers P1 / P2 each 1μL (10 umol / L), add ddH2O to a total volume of 25μL, and amplify on a PE 2400 PCR machine. The PCR reaction conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 60 sec, annealing at 70°C for 60 sec, extension at 72°C for 60 sec, a total of 35 cycles; extension at 72°C for 7 min. The amplified products were detected by electrophoresis.

[0042] 2. Test results

[0043] Specificity of detection: such as figure 1 As shown, in addition to the DNA of 25 Colletotrichum gloeosporioides from Fuzhou and Zhangzhou in our province, which can specifically amplify the 304 bp product, the detection of 3 orchid Fusarium wilt and 10 different fungal DNAs failed to expand. Add any product, with strong specificity.

Embodiment 2

[0044] Example 2: Detection of sensitivity of primers to Colletotrichum gloeosporioides

[0045] 1. DNA concentration dilution: The extracted genomic DNA of Colletotrichum gloeosporioides is diluted by serial concentration after measuring the concentration by spectrophotometer.

[0046] 2. Sensitivity detection of Colletotrichum gloeosporioides

[0047] 25μl PCR reaction system, including Taq PCR Master Mix 12.5μL, serial concentration template DNA, primers P1 / P2 each 1μL (10 umol / L), add ddH2O to a total volume of 25μL, and amplify on a PE 2400 PCR machine. The PCR reaction conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 60 sec, annealing at 70°C for 60 sec, extension at 72°C for 60 sec, a total of 35 cycles; extension at 72°C for 7 min. The amplified products were detected by electrophoresis.

[0048] 3. Test results: such as figure 2 As shown, in a 25 μl reaction system, 20 fg of the genomic DNA of Colletotrichum gloeosporioides can obtain obviou...

Embodiment 3

[0049] Example 3: Detection of Colletotrichum gloeosporioides in diseased plants.

[0050] 1. Sample collection: Plant tissue samples were collected from the orchid planting bases in Fuzhou and Zhangzhou, Fujian Province.

[0051] 2. DNA extraction and detection

[0052] The diseased plant tissues used CTAB method to extract DNA, and carried out PCR amplification according to the above-mentioned method. The PCR reaction system was 25μL, including Taq PCR Master Mix 12.5μL, 200ng template DNA, primers P1 / P2 each 1μL (10 umol / L), plus ddH2O to a total volume of 25μL amplified on a PE 2400 PCR machine. The PCR reaction conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 60 sec, annealing at 70°C for 60 sec, extension at 72°C for 60 sec, a total of 35 cycles; extension at 72°C for 7 min. The amplified products were detected by electrophoresis.

[0053] 3. Test results

[0054] See the result image 3 There is a clear specific band with a molecular weight of ...

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Abstract

The invention relates to a primer for detecting orchid colletotrichum gloeosporioides molecules and a using method of the primer, which is specially used for detecting orchid colletotrichum gloeosporioides specific molecules and belongs to the fields of detection, identification and prevention and treatment of crop diseases. A pair of specific primers of orchid colletotrichum gloeosporioides comprising an upstream primer P1:5'-GGCCTCCCGCCTCCGGGCGGGTC-3' and a downstream primer P2:5'-TGAGGGCCTACATCAGCT-3' are subjected to polymerase chain reaction (PCR) amplification and agarose gel electrophoresis and can specifically amplify a specific amplification product with the segment length of 304bp in a plant infected with orchid colletotrichum gloeosporioides with pure DNA and germs and a culture medium. The specific molecule detection primer and the using method thereof can be used for detecting the orchid colletotrichum gloeosporioides in the plant infected with the orchid colletotrichum gloeosporioides and the culture medium quickly, sensitively and specifically, can also be used for performing early diagnosis on field diseases and monitoring and identifying germs, and provides reliable technological and theoretical basis for preventing and treating diseases caused by the orchid colletotrichum gloeosporioides.

Description

technical field [0001] The invention relates to a primer for molecular detection of Gynosporum anthracnose and its usage, which is specially used for the rapid molecular detection of Gylospora anthracnose, and can be used for early diagnosis of Gylospora anthracnose and the monitoring and identification of pathogens in the field, and belongs to crops The field of disease detection, identification and control technology. Background technique [0002] Orchids are one of the main flower products in Fujian Province that are exported to earn foreign exchange, bringing huge benefits to growers. Jianlan in Fujian ( Cymbidium ensifolium ) is the ancestor of Guolan, and Molan ( Cymbidium sinense ), Hanlan ( Cymbidium kanran ), Chunlan ( Gymbidium goeringii ), Cymbidium ( Cymbidium faberi ) belongs to Orchidaceae (Orchidaceae) Cymbidium ) Perennial monocotyledonous herbaceous plant, is a precious garden ornamental plant. It has a long history of cultivation among the peo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 姚锦爱余德亿陈峰黄鹏鹿连明
Owner INST OF PLANT PROTECTION FAAS
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