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Simultaneous detection of aflatoxin production and nor-1 gene transcription rt-pcr kit and its detection method

A RT-PCR, aflatoxin technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as falsehood, and achieve reliable results, reliable ratio results, and low cost.

Active Publication Date: 2021-06-22
OIL CROPS RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But the weak point of this kind of method is: just have at least about 20% bacterial strains not produce toxin naturally because of the deletion of the toxin-related gene except detection gene under natural state, but detection gene can still be expressed, thereby causes this kind of strain. method spurious results

Method used

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  • Simultaneous detection of aflatoxin production and nor-1 gene transcription rt-pcr kit and its detection method
  • Simultaneous detection of aflatoxin production and nor-1 gene transcription rt-pcr kit and its detection method
  • Simultaneous detection of aflatoxin production and nor-1 gene transcription rt-pcr kit and its detection method

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Experimental program
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Effect test

Embodiment 1

[0037] 1. The research has proved that the ratio of aflatoxin production to Nor-1 gene transcription has very good relative stability

[0038] Weigh 1g NaNO 3 , 1g K 2 HPO 4 , 0.5g MgSO 4 ·7H 2 O, 0.5g KCl, 0.01g FeSO 4 , 30 g of glucose and 20 g of agar powder were made up to a total volume of 1000 ml with deionized water, and sterilized by high-temperature steam at 121° C. for 30 minutes to prepare a CDA medium. The Aspergillus flavus strain was cultivated for 10 days at 28° C. and 90% humidity with CDA solid culture, and then the culture plate was washed with 20% Tween-20 to obtain the Aspergillus flavus spore solution. The Aspergillus flavus spore solution was oscillated evenly with a vortex shaker by the hemocytometer plate counting method, and the Aspergillus flavus spore solution was microscopically counted with a microscope.

[0039] Put 15mL potato dextrose liquid culture medium in a 50mL Erlenmeyer flask, autoclave at 121°C for 30min, add Aspergillus flavus spo...

Embodiment 2

[0046] The establishment of the simultaneous detection RT-PCR method of embodiment 2 aflatoxin output and Nor-1 gene transcription amount

[0047] Using the existing phage VHH 2-5 displaying aflatoxin anti-idiotypic nanobody on the surface and the nor-1 gene DNA fragment Tq-nor1, a RT-PCR method for simultaneous detection of aflatoxin production and Nor-1 gene transcription was established According to the above detection results as the scientific basis for the identification and evaluation of the virulence of Aspergillus flavus strains, it provides method support for the rapid identification and evaluation of the virulence of Aspergillus flavus strains. These specific steps are as follows.

[0048] The phage VHH 2-5 displaying aflatoxin anti-idiotypic nanobodies on the surface was developed by the Quality Inspection Center of the Institute of Oil Crops, Chinese Academy of Agricultural Sciences, and has been published in the journal literature "Yanru Wang; Peiwu Li; ZuzanaMajk...

Embodiment 3

[0101] 1. The comparative analysis of the virulence of Aspergillus flavus was evaluated by Nor-1 transcript and AFT / Nor-1 (ratio of toxin production and nor-1 transcript) respectively

[0102] Through the comparative analysis of the results, we found that if the virulence of Aspergillus flavus was evaluated only from the amount of nor-1 transcript, the identification result was unreliable. For example, in Aspergillus flavus strains Pc124-2 and Pc34-1, the logarithms of the copy numbers of nor-1 gene expression were 7.85±0.52 and 6.75±0.58, respectively, and the expression levels of nor-1 gene in the two strains were comparable; The yield of aspergillus toxin was 66.5±4.93ng / mL, and the amount of aflatoxin produced by Pc34-1 was only 19.69±2.27ng / mL; in addition, no aflatoxin was detected in CY1, CY2, Pg28-1 and Pc321-1-3 strains. Aspergillus-producing, however, Pg28-1 and Pc321-1-3 expressed even higher levels of the nor-1 gene than some of the aflatoxin-producing strains.

...

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Abstract

The invention belongs to the field of biology, and in particular relates to an RT-PCR kit for synchronous detection of aflatoxin output and Nor-1 gene transcription and a detection method thereof. The present invention has established the synchronous RT-PCR detection method that synchronously detects the toxin production amount of the Aspergillus flavus strain and the nor-1 gene transcription amount, utilizes described method to detect the toxin production amount of the gained Aspergillus flavus strain, the nor-1 gene transcription amount and adopts HPLC quantification There is a good linear relationship between the results of aflatoxin and Nanodrop quantification of the amount of Nor-1 gene transcription. Therefore, the ratio of AFT / Nor-1 obtained based on the simultaneous detection RT-PCR method is reliable and accurate, and can be used to determine the production of Aspergillus flavus strains. The identification index of virulence level realizes the rapid and accurate identification and evaluation of the virulence of Aspergillus flavus strains, which is of great significance for the early warning and prevention and control of aflatoxin pollution.

Description

technical field [0001] The invention belongs to the field of biology, and in particular relates to a RT-PCR kit for simultaneous detection of aflatoxin output and Nor-1 gene transcription and a detection method thereof. Background technique [0002] Aflatoxin is the most toxic type of mycotoxin found so far. Taking aflatoxin B1 as an example, its toxicity is 10 times that of potassium cyanide and 68 times that of arsenic. It is listed as a class I carcinogen by the International Cancer Organization. Aflatoxins can easily contaminate grain and oil crops such as peanuts, corn, and rice, and also contaminate many plant products such as walnuts, pistachios, and Chinese herbal medicines. There have been many cases of human and livestock poisoning incidents caused by aflatoxin at home and abroad. According to the latest report from the International Agency for Research on Cancer (IARC), about 500 million people in developing countries alone are at risk of exposure to aflatoxins. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2531/113C12Q2537/143C12Q2563/107C12Q2545/114
Inventor 李培武李慧张奇唐晓倩白艺珍张文
Owner OIL CROPS RES INST CHINESE ACAD OF AGRI SCI
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