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PCR (Polymerase Chain Reaction) detection primer for phytophthora capsici leonian, kit containing primer and application thereof

A technology for Phytophthora capsicum and detection primers, which is applied in the field of PCR detection primers for Phytophthora capsicum, can solve the problems of no specific Phytophthora capsicum detection primers related reports, small sequence differences, and low resolution, etc., and achieves good specificity. , easy operation, high sensitivity effect

Inactive Publication Date: 2012-08-22
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research results of molecular detection techniques for Phytophthora sojae, Phytophthora infestans, Phytophthora capsici, Phytophthora parasitica, and Phytophthora winteritiva show that the differences in ITS sequences between some Phytophthora species are very small, and the resolution is not high
[0005] Enl gene is an enolase (enolase) encoding gene, which widely exists in eukaryotes, and there is no relevant report on the specific detection primers for Phytophthora capsici designed for this target gene

Method used

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  • PCR (Polymerase Chain Reaction) detection primer for phytophthora capsici leonian, kit containing primer and application thereof
  • PCR (Polymerase Chain Reaction) detection primer for phytophthora capsici leonian, kit containing primer and application thereof
  • PCR (Polymerase Chain Reaction) detection primer for phytophthora capsici leonian, kit containing primer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 is used to detect the synthesis of the PCR primer of Phytophthora capsici

[0038] Select the Enl and Ypt gene sequences of Phytophthora capsici and other Phytophthora from the GenBank database, and the involved sequences are shown in Table 2:

[0039] Table 2 Enl and Ypt gene sequences involved in the present invention

[0040]

[0041] Using DNAMAN and other biological software to compare and analyze the above-mentioned Enl and Ypt gene sequences, design Phytophthora capsici-specific primers located on Enl and Ypt genes, and obtain four pairs of specific primers, the sequences of which are shown in Table 1:

[0042] Table 1 Phytophthora capsici specific primer sequence involved in the present invention

[0043]

[0044]

[0045] *W is A or T.

[0046] The primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

Embodiment 2

[0047] Example 2 Utilize PCR primers Enl4s / Enl1a to detect the specificity and sensitivity analysis of Phytophthora capsici

[0048] 1.1 Sample source:

[0049] The six strains of Phytophthora capsici used in this example, Phytophthora infestans, Phytophthora cucumber, Phytophthora sojae, Phytophthora falciparum, Phytophthora lychee, Pythium ultima, Pythium maloestrogens, Phytophthora spinosa, Pythium solani, Fusarium, etc. (Table 3) are preserved in the State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences and the laboratory of Professor Liu Xili, China Agricultural University, and some pathogens were donated by the laboratory of Professor Zhang Xiuguo, Shandong Agricultural University.

[0050] Table 3 Sources of samples used for PCR testing

[0051]

[0052]

[0053] 1.2 DNA extraction:

[0054] Refer to the NaOH method described in Wang et al. (Nucleic Acids Research, 1993, 21:4153-4154), slightly modified to extract DNA from plan...

Embodiment 3

[0065] Example 3 Utilize PCR primers Enl2s / Enl3a to detect the specificity and sensitivity analysis of Phytophthora capsici

[0066] The primers used in the PCR reaction are Enl2s and Enl3a, the PCR amplification procedure and the detection method of the amplified product are the same as in Example 2, and the specificity and sensitivity analysis methods of the primers are the same as in Example 2.

[0067] After the amplification reaction, the amplified product was detected by electrophoresis on a 1% agarose gel. If a band of about 300bp appears, it proves that the detected pathogen is Phytophthora capsici. The results of electrophoresis were as image 3 As shown, lanes 1-6 are samples of Phytophthora capsici, and DNA bands of about 300 bp can be amplified, while no bands appear in other species, Pythium species and Fusarium species. The above results indicated that the primers Enl2s and Enl3a had strong specificity.

[0068] The PCR amplification reaction was carried out u...

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Abstract

The invention provides a PCR (Polymerase Chain Reaction) detection primer for phytophthora capsici leonian. The primer comprises Enl4s and Enl1a (as shown in Seq ID No.1 and 2). Four pairs of specific primers capable of quickly detecting phytophthora capsici leonian germs can be obtained based on the sequence difference between the Enl and Ypt genes of the phytophthora capsici leonian and other phytophthora nicotianaes: Enl2s / Enl3a, Enl4s / Enl1a, Ypt2s / Ypt3a and Ypt2s / Ypt5a, wherein the sensitivity of Enl4s / Enl1a is the highest. A PCR detection system is established on the basis, which is capable of quickly and accurately detecting the phytophthora capsici leonian from complex pathogenic bacteria environments in the tissues of plants having diseases and soil. A detection kit established according to the method is simple and convenient for operation, good in specificity and high in sensitivity; and the detection kit is capable of detecting the propagules in various forms of the phytophthora capsici leonian, such as hypha, oospore, zoospore and the like, thereby having great significance in the aspects of early warning on the epidemic situation of the phytophthora capsici leonian, pathogen supervision on the epidemic area and the like.

Description

technical field [0001] The invention relates to the detection of Phytophthora capsici, in particular to a PCR detection primer for Phytophthora capsici, a kit containing the primer and application thereof. 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 ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 孙翔郭良栋
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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