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 and high sensitivity

Inactive Publication Date: 2005-05-25
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Examples

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Effect test

Embodiment 1

[0038] Detect Phytophthora soybean in the soil sample of embodiment 1

[0039] Phytophthora soybean detection kit, including the following components:

[0040] Specific primer sequences for Phytophthora sojae

[0041] Upstream (18bp): 5'CTGGATCATGAGCCCACT 3'(Pso1)

[0042] Downstream (16bp): 5'GCAGCCCGAAGGCCAC 3'(Pso2)

[0043] Kit reaction system

[0044] 1mL detection solution includes: 0.05mmol Tris.Cl, 0.125mmol KCl, 0.005mmol MgCl 2 , 0.01mmoldNTPs, upstream and downstream primers 0.25mmol, 0.1mgBSA, Taq DNA polymerase 100 units, add ultrapure water to prepare 1mL detection solution. The solution was stored in a -20°C freezer. The storage period is 1 year. Example 1 Detection of Phytophthora soybean from soil samples of imported soybeans with bacteria:

[0045] The method that the above-mentioned Phytophthora soybean detection kit is used to detect Phytophthora soybean includes:

[0046] 1) Enrichment of oospores in soil:

[0047] Take 20-100 grams of the soil sam...

example 2

[0059] Example 2 Detection of zoospores of Phytophthora sojae from polluted water

[0060] 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 Phytophthora sojae can be amplified. figure 2

[0061] Note: When using this method for detection, it should be noted that it is best to crush the sample before loading, and the crushed solution should not be placed for a long time. The detection sensitivity of this method is 0.1-1 zoospore per milliliter of polluted water.

example 3

[0062] Example 3 Identification of Phytophthora sojae from diseased soybean tissues

[0063] 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 detection kit for phytophthora.sojae of the invention belongs to the field of crop disease prevention and treatment and plant quarantine. A pair of specific oligonucleotide primers were designed according to the ITS gene sequence of Phytophthora sojae. The kit includes: 1mL detection solution includes: 0.05mmol Tris.Cl, 0.125mmol KCl, 0.005mmol MgCl 2 , 0.01mmoldNTPs, upstream and downstream primers 0.25mmol, 0.1mgBSA, Taq DNA polymerase 100 units, add ultrapure water to prepare 1mL detection solution. The Phytophthora soybean detection kit based on this primer has strong specificity, sensitivity and stability, and provides a rapid, sensitive and specific technical method for the detection of Phytophthora soybean. The picture shows the specific PCR product of 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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Patent Type & Authority Patents(China)
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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