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LAMP primer for detecting Phytophthora melonis and kit containing LAMP primer

A technology of Phytophthora cucumber and LAMP-PCR, which is applied in the detection field of Phytophthora cucumber, can solve the problems of large investment, high requirements for equipment and technology, and obstacles to the promotion of technology at the grassroots level. The effect of promotion

Inactive Publication Date: 2014-07-23
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, most of the conventional PCR methods are used for the detection of plant pathogenic bacteria, which has high requirements for equipment (such as PCR instrument, gel imaging system) and large investment, which has caused great obstacles to the promotion of the technology at the grassroots level.

Method used

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  • LAMP primer for detecting Phytophthora melonis and kit containing LAMP primer
  • LAMP primer for detecting Phytophthora melonis and kit containing LAMP primer
  • LAMP primer for detecting Phytophthora melonis and kit containing LAMP primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 is used to detect the synthesis of the LAMP primer set of Phytophthora cucumber

[0030] 1.1 Acquisition of Phytophthora melonis ITS+5.8S area

[0031] Cucumber mold (P.melonis) tested was cultured on oat plate medium at 25°C for 5-7 days, mycelial DNA was extracted by CTAB method, and stored at -20°C for future use.

[0032] The ITS+5.8S region was amplified using the primer pair ITS1 and ITS4 (ITS1: 5'-TCCGTAGGTGAACCTGCGG-3', ITS4: 5'-TCCTCCGCTTATTGATATGC-3', White et al., 1990). The amplification reaction was carried out in a 25 μL reaction system. The reaction system is as follows: PCR buffer (20mM KCl, 10mM (NH 4 ) 2 SO 4 ,2mM MgCl 2 , 20mM Tris-HCl, pH8.4), 200μM each of the four deoxyribonucleic acid triphosphates, 15pmol each of the primers, about 100ng of DNA template, and 2.5U of Taq DNA polymerase (Biocolor BioScience & Technolgy Company, Shanghai, China). The thermal cycle reaction program was set as follows: initial denaturation at 95°C for ...

Embodiment 2

[0040] Example 2 Utilize LAMP primer set to detect the specificity analysis of cucumber Phytophthora

[0041] 1.1 Reagents and equipment

[0042] The water bath and the LAMP-PCR kit were purchased from Guangdong Huafeng Biotechnology Co., Ltd.

[0043] 1.2 Sample source

[0044] The two strains of Phytophthora cucumber used in this example, Phytophthora capsici, Phytophthora infestans, Phytophthora sojae, Phytophthora litchi, Pythium ultima, Pythium maloestrogens, Pythium terrestriale, Fusarium etc. ( Table 1) are preserved in the State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences and the laboratory of Professor Xili Liu, China Agricultural University, and some bacteria were donated by the laboratory of Professor Zhang Xiuguo, Shandong Agricultural University.

[0045] Table 1 Sources of samples used for LAMP-PCR detection

[0046]

[0047] 1.3 DNA extraction

[0048] Refer to the NaOH method described in Wang et al. (Nucleic Acids ...

Embodiment 3

[0058] Example 3 Detection of Infection of Phytophthora cucumber in Diseased Tissues Using LAMP Primer Set

[0059] 1.1 Extraction of DNA from cucumber diseased tissues

[0060] Transfer Phytophthora cucumber to the plate of oat medium, culture in the dark at 25°C for 2-3 days, use a puncher to take colonies (1cm×2cm) from the edge of the colony, and inoculate them on cucumber seedlings (4-6 leaves ) leaves and stem bases, 7 days after inoculation, the disease effect is as follows image 3 shown. Then cut out different diseased tissues respectively, refer to the NaOH method described in Wang et al. (Nucleic Acids Research, 1993, 21:4153-4154), and slightly improve the extraction of DNA from plant tissues: take an appropriate amount of diseased leaves and stems, and 1mg of diseased tissues Add 10μl of 0.5mol / L NaOH to the solution, grind it thoroughly, transfer it to a 1.5mL centrifuge tube, and centrifuge at 10,000rpm for 5 minutes, take 5μl of the supernatant and add 495μl ...

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Abstract

The invention provides an LAMP primer for detecting Phytophthora melonis and a kit containing the LAMP primer. The primer comprises FIP, BIP, F3 and B3 (as shown in Seq ID No.1-4). According to the invention, an LAMP primer constant-temperature amplification technology is used for the fast detection of Phytophthora melonis pathogens, thus the Phytophthora melonis can be accurately detected from complicated pathogenic bacteria contained in pathogenetic plant tissues and soil. Compared with the conventional PCR (Polymerase Chain Reaction) method, the method provided by the invention has higher specificity and sensitivity, can be used for detecting propagules, such as hypha, oospore and zoospore which have various forms, of the Phytophthora melonis, has improtant significance in the aspects of the early prewarning of a Phytophthora melonis epidemic situation, the pathogeny monitoring of an epidemic area and the like, and is convenient to popularize and use on a grass root because large instrument investment is avoided.

Description

technical field [0001] The invention relates to the detection of Phytophthora cucumber, in particular to a LAMP primer for detecting Phytophthora cucumber and a kit containing the primer. Background technique [0002] Phytopathogenic oomycetes are an important class of plant pathogens, which can infect and harm a variety of plants and cause devastating diseases of a variety of plants, such as Phytophthora infestans, P. capsici, soybean blight P. sojae, P. parasitic, P. cinnamomi and P. hibemalis cause important diseases of potato, pepper, soybean, tobacco, camphor tree and citrus respectively , Serious years or even extinct production. This type of fungus mainly uses mycelium or oospores to pass through the unfavorable environment in the diseased residue or soil as the primary source of infection. Under suitable conditions, mycelium or oospores can germinate to produce sporangia-release zoospores , and then spread and infect with the help of rainwater or airflow to cause p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 孙翔郭良栋季妞妞
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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