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Loop-mediated isothermal amplification (LAMP) primers for detecting Phytophthora capsici and kit comprising primers

A technology of Phytophthora capsicum and LAMP-PCR is applied in the field of LAMP primers and kits containing the primers, which can solve the problems of large investment, high requirements for instruments and equipment, and obstacles to the promotion of technical bases, and achieves the elimination of instrument investment, specificity and efficiency. High sensitivity, easy to promote and use

Inactive Publication Date: 2012-07-18
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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  • Loop-mediated isothermal amplification (LAMP) primers for detecting Phytophthora capsici and kit comprising primers
  • Loop-mediated isothermal amplification (LAMP) primers for detecting Phytophthora capsici and kit comprising primers
  • Loop-mediated isothermal amplification (LAMP) primers for detecting Phytophthora capsici and kit comprising primers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 is used to detect the synthesis of the LAMP primer set of Phytophthora capsici

[0027] According to the gene sequence of Phytophthora capsici (P.capsici) ITS+5.8S region, the LAMP primer set for detecting Phytophthora capsici was designed, which included:

[0028] Outer forward primer F3: 5'-AACGCATATTGCACTTCCG-3';

[0029] Outer reverse primer B3: 5'-GCCTCCACAACCAGCAAG-3';

[0030] Inner forward primer FIP: 5'-CATCCTCACCGACTACACGGCCTGGGAGTATGCCTGTATCAG-3';

[0031] Inner forward primer BIP: 5'-TGTTGTCCTTCGGGTCGACTGTACCACGCTTTTCGAGCAA-3'.

[0032] The primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

Embodiment 2

[0033] Example 2 Utilizes LAMP primer set to detect the specificity analysis of Phytophthora capsici

[0034] 1.1 Reagents and equipment:

[0035] The water bath and the LAMP-PCR kit were purchased from Japan Rongken Company.

[0036] 1.2 Sample source:

[0037] The six strains of Phytophthora capsici used in the present embodiment, Phytophthora infestans, Phytophthora cucumber, Phytophthora sojae, Phytophthora falciparum, Pythium ultima, Pythium maloestrogens, Pythium thorns, Pythium terrestrial, 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.

[0038] Table 1 Sources of samples used for LAMP-PCR detection

[0039]

[0040] 1.3 DNA extraction:

[0041] Refer to the NaOH method described in Wang et al. (N...

Embodiment 3

[0049] Example 3 Sensitivity analysis of detecting Phytophthora capsici using LAMP primer set

[0050] 1.1 DNA sample concentration:

[0051] The DNA concentration of the Phytophthora capsici sample extracted in Example 2 was detected by NanoVue (General Electric Company), and it was 104.5 μg / ml.

[0052] 1.2 Sensitivity detection of LAMP primer set:

[0053] Carry out 10 times serial dilution of DNA sample, take 10 1 、10 4 、10 8 、10 9 、10 10 、10 11 and 10 12 Double-diluted DNA samples were subjected to LAMP constant temperature amplification reaction. Reaction system and reaction condition are with embodiment 2.

[0054] 1.3 Results:

[0055] The above amplification products were subjected to agarose gel electrophoresis, and the electrophoresis detection results were as follows: image 3 As shown, LAMP was able to detect a dilution of 10 9 times the DNA sample. When the DNA sample was diluted to 10 10 When it is more than a factor of 1, detection cannot be ensur...

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Abstract

The invention provides loop-mediated isothermal amplification (LAMP) primers for detecting Phytophthora capsici and a kit comprising the primers. The primers comprise FIP, BIP, F3 and B3 (shown as Seq ID No.1-4). An LAMP primer technology is used for quickly detecting the Phytophthora capsici, and the Phytophthora capsici can be accurately detected in a complicated pathogenic bacteria environmentin diseased plant tissues and soil. The method has higher specificity and sensitivity than the conventional PCR method, propagules of the Phytophthora capsici in various forms, such as mycelia, oospores and zoospores can be detected, and the method has significance in aspects of early warning of Phytophthora capsici epidemics and monitoring pathogeny in epidemic areas; meanwhile, expensive instruments can be avoided, and the method is easily promoted and used in basic level.

Description

technical field [0001] The invention relates to the detection of Phytophthora capsici, in particular to a LAMP primer for detecting Phytophthora capsici 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 pla...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/645
Inventor 孙翔郭良栋
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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