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Immunomagnetic separation-ELISA detection method of alicyclobacillus in fruit juice

A technology of bacillus and detection method, which is applied in the detection field of food microorganism detection, can solve the problems of high detection limit, poor timeliness and reliability, etc., achieves the effect of less sample consumption, guaranteed identification and separation, and fast speed

Active Publication Date: 2014-09-10
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a relatively low detection limit and fast and accurate ELISA detection method for Alicyclobacillus in fruit juice, to solve the relatively high detection limit, timeliness and reliability of the prior art. relatively poor problem

Method used

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  • Immunomagnetic separation-ELISA detection method of alicyclobacillus in fruit juice
  • Immunomagnetic separation-ELISA detection method of alicyclobacillus in fruit juice
  • Immunomagnetic separation-ELISA detection method of alicyclobacillus in fruit juice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] (1) Silanization reaction of magnetic microspheres

[0074] Dissolve 9.5g of sodium silicate in 200mL of deionized water and adjust the pH value to 12-13 with hydrochloric acid, add 5g of Fe to the sodium silicate solution 3 o 4 Ultrasonic treatment with magnetic core for 30 minutes, adjust the pH of the reaction system to 6-7 with hydrochloric acid in a water bath at 80-90°C, obtain microspheres by magnetic separation and wash with water to obtain silanized magnetic microspheres with a neutral pH;

[0075] (2) Amination modification of magnetic microspheres

[0076] Add 2 grams of silanized magnetic microsphere suspension, 1mL deionized water and 10mL APTES to 150mL methanol, add 150mL glycerin after ultrasonic treatment for 30min, fully react at 85-90℃ for 3h, obtain microspheres by magnetic separation, and use methanol and deionized water Washing three times respectively, freeze-drying to obtain aminated magnetic microspheres, and the particle diameter of the amina...

Embodiment 2

[0082] This example differs from example 1 in that the Fe used in this example 3 o 4 The magnetic core is prepared by the following method:

[0083] 3.4g of FeCl 3 ·6H 2 O with 1.2g FeCl 2 4H 2 O was added to 800mL deionized water, and 4g PEG-4000 was added, fully stirred and dissolved under the protection of nitrogen, and ammonia solution was added dropwise to make the pH of the reaction system greater than 10; after that, the reaction system was fully matured in a water bath at 75-85°C for 20-40min , the microspheres were obtained by magnetic separation and washed with water to obtain Fe 3 o 4 Magnetic nucleus; the Fe was detected 3 o 4 The magnetic core particle size is 15-25nm, and the magnetic saturation magnetization is 72.18emu / g.

Embodiment 3

[0085] The difference between this example and Example 1 is that the immunomagnetic microspheres of this example are blocked by a blocking solution: every 100ml of PBS (0.01M, pH7.4) buffer contains 5g of BSA and 0.05ml of Tween-20 The mixed system is the blocking solution, and the immunomagnetic microspheres adsorbing antibodies are added to act for 60 minutes at 37°C, and then magnetically separated and fully washed to obtain the immunomagnetic microspheres after blocking treatment.

[0086] One, utilize the immunomagnetic microsphere prepared in embodiment 3 to separate Alicyclobacillus:

[0087] The specific operation steps are: prepare 10 mL of the concentration gradient samples shown in Table 1, add 20 mg of immunomagnetic microspheres to each sample to separate and remove the target bacteria: 120-180 rpm, reaction adsorption time 60 min; magnetic separation and adsorption of bacteria The immunomagnetic microbeads were washed with sterile water and suspended in 200 μL of P...

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Abstract

The invention discloses an immunomagnetic separation-ELISA detection method of alicyclobacillus in fruit juice. The method includes the steps: separating and enriching the alicyclobacillus in the fruit juice sample by using alicyclobacillus immunomagnetic microspheres, so as to obtain an enriched sample to be detected; and detecting whether the alicyclobacillus exists in the enriched sample to be detected by using an ELISA detection method, wherein the used alicyclobacillus immunomagnetic microspheres are obtained by coating aminated magnetic microspheres with oxidized alicyclobacillus specific antibodies. Through antigen-antibody specific recognition, the alicyclobacillus is adsorbed to the surface of the immunomagnetic microspheres, the alicyclobacillus sample is obtained due to separation and enrichment under the action of a magnetic field, and a target thallus is identified with an ELISA detection system. Compared with a conventional ELISA detection method, the method has the characteristics of low detection limit, and more reliable timeliness and accuracy.

Description

technical field [0001] The invention belongs to the detection technical field of food microorganism detection, and in particular relates to an immunomagnetic separation-ELISA detection method for Alicyclobacillus in fruit juice. Background technique [0002] The ELISA detection method of Alicyclobacillus in existing fruit juice mainly contains: [0003] Document 1: Zhouli Wang, Tianli Yue, Yahong Yuan, et al. Development of Polyclonal Antibody-Based Indirect Enzyme-Linked Immunosorbent Assay for the Detection of Alicyclobacillus Strains in Apple Juice. Journal of food science, 2012, 77: M643-M649 published ELISA detection of Alicyclobacillus in fruit juice; and [0004] Document 2: Jianke Li, Kai Xia, Chaozhou Yu. Detection of Alicyclobacillus acidoterrestris in apple juice concentrate by enzyme-linked immunosorbent assay. Food control, 2013, 30:251-254. ELISA detection of Alicyclobacillus disclosed in. [0005] The detection limit of the ELISA detection method disclosed i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/531
Inventor 岳田利王周利袁亚宏蔡瑞牛晨郭彩霞
Owner NORTHWEST A & F UNIV
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