CN102758025A - Reagent kit for fast detection of drainage oil and method for fast detection of drainage oil - Google Patents

Abstract

The invention discloses a reagent kit for fast detection of drainage oil and a method for fast detection of the drainage oil. The reagent kit comprises a deoxyribonucleic acid (DNA) extraction buffer, thermus aquaticus DNA (Taq DNA) polymerase, a polymerase chain reaction (PCR) solution and a sample loading buffer. According to the reagent kit, on the basis of existing research, key dominant factors such as extraction reagents and reaction conditions in the whole process from extraction of drainage oil genes to PCR detection are improved and optimized, and the whole set of drainage oil detection reagent kit which is high in stability, fast and simple is produced. According to the reagent kit and the detection method, constituents of the drainage oil are identified from the point of genes, the detection of the drainage oil has the advantages of being high in specificity, high in sensitivity, good in method reproducibility, fast and simple to detect and the like, and the gap of existing detection methods is filled. The reagent kit and the detection method have important popularization and application values and are important to effective supervision of the drainage oil and protection of benefit of consumers.

Description

The test kit of rapid detection sewer oil and detection method thereof

Technical field

The present invention relates to a kind of test kit and detection method thereof of rapid detection sewer oil.

Background technology

At present; The quality of edible oil is very different on the market; Sub-fraction grease processing merchant is in order to practice thrift cost, to seek exorbitant profit, and sewer oil is refined in illegal processing, or in oil with common edible, mixes sewer oil and cheat the human consumer; These malpractices have come hidden danger for the food securing band, serious harm human consumer's health and interests.

Sewer oil is quality, health extreme difference, and the inedible oil of index severe overweights such as peroxide value, acid value, moisture, carbonyl valency, mda generally is divided into 3 types:

The one, the sewer oil of narrow sense, be about in the water drain greasy swimmer or with leftovers, the oil of leftovers (common name swill) of hotel, restaurant through simply processing, extracting;

The 2nd, animal meat inferior, pluck, the processing of animal sebum and the oil that refines the back output;

The 3rd, after the oily access times that are used for fried food product surpass specified requirement, be repeated again to use or toward wherein adding the oil of reusing behind some fresh oils.

Heavy metal in the sewer oil, toxin severe overweights such as (like propenal), peroxide value generally is higher than 0.4%, and considerably beyond national standard 0.15%, long-term absorption can make cell function depleted, brings out multiple disease, even carcinogenic.Edible peroxide value once took place in Japan is that 7.5% grease inferior causes collective's acute poisoning incident.

At present domestic discriminating and detection to sewer oil mainly is to carry out from sense organ and two aspects of physical and chemical inspection.Physics and chemistry detects checks indexs such as moisture content, proportion, refractive index, saponification value, acid number, carbonyl value, peroxide value, iodine number heavy metal, the relative degree of unsaturation of lipid acid, SUV, residue detection, oxidation products respectively.Sewer oil is owing to through behind the refinery practices such as washing, distillation, decolouring, deodorization, after perhaps mixing with common edible vegetable oil, be difficult to distinguish with conventional organoleptic analysis or physical and chemical index etc.

The at present domestic detection method that effectively to differentiate sewer oil that still lacks.

Summary of the invention

Based on this, the present invention according to sewer oil process raw material and there is bigger difference in the oil with common edible raw material, a kind of test kit and detection method thereof of rapid detection sewer oil is provided.Utilize test kit that the gene element in the sewer oil is detected, judge processing raw material of sewer oil, thereby reach the purpose of rapid detection sewer oil through detected result.

A kind of test kit of rapid detection sewer oil comprises:

(1) DNA extraction damping fluid

Consisting of of said DNA extraction damping fluid: CTAB (being called for short CTAB), 90～110nmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride (being called for short Tris-HCl), 15～25mmol/L EDTA Disodium (ethylenediaminetetraacetic acid ₂Na), 1.3～1.5mol/L sodium-chlor (being called for short NaCl), 0.09～0.12g/L Proteinase K;

(2) Taq archaeal dna polymerase, the concentration of said Taq archaeal dna polymerase are 125IU;

(3) PCR reaction solution

Consisting of of said PCR reaction solution: 2.7～3.3mmol/L magnesium chloride, 0.3～0.5 μ mol/L primer, the triphosphate deoxy-nucleotide of 0.3～0.5mmol/L;

Said primer is for to the universal primer SEQ ID NO:1 of animal derived gene order and SEQ ID NO:2 and the universal primer SEQ ID NO:3 and the SEQ ID NO:4 that are directed against plant-derived gene order;

(4) sample-loading buffer

Consisting of of said sample-loading buffer: 2.0～3.0g/L tetrabromophenol sulfonphthalein, 350～450g/L sucrose.

Among some embodiment, said primer also comprises the primer to the chondriogen sequence of ox, sheep, pig or chicken therein.

Among embodiment, said primer to ox chondriogen sequence is SEQ ID NO:5 and SEQ ID NO:6 therein.

Among embodiment, said primer to sheep chondriogen sequence is SEQ ID NO:7 and SEQ ID NO:8 therein.

Among embodiment, said primer to the porcine mtdna gene order is SEQ ID NO:9 and SEQ ID NO:10 therein.

Among embodiment, said primer to chicken chondriogen sequence is SEQ ID NO:11 and SEQ ID NO:12 therein.

Therein among embodiment, said primer also comprises to the primer SEQ ID NO:13 of the native gene sequence of corn and SEQ ID NO:14, to the primer SEQ ID NO:15 of the native gene sequence of soybean and SEQ ID NO:16, to the primer SEQ ID NO:17 of the native gene sequence of rice and SEQ IDNO:18, to the primer SEQ ID NO:19 and the SEQ ID NO:20 of the native gene sequence of peanut or be directed against the primer SEQ ID NO:21 and the SEQ ID NO:22 of the native gene sequence of Semen Brassicae campestris.

A kind of method of rapid detection sewer oil adopts the mentioned reagent box, may further comprise the steps:

(1) preparation sample DNA

Get centrifuge tube, every pipe adds 20～50mL oil sample and equal-volume distilled water, the 30～60min that vibrates on the shaker, and high speed centrifugation 15～20min discards the upper strata grease, keeps lower floor's water.In centrifuge tube, add 20～50mL oil sample again, continue vibration, centrifugal, said process repeats 7～8 times, and the volume of the oil sample that extracts is about 300～400mL.After being extracted into lower floor's water muddiness, high speed centrifugation discards the upper strata grease, water is transferred in the ware of plane concentrate drying, about 3～5 hours of dry concentration process; After the thorough drying, in the ware of plane, add the DNA extraction damping fluid of 2～3mL, behind the mixing, shift suspension liquid in centrifuge tube.Place 65 ℃ to place 45～60min centrifuge tube, shake frequently, make the abundant cracking of sample.Use chloroform: the primary isoamyl alcohol volume ratio is that 24: 1 mixed solution extracts, and is centrifugal.Get supernatant, add isopropanol precipitating nucleic acid, placed 2 hours for-20 ℃, make Virahol and nucleic acid fully react, high speed centrifugation obtains the DNA deposition.Wash throw out with 70% ethanolic soln again, throw out is dissolved in water, and promptly gets sample DNA;

(2) pcr amplification reaction

Get the PCR reaction solution, the Taq archaeal dna polymerase places reaction tubes, mixing adds sample DNA; Centrifugal being placed on the amplification appearance increased reaction parameter: behind 95 ℃ of preparatory sex change 5min, by 94 ℃ of 30sec; 55 ℃ of 45sec; 72 ℃ of 60sec programs are carried out 40 circulations, and last 72 ℃ are extended 10min, finish reaction in 4 ℃;

(3) product detects

Reaction product adds a kind damping fluid, fully point sample behind the mixing; With DNA Marker as molecular weight standard; With containing painted 1.5% agarose gel electrophoresis of the own ingot of bromination, after the end, put under the uv lamp and observe; Occur the target specific band for containing certain species gene element, otherwise for not containing this species gene composition.

It is complicated to the present invention is directed to sewer oil matrix, and interfering factors is many, and problem such as the gene degraded is more serious in the sewer oil, and the process for extracting of sewer oil DNA has efficiently been set up in exploitation, successfully from tens of parts of sewer oil samples, extracts the DNA that has obtained better quality.Again further according to the kind of the gene element that possibly contain in the sewer oil; Designs specificity, narrow spectrum primer sequence carry out PCR reaction amplification, extract animal derived gene element and plant-derived gene element in the sewer oil sample according to the detected result Rapid identification.

Sewer oil DNA extraction method among the present invention has increased the volume that extracts sample; Adopt the above sewer oil sample of 300mL to extract; Adopt distilled water and the well-mixed mode of sewer oil to extract the gene element in the sewer oil; Utilize the DNA extraction damping fluid to make the lysis in the sewer oil, discharge nucleic acid.Remove the albumen in the solution with chloroform, primary isoamyl alcohol mixing solutions, under coldcondition, make the DNA deposition with Virahol at last, obtain the sewer oil DNA of purifying.After obtaining sewer oil DNA, the screening Auele Specific Primer detects the gene element in the extraction sewer oil, at first detects animal native gene composition and plant endogenous gene element in the sewer oil.After detecting the animal native gene, again according to the specific animal species gene amplification primer that designs, the concrete discriminating is the gene order of species such as ox, sheep or pig; After detecting plant endogenous gene,, differentiate accurately whether oil sample sign kind to be measured is consistent with the gene element kind again according to the plant species gene amplification primer of species specificities such as the soybean that designs, corn, peanut, Semen Brassicae campestris.Detected result shows that the quality of the sewer oil DNA of extraction satisfies PCR and detects requirement, and the amplimer specificity of employing is good, highly sensitive, can in the sewer oil sample, detect animal native gene composition and plant endogenous gene element.

Contriver of the present invention explores the method for quick of having set up sewer oil; On the basis of existing research; Improve and optimized from the PCR that extracts of sewer oil gene and detected the crucial controlled factors such as extraction reagent and reaction conditions the whole process, set up stable height, fast and convenient a whole set of sewer oil detection kit.The present invention differentiates the trench oil component from the angle of gene, detects to sewer oil to have high specificity, highly sensitive, method favorable reproducibility, detect advantages such as fast and convenient, remedied the blank of current detection method.

The inventive method has great application value, and for effective supervision sewer oil, protection consumers in general interests are significant.The present invention can be widely used in the aspects such as extraction, grease cultivar identification and exploitation grease nucleic acid quick detection kit of lipa gene, has good economy and society benefit.

Description of drawings

Fig. 1 is the pcr amplification product electrophoretogram of animal native gene in the doubtful sewer oil sample of embodiment 2; Wherein: M is DNA Marker; P1, the positive contrast of P2 (beef DNA is diluted to 50ng/ μ L); 1,2 negative contrasts (maize dna is diluted to 50ng/ μ L); 3～10: the doubtful sewer oil sample DNA that the inventive method is extracted; 11, human consumption soybean oil samples (Jin Longyu, 20111209); 12, edible corn oil samples (Jin Longyu, 20120309); 13, edible peanut oil samples (Ji spends 20120108 recklessly); 14, edible rapeseed oil sample (South Sea grease, 20120215); 15, edible sunflower seed oil sample (many power, 20120318); CK1, CK2: blank;

Fig. 2 is the pcr amplification product electrophoretogram of plant endogenous gene in the doubtful sewer oil sample among the embodiment 2; Wherein: M is DNA Marker; P1, the positive contrast of P2 (maize dna is diluted to 50ng/ μ L); 1～8: the doubtful sewer oil sample DNA that the inventive method is extracted; 9, beef DNA is diluted to 50ng/ μ L; 10, mutton DNA is diluted to 50ng/ μ L; 11, pork DNA is diluted to 50ng/ μ L; 12, human consumption soybean oil samples (Jin Longyu, 20111209); 13, edible corn oil samples (Jin Longyu, 20120309); 14, edible peanut oil samples (Ji spends 20120108 recklessly); 15, edible rapeseed oil sample (South Sea grease, 20120215); CK1, CK2: blank;

Fig. 3 is the pcr amplification product electrophoretogram of ox native gene in the doubtful sewer oil sample among the embodiment 2; Wherein: M is DNA Marker; P1, the positive contrast of P2 (beef DNA is diluted to 50ng/ μ L); 1～5: detect the doubtful sewer oil sample DNA that contains the animal native gene, sample number into spectrum is respectively sewer oil 1,2,4,6,7; 6, maize dna; 7, soy bean DNA; 8, mutton DNA; 9, pork DNA; 10, chicken DNA; 11, human consumption soybean oil samples (Jin Longyu, 20111209); 12, edible corn oil samples (Jin Longyu, 20120309); 13, edible peanut oil samples (Ji spends 20120108 recklessly); 14, edible rapeseed oil sample (South Sea grease, 20120215); 15, edible sunflower seed oil sample (many power, 20120318); CK1, CK2: blank.

Embodiment

Specify the present invention below in conjunction with specific embodiment.

The test kit of embodiment 1 rapid detection sewer oil

Comprise following composition (can supply to carry out 50 parts of PCR reactions):

(1) DNA extraction damping fluid (100mL)

Take by weighing 2.00mg CTAB (cetyl trimethylammonium bromide), 1.21mg Tris.HCl (Tri(Hydroxymethyl) Amino Methane Hydrochloride), 0.58g EDTA2Na (EDTA Disodium) and 8.18g NaCl respectively in volumetric flask; Water is settled to 100mL, mixing.Through 120 ℃, 30min, after the high-temperature sterilization sterilization, add the Proteinase K (available from the precious biotech firm in Dalian) that 10.00mg purchases again;

(2) Taq archaeal dna polymerase (25 μ l);

Purchase Taq archaeal dna polymerase 25 μ l 125IU (available from the precious biotech firm in Dalian) altogether;

(3) PCR reaction solution (1000 μ l)

At first prepare primer, the primer concentration of preparation is 100nmol/L.

Prepare primer according to this area ordinary method.The specification sheets of going up according to manufacturers at automatic dna synthesizer (adopting the full-automatic dna synthesizer of ABI3949 type of American AB I company) synthesizes.Synthetic respectively following primer:

Animal gene composition universal primer, forward: 5 '-aagacgagaagacccttggacttta-3 ' (SEQ ID NO.1);

Animal gene composition universal primer, reverse: 5 '-gattgcgctgttatccctagggta-3 ' (SEQ ID NO.2);

Plant gene composition universal primer, forward: 5 '-cgaaatcggtagacgctacg-3 ' (SEQ ID NO.3);

Plant gene composition universal primer, reverse: 5 '-ttccattgagtctctgcacct-3 ' (SEQ ID NO.4);

The forward primer of ox chondriogen: 5 '-gccatatactctccttggtgaca-3 ' (SEQ ID NO.5);

The reverse primer of ox chondriogen: 5 '-gtaggcttgggaatagtacga-3 ' (SEQ ID NO.6);

The forward primer of sheep chondriogen: 5 '-tattaggcctcccccttgtt-3 ' (SEQ ID NO.7);

The reverse primer of sheep chondriogen: 5 '-ccctgctcataagggaatagcc-3 ' (SEQ ID NO.8);

The forward primer of porcine mtdna gene: 5 '-atgaaacattggagtagtcctactatttacc-3 ' (SEQ ID NO.9);

The reverse primer of porcine mtdna gene: 5 '-ctacgaggtctgttccgatataagg-3 ' (SEQ ID NO.10);

The forward primer of chicken chondriogen: 5 '-gggacaccctcccccttaatgaca-3 ' (SEQ ID NO.11);

The reverse primer of chicken chondriogen: 5 '-ggagggctggaagaaggagtg-3 ' (SEQ ID NO.12);

The forward primer of corn native gene: 5 '-ccgctgtatcacaagggctggtacc-3 ' (SEQ ID NO.13);

The reverse primer of corn native gene: 5 '-ggagcccgtgtagagcatgacgatc-3 ' (SEQ ID NO.14);

The forward primer of soybean native gene: 5 '-gccctctactccacccccatcc-3 ' (SEQ ID NO.15);

The reverse primer of soybean native gene: 5 '-gcccatctgcaagcctttttgtg-3 ' (SEQ ID NO.16);

The forward primer of rice native gene: 5 '-ttgcgcctgaacggatat-3 ' (SEQ ID NO.17);

The reverse primer of rice native gene: 5 '-ggagaagcactggacgagg-3 ' (SEQ ID NO.18);

The forward primer of peanut native gene: 5 '-tatgatcctcgttgtgtctatgac-3 ' (SEQ ID NO.19);

The reverse primer of peanut native gene: 5 '-tctccagtcttcttctctttcac-3 ' (SEQ ID NO.20);

The forward primer of Semen Brassicae campestris native gene: 5 '-ccagttcttggagccgcttga-3 ' (SEQ ID NO.21);

The reverse primer of Semen Brassicae campestris native gene: 5 '-aagggccagtccaaatgcaga-3 ' (SEQ ID NO.22);

The forward primer of yam native gene: 5 '-tgacctggacaccacagttat-3 ' (SEQ ID NO.23);

The reverse primer of yam native gene: 5 '-gtggatttcaggagttcttcga-3 ' (SEQ ID NO.24);

Take by weighing 0.285mg MgCl ₂, with 500 μ L water dissolution,, after the high-temperature sterilization sterilization, add a pair of primer of 0.5 μ mol and the triphosphate deoxy-nucleotide of 0.5mmol respectively through 120 ℃, 30min, be settled to 1000 μ L with aqua sterilisa;

(4) sample-loading buffer (100 μ l)

Take by weighing 2.5g tetrabromophenol sulfonphthalein, 400g sucrose respectively in the 1L volumetric flask, water constant volume, mixing.

Embodiment 2 utilizes the test kit of embodiment 1 to detect the gene element in the sewer oil

Detection method may further comprise the steps:

(1) preparation of sample DNA

The doubtful sewer oil of a, food plant oil samples: measure 300～500mL sample;

Get centrifuge tube, every pipe adds 50mL oil sample and 50mL distilled water, the 60min that vibrates on the shaker, and the centrifugal 10min of 12000r/min discards the upper strata grease, keeps lower floor's water.In centrifuge tube, add the 50mL oil sample again, continue vibration, centrifugal, said process repeats 8 times, and the volume of the oil sample that extracts is about 400mL.After being extracted into lower floor's water muddiness, the centrifugal 10min of 12000r/min discards the upper strata grease, water is transferred in the ware of plane concentrate drying, about 3 hours of dry concentration process; After the thorough drying, in the ware of plane, add the DNA extraction damping fluid in the 2mL test kit, behind the mixing, shift suspension liquid in centrifuge tube.Place 65 ℃ to place 60min centrifuge tube, shake frequently, make the abundant cracking of sample.Use chloroform: the primary isoamyl alcohol volume ratio is that 24: 1 mixed solution extracts, careful mixing two phases, the centrifugal 10min of 12000r/min.Supernatant is moved in the new centrifuge tube, add the Virahol of the precooling of 2 times of volumes, fully mixing was placed 2 hours for-20 ℃, made Virahol and nucleic acid fully react, and the centrifugal 10min of 12000r/min obtains DNA and precipitates.Wash throw out 2 times with 70% ethanolic soln again, the centrifugal 12000r/min of room temperature, 10min collects DNA, removes ethanol as far as possible.With 100 μ l distilled water dissolving DNAs depositions, DNA is dissolved fully after, promptly get sample DNA, place-20 ℃ of preservations.

B animal meat (beef, mutton, pork, chicken), frequently seen plants species (corn, soybean, peanut, Semen Brassicae campestris, yam) sample:, use the solid kibbler crossed through 120 ℃, 30min autoclave sterilization or mortar to pulverize and process powder appearance respectively with test materialss such as beef, mutton, pork, chicken, rice, corn, soybean, peanut, Semen Brassicae campestris, yams.

Take by weighing sample after 100mg grinds respectively in centrifuge tube, add the DNA extraction damping fluid in the 1mL test kit, mixing.Place 65 ℃ to place 60min centrifuge tube, shake frequently, make the abundant cracking of sample.Use chloroform: the primary isoamyl alcohol volume ratio is that 24: 1 mixed solution extracts, careful mixing two phases, the centrifugal 5min of 12000r/min.Supernatant is moved in the 1.5mL centrifuge tube, add the Virahol of 2 times of volume precoolings, abundant mixing, the centrifugal 5min of 12000r/min obtains the DNA deposition.Wash throw out 2 times with 70% ethanolic soln again, the centrifugal 12000r/min of room temperature, 5min collects DNA, removes ethanol as far as possible.With 100 μ l distilled water dissolving DNAs depositions, DNA is dissolved fully after, promptly get sample DNA, place-20 ℃ of preservations.

(2) purity and the content of inspection DNA

In quartz colorimetric utensil, add the DNA sample (5 μ l DNA samples add water 45 μ l mixings) of 50 μ l, measure the ultraviolet absorption value of DNA sample respectively, and calculate A at 260nm and 280nm place by 10 times of dilutions ₂₆₀/ A ₂₈₀Value.A ₂₆₀/ A ₂₈₀Should be between 1.7～1.9, be lower than 1.7 and show and retain remarkable protein in the prepared product, should extract DNA again; Show in the DNA sample greater than 1.9 and to contain RNA that should add the RNA enzyme and digest this moment.

Calculate DNA concentration: 10 * A ₂₆₀(μ g/ μ l).

Doubtful sewer oil sample, edible vegetable oil, common meat and plant tissue DNA extract the result and see table 1.

Table 1 sample DNA extracts the result

Can know that by table 1 result the requirement that the DNA quality that obtains better can satisfy the PCR reaction of extracting can obtain target P CR product when carrying out the PCR reaction.Because dna content is few and fragment is little in the oil sample, through repeatedly obtaining the DNA more than the 50ng/ μ L behind the enriching and purifying, the sample DNA process for extracting of illustrative embodiment 2 can extract grease DNA effectively, can further carry out the PCR reaction.

(3) pcr amplification reaction (selecting amplimer) according to detecting gene type

Get PCR reaction solution 906.3 μ l, Taq archaeal dna polymerase 5.7 μ l in centrifuge tube; Mixing; Install to by the every pipe branch of 48.0 μ l in the reaction tubes (wherein positive control, negative control, each two repetition of blank sample, the repetition of all the other homogeneous) of 19 numbers of finishing, add the DNA sample that 2.0 μ l contain target gene fragment in the heliotropism control tube; In the negative control pipe, add the control sample DNA that 2.0 μ l do not contain goal gene; In the blank pipe, add distilled water, in all the other each reaction tubess, add 2.0 μ l sample DNA (reaction system is 50 μ l) to be analyzed again, centrifugal being placed on the pcr amplification appearance increased.Reaction parameter is: behind 94 ℃ of preparatory sex change 5min, by 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 60sec programs are carried out 40 circulations, and last 72 ℃ are extended 5min, finish reaction in 4 ℃.

(4) product detects

Get 10.0 μ l PCR reaction product, add 2.0 μ l sample-loading buffers, fully point sample behind the mixing; As molecular weight standard, (accurately take by weighing 1 * TAE (0.04mol/L Tris-HCl, 0.02mol/L NaAC that the 0.15g agarose dissolves in 100mL with DNAMarker with containing painted 1.5% sepharose of ethidium bromide; 2mmol/LEDTA pH 8.0) in, put to heat on the microwave oven and also shake frequently to fusing fully, room temperature is chilled to 50～60 ℃; The 10mg/mL EB that adds 1 μ L shakes up immediately, pours into rapidly and seals in the electrophoresis chamber of mouth, more than the room temperature held 30min with the adhesive waterproof tape of the bacterium of going out; Extract comb) 3V/cm, electrophoresis 1h finishes electrophoresis; Place under the uv lamp and to observe, occur the target specific band for containing certain type gene composition, otherwise for not containing certain type gene composition.

(5) the animal native gene detects in the doubtful sewer oil sample

Present embodiment has at first carried out the animal native gene to 8 parts of doubtful sewer oil dna samples and edible vegetable oil dna sample and has detected, with beef DNA as positive control, with the maize dna that do not contain the animal native gene as negative control.Detected result is seen accompanying drawing 1, can be known by Fig. 1, and the positive, feminine gender and blank are normal, and the safety of detected result is described.There are 5 duplicate samples (sample number into spectrum is respectively sewer oil 1,2,4,6,7) to detect in 8 parts of doubtful sewer oil samples and contain animal native gene composition; And all do not detect animal native gene composition in VT 18, Semen Maydis oil, peanut oil, rapeseed oil and the sunflower seed oil sample; The specificity of the primer in illustrative embodiment 1 test kit is good, discrimination is high, and the sample that only contains animal derived materials can be detected.

The detected result explanation has 5 parts to contain the animal gene composition in 8 parts of doubtful sewer oil samples.

(6) plant endogenous gene test in the doubtful sewer oil sample

After having obtained animal native gene amplification; This test has been carried out plant endogenous gene test to 8 parts of doubtful sewer oil dna samples and edible vegetable oil dna sample etc. again; With maize dna as positive control, with beef, mutton, pork DNA as negative control.Detected result is seen accompanying drawing 2, can be known by Fig. 2, and the positive, feminine gender and blank are normal, and the safety of detected result is described.There are 4 duplicate samples (sample number into spectrum is respectively sewer oil 2,5,6,8) to detect in 8 parts of doubtful sewer oil samples and contain plant endogenous gene element; And all do not detect plant endogenous gene element in the beef, mutton, pork DNA sample; The specificity of the primer of the test kit of illustrative embodiment 1 is good and discrimination is high, and the sample that only contains plant-derived composition can detect.

The detected result explanation has 4 parts to contain the plant gene composition in 8 parts of doubtful sewer oil samples.

(7) the ox native gene detects in the doubtful sewer oil sample

In preliminary 5 parts of sewer oil samples that contain the animal native gene of confirming, present embodiment has further carried out the test experience of ox native gene to above-mentioned 5 parts of sewer oil dna samples.With beef DNA as positive control, with the maize dna that do not contain the cow genome composition, soy bean DNA as negative control.Detected result is seen accompanying drawing 3, can be known by Fig. 3, and the positive, feminine gender and blank are normal, and the safety of detected result is described.There are 2 duplicate samples (sample number into spectrum is respectively sewer oil 1,2) to detect in 5 parts of doubtful sewer oil samples and contain ox native gene composition; And all do not detect ox native gene composition in pork, mutton, chicken, VT 18, Semen Maydis oil, peanut oil, rapeseed oil and the sunflower seed oil sample; The specificity of the primer of the test kit of illustrative embodiment 1 is good and discrimination is high, can accurately detect oil sample to be measured and whether contain ox source property gene element.

The sensitivity experiment of the test kit of embodiment 3 embodiment 1

Present embodiment is allocated the above-mentioned sewer oil sample 1 that contains ox native gene composition that detects and abundant mixing by 50: 50,30: 70,10: 90,5: 95,1: 99 volume ratio with common edible soybean oil, carries out DNA extraction and pcr amplification according to the method for embodiment 2.

Detected result shows; The test kit of embodiment 1 can detect the sewer oil sample and mix than animal native gene and the ox native gene in the oil sample that mix that is 50: 50,30: 70,10: 90,5: 95 with the vegetables oil sample; But can not detect the doping ratio and be 1: 99 oil sample, explain that this test kit can detect the oil sample that lower bound is a doping sewer oil 5%.

The repeated experiment of the test kit of embodiment 4 embodiment 1

Present embodiment has carried out the method repeated experiment to the doubtful sewer oil sample of 20 parts of on-the-spot random checkings.Experimental result is seen shown in the table 2.

20 parts of sewer oil test result of samples in table 2 repeated experiment

Experimental result shows; Can application implementation the test kit of example 1 in 20 parts of sewer oil samples, detect the animal native gene; Compositions such as plant endogenous gene, rice native gene, corn native gene, pig native gene, ox native gene, chicken native gene; Repeat 3 experiments, experimental result is consistent, and the inventive method good reproducibility is described.Through the experiment proof; The inventive method can detect animal and plant species gene orders such as ox, pig, chicken, rice, corn in doubtful sewer oil sample; As in the sample of sign vegetables oil such as VT 18, detecting animal derived materials or different types of plant-derived composition, explain that the target oil sample maybe be for mingling oil or sewer oil.Presentation of results the inventive method is applicable to the detection discriminating of sewer oil, and method has good application value.

The above embodiment has only expressed several kinds of embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art under the prerequisite that does not break away from the present invention's design, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with accompanying claims.

Claims (8)

1. the test kit of a rapid detection sewer oil is characterized in that, comprising:

(1) DNA extraction damping fluid

Consisting of of said DNA extraction damping fluid: 15～25mg/L CTAB, 90～110nmol/L Tris-HCl, 15～25mmol/L EDTA ₂Na, 1.3～1.5mol/L NaCl, 0.09～0.12g/L Proteinase K;

(2) Taq archaeal dna polymerase, the concentration of said Taq archaeal dna polymerase are 125IU;

(3) PCR reaction solution

Consisting of of said PCR reaction solution: 2.7～3.3mmol/L MgCl ₂, 0.3～0.5 μ mol/L primer, 0.3～0.5mmol/L dNTPs;

Said primer is universal primer SEQ ID NO:1 and SEQ ID NO:2 to animal endogenous gene sequence, and the universal primer SEQ ID NO:3 and the SEQ ID NO:4 that are directed against plant endogenous property gene order;

(4) sample-loading buffer

Consisting of of said sample-loading buffer: 2.0～3.0g/L tetrabromophenol sulfonphthalein, 350～450g/L sucrose.

2. the test kit of rapid detection sewer oil according to claim 1 is characterized in that, said primer also comprises the primer to the chondriogen sequence of ox, sheep, pig or chicken.

3. the test kit of rapid detection sewer oil according to claim 2 is characterized in that, said primer to ox chondriogen sequence is SEQ ID NO:5 and SEQ ID NO:6.

4. the test kit of rapid detection sewer oil according to claim 2 is characterized in that, said primer to sheep chondriogen sequence is SEQ ID NO:7 and SEQ ID NO:8.

5. the test kit of rapid detection sewer oil according to claim 2 is characterized in that, said primer to the porcine mtdna gene order is SEQ ID NO:9 and SEQ ID NO:10.

6. the test kit of rapid detection sewer oil according to claim 2 is characterized in that, said primer to chicken chondriogen sequence is SEQ ID NO:11 and SEQ ID NO:12.

7. according to the test kit of each described rapid detection sewer oil of claim 1-6; It is characterized in that said primer also comprises to the primer SEQ ID NO:13 of the native gene sequence of corn and SEQ ID NO:14, to the primer SEQ ID NO:15 of the native gene sequence of soybean and SEQ ID NO:16, to the primer SEQ ID NO:17 of the native gene sequence of rice and SEQ ID NO:18, to the primer SEQ ID NO:19 and the SEQ ID NO:20 of the native gene sequence of peanut or be directed against the primer SEQ ID NO:21 and the SEQ ID NO:22 of the native gene sequence of Semen Brassicae campestris.

8. the method for a rapid detection sewer oil is characterized in that, adopts each described test kit of claim 1-7, may further comprise the steps:

(1) preparation sample DNA

Get 20～50mL oil sample and equal-volume distilled water, it is centrifugal to vibrate, and discards the upper strata grease, adds 20～50mL oil sample again, and it is centrifugal to continue vibration, repeat 7～8 times.After treating lower floor's water muddiness, high speed centrifugation discards the upper strata grease, obtains 300～400mL oil sample; The water concentrate drying after 3～5 hours, is added the DNA extraction damping fluid of 2～3mL, behind the mixing, place 65 ℃ to place 45～60min; Shake frequently, the use volume ratio is 24: 1 a chloroform: the primary isoamyl alcohol mixed solution extracts, and is centrifugal, gets supernatant; Add Virahol, placed 2～3 hours for-20 ℃, high speed centrifugation obtains the DNA deposition; Wash throw out with 70% ethanolic soln again, throw out is dissolved in water, and promptly gets sample DNA;

(2) pcr amplification reaction

Get the PCR reaction solution, the Taq archaeal dna polymerase places reaction tubes, mixing adds sample DNA; Centrifugal being placed on the amplification appearance increased reaction parameter: behind 95 ℃ of preparatory sex change 5min, by 94 ℃ of 30sec; 57 ℃ of 45sec; 72 ℃ of 60sec programs are carried out 40 circulations, and last 72 ℃ are extended 10min, finish reaction in 4 ℃.

(3) product detects

Reaction product adds a kind damping fluid, fully point sample behind the mixing; With DNA Marker as molecular weight standard; With containing painted 1.5% agarose gel electrophoresis of the own ingot of bromination, after the end, put under the uv lamp and observe; Occur the target specific band for containing certain species gene element, otherwise for not containing this species gene composition.

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