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Method for rapidly detecting alternaria tenuissima ketone acid

A technology of finely interleaved strobiluronic acid and strobiluronic acid, which is applied in the field of food testing, can solve the problems of being unsuitable for large-scale and rapid on-site testing, high quality requirements, and expensive equipment, and achieves continuous luminescence. The effect of long time, good specificity and high intensity of luminescent signal

Inactive Publication Date: 2017-05-10
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Commonly used detection methods for TA include gas chromatography, liquid chromatography, liquid chromatography-mass spectrometry, etc., but these methods require high quality equipment and personnel, are time-consuming, and expensive, and are not suitable for large-scale and rapid on-site detection , so there is an urgent need for a simple, rapid and highly sensitive detection method

Method used

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  • Method for rapidly detecting alternaria tenuissima ketone acid
  • Method for rapidly detecting alternaria tenuissima ketone acid
  • Method for rapidly detecting alternaria tenuissima ketone acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Polyclonal Antibody to Alternaria Acid

[0044] Take 6-8 week-old Balb / c female mice and immunize them by subcutaneous injection on the back of the neck. For the first immunization, TAO-KLH was mixed with an equal volume of Freund's complete adjuvant and completely emulsified; 21 days later, the booster was immunized with the above-mentioned TAO-KLH mixed with Freund's incomplete adjuvant; the booster was given every 2 weeks. Blood was collected from the orbit 7 days after the fifth immunization. The supernatant was collected by centrifugation, mixed with an equal volume of glycerol, and stored in a -20°C refrigerator.

[0045] Potency Determination of Polyclonal Antibody to Alternaria tenoneric Acid

[0046] At intervals of 7 days after immunization, blood was collected by docking the tail, and the serum titer was detected by indirect ELISA. by OD 450nm The dilution factor when the value is close to 1.0 is taken as the serum titer. Titer changes as figure 1 shown...

Embodiment 2

[0048] (1) Take the ELISA plate, add alternarione acid and BSA conjugate (TAO-BSA) diluted in carbonate buffer to each well, incubate at 4°C for 12 hours, and then wash the plate with PBST buffer Twice, pat dry, then add 5.0% skimmed milk powder to each well at 200 μL / well, block at 37°C for 60 min, wash the plate twice with PBST, and pat dry; obtain a TAO-BSA-coated ELISA plate with a coating concentration of 1.0 μg / mL, the coating amount is 100 μL / well; the concentration of the carbonate buffer is 0.05mol / L, and the pH is 9.6; the concentration of the PBST buffer is 0.01mol / L, and the pH is 7.4;

[0049] (2) Dilute the 1.0mg / mL alternaria tenoneric acid standard solution with PBS buffer solution to 7 concentrations of 0.018, 0.054, 0.164, 0.494, 1.481, 4.444, 13.333, 40ng / mL, and then Each 50 μL of the Alternaria tenoic acid standard solution of different concentrations was added to the standard well of the microplate in step (1), and then 50 μL of the preparation prepared i...

Embodiment 3

[0051] Influence of the pH value of PBS buffer on the detection results

[0052] Since the pH of the reaction system can directly affect the binding between the antigen and the antibody, thereby affecting the detection limit and sensitivity of the method, the pH of the diluted PBS buffer was adjusted to 5.8, 6.4, 7.4, and 8.0, and the remaining steps were performed according to Example 2. Experimental results such as figure 2 shown by figure 2 It can be seen that IC 50 As the pH increases, it first decreases slowly and then increases, and the RLU max Rise first and then fall, RLU max / IC 50 It is the highest when the pH is 7.4, so the pH of the PBS buffer is selected to be 7.4.

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Abstract

The invention relates to a method for rapidly detecting alternaria tenuissima ketone acid. The method is based on a horse radish peroxidase catalysis luminol-hydrogen peroxide luminescence system, indirect competitive chemiluminescent enzyme-linked immunosorbent assay of the alternaria tenuissima ketone acid is built, and the method has the advantages of high sensitivity of a chemiluminescence and high specificity of an immunoassay. The detection range of the method is 0.032ng / mL-30.244ng / mL, IC50 (half maximal inhibitory concentration) is 0.973ng / mL, lowest detection limit is 0.010ng / mL, a linear regression equation is y=49.82-20.14x, and R20=0.9966. Compared with a traditional ELISA (enzyme-linked immunosorbent assay) method, the IC50 and the lowest detection limit are lower and high in specificity, duration time of light-emitting signals is long and can reach several hours, linear detection range is wider, and the method provides bases for research of limit standards of the alternaria tenuissima ketone acid, detection of pollution level of the alternaria tenuissima ketone acid and risk analysis.

Description

technical field [0001] The invention belongs to the field of food detection and relates to a method for rapidly detecting alternaria gracilis acid. Background technique [0002] Alternariol (tenμazonic acid, TA) is one of the toxic metabolites produced by Alternaria, Magnaporthe oryzae, Pointer sorghum, etc. under specific conditions. ), Alternariol monomethyl ether (Alternariol monomethyl ether, AME), an Alternaria mycotoxin discovered. TA is a natural poison widely present in grains, vegetables, and fruits. It can be produced during growth, processing, and storage. TA contamination has been found in agricultural products such as wheat, tomato paste, edible oil, and wine. Among the various Alternaria mycotoxins, TA is the most toxic, and long-term consumption of food contaminated by TA may seriously endanger human health. The oral semi-lethal dose of male mice is 182mg / kg, and the oral semi-lethal dose of female mice is 81mg / kg. Some research reports pointed out that whe...

Claims

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Application Information

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IPC IPC(8): G01N33/535G01N33/543G01N21/76
CPCG01N33/535G01N21/76G01N33/543G01N2333/37
Inventor 马良钟红张宇昊
Owner SOUTHWEST UNIVERSITY
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