A rapid genetic screening method for the simultaneous identification of multiple heat-resistant bacteria in fruit juice beverages

A technology for heat-resistant bacteria and beverages, applied in biochemical equipment and methods, and microbial measurement/inspection, etc., can solve the problems of cumbersome, time-consuming and labor-intensive detection technology for heat-resistant bacteria, so as to save detection time and achieve accurate detection results , The effect of saving testing cost

Active Publication Date: 2018-11-13
HANGZHOU DIANZI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to address the defects of the prior art, overcome the cumbersome operation, time-consuming and labor-intensive detection technology of the main heat-resistant bacteria in fruit juice drinks, and provide a rapid genetic screening method for simultaneously identifying multiple heat-resistant bacteria in fruit juice drinks , using a primer system composed of 4 pairs of primers, through the combination of high-throughput multiplex PCR technology and high-resolution capillary electrophoresis separation technology, the detection of heat-resistant bacteria contamination in fruit juice beverages is realized

Method used

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  • A rapid genetic screening method for the simultaneous identification of multiple heat-resistant bacteria in fruit juice beverages
  • A rapid genetic screening method for the simultaneous identification of multiple heat-resistant bacteria in fruit juice beverages
  • A rapid genetic screening method for the simultaneous identification of multiple heat-resistant bacteria in fruit juice beverages

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Experimental program
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Effect test

Embodiment 1

[0036] (1) Cultivation of heat-resistant bacteria in juice drinks and extraction of DNA templates:

[0037] Pick single colonies of Alicyclobacillus acidoterrestris, Cladosporium sphaerospermum, Neosartoria fischeri, and Byssochlamys spectabilis grown on the plate medium and add them to 10 mL of finished apple juice , mix well to make a bacterial suspension, take 1mL of the bacterial suspension and add it to a 1.5ml centrifuge tube, centrifuge at 13,000×g for 2min, collect the cell pellet, remove the supernatant; add 480ul of 50mM EDTA to completely resuspend the cells; add 120ul 10mg / ml lysobacteria, incubate at 37°C for 60min, centrifuge at 13,000×g for 2min, and remove the supernatant. Extract the DNA template of fruit juice drink thermostable 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 extraction under the condition of 260nm wavelength, t...

Embodiment 2

[0045] Take 10 bottles of 500mL unsterilized fruit juice from the production workshop of a fruit juice beverage factory, and use a filter membrane with a pore size of 0.45μm to filter twice, each time using one filter membrane to filter 250mL, and one filter membrane with juice beverage acid soil Alicyclobacillus acidoterrestris detection medium MRS-agar at 26°C, 15 colonies were detected after 7 days of anaerobic culture, and used as experimental control; for another piece of filter membrane, use 1mL sterile water Shake and wash, collect the washing liquid into a 1.5ml centrifuge tube, 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 2 min, and remove the supernatant.

[0046] Afterwards, extract the DNA template of the fruit juice drink heat-resistant bacteria according to the instructions of the G...

Embodiment 3

[0049] Lactic acid bacteria (Lactobacillus) is another common contaminating bacteria in fruit juice drinks. This example can prove that the present invention is to acid soil cycloaliphatic bacillus (Alicyclobacillus acidoterrestris), ball spore branch spore (Cladosporium sphaerospermum), spoilage mold (Neosartoria fischeri), the detection specificity of silk mold flower (Byssochlamysspectabilis), i.e. the detection of the present invention Method In the case of only lactic acid bacteria (Lactobacillus) exists, there is no characteristic peak, and the presence of lactic acid bacteria (Lactobacillus) will not interfere with the detection.

[0050] In this example, common heat-resistant bacteria Lactobacillus brevis, Lactobacillus casei, Lactobacillus coryniformi, and Lactobacillus plantarum were used as interfering bacteria.

[0051] The operating steps of the present invention are as described in Embodiment 1 or 2.

[0052] (1) Cultivation of contaminated bacteria and extracti...

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Abstract

The invention discloses a rapid gene screening method for simultaneously identifying multiple heat-resistant bacteria in fruit juice drinks. The method uses a primer system consisting of 4 pairs of primers, and combines high-throughput multiplex PCR technology with high-resolution capillary electrophoresis separation technology to detect the presence of heat-resistant bacteria in fruit juice beverages. . Compared with other current molecular biology detection methods based on PCR technology, the present invention can integrate and detect 4 kinds of fruit juice beverage heat-resistant bacteria, cycloaliphatic bacillus, cladosporium, spoilage mold, and silk mold in one reaction. Flowers, saving the detection cost and detection time, the detection time was shortened from 7 to 14 days to 8 hours.

Description

technical field [0001] The invention belongs to a detection method for heat-resistant bacteria in fruit juice drinks, in particular to a rapid screening method for genes of heat-resistant bacteria in several common fruit juice drinks based on multiple PCR technology and capillary electrophoresis separation technology, and relates to a simultaneous identification of fruit juice drinks A rapid genetic screening method for a variety of thermotolerant bacteria in China. Background technique [0002] Juice is one of the favorite liquid beverages in people's life. 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. Juice is a juice product obtained from fruit as raw material through physical methods such as pressing, centrifugation, extraction, etc. Juice is divided into clarified juice and turbid juice according to its shape. Clarified juice...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686C12Q1/04
CPCC12Q1/686C12Q2531/119C12Q2565/125
Inventor 沈洁
Owner HANGZHOU DIANZI UNIV
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