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Microdroplet digital PCR detection method for simultaneously detecting fungi producing aflatoxin and ochratoxin

A technology of micro-droplet digital and Aspergillus flavus, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problems of many types of bacteria, time-consuming, inaccurate species identification, etc.

Active Publication Date: 2021-05-14
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, conventional morphological detection methods have certain defects, such as many types of bacteria, complex morphological characteristics, some morphological characteristics and physiological and biochemical indicators are unstable with changes in the environment, inaccurate identification at the species level, and time-consuming, etc. , researchers at home and abroad have developed a series of precise detection systems for multiple toxins that combine pretreatment products such as immunoaffinity columns with chromatography-mass spectrometry, effectively improving the detection ability of mycotoxins in food and feed
However, the existing instrument methods only target the toxin itself, and cannot predict the toxin-producing potential of the sample before or in the early stage of toxin formation, and effectively pre-intervention toxin hazards
Generally speaking, if the toxin has been detected from food or feed, the contamination is already very serious, and the infected product can only be destroyed and disposed of, and the economic loss and harm to the people who ingest the contaminated product cannot be recovered.

Method used

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  • Microdroplet digital PCR detection method for simultaneously detecting fungi producing aflatoxin and ochratoxin
  • Microdroplet digital PCR detection method for simultaneously detecting fungi producing aflatoxin and ochratoxin
  • Microdroplet digital PCR detection method for simultaneously detecting fungi producing aflatoxin and ochratoxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] In this example, the specificity of the primers and probes of Aspergillus flavus and Aspergillus ochrae was verified through the following experiments.

[0039] The main testing instruments used:

[0040] Micropipettes (10 µL, 100 µL, 1000 µL, Eppendorf), Electronic Pipette pipettes (RAININ), centrifuges (5804R, Eppendorf), nucleic acid and protein analyzers (DU640, Beckman), microdrop digital PCR instruments ( Bio-rad) etc.

[0041] Main reagents for detection:

[0042] Brain Heart Infusion Broth (BHI) (Beijing Land Bridge Technology Co., Ltd.), Droplet Generation Oil for Probes (Bio-rad), ddPCR TM Supermix for probes (Bio-rad), the primer probe was synthesized by Sangon Bioengineering Co., Ltd.

[0043] The main steps of detection:

[0044] (1) System configuration

[0045] Droplet digital PCR amplification system: Bio-Rad digital PCR platform reaction system has a total volume of 20 µL, including ddPCR TM Supermix for probes10 µL, upstream and downstream primer...

Embodiment 2

[0062] In this example, based on the detection of the nucleic acids of Aspergillus flavus and Aspergillus ochratum, the multiple systems were screened through the following experiments.

[0063] The reaction system of the double droplet digital PCR method can be determined by adjusting the concentration of primers and probes, annealing temperature and other conditions.

[0064] The specific primer pair and probe sequence used are the same as those in Example 1.

[0065] The main detection instruments used and the main reagents used for detection are the same as in Example 1.

[0066] Experimental steps:

[0067] (1) Mix the two sets of primers according to the ratio of upstream primers: downstream primers: probes = 18:18:5 to form F / R / P master mix 1;

[0068] (2) Will QUR / Och The plasmids were mixed in the ratio of 1:1 to DNA master mix 2;

[0069] (3) Remix the premix 1 in (1) according to the ratio of AflR / Och of 3:1, 2:1, 1:1, 1:2, 1:3 to form the premix 3 again; ...

Embodiment 3

[0075] The inventors of the present invention conducted experiments on the sensitivity and repeatability of the multiplex system through the following steps.

[0076] Combine the two plasmid DNA templates according to QUR / Och After the ratio in the system was mixed to make a premixed solution with a concentration of 20 ng / µL, the premixed solution, 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 A total of 10 gradients were diluted and amplified in the double droplet digital PCR reaction system that had been optimized and screened. The detailed steps were the same as in Example 2. In addition, in order to investigate the stability of the optimized system, three sets of parallel experiments were set up. At the same time, in order to evaluate the difference caused by the operation of personnel to the experimental results, repeated experiments among personnel were carried out.

[0077] Amplification results such as image 3 As shown, the sensitivity ...

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PUM

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Abstract

The invention relates to a primer and a probe for simultaneously detecting aspergillus flavus and aspergillus ochraceus. The invention further relates to a double microdroplet digital PCR detection method for determining the aspergillus flavus and the aspergillus ochraceus in the food. The method comprises the step of using the specific oligonucleotide primer and the probe for detecting the aspergillus flavus and the aspergillus ochraceus. The invention further relates to a double microdroplet digital PCR detection kit for the aspergillus flavus and the aspergillus ochraceus in the food. The kit comprises the specific oligonucleotide primer and the probe. By using the double microdroplet digital PCR detection method, the aspergillus flavus and the aspergillus ochraceus can be simply, quickly, specifically and sensitively detected at the same time.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to oligonucleotide primers and probes for the rapid detection of Aspergillus flavus and Ochraus japonicus with high specificity and high sensitivity and a double microbe containing the primer pair and probe A droplet digital PCR detection kit, a double droplet digital PCR detection method for determining Aspergillus flavus and A. Background technique [0002] Fungi are a class of microorganisms that have cell walls, do not contain chlorophyll, have no roots, leaves and stems, live in a saprophytic or parasitic manner, and can reproduce sexually or asexually. Mycotoxins are toxic secondary metabolites produced by fungi, which widely contaminate plant products such as crops, food and feed. Fungi and the mycotoxins they produce have the characteristics of strong toxicity, stable structure, and small molecular weight. They are widely distributed in nature and have mo...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2600/16C12Q2563/159C12Q2561/101C12Q2563/107C12Q2537/143
Inventor 王娉赵晓美陈颖张庆
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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