Phytophthora sojae molecule detecting primer and detection reagent kit thereof

A technology for root rot fungus and Phytophthora sojae, applied in microorganisms, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of low sensitivity and poor specificity, and achieve simple and fast operation, good practicability, fast and reliable. The effect of detection and identification

Inactive Publication Date: 2009-09-02
INST OF PLANT PROTECTION FAAS
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a kind of molecular detection primer of Phytophthora soybean root rot and detection kit thereof, for the biological detection method of Phytophthora soybean root rot in the prior art requires a long period, the current Phytophthora soybean root The molecular detection method of Phytophthora sojae has poor specificity and low sensitivity. By determining the ITS base sequence of Phytophthora soybean root rot and other Phytophthora and performing multiple comparison analysis, the ITS base specificity of Phytophthora soybean root rot A pair of highly specific primers were design

Method used

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  • Phytophthora sojae molecule detecting primer and detection reagent kit thereof
  • Phytophthora sojae molecule detecting primer and detection reagent kit thereof
  • Phytophthora sojae molecule detecting primer and detection reagent kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: Primer is to the specific amplification of soybean Phytophthora root rot bacterium

[0044] 1. Specific detection of soybean Phytophthora root rot

[0045] PCR reaction system 25μl, including 2.5μl 10×PCR reaction buffer, 5mM MgCl 2 2.5μl, 10mM dNTPs2.0μl, 1.0U Taq DNA polymerase, 20pmol / μl primer PSD11 / PSD12 each 0.2μl, weight concentration 99.5% DMSO0.5μl and 10ng template DNA,, d.d.H 2 O to make up 25 μl. The PCR amplification program was: denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 68°C for 30 sec, extension at 72°C for 1 min, 35 cycles, and finally extension at 72°C for 10 min.

[0046] 2. Test results

[0047] Specificity of detection: In addition to the 382bp product that can be specifically amplified from the DNA of Phytophthora root rot of soybean from Fujian, Heilongjiang and other provinces in my country and abroad, other 13 species of Phytophthora, downy mildew, and rot of oomycetes were detected. Mold and o...

Embodiment 2

[0048] Example 2: Detection of Phytophthora sojae root rot in diseased plant tissues.

[0049] 1. Sample collection: Plant tissue samples were collected from the soybean vegetable base in Bangshan Town, Longhai City, Fujian Province

[0050] 2. DNA extraction and detection

[0051] The diseased plant tissue was extracted with the CTAB method, and PCR amplification was carried out according to the method implemented in the above kit. The PCR reaction system was 25 μl, including 2.5 μl 10×PCR reaction buffer, 5 mM MgCl 2 2.5μl, 10mM dNTPs 2.0μl, 1.0UTaq DNA polymerase, 20pmol / μl primer PSD11 / PSD12 0.2μl each, weight concentration 99.5% DMSO 0.5μl and 10ng template DNA, d.d.H2O make up 25μl. The PCR amplification program was: denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 68°C for 30 sec, extension at 72°C for 1 min, 35 cycles, and finally extension at 72°C for 10 min. Amplified products were detected by electrophoresis.

[0052] 3. Test results ...

Embodiment 3

[0054] Example 3: Detection of Phytophthora soybean root rot in soil samples.

[0055] 1. Sample collection: Soil samples were collected from soybean vegetable base in Fugong Town, Longhai City, Fujian Province

[0056] 2. DNA extraction and detection

[0057] (1) Extract DNA from diseased soil samples:

[0058] Take the sieved soil, freeze and dry it for 24-48 hours, add a small amount of quartz sand, pour liquid nitrogen into it and grind it thoroughly, divide the ground soil fine powder into 1.5ml centrifuge tubes, add 500μl weight concentration of 0.4% skimmed milk powder to each tube solution, vortexed to mix. Centrifuge at 12000rpm for 15min. Take the supernatant and add an equal volume of proteinase K buffer to a final concentration of 10 μg / ml proteinase K, and bathe in water at 55°C for 1-3h. After the water bath, add 1 / 2 volume of 7.5M NH 4 AC solution, mix up and down. Centrifuge at 12000rpm for 15min. Aspirate the supernatant and add 2 times the volume of ab...

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Abstract

The invention provides a phytophthora sojae molecule detecting primer and a detection reagent kit thereof. The primer comprises a forward primer PSD11 and a backward primer PSD12; and the detection reagent kit comprises PCR buffer solution, MgCl2, dNTPs, Taq polymerase, positive control DNA, DMSO, PSD11/PSD12 and ultrapure water. The detection reagent kit based on the primer specifically amplifies specific amplifying products with the fragment length of 382bp in phytophthora sojae pure DNA, bacteria-bearing pathological tissues and soil. The specific molecule detecting primer and the detection reagent kit thereof can be applied to the rapid, sensitive and specific detection for plant tissues infected with phytophthora sojae and the phytophthora sojae in soil and can be also applied to the early diagnosis of field diseases and the high-sensitive and rapid molecule detection and identification for the phytophthora sojae carried by soybean exporting or importing from customs.

Description

technical field [0001] The invention relates to the field of crop disease detection and identification and plant quarantine, and more specifically relates to a molecular detection primer for Phytophthora soybean root rot and a detection kit thereof. Background technique [0002] Soybean Phytophthora root rot caused by Phytophthora sojae is a devastating soil-borne disease, which is listed as Class I external inspection object in my country. The disease was first detected in Heilongjiang Province, my country in 1991. It was found that it has been expanding year by year since then. Soybean blight was discovered in Zhangzhou area in Fujian Province in 1997, and Phytophthora sojae was isolated in Zhangzhou area in 2002. At present, the disease has posed a threat to soybean production in my country. Soybean Phytophthora root rot is a soil-borne disease. The pathogen can be transmitted from the diseased area to the non-infected area through rain, soil, diseased residues, etc., and ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/645
Inventor 陈庆河翁启勇李本金兰成忠赵健
Owner INST OF PLANT PROTECTION FAAS
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