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Rapid identification method for pathogenic fungi

A technology of pathogenic fungi and fungi, which is applied in the direction of biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of high reagent and labor costs, long time-consuming, and many fungal hyphae, etc., to save time and cost Low, easy-to-operate effect

Active Publication Date: 2013-10-09
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

In this traditional method, the time for fungal culture is relatively long, and a large amount of fungal mycelia is required. The method of extracting fungal DNA takes a long time, consumes high reagent and labor costs, and the steps are cumbersome. Fungal DNA is amplified by PCR. After connecting the vector and transforming the competent bacteria, it will take at least 2 days for the identification of the obtained clones. In addition to the subsequent sequencing time, the traditional identification method will take at least 2 weeks.

Method used

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  • Rapid identification method for pathogenic fungi
  • Rapid identification method for pathogenic fungi
  • Rapid identification method for pathogenic fungi

Examples

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Embodiment 1

[0035] A method for rapidly identifying pathogenic fungi, specifically comprising the steps of:

[0036] (1) Cultivate the fungus to be identified: inoculate the fungus to be identified on PDA medium, and culture in dark at 28°C until the colony grows to 2-3 cm to obtain fungal mycelium.

[0037] (2) DNA extraction of fungi by microwave method: use an inoculation needle to take 2 mg of fresh mycelium (that is, the above-mentioned fungal mycelium) grown on PDA medium into a sterilized 1.5ml centrifuge tube, add 1×TE buffer After oscillating for a few seconds, place in a microwave oven, microwave at 750W for 2 minutes, take it out and put it on ice for 7 minutes, then centrifuge (10000rpm, 1.5min) and transfer the supernatant to a fresh centrifuge tube In the process, the fungal DNA extract was obtained.

[0038] (3) Use fungal universal primers ITS1 and ITS4 to perform PCR amplification on the fungal DNA extract. The PCR amplification system is: 10×Taq buffer 2 μl, 10 pmol of ...

Embodiment 2

[0049] The identification objects in this example include fungal hyphae (mycelium of fungi), sclerotia and conidia, including three main steps of extraction of fungal DNA, amplification of specific PCR fragments, and sequence determination of amplified fragments.

[0050] A method for quickly identifying pathogenic fungi, the specific operation steps are roughly the same as in Example 1, the difference is: (1) Cultivate the fungi to be identified: Aspergillus flavus, Pyrophyllum, Alternaria, Fusarium and white silk fungus were respectively inoculated on PDA medium, cultured in dark at 28°C for 1 to 4 days, until the colony grew to 2-3cm, and Aspergillus flavus, Pyrophyllum, Alternaria, Fusarium and The hyphae of white silk fungus (i.e. fungal mycelium); at the same time, inoculate peanut white silk fungus on PDA medium and culture it in dark at 28°C until 10d to obtain the sclerotium of peanut white silk fungus; Culture medium (CZA) medium was cultivated in the dark at 28°C fo...

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Abstract

The invention relates to a rapid identification method for pathogenic fungi, which specially comprises the following steps: (1) culturing fungi on a solid culture medium; (2) taking fresh fungi, and extracting the DNA of the fungi by using a microwave method; (3) amplifying DNA fragments between ribosomal spacers of the fungi by using ITS1 and ITS4 primers through PCR (polymerase chain reaction); (4) after recovering the PCR fragments, carrying out sequence determination on the fragments; and (5) carrying out comparison on the obtained sequences by using the Blast of NCBI. According to the invention, the identification of unknown pathogenic fungi can be performed within a shorter time and under relatively simple experimental conditions, thereby facilitating the rapid diagnosis for diseases occurring in fields so as to guide the prevention and control of diseases. According to the method, through the identification on 21 kinds of pathogenic fungi on different crops, and the coincidence rate is 100% through sequence comparison. The method is not only applicable to the identification on fresh mycelia, but also is applicable to the identification on conidium and sclerotium of pathogenic fungi, and has the characteristics of rapidness and high efficiency.

Description

technical field [0001] The invention belongs to the field of identification of pathogenic fungi, and in particular relates to a method for rapidly identifying pathogenic fungi. Background technique [0002] Peanut pathogenic fungi harm peanuts, causing yield loss and affecting product quality. In order to effectively prevent and control the disease, it is necessary to quickly identify the pathogen of peanut and prevent and control the disease in a timely and targeted manner. At present, in addition to the traditional morphological identification of fungi, in order to obtain more accurate information, it generally relies on the understanding of fungal genomic DNA information, and the PCR obtained by amplifying the transcriptional spacer in the ribosomal DNA of fungal genomic ribosomal DNA through common fungal PCR primers The product was subjected to sequence analysis. The conventional fungal DNA extraction method is as follows: culture the liquid for 5-7 days to obtain the...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 晏立英廖伯寿雷永黄家权万丽云温少华
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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