Primer set, kit and detection method for multiplex PCR detection of aflatoxin-producing fungi

Abstract

The invention relates to the field of biotechnology and provides a multiple PCR detection primer group, a kit and a detection method for fungus producing aflatoxin. According to the invention, through combined utilization of a fungus universal molecular marker sequence (internal transcribed spacer, ITS) and three key genes, comprising an aflatoxin production regulating gene (aflR), a sterigmatocystin-o-methoxy transferase gene (omt-1), and versicolorin A dehydrogenase gene (ver-1), in the biochemically synthetic process of the aflatoxin, the multiple PCR detection primer group, the kit and the detection method have the technical effects of wide coverage range, strong specificity and high sensitivity during the detection of the fungus producing aflatoxin. The primer group, the kit and the detection method are short in detection time, are simple in operation and are low in device requirement, can save large amount of manpower, materials and costs, are suitable for quick detection and are easy to promote in practical production.

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to a primer set, a kit and a detection method for rapid detection and identification of toxin-producing fungi by using multiple PCR technology. Specifically, the present invention relates to a primer set, a kit and a detection method for multiplex PCR detection of aflatoxin-producing fungi. Background technique [0002] Aflatoxin (AFT) is a class of secondary metabolites with similar chemical structures mainly produced by fungi such as A.flavus, A.parasiticus and A.nomius. As many as 18 different aflatoxins have been isolated, including AFB1, AFB2, AFG1, AFG2, AFM1, AFM2, AFB2a, AFG2a, AFBM2a and AFGM2a, among which AFB1 is the most toxic and the most stable in chemical properties. It has a strong Carcinogenicity, mutagenicity and teratogenicity, it is an internationally recognized class A carcinogen. A large number of investigations and studies have confirmed that the main targe...

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Problems solved by technology

When the template DNA concentration was reduced to 0.1ng / μL, the aflR gene amplified fragment completely disappeared, indicating that the overall sensitivity of the system was 2ng / μL (see the specific description below for the sensitivity calculation method), considering the extremely strong toxicity of aflatoxin and carcinogenicity, this sensitivity still needs to be improved

In addition, CN103103258A also discloses a method for analyzing aflatoxin in peanut meal by multiplex PCR technology. However, the sensitivity of the method is not mentioned in CN103103258A, so it is difficult to know whether it can meet the actual detection requirements. In fact, CN103103258A The four pairs of primers used in Qin Wenyan et al. (Multiple PCR detection of aflatoxin-producing fungi that can contaminate food and feed, "Acta Mycophyta" 26(3): 448-454, 2007) are completely identical. The same, so it is speculated that its sensitivity is also at most 2ng / μL

[0009] It can be seen that the existing technology cannot fully meet the needs of the detection of aflatoxin-producing fungi

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