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Primer, kit and detection method for detecting phytophthora infestans

A technology of Phytophthora infestans and kits, applied in the biological field, can solve the problems of less research, small mitochondrial gene variation, and unsuitable molecular detection targets, etc., and achieve the effects of strong specificity, high accuracy, and improved detection sensitivity

Inactive Publication Date: 2010-12-22
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are relatively few studies on the Lpv gene in other Phytophthora except Phytophthora cryptophytophthora. Unlike the ITS sequence, almost all Phytophthora species can obtain their sequences from GenBank
However, the mitochondrial genes have little variation among different Phytophthora species, and the GC content is relatively high, some even reach 89%, which is not suitable for molecular detection targets of most Phytophthora species

Method used

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  • Primer, kit and detection method for detecting phytophthora infestans
  • Primer, kit and detection method for detecting phytophthora infestans
  • Primer, kit and detection method for detecting phytophthora infestans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Designing, synthesizing primers and establishing a PCR reaction system for the Phytophthora infestans detection kit

[0030] 1. Primer design and synthesis

[0031] Download the partial sequence of the Ypt1 gene of P.infestans from GenBank (accession number: DQ162961), and compare with the sequence of more than 30 species of Phytophthora registered in GenBank. By comparison, the primer PiF / PiR was designed according to the unique fragment of Phytophthora infestans:

[0032] PiF: 5'-GACTTTGTGAGTGTCTAACATA-3' (SEQ ID NO.1);

[0033] PiR: 5'-CAAGACGAGCGCACCTATCG-3' (SEQ ID NO. 2).

[0034] At the same time, the Ypt1 universal primer Yph1F / Yph1R of the genus Phytophthora was also designed as a round of reaction primers for nested PCR:

[0035] Yph1F (20bp): 5'-CGACCATTGGCGTGGACTTT-3' (SEQ ID NO.3);

[0036] Yph1R (20bp): 5'-ACGTTCTCGCAGGCGTATCT-3' (SEQ ID NO. 4).

[0037] All primers were synthesized by Invitrogen.

[0038] 2. Establish a conventional PCR reaction system

[00...

Embodiment 2

[0042] Example 2: Preparation of DNA template

[0043] The DNA of various samples is extracted as a template for PCR reaction, the specific process is as follows:

[0044] 1. DNA extraction of mycelium powder

[0045] Refer to Sambrook's method and make a slight improvement. Take a small amount of mycelium powder, add 900μl of 2% CTAB extract and 90μl of 10% SDS, vortex and mix, in a 55℃ water bath for 1h, inverted several times every 10min. Centrifuge at 12000rpm for 10min, take the supernatant and add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), mix by inversion, and centrifuge at 12000rpm for 10min; transfer the supernatant to a new tube, add an equal volume of chloroform, and gently mix upside down Evenly, centrifuge at 12000rpm for 5min. Transfer the supernatant to a new tube, add 2 volumes of absolute ethanol and 1 / 10 volume of 3M NaAc (pH 5.2), and precipitate at -20°C (>1h). Centrifuge at 12000 rpm for 10 min, decant the supernatant, wash the pellet twi...

Embodiment 3

[0055] Example 3: Detection of the specificity and sensitivity of primers

[0056] 1. Specific detection

[0057] The specific primers of Phytophthora infestans of the present invention were used to perform PCR amplification on the Phytophthora infestans strains and other Phytophthora and fungal strains in Table 1. As a result, among all the tested strains, only Phytophthora infestans could be amplified Specific fragment with a molecular weight of 369bp (see figure 1 ), indicating that the primer has good specificity.

[0058] Table 1. Fungi and oomycetes for specific analysis of detection primers (PiF / PiR)

[0059]

[0060]

[0061]

[0062] In the above table: + means a specific amplified band with primer Yph1F / 1R or PiF / PiR;-means no amplified product.

[0063] 2. Sensitivity detection

[0064] When the 25μl reaction system contains 100pg of genomic DNA, the primers PiF and PiR can still be stably amplified to obtain a 369bp specific band (see Figure 2a ). Using the universal prim...

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PUM

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Abstract

The invention provides a primer, a test kit containing the primer and a detection method for detecting phytophthora infestans, the utilization of the primer and the detection method for detecting the phytophthora infestans can realize high accuracy, strong specificity and high sensitivity, the method of the invention has the advantages of accuracy, speediness, simplicity, easy operation and the like and can identify pathogens at the early stage of infection of the disease and simultaneously detect the pathogens in field soil, and the invention has great significance for preventing and treating late bright of potatoes and tomatoes. Simultaneously, the invention also has the great significance for reducing the blind use of pesticides, reducing the production cost and reducing the environmental pollution of the pesticides.

Description

Technical field [0001] The invention relates to a detection primer for Phytophthora infestans, a detection kit containing the primer and a detection method, and belongs to the field of biotechnology. Background technique [0002] Late blight caused by Pyhtophthora infestans (Mont.) de Bary is one of the devastating diseases in potato and tomato production in the world. Under the conditions that the temperature is suitable for the disease to occur, the disease can cause a substantial reduction in crop yield or even no harvest. The traditional methods of detecting plant diseases include directly observing the symptoms of plant tissues or performing morphological identification after the pathogen is isolated. However, direct observation will miss the absence of symptoms at the beginning of the onset, and the disease caused by Phytophthora spp., Fusarium spp. and Rhizoctonia spp. can easily be confused with the disease symptoms caused by Pythium spp., Fusarium spp. and Rhizoctonia s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 王源超郑小波张正光孟军
Owner NANJING AGRICULTURAL UNIVERSITY
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