Method for quantitatively detecting ustilaginoidea virens from seed-borne and soil-borne media

A technology for quantitative detection of rice false smut bacteria, applied in the field of microorganisms, can solve the problems of unsuitable for large-scale rapid application in the field, long detection time, high detection cost, etc., and achieve high specificity, simple method, and high sensitivity

Inactive Publication Date: 2014-09-10
CHINA AGRI UNIV
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Problems solved by technology

[0008] In recent years, there have been reports that PCR-based methods have been successfully used in the detection of rice false smut. Conventional PCR methods have greatly improved the operating time and detection specificity and sensitivity compared with other methods. However, PCR detection technology requires special Equipment and testing costs are high, and its testing time is still relatively long (2 to 3 hours), which is not suitable for large-scale rapid application in the field

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  • Method for quantitatively detecting ustilaginoidea virens from seed-borne and soil-borne media
  • Method for quantitatively detecting ustilaginoidea virens from seed-borne and soil-borne media
  • Method for quantitatively detecting ustilaginoidea virens from seed-borne and soil-borne media

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Experimental program
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Effect test

Embodiment 1

[0040] 1. Pre-treat the tested samples and quickly extract the DNA of the tested samples

[0041] (1) Seed DNA extraction steps: Weigh the rice seeds according to the thousand-grain weight standard, collect the seed shells after threshing, and operate according to the operating instructions of the Promega Plant Genomic DNA Extraction Kit (Promega (Beijing) Biotechnology Co., Ltd.) .

[0042] (2) Soil DNA extraction step: add 2ml SPCB buffer (100mmol / LTris-HCl, 100mmol / L EDTA, 120mM Na 3 PO 4 , mass fraction is 2% CTAB, 1.5M NaCl, pH8.0, volume percentage is 2% mercaptoethanol), the ratio of 100 μ g / ml proteinase K, 37 ℃ process 30-60min, then in final concentration 1% ( mass percent concentration) in SDS solution at 65°C for 1-2 hours, centrifuge (at room temperature) to take the supernatant, and extract once with an equal volume of chloroform to remove the protein. If the interface between water and chloroform solution is not clear after extraction, Consider re-extracting ...

Embodiment 2

[0059] Specificity analysis of embodiment 2 RealAmp detection

[0060] Other rice pathogens used for specific analysis are rice blast (Magnaporthe grisea), sheath blight (Thanatephorus cucumeris), rice bakanae (Gibberella moniliformis), bacterial blight (Xanthomonas oryzae pv.oryzae), bacterial Stripe (Xanthomonas oryzae pv.oryzicola), and the insecticidal fungus Metarhizium anisopliae (see Table 1 for details).

[0061] The product electrophoresis detection results after constant temperature amplification showed that only the template was rice smut (Ustilaginoidea virens) genomic DNA showing gradient bands, and the rest of the pathogenic DNA had no gradient bands (see figure 1 ); All bacterial strains adopt the nested PCR detection of rice false smut bacterium, and the result shows, only has the specific band of 232bp amplified by the rice false smut genomic DNA, and other controls have no amplified product (see figure 2 ); the ESE-Quant Tube Scanner instrument has only one...

Embodiment 3

[0066] The analysis of embodiment 3 seed carrier detection sensitivity and standard curve construction thereof

[0067] Use the pMD-18-T-UV plasmid DNA containing the RealAmp amplification region as a template, and use the healthy testa DNA as water to dilute the pMD-18-T-UV plasmid DNA sequentially from 38ng / μl by 10-fold gradient serial dilution method to 3.8×10 -6 ng / μl, sterile water was used as negative control. The results showed that, RealAmp can be obtained from pMD-18-T-UV plasmid DNA concentration is 38ng / μl~3.8×10 -4 The products amplified from template DNA with 6 orders of magnitude such as ng / μl were all green after staining with SYBR GreenI, and the negative control was colored orange, indicating that the lower limit of detection of Aspergillus oryzae DNA by RealAmp is about 3.8×10 -4 ng / μl (see Figure 4 );

[0068] The amplification curves of ESE-Quant Tube Scanner showed a highly linear relationship between different concentrations of starting template DNA...

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Abstract

The invention discloses a method for quantitatively detecting ustilaginoidea virens from seed-borne and soil-borne media. The method comprises the steps of (1) pre-treating a detected sample, and quickly extracting DNA of the detected sample; (2) constructing a standard curve; and (3) performing reaction by using a fluorescent nucleic acid thermostatic amplification detection technology. The method can be used for quantitatively detecting the ustilaginoidea virens, overcomes the defects of the conventional first generation LAMP (loop-mediated isothermal amplification) detection method, and has the advantages of high sensitivity, high specificity, low pollution, stable reaction and the like. By adopting the strategy of sensitively detecting the reaction product by adopting a closed-tube detection technique, the method disclosed by the invention is unlikely to be affected by pollutants and can be used for detecting a soil sample on site and analyzing and judging the reaction product, so that the method is extremely simple and suitable for being widely popularized and applied.

Description

technical field [0001] The invention relates to the field of microbes, in particular to a method for quantitatively detecting rice smut bacteria from seed-borne and soil-borne media. Background technique [0002] Rice false smut is a fungal disease of rice ears caused by the infection of Ustilaginoidea virens (Villosiclava virens). Pathogen infection leads to the formation of pathogen colonies on rice florets that are several times the size of rice grains, namely Rice curlers. In recent years, with the promotion and large-scale planting of high-quality hybrid rice, the application of a large amount of nitrogen fertilizer has created favorable conditions for the occurrence and prevalence of rice false smut. one of the diseases. The occurrence of rice false smut not only has a great impact on the yield of rice, but also the rice false smut can produce a large amount of toxins, which have a strong inhibitory effect on the cell division of humans, animals and plants. Therefor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06
CPCC12Q1/6851C12Q2545/114C12Q2547/107C12Q2563/107
Inventor 孙文献韩彦卿彭军张楠方安菲张亢
Owner CHINA AGRI UNIV
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