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Chemiluminiscence kit for detecting Enniatins and application of chemiluminiscence kit

A chemiluminescence reagent, the technology of Fusarium spp., used in chemiluminescence/bioluminescence, analysis by chemical reaction of materials, chemical instruments and methods, etc., can solve the problem of detection of Fusarium spp. by chemiluminescence In order to achieve the effect of expanding the effective reaction volume, high sensitivity and specificity, and high crossover rate

Pending Publication Date: 2022-02-08
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] 1. Large-scale precision analysis equipment is required, including high-performance liquid chromatography, mass spectrometer, etc., which cannot be equipped by grass-roots laboratories;
[0013] 2. It requires a complex and time-consuming sample pretreatment process;
[0014] 3. The throughput of sample detection is low, and only one sample can be detected at a time;
[0015] 4. Skilled technicians are required. The operation of precision instruments requires personnel training. More importantly, the analysis of test data requires skilled scientific research and technical personnel. It is difficult for ordinary grassroots technicians to meet this requirement.
[0020] In summary, no literature or patents have been found on the detection of enfusarin by chemiluminescence

Method used

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  • Chemiluminiscence kit for detecting Enniatins and application of chemiluminiscence kit
  • Chemiluminiscence kit for detecting Enniatins and application of chemiluminiscence kit
  • Chemiluminiscence kit for detecting Enniatins and application of chemiluminiscence kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1, the preparation of enfusarin monoclonal antibody can be carried out with reference to the conventional monoclonal antibody method, specifically the following steps are carried out in sequence:

[0085] 1. Synthesis of enfusarin immunogen:

[0086]Carbodiimide method (DCC method) is used to couple enfusarin BENB with carrier protein BSA, and enfusarin BENB is any of the following: enfusarin A (ENA), enfusarium Enzyme A1 (ENA1), Enfusarin B (ENB), Enfusarin B1 (ENB1);

[0087] 1) Dissolving carrier protein bovine serum albumin BSA in 0.1-1M MES buffer solution containing 0.05-1M NaCl at pH 4-7, with a final concentration of 2-15mg / ml, to obtain carrier protein bovine serum albumin solution;

[0088] The 0.1-1M MES buffer solution with pH 4-7 containing 0.05-1M NaCl is: add NaCl to the 0.1-1M MES buffer solution (PH4-7) until the concentration is 0.05-1M.

[0089] The final preferred condition is 0.25M MES buffer at pH6, containing NaCl with a final concentrat...

Embodiment 2

[0107] Example 2: Preparation of Enfusarin Chemiluminescent Kit

[0108] 1. Preparation of enfusarin calibrator:

[0109] 1) Preparation of calibrator buffer: weigh 5g Tris base, 9gNaCl, 0.6g Proclin-300, add 900ml of purified water, adjust the pH to pH7.2 with citric acid, add 5g of rabbit serum albumin, and dilute the purified water to 1000ml. After filtering with a 0.22um filter membrane, store it as a calibrator buffer at 2-8°C.

[0110] 2) Dissolve the pure product of enfusarin BENB in ​​dimethyl sulfoxide DMSO to make a stock solution of 1 ug / ml, and then dilute it with calibrator buffer to 2.5 ng / ml, 5 ng / ml, 10 ng / ml, 20ng / ml, 40ng / ml, 80ng / ml, 200ng / ml, plus 0 point, a total of 8 calibrator gradients.

[0111] 2. Preparation of enfusarin magnetic bead conjugates

[0112] 1) Add 500ul 10% (v / v) magnetic beads into a 2ml centrifuge tube, remove the supernatant by magnetic separation;

[0113] 2) Add 1ml of absolute ethanol, mix evenly and remove the supernatant by m...

Embodiment 3

[0137] Embodiment 3: Utilize the kit of the present invention to detect the enfusarin content in the wheat sample

[0138] The kit of the invention is used to test the recovery efficiency of standard addition in wheat samples, and to test the precision and accuracy of the kit.

[0139] Accuracy refers to the degree of agreement between the measured value and the true value, and the accuracy of the kit is often expressed by the recovery rate; precision refers to the repeatability, which refers to the variation when testing the same sample, and is often expressed by the coefficient of variation.

[0140] The detection steps include:

[0141] 1. Weigh 50g of wheat sample, and crush it with a small steel mill at high speed (it can pass through a 200-mesh sieve);

[0142] 2. According to the final concentration of 1 μg / kg, 10 μg / kg and 100 μg / kg, add enfusarin B standard solution to the pulverized wheat sample obtained in step 1;

[0143] 3. Take 5g of the product obtained in ste...

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Abstract

The invention belongs to the field of food safety detection, and particularly relates to a chemiluminescence kit for detecting Enniatins and application of the chemiluminescence kit. The invention discloses a chemiluminescence kit for detecting Enniatins, which comprises a cleaning solution and further comprises the following reagents: an Enniatins calibrator, an Enniatins derivative magnetic bead connector, an enzyme conjugate and a chemiluminescence substrate. The invention also provides application of the chemiluminescence kit for detecting the Enniatins. The chemiluminescence kit is used for detecting the total amount of four Enniatins in a sample. The four kinds of Enniatins are an Enniatins A (ENA), an Enniatins A1 (ENA1), an Enniatins B (ENB) and an Enniatins B1 (ENB1).

Description

technical field [0001] The invention belongs to the field of food safety detection, and in particular relates to a chemiluminescence kit for detecting enfusarin and an application thereof. Background technique [0002] Enniatins (ENNs) are mainly mycotoxins produced by certain species of Fusarium fungus infecting grains such as wheat, barley, rye and oats under humid and low temperature conditions. ENNs are a class of structurally similar mycotoxins that were first discovered in F. oxysporum and subsequently in F. acuminatum, F. avenaceum and F. equiseti ( F. equiseti). So far, more than 20 analogues of ENNs have been found, but only 4 ENNs [enfusarin A (ENA), enfusarin A1 (ENA1), enfusarin B (ENB), Enfusarin B1 (ENB1)] is often detected in food and feed. [0003] ENNs are typical cyclic hexaester peptides containing variable hydroxy acid residues and N-methyl amino acid residues. ENNs have different aliphatic amino acid residues on N-methyl amino acid residues. These r...

Claims

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Application Information

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IPC IPC(8): G01N21/76C07K16/44C12N5/20
CPCG01N21/763C07K16/44G01N2021/757
Inventor 吉小凤杨华肖英平王小骊汪建妹
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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