CN106555000A - A kind of method of plant derived component in plant identification protein beverage - Google Patents

A kind of method of plant derived component in plant identification protein beverage Download PDF

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CN106555000A
CN106555000A CN201610987090.7A CN201610987090A CN106555000A CN 106555000 A CN106555000 A CN 106555000A CN 201610987090 A CN201610987090 A CN 201610987090A CN 106555000 A CN106555000 A CN 106555000A
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plant
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its2
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杨硕
江丰
张莉
李鑫
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Hubei Food Quality And Safety Supervision And Inspection Institute
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Hubei Food Quality And Safety Supervision And Inspection Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

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Abstract

The present invention provides plant derived component analysis method in a kind of vegetable protein beverage combined based on semiconductor chip sequencing technologies and digital round pcr, and step is：1）Extract the genomic DNA of biotic component in vegetable protein beverage；2）Enter performing PCR amplification to ITS2 fragments with fusion primer pair；3）The DNA fragmentation that amplification is obtained carries out high-flux sequence with ion torrent platforms；4）Plug-in unit is analyzed using plant source ITS2 bar code datas storehouse is carried in the platform, or the sequence application BLAST methods of acquisition are carried out into result identification；5）Digital pcr checking sequencing result quantitative analyses；6）The species of content proportion >=5% are classified as the plant derived component of the sample detection by comprehensive sequencing comparison result and quantitative result, each species DNA copy number proportion in analytical calculation inspection product nucleic acid.Its high flux, automatization, cycle is short, provide strong technical support for food safety Regulation.

Description

A kind of method of plant derived component in plant identification protein beverage
Technical field
The present invention relates to a kind of plant derived component detection method, more particularly to it is a kind of based on high throughput sequencing technologies sum Method of analyzing organism components in the vegetable protein beverage that word PCR combines.
Background technology
Due to " natural, green, nutrition, health " category feature that vegetable protein beverage innately possesses, meet beverage market Trend and trend, are increasingly liked by consumer, have become product indispensable on beverage market.Chinese food work What industry association issued《2015 annual beverage industry overall operation reports》In point out that China's beverage industry 2015 is annual accumulative total 17661.0 ten thousand tons of yield, and health type beverage proportion constantly rising.《12 development of food industry are planned》In it is proposed that, Vegetable protein beverage to be greatly developed, can reach the 15% of beverage total output to protein beverage production ratio in 2015.With city The rise of field rush of demand and cost of material, some manufacturing enterprises are mixed in vegetable protein beverage to reduce production cost Cheap vegetable protein powder, or mix the plant material of non-product mark with low cost, adulterates, adulteration etc. is asked Topic becomes increasingly conspicuous, and lacks the plant derived component to such product as food safety Regulation testing agency and differentiate effective The method of inspection and judgment basis.At present, only almond milk and walnut Lu (breast) this two classes product have the true and false to distinguish in execution standard Other index, but check item setup excessively single in standard, merely with several characteristic fatty acids containing in total fatty acids Amount is also not rigorous enough to judge the method for the true and false of almond milk and walnut Lu (breast), because single fat acid is in total fatty acids Content may be relevant with kind, Maturity, the place of production, it is impossible to is not only inconsistent standardization requirement with certain content of fatty acid and just predicates product Product mix puppet.In addition, the regulation that the content of single fat acid can also be artificial in product, therefore, can not judge to meet mark In standard, the product of content of fatty acid is exactly genuine almond milk and walnut Lu (breast).In sum, due to not yet setting up vegetable protein In beverage, the universal method of plant derived component identification, makes vegetable protein beverage adulteration problem detect and judge, this The development and the supervision of relevant departments of strong influence the industry.Set up plant derived component mirror in vegetable protein beverage Fixed method, can provide strong technical support for food safety Regulation, and becoming strike, vegetable protein beverage is adulterated has by force The technological means of power.Therefore it is badly in need of formulating the corresponding method of inspection.
Based on the grand genome research method of high throughput sequencing technologies be at present understanding, the biological hybrid architecture of analysis and Function is most effective, one of most important method.Metagenomics, i.e., to micropopulation gene order-checking in environmental sample, from And obtain sequence, microbial diversity and their relations and environment between of required function gene.Its research method is mainly wrapped Include relatively independent but the two of close complementary kinds of means：The measure of the evolution DNA molecular marker sequence of Jing amplifications and all genes The parsing of group.The former carries out the amplification of DNA molecular marker fragment using specific primer, and recognizes biome by being sequenced Species composition and quantitative its relative abundance；The latter then in measure system all of DNA sequence, can provide in theory including In evolutionary process, molecular marker is in interior all genomic informations.Therefore grand genome research method can it is more objective, comprehensive, The 26S Proteasome Structure and Function of mixed biologic sample is analyzed rapidly.Sending out recently as high throughput sequencing technologies and field of molecular marker Exhibition, grand genome research direction progressively penetrate into each research field, including soil, ocean, human oral cavity and gastrointestinal tract etc. are raw In thing group.
At present with regard to adulteration authentication method in food, what domestic literature record was more is protein electrophorese technology, exempts from Epidemiology technology, chromatograph and mass-spectrometric technique, and basic molecular biology detection technique.There is pre-treatment pole in protein electrophorese technology Its complexity, comparison data storehouse imperfection, automaticity is poor, flux is low, be related to the defects such as multi-biological toxicity reagent.Immunity Technology is to determine characteristic protein using ELISA euzymelinked immunosorbent assay (ELISA), has been applied in terms of meat productss identification, but at present not yet There is the report synthesized for the monoclonal antibody of all kinds of plant derived component characteristic proteins, therefore the method is in vegetable protein beverage It is still infeasible.Chromatograph, mass-spectrometric technique be by detect characteristic material, such as by the detection to soybean isoflavone, can be preliminary Whether judge in Walnut Milk mixed with Soybean Milk, the method poor accuracy, detect limit for height.Basic molecular biology technology such as qualitative PCR Or LAMP methods, the method is cumbersome, and false Yin/Yang rate is high, it is often more important that be only capable of in unilateral detection product with the presence or absence of certain Plant plant derived component, it is impossible to which " blind sieve " goes out whole plant derived components in product, cannot also find new dopant species, not be inconsistent Close the logical thinking of species identification, it is difficult to as arbitration or standard method.In addition, judge product with the presence or absence of it is premeditated fake, Mix puppet or technique brings aspect also no corresponding detection method into.
The content of the invention
For above-mentioned situation, instant invention overcomes shortcoming of the prior art, there is provided a kind of to be surveyed based on semiconductor chip Plant derived component analysis method in the vegetable protein beverage that sequence technology and digital round pcr combine.
The present invention is achieved by the following technical solutions：
1) vegetable protein beverage carries out sample pre-treatments；
2) nucleic acid extraction；
3) DNA concentration is determined；
4) enter performing PCR amplification to ITS2 fragments：The joint of sequencing and the bar code sequence Barcode for distinguishing sample are added Fusion primer pair is obtained in initial primers, initial primers sequence is：
F：5′-ATGCGATACTTGGTGTGAAT-3′；
R：5′-GACGCTTCTCCAGACTACAAT-3′；
The amplicon obtained after ITS2 amplifications can be used as the library of upper machine after purification, dilution, equal-volume mixing；
5) the ITS2 fragments that amplification is obtained are carried out into high-flux sequence；
6) using carry in ion torrent platforms plant source ITS2 bar code datas storehouse analysis plug-in unit, or by obtain Sequence application BLAST methods carry out result identification；Analysis software is carried using platform and counts effective fragment reading；
7) effectively number of fragments is counted：In a sample can tentatively being judged, the proportion relation between plant species is detected；
8) digital pcr checking sequencing result quantitative analyses；
9) result report：Comprehensive sequencing comparison result and quantitative result, each species DNA copy in analytical calculation inspection product nucleic acid The species of content proportion >=5% are classified as the plant derived component of the sample detection by number proportion.
Vegetable protein beverage refers to Walnut Milk, peanut emulsion, Soybean Milk, almond milk, sesame milk etc. and containing mentioned component Compound plant protein beverage.
Specially：
First, pre-treatment
Inspection product are fully shaken up, 30-40ml is drawn into centrifuge tube, 12000rpm, 3min abandon supernatant, leave and take 100mg and sink Form sediment stand-by；
It is optional：Vacuum freeze-drying method will examine product lyophilizing, take 100mg dried objects stand-by.
Explanation：Beverage class product substrate character is more single, generally at room temperature, sample is fully mixed, leave and take 30- 40mL samples are centrifuged, and take precipitation, if the not enough repeatable centrifugation of precipitation capacity is once, centrifugal takes short magnetic bead with downstream Method is extracted nuclei aoid methods combination and 10 parts of samples of number, and no cross contamination risk can be processed in 2 hours, suitable to most of inspection product With.Sample concentration can be dried by the relatively low sample of certain plants tissue concentration using vacuum freeze-drying method.If centrifugation is without plant Tissue precipitation, can be used directly CTAB methods and extracts sample nucleic.
2nd, nucleic acid extraction
1st, filter membrane method：DNA is extracted using business-like universal test kit, concrete operation step is with reference to related kit Description, can be adjusted during use as needed.
2nd, paramagnetic particle method：Using business-like special plant nucleic acid extraction kit, carried using full-automatic nucleic acid extraction instrument DNA is taken, concrete operation step is with reference to related kit and equipment operating manual.
3rd, DNA concentration is determined
2.5 μ L of DNA profiling are drawn, is put in full-automatic nucleic acid-protein concentration mensuration instrument, calculate concentration, concentration is in 10- 100ng/ μ L, A260/A280 between 1.6-2.0, suitable subsequent experimental.
4th, qualitative PCR (ITS2 gene amplifications)
1st, reaction system：2.5 μ L of PCR Buffer (10 ×), MgCl22 μ L (25mmol/L), 2 μ L of dNTPs mixture (2.5mmol/L), each 1.0 μ L of upstream and downstream primer (10 μm of ol/L), template DNA, Taq archaeal dna polymerase 1.0U, plus it is aseptic Distilled water is adjusted to 25 μ L also dependent on concrete condition.2nd, reaction condition：Jing refers to lot of documents, improves amplification efficiency And non-specific amplification is reduced, progressively grope PCR reaction annealing temperatures and extend duration.I.e. 94 DEG C, 5min, 94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 50s, 40 circulations；72 DEG C of extension 10min, 12 DEG C of holdings.
3rd, merge primer sequence：ITS is one of most widely used molecular marker, great Liang Guo during botanical system development is studied Inside and outside pertinent literature shows to identify very ripe as plant species using ITS2 sequences.High-flux sequence pre-treatment step compared with It is many, it is to reduce downstream experimental implementation, increases easily repeatability, can be in design of primers link by the joint being sequenced and differentiation sample Bar code sequence (Barcode) is added in initial primers to upper, and by this method, the amplicon obtained after ITS2 amplifications is passed through Purification, dilution, can be used as the library of machine after equal-volume mixing, using this design of primers scheme, both high degrees Troublesome operation and the detected downstream time of library preparation are reduced, again can be disposable with the maximized capacity for utilizing a chip Most samples are measured, secondary sequencing high flux advantage is embodied.Initial primers sequence is：F：5′- ATGCGATACTTGGTGTGAAT-3 ', R：5′-GACGCTTCTCCAGACTAC AAT-3′.
4th, amplicon purification：Take agarose gel electrophoresis method for detecting detection PCR primer.After electrophoresis, negative control should be without bar Band, purpose product such as band are single, can direct Sequencing after purification；Generally there is the miscellaneous band of non-targeted near 100bp after electrophoresis, rising Eliminated after high annealing temperature not yet, this non-specific amplification possible cause Jing analyses have two, and one is due to adopting fusion to draw Thing is expanded, and has a great role to reducing downstream process, but the joint that adds on original upstream and downstream primer and bar code sequence (Barcode) length of 30bp or so is increased, these fragments itself can not be specifically bound with target area, but plant gene Pack section is very big, and these fragments have the possibility with nontarget area specific binding, so generate miscellaneous band；Two allow for drink , there is a large amount of fractures, the DNA fragmentation of degraded in material product deep processing itself, low-abundance feature, some wheels of Jing expand in judgement sample After increasing, concentration is increased sharply, and produces miscellaneous band.Non-specific amplification seriously reduces template quality, brings to downstream experiment and has a strong impact on, institute Seem very necessary with amplicon purification, there are two kinds of way of purification：One is just to be carried nucleic acid Jing original primers to carry out first round expansion Increase, gained amplicon is carried out into the second wheel amplification as template with fusion primer then, non-specific amplification can be so reduced Situation, two be if template concentrations meet require, rubber tapping purification is directly carried out after amplification, from corresponding DNA reclaim reagents Target stripe (430bp) is reclaimed stand-by by box, and this process can lose about 30%DNA.
5th, high flux gene sequencing
1st, template is diluted, generates library：By upstream, the good DNA sample of purification is determined after concentration, is diluted to respective concentration Equal-volume mixes, and makes sequencing library, as every library sequence all carries particular bar, has ten kinds if ten parts of samples Bar code, the sequence with same bar code divide a sample into；
2nd, sequencing data splicing：Splicing software is carried using server to splice base sequence；
3rd, sequence is submitted to and is compared：Analyze using plant source ITS2 bar code datas storehouse is carried in ion torrent platforms Plug-in unit, or the sequence application BLAST methods of acquisition are carried out into result identification.Analysis software is carried using platform and counts effective fragment Reading.
4th, effective number of fragments statistics：In a sample can tentatively being judged, the proportion relation between plant species is detected. 6th, digital pcr checking sequencing result quantitative analyses
1st, reaction system：Premixed liquid 10 μ L, primer 900nM, probe 250nM, primary template DNA, plus aseptic double-distilled water is extremely 20μL；
2nd, reaction condition：95 DEG C, 10min；94 DEG C, 30s, Tm (annealing temperature presses the setting of genes of interest appropraite condition), 60s, 40 circulations；98 DEG C, 10min；4 DEG C of holdings, temperature rate are adjusted to 2 DEG C/s.
3rd, primer sequence：Corresponding primer and probe sequence are designed according to the species that sequencing examination goes out.
7th, result and report
Comprehensive and quantitative result and sequencing comparison result, each species DNA copy number proportion in analytical calculation inspection product nucleic acid, In view of in beverage actual production, production line has the intersection of various species or brings on a small quantity, and the species of content proportion >=5% are arranged For the plant derived component of the sample detection.
The experimental program that high flux gene sequencing is combined with absolute quantitation digital pcr technology that the present invention is adopted is contrasted Existing method has following clear superiority：First, with characteristic protein (protein electrophorese technology, immunological technique, chromatograph and mass spectrum Technology is all based on specific proteinses) detection compares, more substantially, the detection of more stable, more sensitive gene level it is more suitable For deep processing, low-abundance beverage Terminal Type product；Second, the directivity detection of qualitative PCR or LAMP methods is compared to, is had only By total to product plant-derived hereditary material sequencing, compare could be most true, complete discriminating product in all plant sources Property composition.3rd, PCR method or LAMP methods need the special analog sample using different melting concns to detect the sensitive of this method Degree is detection limit, and gene sequencing or digital pcr both of which are method sensitivity to be brought up to single base in theory Or the level of 1 copy, grope the quantitative detection limit of new method, further method for reducing checking completely without specially spending a lot of time and energy Cycle.4th, the semiconductor chip sequencing technologies based on current main flow are mutually tied with the digital pcr technology of achievable absolute quantitation Close, possess 2 points of advantages, one be as already described above, in product plant derived component detection by quantitative for when product detect it is non-identifying During composition, actually or deliberate to fake, mix in the property judgement that puppet technique is brought into, there is very necessary realistic meaning, therefore Composition accurate quantitative analysis must will be detected after high-flux sequence examination goes out composition, accurately judge the property of product doping；Two is to the greatest extent Pipe high-flux sequence result, can provide the bar number of the aligned fragment (reads) of each composition in sample using bioinformatics means, But this kind of data limit and compare the reasons such as the Systematic Errors of plug-in unit due to the experimental principle that experiment itself is sequenced, and are only capable of doing one Determine the reference of degree, it is impossible to as final accurate judgment basis.Therefore the digital pcr skill of the accurate absolute quantitation of energy must be adopted Art determines detection component content, is mutually authenticated with sequencing result, comprehensive analysis, could ensure data reliability to greatest extent, Strong technical support is provided for foods supervision.5th, two kinds of high-end molecular biology experiment technologies that this programme is adopted coordinate to be made With, can farthest realize that large sample, high flux, automatization, standardized method are required, shorten identification response cycle, The high timeliness of the supervision inspection extremely to emphasize provides method guarantee.6th, creative applies to fusion primer technique In the sequencing upstream experiment of beverage products, the experimental implementation of downstream library preparation is greatly simplify, and while realizes one Chip detects the experimental program for being mixed with tens of parts of sample of nucleic acid simultaneously.
Appendix A
Frequently seen plants protein beverage plant derived component primer, probe sequence
A.1 Semen sojae atricolor
5-GCCCTCTACTCCACCCCCA-3
5-GCCCATCTGCAAGCCTTTTT-3
5-HEX(FAM)-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ-3
A.2 Semen arachidis hypogaeae
5-GCAACAGGAGCAACAGTTCAAG-3
5-CGCTGTGGTGCCCTAAGG-3
5-FAM(HEX/VIC)-AGCTCAGGAACTTGCCTCAACAGTGCG-BHQ-3
A.3 Semen Juglandiss
5-CGCGCAGAGAAAGCAGAG-3
5-GACTCATGTCTCGACCTAATGCT-3
5-FAM(HEX/VIC)-TTGTGCCTCTGTTGCTCCTCTTCCC-BHQ-3
A.4 Semen Armeniacae Amarum
5-TTTGGTTGAAGGAGATGCTC-3
5-TAGTTGCTGGTGCTCTTTATG-3
5-FAM(HEX/VIC)-TCCATCAGCAGATGCCAAC-BHQ-3
A.5 Semen Sesami
5-CCAGAGGGCTAGGGACCTTC-3
5-CTCGGAATTGGCATTGCTG-3
5-FAM(HEX/VIC)-TCGCAGGTGCAACATGCGACC-BHQ-3
A.6 hazelnut
5-CCCCGTGTTTGTGATA-3
5-ATGATAATAAGCGATACTGTGAT-3
5-FAM(HEX/VIC)-TCCCGTTCTCGTCCCTGCGCT-BHQ-3
Description of the drawings
Fig. 1 is that vegetable protein beverage plant derived component identifies testing procedure figure；
Fig. 2 is electrophoretogram before purification after ITS2 gene amplifications in Walnut Milk；
Fig. 3 is electrophoretogram after purification after ITS2 gene amplifications in Walnut Milk；
Fig. 4 is sequencing result figure after ITS2 gene amplifications in Walnut Milk；
Fig. 5 is Semen sojae atricolor positive control digital pcr result figure；
Fig. 6 is Semen Juglandiss positive control digital pcr result figure；
Fig. 7 is that 3# samples detect Semen Juglandiss digital pcr result；
Fig. 8 is that 8# samples detect Semen Juglandiss and Semen sojae atricolor digital pcr result；
Fig. 9 is that 2# samples detect Semen Juglandiss and Semen sojae atricolor digital pcr result；
Figure 10 is that 1# samples detect Semen Juglandiss and Semen sojae atricolor digital pcr result；
Figure 11 is that 7# samples detect Semen Juglandiss and Semen sojae atricolor digital pcr result；
Figure 12 is that 1# samples detect very small amount Semen Armeniacae Amarum (0.035cope/ μ L) digital pcr result；
Specific embodiment
By combination accompanying drawing described further below it will be further appreciated that the features and advantages of the invention.The enforcement for being provided Example is only the explanation to the inventive method, and limits remaining content of present invention announcement never in any form.
Equipment and material
In addition to Microbiological Lab's conventional sterilant and culture device, other equipment and material are as follows：
1st, High speed refrigerated centrifuge (centrifugal force >=16000g).
2nd, thermal cycler.
3rd, digital pcr instrument.
4th, balance：Sensibility reciprocal 0.1g.
5th, quasiconductor sequenator.
6th, full-automatic nucleic acid-protein analyser.
7th, full-automatic nucleic acid-protein concentration mensuration instrument (or ultraviolet spectrophotometer).
8th, pure water meter.
9th, Horizontal electrophoresis tank, electrophresis apparatuses.
10th, micropipettor (1.0-1000 μ L).
11st, vacuum freeze drier (optional).
12nd, full-automatic nucleic acid extraction system (optional).
13rd, -80 DEG C of refrigerators (optional).
Reagent
1st, DNA extraction kit.
2nd, tbe buffer liquid.(Tris base：54g, Na2EDTA·2H2O：3.72g, boric acid 27.5g, is settled to 1L)
3rd, PCR premixed liquids.
4th, DNA purified reagent.
5th, 70% ethanol.
6th, DNA recovery purifyings test kit.
7th, high flux gene sequencing kit.
Embodiment：
Attached sample message is shown in Table 1：
1 sample message of table
1. ITS2 gene amplifications in Walnut Milk：
(1) reaction system：2.5 μ L of PCR Buffer (10 ×), MgCl22 μ L (25mmol/L), 2 μ L of dNTPs mixture (2.5mmol/L), each 1.0 μ L of upstream and downstream primer (10 μm of ol/L), template DNA, Taq archaeal dna polymerase 1.0U, plus it is aseptic Distilled water is to 25 μ L
(2) primer liquid：Ten pairs of fusion primers are respectively：
1#:
F:5’-3’：CCATCTCATCCCTGCGTGTCTCCGACTCAG CTAAGGTAAC
GAT ATGCGATACTTGGTGTGAAT
R:5’-3’：CCTCTCTATGGGCAGTCGGTGATTCCTCCGCTTATTGATATGC
2#：
F:5’-3’：CCATCTCATCCCTGCGTGTCTCCGACTCAG TAAGGAGAAC GAT
ATGCGATACTTGGTGTGAAT
R:5’-3’：CCTCTCTATGGGCAGTCGGTGATTCCTCCGCTTATTGATATGC
3#：
F:5’-3’：CCATCTCATCCCTGCGTGTCTCCGACTCAG AAGAGGATTC GAT
ATGCGATACTTGGTGTGAAT
R:5’-3’：CCTCTCTATGGGCAGTCGGTGATTCCTCCGCTTATTGATATGC
4#：
F:5’-3’：CCATCTCATCCCTGCGTGTCTCCGACTCAG TACCAAGATC
GATATGCGATACTTGGTGTGAAT
R:5’-3’：CCTCTCTATGGGCAGTCGGTGATTCCTCCGCTTATTGATATGC
5#:
F:5’-3’：CCATCTCATCCCTGCGTGTCTCCGACTCAG CAGAAGGAAC
GATATGCGATACTTGGTGTGAAT
R:5’-3’：CCTCTCTATGGGCAGTCGGTGATTCCTCCGCTTATTGATATGC
6#:
F:5’-3’：CCATCTCATCCCTGCGTGTCTCCGACTCAGCTGCAAGTTCGA
TATGCGATACTTGGTGTGAAT
R:5’-3’：CCTCTCTATGGGCAGTCGGTGATTCCTCCGCTTATTGATATGC
7#：
F:5’-3’：CCATCTCATCCCTGCGTGTCTCCGACTCAG TTCGTGATTC
GATATGCGATACTTGGTGTGAAT
R:5’-3’：CCTCTCTATGGGCAGTCGGTGATTCCTCCGCTTATTGATATGC
8#：
F:5’-3’：CCATCTCATCCCTGCGTGTCTCCGACTCAG TTCCGATAAC
GATATGCGATACTTGGTGTGAAT
R:5’-3’：CCTCTCTATGGGCAGTCGGTGATTCCTCCGCTTATTGATATGC
9#：
F:5’-3’：CCATCTCATCCCTGCGTGTCTCCGACTCAGTGAGCGGAAC
GATATGCGATACTTGGTGTGAAT
R:5’-3’：CCTCTCTATGGGCAGTCGGTGATTCCTCCGCTTATTGATATGC
10#：
F:5’-3’：CCATCTCATCCCTGCGTGTCTCCGACTCAGCTGACCGAACG
AT ATGCGATACTTGGTGTGAAT-3’
R:5’-3’：CCTCTCTATGGGCAGTCGGTGATTCCTCCGCTTATTGATATGC
(3) reaction condition：94 DEG C, 5min, 94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 50s, 40 circulations；72 DEG C of extensions 10min, 12 DEG C of holdings.
(4) amplification：
Fig. 2 is the nucleic acid that 10 parts of inspection product are extracted, and as the ITS2 fusion primer amplification glue figures of template, target stripe is There is non-specific amplification situation in 430bp, most of templates, taking rubber tapping purification to reclaim nucleic acid carries out downstream experiment.
Fig. 3 is the electrophoretogram Jing after rubber tapping purification is reclaimed, and shows only have target stripe, nothing but specific band, under satisfaction Trip library construction is required.
2nd, experimental result is sequenced
Fig. 4 is the running quality report of epicycle experiment, the every major parameter of report show epicycle experimental data effectiveness compared with Height, up to 81%, effective fragment major part is distributed in 400bp to chip libraries loading, effective fragment distribution between different barcode It is more uniform, illustrate for the nucleic acid of tens of parts of inspection product to be blended in the experimental strategy analyze on a chip feasible.
3rd, sequencing compares statistical result：
Table 2 shows, wherein No. 1 sample show denier Semen Armeniacae Amarum into Point.
2 species identification of table and effective fragment statistics
4. digital pcr experiment：
(1) experimental design：There is the detection of three kinds of plant species by sequencing result reflection, be Semen Juglandiss, Semen sojae atricolor and Semen Armeniacae Amarum respectively, Jing consults primer, probe sequence and the reaction condition of amplification Semen sojae atricolor, Semen Juglandiss and Semen Armeniacae Amarum, it is found that reaction annealing temperature is consistent, therefore adopt Detected with dual digital pcr, more intuitively observed in the original nucleic acid of species, the proportion relation of each copy number.Due to the number for being used Word PCR is double sense channels, so in probe design, reporter group is respectively adopted FAM and VIC (HEX), quencher BHQ, can visually detect in a nucleic acid, the relation between different plant species nucleic acid copies, so as to be inferred in sample it is each into The accounting relation divided, and be mutually authenticated with effectively fragment reading in sequencing, comprehensive analysis obtains reliable conclusion.If other feelings Condition, can design primer one by one using substance digital pcr and detect each species DNA initial copies, such as：There is a sample in this experiment Sequencing result shows also almond component, and its amplification condition has larger difference with other compositions, therefore is examined using substance digital pcr Survey.
(2) reaction condition
With reference to related content in 1961 series standards of SN/T, Semen sojae atricolor ＆ walnut content double check experiment conditions are：95 DEG C, 10min, 94 DEG C, 30s, 60 DEG C, 1min, 40 circulations；98 DEG C, 10min, 12 DEG C of holdings.Temperature rate is tuned into 2 DEG C/s.
Semen Armeniacae Amarum content detection experiment condition is：95 DEG C, 15s, 60 DEG C, 40s, 45 circulations；98 DEG C, 10min, 12 DEG C of holdings. Temperature rate is tuned into 2 DEG C/s.
(3) primed probe
With reference to related content in 1961 series standards of SN/T, appendix A is seen.
(4) result：Fig. 7-12 is digital pcr X-Y scheme, and green is Semen Juglandiss positive microdroplet, and blue signal is Semen sojae atricolor sun Property microdroplet, black signal is full feminine gender microdroplet, and orange red chrominance signal is full positive microdroplet.Analysis software can be directly anti-according to each fluorescence Intensity is answered to calculate in every 20 μ L systems, the copy number of each species obtains final product copy number/μ L divided by sample-adding amount.
5. conclusion：Comprehensive high-flux sequence result and digital pcr result, in terms of detection composition, performance is consistent, but quantitatively As a result need to be defined by digital pcr result, because digital pcr amplification uses initial sample nucleic acid, without following amplification, its survey The absolute copy number for calculating is the original nucleic acid content of each composition in sample.Have 4 parts of samples detection certain proportions (>5%) it is non- Product identification composition.It is shown in Table 3
The result that 3 comprehensive high-flux sequence of table is drawn with digital pcr result
Sample number into spectrum Product identification composition Detection composition
1 Semen Juglandiss Semen Juglandiss, Semen sojae atricolor, Semen Armeniacae Amarum
2 Semen Juglandiss Semen Juglandiss, Semen sojae atricolor
7 Semen Juglandiss Semen Juglandiss, Semen sojae atricolor
8 Semen Juglandiss Semen Juglandiss, Semen sojae atricolor
SEQUENCE LISTING
<110>Hubei Province Safety of Food Quality supervision and inspection academy
<120>A kind of method of plant derived component in plant identification protein beverage
<160> 22
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>ITS2 fragments enter performing PCR amplification initial primers F
<400> 1
atgcgatact tggtgtgaat 20
<210> 2
<211> 21
<212> DNA
<213>ITS2 fragments enter performing PCR amplification initial primers R
<400> 2
gacgcttctc cagactacaa t 21
<210> 3
<211> 63
<212> DNA
<213>1# merges primers F
<400> 3
ccatctcatc cctgcgtgtc tccgactcag ctaaggtaac gatatgcgat acttggtgtg 60
aat 63
<210> 4
<211> 43
<212> DNA
<213>1# merges primer R
<400> 4
cctctctatg ggcagtcggt gattcctccg cttattgata tgc 43
<210> 5
<211> 63
<212> DNA
<213>2# merges primers F
<400> 5
ccatctcatc cctgcgtgtc tccgactcag taaggagaac gatatgcgat acttggtgtg 60
aat 63
<210> 6
<211> 43
<212> DNA
<213>2# merges primer R
<400> 6
cctctctatg ggcagtcggt gattcctccg cttattgata tgc 43
<210> 7
<211> 63
<212> DNA
<213>3# merges primers F
<400> 7
ccatctcatc cctgcgtgtc tccgactcag aagaggattc gatatgcgat acttggtgtg 60
aat 63
<210> 8
<211> 43
<212> DNA
<213>3# merges primer R
<400> 8
cctctctatg ggcagtcggt gattcctccg cttattgata tgc 43
<210> 9
<211> 63
<212> DNA
<213>4# merges primers F
<400> 9
ccatctcatc cctgcgtgtc tccgactcag taccaagatc gatatgcgat acttggtgtg 60
aat 63
<210> 10
<211> 43
<212> DNA
<213>4# merges primer R
<400> 10
cctctctatg ggcagtcggt gattcctccg cttattgata tgc 43
<210> 11
<211> 63
<212> DNA
<213>5# merges primers F
<400> 11
ccatctcatc cctgcgtgtc tccgactcag cagaaggaac gatatgcgat acttggtgtg 60
aat 63
<210> 12
<211> 43
<212> DNA
<213>5# merges primer R
<400> 12
cctctctatg ggcagtcggt gattcctccg cttattgata tgc 43
<210> 13
<211> 63
<212> DNA
<213>6# merges primers F
<400> 13
ccatctcatc cctgcgtgtc tccgactcag ctgcaagttc gatatgcgat acttggtgtg 60
aat 63
<210> 14
<211> 43
<212> DNA
<213>6# merges primer R
<400> 14
cctctctatg ggcagtcggt gattcctccg cttattgata tgc 43
<210> 15
<211> 63
<212> DNA
<213>7# merges primers F
<400> 15
ccatctcatc cctgcgtgtc tccgactcag ttcgtgattc gatatgcgat acttggtgtg 60
aat 63
<210> 16
<211> 43
<212> DNA
<213>7# merges primer R
<400> 16
cctctctatg ggcagtcggt gattcctccg cttattgata tgc 43
<210> 17
<211> 63
<212> DNA
<213>8# merges primers F
<400> 17
ccatctcatc cctgcgtgtc tccgactcag ttccgataac gatatgcgat acttggtgtg 60
aat 63
<210> 18
<211> 43
<212> DNA
<213>8# merges primer R
<400> 18
cctctctatg ggcagtcggt gattcctccg cttattgata tgc 43
<210> 19
<211> 63
<212> DNA
<213>9# merges primers F
<400> 19
ccatctcatc cctgcgtgtc tccgactcag tgagcggaac gatatgcgat acttggtgtg 60
aat 63
<210> 20
<211> 43
<212> DNA
<213>9# merges primer R
<400> 20
cctctctatg ggcagtcggt gattcctccg cttattgata tgc 43
<210> 21
<211> 63
<212> DNA
<213>10# merges primers F
<400> 21
ccatctcatc cctgcgtgtc tccgactcag ctgaccgaac gatatgcgat acttggtgtg 60
aat 63
<210> 22
<211> 43
<212> DNA
<213>10# merges primer R
<400> 22
cctctctatg ggcagtcggt gattcctccg cttattgata tgc 43

Claims (1)

1. in a kind of vegetable protein beverage combined based on semiconductor chip sequencing technologies and digital round pcr it is plant-derived into Divide analysis method, it is characterised in that comprise the steps of：
1) vegetable protein beverage carries out sample pre-treatments；
2）Nucleic acid extraction；
3）DNA concentration is determined；
4）Enter performing PCR amplification to ITS2 fragments：The joint of sequencing and the bar code sequence Barcode for distinguishing sample are added in just Fusion primer pair is obtained in beginning primer pair, initial primers sequence is：
F：5′-ATGCGATACTTGGTGTGAAT-3′；
R：5′-GACGCTTCTCCAGACTACAAT-3′；
The amplicon obtained after ITS2 amplifications can be used as the library of upper machine after purification, dilution, equal-volume mixing；
5）The ITS2 fragments that amplification is obtained are carried out into high-flux sequence with ion torrent platforms；
6）Plug-in unit, or the sequence that will be obtained are analyzed using plant source ITS2 bar code datas storehouse is carried in ion torrent platforms Result identification is carried out using BLAST methods；
7）Effectively number of fragments is counted：Analysis software is carried using platform and counts effective fragment reading, it is preliminary to judge a sample In, detect the proportion relation between plant species；
8）Digital pcr checking sequencing result quantitative analyses；
9）As a result report：Comprehensive sequencing comparison result and quantitative result, each species DNA copy number institute in analytical calculation inspection product nucleic acid The species of content proportion >=5% are classified as the plant derived component of the sample detection by accounting weight.
CN201610987090.7A 2016-11-10 2016-11-10 A kind of method of plant derived component in plant identification protein beverage Pending CN106555000A (en)

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CN112813149A (en) * 2021-03-01 2021-05-18 中国检验检疫科学研究院 Method for identifying plant-derived components in five-spice powder

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CN109943627A (en) * 2019-05-07 2019-06-28 广东出入境检验检疫局检验检疫技术中心 Utilize the method for peanut ingredient in dual digital pcr quantitative detection sesame paste and sesame Rong
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CN112813149A (en) * 2021-03-01 2021-05-18 中国检验检疫科学研究院 Method for identifying plant-derived components in five-spice powder

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Application publication date: 20170405