CN103074433A - Turtle shell DNA detection kit and identification method - Google Patents
Turtle shell DNA detection kit and identification method Download PDFInfo
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- CN103074433A CN103074433A CN2013100279180A CN201310027918A CN103074433A CN 103074433 A CN103074433 A CN 103074433A CN 2013100279180 A CN2013100279180 A CN 2013100279180A CN 201310027918 A CN201310027918 A CN 201310027918A CN 103074433 A CN103074433 A CN 103074433A
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Abstract
The invention relates to the technical field of traditional Chinese medicine identification, in particular to a turtle shell DNA (deoxyribonucleic acid) detection kit and an identification method. The turtle shell DNA detection kit comprises a sample pretreatment liquid, a decalcification liquid, a mitochondrial DNA extraction system, a PCR (polymerase chain reaction) system and a result observation system. The turtle shell DNA identification method comprises the steps of pretreating a detection sample, extracting mitochondrial DNA, conducting PCR primer design and synthesis, establishing PCR, and judging a result. A judgment standard is that a turtle shell is authentic if 1000bp and 500bp bands appear simultaneously, and the turtle shell is fake if only one or no band appears. The established multi-PCR identification method has the advantages of simplicity, convenience, quickness, reliable detection result and the like, and can differentiate and identify specificities of the turtle shell and the fake turtle shell accurately and simultaneously.
Description
Technical field
The present invention relates to the Materia Medica Identification technology, specifically a kind of turtle shell DNA detection test kit and authentication method.
Background technology
Turtle shell is the carapace of the animal soft-shelled turtle (Trionyx sinensis Wiegamann) of Trionychidae, has another name called: Bie Jia, group's crust, Shang Jia, cuckold lid, soft-shelled turtle shell, soft-shelled turtle lid, water fish shell etc.Its former animal is Trionyx sinensis (Wiegmann), is commonly called as the soft-shelled turtle, soft-shelled turtle, cuckold etc., belongs to reptilia (Repitlia); Chelonia (Testudinata); Trionychidae (Tironychidae); Soft-shelled turtle belongs to (Peodiscus).In China other areas except Tibet, Qinghai and Xinjiang distribution is arranged all, the most common with the Yangtze valley and South China.China's Trionyx sinensis (Wiegmann) cultured output occupies first of the world, but since the nineties in 20th century, smuggle in a large number soft-shelled turtle overseas and pour in many China, add that each plant introduces a fine variety, falls to plant extremely lack of standardization each other, do not pay attention to cultivating kind, serious harm the germ plasm resource of Trionyx sinensis (Wiegmann), germplasm is the source of industry, have influence on the resource of turtle shell, have influence on the medication of turtle shell.
The turtle shell nature and flavor are salty, be slightly cold, and return liver, kidney channel.Begin to be stated from Shennong's Herbal, classify middle product as.Contain Multiple components in the turtle shell, mainly contain gelatin, Keratin sulfate, iodine matter, vitamins D, calcium phosphate, turtle shell polysaccharide, multiple amino acids and various trace elements etc.Abundant composition makes the pharmaceutical use of turtle shell very high.Have nourishing and suppressing Yangly, bring down a fever except steaming, the functions such as softening and resolving hard mass are used for fever due to yin deficiency, hectic fever due to yin labor heat, and hyperactivity of YANG due to deficiency of YIN is had a dizzy spell, stirring-up of pathogenic wind in the interior resulting from deficiency, brothers Chi Zong, through Bi ， Disorder lump in the abdomen, the chronic malaria malaria is female.Be one of conventional Chinese medicine, extensive in clinical application, except clinical prescription uses, still in several Chinese patent medicines such as FUFANG BIEJIA RUANGAN PIAN, ginseng biejiajian pills, Shengui antler ball, use." discrimination method in the Chinese pharmacopoeia (2010, an one) is: character identification and extract are differentiated.Microscopical identification, physics and chemistry discriminating, spectrum discriminating, chromatographic identification, biochemical discriminating etc. are still arranged in the document, but these methods can not be differentiated fast and effectively to turtle shell still.
Molecular genetic marker technique begins to permeate with the field of Chinese medicines, and rapid fusion, development, wherein to have specificity aspect the evaluation of Chinese medicinal materials (except mineral drug) strong for dna fingerprinting, accuracy is high, the advantages such as good reproducibility, the imperfection on macroscopical identification level the identification and assessment of Chinese medicines before molecular level has solved.Modern molecular biology progressively enlarges in the field of quality control of Chinese medicine, and method is perfect gradually, has greatly accelerated the process of the modernization of Chinese medicine.
Plastosome is a kind of organoid that is present in the eukaryotic cell, Mitochondrial DNA (mitochondrial DNA, mtDNA) be the genetic material of the double-stranded circular of a covalency closure, have molecular weight little, simple in structure, be easy to separate, copy number is high, the characteristics such as mutation rate is high, rate of evolution is fast, matrocliny, inorganization specificity, high conservative property.Cytochrome b (cytochrome b wherein, cyt b) gene and cytochrome C oxidase subunit base I (cytochrome c oxidase subunit I, CO I) gene is present in all animals, rate of evolution is moderate, a short fragment just can comprise from planting interior to information planting, and its dna sequence dna seldom exists insertion and disappearance, can guarantee enough to hand over different, in the analysator and sibling species between the suitable dna fragmentation of genetic diversity and evolutionary relationship, can be applicable to the research of biological classification, phylogeny and genetic diversity.The gene of these two fragments can be used as in the animal species identifying mark and study, and therefore, the identification and assessment of Chinese medicines take the Mitochondrial DNA characterization of molecules as genetic marker is more accurate, reliable.
We rely on Science and Technology Department of Jilin Province (in January, 2008 project verification) and Jilin Province education department to subsidize problem (in January, 2010 project verification): " research and development of Chinese medicine dna fingerprint detection kit " (in May, 2012, achievement was identified in the Science and Technology Department of Jilin Province), set up the dna fingerprint feature of Animal Medicine material based on Mitochondrial DNA, develop goes out the ermine heart, deer whip and three kinds of Chinese medicinal materials DNA of pilose antler identification kit.
The ermine heart belongs to thin precious medicinal material, is the main ingredient in the sharp heart ball of office of national Bureau of Drugs Supervision standard (former Ministry of Health ministerial standard).At present, there is no the legal discrimination method of the ermine heart in the national standard, to the composition Study of the ermine heart and differentiate in the domestic and foreign literature and also have no report, the parts of generic medicinal plants discrimination method can't be distinguished the heart of the ermine heart and easily mixed animal, so brought very large difficulty for the quality control of the ermine heart and preparation thereof.Seminar is under the full gene group prerequisite of finding ermine heart Mitochondrial DNA, utilize the advantage of ermine heart mtDNA uniqueness, use the genome partial sequence of DNA cloning technology and sequencing technologies research ermine heart Mitochondrial DNA, the oligonucleotide fragment of design can special evaluation heart, the duck heart, the goose heart and the rabbit heart, a kind of easy, quick, special evaluation ermine heart patented technology (mink heart DNA detection kit and authentication method, patent of invention: ZL200810051643.3 have successfully been invented; 2011.4 authorize).Relevant achievement is published in Jilin University's journal " ermine is organized Mitochondrial DNA and cytochrome b identification and characterization " [2008,34 (5): 790-793], Chinese Pharmaceutical Journal " mink myocardial mitochondria mtDNA identifies and the RFLP feature " [2012,4 (3): 182-185].
On this basis, seminar's applied molecular biology is studied the authenticate technology of turtle shell with the theory that merges mutually of identification and assessment of Chinese medicines, mainly uses the high conservative property of cyt b and the specificity of Mitochondrial DNA turtle shell is identified.Adopt the salting-out process method from turtle shell, to extract Mitochondrial DNA, utilize the relevant information among the Genbank, according to the difference of Trionyx sinensis (Wiegmann) with the mtdna sequence of other relevant animals, choose cyt b gene order, use a pair of Auele Specific Primer of primer premier5.0 primer-design software design turtle shell, carry out PCR (polymerase chain reaction) amplification, this method can be identified turtle shell from molecular level provides foundation.Relevant achievement is published in Chinese Pharmaceutical Journal " research of Chinese medicinal materials tortoise plastron cytochrome b specificity identification " [2012,4 (3): 182-185].
The people such as domestic cuckoo pass through cytochrome C oxidase subunit base I (cytochrome c oxidase subunit I, CO I) gene order comparative study is found, variation is very little in the Trionyx sinensis (Wiegmann) CO I gene order kind, there is more variant sites between kind, can be used as the DNA bar codes technique and differentiate turtle shell [cuckoo, Cui Lina, Zhang Hui, Deng: the dna molecular of turtle shell and adulterant thereof is identified. World Science technology-Chinese materia medica modernization, 2011,13 (2): 429-434.].Experimental results show that Trionyx sinensis (Wiegmann) CO I gene can be used as the discriminating target position.But the DNA bar codes technique requires to select at least two gene locuss, selects separately a site can not avoid the variation that suddenlys change and cause.The people such as Liu Zhongquan utilize the locus specificity round pcr, cooperation sequencing technologies discriminating turtle shell and equal animal thereof [Liu Chongquan, etc. the locus specificity PCR identification research of Chinese medicinal materials turtle shell. Chinese medicinal materials, 2001,32 (8): 736-739.; Wu Ping, etc. with PCR products bidirectional sequencing technical evaluation Chinese medicinal materials turtle shell. China Medicine University's journal, 1998,29 (1): 28-30.].Experimental results show that some gene locuss of Trionyx sinensis (Wiegmann) can differentiate by specific PCR and sequencing technologies.
On this basis, patent of the present invention is differentiated based on selecting simultaneously two specific sites to be used for Trionyx sinensis (Wiegmann).From Trionyx sinensis (Wiegmann) CO I gene and cyt b gene, adopt two genes of high-throughput techniques analysis to have the sequence fragment of discriminating as differential point.Set up Multiplex PCR (claiming again composite PCR) reaction system.Multiplex PCR has the high efficiency characteristics, namely detects simultaneously multiple goal gene in same PCR reaction tubes, or the goal gene that a plurality of types are arranged is carried out somatotype, particularly just can detect multiple project with a sample.Because the turtle shell type is more, has more variant sites between kind, multiplex PCR can improve its recall rate and identify simultaneously its type and sudden change etc.In addition, multiplex PCR has the economical and convenient characteristic, and multiple goal gene detects in same reaction tubes simultaneously, will save time greatly, saves reagent, the reduction of expenditure spending.Materia Medica Identification had the advantages such as the sample of saving, the characteristics that have saving time, reduce cost and raise the efficiency.
Summary of the invention
Patent of the present invention adopts bioinformatics technique that Trionyx sinensis (Wiegmann) turtle shell Mitochondrial DNA genome is screened, utilize the relevant information among the Genbank, according to the difference of Trionyx sinensis (Wiegmann) with the mtdna sequence of other relevant animals, choose COX I and cyt b gene order, use primer premier5.0 design software, design can be distinguished the specific DNA sequences of turtle shell and adulterant effectively, utilize multiple PCR technique in a reaction system, to add two pairs of Auele Specific Primers, for a template two the discrepant purpose fragments of tool that can increase.Provide a kind of and can accurately differentiate turtle shell and adulterant specific molecular sign, turtle shell easy to use and adulterant multiple PCR detection kit, the characteristics that have saving time, reduce cost and raise the efficiency, and provide that it is easy, quick, the reliable authentication method of detected result.
The objective of the invention is to be realized by following technical scheme:
A kind of discriminating turtle shell and adulterant multiple PCR detection kit is characterized in that it comprises:
1. sample pretreatment solution and decalcifying Fluid
Every sample is got 2g, scrubs clean to rinse well with pretreatment liquid (mainly containing deionized water) afterwards, and drying at room temperature is more than ultra violet lamp 30min.Use mortar that sample is ground to 2～3mm, place centrifuge tube, every part by mass volume ratio adding in 1: 20 respective volume decalcifying Fluid (mainly containing 0.5mol/L pH8.0EDTA), 56C water-bath 48h～60h, rotating speed 100r/min.Every 12h changes once fresh decalcifying Fluid.The centrifugal 5min of 8000r/min abandons supernatant after the decalcification, precipitates stand-by.
2. Mitochondrial DNA extracts system
(1) lysate adds 5ml lysate [10mmol/L Tris-HCl (pH8.0) in the process sample of pre-treatment, 10m mol/L EDTA (pH8.0), 100m mol/L NaCl, 2%SDS, 40 μ g/ml Proteinase Ks, 0.039mol/L DTT], place water-bath, 56 ℃ of water-baths are spent the night, rotating speed 100r/min.
(2) precipitated liquid directly adds 2ml precipitated liquid (mainly containing NaAC) in the sample after the cracking, abundant mixing, and 6,000r/min4 ℃ of centrifugal 10min draws supernatant, abandons precipitation.The Virahol that adds isopyknic precooling in the supernatant, behind the mixing, low temperature is placed 20～30min, and 12,000r/min4 ℃ of centrifugal 20min abandons supernatant, stays precipitation.
(3) washings uses washings (mainly containing 70% ethanol) washing precipitation two to three times, and is drying precipitated, is dissolved in the sterilization distilled water, and-80 ℃ save backup.
3.PCR reaction system
(a) the identification reaction system totally is 100 μ L, wherein contains damping fluid 10 μ L, 12.5mM dNTP4～10 μ L, 0.1mM each 1.5～4.0 μ L of turtle shell COX I primer and cyt b primer, Taq archaeal dna polymerase 2～10 μ L, testing sample DNA2～5 μ L, remaining is bi-distilled water;
(b) the positive control reaction system totally is 100 μ L, wherein contains damping fluid 10 μ L, 12.5mM dNTP4～10 μ L, 0.1mM each 1.5～4.0 μ L of turtle shell COX I primer and cyt b primer, Taq archaeal dna polymerase 2～10 μ L, Trionyx sinensis (Wiegmann) turtle shell DNA2～5 μ L, remaining is bi-distilled water;
(c) the negative control reaction system totally is 100 μ L, contains damping fluid 10 μ L, 12.5mM dNTP4～10 μ L, 0.1mM each 1.5～4.0 μ L of turtle shell COX I primer and cyt b primer, Taq archaeal dna polymerase 2～10 μ L, adulterant DNA2～5 μ L, remaining is bi-distilled water;
(d) the design response procedures will be differentiated respectively (a) identification reaction system, (b) positive control reaction system of turtle shell and adulterant PCR detection kit and (c) in each 100 μ L adding reaction tubes of total system of negative control reaction system, (market is on sale to place PCR reaction instrument, disposable type) undertaken by follow procedure in: 94 ℃ of denaturation 3min～7min, 94 ℃ of 30s, 56 ℃ of 1min～30s of annealing temperature, 72 ℃ of 1min～30s, 40 circulations, 72 ℃ are extended 5min～8min, 4 ℃.
4. observe system as a result
Prepare with 1.5 sepharoses (market is on sale, can buy), electrophoresis, DNA Marker (the standard molecular weight of molecular weight 100～1500bp, market is on sale) do reference, the observation and analysis result of gel imaging analysis system (market is on sale, can buy).
Turtle shell DNA authentication method is characterized in that it comprises following steps:
1. detection Sample pretreatment
Each sample is got 2g, scrubs clean to rinse well with pretreatment liquid afterwards, and drying at room temperature is more than ultra violet lamp 30min.Use mortar that sample is ground to 2～3mm, place centrifuge tube, press mass volume ratio adding in 1: 20 respective volume decalcifying Fluid, 56 ℃ of water-bath 48h～60h, rotating speed 100r/min for every part.Every 12h changes once fresh decalcifying Fluid.The centrifugal 5min of 8000r/min abandons supernatant after the decalcification, precipitates stand-by.
2. Mitochondrial DNA extracts
(1) the sample cracking places water-bath to through adding the 5ml lysate in the sample of pre-treatment, and 56 ℃ of water-baths are spent the night, rotating speed 100r/min.
(2) add the 2ml precipitated liquid in the direct sample after cracking of precipitation, abundant mixing, 6,000r/min4 ℃ of centrifugal 10min draws supernatant, abandons precipitation.The Virahol that adds isopyknic precooling in the supernatant, behind the mixing, low temperature is placed 20～30min, and 12,000r/min4 ℃ of centrifugal 20min abandons supernatant, stays precipitation.
(3) washings washing precipitation two to three times is used in washing, and is drying precipitated, is dissolved in the sterilization distilled water, and-80 ℃ of preservations are as detecting dna profiling.
3.PCR design of primers is with synthetic
(1) two pairs of special Oligonucleolide primers of design turtle shell Mitochondrial DNA, contain the turtle shell dna sequence dna, following Primer1, Primer2:
Primer1(COX I)
5′ATACATCCGATACAACAGC 3′
5′GATAAATGGGAGATAAGTG 3′
Primer2(cyt b)
5′ATCATCCGATCAATAACAG 3′
5′AGGGAGAATAATAAAGTGAAA 3′
(2) two pairs of special Oligonucleolide primers of synthetic turtle shell Mitochondrial DNA adopt ABI3900 high-throughput synthesizer.(entrusting Shanghai to give birth to the worker finishes)
4. set up the PCR reaction
(a) identification reaction totally is 100 μ L, wherein contains damping fluid 10 μ L, 12.5mM dNTP4～10 μ L, and each 1.5～4.0 μ L of 0.1mM primer 1 and primer 2, Taq archaeal dna polymerase 2～10 μ L, testing sample DNA2～5 μ L, remaining is bi-distilled water.
(b) the positive control reaction totally is 100 μ L, wherein contains damping fluid 10 μ L, 12.5mM dNTP4～10 μ L, 0.1mM each 1.5～4.0 μ L of turtle shell COX I primer and cyt b primer, Taq archaeal dna polymerase 2～10 μ L, Trionyx sinensis (Wiegmann) turtle shell DNA2～5 μ L, remaining is bi-distilled water.
(c) the negative control reaction totally is 100 μ L, contains damping fluid 10 μ L, 12.5mM dNTP4～10 μ L, 0.1mM each 1.5～4.0 μ L of turtle shell COX I primer and cyt b primer, Taq archaeal dna polymerase 2～10 μ L, adulterant DNA2～5 μ L, remaining is bi-distilled water.
(d) the design response procedures will be differentiated respectively (a) identification reaction system, (b) positive control reaction system of turtle shell and adulterant PCR detection kit and (c) in each 100 μ L adding reaction tubes of total system of negative control reaction system, (market is on sale to place PCR reaction instrument, disposable type) undertaken by follow procedure in: 94 ℃ of denaturation 3min～7min, 94 ℃ of 30s, 56 ℃ of 1min～30s of annealing temperature, 72 ℃ of 1min～30s, 40 circulations, 72 ℃ are extended 5min～8min, 4 ℃.
5. the result judges
Product 1.5-1.8% sepharose (market is on sale) electrophoresis, DNA Marker (the standard molecular weight of molecular weight 100bp～1500bp, market is on sale) do reference, the observation and analysis result of gel imaging analysis system (market is on sale, can buy).1000bp and 500bp band occurring simultaneously is the certified products turtle shell, only occurs one or adulterant turtle shell (Fig. 1) do not occur being.
Discriminating turtle shell of the present invention and adulterant identification kit are utilize to differentiate turtle shell and adulterant Mitochondrial DNA and the species specificity site that cytochrome b and c have, and can accurately distinguish simultaneously the specificity of discriminating turtle shell and adulterant; The multiple PCR identification method of setting up has the advantages such as easy, quick, that detected result is reliable.
Description of drawings
Fig. 1 is turtle shell test kit detected result synoptic diagram.
Wherein: 1. be certified products turtle shell result, amplify simultaneously 1000bp and 500bp; 2. positive contrast amplifies 1000bp and 500bp simultaneously; 3. be standard molecular weight; 4. 1 amplified production does not appear or only occurs in negative result.
Fig. 2 embodiment one turtle shell test kit detected result synoptic diagram.
Wherein: 1. be certified products turtle shell result, amplify simultaneously 1000bp and 500bp; 2. positive contrast amplifies 1000bp and 500bp simultaneously; 3. be standard molecular weight; 4. 1 amplified production does not appear or only occurs in negative result.5. negative contrast.Two pairs of commercially available turtle shell medicinal materials of Fig. 3 embodiment carry out the qualification result synoptic diagram.
Wherein: 1. positive contrast amplifies 1000bp and 500bp; 2. be certified products turtle shell result, amplify 1000bp and 500bp; 3: Burma's Testudo elongata first; 4: the Testudo impressa (Guenther) first; 5: yellow volume closed shell tortoise plastron; 6: the Mauremys mutica first; 7: the Cuora trifasciata first; 8. be standard molecular weight; 9. negative result
Embodiment
The invention will be further described below in conjunction with embodiment, below embodiment only be used for explanation the present invention and be not limitation of the present invention.
Embodiment one
1. detection Sample pretreatment
Test sample (two kinds, 1 is the turtle shell certified products, 2 are the flower tortoise, the turtle shell adulterant provides and identifies by Nat'l Pharmaceutical ﹠ Biological Products Control Institute) is got 2g for every part, scrubs clean to rinse well with pretreatment liquid afterwards, and drying at room temperature is more than ultra violet lamp 30min.Use mortar that sample is ground to 2～3mm, place centrifuge tube, press mass volume ratio adding in 1: 20 respective volume decalcifying Fluid, 56 ℃ of water-bath 48hh, rotating speed 100r/min for every part.Every 12h changes once fresh decalcifying Fluid.The centrifugal 5min of 8000r/min abandons supernatant after the decalcification, precipitates stand-by.
2. Mitochondrial DNA extracts
(1) the sample cracking places water-bath to through adding the 5ml lysate in the sample of pre-treatment, and 56 ℃ of water-baths are spent the night, rotating speed 100r/min.
(2) add the 2ml precipitated liquid in the direct sample after cracking of precipitation, abundant mixing, 6,000r/min4 ℃ of centrifugal 10min draws supernatant, abandons precipitation.The Virahol that adds isopyknic precooling in the supernatant, behind the mixing, low temperature is placed 30min, and 12,000r/min4 ℃ of centrifugal 20min abandons supernatant, stays precipitation.
(3) washings washing precipitation two to three times is used in washing, and is drying precipitated, is dissolved in the sterilization distilled water, and-80 ℃ of preservations are as detecting dna profiling.
3.PCR design of primers is with synthetic
(1) two pairs of special Oligonucleolide primers of design turtle shell Mitochondrial DNA, contain the turtle shell dna sequence dna, following Primer1, Primer2:
Primer1(COX I)
5′ATACATCCGATACAACAGC 3′
5′GATAAATGGGAGATAAGTG 3′
Primer2(cyt b)
5′ATCATCCGATCAATAACAG 3′
5′AGGGAGAATAATAAAGTGAAA 3′
(2) two pairs of special Oligonucleolide primers of synthetic turtle shell Mitochondrial DNA adopt ABI3900 high-throughput synthesizer.(entrusting Shanghai to give birth to the worker finishes)
4. set up the PCR reaction
(a) identification reaction totally is 100 μ L, wherein contains damping fluid 10 μ L, 12.5mM dNTP4 μ L, and each 2.0 μ L of 0.1mM primer 1 and primer 2, Taq archaeal dna polymerase 2 μ L, testing sample DNA2 μ L, remaining is bi-distilled water.
(b) the positive control reaction totally is 100 μ L, wherein contains damping fluid 10 μ L, 12.5mM dNTP4 μ L, and each 2.0 μ L of 0.1mM turtle shell COX I primer and cyt b primer, Taq archaeal dna polymerase 2 μ L, Trionyx sinensis (Wiegmann) turtle shell DNA2 μ L, remaining is bi-distilled water.
(c) the negative control reaction totally is 100 μ L, contains damping fluid 10 μ L, 12.5mM dNTP4 μ L, and each 2.0 μ L of 0.1mM turtle shell COX I primer and cyt b primer, Taq archaeal dna polymerase 2 μ L, adulterant DNA2 μ L, remaining is bi-distilled water.
(d) the design response procedures will be differentiated respectively (a) identification reaction system, (b) positive control reaction system of turtle shell and adulterant PCR detection kit and (c) in each 100 μ L adding reaction tubes of total system of negative control reaction system, (market is on sale to place PCR reaction instrument, disposable type) undertaken by follow procedure in: 94 ℃ of denaturation 5min, 94 ℃ of 30s, 56 ℃ of 30s of annealing temperature, 72 ℃ of 1min, 40 circulations, 72 ℃ are extended 5min, 4 ℃.
5. the result judges
PCR product 1.5 agarose gel electrophoresis, the DNA Marker of standard molecular weight 100bp～1500bp does reference, gel imaging analysis systematic observation and analytical results (Fig. 2).
2 pairs of commercially available turtle shell medicinal materials of embodiment are identified
1. material
5 kinds in commercially available turtle shell sample, a kind of turtle shell certified products (provide and identify by the drug inspection office, Jilin)
2. method
Get 2g 2.1 detect every part of test sample of Sample pretreatment, scrub clean and rinse well with pretreatment liquid afterwards, drying at room temperature is more than ultra violet lamp 30min.Use mortar that sample is ground to 2～3mm, place centrifuge tube, press mass volume ratio adding in 1: 20 respective volume decalcifying Fluid, 56 ℃ of water-bath 48hh, rotating speed 100r/min for every part.Every 12h changes once fresh decalcifying Fluid.The centrifugal 5min of 8000r/min abandons supernatant after the decalcification, precipitates stand-by.
2.2 Mitochondrial DNA extracts
(1) the sample cracking places water-bath to through adding the 5ml lysate in the sample of pre-treatment, and 56 ℃ of water-baths are spent the night, rotating speed 100r/min.
(2) add the 2ml precipitated liquid in the direct sample after cracking of precipitation, abundant mixing, 6,000r/min4 ℃ of centrifugal 10min draws supernatant, abandons precipitation.The Virahol that adds isopyknic precooling in the supernatant, behind the mixing, low temperature is placed 30min, and 12,000r/min4 ℃ of centrifugal 20min abandons supernatant, stays precipitation.
(3) washings washing precipitation two to three times is used in washing, and is drying precipitated, is dissolved in the sterilization distilled water, and-80 ℃ of preservations are as detecting dna profiling.
2.3PCR design of primers is with synthetic
(1) two pairs of special Oligonucleolide primers of design turtle shell Mitochondrial DNA, contain the turtle shell dna sequence dna, following Primer1, Primer2:
Primer1(COX I)
5′ATACATCCGATACAACAGC 3′
5′GATAAATGGGAGATAAGTG 3′
Primer2(cyt b)
5′ATCATCCGATCAATAACAG 3′
5′AGGGAGAATAATAAAGTGAAA 3′
(2) two pairs of special Oligonucleolide primers of synthetic turtle shell Mitochondrial DNA adopt ABI3900 high-throughput synthesizer (entrusting Shanghai to give birth to the worker finishes).
2.4 set up the PCR reaction
(a) identification reaction: totally be 100 μ L, wherein contain damping fluid 10 μ L, 12.5mM dNTP4 μ L, each 2.0 μ L of 0.1mM primer 1 and primer 2, Taq archaeal dna polymerase 2 μ L, testing sample DNA2 μ L, remaining is bi-distilled water.
(b) positive control reaction: totally be 100 μ L, wherein contain damping fluid 10 μ L, 12.5mM dNTP4 μ L, each 2.0 μ L of 0.1mM turtle shell COX I primer and cyt b primer, Taq archaeal dna polymerase 2 μ L, Trionyx sinensis (Wiegmann) turtle shell DNA2 μ L, remaining is bi-distilled water.
(c) negative control reaction: totally be 100 μ L, contain damping fluid 10 μ L, 12.5mM dNTP4 μ L, each 2.0 μ L of 0.1mM turtle shell COX I primer and cyt b primer, Taq archaeal dna polymerase 2 μ L, adulterant DNA2 μ L, remaining is bi-distilled water.
(d) the design response procedures will be differentiated respectively (a) identification reaction system, (b) positive control reaction system of turtle shell and adulterant PCR detection kit and (c) in each 100 μ L adding reaction tubes of total system of negative control reaction system, (market is on sale to place PCR reaction instrument, disposable type) undertaken by follow procedure in: 94 ℃ of denaturation 5min, 94 ℃ of 30s, 56 ℃ of 30s of annealing temperature, 72 ℃ of 1min, 40 circulations, 72 ℃ are extended 5min, 4 ℃.
3. the result judges
PCR product 1.5 agarose gel electrophoresis, the DNA Marker of standard molecular weight 100bp～1500bp does reference, gel imaging analysis systematic observation and analytical results (Fig. 3).
Claims (5)
1. a turtle shell DNA detection test kit is characterized in that, it comprises sample pretreatment solution and decalcifying Fluid, Mitochondrial DNA and extracts system, PCR reaction system, observe system as a result.
(1) sample pretreatment solution and decalcifying Fluid
(a) pretreatment liquid: deionized water.
(b) decalcifying Fluid: 0.5mol/L pH8.0EDTA.
(2) Mitochondrial DNA extracts system
(a) lysate: 10m mol/L Tris-HCl (pH8.0), 10m mol/L EDTA (pH8.0), 100m mol/L NaCl, 2%SDS, 40 μ g/ml Proteinase Ks, 0.039mol/L DTT.
(b) precipitated liquid: saturated NaAC.
(c) washings: 70% ethanol.
(3) PCR reaction system
(a) identification reaction system: totally be 100 μ L, wherein contain damping fluid 10 μ L, 12.5mM dNTP4～10 μ L, 0.1mM each 1.5～4.0 μ L of turtle shell COX I primer and cyt b primer, Taq archaeal dna polymerase 2～10 μ L, testing sample DNA2～5 μ L, remaining is bi-distilled water;
(b) positive control reaction system: totally be 100 μ L, wherein contain damping fluid 10 μ L, 12.5mM dNTP4～10 μ L, 0.1mM each 1.5～4.0 μ L of turtle shell COX I primer and cyt b primer, Taq archaeal dna polymerase 2～10 μ L, Trionyx sinensis (Wiegmann) turtle shell DNA2～5 μ L, remaining is bi-distilled water;
(c) negative control reaction system: totally be 100 μ L, contain damping fluid 10 μ L, 12.5mM dNTP4～10 μ L, 0.1mM each 1.5～4.0 μ L of turtle shell COX I primer and cyt b primer, Taq archaeal dna polymerase 2～10 μ L, adulterant DNA2～5 μ L, remaining is bi-distilled water;
(4) observe system as a result
(a) sepharose: 1.5% agarose.
(b) the DNA Marker of standard molecular weight: 100bp～1500bp (can select the standard molecular weight of other kind, what require maximum can not surpass 2000bp, contain 1000bp and 500bp).
2. turtle shell DNA authentication method is characterized in that, its inclusion test Sample pretreatment, Mitochondrial DNA extract, the PCR design of primers with synthesize, set up the PCR reaction, the result judges five steps.
(1) detects Sample pretreatment
Each sample is got 2g, scrubs clean to rinse well with pretreatment liquid afterwards, and drying at room temperature is more than ultra violet lamp 30min.Use mortar that sample is ground to 2～3mm, place centrifuge tube, press mass volume ratio adding in 1: 20 respective volume decalcifying Fluid, 56 ℃ of water-bath 48h～60h, rotating speed 100r/min for every part.Every 12h changes once fresh decalcifying Fluid.The centrifugal 5min of 8000r/min abandons supernatant after the decalcification, precipitates stand-by.
(2) Mitochondrial DNA extracts
(a) the sample cracking places water-bath to through adding the 5ml lysate in the sample of pre-treatment, and 56 ℃ of water-baths are spent the night, rotating speed 100r/min.
(b) add the 2ml precipitated liquid in the direct sample after cracking of precipitation, abundant mixing, 6,000r/min4 ℃ of centrifugal 10min draws supernatant, abandons precipitation.The Virahol that adds isopyknic precooling in the supernatant, behind the mixing, low temperature is placed 20～30min, and 12,000r/min4 ℃ of centrifugal 20min abandons supernatant, stays precipitation.
(c) washings washing precipitation two to three times is used in washing, and is drying precipitated, is dissolved in the sterilization distilled water, and-80 ℃ of preservations are as detecting dna profiling.
(3) the PCR design of primers is with synthetic
(a) two pairs of special Oligonucleolide primers of design turtle shell Mitochondrial DNA, contain the turtle shell dna sequence dna, following Primer1, Primer2:
Primerl(COX I)
5′ATACATCCGATACAACAGC 3′
5′GATAAATGGGAGATAAGTG 3′
Primer2(cyt b)
5′ATCATCCGATCAATAACAG 3′
5′AGGGAGAATAATAAAGTGAAA 3′
(b) two pairs of special Oligonucleolide primers of synthetic turtle shell Mitochondrial DNA adopt ABI3900 high-throughput synthesizer (entrusting Shanghai to give birth to the worker finishes).
(4) set up the PCR reaction
(a) identification reaction totally is 100 μ L, wherein contains damping fluid 10 μ L, 12.5mM dNTP4～10 μ L, and each 1.5～4.0 μ L of 0.1mM primer 1 and primer 2, Taq archaeal dna polymerase 2～10 μ L, testing sample DNA2～5 μ L, remaining is bi-distilled water.
(b) the positive control reaction totally is 100 μ L, wherein contains damping fluid 10 μ L, 12.5mM dNTP4～10 μ L, 0.1mM each 1.5～4.0 μ L of turtle shell COX I primer and cyt b primer, Taq archaeal dna polymerase 2～10 μ L, Trionyx sinensis (Wiegmann) turtle shell DNA2～5 μ L, remaining is bi-distilled water.
(c) the negative control reaction totally is 100 μ L, contains damping fluid 10 μ L, 12.5mM dNTP4～10 μ L, 0.1mM each 1.5～4.0 μ L of turtle shell COX I primer and cyt b primer, Taq archaeal dna polymerase 2～10 μ L, adulterant DNA2～5 μ L, remaining is bi-distilled water.
(d) the design response procedures will be differentiated respectively (a) identification reaction system, (b) positive control reaction system of turtle shell and adulterant PCR detection kit and (c) in each 100 μ L adding reaction tubes of total system of negative control reaction system, (market is on sale to place PCR reaction instrument, disposable type) undertaken by follow procedure in: 94 ℃ of denaturation 5min, 94 ℃ of 30s, 56 ℃ of 30s of annealing temperature, 72 ℃ of 1min, 40 circulations, 72 ℃ are extended 5min, 4 ℃.
(5) result judges
Product 1.5 agarose gel electrophoresis, the DNA Marker of molecular weight 100bp～1500bp (standard molecular weight, market is on sale) does reference, the observation and analysis result of gel imaging analysis system (market is on sale, can buy).1000bp and 500bp band occurring simultaneously is the certified products turtle shell, only occurs one or the adulterant turtle shell do not occur being.
3. such as claim 1 and a kind of turtle shell DNA detection test kit claimed in claim 2 and authentication method, it is characterized in that described turtle shell DNA detection test kit and authentication method are the Trionyx sinensis (Wiegmann) turtle shell.
4. the authentication method of turtle shell DNA detection test kit as claimed in claim 2, it is characterized in that, the result identifies it is utilize comparative sample pcr amplified fragment DNA size and DNA standard molecular weight identical, and criterion is in full accord carries out authenticity to the turtle shell sample according to occurring.
5. a claim 1 and a kind of turtle shell DNA detection test kit claimed in claim 2 and authentication method can be applicable to the authenticity of turtle shell sample.
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