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Soybean phytophthora testing reagent kit and its test method

A detection kit, Phytophthora soybean technology, applied in the biological field, can solve the problems of long cycle and low sensitivity, and achieve the effect of high accuracy, high sensitivity and high sensitivity

Inactive Publication Date: 2003-11-05
NANJING AGRICULTURAL UNIVERSITY
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Technical problem The purpose of the present invention is to solve the problems of long period (15-20 days) and low sensitivity (50-100 oospores / gram soil) required by the biological detection method of Phytophthora soybean in the prior art, and provide Phytophthora soybean The detection kit and detection method thereof are used for PCR detection of Phytophthora soybean, with high accuracy and high sensitivity

Method used

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  • Soybean phytophthora testing reagent kit and its test method
  • Soybean phytophthora testing reagent kit and its test method
  • Soybean phytophthora testing reagent kit and its test method

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example 2

[0042] Electrophoresis detection of amplified products: take 10 μL of PCR amplified products, perform electrophoresis on 1% agarose gel with a voltage of 50-100V, and detect the results under ultraviolet light after 30 minutes. If there is a DNA band with a molecular weight of about 330bp, it proves that the detected pathogen is Phytophthora sojae. figure 1 Example 2 Detection of zoospores of Phytophthora sojae from polluted water

[0043] 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 For 10 sec, take 1uL of the zoospore fragmentation solution as a template to obtain 100% specific amplification products. Wherein, 2-3 μl template PCR template is used for gene amplification according to the method of Example 1, and a unique fragment of Phyt...

example 3

[0045] After sterilizing the soybean leaves or rhizome parts with water-soaked lesions with 70% alcohol, extract DNA by CTAB method or alkali lysis method, draw 1uL DNA solution, carry out PCR amplification according to the method of Example 1, and electrophoresis detection amplification If the product is a disease caused by Phytophthora soybean infection, a clear specific band with a molecular weight of 330bp can be seen, and the results are shown in image 3 .

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Abstract

The present invention belongs to the domain of crop disease prevention and treatment and plant quarantine. Based on soybean phytophthora sojae ITS gene sequence, one pair of specific oligomeric nucleotide primers are designed, and the reagent kit includes Tris. Cl 0.05 mmol, KCl 0.125 mmol, MgCl2 0.005 mmol, dNTPs 0.01 mmol, upstream and downstream primer 0.25 mmol, BSA 0.1 mg, and Taq DNA polymerase 100 U, which are mixed with pure water to prepare 1 ml of the test solution. The soybean phytophthora sojae testing reagent kit based on the primer has powerful specificity, high sensitivity and stability and may be used in fast, sensitive and specific test of soybean phytophthora sojae.

Description

(1) Technical field [0001] The detection kit for Phytophthora soybean and the detection method thereof of the present invention belong to the field of biotechnology. It is specially used for the high-sensitivity and rapid detection of Phytophthora sojae carried by soybeans imported and exported by customs, and can also be used for early diagnosis of soybean blight in the field and monitoring of pathogens. (2) Background technology [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 state...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王源超郑小波王立安张文利
Owner NANJING AGRICULTURAL UNIVERSITY
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