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A primer pair, kit and detection method for rapid detection of camelsporium coffee rust

The technology of primer pair and kit is applied in the field of primer pairs for rapid detection of C. caffeine rust, and achieves the effects of overcoming cumbersome steps, high sensitivity and rapid sensitivity.

Active Publication Date: 2019-05-07
ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no simple, accurate and sensitive molecular detection technology for the detection of camelsporium coffee rust in China.

Method used

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  • A primer pair, kit and detection method for rapid detection of camelsporium coffee rust
  • A primer pair, kit and detection method for rapid detection of camelsporium coffee rust
  • A primer pair, kit and detection method for rapid detection of camelsporium coffee rust

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. Primer design:

[0028] According to the ITS sequence (SEQ ID NO: 3) of the coffee camel rust strain measured by 4 laboratories and the NCBI website (http: / / www.ncbi.nlm.nih.gov / ) all the coffee camel rust fungus Hemileia vastatrix ITS gene sequence (EF394118~EF394132.1, DQ022191.1) and other highly homologous ITS sequences (Chrysomyxa empetri, Cronartiumquercuum, Endocronartium harknessii, Cronartium quercuum f.sp.banksianae, Puccinia andropogonis var. 、Melampsoridium hiratsukanum、Melampsoridium betulinum、Melampsoridium alni、Puccinia allii、Cronartiumcoleosporioides、Chrysomyxa chiogenis、Pucciniastrum boehmeriae、Pucciniastrumactinidiae、Pucciniastrum miyabeanum、Pucciniastrum hikosanense、Pucciniastrumkusanoi)之间的差异位点区域,设计咖啡驼孢锈菌的特异性引物, That is the primer pair Hv-ITS-F / R of the present invention, specifically: upstream forward primer Hv-ITS-F sequence: 5'-GGTACACCTGTTTGAGAGTATG-3'; downstream reverse primer Hv-ITS-R sequence: 5' -CAAAATATGTCATACCTCTCATTCT-3'.

[0029] 2....

Embodiment 2

[0031] Embodiment 2: The specificity of primer pair Hv-ITS-F / R detection camelsporium coffeae rust

[0032] The specificity of the primers was verified with the following strains: 4 isolates from different regions of my country (1 strain in Malipo County, Yunnan, 1 strain in Ruili, Yunnan, 2 strains in Mangshi, Yunnan), 1 strain of coffee anthracnose, 1 One strain of coffee rust hyperparasite, one strain of coffee leaf spot fungus, one strain of plumeria rust fungus, and one strain each of sugarcane red rot fungus, smut fungus, ring spot fungus, and sisal zebra spot fungus.

[0033] The DNA of the above-mentioned strains was extracted using a fungal DNA extraction kit (E.Z.N.A Fungal DNA kit, Omega Company, the United States). Among them, the uredospores of camelsporium coffee and plumeria rust were directly used (collecting severely rusted leaves, in the experiment chamber using an inoculation blade to scrape off the piles of uredospores attached to its surface) to extract DNA...

Embodiment 3

[0040] Embodiment 3: The sensitivity of primers to Hv-ITS-F / R detection camelsporium coffee rust

[0041] Adjust the genomic DNA concentration of the camelspora coffee rust strain to 10 ng / μL, and gradually dilute it down to 10 by the order of 10 -5 ng / μL was used as a template for PCR amplification. The PCR system and reaction procedure were the same as in Example 2, and the PCR product was detected by 1.0% agarose gel electrophoresis. see results figure 2 . like figure 2 Shown, when the DNA concentration of camelsporium coffeae was greater than or equal to 10pg, utilize the primer pair Hv-ITS-F / R to obtain the DNA band of 396bp specific amplification, but after the concentration is less than 10pg, then amplify No band can be added, which shows that the primer pair can detect the concentration of the genomic DNA of camelsporium coffeae ≥ 10pg.

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Abstract

The invention provides a primer pair used for rapid detection of hemileia vastatrix. The sequence of the primer pair is shown as SEQIDNO:1 and SEQIDNO:2. The invention further provides a kit containing the primer pair used for rapid detection of hemileia vastatrix, and a method of using the primer pair for rapid detection of hemileia vastatrix. The primer pair provided by the invention can successfully detect hemileia vastatrix when the concentration of a to-be-detection sample reaches 10 pg, has relatively high sensitivity, and meets requirements of daily detection. At the same time, the time for identification of hemileia vastatrix can be shortened by the primer pair, hemileia vastatrix can be identified quickly, and the problems of tedious steps, long identification period and the like of the traditional identification method can be overcome. The primer pair used for detection has the characteristics of high sensitivity and strong specificity, and the detection method has the advantages of high efficiency, high speed and high sensitivity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer pair, a kit and a detection method for rapid detection of coffee camel rust (Hemileia vastatrix). Background technique [0002] Coffee is a plant of the genus Coffea of ​​the family Rubiaceae (Bridson and Verdcourt, 1988). Among the world's three major beverage crops (coffee, tea, cocoa), coffee's output, output value and consumption all rank the top of the three major beverage crops. Camelina coffee is an obligate parasitic pathogen that infects coffee leaves (Muthappa, 1975; Vallega and Chiarappa, 1964; Bhat et al., 2000), causing yellow rust on coffee leaves. Under favorable climatic conditions, the disease can cause leaf loss of up to 50% and berry loss of up to 70% (Daivasikamani S, and Rajanaika., 2009). The disease was first reported in India in 1869. Subsequently, the disease occurred in large numbers in Sri Lanka and Indonesia, causing devastating disasters to th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895
Inventor 吴伟怀易克贤贺春萍习金根郑金龙梁艳琼郑肖兰李锐邓彪龙
Owner ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI