Detection reagent kit and detection method of soybean phytophthora

A technology of Phytophthora sojae and a kit, which is applied in the biological field, can solve the problems of long cycle time and poor specificity, and achieve the effects of high accuracy, high sensitivity, and high sensitivity

Inactive Publication Date: 2010-08-18
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0008] The technical problem to be solved by the present invention is to solve the problems of the required cycle length (15-20 days) and poor specificity (50-100 oospores / gram soil) of the biological detection method of Phytophthora soybean in the prior art, for this reason, The invention provides detection primers, a kit and a method for Phytophthora soybean, and performs PCR detection on Phytophthora soybean, which has strong practicability, high accuracy and sensitivity

Method used

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  • Detection reagent kit and detection method of soybean phytophthora
  • Detection reagent kit and detection method of soybean phytophthora
  • Detection reagent kit and detection method of soybean phytophthora

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Experimental program
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Effect test

Embodiment 1

[0029] Embodiment 1: Design, synthesize primer and establish the PCR reaction system of soybean Phytophthora detection kit

[0030] 1. Primer design and synthesis

[0031] Design of PsYpt3F / PsYpt2R specific primers for Phytophthora sojae:

[0032] PsYpt3F (20bp): 5'-TACCAATAATCAGAAGCGTA-3' (SEQ ID NO.1);

[0033] PsYpt2R (18bp): 5'-CCTTGTCTGCCCTCTCGA-3' (SEQ ID NO. 2).

[0034] At the same time, the Ypt1 universal primer Yph1F / Yph1R of Phytophthora (see the nucleotide sequence table SEQ ID NO.3 and SEQ ID NO.4 for details) was also designed as a round reaction primer 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 Company.

[0038] 2. Establish PCR reaction system

[0039] 1. Establish a conventional PCR reaction system

[0040] 1mL conventional PCR reaction system includes: several templates, 0.05mmol Tris.Cl, 0.125mmol K...

Embodiment 2

[0045] Example 2: Detection of Phytophthora sojae in soil samples

[0046] Phytophthora sojae was detected from soil samples of imported soybeans with fungus at the customs. The detection process is as follows:

[0047] 1. Enrichment of oospores in soil:

[0048] Take 20-100 grams of the soil sample to be tested, grind it, first use a 200-mesh sieve to remove larger soil particles, then filter through a 400, 500, and 800-mesh sieve, and wash it repeatedly with 3-10 liters of water at the same time. Collect oospores on the net and suspend with 1ml sterile water. Since oospores cannot pass through the 800-mesh sieve, such treatment can achieve the effect of enriching oospores.

[0049] 2. DNA extraction from trace oospores:

[0050] Transfer the oospores suspended in sterile water to a 1.5mL centrifuge tube, at 12000r.min -1 Centrifuge at high speed for 5 minutes and pour off the liquid;

[0051] Add 50 μL CTAB buffer, grind, then add 500 μL CTAB buffer, and bathe in water ...

example 3

[0064] Example 3: Detection of Zoospores of Phytophthora sojae from Contaminated Water

[0065] Take 500mL of irrigation water contaminated by Phytophthora sojae, centrifuge it for 20min under the centrifugal force of 6000g, discard the supernatant, suspend the precipitated zoospores with 100uL water, transfer them into a 1.5mL centrifuge tube, add 0.05g of quartz sand, and vortex 10sec, take 1uL zoospore fragmentation liquid as template, carry out gene amplification according to the method for embodiment 2, can amplify the unique fragment of Phytophthora sojae, the results are shown in Figure 3a and Figure 3b . in, Figure 3a Sensitivity of primers PsYpt3F / PsYpt2R to amplify different concentrations of zoospores; Figure 3b The sensitivity of the nested PCR reaction to amplify different concentrations of zoospores for the combination of primers Yph1F / Yph2R and PsYpt3F / PsYpt2R; lane 1 is a 2000bp DNA marker; lane 2 is a negative control; lanes 3-6 are 25μl reaction system...

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Abstract

The invention relates to a detection reagent kit and a detection method of soybean phytophthora, solving the problems of long required period (15-20 days) and poor specificity (50-100 oospores / gram of soil) of the biological detection method of the soybean phytophthora in the prior art and belonging to the fields of crop diseases prevention and treatment and plant quarantine. A primer, the reagent kit and the method adopted in the invention have the advantages of strong practicality and high accuracy and sensitivity when used for carrying out PCR (Polymerase Chain Reaction) detection on the soybean phytophthora.

Description

technical field [0001] The invention relates to a detection kit for Phytophthora soybean and a detection method thereof, belonging to the field of biotechnology. Background technique [0002] Soybean Phytophthora root rot is caused by Phytophothora sojae, which is a widely distributed and extremely serious soil-borne disease. It is the A1 entry plant quarantine object announced by our country. The disease was first discovered in Indiana, USA in 1948. After the public report in 1955, it was discovered successively in 15 countries including Brazil, Argentina and Canada, the main soybean producing countries in the world. In the three years from 1989 to 1991 alone, soybean blight caused 2.79 million tons of yield loss in 12 states in the north-central United States, with an economic value of up to 560 million U.S. dollars. [0003] In 1989, Shen Chongyao of Beijing Agricultural University and others first discovered Phytophthora sojae in Northeast my country. At present, Phyto...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 王源超郑小波王颖董莎萌
Owner NANJING AGRICULTURAL UNIVERSITY
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