CN103361406A - Method for marking and distinguishing oil products and detecting quality of oil products - Google Patents
Method for marking and distinguishing oil products and detecting quality of oil products Download PDFInfo
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Abstract
The invention relates to the field of detection and analysis, and in particular relates to a method for marking and distinguishing oil products and detecting the quality of the oil products by using nucleic acid sequence information in the oil products. According to the method, a marker is obtained by measuring at least part of sequence information of at least part of nucleic acid molecules and fragments and marking the oil products on the basis of the sequence information. The oil products are marked and distinguished and the quality of the oil products is detected on the basis of the marker. The method is high in sensitivity and strong in practicability and can realize a high flux.
Description
Technical field
The present invention relates to detect analysis field, be specifically related to oil product is classified and the method for quality examination, more specifically relate to the genetic material that utilizes in the oil product oil product is identified, distinguish and the method for quality examination.
Background technology
Oil product, edible oil for example, the existing chemical analysis indexs such as acid numbers, peroxide value, solvent residual amount that rely on of criteria for classification more.But these indexs are for the discrimination of different classes of oil product Shortcomings also.For example, the sewer oil of discussing warmly on the present society can not well be distinguished under the system of existing criteria for classification.
Sewer oil (also claiming hogwash fat or swill oil) is people's common names for all kinds of greases inferior in life.The sewer oil of narrow sense refers to that the cooking Residual oil, residue leftovers, dishwashing detergent water of eating and drinking establishment etc. flows into trench, pulp water through oil trap separates, fishes for oil reservoir, the grease that forms by operation refining treatment such as thermal dehydration, decolouring, deodorizing, deacidifications again belongs to the catering trade waste grease.The sewer oil of broad sense also comprises the eating and drinking establishment, should abandon the bellding that changes after repeatedly using with oily enterprise etc., frying oil etc. except the waste grease of catering trade, its source, processing, sale and use and all do not meet the food sanitation requirement.
Sewer oil has passed through the Sewage Environment of high temperature frying and water drain, be hydrolyzed, oxidation, the reaction such as become sour, variation has occured in composition, can produce many hazardous and noxious substances, add chemical substance and a large amount of harmful microorganism of washing composition and so on, long-term eating can be brought out various diseases even cancer, and the healthy of people in serious threat.Although the sewer oil main component also is triglyceride level, but Duoed many causing a disease even carcinogenic toxicants than the certified products edible oil, if edible trench can cause obvious injury to human body for a long time, such as dysplasia, easily suffer from enteritis, diarrhoea, and with the generation of the pathologies such as liver, the heart, kidney enlargement and fatty liver, even bring out cancer; Sewer oil contains the poisonous and harmful substances such as nitrate and nitrite, heavy metal, aflatoxin, and wherein flavacin is a kind of strong carcinogenic substance, and its toxicity is 100 times of highly toxic substance arsenic, and the harm of visible sewer oil is big.
The sewer oil comparison of ingredients is complicated, and the specificity physical and chemical index of its detection also is in the investigation stage, and accurately qualitative, quantitative is relatively more difficult.5 kinds of sewer oil detection methods of 7 technical body developments have been collected by the Ministry of Health, but after being expounded through peer review, find that these method specificitys are not strong.So-called specificity is not strong, refers to which these methods do not tell well is sewer oil, and which is qualified edible oil.
Sewer oil is an example, and other oil products are faced with equally to different mass, different qualities, even the true and false good of inferior quality oil product demand distinguishing and detect.
Summary of the invention
The present invention is intended at least part of solution problems of the prior art.For this reason, the invention provides for oil product identify, differentiation and the method for quality and a kind of marker of oil product.
One aspect of the present invention relates to a kind of identification method of oil product, according to embodiments of the invention, the method may further comprise the steps: at least part of sequence information of at least part of nucleic acid molecule or fragment in the order-checking acquisition oil product, based on this sequence information oil product is identified, and obtain marker.Contain some in the oil manufacture raw material from nucleic acid molecule or the fragment of animals and plants or microorganism, under existing technical qualification, the nucleic acid sequence information of these nucleic acid molecule or fragment can be identified by the means of order-checking more.Based on the nucleic acid sequence information of these single bases that are accurate to, can make the specificity marker of oil product, identify for the oil product of particular category, particular batch or level, it is strong to have accuracy, the high and general advantage of suitability of susceptibility.
Alternatively, the method further may further comprise the steps: at least a portion of sequence information and the reference nucleic acid sequence of known species are compared, obtain the kind information of at least a portion nucleic acid molecule in the oil product according to comparison result, and based on this kind information oil product is identified, obtain marker.In such cases, can judge the Species origin of oil product amplifying nucleic acid molecule or fragment based on nucleic acid sequence information, based on the Species origin specificity oil product is identified, this identification method is substantial to the sign of oil product raw material sources, can enrich original raw material information, detect other species information of in production process, sneaking into, in sign, reach production technique is controlled and antipollution effect.
According to embodiments of the invention, also comprise the step that the nucleic acid molecule in the oil product or fragment are increased in the aforesaid method step.The further embodiment according to the present invention, this type of amplification comprises polymerase chain reaction (PCR) and/or multiple displacement amplification reaction.Alternatively, PCR selects degenerated primer amplification (DOP-PCR).The step of amplification can increase the nucleic acid molecule that is separated to or the amount of fragment, reduces the usage quantity of oil product to be identified.
According to embodiments of the invention, also comprise in the aforesaid method step nucleic acid molecule or fragment are carried out the step that the target area is selected.The further embodiment according to the present invention, described nucleic acid molecule or fragment are carried out that the target area selects is for target area design primer, obtains nucleic acid molecule or fragment through selecting by the PCR reaction.According to other embodiments of the invention, the selection of described target area is for target area design complementary probe, the nucleic acid molecule in target acquisition zone or fragment.The target area is selected to take full advantage of known species gene group information resources, the prerequisite of selecting interested genetic locus to choose as marker, and the workload of reduction order-checking process is raised the efficiency.
The present invention provides a kind of differentiating method of oil product on the other hand, and according to embodiments of the invention, it may further comprise the steps: the identification method of complying with above-mentioned oil product obtains the marker of different oil products, according to the difference of marker oil product is distinguished.Nucleic acid sequence information, it is accurate to the resolving power of single base, and nucleic acid molecule is rich, through suitable selection, will form the specific identification code of oil product (marker).Based on the kind information that nucleic acid sequence information obtains, be different from the rough kind that the obtains classification based on raw material, be the more accurately note for the kind information that comprises in the oil product, more near the True composition of aspect, reaction oil product kind source.Based on the marker of being established by nucleic acid sequence information, distinguish for oil product, be fully take into account different sorts, classification or batch oil product itself carry complicacy and the specificity of genetic information, have more accurate resolving power.This method can also be carried out substantial differentiation to the oil product that is designated same materials, can fill in false oil product by the identification division raw material information.Simultaneously, also can detect oil product from the angle of source of species and whether have external pollution.
The present invention also provides a kind of quality determining method of oil product, and according to embodiments of the invention, the method is to obtain marker by above-mentioned identification method, then based on this marker the quality of oil product is judged.According to some embodiments of the present invention, after obtaining marker, to be compared by the kind information of the nucleic acid sequence information marker in the oil product or kind message identification thing and known raw material or the nucleic acid sequence information in the raw material, to determine the quality of this oil product.The method is that the aspect from nucleic acid compares oil product and raw material, to judge the quality of oil product.The variation of the nucleic acid sequence information aspect that this determination methods is introduced according to the technological process of Oil Production changes according to these, the fake and forged oil product that identification is produced through improper channel or underproof technological process.From another aspect, by the method can the characterization processes flow process unusual fluctuation, be conducive to carry out early warning and the quality control of risk.
The further embodiment according to the present invention, the quality determining method of above-mentioned oil product can be concrete may further comprise the steps: the nucleic acid molecule of at least a portion or fragment in the separating oil; Obtain nucleic acid molecule or the fragment of target area by PCR; To checking order in the nucleic acid molecule of at least part of target area or the fragment, obtain nucleic acid sequence information; At least a portion of this nucleic acid sequence information and the reference sequences of known species are compared; Species under definite kernel acid molecule or the fragment, and according to species information the quality of this oil product is judged.In the above-mentioned steps, the order of part steps can be changed.
The further embodiment according to the present invention, for specific testing goal, the target area is chosen as corresponding nucleic acid molecule or the fragment of conservative region of CYTB (cytochrome b) gene that comprises at least a portion pig, ox, sheep, chicken and duck, for example, select such as forward primer: 5 '-TCTTGCAAATCCTAACAGGC-3 ' reverse primer: one couple of PCR primers increases and can obtain similar conservative region the 5 ' CGTTTGCATGTAGATAGCGA-3 '.
According to embodiments of the invention, for specific testing goal, the target area is chosen as the conservative region of rbcl (the RuBisCO large subunit) gene that contains at least a portion sunflower, rape, soybean and olive.For example, select forward primer: the such one couple of PCR primers of 5 '-ATGTCACCACAAACAGAAAC-3 ' reverse primer: 5 '-TCGCATGTACCTGCAGTAGC-3 ' increases and can obtain similar conservative region.
According to other embodiments of the present invention, comprise the conservative region of CYTB gene of above-mentioned at least a portion and the conservative region of at least a portion rbcl gene in the target area.
The present invention also provides a kind of marker of oil product, and it is to obtain by the method that above-mentioned oil product identifies.
The present invention provides again a kind of separation method of nucleic acid in addition, mainly for be the sample of oil product class, and be applied to the link that relates to nucleic acid extraction in the above-mentioned the whole bag of tricks of mentioning, according to embodiments of the invention, it comprises the steps: oil phase and water mixing, centrifugal, abandon upper oil phase; Repeat above-mentioned steps 20-30 time, the water of collecting is used for nucleic acid extraction.The method farthest is separated to the aqueous phase that is easy to carry out further separate nucleic acid with the residual DNA in the oil product by repeated multiple times fully emulsified of oil phase and water.
Additional aspect of the present invention and advantage in the following description part provide, and part will become obviously from the following description, or recognize by practice of the present invention.
Description of drawings
Above-mentioned and/or additional aspect of the present invention and advantage are from obviously and easily understanding becoming the description of embodiment in conjunction with following accompanying drawing, wherein:
Fig. 1 is the schematic flow sheet of an embodiment of quality determining method of the present invention;
Fig. 2 is the pcr amplification product electrophorogram of one embodiment of the invention amplification target area (gene rbcL);
The pcr amplification product electrophorogram of Fig. 3 one embodiment of the invention amplification target area (gene C YTB).
Fig. 4 is the sequencing result demonstration figure based on the order-checking of sanger method.
Embodiment
The below describes embodiments of the invention in detail, and the example of described embodiment is shown in the drawings, and wherein identical or similar label represents identical or similar element or the element with identical or similar functions from start to finish.Be exemplary below by the embodiment that is described with reference to the drawings, only be used for explaining the present invention, and can not be interpreted as limitation of the present invention.Term " first ", " second " etc. only are used for convenient description purpose, and can not be interpreted as indication or hint relative importance.In description of the invention, except as otherwise noted, the implication of " a plurality of " is two or more.
Owing to relate to the part technical term among the present invention, may bring conceptual confusion, hereby carry out lexical or textual analysis, the concept that other do not relate to should be with those of ordinary skills' the standard that is generally understood as.
Oil product: and be not particularly limited, make a general reference the hydrophobic nature material that all can be referred to as oils, it is generally liquid state at normal temperatures.For example comprise vegetables oil, animal oil, mineral oil, volatile oil.Whether can be edible according to them, can be divided into edible oil and inedible oil again.Wherein, vegetables oil and animal main body of oil are the glyceryl ester of lipid acid; The mineral main body of oil is hydrocarbon polymer, such as oil, shale wet goods.Great majority have volatility, can distill or fractionation, are processed into gasoline, kerosene, lubricated wet goods; The essence main body of oil is terpenes, is a kind of special vegetables oil, because having volatility and fragrance, is mainly used in preparing spices.In the situation of part, the present invention can use " oil phase ", " sample " or " sample " to come acute pyogenic infection of finger tip oil product or treated oil product.
Nucleic acid: can be thymus nucleic acid (DNA), also can be Yeast Nucleic Acid (RNA), preferred DNA.For RNA, can be converted into the DNA with corresponding sequence by conventional means, carry out subsequent detection and analysis.
Kind: mainly refer to the ownership of species among the present invention, use with " species " in the part situation.The species indication relates to animals and plants, microorganism, virus etc.
Kind information: refer to belong to summing up of this information about species set.For example, may detect the nucleic acid that contains 10 species in certain oil product.Kind information namely refers at least one in these 10 species classification set so.Among the present invention, the meaning that " kind information " is identical with " species information " expression.
Order-checking: the process that obtains sample nucleic acid sequence information.Order-checking can be undertaken by existing any sequence measurement, includes but not limited to dideoxy chain termination; Preferred high-throughout sequence measurement includes but not limited to s-generation sequencing technologies or single-molecule sequencing technology.
Described s-generation order-checking platform (Metzker ML.Seq uencing technologies-the next generation.Nat Rev Genet.2010 Jan; 11 (1): 31-46) include but not limited to Illumina-Solexa (Miseq, GA
TM, HiSeq2000
TM, HiSeq2500
TMDeng), Life Technologies-Solid, Life Technologies-Ion Torrent/Proton and Roche-454 (tetra-sodium order-checking) order-checking platform; Single-molecule sequencing platform (technology) includes but not limited to the true single-molecule sequencing technology (True Single Molecule DNA sequencing) of Helicos company, Pacific Biosciences company unit molecule check order in real time (single molecule real-time (SMRTTM)), and (Rusk, Nicole (2009-04-01) the .Cheap Third-Generation Sequencing.Nature Methods6 (4): 2446) such as nanoporous sequencing technologies of Oxford Nanopore Technologies company.
Along with the continuous evolution of sequencing technologies, those skilled in the art can be understood that sequence measurement and the device that can also adopt other check order.
Sequence alignment (comparison): refer to the process that one or more nucleotide sequences and reference sequences compare.Concrete common for one section short nucleotide sequence (as reading order) with compare with reference to genome sequence, with definite than the position of short nucleic acid sequences on the reference genome.When utilizing computer to carry out sequence alignment, sequence alignment can be by any sequence alignment program, NCBI BLAST, ELAND (efficient local alignment ofnucleotide data), SOAP (Sho Oligonucleotide Analysis Package) and BWA (Burrows-Wheeler Aligner) supervisor carry out.Compare successful identification standard and be divided into again not fault-tolerant comparison (100% coupling) and partial fault-tolerance comparison (coupling less than 100%).
Reference sequences: at least part of genome sequence of any known species.It is as a reference, by with the comparing of reference sequences, judge according to similarity, can infer unknown nucleotide sequence to be detected and whether belong to these species.
Sequence label (index): be one section length-specific, play the nucleotide sequence of logo role.When dna molecular to be measured during from a plurality of tested sample, each sample can be coupled with different sequence labels, to be used for carrying out in the order-checking process differentiation (the Micah Hamady of sample, Jeffrey JWalker, J Kirk Harris et al.Error-corecting barcoded primers forpyrosequencing hundreds of samples in multiplex.Nature Methods, 2008, March, Vol.5 No.3), thus realize simultaneously a plurality of samples being checked order.Sequence label is in order to distinguish different sequences, but do not affect other functions of the dna molecular that adds sequence label.
The identification method of oil product and marker
One aspect of the present invention provides a kind of method and marker that oil product is identified.
According to embodiments of the invention, the method comprises the steps: to check order and obtains at least part of sequence information of at least part of nucleic acid molecule in the oil product or fragment, based on this sequence information oil product is identified, and obtains marker.And marker of the present invention namely is to produce by above-mentioned method.Alternatively, the method further may further comprise the steps: at least a portion of sequence information and the reference nucleic acid sequence of known species are compared, obtain the kind information of at least a portion nucleic acid molecule in the oil product according to comparison result, and based on this kind information oil product is identified, obtain marker.
Among the present invention, the nucleic acid molecule in the described oil product or fragment refer under the standing state of oil product, are present in wherein nucleic acid molecule or fragment.The source of nucleic acid molecule or fragment may be to exist in the raw materials for production, the course of processing is introduced, and in the course of processing of oil product, do not eliminated, be derived from various organisms (animal and plant, microorganism, virus etc.) nucleic acid molecule or fragment with what various forms existed.
Among the present invention, the order-checking process is used different order-checking steps for sample of different nature.Complete order-checking step generally comprises the extraction of sample nucleic acid, the preparation of nucleic acid library and the step of upper machine order-checking.With regard to existing technique means, manually order-checking is eliminated substantially, and in the present invention, the indication order-checking also mainly refers to rely on the instrument of automatic/semi-automaticization to finish.Can introduce in detail the step about preparation and the order-checking of upper machine of sample preparation, nucleic acid library in the operation instruction of necessary instrument.The various expository materials that specifically provide with reference to each sequenator producer are provided.The producer that produces sequenator comprises, illumina inc, and Life Technologies, Roche etc. do not enumerate herein one by one.Those skilled in the art should be understood that, if the sample that the present invention is directed to is treated sample, for example finished the nucleic acid samples of separate nucleic acid, or be to have finished to build the nucleic acid samples that prepare in the storehouse, step accordingly can directly check order sample.Those skilled in the art should also be understood that order-checking can carry out by single, also can carry out a plurality of samples are parallel simultaneously by several machines.Those skilled in the art will also be understood that, order-checking can realize the parallel order-checking of a plurality of samples in the once sequencing operational process by classification and the recognition reaction of sequence label to sample, and the once sequencing process can produce different samples, or the same sample different batches, the sequencing result of different zones.
According to one embodiment of present invention, also comprise the step that the nucleic acid molecule in the oil product or fragment are increased before the order-checking.Preferably, amplification occurs in nucleic acid molecule after separating from oil product.Amplification can be the polymerase chain reaction (more specifically, for example, degenerated primer increases (DOP-PCR)) and/or multiple displacement amplification etc., purpose be the to double total amount of oil product amplifying nucleic acid molecule, be conducive to use less oil product amount, can obtain the nucleic acid quantity of the step operational requirements such as follow-up order-checking.
Generally, the amount of nucleic acid molecule is many, and quantity of information is unusually abundant, for example the size of wheat cdna group is about 5 times of human genome, mean the base sequence that contains the 15G that has an appointment, and, the not only nucleic acid sequence information of species usually contained in the oil product.Although it is possible obtaining all these nucleotide sequences by order-checking, dispensable.The present invention considers this a bit, the step that can optionally use a target area to select.So-called " target area selection " namely is that nucleic acid molecule or fragment are screened, and selects interested, significant zone.
According to one embodiment of present invention, described nucleic acid molecule or fragment are carried out that the target area selects is for target area design primer, obtains nucleic acid molecule or fragment through selecting by the PCR reaction.How right primer can be, like this by the method for multiplex PCR, just consisted of the disposable selection to a plurality of target areas.This selection can be for a plurality of targets, and these realizations of goal are distinguished.For example by sequence label (index), these targets can be made a distinction in the order-checking process completely.By designing different affinity molecule parts, be equipped with different affinity receptors, also can different targets be made a distinction in the choice phase by corresponding affine absorption means.By independent chosen process repeatedly, certain different target areas can being divided come.
According to another embodiment of the invention, the selection of described target area is for target area design complementary probe, the nucleic acid molecule in target acquisition zone or fragment.This catching equally can be for simple target or a plurality of target.For example by sequence label, these targets can be made a distinction in the order-checking process completely.By designing different affinity molecule parts, be equipped with different affinity receptors, also can different targets be made a distinction at acquisition phase by corresponding affine absorption means.At present, the chip design manufacturers such as Agilent, Nimblegen all provide the service of custom chip, can selected target the zone, under the help of this type of manufacturer, finish capture probe, even the customization of chip.
Among the present invention, described based on sequence information, refer to directly utilize sequence information as marker, perhaps utilize other information of being derived by sequence information as marker.In one embodiment of the invention, the contriver compares at least a portion of sequence information and the reference nucleic acid sequence of known species, kind information according at least a portion nucleic acid molecule in the comparison result acquisition oil product identifies oil product based on this kind information, obtains marker.Marker herein is the kind marker, namely is based on a kind of marker of deriving that sequence information obtains.Using the kind marker, is the external reflection of sequence information on the one hand, also makes things convenient on the other hand sign, also relatively convenient between the marker.
The differentiating method of oil product
Another aspect of the present invention also provides a kind of differentiating method of oil product, according to embodiments of the invention, may further comprise the steps: the identification method of complying with above-mentioned oil product obtains the marker of different oil products, according to the difference of marker oil product is distinguished.Differentiation of the present invention can be the differentiation of the oil product of different sorts, different levels, quality, also the differentiation of the oil product of the one species of different batches.The so-called differentiation is intended to Recognition Different.Can be to the quality of different oil products after the difference identification, characteristic compares.The method also is convenient to the reason that observation analysis difference causes for the same class oil product of different batches, be conducive to the improvement of quality system and technique.Therefore, the method or a kind of quality determining method, the quality of quality is differentiated in the differentiation by difference.In addition, according to different purposes, the method can also be a kind of pollutent method for detecting, quality controlling means etc.
Nucleic acid sequence information directly has unique advantage as marker, it has the resolving power that is accurate to single base, after the bar code scan in modern times and recognition technology are combined, can be used as the identity label of oil product fully, can realize distinguishing accurately to different oil products by this label.
Kind information is then accepted by popular as marker, and understands easily, and is more eye-catching.Because its kind information is to get by nucleic acid sequence information, also there is high accuracy.Under without a lot of situations, utilize kind information to distinguish and namely can satisfy the requirement that oil product is distinguished.
The quality determining method of oil product
Another aspect of the present invention also provides a kind of quality determining method of oil product, according to embodiments of the invention, may further comprise the steps: the method for complying with above-mentioned oil product sign obtains the marker of oil product, judges the quality of oil product according to marker.So-called quality examination is whether digital examination meets relevant standard with the checking oil quality.Setting up of standard is flexibly, need to distinguish different situations, for example for the ease of the purpose of control quality, take into account ageing, needs of convenience etc.According to embodiments of the invention, can be with the detecting as standard of specific nucleic acid sequence, also can with the limit value of the detected level of specific nucleic acid sequence as standard, perhaps can will detect the kind of nucleotide sequence as standard again.According to embodiments of the invention, for the ease of quality is estimated qualitatively, with the kind information as standard.By obtain the comparison of kind information and predefined kind information standard based on nucleic acid sequence information, differentiate the quality of oil quality.According to embodiments of the invention, obtain kind information by nucleic acid sequence information, be to obtain by comparing with the reference sequences of species.According to the principle of same species consensus nucleic acid sequence (get rid of polymorphism may), utilize the genome sequence column information of already present species in the existing database, can find by the means of sequence alignment the species under this nucleotide sequence.
The acquisition of standard can be passed through number of ways.For example, for normal vegetables oil, based on the judgement of raw material, it may contain the plant nucleic acid sequence, and the kind information that it contains only may be plant.So, can be directly with this kind information of plant as standard, if the nucleotide sequence of oil product to be measured inspection beyond the plant indicating that then there are some problems in the quality aspect, but problem might not be exactly defective.
According to some embodiments of the present invention, after obtaining marker, to be compared by the kind information of the nucleic acid sequence information marker in the oil product or kind message identification thing and known raw material or the nucleic acid sequence information in the raw material, to determine the quality of this oil product.Because the kind information of oil product amplifying nucleic acid can be inferred by raw material sources, if there is situation about not being inconsistent, mean that then the quality of oil product is subject to the impact of foeign element, there is the step of introducing unexpected species in the technical process of for example producing.Based on this principle, can estimate quality, source, the production technique of oil product; Perhaps be used for differentiating and distinguishing some fake and forged oil products, the source of this type of oil product is normally abnormal, can introduce corresponding pollutent (specific Species composition).
According to a specific embodiment of the present invention, as shown in Figure 1, the quality determining method of above-mentioned oil product can comprise nucleic acid molecule or the fragment of at least a portion in the step 100:110 separating oil more specifically; 120 nucleic acid molecule or fragments by PCR acquisition target area; At least a portion nucleic acid molecule that the 130 pairs of separation obtain or at least a portion in the fragment check order, and obtain nucleic acid sequence information; 140 compare the nucleic acid sequence information of at least a portion and the reference sequences of known species; Species under 150 definite kernel acid molecules or the fragment; And 160 judge the quality of this oil product according to species information.Wherein it has comprised the step that method of using PCR is selected the target area for this.The purpose that target area selective basis oil quality detects is different, has different choice criteria.For example detect whether sewer oil or contain the sewer oil composition of this oil product, namely need to consider the characteristics of sewer oil.Sewer oil refers to that the cooking Residual oil, residue leftovers, dishwashing detergent water of eating and drinking establishment etc. flows into trench, pulp water through oil trap separates, fishes for oil reservoir, the grease that forms by operation refining treatment such as thermal dehydration, decolouring, deodorizing, deacidifications more also comprises the eating and drinking establishment, should abandon the bellding that changes after repeatedly using with oily enterprise etc., frying oil etc.All contain various animal raw materials in these raw materials, although passed through layer by layer processing, but nucleic acid component wherein can thoroughly not degraded, and at present on the market suitable edible oil mostly is vegetables oil or the single animal raw fat of raw material, can not contain complicated animality genomic constitution.
Therefore, if based on the purpose of distinguishing sewer oil and food oil, just can set up similar following target area screening criteria:
The nucleic acid sequence information of target area is the property of there are differences between edible oil and sewer oil, and otherness is enough to distinguish edible oil and sewer oil.
The target area choose the principle of also wanting considering efficiency preferential, namely select the least possible target area.
According to a particular embodiment of the invention, the present invention proposes the method for selecting nucleotide sequence conservative region between many species.This zone should have following characteristics: although a) be conserved sequence between each species, the polymorphism between its species still can be distinguished different plant species; B) or the target area such as being convenient to carry out that the pcr amplification probe is caught catches operation.More specifically, the screening of target area can comprise following step a1) reference, obtain more typical gene in a plurality of animal and plants; B1) a plurality of candidate genes are compared through blast and GenBank database; C1) institute's selected episode is carried out blast comparison specificity and conservative property.
The embodiment more specifically according to the present invention filters out the target area of at least part of CYTB of comprising (cytochrome b) gene order in the animals such as pig, ox, sheep chicken, duck; And at least part of rbcL (RuBisCO large subunit) gene order that comprises in the plants such as sunflower, rape, soybean and olive.This gene has in each kind of plant, and sequence difference to some extent, does not know how to provide accurately gene I/D) the target area.These two zones can be used separately, also can jointly use, and mutually replenish checking.
CYTB gene and plant (sunflower, rape, soybean and olive etc.) gene specific is larger, and in animal (pig, ox, sheep, chicken etc.) good conservative property is arranged, and is the peculiar gene of animal so select this gene.Plant rbcL gene higher conservative property (wherein have the rbcL gene to account for 94.5% in the angiosperm, have the rbcL gene to account for 98.7% in the gymnosperm) and can be used in discriminating between species in plant is so select this gene to ask plant peculiar constitutive gene.
The target area need to be caught the target area after determining.When the less and complexity in target area is lower, select the method for PCR to catch still comparatively easily.The design of PCR primer then belongs to those skilled in the art's basic operating technical ability, and some softwares are also arranged, Primer Primer for example, the selective aided design of carrying out such as Primer Express.
The further embodiment according to the present invention, for the primer of CYTB design such as forward primer (SEQ ID NO:1): 5 '-TCTTGCAAATCCTAACAGGC-3 ' reverse primer (SEQID NO:2): 5 '-CGTTTGCATGTAGATAGCGA-3 '; For the primer of rbcL design such as forward primer: (SEQ ID NO:3) 5 '-ATGTCACCACAAACAGAAAC-3 ' reverse primer (SEQ ID NO:4): 5 '-TCGCATGTACCTGCAGTAGC-3 '.
The separation method of oil product nucleic acid
The present invention also provides a kind of separation method of nucleic acid, mainly for be the sample of oil product class, and be applied to the link that relates to nucleic acid extraction in the above-mentioned the whole bag of tricks of mentioning, according to embodiments of the invention, it comprises the steps: oil phase and water mixing, centrifugal, abandon upper oil phase; Repeat above-mentioned steps 20-30 time, the water of collecting is used for nucleic acid extraction.Among the present invention, oil phase refers to water-fast part.Water refers to the solution for the dissolving contained nucleic acid molecule of oil phase or fragment.According to embodiments of the invention, water be for nucleic acid molecule or fragment can the stable existence environment solution, or claim damping fluid.For example, when for separating of DNA, the optional TE damping fluid of water.Centrifugation step is suitably selected centrifugal speed and time depending on the size of selected centrifuge tube, should consider in the applicable short period of time water oil content phase will not considered yet nucleic acid molecule is caused larger destruction, guarantees its integrity, obtains a relative balance with this.For example, during applicable 1.5ml centrifuge tube, the optional 10000-15000rpm of centrifugal speed.Centrifugation time 3-10 minute.Pass through aforesaid method, with nucleic acid molecule or fragment contain nucleic acid molecule or the separating substances of fragment to water, further extraction step, then can be with reference to relevant experiment guide, in one embodiment of the invention, the nucleic acid of aqueous phase extracts in following step: the water that will be dissolved with nucleic acid molecule adds lysate according to 1: 2 ratio, fully mixing is 5 minutes, isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1) extracting once, carry out centrifugal layering, centrifugal speed can be controlled at 8000-14000rpm, centrifugation time 3-10 minute, adds precipitation agent to aqueous phase, precipitation agent can add isopyknic Virahol (the perhaps dehydrated alcohol of 2 times of volumes),-20 degree precipitation 2h (precipitation of perhaps spending the night), centrifugal collection DNA precipitation, the optional 10000-15000rpm of centrifugal speed.Centrifugation time 30 minutes.80% washing with alcohol precipitation 1-2 time is dissolved in the 20ulTE damping fluid after drying.
Below with reference to specific embodiment, the present invention will be described, need to prove, these embodiment only are illustrative, and can not be interpreted as limitation of the present invention.
If do not specialize, the conventional means that the technique means that adopts among the embodiment is well known to those skilled in the art can carry out with reference to " molecular cloning experiment guide " third edition or related products, and the reagent that adopts and product also are and can commercial obtain.Various processes and the method do not described in detail are ordinary methods as known in the art, the source of agents useful for same, trade(brand)name and be necessary to list its moiety person, all when occurring first, indicate, thereafter used identical reagent if no special instructions, all identical with the content of indicating first.
Experiment material
The simulation sewer oil: get the oil reservoir in the 50ml swill, through 4 layers of filtered through gauze, centrifugal 10 minutes of 1000rpm gets the supernatant layer.
Lysate and DNA extraction method reference " PCR of the extraction of DNA and ox, sheep derived material detects in the animal grease ".
10xBuffer, dNTP (10mM) and Taq enzyme (Taq DNA Polymerase) are all from takara company.
The precious biological PMD18-T Vector article No. D101A of TA clone's test kit title.
Embodiment 1 sewer oil detects
(1) extraction of sewer oil amplifying nucleic acid:
In the 2ml centrifuge tube, add 1.5ml simulation sewer oil and 300ulTE damping fluid, concussion mixing 5min, the centrifugal 5min of 13000rpm abandons upper oil phase, adds sewer oil 1.5ml again, repeatable operation 30 times, the water of collecting is used for nucleic acid extraction.The 300ul aqueous phase adds the 600ul lysate, and the whirlpool mixing adds 900ul chloroform/primary isoamyl alcohol (24: 1), concuss 5min, and the centrifugal 5min of 13000rpm gets the Virahol of 1 times of volume precooling of upper water addition, places 1h for-20 ℃.The centrifugal 30min of 13000rpm abandons supernatant, adds 75% ethanol of 1ml precooling, and the centrifugal 5min of 13000rpm removes supernatant, and drying at room temperature adds the 20ulTE dissolving.
(2) target area amplification
Primer:
The CYTB primer
Forward primer: 5 '-TCTTGCAAATCCTAACAGGC-3 '
Reverse primer: 5 '-CGTTTGCATGTAGATAGCGA-3 ';
The primer of rbcL design:
Forward primer: 5 '-ATGTCACCACAAACAGAAAC-3 '
Reverse primer: 5 '-TCGCATGTACCTGCAGTAGC-3 '.
Pcr amplification system: 25ul reaction system:
The pcr amplification program:
Amplification as shown in Figures 2 and 3, the CYTB zone is that size be that the band of 720bp, rbcL are the big or small band of 120bp that is.
(3) TA clone and sanger order-checking
The a.PCR product is connected with carrier
(take the PMD18-T Vector of TAKARA as example) is as follows for linked system:
| PCR product | 4.5ul |
| Solution I | 5ul |
| PMD18-T Vector | 0.5ul |
| Total Volume | 10ul |
16 ℃ of connections of PCR instrument 1 hour.
B. connect product transformed competence colibacillus cell
It is dull and stereotyped to take out Amp+LB from 4 ℃ of refrigerators, places 30 minutes in 37 ℃ of constant incubators.The dull and stereotyped every plate of 90mm adds 10 μ L IPTG, 60 μ L X-gal (X-gal is 5-bromo-4-chloro-3-indoles-β-D galactoside); The dull and stereotyped every plate of 150mm adds 20 μ L IPTG, 100 μ L X-gal.Evenly be coated with spreader.(IPTG is isopropylthio-β-D-galactoside, and its final concentration is that the final concentration of 20%, X-gal is 2%).
Get the competent cell 50 μ L of a fresh preparation of pipe or commerce-change, add and connect product 10 μ L, behind the mixing, ice bath 30 minutes.42 ℃ of heat shocks 90 seconds, ice bath 10 minutes.
Add 800 μ L without the LB liquid nutrient medium of Amp, 37 ℃, 220rpm recovery cell 1 hour.Get whole converted product 2000rpm, centrifugal 3min abandons supernatant and remains 100 μ L, evenly coats on the LB flat board of Amp+, and 37 ℃ of inversion incubators were cultivated 15-16 hour.
Take out the flat board of incubated overnight, observe the growing state of bacterial plaque.Under normal circumstances, can grow hundreds of of blue hickie list bacterium colonies of every plate.Every plate is chosen 80 hickies, carries out the order-checking of sanger method.
The c.sanger order-checking
Sequenator: the 3730XL sequenator of ABI company
Reagent:
Terminator v3.1 Cycle Sequencing Kits
Sequence measurement: carry out according to 3730XL sequenator service manual.
Utilize the A of PCR product end of Taq enzymatic amplification and the T pairing of T carrier to reprint, the mono-clonal of cultivating after screening is reprinted carries out the order-checking of sanger method, reads the base sequence of Insert Fragment, is used for gene identification.
(4) analysis of biological information and result judge
By sanger method order-checking, obtain the collection of illustrative plates that checks order, as shown in Figure 4, as remove the base ordering that carrier sequence and inferior quality sequence obtain the purpose zone.The available sequences that obtains is carried out the blast comparison on NCBI (http://misuse.ncbi.nih.gov/), find species information corresponding to goal gene.
Detected result for sewer oil to be measured is as shown in the table:
| The target area | Species in the comparison |
| CYTB | Wild boar |
| rbcL | Nicotiana, Solanum, capsicum, angelica sweet potato, Btassica etc. |
By treating the detection of surveying sewer oil CYTB and two genes of rbcL, contain a large amount of genes from pig and other plant species in the finding illegal cooking oil, and these should not appear in the qualified vegetables oil, analysis to the plant gene composition, plant gene residual in the finding illegal cooking oil is complicated, on the result of comparison, it is not this gene that has in the qualified vegetables oil that the plant gene in the sewer oil contains a lot, from then on can judge the composition of edible oil.
In the description of this specification sheets, the description of reference term " embodiment ", " some embodiment ", " example ", " concrete example " or " some examples " etc. means to be contained at least one embodiment of the present invention or the example in conjunction with specific features, structure, material or the characteristics of this embodiment or example description.In this manual, the schematic statement of above-mentioned term not necessarily referred to identical embodiment or example.And the specific features of description, structure, material or characteristics can be with suitable mode combinations in any one or more embodiment or example.
Although illustrated and described embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple variation, modification, replacement and modification to these embodiment in the situation that does not break away from principle of the present invention and aim, scope of the present invention is limited by claim and equivalent thereof.
Claims (15)
1. the identification method of an oil product is characterized in that, may further comprise the steps:
At least part of sequence information of at least part of nucleic acid molecule or fragment identifies oil product based on this sequence information in the order-checking acquisition oil product, obtains marker.
2. method according to claim 1 further comprises
At least a portion of sequence information and the reference nucleic acid sequence of known species are compared,
According to the kind information of at least a portion nucleic acid molecule in the comparison result acquisition oil product,
Based on this kind information oil product is identified, obtain marker.
3. the differentiating method of an oil product is characterized in that, may further comprise the steps:
Obtain the marker of different oil products according to claim 1 or 2 described methods,
Difference according to marker is distinguished oil product.
4. the quality determining method of an oil product may further comprise the steps:
According to the marker of claim 1 or 2 described methods acquisition oil products,
According to this marker oil quality is judged.
5. each described method is characterized in that according to claim 1-4, also comprises the step that the nucleic acid molecule in the oil product or fragment are increased.
6. method according to claim 5 is characterized in that, described amplification comprises polymerase chain reaction and/or multiple displacement amplification reaction.
7. each described method is characterized in that according to claim 1-4, also comprises nucleic acid molecule or fragment are carried out the step that the target area is selected.
8. method according to claim 7 is characterized in that, described nucleic acid molecule or fragment are carried out that the target area selects is for target area design primer, obtains nucleic acid molecule or fragment through selecting by the PCR reaction.
9. method according to claim 7 is characterized in that, the described target area is selected is for target area design complementary probe, the nucleic acid molecule in target acquisition zone or fragment.
10. the quality determining method of an oil product is characterized in that, may further comprise the steps:
The nucleic acid molecule of at least a portion or fragment in the separating oil,
By nucleic acid molecule or the fragment of PCR acquisition target area,
To checking order in the nucleic acid molecule of at least part of target area or the fragment, obtain nucleic acid sequence information,
At least a portion of this nucleic acid sequence information and the reference sequences of known species are compared,
The kind information of definite kernel acid molecule or fragment,
According to kind information the quality of this oil product is judged.
11. method according to claim 10 is characterized in that, the step of described isolating nucleic acid may further comprise the steps: oil phase and water mixing is centrifugal, abandon upper oil phase; Repeat above-mentioned steps 20-30 time, the water of collecting is used for nucleic acid extraction.
12. method according to claim 10 is characterized in that, described target area is the conservative region that comprises at least a portion CYTB (cytochrome b) gene, and/or
The conservative region of at least a portion plant rbcl (RuBisCO large subunit) gene.
13. method according to claim 12 is characterized in that, the primer of described PCR is:
The primer of the conservative region of at least a portion CYTB (cytochrome b) gene:
Forward primer: 5 '-TCTTGCAAATCCTAACAGGC-3 '
Reverse primer: 5 '-CGTTTGCATGTAGATAGCGA-3 '
The primer of the conservative region of at least a portion plant rbcl (RuBisCO large subunit) gene:
Forward primer: 5 '-ATGTCACCACAAACAGAAAC-3 '
Reverse primer: 5 '-TCGCATGTACCTGCAGTAGC-3 '.
14. the marker of an oil product, it obtains by claim 1 or 2 described methods.
15. the separation method of an oil product amplifying nucleic acid is characterized in that, may further comprise the steps:
With oil phase and water mixing, centrifugal, abandon upper oil phase; Repeat above-mentioned steps 20-30 time, the water of collecting is used for nucleic acid extraction.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105112498A (en) * | 2014-10-10 | 2015-12-02 | 青岛科技大学 | Method for detecting waste oil by using exogenous animal mitochondrial DNA |
| CN108070587A (en) * | 2018-02-11 | 2018-05-25 | 云南省烟草农业科学研究院 | The specific primer pair of plant derived component and its application in a kind of identification food |
| CN110333688A (en) * | 2019-08-06 | 2019-10-15 | 合肥创博信息科技有限公司 | A kind of Loss of Oil Products at Gas Station source real time monitoring system |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906471A (en) * | 2010-07-13 | 2010-12-08 | 上海之江生物医药科技有限公司 | Swill-cooked dirty oil detecting method |
-
2012
- 2012-04-01 CN CN2012100959705A patent/CN103361406A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906471A (en) * | 2010-07-13 | 2010-12-08 | 上海之江生物医药科技有限公司 | Swill-cooked dirty oil detecting method |
Non-Patent Citations (2)
| Title |
|---|
| S KUMAR, T KAHLON AND S CHAUDHARY: "A rapid screening for adulterants in olive oil using DNA barcodes", 《FOOD CHEMISTRY》 * |
| 唐建阳和周先治: "植物DNA条形码研究现状及应用前景", 《中国农学通报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105112498A (en) * | 2014-10-10 | 2015-12-02 | 青岛科技大学 | Method for detecting waste oil by using exogenous animal mitochondrial DNA |
| CN108070587A (en) * | 2018-02-11 | 2018-05-25 | 云南省烟草农业科学研究院 | The specific primer pair of plant derived component and its application in a kind of identification food |
| CN110333688A (en) * | 2019-08-06 | 2019-10-15 | 合肥创博信息科技有限公司 | A kind of Loss of Oil Products at Gas Station source real time monitoring system |
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