Aflatoxin detection reagent kit based on loop-mediated isothermal gene amplification method

A technology for isothermal gene amplification and detection kits, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of difficulty in large-scale promotion, high equipment and reaction conditions, and increased detection costs. Achieve stable test results, ensure high specificity, and reduce costs

Inactive Publication Date: 2012-07-18
万俊松
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  • Abstract
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Problems solved by technology

However, the processing methods of these methods for detecting mold are limited to traditional fungal culture methods and biological polymerase chain reaction (PCR). The former is very cumbersome to operate and takes 4 to 7 days. The conditions are relatively high, and the detection cost is also increased accordingly, making it difficult to achieve large-scale promotion

Method used

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  • Aflatoxin detection reagent kit based on loop-mediated isothermal gene amplification method
  • Aflatoxin detection reagent kit based on loop-mediated isothermal gene amplification method
  • Aflatoxin detection reagent kit based on loop-mediated isothermal gene amplification method

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Effect test

Embodiment 1

[0033] Based on the Aspergillus flavus ver-1 gene sequence as the amplification template, a set of specific primers F3, B3, FIP and BIP were designed at 25bp to 221bp of the sequence using software and visual inspection. Each primer sequence. For specific results, see figure 1 ,exist figure 1 Among them, F3 and B3 are outer primers, also called short primers, and FIP and BIP are inner primers, also called long primers. Among them, BIP is formed by connecting B2 and BIC, while FIP is formed by connecting F2 and FIC. Four primers identify 6 loci of the target gene.

Embodiment 2

[0035] The three different lysis methods of liquid nitrogen extraction, microwave thermal shock method and simple extraction method can successfully lyse the cells. In comparison, the simple extraction method is simple and easy to operate, and the effect is stable. For this test, three methods were used to lyse the cells. Cell making template LAMP amplification electrophoresis results are as follows figure 2 ,exist figure 2 Among them, 1 represents the LAMP reaction result of the bacteriolysis method of the liquid nitrogen extraction method, 2 represents the LAMP reaction result of the microwave thermal vibration method of lysis method, 3 represents the LAMP reaction result of the traditional reaction method, and 4 represents the no-template control.

Embodiment 3

[0037] The fungi were placed in a water bath at 100°C for 1 min, 2 min, 3 min, 4 min, 5 min, 6 min and 7 min respectively, centrifuged at 10,000 r / min for 1 min, and the supernatant was taken as a template for LAMP detection. Repeated tests showed that the bacteriostasis effect was stable in 5 minutes. For specific results, see image 3 , in the figure, 1~7 represent the reaction display at 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, and 7 minutes respectively, and 8 represents the control without template.

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Abstract

An aflatoxin detection reagent kit based on a loop-mediated isothermal gene amplification method is based on the basic principle of loop-mediated isothermal amplification (LAMP). The loop-mediated isothermal gene amplification method includes particular steps: preparing a culture medium of PDA (potato dextrose agar); cultivating strains; designing a loop-mediated isothermal gene amplification (LAMP) primer; extracting a fungus DNA (deoxyribose nucleic acid) template; and proportionally preparing, adding a PCR (polymerase chain reaction) tube and realizing water bath heat-insulation reaction, agarose gel electrophoresis and the like. The aflatoxin detection reagent kit is combined with a method (GB\T13092-2006) for detecting the total number of mold in feed, the concentration of selected fungus liquid is detected by an MPN (most probable number) method, and the aflatoxin detection reagent kit is prepared according to the method. When used for detecting aflatoxin fungi in the feed and food, the reagent kit is high in specificity and sensitivity, quick in detection and low in cost, can be used for detecting a plurality of samples at the same time, and is applicable to sanitation examination for primary-level feed and worthy of being widely popularized.

Description

technical field [0001] The invention relates to the field of gene detection, specifically a kind of aflatoxin detection reagent based on the constant temperature gene amplification method (LAMP) based on the loop media constant temperature gene amplification method (LAMP), which has the advantages of fast speed and strong specificity. box. Background technique [0002] Aflatoxin (AF for short) is a mycotoxin, mainly produced by Aspergillus flavus and Aspergillus parasitica, which affects the immune system of the body, causes mutagenesis, teratogenicity, and carcinogenicity, and poses a serious threat to animal and human health. . my country's latest feed hygiene standards have strict requirements on mold and toxins. The current detection methods of aflatoxin include thin layer chromatography (TLC), high performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA) and so on. However, the processing methods of these methods for detecting mold are limi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 万俊松
Owner 万俊松
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