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Tracing method of probiotics in dairy product

A technology of probiotics and dairy products, applied in the field of detection, can solve the problems of not including probiotics, poor DNA extraction effect, unsatisfactory test results, etc., and achieve the effect of improving the effect

Inactive Publication Date: 2017-08-18
PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But its DNA extraction effect is not good, the test results are not ideal, and it cannot include all probiotics

Method used

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  • Tracing method of probiotics in dairy product
  • Tracing method of probiotics in dairy product
  • Tracing method of probiotics in dairy product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1: Different primers detect probiotic standard

[0084] A traceability method for probiotics in dairy products, comprising the following steps:

[0085] (1) Sample pretreatment: Take 1g of mixed probiotic standard, dissolve in 10mL ddH 2 In O, add 1 mL of 18% sodium citrate and 500 μL of 1mol / L sodium hydroxide, mix and centrifuge at 12,000 r / min for 10 min, discard the supernatant, and add 180 μL of lysozyme to the precipitate for incubation;

[0086] (2) The processed samples were extracted with Tiangen Bacteria Genomic DNA Extraction Kit to extract the whole genome DNA of probiotics;

[0087] (3) 16S rRNA PCR amplification: Aiming at the v1-v3 region of bacterial 16s rDNA, use SEQ ID NO.1-6 primers to perform PCR amplification respectively;

[0088] The nucleotide sequence of the primer is as follows:

[0089] 27F (SEQ ID NO.1): 5'-AGAGTTTGATCCTGGCTCAG-3';

[0090] 543R (SEQ ID NO.2): 5'-ATTACCGCGGCTGCTGG-3';

[0091] 27F' (SEQ ID NO.3): 5'-AGRGTTYGATYCT...

Embodiment 2

[0108] Example 2: Analysis of 16S rDNA library construction of standard probiotics

[0109] (1) PCR product purification

[0110] After the electrophoresis detection is correct, use AxyPrep TM PCR Clean-Up Kit is used for column purification and recovery of PCR products. First add 3 volumes of buffer buffer PCR-A to the PCR product (if the buffer PCR-A is less than 100uL, add to 100uL); put the preparation tube into a 2mL centrifuge tube, add 700μL buffer W2, 12,000Xg for 1min , discard the filtrate; put the preparation tube into a centrifuge tube, add 400 μL of buffer buffer W2, and centrifuge at 12,000 rpm for 1 min; discard the filtrate, and spin at 12,000 rpm for 1 min to elute the DNA.

[0111] (2) Mixed library and on-machine sequencing

[0112] The 16S rRNA sequences corresponding to the recovered samples were uniformly mixed, the library was constructed using the Illumina DNA Fragment Library Construction Kit, the library was quantified by the Qubit method, and the ...

Embodiment 3

[0122] Example 3: Detection of actual samples with degenerate primers

[0123] A traceability method for probiotics in dairy products, comprising the following steps:

[0124] (1) Sample pretreatment: Take 1g of mixed probiotic standard, dissolve in 10mL ddH 2 In O, add 1 mL of 18% sodium citrate and 500 μL of 1mol / L sodium hydroxide, mix and centrifuge at 12,000 r / min for 10 min, discard the supernatant, and add 180 μL of lysozyme to the precipitate for incubation;

[0125] (2) The processed samples were extracted with Tiangen Bacteria Genomic DNA Extraction Kit to extract the whole genome DNA of probiotics;

[0126] (3) 16S rRNA PCR amplification: Aiming at the v1-v3 region of bacterial 16s rDNA, use SEQ ID NO.3-4 primers to perform PCR amplification respectively;

[0127] The nucleotide sequence of the primer is as follows:

[0128] 27F' (SEQ ID NO.3): 5'-AGRGTTYGATYCTGGCTCAG-3';

[0129] 543R' (SEQ ID NO.4): 5'-ATTACCGCGGCTGCTGG-3';

[0130] The specific amplification...

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Abstract

The invention belongs to the technical field of detection, relates to a tracing method of probiotics in a dairy product, and particularly relates to a tracing method of probiotics in the dairy product on an Illumina Miseq sequencing platform. The method comprises the following steps: (1) sample pretreatment: adding sodium citrate and sodium hydroxide into a to-be-detected sample, mixing and centrifuging, discarding supernatant, and adding lysozyme into precipitates for incubation; (2) genomic DNA extraction of the pretreated sample; (3) 16S rRNA PCR amplification: aiming at v1-v3 regions and v5-v6 regions of bacterium 16s rDNA, and designing a primer for PCR amplification; and (4) Illumina Miseq sequencing: performing Illumina Miseq sequencing to the PCR product. Analysis on bacterial strain types and abundance of probiotics contained in food of market fermented dairy products, probiotic milk powder and the like is conducted by utilizing the Illumina Miseq sequencing platform, showing that the practical abundance of probiotics in the solid probiotic products is basically identical with the labeling, and the practical sequencing in the fermented type probiotic product has great difference from that of the labeled bacterial strain.

Description

technical field [0001] The invention relates to the technical field of detection, and relates to a traceability method for probiotics in dairy products, in particular to a traceability method for probiotics in dairy products 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. Probiotic products mostly use Lactobacillus, Streptococcus, and Bifidobacterium in lactic acid bacteria. strains. With the progress of probiotic research and the popularization of knowledge, the p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6869C12Q2535/122
Inventor 杨捷琳蒋原蒋静杨惠琴
Owner PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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