Method for detecting probiotics in milk powder by Illumina Miseq sequencing platform

A sequencing platform and probiotic technology, which can be used in biochemical equipment and methods, microbial determination/inspection, etc., and can solve the problems of high cost, low sequencing throughput, and inaccurate information.

Inactive Publication Date: 2017-05-24
PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] Previous research on microbial diversity usually uses Sanger first-generation sequencing technology, which mainly uses DNA single strands as templates, adds fluorescent markers and nucleotides for polymerase chain reaction amplification, separates and purifies by gel electrophoresis, and then performs fluorescence detection. Obtain the base composition sequence, but its cost is high, the sequencing throughput is low, and the information is inaccurate

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  • Method for detecting probiotics in milk powder by Illumina Miseq sequencing platform
  • Method for detecting probiotics in milk powder by Illumina Miseq sequencing platform
  • Method for detecting probiotics in milk powder by Illumina Miseq sequencing platform

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Embodiment 1

[0070] Embodiment 1: prepare to detect milk powder sample

[0071] The described method utilizing Illumina Miseq sequencing platform to detect probiotics in milk powder comprises the steps:

[0072] (1) Extraction of genomic DNA from milk powder: after brewing the milk powder, perform the first centrifugation at 4000r / min for 5min, take the supernatant, then perform the second centrifugation at 12000r / min for 10min, take the precipitate, and add 200μL lysis buffer Shake until the cells are completely suspended, then add lysozyme to react at 37°C for 40 minutes, and then extract the genomic DNA;

[0073] (2) PCR amplification: for the v1-v3 region of bacterial 16s rDNA, select specific primers 27F and 534R for PCR amplification, and the PCR amplification reaction system is as follows:

[0074]

[0075]

[0076] A set of negative controls with water as the sample.

[0077] The reaction conditions are as follows:

[0078]

[0079] (3) Illumina Miseq sequencing: PCR pr...

Embodiment 2

[0080] Embodiment 2: prepare to detect milk powder sample

[0081] The described method utilizing Illumina Miseq sequencing platform to detect probiotics in milk powder comprises the steps:

[0082] (1) Extraction of genomic DNA from milk powder: After brewing the milk powder, centrifuge at 2000r / min for 10min for the first time, take the supernatant, then centrifuge at 15000r / min for 5min for the second time, take the precipitate, and add 100μL lysis buffer Shake until the bacteria are completely suspended, then add lysozyme to react at 35°C for 60 minutes, and then extract the genomic DNA;

[0083] (2) PCR amplification: For the v1-v3 region of bacterial 16s rDNA, specific primers 27F and 519R were selected for PCR amplification. The PCR amplification reaction system was as follows:

[0084]

[0085] A set of negative controls with water as the sample.

[0086] The reaction conditions are as follows:

[0087]

[0088] (3) Illumina Miseq sequencing: PCR products were s...

Embodiment 3

[0089] Embodiment 3: prepare to detect milk powder sample

[0090] The described method utilizing Illumina Miseq sequencing platform to detect probiotics in milk powder comprises the steps:

[0091] (1) Extraction of genomic DNA from milk powder: after brewing the milk powder, centrifuge for the first time at 5000r / min for 1min, take the supernatant, then centrifuge for the second time at 10000r / min for 5min, take the precipitate, and add 500μL lysis buffer Shake until the bacteria are completely suspended, then add lysozyme to react at 40°C for 10 minutes, and then extract the genomic DNA;

[0092] (2) PCR amplification: for the v1-v3 region of bacterial 16s rDNA, select specific primers 27F and 530R for PCR amplification. The PCR amplification reaction system is as follows:

[0093]

[0094] A set of negative controls with water as the sample.

[0095] The reaction conditions are as follows:

[0096]

[0097] (3) Illumina Miseq sequencing: PCR products were subjecte...

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Abstract

The invention relates to the technical field of milk powder detection, and particularly relates to a method for detecting probiotics in milk powder by an Illumina Miseq sequencing platform. The method comprises the following steps of: (1) extraction of DNA (deoxyribonucleic acid) of a milk powder genome, i.e., after brewing the milk powder, carrying out primary centrifugation to take supernatant, then carrying out secondary centrifugation to take precipitates, adding buffer solution to oscillate, and then after adding lysozyme to perform a reaction, carrying out extraction of the DNA of the genome; (2) PCR (Polymerase Chain Reaction) amplification, i.e., aiming at v1 to v3 regions of 16s rDNA of a bacterium, selecting a specific primer to carry out PCR amplification; (3) Illumina Miseq sequencing, i.e., carrying out Illumina Miseq sequencing on a PCR product. The Illumina Miseq sequencing method effectively avoids the defects of low flux, complex operation, low accuracy and the like by analysis, not only can simultaneous sequencing on a plurality of variable regions of a plurality of samples be implemented, but also both a sequencing speed and a sequencing flux are further, and a detection result is high in credibility.

Description

technical field [0001] The invention relates to the technical field of milk powder detection, and relates to a method for detecting probiotics in milk powder, in particular to a method for detecting probiotics in milk powder by an Illumina Miseq sequencing platform. Background technique [0002] The composition of intestinal flora is closely related to human nutrition, immunity, cancer and aging. It promotes the proliferation of beneficial bacteria in the intestine and inhibits the growth of harmful bacteria, which is of great significance for preventing diseases caused by poor living habits. The application of probiotics has also become more and more extensive with the deepening of research. At present, the microorganisms used as probiotics are all derived from humans or animals, and probiotic products mostly use bacteria of the two genera of lactic acid bacteria and bifidobacteria. With the progress of probiotics research and the popularization of knowledge, the public ten...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6869C12Q2531/113
Inventor 杨捷琳袁辰刚何宇平王敏窦同海陈雯雯王曦路
Owner PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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