Kit for quantitatively detecting Ustilaginoidea virens

A technology for the quantitative detection of rice false smut bacteria, applied in the biological field, can solve the problems of no specific morphological characteristics, small rice smut spores, slow growth rate, etc.

Inactive Publication Date: 2015-05-20
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Traditionally, isolation culture and microscopic examination have also been used to detect the number of rice smut spores in the field; however, there are many kinds of microorganisms in the farmland environment, and the rice smut spores are small, have no specific morphological characteristics, and grow slowly after germination. The traditional method of quantitative detection of rice smut is not only time-consuming and laborious, but also cannot accurately identify and quantify the pathogen

Method used

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  • Kit for quantitatively detecting Ustilaginoidea virens
  • Kit for quantitatively detecting Ustilaginoidea virens
  • Kit for quantitatively detecting Ustilaginoidea virens

Examples

Experimental program
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Effect test

Embodiment 1

[0016] Embodiment 1: the spore quantitative detection of rice false smut

[0017] Genome extraction of spore suspension: Dilute the conidia of rice mistletoe with TE solution to obtain 4.2×10 7 , 4.2×10 6 , 4.2×10 5 , 4.2×10 4 , 4.2×10 3 , 4.2×10 2 , 4.2×10 / mL spore solution. Genomic DNA was extracted by taking 400 μL of the spore suspension, and finally obtaining 50 μL of each DNA sample solution. Take 2 μL respectively as a template for real-time PCR quantitative detection.

[0018] Oryza spp. spore DNA extraction: Add 0.4 g of pickled beads to each sample, put it in a Fast-prep instrument, process the sample for 40 s at a speed of 6 m / s, and repeat three times. Add 400 μL of DNA extraction buffer (10mM Tris HCL, pH7.5; 0.1mM EDTA, 6% SDS, 2% β-mercaptoethanol), mix well, and place in a 65°C water bath for 1 hour. Add 800μL of chloroformphenol (1:1), mix well, centrifuge at 12000g for 10min, and transfer the supernatant to a new centrifuge tube. Add 1 μL of glycogen...

Embodiment 2

[0021] Example 2: Quantitative detection of samples captured by airborne spores

[0022] Quantitative detection of spores of rice false smut in the spore capture samples from August 21 to September 10, 2012 in the rice-growing area of ​​Ninghai County, Zhejiang Province. The 7-day spore capture instrument of Burkard Company was used for spore capture, and the polyester film was divided into small pieces according to the day, and loaded into a centrifuge tube with 400 μL of TE.

[0023] DNA extraction: Add 0.4 g of acid-washed beads to each sample, place in the Fast-prep instrument, process the sample for 40 s at a speed of 6 m / s, and repeat three times. Add 400 μL of DNA extraction buffer (10mM Tris HCL, pH7.5; 0.1mM EDTA, 6% SDS, 2% β-mercaptoethanol), mix well, and place in a 65°C water bath for 1 hour. Add 800μL of chloroformphenol (1:1), mix well, centrifuge at 12000g for 10min, and transfer the supernatant to a new centrifuge tube. Add 1 μL of glycogen (20 μg / μL), 40 μL...

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Abstract

The invention provides a kit for quantitatively detecting Ustilaginoidea virens. The kit comprises an upstream primer UvP292, a downstream primer UvP397, a fluorescence probe UvP333, 10*PCR buffer solution, 2mmol/L of dNTPs, 25mmol/L of Mg<2+> and TaqDNA polymerase, wherein eh sequence of the upstream primer UvP292 is 5'-GAACCAAGAGATCCGTTGTTGAA-3', and the sequence of the downstream primer UvP397 is 5'-GCCGGAGGATACAACCAAAA-3' The kit can highly sensitively detect Ustilaginoidea virens spores surrounding the rice field, especially spores in air surrounding the rice filed, and well facilitates the epidemiology research of the Ustilaginoidea virens.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a technique for quantitatively detecting the number of spores of rice smut bacteria. Background technique [0002] Rice false smut (Ustilaginoidea virens (cooke) Tak) is a worldwide fungal disease. In recent years, with the promotion of susceptible varieties such as hybrid japonica rice and the improvement of soil fertility, the occurrence area and incidence of rice false smut have increased, and even caused serious economic losses in some areas. The occurrence and prevalence of rice false smut is closely related to the amount of bacteria in the field. Quantitative detection of the source of rice false smut in the field is conducive to revealing the prevalence of rice false smut and guiding disease control. Traditionally, isolation culture and microscopic examination have also been used to detect the number of rice smut spores in the field; however, there are many kinds of microorg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/645
CPCC12Q1/6895
Inventor 张震柴荣耀王教瑜王艳丽姜华邱还萍毛雪芹杜新法孙国昌
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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