CN107058482A - The source tracing method of probiotics in a kind of dairy products - Google Patents
The source tracing method of probiotics in a kind of dairy products Download PDFInfo
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- CN107058482A CN107058482A CN201611213301.8A CN201611213301A CN107058482A CN 107058482 A CN107058482 A CN 107058482A CN 201611213301 A CN201611213301 A CN 201611213301A CN 107058482 A CN107058482 A CN 107058482A
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Abstract
The present invention relates to detection technique field, it is related to a kind of source tracing method of probiotics in dairy products, more particularly to a kind of Illumina Miseq microarray datasets to the source tracing method of probiotics in dairy products.Methods described comprises the following steps：(1) sample pre-treatments：Sodium citrate and sodium hydroxide are added into detected sample, supernatant is abandoned in centrifugation after mixing, lysozyme is added into precipitation and is incubated；(2) extraction of the genomic DNA of sample after handling；(3) 16S rRNA PCR are expanded：Directed toward bacteria 16s rDNA v1 v3 areas and v5 v6 areas, design primer enters performing PCR amplification；(4) Illumina Miseq are sequenced：PCR primer is subjected to Illumina Miseq sequencings.The present invention has carried out the analysis of bacterial strain species and abundance using Illumina Miseq microarray datasets to the probiotics contained by the food such as commercially available fermented dairy product and probiotics milk powder, show that the actual abundance of probiotics and sign are basically identical in solid probiotic products, and the bacterial strain for being actually sequenced and indicating in fermented type probiotic products is compared and differed greatly.
Description
Technical field
The present invention relates to detection technique field, it is related to a kind of source tracing method of probiotics in dairy products, more particularly to it is a kind of
Source tracing method of the Illumina Miseq microarray datasets to probiotics in dairy products.
Background technology
The composition of gut flora has close relationship to human nutrition, immune, cancer and aging etc., promotes to have in enteron aisle
The propagation of beneficial bacteria, suppresses the growth of harmful bacteria, for preventing disease caused by being not good at due to habits and customs to have important meaning
Justice.The application of probiotics is also with the deeply more and more extensive of research, and at present, the microorganism for being used as probiotics is derived from people
Or animal, probiotic composition use lactic acid bacteria in lactobacillus, streptococcus, the bacterial strain of Bifidobacterium.With probiotics
The progress of research and popularization of knowledge, masses, which tend to buy, indicates the milk powder for having added probiotics or food.And probiotics is different
In other nutrition fortifiers, relatively stringent is required to production, Conservation environment condition, is detected at present for the probiotics in food
Lack quick, accurate detection mode.
Probiotics fermention breast is one of most common micro-ecological foods in the market, and adds the formula milk of probiotics
It is then the most micro-ecological foods of infants.Because probiotics fermention breast in the huge market demand of China, Chinese market
Numerous with formula milk brand, the probiotics strain added in product should meet Ministry of Public Health's issue《Available for the bacterium in food
Plant list》With《Strain list available for infant food》, the former is that regulation arrives strain, and the latter is that regulation arrives bacterial strain.According to system
Meter, conventional business strain includes streptococcus thermophilus (Streptococcus thermophilus), lactobacillus bulgaricus
(Lactobacillus bulgaricus), lactobacillus acidophilus (Lactobacillus acidophilus), Bifidobacterium
And Lactobacillus casei (Lactobacilus casei) etc. (Bifidobacterium).
According to the Ministry of Public Health announce 2011 No. 25《Strain list available for infant food》, baby can be added at present
Strain in baby food has lactobacillus acidophilus NCFM, animal bifidobacteria Bb-12, bifidobacterium lactis HN019, bifidobacterium lactis
Bi-07, Lactobacillus rhamnosus HN001, seven kinds of Lactobacillus rhamnosus LGG, wherein lactobacillus acidophilus NCFM are only limited the use of in more than 1 years old
The food of child.Wherein, animal bifidobacteria is one of dominant bacteria in people and many mammalian guts, due to suppression
Spoilage organisms and opportunistic bacteria growing, synthetic vitamin, raising immunity of organisms and the anti-ageing effect of waiting for a long time, therefore extensively should
With.In baby milk powder in the market, the probiotics strain of product indicia addition is mainly animal bifidobacteria Bb-12.
Microorganisms diversity is mainly single-stranded for template with DNA usually using Sanger first generation sequencing technologies before this,
Add fluorescence labeling and nucleotides carries out PCR amplification, fluorescence is carried out after being isolated and purified by gel electrophoresis
Detection, obtains base composition sequence, but its cost is high, sequencing throughput is low, information is inaccurate.
Flora ecology point is carried out to probiotics complex microorganism colony using 16s rDNA different variable regions sequencing
Analysis, can obtain the species and frequency distribution information of various microorganisms, to ensure " identifying ", " bill of lading is consistent ".MiSeq is high
Flux microarray dataset can be realized, and many variable regions of Multi-example are sequenced simultaneously, and this technology is larger to improve sequencing speed
And flux, this current platform has been used in the research of microbial diversity structure of community.
2014 Vol.33 No.5 Serial No.267 brewage in China, burnt brilliant triumphant etc. to have delivered that " IllumniaMiSeq is put down
Platform high coverage rate determines the bacterial micro-organism diversity in cheese " determined using second generation sequencing Illumina MiSeq methods
Bacterial micro-organism diversity in natural maturity cheese from 4, Inner Mongol different regions.Illumnia MiSeq methods can
Fast and effeciently to determine composition structure and its distribution of microorganism.Analyze strain classification and abundance point in 4 kinds of cheese samples
Cloth, group's composition, different sample room species difference and evolutionary relationship etc., comprehensively present the various of bacterial micro-organism in cheese
Property distribution.But its DNA extraction effect is not good, and the result of test is unsatisfactory, all probiotics can not be included.
Therefore, the probiotics in dairy products can be determined by how finding a kind of method, to microorganism group in product into progress
Analyzing in detail, further obtains different sample room microorganism differences.
The content of the invention
In view of the shortcomings of the prior art and actual demand, the present invention provides a kind of side of tracing to the source of probiotics in dairy products
Method, by the probiotic group in Illumina Miseq platform assay dairy products into, to microorganism group in product into carry out it is detailed
Analysis, further obtains the information of different sample room microorganism quality and quantities.
For up to this purpose, the present invention uses following technical scheme：
On the one hand, the present invention provides a kind of method for detecting the probiotics in milk powder, and especially one kind utilizes Illumina
Miseq microarray datasets are come the method that detects probiotics in milk powder, it is characterised in that comprise the following steps：
(1) sample pre-treatments：Sodium citrate and sodium hydroxide are added into detected sample, supernatant is abandoned in centrifugation after mixing,
Lysozyme is added into precipitation to be incubated；
(2) extraction of the genomic DNA of sample after handling；
(3) 16S rRNA PCR are expanded：Directed toward bacteria 16s rDNA v1-v3 areas and v5-v6 areas, design primer is carried out
PCR is expanded；
(4) Illumina Miseq are sequenced：PCR primer is subjected to Illumina Miseq sequencings.
The present invention by the way that sample is carried out into pre-treatment, by add the sodium citrate and sodium hydroxide of certain concentration mix from
The heart, coordinates lysozyme to be incubated so that pre-treatment substantially increases the effect of DNA extractions, and wherein sodium citrate and sodium hydroxide delays
Fliud flushing assists in removing impurity, and for removing most of fat and protein in sample, lysozyme is used to crack Gram-positive
Cell wall.
According to the present invention, the final concentration of 0.01-0.2mol/L, preferably 0.02- of step (1) described sodium citrate
0.1mol/L, for example, can be 0.01mol/L, 0.02mol/L, 0.03mol/L, 0.04mol/L, 0.05mol/L, 0.06mol/
L、0.08mol/L、0.09mol/L、0.1mol/L、0.12mol/L、0.15mol/L、0.16mol/L、0.18mol/L、
0.2mol/L, more preferably 0.03-0.08mol/L, most preferably 0.05-0.07mol/L, more preferably 3%, with
And the specific point value between above-mentioned numerical value, as space is limited and for concise consideration, scope of the invention no longer described in exclusive list
Including specific point value.
After " final concentration " of the present invention is addition testing sample, the concentration in reaction system.
According to the present invention, the final concentration of 0.01-0.5mol/L of step (1) described sodium hydroxide for example can be
0.01mol/L、0.02mol/L、0.03mol/L、0.04mol/L、0.05mol/L、0.06mol/L、0.08mol/L、
0.09mol/L、0.1mol/L、0.12mol/L、0.15mol/L、0.16mol/L、0.18mol/L、0.2mol/L、0.22mol/
L、0.23mol/L、0.25mol/L、0.26mol/L、0.28mol/L、0.3mol/L、0.32mol/L、0.35mol/L、
0.36mol/L, 0.38mol/L, 0.4mol/L, 0.42mol/L, 0.43mol/L, 0.45mol/L, 0.48mol/L or 0.5mol/
L, preferably 0.05-0.3mol/L, more preferably 0.08-0.2mol/L, most preferably 0.1mol/L, more preferably
Specific point value between 3%, and above-mentioned numerical value, as space is limited and for concise consideration, the present invention no longer exclusive list institute
State the specific point value that scope includes.
Preferably, the rotating speed of step (1) described centrifugation be 10000-15000r/min, for example can be 10000r/min,
10500r/min、11000r/min、11500r/min、12000r/min、12500r/min、13000r/min、13500r/min、
14000r/min, 14500r/min or 15000r/min, preferably 11000-13000r/min, more preferably 12000r/
Min, the specific point value between more preferably 3%, and above-mentioned numerical value, as space is limited and for concise consideration, this hair
The specific point value that scope described in bright no longer exclusive list includes.
Preferably, the time of step (1) described centrifugation be 5-20min, for example can be 5min, 6min, 7min, 8min,
9min, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min or 20min, preferably
For 8-15min, more preferably 10min, the specific point value between more preferably 3%, and above-mentioned numerical value is limited to a piece
Width and the consideration for simplicity, the present invention specific point value that no longer scope described in exclusive list includes.
According to the present invention, the concentration of the lysozyme added in the present invention is 50mg/mL, step (1) described lysozyme
The addition amount of being is 50-300 μ L, for example, can be 50 μ L, 60 μ L, 70 μ L, 80 μ L, 90 μ L, 100 μ L, 110 μ L, 120 μ L, 130
μ L, 150 μ L, 160 μ L, 180 μ L, 200 μ L, 210 μ L, 230 μ L, 250 μ L, 260 μ L, 270 μ L, 280 μ L, 290 μ L or 300 μ L,
Preferably 100-200 μ L, more preferably 180 μ L, and the specific point value between above-mentioned numerical value, as space is limited and for letter
Bright consideration, the present invention specific point value that no longer scope described in exclusive list includes.
Preferably, step (1) reaction temperature for adding lysozyme is 35-40 DEG C, for example can be 35 DEG C, 36 DEG C,
It is 37 DEG C, 38 DEG C, 39 DEG C or 40 DEG C, preferably 36-38 DEG C, more preferably specific between 37 DEG C, and above-mentioned numerical value
Value, as space is limited and for concise consideration, the present invention specific point value that no longer scope described in exclusive list includes.
Preferably, step (1) it is described add lysozyme reaction time be 10-60min, for example can be 10min,
12min, 13min, 15min, 18min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min or
Specific point value between 60min, preferably 25-50min, more preferably 40min, and above-mentioned numerical value, as space is limited and
For concise consideration, the present invention specific point value that no longer scope described in exclusive list includes.
According to the present invention, the nucleotide sequence of the primer is as shown in SEQ ID NO.1-6, shown in SEQ ID NO.1-4
Nucleotide sequence it is amplifiable go out probiotics 16s rDNA v1-v3 areas, the nucleotide sequence shown in SEQ ID NO.5-6 can expand
Increase the v5-v6 areas for probiotics 16s rDNA, the preferably nucleotide sequence shown in SEQ ID NO.3-4, the SEQ ID
Nucleotide sequence shown in NO.3-4 is the degenerate primer designed according to 16s rDNA v1-v3 areas, described nucleotide sequence
It is as follows：
27F(SEQ ID NO.1)：5’-AGAGTTTGATCCTGGCTCAG-3’；
543R(SEQ ID NO.2)：5’-ATTACCGCGGCTGCTGG-3’；
27F’(SEQ ID NO.3)：5’-AGRGTTYGATYCTGGCTCAG-3’；
543R’(SEQ ID NO.4)：5’-ATTACCGCGGCTGCTGG-3’；
786F(SEQ ID NO.5)：5’-GATTAGATACCCTGGTAGT-3’；
1079R(SEQ ID NO.6)：5’-TCACGACACGAGCTGACGAC-3’.
Preferably, the condition of step (2) the PCR amplifications, with the amplification condition of the primer shown in SEQ ID NO.1-4
For：(a) 50 DEG C of pre-degenerations 2min, 95 DEG C of pre-degeneration 3min；(b) 95 DEG C of denaturation 20s, 58 DEG C of annealing 30s, 72 DEG C of extension 2min,
Totally 30 circulations；(c) 4 DEG C of preservations.
Preferably, it is with the amplification condition of the primer shown in SEQ ID NO.5-6：(a) 50 DEG C of pre-degeneration 2min, 95 DEG C pre-
It is denatured 3min；(b) 95 DEG C of denaturation 15s, 55 DEG C of annealing 20s, 72 DEG C of extension 2min, totally 30 circulations；(c) 4 DEG C of preservations.
Preferably, the method for described source tracing method, comprises the following steps：
(1) sample pre-treatments：Sodium citrate and sodium hydroxide, the final concentration of the sour sodium of the lemon are added into detected sample
For 0.01-0.3mol/L, the final concentration of 0.01-0.5mol/L of the sodium hydroxide, after mixing, 10000-15000r/min from
Heart 5-20min abandons supernatant, and lysozyme, 35-40 DEG C of incubation 1-10h are added into precipitation；
(2) extraction of the genomic DNA of sample after handling；
(3) 16S rRNA PCR are expanded：Directed toward bacteria 16s rDNA v1-v3 areas, enter performing PCR from specific primer and expand
Increase, the condition of PCR amplifications is：(a) 50 DEG C of pre-degenerations 2min, 95 DEG C of pre-degeneration 3min；(b) 95 DEG C of denaturation 20s, 58 DEG C of annealing
30s, 72 DEG C of extension 2min, totally 30 circulations；(c) 4 DEG C of preservations；
(4) Illumina Miseq are sequenced：PCR primer is subjected to Illumina Miseq sequencings.
Second aspect, the present invention provides a kind of specific PCR amplimer of the source tracing method of probiotics in dairy products, institute
Stating the sequence of pcr amplification primer thing includes：Nucleotide sequence shown in SEQ ID NO.1-6, nucleosides preferably shown in SEQ ID NO.3
The anti-sense primer of nucleotide sequence shown in the sense primer and SEQ ID NO.4 of acid sequence.
27F(SEQ ID NO.1)：5’-AGAGTTTGATCCTGGCTCAG-3’；
543R(SEQ ID NO.2)：5’-ATTACCGCGGCTGCTGG-3’；
27F’(SEQ ID NO.3)：5’-AGRGTTYGATYCTGGCTCAG-3’；
543R’(SEQ ID NO.4)：5’-ATTACCGCGGCTGCTGG-3’；
786F(SEQ ID NO.5)：5’-GATTAGATACCCTGGTAGT-3’；
1079R(SEQ ID NO.6)：5’-TCACGACACGAGCTGACGAC-3’.
Preferably, the primer is used for the v1-v3 areas and v5-v6 areas for expanding the 16s rDNA of probiotics in dairy products.
(1) sample pre-treatments：Sodium citrate and sodium hydroxide are added into detected sample, supernatant is abandoned in centrifugation after mixing,
Lysozyme is added into precipitation to be incubated；
(2) extraction of the genomic DNA of sample after handling；
(3) 16S rRNA PCR are expanded：Directed toward bacteria 16s rDNA v1-v3 areas and v5-v6 areas, design primer is carried out
PCR is expanded；
(4) Illumina Miseq are sequenced：PCR primer is subjected to Illumina Miseq sequencings.
According to the present invention, the final concentration of 0.01-0.2mol/L, preferably 0.02- of step (1) described sodium citrate
0.1mol/L, for example, can be 0.01mol/L, 0.02mol/L, 0.03mol/L, 0.04mol/L, 0.05mol/L, 0.06mol/
L、0.08mol/L、0.09mol/L、0.1mol/L、0.12mol/L、0.15mol/L、0.16mol/L、0.18mol/L、
0.2mol/L, more preferably 0.03-0.08mol/L, most preferably 0.05-0.07mol/L, more preferably 3%, with
And the specific point value between above-mentioned numerical value, as space is limited and for concise consideration, scope of the invention no longer described in exclusive list
Including specific point value.
After " final concentration " of the present invention is addition testing sample, the concentration in reaction system.
According to the present invention, the final concentration of 0.01-0.5mol/L of step (1) described sodium hydroxide for example can be
0.01mol/L、0.02mol/L、0.03mol/L、0.04mol/L、0.05mol/L、0.06mol/L、0.08mol/L、
0.09mol/L、0.1mol/L、0.12mol/L、0.15mol/L、0.16mol/L、0.18mol/L、0.2mol/L、0.22mol/
L、0.23mol/L、0.25mol/L、0.26mol/L、0.28mol/L、0.3mol/L、0.32mol/L、0.35mol/L、
0.36mol/L, 0.38mol/L, 0.4mol/L, 0.42mol/L, 0.43mol/L, 0.45mol/L, 0.48mol/L or 0.5mol/
L, preferably 0.05-0.3mol/L, more preferably 0.08-0.2mol/L, most preferably 0.1mol/L, more preferably
Specific point value between 3%, and above-mentioned numerical value, as space is limited and for concise consideration, the present invention no longer exclusive list institute
State the specific point value that scope includes.
Preferably, the rotating speed of step (1) described centrifugation be 10000-15000r/min, for example can be 10000r/min,
10500r/min、11000r/min、11500r/min、12000r/min、12500r/min、13000r/min、13500r/min、
14000r/min, 14500r/min or 15000r/min, preferably 11000-13000r/min, more preferably 12000r/
Min, the specific point value between more preferably 3%, and above-mentioned numerical value, as space is limited and for concise consideration, this hair
The specific point value that scope described in bright no longer exclusive list includes.
Preferably, the time of step (1) described centrifugation be 5-20min, for example can be 5min, 6min, 7min, 8min,
9min, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min or 20min, preferably
For 8-15min, more preferably 10min, the specific point value between more preferably 3%, and above-mentioned numerical value is limited to a piece
Width and the consideration for simplicity, the present invention specific point value that no longer scope described in exclusive list includes.
According to the present invention, the concentration of the lysozyme added in the present invention is 50mg/mL, step (1) described lysozyme
Addition be 50-300 μ L, for example can be 50 μ L, 60 μ L, 70 μ L, 80 μ L, 90 μ L, 100 μ L, 110 μ L, 120 μ L, 130 μ L,
150 μ L, 160 μ L, 180 μ L, 200 μ L, 210 μ L, 230 μ L, 250 μ L, 260 μ L, 270 μ L, 280 μ L, 290 μ L or 300 μ L, preferably
For 100-200 μ L, more preferably 180 μ L, and the specific point value between above-mentioned numerical value, as space is limited and for concise
Consider, the present invention specific point value that no longer scope described in exclusive list includes.Preferably, step (1) is described adds lysozyme
Reaction temperature is 35-40 DEG C, for example, can be 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C or 40 DEG C, preferably 36-38 DEG C, enter one
Step is preferably the specific point value between 37 DEG C, and above-mentioned numerical value, and as space is limited and for concise consideration, the present invention is no longer poor
The specific point value that the scope includes is enumerated to the greatest extent.
Preferably, step (1) it is described add lysozyme reaction time be 10-60min, for example can be 10min,
12min, 13min, 15min, 18min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min or
Specific point value between 60min, preferably 25-50min, more preferably 40min, and above-mentioned numerical value, as space is limited and
For concise consideration, the present invention specific point value that no longer scope described in exclusive list includes.
Preferably, the condition of step (2) the PCR amplifications, with the amplification condition of the primer shown in SEQ ID NO.1-4
For：(a) 50 DEG C of pre-degenerations 2min, 95 DEG C of pre-degeneration 3min；(b) 95 DEG C of denaturation 20s, 58 DEG C of annealing 30s, 72 DEG C of extension 2min,
Totally 30 circulations；(c) 4 DEG C of preservations.
Preferably, it is with the amplification condition of the primer shown in SEQ ID NO.5-6：(a) 50 DEG C of pre-degeneration 2min, 95 DEG C pre-
It is denatured 3min；(b) 95 DEG C of denaturation 15s, 55 DEG C of annealing 20s, 72 DEG C of extension 2min, totally 30 circulations；(c) 4 DEG C of preservations.
Preferably, the method for described source tracing method, comprises the following steps：
(1) sample pre-treatments：Sodium citrate and sodium hydroxide are added into detected sample, the end of the sodium citrate is dense
Spend for 0.01-0.3mol/L, the final concentration of 0.01-0.5mol/L of the sodium hydroxide, after mixing, 10000-15000r/min
Centrifugation 5-20min abandons supernatant, and lysozyme, 35-40 DEG C of incubation 1-10h are added into precipitation；
(2) extraction of the genomic DNA of sample after handling；
(3) 16S rRNA PCR are expanded：Directed toward bacteria 16s rDNA v1-v3 areas, enter performing PCR from specific primer and expand
Increase, the condition of PCR amplifications is：(a) 50 DEG C of pre-degenerations 2min, 95 DEG C of pre-degeneration 3min；(b) 95 DEG C of denaturation 20s, 58 DEG C of annealing
30s, 72 DEG C of extension 2min, totally 30 circulations；(c) 4 DEG C of preservations；
(4) Illumina Miseq are sequenced：PCR primer is subjected to Illumina Miseq sequencings.
Compared with prior art, the present invention has the advantages that：
(1) present invention is by by sample pre-treatments step, especially by the sodium citrate and hydrogen for adding certain concentration
Sodium oxide molybdena mixes centrifugation, coordinates lysozyme to be incubated, and improves the effect of DNA extractions, is conducive to follow-up storehouse of building to detect；
(2) present invention shows 27F ' (SEQ ID NO.3) and 543R ' that the present invention is designed by the comparison of multipair primer
(SEQ ID NO.4) resolution ratio and amplification efficiency are balanced, can carry out effective district to the probiotics in sample and assign to kind；
(3) present invention utilizes Illumina Miseq microarray datasets to the food such as commercially available fermented dairy product and probiotics milk powder
Contained probiotics has carried out the analysis of bacterial strain species and abundance, and analysis result shows that probiotics is actual in solid probiotic products
Abundance and sign are basically identical, and the bacterial strain for being actually sequenced and indicating in fermented type probiotic products is compared and differed greatly, and deposit
In product indicia mistake, the bacterial strain such as Bifidobacterium is the problems such as abundance is relatively low in the product.
Brief description of the drawings
Fig. 1 is the electrophoretogram that different primers of the present invention expand probiotics standard items DNA, wherein, marker is DL2000 points
Protonatomic mass standard, band is followed successively by 100,250,500,750,1000 from down to up and 2000bp, a are 27F (SEQ ID NO.1)
With 543R (SEQ ID NO.2) amplification, b is 27F ' (SEQ ID NO.3) and 543R ' (SEQ ID NO.4) amplification
As a result, c is 786F (SEQ ID NO.5) and 1079R (SEQ ID NO.6) amplification, and con is positive control, and NTC is the moon
Property control；
Fig. 2 is the DNA that 27F ' of the present invention (SEQ ID NO.3) and 543R ' (SEQ ID NO.4) primer expand actual sample
Electrophoretogram, wherein, Fig. 2 (A) is that sample 1-12, Fig. 2 (B) are that sample 13-24, Fig. 2 (C) are that sample 25-36, marker are
DL2000 molecular mass standards, band be followed successively by from down to up 100,250,500,750,1000 and 2000bp, 1-36 represent not
Same detection sample, N is blank control, and P is positive control；
Fig. 3 is the distributed number of 1~No. 16 sample sequencing sequence of the invention；
Fig. 4 is the distributed number of 17~No. 36 sample sequencing sequences of the invention.
Embodiment
Further to illustrate the technological means and its effect of the invention taken, below in conjunction with accompanying drawing and by specific real
Mode is applied to further illustrate technical scheme, but the present invention is not limited in scope of embodiments.
In the examples where no specific technique or condition is specified, according to the technology or condition described by document in the art,
Or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be can be by regular channel commercially available from
The conventional products of acquisition.
Experiment material：
Mixing probiotics standard items (bite Lactobacillus lactis, bifidobacterium lactis, Lactobacillus rhamnosus, streptococcus thermophilus, plant breast
Bacillus, bifidobacterium longum, lactobacillus paracasei), red Knicks company provides；Acidified milk, baby formula milk powder and containing prebiotic
Bacterium food totally 36, is purchased from the major supermarkets in Shanghai, and all samples are all indicated containing profitable probliotics, wherein 15 kinds of domestic brand, are entered
21 kinds of brand of mouth, covers different manufacturing enterprises totally 26.Record species composition of all samples sign etc..
The water of trisodium citrate two, sodium hydroxide, absolute ethyl alcohol, chloroform, phenol chloroform be purchased from raw work bioengineering (on
Sea) limited company；Tiangen bacterial genomes DNA extraction kits (Cat.：DP302-02), Proteinase K (20mg/
ML), lysozyme soln (50mg/mL), agarose, ethidium bromide EB, 1 × TE (Cat.：DP324-03)、50×TAE
Electrophoresis buffer solutions (Fermentas), DL 2000TM DNA Marker Tiangeng biochemical technologies Co., Ltd；
TaKaRa Ex TaqTM PCR reagents；AxyPrepTMPCR Clean-Up Kit(Cat.：AP-PCR-50), DNA glue reclaims are tried
Agent box (Cat：AP-Gx-500) DNA glue reclaims kit：) Axygen companies of the U.S.；
Laboratory apparatus：
Illumina companies of the Illumina Miseq high-flux sequence instrument U.S.；
Tanon2500 gel analysis imaging systems Shanghai Tian Neng companies；
BIO-RAD companies of the CFX-96 quantitative real time PCR Instruments U.S.；
Eppendorf Centrifuge 5415R centrifuges, Thermomixer Comfort constant temperature oscillation couveuses,
Eppendorf Mastercycle Gradient PCR instrument Ai Bende companies.
Embodiment 1：Different primer detection probiotics standard items
The source tracing method of probiotics in a kind of dairy products, it is characterised in that comprise the following steps：
(1) sample pre-treatments：Take 1g to mix probiotics standard items, be dissolved in 10mL ddH2In O, the lemons of 1 mL 18% are added
Sour sodium and 500 μ L 1mol/L sodium hydroxides, 12000r/min centrifuges 10min and abandons supernatant after mixing, and 180 μ L are added into precipitation
Lysozyme is incubated；
(2) sample extracts probiotics complete genome DNA with Tiangen bacterial genomes DNA extraction kit after handling；
(3) 16S rRNA PCR are expanded：Directed toward bacteria 16s rDNA v1-v3 areas, are distinguished with SEQ ID NO.1-6 primers
Enter performing PCR amplification；
The nucleotide sequence of described primer is as follows：
27F(SEQ ID NO.1)：5’-AGAGTTTGATCCTGGCTCAG-3’；
543R(SEQ ID NO.2)：5’-ATTACCGCGGCTGCTGG-3’；
27F’(SEQ ID NO.3)：5’-AGRGTTYGATYCTGGCTCAG-3’；
543R’(SEQ ID NO.4)：5’-ATTACCGCGGCTGCTGG-3’；
786F(SEQ ID NO.5)：5’-GATTAGATACCCTGGTAGT-3’；
1079R(SEQ ID NO.6)：5’-TCACGACACGAGCTGACGAC-3’；
Specific amplification condition is as follows：
One group of negative control using water as sample.
27F (SEQ ID NO.1) and 543R (SEQ ID NO.2)
27F ' (SEQ ID NO.3) and 543R ' (SEQ ID NO.4) amplification 16S rRNA V1-V3 areas,
This two groups reaction condition is as follows：
786F (SEQ ID NO.5) and 1079R (SEQ ID NO.6) amplification 16S rRNA V5-V6 areas, the reaction of this group
Condition is as follows：
(3) Illumina Miseq are sequenced：PCR primer is subjected to Illumina Miseq sequencings.
1% Ago-Gel is prepared, 5 μ L PCR primers are taken, using the molecular weight standards of DL 2000 as Marker, regulation electricity
It is pressed in after 120V, electrophoresis 30min, result figure is as shown in figure 1, all amplified productions can obtain one clearly on Ago-Gel
Clear fine and close band, and it is consistent with expected clip size, 3 parts of standard items DNA use 27F (SEQ ID NO.1) and 543R (SEQ
ID NO.2), 27F ' (SEQ ID NO.3) and 543R ' (SEQ ID NO.4) amplification, band 508bp occur；Use 786F
(SEQ ID NO.5) and 1079R (SEQ ID NO.6) primer is expanded, and band occurs in 294bp, and positive control and feminine gender are right
Unanimously illustrate that epicycle is expanded qualified with expected according to result, can proceed with and build storehouse experiment.
Embodiment 2：Probiotics standard items 16S rDNA build storehouse analysis
(1) PCR primer is purified
After electrophoresis detection is errorless, AxyPrep is usedTMPCR Clean-Up Kit carry out post purifying to PCR primer and reclaimed.First
Add the buffer solution buffer solution PCR-A (if buffer solution PCR-A adds to 100uL less than 100uL) of 3 volumes in PCR primer；Will system
Standby pipe is put into 2mL centrifuge tubes, plus 700 μ L buffer solutions W2,12,000Xg 1min, abandons filtrate；Pipe will be prepared and be put into centrifuge tube
In, add 400 μ L buffer solution buffer solutions W2,12,000rpm centrifugation 1min；Filtrate is abandoned, dally 12,000rpm 1min, elution
DNA。
(2) mixing library and the sequencing of upper machine
Each sample reclaimed correspondence 16S rRNA sequences are uniformly mixed, storehouse reagent is built using Illumina DNA fragmentations
Box builds storehouse, the quantitative library of Qubit methods, and uses Q-PCR detections library effective percentage.
Mixing library (10nmol/L) is progressively quantitatively diluted to the upper machine high-flux sequence of progress after 5pmol/L, sequencing side
Method is synthesis sequencing, and measured length is 2 × 300bp.
(3) data analysis
Obtained raw image data is sequenced to Illumina Miseq using FastQC and CASAVA v1.8.2 softwares to enter
The identification of row base is converted into sequence data and output statistics.By biological information statistical method, the base of sequencing reading length is counted
Distribution.Using QIIME v1.8.0 softwares, the sequence for splicing completion is made a distinction according to barcode labels, retained and label
The sequence being consistent, removes joint sequence and the relatively low reading of sequencing quality is long, finally removes two ends primer sequence, sequence label and embedding
Fit sequence.OTU clusters are carried out using pick_open_reference modes in QIIME analysis process, clustering algorithm is used
Uclust softwares are 97% pair of all sequences progress OTU division according to similarity threshold and carry out biological information statistical analysis；Most
The 16S databases in RDP Classifier softwares are utilized afterwards, and species annotation is carried out after filtering OUT under the conditions of confidence level 0.9.
The abundance situation (reading long quantity) of each reference culture see the table below 1 and Fig. 2 in standard items sequencing result：
Table 1
From result, V1-V3 areas analysis result analyzes result difference than larger with V5-V6 areas, on V1-V3 sections,
Lactobacillus acidophilus, Lactobacillus rhamnosus, streptococcus thermophilus, Lactobacillus plantarum, lactobacillus paracasei and bifid bar in standard items
Pseudomonas has good differentiation, but because 27F universal primer has 3 mispairing to Bifidobacterium, bifidobacterium species atcc is divided
Distinguish bad；And the degenerate primer 27F ' (SEQ ID NO.3) and 543R ' (SEQ ID NO.4) designed according to V1-V3 sections, energy
Bifidobacterium species atcc is distinguished well, solves mismatch problems.V5-V6 areas its amplification efficiency of universal primer is more general than V1-V3 area to be drawn
Thing is high, but resolution ratio is relatively low.
The long quantity precentagewise of reading corresponding to each reference culture in every group of sequencing result is indicated, with standard items
In each reference culture actual quantity percentage be compared, using 27F (SEQ ID NO.1) and 543R (SEQ ID NO.2) with
Two groups of primer sequencing comparison results of 786F (SEQ ID NO.5) and 1079R (SEQ ID NO.6) and standard items bacterial strain quantitative proportion
It is more larger than difference, illustrate to be sequenced again using the amplification of these two pair primer, it is impossible to reflect actual sample bacterial strain information；And use V1-
Bacterial strain abundance and standard obtained by V3 degenerate primers 27F ' (SEQ ID NO.3) and 543R ' (SEQ ID NO.4) amplification sequencings
Product bacterial strain quantitative proportion is substantially suitable, therefore in the sequencing of actual sample, should select V1-V3 degenerate primers 27F ' (SEQ ID
) and 543R ' (SEQ ID NO.4) is used as the amplimer of early stage NO.3.
Embodiment 3：Actual sample is detected with degenerate primer
The source tracing method of probiotics in a kind of dairy products, it is characterised in that comprise the following steps：
(1) sample pre-treatments：Take 1g to mix probiotics standard items, be dissolved in 10mL ddH2In O, the lemons of 1mL 18% are added
Sour sodium and 500 μ L 1mol/L sodium hydroxides, 12000r/min centrifuges 10min and abandons supernatant after mixing, and 180 μ L are added into precipitation
Lysozyme is incubated；
(2) sample extracts probiotics complete genome DNA with Tiangen bacterial genomes DNA extraction kit after handling；
(3) 16S rRNA PCR are expanded：Directed toward bacteria 16s rDNA v1-v3 areas, are distinguished with SEQ ID NO.3-4 primers
Enter performing PCR amplification；
The nucleotide sequence of described primer is as follows：
27F’(SEQ ID NO.3)：5’-AGRGTTYGATYCTGGCTCAG-3’；
543R’(SEQ ID NO.4)：5’-ATTACCGCGGCTGCTGG-3’；
Specific amplification condition is as follows：
One group of negative control using water as sample.
27F ' (SEQ ID NO.3) and 543R ' (SEQ ID NO.4) amplification 16S rRNA V1-V3 areas,
This two groups reaction condition is as follows：
(3) Illumina Miseq are sequenced：PCR primer is subjected to Illumina Miseq sequencings.
All PCR primers are detected using 1% agarose gel electrophoresis, using the molecular weight standards of DL 2000 as Marker, are adjusted
Economize on electricity is pressed in after 120V, electrophoresis 30min, shown in result figure such as Fig. 2 (A) -2 (C), it is seen then that 36 kinds of in the market purchase are containing prebiotic
Bacterium sample, as shown in table 2, the 16S v1-v3 intervals in the sample occur only specific species through augmentation detection at 508bp
One and clearly fluorescent bands, PCR primer result is the positive, illustrate the degenerate primer 27F ' (SEQ ID NO.3) that designs and
543R ' (SEQ ID NO.4) can be used for amplification target dna.
Table 2
Embodiment 4：The 16S rDNA of actual sample build storehouse analysis
(1) PCR primer is purified
After electrophoresis detection is errorless, AxyPrep is usedTMPCR Clean-Up Kit carry out post purifying to PCR primer and reclaimed.First
Add the buffer solution PCR-A (if buffer solution PCR-A adds to 100uL less than 100uL) of 3 volumes in PCR primer；Pipe will be prepared to put
Enter in 2mL centrifuge tubes, plus 700 μ L buffer solutions W2,12,000Xg 1min, abandon filtrate；Pipe will be prepared to be put into centrifuge tube, added
400 μ L buffer solutions W2,12,000rpm centrifugation 1min；Filtrate is abandoned, dally 12,000rpm 1min, eluted dna.
(2) mixing library and the sequencing of upper machine
Each sample reclaimed correspondence 16S rRNA sequences are uniformly mixed, storehouse reagent is built using Illumina DNA fragmentations
Box builds storehouse, the quantitative library of Qubit methods, and uses Q-PCR detections library effective percentage.
Mixing library (10nmol/L) is progressively quantitatively diluted to the upper machine high-flux sequence of progress after 5pmol/L, sequencing side
Method is synthesis sequencing, and measured length is 2 × 300bp.
(3) data analysis
The primary data obtained through high-flux sequence handles to obtain by Quality Control can be used for the dry of later stage analysis of biological information
The long sequence of net reading, then distinguishes the clean aggregate sample read in long sequence according to the corresponding primer bar code sequence of different samples
This, using Bowtie2 softwares to sequence carry out sign positioning, then with RDP ribosomes database projects (Ribosomal
Database Project) in the existing sequence of 16S databases be compared, only when in two ends and the database of the sequence
Sequence when matching completely the sequence could be retained.Carried out according to the method described above to obtained total sequence is sequenced in 36 samples
Gene sequencing, obtains the mark sequence matched with RDP databases.
The sequence that can be compared with bacterium of the same race will be merged into a class, according to mark sequence of this method to all samples
Row carry out statistic of classification by bacterial species, by counting the quantity i.e. sequence quantity that a certain " sequence " occurs in whole sequencing,
Strain corresponding to " sequence " actual distribution situation in the sample is just capable of detecting when, sequence number numerical quantity is higher, illustrates the bacterium
Plant RNA abundance high.Probiotics abundance (sequence quantity) situation is shown in Table 3-6 in 36 samples.
Table 3
Table 4
Table 5
Table 6
1~No. 16 sample is acidified milk, and this kind of product needs to use lactobacillus-fermented to produce yogurt in production, industrially
Conventional fermentation strain is generally streptococcus thermophilus, lactobacillus bulgaricus, bites Lactobacillus lactis etc..From 1~No. 16 sample 16S
From the point of view of rDNA analysis results, this class product has the conventional fermented bacterium of industry, from Fig. 4 it can be seen that, in addition to No. 15 samples, protect
Plus Leah lactobacillus, streptococcus thermophilus and abundance of the Lactobacillus lactis in this kind of sample is bitten all in front three, illustrate this kind of product
The composition of middle probiotics has higher similarity.More effect of above-mentioned three kinds of strains is that milk raw material is fermented, and right
Human body acts on bigger probiotics such as Bifidobacterium, then universal content is relatively low.
Fig. 3-4 is visible, 1, No. 5 samples do not detect Bifidobacterium in product indicia；No. 8 sample markers have not tally bifid
Bacillus, but actually detected actually animal bifidobacteria；And abundance of the Bifidobacterium in this kind of product is well below other bacterial strains,
May it is original be because Bifidobacterium be obligate anaerobe, it is more harsh to growth conditions, in production storage not
Easily survival.
In 16 acidified milk samples selected by the present invention, the actual bacterial strain for being sequenced and indicating, which is compared, all to be had differences,
Mainly have it is following some：First, sign bacterial strain is not comprehensive enough, and less than actual bacterial strain, such as sample 14,15 only indicates biodiasmin；
2nd, sign strain name is general, only to category, less than kind, and such as sample 3,4 only indicates Bifidobacterium, indicates not specific enough；3rd, bacterium
Animal bifidobacteria is denoted as bifidobacterium bifidum by strain title sign mistake, sample 8；4th, sign bacterial strain is in actually detected
Missing.
17~No. 32 samples are probiotics milk powder, and 33~No. 36 samples are probiotics solid food.This kind of product be sequenced with
It is smaller that the bacterial strain of sign compares difference.China has strict demand to the edible probiotics of infant, and regulation arrives " strain ".Newborn bifid
Bacillus is a subspecies of animal bifidobacteria, and both bacteriums are minimum in the interval differences of 16S rRNAV1-V3, only to V1-V3
Interval sequencing is difficult to separate them, therefore bifidobacterium lactis and animal bifidobacteria are attributed into a class in the present invention united
Meter analysis.It is prebiotic in other samples from Fig. 3-4 as can be seen that removing No. 36 sample marker strain bifidobacteriums not specific to kind
The actual abundance of bacterium is basically identical with indicating.Possible cause is due to that fermented type probiotic composition is short to sales cycle from producing, and
Probiotic group is difficult supervision detection into being compound mostly；And in probiotics milk powder add bacterial strain by strict regulations, composition it is single and
Product is more easy to storage.
Using Illumina Miseq microarray datasets to the benefit contained by the food such as commercially available fermented dairy product and probiotics milk powder
Raw bacterium has carried out the analysis of bacterial strain species and abundance, and analysis result shows the actual abundance of probiotics and mark in solid probiotic products
Show basically identical, and actually sequencing and the bacterial strain of sign are compared and differed greatly in fermented type probiotic products, and there is product mark
Show mistake, the bacterial strain such as Bifidobacterium is the problems such as abundance is relatively low in the product.Test result indicates that the V1-V3 degenerate primers of design
27F ' (SEQ ID NO.3) and 543R ' (SEQ ID NO.4) resolution ratio and amplification efficiency are balanced, can be to the probiotics in sample
Carry out effective district and assign to kind.
Applicant states that the present invention illustrates the method detailed of the present invention, but not office of the invention by above-described embodiment
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implemented.Art
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., within the scope of all falling within protection scope of the present invention and being open.
SEQUENCE LISTING
<110>Shanghai inspection and quarantine bureau animals and plants and Food Inspection center
<120>The source tracing method of probiotics in a kind of dairy products
<130> 2016
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial synthesized sequence
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 17
<212> DNA
<213>Artificial synthesized sequence
<400> 2
attaccgcgg ctgctgg 17
<210> 3
<211> 20
<212> DNA
<213>Artificial synthesized sequence
<400> 3
agrgttygat yctggctcag 20
<210> 4
<211> 17
<212> DNA
<213>Artificial synthesized sequence
<400> 4
attaccgcgg ctgctgg 17
<210> 5
<211> 19
<212> DNA
<213>Artificial synthesized sequence
<400> 5
gattagatac cctggtagt 19
<210> 6
<211> 20
<212> DNA
<213>Artificial synthesized sequence
<400> 6
tcacgacacg agctgacgac 20
Claims (9)
1. the source tracing method of probiotics in a kind of dairy products, it is characterised in that comprise the following steps：
(1) sample pre-treatments：Sodium citrate and sodium hydroxide are added into detected sample, supernatant, Xiang Chen are abandoned in centrifugation after mixing
Lysozyme is added in shallow lake to be incubated；
(2) extraction of the genomic DNA of sample after handling；
(3) 16S rRNA PCR are expanded：Directed toward bacteria 16s rDNA v1-v3 areas and v5-v6 areas, design primer enters performing PCR expansion
Increase；
(4) Illumina Miseq are sequenced：PCR primer is subjected to Illumina Miseq sequencings.
2. according to the method described in claim 1, it is characterised in that the final concentration of 0.01- of step (1) described sodium citrate
0.2mol/L, preferably 0.02-0.08mol/L, more preferably 0.03-0.08mol/L, most preferably 0.05-
0.07mol/L；
Preferably, the final concentration of 0.01-0.5mol/L, preferably 0.05-0.3mol/L of step (1) described sodium hydroxide, enter
One step is preferably 0.08-0.2mol/L, most preferably 0.1mol/L.
3. method according to claim 1 or 2, it is characterised in that the rotating speed of step (1) described centrifugation is 10000-
15000r/min, preferably 11000-13000r/min, more preferably 12000r/min；
Preferably, the time of step (1) described centrifugation is 5-20min, more preferably preferably 8-15min, 10min.
4. the method according to any one of claim 1-4, it is characterised in that the addition of step (1) described lysozyme
For 50-300 μ L, preferably 100-200 μ L, more preferably 180 μ L；
Preferably, step (1) it is described add lysozyme reaction temperature be 35-40 DEG C, preferably 36-38 DEG C, further preferably
For 37 DEG C；
Preferably, step (1) it is described add lysozyme reaction time be 10-60min, preferably 25-50min, further it is excellent
Elect 40min as.
5. the method according to any one of claim 1-4, it is characterised in that the core of step (2) described specific primer
Nucleotide sequence is as shown in SEQ ID NO.1-6, the nucleotide sequence preferably shown in SEQ ID NO.3-4.
6. the method according to any one of claim 1-5, it is characterised in that the condition of step (2) the PCR amplifications,
Amplification condition with the primer shown in SEQ ID NO.1-4 is：(a) 50 DEG C of pre-degenerations 2min, 95 DEG C of pre-degeneration 3min；(b)95
DEG C denaturation 20s, 58 DEG C annealing 30s, 72 DEG C extension 2min, totally 30 circulation；(c) 4 DEG C of preservations；
Preferably, it is with the amplification condition of the primer shown in SEQ ID NO.5-6：(a) 50 DEG C of pre-degeneration 2min, 95 DEG C of pre-degenerations
3min；(b) 95 DEG C of denaturation 15s, 55 DEG C of annealing 20s, 72 DEG C of extension 2min, totally 30 circulations；(c) 4 DEG C of preservations.
7. the source tracing method according to any one of claim 1-6, it is characterised in that comprise the following steps：
(1) sample pre-treatments：Add sodium citrate and sodium hydroxide into detected sample, the sour sodium of the lemon it is final concentration of
0.01-0.3mol/L, the final concentration of 0.01-0.5mol/L of the sodium hydroxide, after mixing, 10000-15000r/min centrifugations
5-20min abandons supernatant, and lysozyme, 35-40 DEG C of incubation 1-10h are added into precipitation；
(2) extraction of the genomic DNA of sample after handling；
(3) 16S rRNA PCR are expanded：Directed toward bacteria 16s rDNA v1-v3 areas, performing PCR amplification is entered from specific primer,
PCR amplification condition be：(a) 50 DEG C of pre-degenerations 2min, 95 DEG C of pre-degeneration 3min；(b) 95 DEG C of denaturation 20s, 58 DEG C of annealing 30s,
72 DEG C of extension 2min, totally 30 circulations；(c) 4 DEG C of preservations；
(4) Illumina Miseq are sequenced：PCR primer is subjected to Illumina Miseq sequencings.
8. the specific PCR amplimer for the source tracing method of probiotics in dairy products, it is characterised in that the PCR amplifications
The sequence of primer includes：Nucleotide sequence shown in SEQ ID NO.1-6, nucleotide sequence preferably shown in SEQ ID NO.3
The anti-sense primer of nucleotide sequence shown in sense primer and SEQ ID NO.4.
9. specific PCR amplimer according to claim 8, it is characterised in that the primer is used to expand dairy products
The 16s rDNA of middle probiotics v1-v3 and V5-V6 areas.
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Application publication date: 20170818 |
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