Synchronous test kit of fungi to produce aflatoxin, ochratoxin and patulin

A technology for ochratoxin and aflatoxin, applied in the field of biological detection, can solve the problems of limited sequence homology, inability to exclude false negative results, no internal reference gene design, etc., to avoid false negatives and false positives, ensure sensitivity, specific effect

Active Publication Date: 2018-06-12
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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  • Application Information

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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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  • Synchronous test kit of fungi to produce aflatoxin, ochratoxin and patulin
  • Synchronous test kit of fungi to produce aflatoxin, ochratoxin and patulin
  • Synchronous test kit of fungi to produce 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 synchronous test kit of fungi to produce aflatoxin, ochratoxin and patulin, which, based on a multiple PCR (polymerase chain reaction) detection method, includes a first primer pair, a second primer pair, a third primer pair and an internal standard primer pair which may carry out PCR in a same reaction tube. A test system herein directly targets at genes necessary to toxin synthesis and has high specificity; internal reference genes are amplified synchronously; positive, negative and blank controls are provided; false negative and false positive are effectively avoided; the PCR index doubling ensures method sensitivity.

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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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)