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Method for rapidly obtaining probiotic drug-resistant gene in sterilized dairy product based on high-throughput sequencing technology

A technology for drug resistance genes and dairy products, applied in the field of biological detection, can solve problems such as inability to achieve detection, and achieve the effect of being suitable for large-scale sample extraction and cheap.

Pending Publication Date: 2022-04-05
SHANGHAI INST OF QUALITY INSPECTION & TECHN RES +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Since the above-mentioned method is mainly aimed at live bacteria isolated from samples and generally can only screen and detect several drug-resistant genes, it cannot detect the drug-resistant genes of lactic acid bacteria in bactericidal products of isolated cultured strains.

Method used

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  • Method for rapidly obtaining probiotic drug-resistant gene in sterilized dairy product based on high-throughput sequencing technology
  • Method for rapidly obtaining probiotic drug-resistant gene in sterilized dairy product based on high-throughput sequencing technology
  • Method for rapidly obtaining probiotic drug-resistant gene in sterilized dairy product based on high-throughput sequencing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 sample total DNA preparation

[0042] 1. The quality of bacterial nucleic acid extraction will directly affect subsequent experiments. It is necessary to consider the particularity of the samples to be tested and the rich diversity of bacterial species. DNA extraction is the basis for subsequent amplification, sequencing and analysis. How to extract samples efficiently and comprehensively The desired DNA in , has a particularly prominent impact on the results. At present, there are various methods of DNA extraction with different principles and each has its own advantages and disadvantages. In this example, 6 representative examples of dairy products with bactericidal probiotics in the market were selected, including 3 examples of fermented milk and 3 examples of milk-containing beverages. The deep-processed food DNA extraction kit method, the magnetic bead method universal genomic DNA extraction kit method and the applicant's improved CTAB method were selected...

Embodiment 2

[0094] Example 2 Using high-throughput sequencing to obtain the sequence of the template DNA fragment

[0095] 1. Fragmentation of the DNA sample extracted in Example 1: about 350bp

[0096] Instrument: Covaris M220

[0097] 2. Construction of PE library

[0098] (1) Connect the "Y" shaped connector;

[0099] (2) Use magnetic beads to screen and remove adapter self-ligation fragments;

[0100] (3) Enrichment of library templates by PCR amplification;

[0101] (4) Sodium hydroxide denatures to produce single-stranded DNA fragments.

[0102] Reagents: TruSeq TM DNA Sample Prep Kit

[0103] 3. Bridge PCR

[0104] (1) One end of the above-mentioned single-stranded DNA fragment is complementary to the base of the primer, and fixed on the chip;

[0105] (2) The other end is randomly complementary to another nearby primer and is also fixed to form a "bridge";

[0106] (3) PCR amplification to generate DNA clusters;

[0107] (4) The DNA amplicon is linearized into a single ...

Embodiment 3

[0115] Example 3 Sequencing sequence statistics and quality control

[0116] (1) Raw sequence statistics

[0117] In the sequencing experiment, multiple DNA samples of Example 2 were used for parallel sequencing, and the sequences in each sample were introduced with an Index tag sequence indicating the source information of the sample. The data of each sample is distinguished according to the Index sequence, and the extracted data is saved in fastq format. In the data results of the MP or PE library, each sample has two files, fq1 and fq2, which contain the reads at both ends of the sequencing, and the order of the reads is strictly consistent and corresponding to each other.

[0118] faSTQ is a text format for storing biological sequences and corresponding quality values ​​in Solexa sequencing technology. Each read contains 4 lines of information, of which the first and third lines are composed of the file identification mark and the read section name (ID) (the first line s...

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Abstract

The invention provides a method for rapidly obtaining drug-resistant genes of probiotics in a sterilized dairy product based on a high-throughput sequencing technology, which comprises the following steps of: extracting DNA (Deoxyribose Nucleic Acid) of the sterilized dairy product, and then analyzing the drug-resistant genes carried by the probiotics in the sterilized dairy product by combining the high-throughput sequencing technology with a bioinformatics method. According to the method for rapidly obtaining the drug-resistant genes of the probiotics in the sterilized dairy product based on the high-throughput sequencing technology, the drug-resistant bacteria genes of the lactic acid bacteria in the sterilized dairy product can be efficiently and rapidly screened, and the method has a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for rapidly obtaining drug-resistant genes of probiotics in sterilized dairy products based on high-throughput sequencing technology. Background technique [0002] The use of antibiotics is one of the foundations of modern medical and health care. It can effectively inhibit the growth of bacteria or kill bacteria. It is widely used in the treatment of human diseases and in fields such as agriculture, animal husbandry, and aquaculture. A serious problem of antibiotic resistance has been produced[GOTHWAL R, SHASHIDHAR T.Antibioticpollution in the environment: a review[J].Clean-Soil,Air,Water,2015,43(4):479-489.DOI:10.1002 / clen.201300989.]. [0003] Lactic acid bacteria are widely used in food, agriculture, pharmaceutical and other industries as starters or probiotic strains. For a long time, the lactic acid bacteria used in fermented products ar...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6869C12Q1/04C12N15/11C12N15/10C12R1/01
Inventor 徐琼俞漪顾晨荣刘洋张娜娜窦同海吴群峰翁史昱钟江祝海军杜雪柯赵磊封金美
Owner SHANGHAI INST OF QUALITY INSPECTION & TECHN RES
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