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Gene rapid screening method for identifying multiple types of thermoduric bacteria in fruit juice beverage synchronously

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

Active Publication Date: 2016-02-10
HANGZHOU DIANZI UNIV
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  • Summary
  • 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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  • Gene rapid screening method for identifying multiple types of thermoduric bacteria in fruit juice beverage synchronously
  • Gene rapid screening method for identifying multiple types of thermoduric bacteria in fruit juice beverage synchronously
  • Gene rapid screening method for identifying multiple types of thermoduric bacteria in fruit juice beverage synchronously

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Experimental program
Comparison scheme
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 Byssochlamysspectabilis grown on the plate medium and add them to 10 mL of finished apple juice, mix Mix evenly 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 resuspend the cells thoroughly; add 120ul of 10mg / ml lysobacteria, incubate at 37°C for 60 minutes, centrifuge at 13,000×g for 2 minutes, and remove the supernatant. Then follow the instructions of the GenomicDNAPurificationKit kit from Qiagen to extract the DNA template of the heat-resistant bacteria in fruit juice drinks, and use a UV spectrophotometer to measure the quality of the extracted DNA at a wavelength of 260nm, dilute the DNA...

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, after 7 days of anaerobic culture, 15 colonies were detected and used as the experimental control; for another piece of filter membrane, use 1mL sterile water to shake 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 minutes, and remove the supernatant.

[0046] Afterwards, extract the DNA template of the fruit juice drink thermostable bacteria according to the instructions of t...

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 sporium cladospore (Cladosporium sphaerospermum), putrefaction mold (Neosartoria fischeri), the detection specificity of silk mold flower (Byssochlamysspectabilis), promptly detection method of the present invention is in When only lactic acid bacteria (Lactobacillus) exists, no characteristic peaks are displayed, 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 ex...

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Abstract

The invention discloses a gene rapid screening method for identifying multiple types of thermoduric bacteria in a fruit juice beverage synchronously. According to the method, four pairs of primers form a primer system, and whether the thermoduric bacteria exist in the fruit juice beverage or not is detected in the mode of combining a high-flux multi-PCR technology with a high-resolution capillary electrophoresis separation technology. Compared with other existing molecular biology detection means based on a PCR technology, four types of fruit juice beverage thermoduric bacteria, namely alicydobadllus addoterrestris, cladosporium sphaerospermum, corruption mold and byssochlamys, can be integrally detected in a reaction, the detection cost is saved, and detection time is saved and shortened to eight hours from the original 7-14 days.

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 Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/686C12Q2531/119C12Q2565/125
Inventor 沈洁
Owner HANGZHOU DIANZI UNIV