A simultaneous detection kit for fungi producing aflatoxin, ochratoxin and patulin

A technology for ochratoxin and aflatoxin, applied in the field of biological detection, can solve the problems of undesigned internal reference gene, unable to exclude false negative results, limited sequence homology, etc., to ensure the sensitivity of the method, avoid false negatives and false positives , the effect of saving labor costs

Active Publication Date: 2021-06-04
厦门市产品质量监督检验院(国家半导体发光器件应用产品质量监督检验中心、国家场(厂)内机动车辆质量监督检验中心)
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Problems solved by technology

[0005] So far, at home and abroad, only the team led by Córdoba of the University of Extremadura in Spain reported a multiplex PCR system for the simultaneous detection of the above three types of toxin-producing bacteria in food in 2013 (LUQUE MI, ANDRADE MJ, RODRIGUEZ A , et al.Development of a multiplex PCR method for the detection of Patulin-, Ochratoxin A-and Aflatoxin-producing molds in foods[J]. FoodAnalytical Methods, 2013, 6(4): 1113-1121.), found after repeated verification There are the following defects: no internal reference gene is designed in the system, and possible false negative results caused by poor sample DNA quality or inappropriate PCR system cannot be ruled out; after comparing the fungal idh gene sequences available in NCBI, the sequence homology is limited , it is necessary to design merger primers to effectively cover different species of toxin-producing bacteria; the omt primers used are prone to non-specific amplification fragments in the DNA of other non-toxigenic bacteria

Method used

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  • A simultaneous detection kit for fungi producing aflatoxin, ochratoxin and patulin
  • A simultaneous detection kit for fungi producing aflatoxin, ochratoxin and patulin
  • A simultaneous detection kit for fungi producing aflatoxin, ochratoxin and patulin

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Embodiment 1

[0032] 1. Extraction of fungal genome

[0033] Toxic positive bacteria confirmed by HPLC (AF producing bacteria Aspergillus flavus CGMCC 3.4408, OTA producing bacteria Aspergillus ochrae CGMCC 3.4411 and PAT producing bacteria Aspergillus clavus CGMCC 3.6890, such as Figure 1 to Figure 3 shown) as a positive control; a strain that does not produce the above three types of toxins (Fusarium graminearum ATCC 46779) as a negative control.

[0034] Inoculate the working bacteria (spores or hyphae) of the above-mentioned strains on a PDA plate with cellophane on the surface, culture at 25°C for 3-4 days, and harvest the mycelium when the mycelium is covered with the culture dish and no spores are produced, refer to the following method Extraction of DNA: Weigh 0.2 g of mycelium, place it in a pre-cooled mortar, and grind it to powder with liquid nitrogen. Then add 4mL DNA extraction solution [0.2M Tris·HCl (pH 7.5), 0.5M NaCl, 10mM EDTA, 1% SDS (w / v)], continue grinding, transfer to...

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Abstract

The invention discloses a simultaneous detection kit for fungi producing aflatoxin, ochratoxin and patulin, which is based on a multiplex PCR detection method and includes a first pair of primers and a second pair of primers capable of performing PCR reactions in the same reaction tube , the third primer pair and the internal standard primer pair. The detection system of the present invention directly targets genes necessary for toxin synthesis, and has strong specificity; internal reference genes are synchronously amplified, and positive, negative and blank controls are set at the same time, effectively avoiding false negatives and false positives; PCR index multiplication ensures the sensitivity of the method.

Description

technical field [0001] The invention belongs to the technical field of detection biology, and in particular relates to a simultaneous detection kit for fungi producing aflatoxin (AF), ochratoxin (OTA) and patulin (PAT). Background technique [0002] AF, OTA and PAT can destroy the normal function of the body and even cause cancer. They are the most concerned and most harmful biological toxins at home and abroad. Penicillium and Aspergillus are the main producers of the above three toxins, among which many species, such as Aspergillus flavus, A. parasiticus, A. ochraceus, A. carbon niger ( A.carbonarius), Aspergillus clavatus (A.clavatus), Penicillium expansum, and Penicillium griseofulvum (P.griseofulvum) produce toxins in the secondary metabolism process, and enter the food chain of humans and animals along with grains, feed, food, etc. Cause huge economic losses and seriously threaten health. [0003] For AF, OTA, and PAT, mature control and detection specifications have...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/16C12Q2600/166
Inventor 谢雪钦高静陈剑林珠方荣谦
Owner 厦门市产品质量监督检验院(国家半导体发光器件应用产品质量监督检验中心、国家场(厂)内机动车辆质量监督检验中心)
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