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Method for detecting sugarcane ratoon stunting disease pathogen

A detection method and sugarcane technology are applied in the field of sugarcane ratoon dwarf pathogen detection, which can solve the problems that the PCR method detection system is not easy to stabilize, is not suitable for rapid detection, and has low detection sensitivity, and achieves easy visual judgment, labor saving, and simple operation. Effect

Inactive Publication Date: 2009-02-11
SUGARCANE RES INST OF YUNNAN ACADEMY OF AGRI SCI
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Problems solved by technology

However, the stem dissection detection method and the phase contrast microscope detection method have poor accuracy, cumbersome operation, low detection sensitivity, and the PCR method detection system is not easy to stabilize, so it is not suitable for rapid detection of large quantities of samples in the field

Method used

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Embodiment Construction

[0012] 1. Sample preparation and tissue imprinting: Take the middle and lower stem nodes of the sugarcane sample to be tested, cut them into 9-11cm long with a machete, drill out the middle part with a rotor, and then cut it into a plane with a sharp blade. Press vertically on the nitrocellulose membrane for 10-15 seconds to obtain blots;

[0013] 2. Blocking: Pour the blocking buffer into a petri dish with a nitrocellulose membrane, and incubate at 37°C for 45 minutes;

[0014] 3. Quickly wash the membrane once with TBST buffer: Pour off the blocking buffer in the petri dish, add TBST buffer, immerse the nitrocellulose membrane in it, turn it gently a few times, and then discard the TBST buffer.

[0015] 4. Adding antibodies: immerse the nitrocellulose membrane in TBST buffer solution containing 0.1% sugarcane perennial dwarf pathogen specific antiserum and 1% skimmed milk powder, incubate at 37°C for 2 hours, wash the membrane 3 times with TBST buffer solution, each time 3m...

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Abstract

The invention provides a method for detecting sugarcane ratoon stunting virus. The method comprises the following steps of marking the tissue spot of a sample to be detected on nitrocellulose films; soaking the films into a sealed buffer solution containing 0.01% of specific antiserum of the sugarcane ratoon stunting virus to incubate; transferring the films to a TBST buffer solution containing 0.1% of alkaline phosphatase labeled antibodies and 1% of skimmed milk powder; transferring the films into the primer / buffer solution (BCIP / NBT) to develop; and determining the result. If the tissue spot appears mauve, the sugarcane ratoon stunting virus exists in the sample; and if no color is developed on the tissue spot, no sugarcane ratoon stunting virus exists in the sample. The method can rapidly and accurately detect the sugarcane ratoon stunting virus, and has the advantages that the detection result is easily judged by the visual observation, the operation is simple, the sampling amount is less, the labor consumption is reduced, the result is reliable, the contamination is prevented, no special instrument is in demand, and the method is suitable for detecting the large-scale field samples.

Description

Technical field: [0001] The invention relates to a fast, accurate and easy-to-operate method for detecting dwarf pathogens of sugarcane perennial roots, which belongs to the field of plant protection. Background technique: [0002] Sugarcane ratoon dwarf disease (RSD) is a worldwide important disease that is ubiquitous in all sugarcane growing areas. Since it was first discovered in Queensland, Australia from 1944 to 1945, 47 countries and regions including the United States, South Africa, Mauritius, India, and Brazil have reported the occurrence of the disease, and it has spread all over the sugarcane areas of the world. The occurrence of this disease was first reported in Taiwan Province of my country in 1945, and sugarcane perennial root dwarf disease was diagnosed in mainland China in 1986. Afterwards, Guangdong, Fujian, Guangxi, and Yunnan conducted general surveys of sugarcane areas in the whole province, and the results showed that this disease existed in all of them....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/544G01N21/78
Inventor 李文凤黄应昆吴才文卢文洁罗志明
Owner SUGARCANE RES INST OF YUNNAN ACADEMY OF AGRI SCI
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