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High-precision specific digital polymerase chain reaction (PCR) detection method for series infectious microbe contamination taking acetobacter aceti as priority in fermented milk

Acetobacter aceti and miscellaneous bacteria contamination technology, applied in the direction of microbial-based methods, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of increasing production costs, waste of production resources such as raw materials and auxiliary materials, etc.

Pending Publication Date: 2019-12-20
河北省食品检验研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The quality control of contaminating bacteria and pathogenic bacteria in fermented milk requires an accurate detection technology system as support. Fermented milk can meet the growth needs of contaminating and pathogenic bacteria due to its nutrient-rich characteristics, and a single test of the final product is likely to cause Waste of production resources such as raw materials and auxiliary materials, thereby increasing production costs

Method used

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  • High-precision specific digital polymerase chain reaction (PCR) detection method for series infectious microbe contamination taking acetobacter aceti as priority in fermented milk
  • High-precision specific digital polymerase chain reaction (PCR) detection method for series infectious microbe contamination taking acetobacter aceti as priority in fermented milk
  • High-precision specific digital polymerase chain reaction (PCR) detection method for series infectious microbe contamination taking acetobacter aceti as priority in fermented milk

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, reagent and apparatus.

[0058] Test reagents include: 2×ddPCR Super Mix for Probes (Bio-Rad, Gemany), Droplet Generation Oil (Bio-Rad, Gemany); Droplet Reader Oil (Bio-Rad, Gemany); Reagent D (Biotecon Diagnostics); primers, reverse primers) were synthesized by Shanghai Bioengineering Company. ; Ezup Column Bacterial Genomic DNA Extraction Kit was purchased from Tiangen Biological Co., Ltd.; Bacterial DNA Extraction Kit by Magnetic Beads: FoodProof Magnetic preparation Kit Ⅳ (Biotecon Diagnostics).

[0059] The medium includes: (1) Acetic acid bacteria liquid medium: glucose 10%, yeast extract 1%, CaCO 32%, distilled water 1.0L, pH 6.8, sterilized at 121°C for 15 minutes. (2) Basic solid medium for acetic acid bacteria: glucose 50g / L, beef extract 5g / L, yeast powder 10g / L, ethanol 10mL / L, agar 15g / L, pH 6.8. (3) Nutrient broth medium: peptone 10.0g / L, beef powder 3.0g / L, sodium chloride 5.0g / L, heated and stirred to dissolve, adjust the pH value to 7.2,...

Embodiment 2

[0061] Embodiment 2, bacterial strain.

[0062] The strains used in this experiment are detailed in Table 3.1. After all the strains are purchased, they are first activated with their respective indicator medium, cultivated at a suitable temperature, and then transferred and passaged three times for later use.

[0063] Table 3.1 Test strains

[0064]

[0065]

Embodiment 3

[0066] Embodiment 3, the optimum method of removing dead bacteria.

[0067] (1) Fully shake the bacterial culture solution transferred three times, draw 100 μL of the enrichment solution and add it to a 2 mL sterilized transparent centrifuge tube;

[0068] (2) Take out Reagent D from -20°C, let it melt at room temperature, take 4 times the volume of Reagent D solution of the above enrichment solution, add it to (1), and incubate at room temperature in the dark for 5 minutes;

[0069] (3) Place the centrifuge tube in a low-temperature centrifuge tube rack, place it directly below a 500W halogen lamp at a distance of 15cm-20cm, and after exposure for 5min, centrifuge at 8000r / min for 5min;

[0070] (4) Carefully remove the supernatant with a pipette, add 100 μL of sterile deionized water to the centrifuge tube and mix thoroughly, and the bacterial suspension can be directly used for genome extraction.

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Abstract

The invention discloses a high-precision specific digital polymerase chain reaction (PCR) detection method for series infectious microbe contamination taking acetobacter aceti as priority in fermentedmilk. The method comprises the steps: firstly, to-be-detected infectious microbes are subjected to dead bacterium de-interference treatment, then bacterial DNA is subjected to low-impurity and high-purity extraction, a specific primer probe of the to-be-detected microbes is researched, developed and designed, a ddPCR system and procedure and the annealing temperature thereof are set, and thus thedetection method meets the requirements of specificity, sensitivity and quantification. The constructed method is good in specificity, has no non-specific amplification, and has good sensitivity andquantitative advantages.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a detection method for harmful bacteria and pathogenic bacteria in food and drinks. Background technique [0002] The quality control of contaminating bacteria and pathogenic bacteria in fermented milk requires an accurate detection technology system as support. Fermented milk can meet the growth needs of contaminating and pathogenic bacteria due to its nutrient-rich characteristics, and a single test of the final product is likely to cause Waste of production resources such as raw materials and auxiliary materials, thereby increasing production costs. Therefore, accurate detection of the number of polluting bacteria and pathogenic bacteria in different production links is the key to process quality control. [0003] Digital PCR (dPCR), as the technical representative of the third generation PCR, was proposed by Vogelstein et al. dPCR is to evenly distribute the re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/689C12Q1/14C12Q1/10C12Q1/06C12R1/42C12R1/445C12R1/02C12R1/01
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2563/159
Inventor 周巍史国华张岩李永波杨岚陈晨王赞
Owner 河北省食品检验研究院
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