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LAMP primer group for detecting hemileia vastatrix and application

A technology of rust cephaeria and a primer set, which is applied in the field of LAMP primer pairs for detecting rust of coffee cephine, can solve the problems of inappropriate primer design, the effect is difficult to achieve the expected effect and the like, and achieves high sensitivity, rapid sensitivity, Overcome the effect of cumbersome steps

Pending Publication Date: 2017-11-17
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, the design of primers with excellent effect is the key to LAMP detection technology. If the primer design is not suitable, the effect is difficult to achieve the expected effect.
There is no report on the application of LAMP detection technology to coffee camel rust (Hemileia vastatrix Berk.et Br.)

Method used

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  • LAMP primer group for detecting hemileia vastatrix and application
  • LAMP primer group for detecting hemileia vastatrix and application
  • LAMP primer group for detecting hemileia vastatrix and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Preparation of LAMP group primers for detection of coffee camelis rust (Hemileia vastatrix)

[0036] 1. Primer design:

[0037] The ITS sequence of Hemileia vastatrix (SEQ ID NO: 5 in the sequence listing) obtained by cloning according to this experiment was compared with the NCBI website ( http: / / www.ncbi.nlm.nih.gov / ) The ITS gene sequence of Hemileiavastatrix is ​​compared with the homologous ITS sequences of other genus or species to obtain specific regions. Using the online software Primer-Primer Explore (Fujitsu Ltd., Tokyo, Japan) ( http: / / primer explorer.jp / e / ) for primer design around the specific region and its flanking sequences (6).

[0038] The primer set of the present invention is designed with sequence 6 in the sequence table as a template, which are respectively:

[0039] F3 upstream primer sequence: GGATCCTTGGCTCTCAC (from the 20th-38th nucleotide at the 5' end of sequence 6 in the sequence listing, that is, the sequence 1 in the sequen...

Embodiment 2

[0048] Embodiment 2, LAMP detects coffee camelid rust (Hemileia vastatrix) optimum reaction temperature condition screening

[0049] Using the primer set prepared in Example 1, LAMP amplification was performed using the DNA of the coffee rust strain as a template.

[0050] Among them, the reaction system of PCR amplification is 25 μl, as follows:

[0051]

[0052]

[0053] The above 25μl LAMP reaction system was quickly placed at different temperatures (54°C, 57°C, 60°C, 63°C, 65°C, 68°C) for LAMP reaction.

[0054]The LAMP reaction results can be judged by two methods: 1) After the reaction is completed, add 0.1 μl SYBR Green I to each reaction system, and directly observe the color of the reaction solution of the sample to be tested through the meat. If the reaction solution of the sample to be tested is green, then Explain that the sample to be tested contains Hemileia vastatrix; on the contrary, if the reaction solution turns orange, it means that the sample to be t...

Embodiment 3

[0055] Example 3: Detection of specificity of LAMP primer sets for Hemileia vastatrix

[0056] Utilize embodiment 1 to prepare gained primer set, with 4 strains isolated from different regions of my country's coffee camel rust (HVL36, Malipo County, Wenshan, Yunnan; HVL51, Malipo County, Yunnan; HVL80, Baoshan, Yunnan; Zhefang, Yunnan; HVL91, 2 strains of coffee anthracnose (Colletotrichum gloeosporioides); 2 strains of Mycoparasites on the coffee rust; 1 strain of Cerospora coffeicola Berket Cooke; and sugarcane Ustilago scitaminea; Pucciniamelanocephala H.&P.Syd.; Colletotrichum falcatum; Coleosporium plumierae; Leptosphaeria sacchari); Phytophthora nicotianae Breda; 1 strain of grape layer rust (Phakopsora ampelopsidis Diet & Syd.). Carry out LAMP reaction for experimental material, analyze the specificity of the primer set provided by the present invention. Above-mentioned bacterial strains are all Collected in the above-mentioned areas according to conventional methods, and ...

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Abstract

The invention discloses an LAMP primer group for detecting hemileia vastatrix and application. The primer group consists of nucleotide shown as a sequence 1 in a sequence table, nucleotide shown as a sequence 2 in the sequence table, nucleotide shown as a sequence 3 in the sequence table and nucleotide shown as a sequence 4 in the sequence table. The detecting method has higher sensitivity and meets the requirement for daily detection. In addition, the time of identifying the hemileia vastatrix can be shortened through the primer group, the hemileia vastatrix can be more quickly identified, and accordingly the shortcomings of complicated steps, long identifying period and the like of a tradition identifying method are overcome.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular, the present invention relates to a LAMP primer pair, a kit and a detection method for detecting Hemileia vastatrix. Background technique [0002] Coffee rust is a worldwide fungal disease, divided into two types: yellow rust and gray rust, the former is caused by Hemileia vastatrix Berk. ) cause [Waller JM (1982). Coffee rust-epidemiology and control. CropProtection 1(4) 385-404]. Among them, coffee yellow rust caused by Hemileia vastatrix Berk.et Br. is the most destructive. The disease has occurred in Africa, the Near East, and coffee-producing regions such as India, Asia, and Australia for a long time. It first appeared in the Western Hemisphere in 1970 and was found in Brazil. [0003] Coffee has been grown in my country for more than 100 years. The earliest recorded occurrence of rust was in Guangxi from 1942 to 1947. At present, the coffee planting area in my country ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119C12Q2563/107
Inventor 吴伟怀易克贤汪涵贺春萍黄兴梁艳琼习金根李锐郑金龙
Owner ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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