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A rapid genetic screening method for simultaneous detection of four contaminating bacteria in juice drinks

A technology for contaminating bacteria and beverages, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of time-consuming and labor-intensive, cumbersome detection technology operation, achieve accurate detection results, save detection time, and save detection costs. Effect

Active Publication Date: 2018-11-13
HANGZHOU DIANZI 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 address the defects of the prior art, overcome the cumbersome operation, time-consuming and labor-intensive shortcomings of several contaminating bacteria detection techniques in fruit juice beverages, and provide a primer system consisting of 4 pairs of primers. Combining PCR technology with high-resolution capillary electrophoresis separation technology, so as to realize the detection of contaminating bacteria in fruit juice beverages

Method used

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  • A rapid genetic screening method for simultaneous detection of four contaminating bacteria in juice drinks
  • A rapid genetic screening method for simultaneous detection of four contaminating bacteria in juice drinks
  • A rapid genetic screening method for simultaneous detection of four contaminating bacteria in juice drinks

Examples

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Effect test

Embodiment 1

[0034] (1) Cultivation of fruit juice beverage contaminated bacteria and DNA template extraction:

[0035] Pick single colonies of Penicillium citrinum, Saccharomyces cereviseae, yeast Dekera naardenensis, and Aspergillus niger grown on the plate medium and add them to 10 mL of finished apple juice, mix well to make a bacterial suspension; take 1 mL of the Add the bacterial suspension into a 1.5ml centrifuge tube, centrifuge at 13,000×g for 2 minutes, collect the cell pellet, and remove the supernatant; add 480ul of 50mM EDTA to resuspend the cells thoroughly; add 120ul of 10mg / ml lysobacterium, incubate at 37°C for 60min, Centrifuge at 13,000×g for 2 minutes, and remove the supernatant. Extract the DNA template of fruit juice beverage contaminating bacteria according to the instructions of the Genomic DNA Purification Kit kit of Qiagen company afterwards, and use the ultraviolet spectrophotometer to measure the DNA quality of extracting under the condition of 260nm wavelength...

Embodiment 2

[0041] Take 10 bottles of 500mL fruit juice in the production workshop of a fruit juice beverage factory, use a filter membrane with a pore size of 0.45 μm to filter twice, and use one filter membrane to filter 250mL each time; use Aspergillus niger detection medium for one filter membrane to detect After 7 days of anaerobic culture at 26°C, 18 colonies were detected, and this was used as an experimental control; the other filter membrane was shaken and washed with 1mL sterile water, and the washing liquid was collected and added to a 1.5ml centrifuge tube In medium, centrifuge at 13,000×g for 2min, collect the cell pellet, remove the supernatant; add 480ul 50mM EDTA to resuspend the cells thoroughly; add 120ul of 10mg / ml lysobacterium, incubate at 37°C for 60min, centrifuge at 13,000×g for 2min, remove the supernatant liquid. Afterwards, the DNA template of the fruit juice beverage contaminating bacteria was extracted according to the instructions of the GenomicDNA Purificati...

Embodiment 3

[0045] Lactic acid bacteria (Lactobacillus) is another common contaminating bacteria in fruit juice drinks. This example can prove the detection specificity of the present invention to Penicillium citrinum, Saccharomyces cereviseae, saccharomyces Dekeranaardenensis, Aspergillus niger, that is, the detection method of the present invention does not show characteristics in the presence of only lactic acid bacteria (Lactobacillus) Peak, which means that the presence of lactic acid bacteria (Lactobacillus) will not interfere with the detection.

[0046] In this example, Lactobacillus brevis, Lactobacillus casei, Lactobacillus coryniformi and Lactobacillus plantarum were used as interfering bacteria. The operating steps are as described in Example 1 or 2.

[0047] (1) Cultivation of contaminated bacteria and extraction of DNA templates:

[0048] Pick single colonies of Lactobacillus brevis, Lactobacillus casei, Lactobacillus coryniformi, and Lactobacillus plantarum grown on the p...

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Abstract

The invention discloses a gene rapid screening method capable of simultaneously detecting contaminating bacteria in fruit juice drinks. The method includes the cultivation of juice beverage contaminating bacteria and DNA template extraction, and then through the combination of high-throughput multiplex PCR technology and high-resolution capillary electrophoresis separation technology, it is possible to detect whether the sample contains of contaminating bacteria. Compared with other current molecular biology detection methods based on PCR technology, the present invention can integrate and detect 4 kinds of fruit juice drink contaminating bacteria in one reaction, saving detection cost and detection time, and the detection time is reduced from 7 to 14 days shortened to 8h.

Description

technical field [0001] The invention belongs to a method for detecting contaminating bacteria in fruit juice drinks, in particular to a method based on multiplex PCR technology and capillary electrophoresis separation technology, and relates to a rapid gene screening method that can detect four kinds of contaminating bacteria in fruit juice drinks at the same time, which is used for several common A rapid genetic screening method for contaminating bacteria in juice drinks. Background technique [0002] Fruit juice is one of the favorite liquid beverages in people's life. It is a juice product obtained by physical methods such as pressing, centrifuging, and extraction, using fruits as raw materials. Fruit juice drinks, especially pure fruit juice, are rich in essential vitamins and trace elements. As consumers become more health conscious, the consumption of juice drinks is increasing. [0003] During the production and processing of fruit juice beverages, some pathogenic m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686C12Q1/04
CPCC12Q1/686C12Q2537/143C12Q2565/125
Inventor 沈洁
Owner HANGZHOU DIANZI UNIV
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