Method for detecting five bacteria in vinegar grains by fluorogenic quantitative PCR (polymerase chain reaction)
A fluorescence quantitative, vinegar-fermented technology, applied in the field of bioengineering, can solve problems such as poor process controllability, batch differences in product quality, and large flavor differences
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Publication Date
- 2013-07-03
- Estimated Expiration
- Not applicable · inactive patent
Smart Images
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Abstract
Description
technical field
[0001] The invention belongs to the technical field of bioengineering, and in particular relates to quantitative research on the application of fluorescent quantitative PCR technology to specific species of microorganisms in solid-state vinegar-making microbial communities. Background technique
[0002] The production of vinegar in my country has a long history. According to different places of origin and varieties, the content of acetic acid in vinegar is also different. For example, Shanxi mature vinegar has a strong sour taste, while Zhenjiang balsamic vinegar has a soft but not sour taste. The differences in vinegar brewing process and taste in different regions have created the "Four Famous Vinegars" of Shanxi Old Mature Vinegar, Zhenjiang Hengshun Vinegar, Fujian Yongchun Old Vinegar, and Baoning Vinegar. However, the fermentation process of many vinegar manufacturers is mainly based on traditional experience, the process controllability is poor, and t...
Examples
Embodiment 1
[0044] Embodiment one: the preparation of standard sample
[0045] 1. Design and synthesize specific primers for Lactobacillus helveticus, Lactobacillus crispatus, Lactobacillus casei, Lactobacillus delbrueckii and Lactobacillus acidophilus.
[0046]
[0047] 2. DNA extraction of Lactobacillus helveticus, Lactobacillus crispatus, Lactobacillus casei, Lactobacillus delbrueckii and Lactobacillus acidophilus.
[0048] The isolated and purified Lactobacillus helveticus, Lactobacillus crispatus, Lactobacillus casei, Lactobacillus delbrueckii and Lactobacillus acidophilus were cultured in shake flasks, and DNA was extracted from their culture fluids using bacterial extraction kits.
[0049] 3. Cloning of the target fragment.
[0050] Using the DNA of Lactobacillus helveticus, Lactobacillus crispatus, Lactobacillus casei, Lactobacillus delbrueckii and Lactobacillus acidophilus as templates, Lhe-F / Lhe-R, Lcr-F / Lcr-R, Lca-F / Lca -R, Lde-F / Lde-R and Lacid-F / Lacid-R are used as prime...
Embodiment 2
[0060] Example 2: Quantitative analysis of Lactobacillus helveticus, Lactobacillus crispatus, Lactobacillus casei, Lactobacillus delbrueckii and Lactobacillus acidophilus in the microbial community of vinegar fermented grains.
[0061] 1. Collection of samples of vinegar fermented grains.
[0062] Collect the samples of vinegar unstrained spirits at different time points during the vinegar fermentation process. If the DNA cannot be extracted in time after the samples are collected, they should be frozen immediately.
[0063] 2. Extraction method of microbial total DNA in vinegar fermented grains.
[0064] Weigh 2g of vinegar unstrained spirits, add liquid nitrogen into a mortar and grind thoroughly, transfer to a 50mL centrifuge tube. Add 6 mL of DNA extraction buffer to the centrifuge tube, then add 100 μL of lysozyme (50 mg / mL), and shake on a shaker at 225 rpm at 37 °C for 30 min. Add 1.5 mL of 10% SDS to the centrifuge tube, bathe in water at 65°C for 2 h, invert gently ...