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Molecular marker for quantifying lactobacillus acidophilus and method for absolutely quantifying bacterial community composition in yellow wine fermentation process

A technology of molecular markers and absolute quantification, applied in the field of bioengineering, can solve the problems of determining the complexity of the original internal standard, the complex microbiome of rice wine, and the inability to use it, and achieve the effect of convenient operation, wide application range, and overcoming the inconvenience of operation

Pending Publication Date: 2022-04-08
SHAOXING UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The method of using the natural internal standard method to quantify the flora proposed by the present invention can effectively solve the technical problem that rice wine cannot be used due to its complicated microbial flora and the complexity of determining the original internal standard

Method used

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  • Molecular marker for quantifying lactobacillus acidophilus and method for absolutely quantifying bacterial community composition in yellow wine fermentation process
  • Molecular marker for quantifying lactobacillus acidophilus and method for absolutely quantifying bacterial community composition in yellow wine fermentation process
  • Molecular marker for quantifying lactobacillus acidophilus and method for absolutely quantifying bacterial community composition in yellow wine fermentation process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1. Design of Lactobacillus acetotolerans-specific primers

[0013] 1. Synthetic primers: based on the Lactobacillus acetotolerans (Lactobacillus acid-tolerant Lactobacillus) genome as the basic data, a specific nucleic acid sequence was screened, and then a specific nucleic acid sequence was found on the genome of Lactobacillus acetotolerans through manual search and comparison, such as SEQ ID NO: 3 Shown), this sequence has only one copy on the Lactobacillusacetotolerans genome, and based on this sequence, primers LA-F (5'-GTGCGTTACGGAGGAGTA-3', SEQ ID NO: 1) were designed on the basis of this sequence; LA-R (5' -CGATCATTGAATCGTCAGG-3', SEQ ID NO: 2), the structure of the primer itself meets the requirements.

[0014] 2. Specificity verification:

[0015] (1) Choose to buy microorganisms in 18 kinds of rice wine for primer specificity test [Lactobacillusplantarum (CGMCC 1.568); Lactobacillus brevis (CGMCC 1.214); Lactobacillus acetotoolerans (ATCC 27745); Lact...

Embodiment 2

[0020] Embodiment 2. A kind of test kit of quantitative acid-resistant Lactobacillus

[0021] The components are: LA-F (5'-GTGCGTTACGGAGGAGTA-3', SEQ ID NO:1), LA-R (5'-CGATCATTGAATCGTCAGG-3', SEQ ID NO:2), AceQ Universal SYBR Green qPCR MasterMix, standard DNA and ddH 2 O. The standard product DNA is a plasmid containing the molecular marker shown in SEQ ID NO:3.

Embodiment 3

[0022] Example 3. Quantitative method of internal standard Lactobacillus acetotolerans

[0023] Using the Lactobacillus acetotolerans genome as a template, use specific primers LA-F, LA-R to amplify the target fragment to obtain the product of SEQ ID NO: 3. The amplified product is cloned into pMD19-T and transformed into Escherichia coliJM109. Colony PCR verifies the transformed Positive clones; use a plasmid extraction kit to extract the plasmid, and quantify the concentration of the plasmid with a micro-ultraviolet spectrophotometer;

[0024] The quantified plasmids were diluted tenfold, and 8 gradients were taken respectively, and the 8 gradients were used for qPCR. PCR system: 1 μL of template, 0.4 μL of upstream and downstream primers, 10 μL of AceQ Universal SYBR Green qPCR Master Mix, supplemented with double distilled water to The final volume is 20 μL; PCR conditions: 98°C for 1 min, 98°C for 10 s, 61°C for 1 min, 40 cycles.

[0025] standard curve as image 3 As s...

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Abstract

The invention discloses a molecular marker for quantifying lactobacillus acidophilus and a method for absolutely quantifying bacterial community composition in a yellow wine fermentation process, and belongs to the technical field of bioengineering. The invention provides application of a molecular marker as shown in SEQ ID NO: 3 as a marker for identifying and specifically quantifying lactobacillus acidophilus, which can effectively solve the technical problem that yellow rice wine cannot be applied due to complex microbiome and complex original internal standard determination, and provides application of the molecular marker as shown in SEQ ID NO: 3 as a marker for identifying and specifically quantifying lactobacillus acidophilus. The specific primer of the internal standard Lactobacillus acetotolans can be used for detecting and quantifying the internal standard in a yellow wine fermentation system. The distribution range of the interior labels is counted, and the result shows that the interior labels are widely distributed in the yellow wine fermentation system. And through absolute quantification of the internal standard, the content of different florae can be calculated, and the method has the characteristics of convenience in operation and wide application range.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a molecular marker for quantifying acid-resistant Lactobacillus and a method for absolutely quantifying the bacterial community composition in the rice wine fermentation process. Background technique [0002] During the production of rice wine, microbial life activities are subject to environmental conditions and the availability of nutrients. Under natural environment or artificial control conditions, fermentative flora may utilize different raw materials to produce various metabolites. Studies have shown that there are specific microbial functional groups in different yellow rice wine fermentation flora, and they are undergoing orderly community succession and functional transformation over time. A large number of studies have found that the function of microbial individuals in the community depends on various interactions with other individuals. During the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12R1/07
CPCY02A50/30
Inventor 彭祺郑华珺林姿辰孟凯
Owner SHAOXING UNIVERSITY
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