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Method for identifying Aspergillus flavus-resistant peanut seeds through real-time quantitative reverse transcription PCR method

A real-time quantitative and reverse transcription technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as yield impact, kernel yield and oil yield decline, loss of nutrition and economic value, etc., to achieve Improve efficiency, shorten detection time, and achieve high accuracy

Inactive Publication Date: 2015-02-04
LIAONING UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to the impact of the disease on the yield, the kernel and oil yields also decreased significantly
The aflatoxin produced by the stubborn and susceptible Aspergillus flavus in the preservation stage is even more harmful, and loses nutritional and economic value due to serious pollution.

Method used

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  • Method for identifying Aspergillus flavus-resistant peanut seeds through real-time quantitative reverse transcription PCR method
  • Method for identifying Aspergillus flavus-resistant peanut seeds through real-time quantitative reverse transcription PCR method
  • Method for identifying Aspergillus flavus-resistant peanut seeds through real-time quantitative reverse transcription PCR method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 A method for identifying anti-Aflatoxin peanut seeds using real-time quantitative reverse transcription PCR

[0034] (1) The steps are as follows:

[0035] 1. Peanut varieties and Aspergillus flavus varieties

[0036]In this experiment, two peanut lines Fuhua 11 and Hongyazi Silihong (provided by the laboratory of Liaoning University School of Environment) were used.

[0037] In this experiment, the A. flavus NRRL3357 (A. flavus sequenced species, USDA Southern Research Center) strain was used to infect peanut seeds. This species is used in laboratory and field studies around the world because of its infestation stability and high production of aflatoxins B1 and B2.

[0038] 2. Reagents and equipment

[0039] Real-time quantitative PCR instrument (StepOnePlus Real-Time PCR System, Applied Biosciences, USA)

[0040] PCR instrument (ABI 2700, USA)

[0041] Filter spin column (Spin-X; Corning Inc., Corning, USA)

[0042] Waters 2695 High Performance Liquid ...

Embodiment 2

[0082] Example 2 A method for identifying anti-Aflatoxin peanut seeds using real-time quantitative reverse transcription PCR

[0083] Proceed as follows:

[0084] 1. Peanut seeds and Aspergillus flavus species

[0085] The detected peanut seeds are: Fuhua 12 (provided by the Sandstorm Institute of Liaoning Academy of Agricultural Sciences) (RX1)

[0086] The comparison of peanut seeds is: Hongyazi four red (S)

[0087] Aspergillus flavus NRRL3357

[0088] 2, reagent and equipment: with embodiment 1

[0089] 3. Sterilization of peanut seeds

[0090] Complete, undamaged and mature peanut seeds were screened from Fuhua 12 and Hongyazi Sizihong. Before inoculation, sterilize the seeds by immersing them in 0.75% sodium hypochlorite solution for 5 minutes, rinse them with sterile water for 1 minute, and rinse them three times in sterile water for 5 minutes each time. Soak in sterile deionized water for 45 min at room temperature to reach a moisture content of 30%.

[0091] 4....

Embodiment 3

[0112] Example 3 A method for identifying anti-Aflatoxin peanut seeds using real-time quantitative reverse transcription PCR

[0113] Proceed as follows:

[0114] 1. Peanut seeds and Aspergillus flavus species

[0115] The detected peanut seeds are: Hongyazi Xiaobaisha variety (Liaoning Zhengye Peanut Seed Capital Center) (RX2)

[0116] The comparison of peanut seeds is: Hongyazi four red (S)

[0117] Aspergillus flavus NRRL3357

[0118] 2, reagent and equipment: with embodiment 1

[0119] 3, peanut seed sterilization: with embodiment 2

[0120] 4. Aspergillus flavus infects peanut seeds: same as in Example 2

[0121] 5. Preparation of total RNA and cDNA synthesis of peanut seeds infected with Aspergillus flavus: same as Example 2

[0122] 6. Real-time quantitative RT-PCR analysis of the expression of peanut resistance gene: same as Example 2

[0123] Using a real-time quantitative PCR instrument, the fluorescence intensity was measured every 0.2°C during the annealing ...

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Abstract

The invention relates to a method for identifying Aspergillus flavus-resistant peanut seeds through a real-time quantitative reverse transcription PCR method. According to the technical scheme, the method comprises the following steps: sterilizing peanut seeds to be detected and contrast peanut seeds; infecting with Aspergillus flavus; sampling and synthesizing cDNA; respectively taking 1 microliter of the cDNA sample, respectively adding corresponding primers, and detecting fluorescence intensity every 0.2 DEG C from 62 DEG C to 95 DEG C in a annealing stage of each amplification by using a real-time quantitative PCR amplifier. The method disclosed by the invention is capable of confirming genes taking part in a resistance mechanism, thereby providing a method for further indentifying the Aspergillus flavus-resistant characteristic of peanut seeds. According to the method, under a non-field experiment condition, the infecting experiment which lasts for 90 days during the early-stage field experiment is shortened to a period of 7 days needed under a laboratory condition, an Aspergillus flavus-resistant peanut variety can be identified within a relatively short period; the detection time is shortened greatly, the efficiency is improved and the accuracy is high.

Description

technical field [0001] The invention belongs to the field of plant physiological lesions and detection, in particular to a real-time quantitative reverse transcription PCR method, through the detection of three stress response processes of peanut genes infected with Aspergillus flavus, further qualitative identification of peanut seeds against Aspergillus flavus resistance. Background technique [0002] Aflatoxin is a secondary metabolite produced by toxin-producing fungi such as Aspergillus flavus and Aspergillus parasitis, and is the most stable mycotoxin found so far. , the most toxic category. Aflatoxin is commonly found in food and feed. Once the feed contaminated by aflatoxin is ingested by livestock, it extends to humans through the food chain. Low doses of different toxins are acutely or slowly toxic to higher vertebrates and other animals. Toxicity, among which AFB1 is the most toxic and strongly carcinogenic, mainly inducing liver cancer. Aflatoxin contamination...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2561/113C12Q2545/113
Inventor 张慧丽陈林张丽男蒋坤
Owner LIAONING UNIVERSITY