CN101974522A - Primer, probe, kit and method for identifying authenticity of cervus elaphus linnaeus - Google Patents
Primer, probe, kit and method for identifying authenticity of cervus elaphus linnaeus Download PDFInfo
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Abstract
The invention relates to a specific oligonucleotide primer, a probe and a real-time fluorescence polymerase chain reaction (PCR) detection method for identifying authenticity of cervus elaphus linnaeus. The method comprises the using of the specific oligonucleotide primer and the probe of the invention. The invention also relates to a PCR detection kit for identifying the authenticity of the cervus elaphus linnaeus. The kit comprises the specific oligonucleotide primer and the probe of the invention. The invention also relates to application of the specific oligonucleotide primer and the probe of the invention to the identification of the authenticity of the cervus elaphus linnaeus. By using the real-time fluorescence PCR detection method of the invention, the authenticity of the cervus elaphus linnaeus can be simply, quickly, specifically and flexibly identified.
Description
Technical field
The invention belongs to biological technical field, particularly, the present invention relates to be used for the specific oligonucleotide primer and the probe of cervus elaphus linnaeus real and fake discrimination, be used to differentiate the real-time fluorescence PCR detection method of the cervus elaphus linnaeus true and false, and described specific oligonucleotide primer and probe or the application of test kit in differentiating the cervus elaphus linnaeus true and false.
Background technology
Pilose antler (Cornu Cervi Pantotrichum) is the young horn of animal in deer family spotted deer (Cervus nippon Temminck) or red deer (Cervus elaphus Linnaeus) the unossified close living fine hair of stag, practises claiming pilose antler of sika, cervus elaphus linnaeus.The pilose antler beginning is stated from Shennong's Herbal, classifies middle product as, has effects such as invigorating kidney-YANG, benefiting essence-blood, strengthening the bones and muscles, is one of famous and precious animal drugs of China.Existing commercially available kind specification is more, because former animal difference is divided two kinds of pilose antler of sika and cervus elaphus linnaeus; Because the collecting method difference, divide again and cut three kinds of young pilose antler, saw young pilose antler and piece of antlers; Because fork branch what and old tender difference, it is multiple to be divided into saddle, two thick sticks, three troubles, lotus flower etc. again.
The kind of deer is a lot of, but the main finger spotted deer and the red deer of pharmaceutical use are arranged.Red deer is the large-scale deer class that is only second to elk, because the bodily form is gained the name like courser.The kind of red deer mainly contains Norway red deer, Ba Bali red deer, Coxica red deer, West Africa red deer, Central European red deer, Scotland red deer, Spain red deer, Eastern Europe red deer, Balkh red deer, Tarim Basin red deer, Kashmir red deer, Tibet red deer, white arm deer.
Because red deer costs an arm and a leg, some lawless persons adopt the means of mixing the spurious with the genuine to reap staggering profits, and have not only endangered human consumer's interests, and have caused and seriously influence making up harmonious market order.The section that often has pseudo-product to make on the market is sold.Be mainly the puppet product that the bone piece of roe deer (Capreolus capreolus), reinder (Alces alces), reinder (Rangifer tarandus) isogonism and other animals of America deer subfamily or gelatin cut into after with the animal skin embedding.
For interests and the good market order of safeguarding the human consumer, domestic and international many scholar's research several different methods the true and false of pilose antler is differentiated.Method commonly used has character identification, microscopical identification, tlc discriminating, spectrography discriminating, ultraviolet spectroscopy discriminating etc.But traditional detection method is subject to the influence of the chemical ingredients that contained in the pilose antler in the different varieties and the place of production since present mix pseudo-level and means more and more brilliant, mix pseudo-composition and become increasingly complex, seriously limited traditional detection method application space.
Protocols in Molecular Biology is with its convenient, accurate, rapid, succinct characteristics, become the focus of Chinese scholars research in recent years, fresh blood has been injected in food false distinguishing researchs such as its true and false for proof food provides truly, reliable foundation, and feeding product kind is differentiated, product is traced to the source.But so far, Protocols in Molecular Biology still is in the preliminary study stage in the application aspect the false distinguishing of Chinese medicinal materials cervus elaphus linnaeus.
At present, rare both at home and abroad report can be quick, simple, special and be differentiated the method for Chinese medicinal materials cervus elaphus linnaeus delicately.
Therefore, the distinguishing method between true and false of the Chinese medicinal materials cervus elaphus linnaeus that this area needs are a kind of fast, specificity is good, highly sensitive carries out the discriminating of the Chinese medicinal materials cervus elaphus linnaeus true and false.
Summary of the invention
One object of the present invention is, the specific oligonucleotide primer and the probe of quick discriminating cervus elaphus linnaeus is provided.
Another object of the present invention is, the real-time fluorescence PCR detection method of the quick discriminating cervus elaphus linnaeus true and false is provided.
A further object of the present invention is, is provided for differentiating fast the test kit of the cervus elaphus linnaeus true and false.
A further object of the present invention is, the application in differentiating the cervus elaphus linnaeus true and false of specific oligonucleotide primer of the present invention and probe is provided.
At the foregoing invention purpose, the invention provides following technical scheme:
According to one embodiment of the invention, the invention provides the specific oligonucleotide primer that is used for the cervus elaphus linnaeus real and fake discrimination to and probe.The present invention detects the basis with the DNA of red deer, and (Displancement loop, D-Loop) region sequence has the characteristics of otherness in different Cervidae species, has cloned red deer Mitochondrial DNA D-ring region sequence according to Mitochondrial DNA (mtDNA) D-ring.According to these sequences Design primer and probe, utilize the cervus elaphus linnaeus composition in the real-time fluorescence PCR method test sample.
Oligonucleolide primers of the present invention is to being to design according to the characteristics that the Mitochondrial DNA D-of different Cervidae species ring district has an otherness with probe.In one embodiment, described primer is to being made up of upstream primer and downstream primer, described upstream primer is Marlu-F:AACACGTGATATAACCTTATGCGC (SEQ ID No.1), and described downstream primer is Marlu-R:TATGTCCTATACACTAACTCATGTGC (SEQ ID No.2); Described probe is Marlu-P:TGTGCTAGAACACGCATGTATAACAGCAC (SEQ ID No.3), holds at 3 ' of probe to be connected with a fluorescent quenching group B HQ1, and 5 ' end is connected with a fluorescence report group FAM.Use primer of the present invention to the combination of probe, under the few situation of sample size, with respect to other primers to still amplifying the purpose fragment special, delicately.
According to another embodiment of the invention, the invention provides the real-time fluorescence PCR detection method of differentiating the cervus elaphus linnaeus true and false, described method comprise use at the specific oligonucleotide primer of cervus elaphus linnaeus to and probe, described primer is to being that the characteristics that Mitochondrial DNA D-ring region sequence according to different Cervidae species has an otherness design.In one embodiment, in the real-time fluorescence PCR detection method of cervus elaphus linnaeus real and fake discrimination of the present invention, employed specific oligonucleotide primer is to being made up of upstream primer and downstream primer, the base sequence of described upstream primer is SEQ ID No.1, and the base sequence of described downstream primer is SEQ ID No.2; The base sequence of described probe is SEQ ID No.3, holds at 3 ' of probe to be connected with a fluorescent quenching group B HQ1, and 5 ' end is connected with a fluorescence report group FAM.The present inventor by a large amount of screening operations determined specific oligonucleotide primer of the present invention to and probe, and set up stable PCR system, thus special and differentiate the true and false of cervus elaphus linnaeus delicately.
In one embodiment, cervus elaphus linnaeus real-time fluorescence PCR detection method of the present invention also further comprises the step of extracting the pilose antler sample total DNA.In one embodiment, in described DNA extraction step, by detecting animal in deer family Mitochondrial DNA D-ring region sequence, the extraction quality of coming the total DNA of specimen.In a preferred embodiment, the universal primer of detection line mitochondrial DNA D-ring region sequence is to being made up of upstream primer and downstream primer, the base sequence of described upstream primer is Luke-F:CATACGCAATYCTACGATCAATTCC (SEQ ID No.4), and the base sequence of described downstream primer is Luke-R:GCTACTARGATTCAGAATAGGCATTG (SEQ ID No.5); The base sequence of described probe is Luke-P:TGGTCGGAATATYATGCTGCGTTGTTTGG (SEQ ID No.6), holds at 3 ' of probe to be connected with a fluorescent quenching group B HQ2, and 5 ' end is connected with a fluorescence report group TAMRA.Described pcr amplification condition is 95 ℃, 10min; 95 ℃ of 15s; 60 ℃, 1min, 40 circulations.
According to another embodiment of the invention, the invention provides the test kit that is used for differentiating fast the cervus elaphus linnaeus true and false, described test kit comprise of the present invention be used for specific oligonucleotide primer that real time fluorescent PCR method differentiates the cervus elaphus linnaeus true and false to and probe and working instructions.In the preferred embodiment of test kit of the present invention, specific oligonucleotide primer of the present invention is to being to design according to the characteristics that the Mitochondrial DNA D-of different Cervidae species ring region sequence has an otherness.In a preferred embodiment, the specific oligonucleotide primer that comprises in the described test kit is to being made up of upstream primer and downstream primer, and the base sequence of described upstream primer is SEQ ID No.1, and the base sequence of described downstream primer is SEQ ID No.2; The base sequence of the probe that comprises in the described test kit is SEQ ID No.3, holds at 3 ' of probe to be connected with a fluorescent quenching group B HQ1, and 5 ' end is connected with a fluorescence report group FAM.In preferred embodiments, described test kit also comprises reagent that is used for the sample DNA extraction and the reagent that is used for the PCR reaction.In a preferred embodiment, comprise description in the working instructions of described test kit to the condition of the pcr amplification that is used for differentiating fast the cervus elaphus linnaeus true and false.In a preferred embodiment, the pcr amplification condition that provides in the specification sheets of described test kit is: 95 ℃, and 10min; 95 ℃ of 15s; 60 ℃, 1min, 40 circulations.
According to another embodiment of the present invention, the invention provides specific oligonucleotide primer of the present invention to the application of probe in differentiating the cervus elaphus linnaeus true and false.In the preferred embodiment according to application of the present invention, described specific oligonucleotide primer is to being made up of upstream primer and downstream primer, and the base sequence of described upstream primer is SEQ ID No.1, and the base sequence of described downstream primer is SEQ IDNo.2; The base sequence of described probe is SEQ ID No.3, holds at 3 ' of probe to be connected with a fluorescent quenching group B HQ1, and 5 ' end is connected with a fluorescence report group FAM.In another embodiment, the present invention also provides the application of test kit of the present invention in differentiating the cervus elaphus linnaeus true and false.Preferably, in above-mentioned application of the present invention, described test kit comprise specific oligonucleotide primer of the present invention to and probe.
The present invention detects the basis with the DNA of cervus elaphus linnaeus, the characteristics that have otherness according to Mitochondrial DNA D-ring region sequence in the different Cervidae species, cloned red deer Mitochondrial DNA D-ring region sequence,, utilized the real-time fluorescence PCR method to differentiate the true and false of cervus elaphus linnaeus according to these sequences Design primer and probe.
Real-time fluorescence quantitative PCR is promptly on the basis of conventional PCR method, add fluorescently-labeled probe or fluorescence dye, accumulation along with the PCR product, the fluorescent signal that probe or dyestuff send strengthens, and the fluorescence monitoring system can receive fluorescent signal, be DNA chain of every generation, just have a fluorescence molecule to form, realized that the accumulation of fluorescent signal and PCR product form fully synchronously.Therefore can monitor whole PCR reaction process in real time, and finally detect the initial copy number of testing sample, thereby can detect cervus elaphus linnaeus composition to be measured.
Real-time fluorescence PCR detection method of the present invention adopts complete stopped pipe to detect, and need not the PCR aftertreatment, has avoided crossed contamination and false positive.Method of the present invention has used dexterously that the DNA of round pcr efficiently increases, the specificity of nucleic acid hybridization and detection technique of fluorescence fast and susceptibility, have simple to operate, time saving and energy saving, reliable results and accurate advantage such as sensitivity.Use real-time fluorescence PCR detection method of the present invention, its simple, quick, special and sensitive characteristics is suitable for the discriminating of the cervus elaphus linnaeus composition true and false on the domestic and international market.
Description of drawings
Fig. 1 is the real-time fluorescence PCR result who shows testing sample DNA extraction effect, wherein uses the animal in deer family universal primer to SEQ ID No.4 and SEQ ID No.5 and probe SEQ ID No.6 detection, and wherein the sample of fluorescence curve is followed successively by 1. spotted deers (1); 2. spotted deer (2); 3. spotted deer (3); 4. spotted deer (4); 5. spotted deer (5); 6. spotted deer (6); 7. spotted deer (7); 8. spotted deer (8); 9. spotted deer (9); 10. spotted deer (10); 11. spotted deer (11); 12. spotted deer (12); 13. spotted deer (13); 14. spotted deer (14); 15. spotted deer (15); 16. spotted deer (16); 17. spotted deer (17); 18. spotted deer (18); 19. spotted deer (19); 20. spotted deer (20); 21. spotted deer (21); 22. spotted deer (22); 23. spotted deer (23); 24. red deer (1); 25. red deer (2); 26. red deer (3); 27. red deer (4); 28. red deer (5); 29. red deer (6); 30. red deer (7); 31. Fallow deer (1); 32. Fallow deer (2); 33. blank (ddH
2O).
Fig. 2 is the result who shows real-time fluorescence PCR specificity discriminating cervus elaphus linnaeus, wherein uses the specific oligonucleotide primer to SEQ ID No.1 and SEQ ID No.2 and probe SEQ ID No.3 detection, and wherein the sample of fluorescence curve is followed successively by 1. red deers (1); 2. red deer (2); 3. red deer (3); 4. red deer (4); 5. red deer (5); 6. red deer (6); 7. red deer (7); 8. spotted deer (1); 9. spotted deer (2); 10. spotted deer (3); 11. spotted deer (4); 12. spotted deer (5); 13. spotted deer (6); 14. spotted deer (7); 15. spotted deer (8); 16. spotted deer (9); 17. spotted deer (10); 18. spotted deer (11); 19. spotted deer (12); 20. spotted deer (13); 21. spotted deer (14); 22. spotted deer (15); 23. spotted deer (16); 24. spotted deer (17); 25. spotted deer (18); 26. spotted deer (19); 27. spotted deer (20); 28. spotted deer (21); 29. spotted deer (22); 30. spotted deer (23); 31. Fallow deer (1); 32. Fallow deer (2); 33. blank (ddH
2O).
Fig. 3 is to be example with the cervus elaphus linnaeus, the result of the Auele Specific Primer of discriminating cervus elaphus linnaeus and the sensitivity of probe combinations, wherein 1-7 is the concentration of carrying out the dna solution of real-time fluorescence PCR amplification, be followed successively by 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 0.1pg/ μ L and blank, repeat 3 times.
Fig. 4 is to be example with cervus elaphus linnaeus, sika deer velvet antler, determine the relative detectability of cervus elaphus linnaeus Auele Specific Primer and probe combinations, wherein 1-5 for sika deer velvet antler DNA with cervus elaphus linnaeus DNA with 10 times of dilutions, make that the cervus elaphus linnaeus dna content is respectively 10ng, 1ng, 100pg, 10pg, 1pg in its PCR reaction system, triplicate, the result who detects then.
Fig. 5 is commercially available pilose antler sample detection result, and wherein fluorescence curve 1 is for red deer positive reference substance, fluorescence curve 2-21 are followed successively by commercially available cervus elaphus linnaeus sample (1)-(20), fluorescence curve 22-38 is followed successively by commercially available cervus elaphus linnaeus sample (1)-(16) and blank (sterilized water).
Embodiment
The present invention is further illustrated for mode by embodiment, but the present invention is not limited only to following examples.
Embodiment 1
The present inventor encircles region sequence by the Mitochondrial DNA D-of the PCR clone and the red deer of having checked order first.
The red deer Mitochondrial DNA D-ring region sequence of present embodiment for obtaining by the PCR cloning and sequencing.
Encircle region sequence has otherness in different animal in deer family characteristics according to Mitochondrial DNA D-, design upstream and downstream primer amplification red deer Mitochondrial DNA D-ring region sequence.Operation instructions according to Wizard GelExtraction Kit (Promega, the U.S.) is carried out purifying, recovery to red deer PCR product.The specification sheets of pressing TaKaRa pMD19-T Vector test kit (TaKaRa, Japan) is connected purified product with pMD19-T Vector, linked system 10 μ L, and its reactive component is: pMD19-T Vector 1 μ L, PCR product 2 μ L, ddH
2O 2 μ L, Solution I 5 μ L are provided with the positive and negative control simultaneously.Linked system is placed room temperature (22-37 ℃) reaction 30min, and reaction places on ice after finishing immediately.Add and connect product in 50 μ LTOP competent cells, flick mixing, ice bath 30min, 42 ℃ of accurate heat shock 90s place 2min on ice immediately, add gone out the brain heart infusion of bacterium of 800 μ L then, 37 ℃, 200r/min shaking culture 1h.5000r/min low-speed centrifugal 1min abandons 600 μ L supernatants, will precipitate mixing, gets 100 μ L mixed solutions and is applied on the nutrient agar plate that Amp+, X-Gal and IPTG handle, and 37 ℃ of incubated overnight are placed on 4h in 4 ℃ of refrigerators.Picking list bacterium colony hickie is cultivated at 37 ℃, 200r/min shaken overnight in the brain heart infusion of the Amp that contains final concentration 200 μ g/mL.
(1) positive colony is identified: the single white clone of picking, carry out bacterium colony PCR reaction, system is 25 μ L, and its component is: reaction 10 * Buffer 2.5 μ L, dNTPs 1 μ L, upstream and downstream primer each 0.5 μ L, Taq enzyme 0.2 μ L, picking list bacterium colony adds water and mends to 25 μ L as template.Response procedures is: 94 ℃ of 5min, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, 40 circulations.The PCR product is identified by agarose gel electrophoresis.
(2) order-checking: after determining positive colony, this bacterium colony of picking is cultivated at 37 ℃, 200r/min shaken overnight in the 5 μ L brain heart infusions that contain final concentration 200 μ g/mL Amp.Get 1mL bacterium liquid and send biotech firm's evaluation of checking order.
The Mitochondrial DNA D-ring region sequence of the red deer that the PCR cloning and sequencing obtains is as follows:
AGACTCAAGG?AAGAAGCCAT?AGCCCCACTA?TCAACACCCA
AAGCTGAAGT?TCTATTTAAA?CTATTCCCTG?ACGCTTATTA
ATATAGTTCC?ATAAAAATCA?AGAACTTTAT?CAGTATTAAA
TTTCCAAAAA?ATCT-AATAT?TTTAATACAG?CTTTCTACTC
AACATCCAAT?TTACATTTTA?CGTCCTACTA?ATTACACAGC
AAAACACGTG?ATATAACCTT?ATGCGCTTGT?AGTACATAAA
ATCAATGTGC?TAGAACACGC?ATGTATAACA?GCACATGAG
TTAGTGTATA?GGACATATTA?TGTATAATAG?TACATAAATC
AATGTATTAG?GACATATTAT?GTATAATAGT?ACATTATATT
ATATGCCCCA?TGCATAAACC?ATGGTTACA(SEQ?ID?No.7)。
Present embodiment uses universal primer to reaching the extraction quality of probe test sample total DNA for passing through.
By detection line mitochondrial DNA D-ring region sequence, extraction quality that can the total DNA of specimen.Taqman fluorescent probe method: the TaqMan technology is a kind of technology of single tube PCR product being carried out the real time fluorescent quantitative detection, in the regular-PCR amplification system, add one and the two fluorescence labeling probes of the special complementary of target-gene sequence, utilize the fluorescent signal accumulation whole PCR process of monitoring in real time, by typical curve unknown template is carried out quantitative analysis at last.Quantitative step: determine that 1. (C represents cycle number (Cycle) to the CT value, and T represents fluorescence thresholding (Threshold), the cycle number that is experienced when promptly the fluorescent signal in each reaction tubes arrives the thresholding of setting; 2. utilize typical curve that unknown sample is carried out quantitative assay.After obtaining the CT value of unknown sample, calculate the initial copy number of this sample from typical curve.There is linear relationship in the logarithm of the CT value of each template and the initial copy number of this template, and promptly initial copy number is many more, and the CT value is more little.Reaction system is: Mastermix 12.5 μ L; Probe (10 μ M) 0.5 μ L; Each 0.5 μ L of upstream and downstream primer (10 μ M); Template DNA 5 μ L; Add ddH
2O to cumulative volume be 25 μ L.Response procedures is 95 ℃ of 10min; 95 ℃ of 15s; 60 ℃ of 1min, 40 circulations.
The employed universal primer that is used to detect cervus elaphus linnaeus total DNA extraction quality is to being made up of upstream primer and downstream primer in the present embodiment, and the base sequence of described upstream primer is SEQ ID No.4, and the base sequence of described downstream primer is SEQ ID No.5; The base sequence of employed probe is SEQ IDNo.6, holds at 3 ' of probe to be connected with a fluorescent quenching group B HQ2, and 5 ' end is connected with a fluorescence report group TAMRA.
In the present embodiment, 7 parts of red deer samples, 23 parts of spotted deer samples, 2 Fen Fallow deer samples have been detected.
Employed detection key instrument:
Micropipet (10 μ L, 100 μ L, 1000 μ L, Eppendorf), quantitative real time PCR Instrument (ABI 7700 Applied Biosystems, USA), high speed tabletop centrifuge (Pico17 Thermo), high speed disintegrator (IKA-WEARKE GERMANY), nucleic acid-protein analyser (DYY-6C Liuyi Instruments Plant, Beijing) etc.
Detect main agents:
Chloroform, Virahol are purchased respectively in the logical company of Beijing six directions; CTAB lysate (20g/L CTAB, 1.4mol/L NaCl, 0.1mol/L Tris, 0.02mol/L Na
2-EDTA), CTAB precipitated liquid (5g/LCTAB, 0.04mol/L NaCl), 1.2mol/L NaCl be this experiment and prepare voluntarily; Fast Start Universal Probe MasterMix (Rox) purchases in Roche Holding Ag; Primer and probe are synthetic etc. by Shanghai AudioCodes bio tech ltd.
Detect key step:
1DNA extracts
Pilose antler sample to be measured is: 7 portions of cervus elaphus linnaeus, 23 parts of sika deer velvet antlers, 2 Fen Fallow pilose antlers.
Take by weighing in the clean 2.0mL centrifuge tube of 0.1g Pilose Antler to, add the 1.5mLCTAB lysate, 65 ℃ of 2h, interval continuous mixing several times; 8000rpm 15min gets in 1mL supernatant liquor to the 1 clean 2.0mL centrifuge tube, adds 700 μ L chloroforms, violent mixing 30s, 14500rpm 10min gets 650 μ L supernatant liquors respectively to clean 2.0mL centrifuge tube, add 1300 μ L CTAB precipitated liquid, violent mixing 30s, room temperature leaves standstill 1h; 14500rpm 20min abandons supernatant liquor, adds 350 μ L 1.2M NaCl, and thermal agitation 30s adds 350 μ L chloroforms again, violent mixing 30s, 14500rpm 10min; Get supernatant liquor 320 μ L respectively, add 0.8 times of volume Virahol, behind the mixing ,-20 ℃ of 1h, 14500rpm 20min abandons supernatant liquor, adds 500 μ L70% ethanol, and behind the mixing, 14500rpm 20min abandons supernatant liquor, dries in the air to air-dry, adds 100 μ L ddH
2The O dissolving, 4 ℃ store for future use.
2 real-time fluorescence PCRs detect the primer and probe
Primer sequence is SEQ ID No.4 and SEQ ID No.5, and probe sequence is SEQ ID No.6, and 3 ' end is connected with a fluorescent quenching group B HQ2, and 5 ' end is connected with a fluorescence report group TAMRA.
3 real-time fluorescence PCR reaction systems:
Fast?Start?Universal?Probe?MasterMix(Rox)12.5μL
Probe (10 μ M) 0.5 μ L
Upstream primer (10 μ M) 0.5 μ L
Downstream primer (10 μ M) 0.5 μ L
Template DNA 5 μ L
Add ddH
2O to cumulative volume be 25 μ L
Annotate: each PCR detects and all sets up corresponding blank (ultrapure water with the preparation reaction system replaces dna profiling, and whether detection reagent is polluted);
4 real-time fluorescence PCR reaction parameters:
95℃ 10min
95℃ 15s
60℃ 1min
40 circulations.
Annotate: different instruments should be done suitable adjustment with each reagent of PCR and reaction parameter.
As shown in Figure 1, all the amplified fluorescence curve can more than baseline, occur during with the DNA of animal in deer family universal primer amplification testing sample, show all testing sample DNA extraction successes.
Embodiment 3
Present embodiment by following test to the primer of red deer to having carried out specificity and sensitivity checking with probe.
By detection line mitochondrial DNA D-ring region sequence, can determine the specificity and the detection sensitivity of red deer primer and probe combinations.Reaction system is: Fast Start Universal Probe MasterMix (Rox) 12.5 μ L; Probe (10 μ M) 0.5 μ L; Each 0.5 μ L of upstream and downstream primer (10 μ M); Template DNA 5 μ L; Add ddH
2O to cumulative volume be 25 μ L.Response procedures is 95 ℃ of 10min; 95 ℃ of 15s; 60 ℃ of 1min, 40 circulations.
The primer and the probe sequence of the employed discriminating cervus elaphus linnaeus true and false are: primer sequence is SEQ ID No.1 and SEQ ID No.2, probe sequence is SEQ ID No.3,3 ' end is connected with a fluorescent quenching group B HQ1, and 5 ' end is connected with a fluorescence report group FAM.
Employed detection key instrument:
Micropipet (10 μ L, 100 μ L, 1000 μ L, Eppendorf), quantitative real time PCR Instrument (ABI 7700 Applied Biosystems, USA)), high speed tabletop centrifuge (Pico17 Thermo), high speed disintegrator (IKA-WEARKE GERMANY), nucleic acid-protein analyser (DYY-6C Liuyi Instruments Plant, Beijing) etc.
Detect main agents:
Chloroform, Virahol are purchased respectively in the logical company of Beijing six directions; CTAB lysate (20g/L CTAB, 1.4mol/L NaCl, 0.1mol/L Tris, 0.02mol/L Na
2-EDTA), CTAB precipitated liquid (5g/LCTAB, 0.04mol/L NaCl), 1.2mol/L NaCl be this experiment and prepare voluntarily; Fast Start Universal Probe MasterMix (Rox) purchases in Roche Holding Ag; Primer and probe are synthetic etc. by Shanghai AudioCodes bio tech ltd.
Detect key step:
1DNA extracts
Detect sample: (1) 7 portion of cervus elaphus linnaeus, 23 parts of sika deer velvet antlers, 2 Fen Fallow pilose antlers are used for specificity analyses; (2) with the red deer be example, it is the concentration of 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 0.1pg/ μ L that the dna solution that extracts is diluted respectively with sterilized water, is used to analyze the absolute sense limit of primer and probe combinations; (3) be example with red deer, spotted deer, with spotted deer DNA with red deer DNA with 10 times of dilutions, make that the red deer dna content is respectively 10ng, 1ng, 100pg, 10pg, 1pg in its PCR reaction system, to determine the relative detectability of red deer Auele Specific Primer and probe combinations.
Take by weighing in the clean 2.0mL centrifuge tube of 0.1g sample powder to, add the 1.5mLCTAB lysate, 65 ℃ of 2h, interval continuous mixing several times; 8000rpm 15min gets in 1mL supernatant liquor to the 1 clean 2.0mL centrifuge tube, adds 700 μ L chloroforms, violent mixing 30s, 14500rpm 10min gets 650 μ L supernatant liquors respectively to clean 2.0mL centrifuge tube, add 1300 μ L CTAB precipitated liquid, violent mixing 30s, room temperature leaves standstill 1h; 14500rpm 20min abandons supernatant liquor, adds 350 μ L 1.2M NaCl, and thermal agitation 30s adds 350 μ L chloroforms again, violent mixing 30s, 14500rpm 10min; Get supernatant liquor 320 μ L respectively, add 0.8 times of volume Virahol, behind the mixing ,-20 ℃ of 1h, 14500rpm 20min abandons supernatant liquor, adds 500 μ L70% ethanol, and behind the mixing, 14500rpm 20min abandons supernatant liquor, dries in the air to air-dry, adds 100 μ L ddH
2The O dissolving, 4 ℃ store for future use.
2 real-time fluorescence PCRs detect the primer and probe
Primer sequence is SEQ ID No.1 and SEQ ID No.2;
Probe sequence is SEQ ID No.3, and 3 ' end is connected with a fluorescent quenching group B HQ1, and 5 ' end is connected with a fluorescence report group FAM.
3 real-time fluorescence PCR reaction systems:
Fast?Start?Universal?Probe?MasterMix(Rox)12.5μL
Probe (10 μ M) 0.5 μ L
Upstream primer (10 μ M) 0.5 μ L
Downstream primer (10 μ M) 0.5 μ L
Template DNA 5 μ L
Add ddH
2O to cumulative volume be 25 μ L
Annotate: each PCR detects and all sets up corresponding blank (ultrapure water with the preparation reaction system replaces dna profiling, and whether detection reagent is polluted);
4 real-time fluorescence PCR reaction parameters:
95℃ 10min
95℃ 15s
60℃ 1min
40 circulations.
Annotate: different instruments should be done suitable adjustment with each reagent of PCR and reaction parameter.
As shown in Figure 2, when utilizing the real-time fluorescence PCR specificity to differentiate the Mitochondrial DNA D-ring region sequence of red deer, typical amplification curve all appears in 7 kinds of red deer samples etc., and other sample: 23 portions of spotted deers, 2 Fen Fallow deer etc. and blank (ddH
2O) amplification curve all do not occur, prove absolutely that the primer probe of this experimental design shows specificity preferably to the red deer sample.
For determining the absolute sense limit of red deer Auele Specific Primer and probe combinations, with the red deer is example, it is the concentration of 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 0.1pg/ μ L that the dna solution that extracts is diluted respectively with sterilized water, carry out the real-time fluorescence PCR amplification by above-mentioned condition respectively, the result as shown in Figure 3.Red deer DNA concentration is that 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ have the specific amplification curve during L, and concentration is reduced to 1pg/ μ L when following, and no specific amplification curve occurs.Experimental result shows that the content that the real-time fluorescence PCR detection method of foundation can detect the red deer composition is 1pg/ μ L.
With red deer, spotted deer is example, with spotted deer DNA with red deer DNA with 10 times of dilutions, make that the red deer dna content is respectively 10ng, 1ng, 100pg, 10pg, 1pg in its PCR reaction system, carry out the real-time fluorescence PCR amplification by above-mentioned condition respectively, to determine the relative detectability (the results are shown in Figure 4) of red deer Auele Specific Primer and probe combinations.Experimental result illustrates that the relative detection of this method detection red deer composition is limited to 10pg.
The present inventor verifies commercially available pilose antler sample by following test.
Choose 36 kinds of pilose antler samples (sample is provided by this laboratory), wherein 20 kinds of cervus elaphus linnaeus samples, 16 parts of sika deer velvet antler samples carry out the real-time fluorescence PCR reaction, whether have feasibility to determine the real time fluorescent PCR method of being set up.
As shown in Figure 5, when utilizing real-time fluorescence PCR detection line mitochondrial DNA D-ring region sequence, red deer positive reference substance, 20 parts of commercially available cervus elaphus linnaeus samples all have typical amplified fluorescence curve more than baseline, and other 16 parts of sika deer velvet antler samples and blank amplification curve show that all at baseline position this method can effectively detect the cervus elaphus linnaeus composition.
Though specific embodiments of the present invention is described, those skilled in the art will appreciate that under the prerequisite that does not depart from scope of the present invention or spirit and can carry out multiple change and modification to the present invention.Thereby, this invention is intended to contain all these changes and modification of dropping in claims and the coordinator scope thereof.
Claims (10)
1. nucleic acid, its base sequence is shown in SEQ ID No.7.
2. be used for real time fluorescent PCR method and differentiate that the specificity nucleotide primer of the cervus elaphus linnaeus true and false is to reaching probe, described primer is to designing based on the described nucleic acid of claim 1 with probe, wherein said primer is to being made up of upstream primer and downstream primer, the base sequence of described upstream primer is SEQ ID No.1, the base sequence of described downstream primer is SEQ ID No.2, the base sequence of described probe is SEQ ID No.3,3 ' end at probe is connected with a fluorescent quenching group B HQ1, and 5 ' end is connected with a fluorescence report group FAM.
3. be used for real time fluorescent PCR method and differentiate the test kit of the cervus elaphus linnaeus true and false, described test kit comprises that specific oligonucleotide primer according to claim 2 is to reaching probe.
4. test kit according to claim 3, described test kit further comprise other universal primer to and probe, described other universal primer is to being that the upstream primer of SEQ ID No.4 and downstream primer that base sequence is SEQ ID No.5 are formed by base sequence, the base sequence of described probe is SEQ ID No.6,3 ' end at probe is connected with a fluorescent quenching group B HQ2, and 5 ' end is connected with a fluorescence report group TAMRA.
5. the real-time fluorescence PCR detection method of cervus elaphus linnaeus real and fake discrimination, wherein said real time fluorescent PCR method is a Taqman fluorescent probe method, and described method comprises uses the described specific oligonucleotide primer of claim 2 to reaching probe or test kit according to claim 3.
6. real-time fluorescence PCR detection method according to claim 5 also comprises the step of extracting the pilose antler sample total DNA.
7. real-time fluorescence PCR detection method according to claim 6, by using universal primer to reaching the extraction quality of probe in detecting sample DNA, described universal primer is to being that the upstream primer of SEQ ID No.4 and downstream primer that base sequence is SEQ ID No.5 are formed by base sequence, the base sequence of described probe is SEQ ID No.6,3 ' end at probe is connected with a fluorescent quenching group B HQ2, and 5 ' end is connected with a fluorescence report group TAMRA.
8. the application of the described nucleic acid of claim 1 in differentiating the cervus elaphus linnaeus true and false.
9. the described specific oligonucleotide primer of claim 2 is to reaching the application of probe in differentiating the cervus elaphus linnaeus true and false.
10. claim 3 or the 4 described test kits application in differentiating the cervus elaphus linnaeus true and false.
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| CN104946730A (en) * | 2014-03-28 | 2015-09-30 | 中国中医科学院中药研究所 | PCR (Polymerase Chain Reaction) specific primers and detection method for authenticating antlers |
| CN106701907A (en) * | 2015-11-18 | 2017-05-24 | 中国检验检疫科学研究院 | Primer, probe, method and kit for detecting cassava-derived ingredients |
| CN107805664A (en) * | 2016-09-07 | 2018-03-16 | 中国检验检疫科学研究院 | GeXP multiplex PCRs differentiate composition, kit and the method for deer kind |
| CN110317881A (en) * | 2019-07-19 | 2019-10-11 | 上海市食品药品检验所 | It is a kind of for identifying the fluorescence PCR detecting method of deer horn |
| CN110331215A (en) * | 2019-07-19 | 2019-10-15 | 上海市食品药品检验所 | A kind of primer combination of probe object and its application for identifying deer horn |
| CN111041108A (en) * | 2020-01-10 | 2020-04-21 | 辽宁省检验检测认证中心 | Primer, kit and detection method for identifying traditional Chinese medicine pilose antler |
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| CN1932041A (en) * | 2005-07-06 | 2007-03-21 | 韩国韩医学研究院 | The primers specific to cervus elaphus, c. nippon, c. canadensis and rangifer tarandus gene and the method to identify cervi parvum cornu species |
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| CN1932041A (en) * | 2005-07-06 | 2007-03-21 | 韩国韩医学研究院 | The primers specific to cervus elaphus, c. nippon, c. canadensis and rangifer tarandus gene and the method to identify cervi parvum cornu species |
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| CN101974522B (en) | 2012-08-08 |
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