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CN101875975A - A PCR-RFLP method for differentiating mutton and duck - Google Patents

️Wed Nov 03 2010

CN101875975A - A PCR-RFLP method for differentiating mutton and duck - Google Patents

A PCR-RFLP method for differentiating mutton and duck Download PDF

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Publication number

CN101875975A

CN101875975A CN 201010226683 CN201010226683A CN101875975A CN 101875975 A CN101875975 A CN 101875975A CN 201010226683 CN201010226683 CN 201010226683 CN 201010226683 A CN201010226683 A CN 201010226683A CN 101875975 A CN101875975 A CN 101875975A Authority

China

Prior art keywords

pcr

mutton

duck

rflp

tube

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2010-07-15

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CN 201010226683

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Chinese (zh)

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冯海永

安添午

何建文

张�浩

赵会静

罗玉柱

韩建林

王继卿

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Institute of Animal Science of CAAS

Gansu Agricultural University

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Institute of Animal Science of CAAS

Gansu Agricultural University

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2010-07-15

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2010-07-15

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2010-11-03

2010-07-15 Application filed by Institute of Animal Science of CAAS, Gansu Agricultural University filed Critical Institute of Animal Science of CAAS

2010-07-15 Priority to CN 201010226683 priority Critical patent/CN101875975A/en

2010-11-03 Publication of CN101875975A publication Critical patent/CN101875975A/en

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Abstract

羊肉和鸭肉鉴别的PCR-RFLP方法属于分子生物学检测领域。它设计了一种羊肉制品肉种来源鉴别的方法，解决了目前羊肉和鸭肉鉴别方法准确性低的缺陷，提供了鉴别羊肉和鸭肉的PCR-RFLP检测技术。可将羊肉和鸭肉按以下方法加以鉴别：提取样品DNA，采用通用引物扩增和凝胶电泳检测，然后分别用Bsu36I和SpeI限制性内切酶进行PCR-RFLP分析，即可区分羊肉和鸭肉。本发明采用PCR-RFLP方法，具有简单、快捷、廉价和准确性高的特点。The PCR-RFLP method for identifying mutton and duck belongs to the field of molecular biology detection. It designs a method for identifying the source of mutton products, which solves the defect of low accuracy of the current identification methods for mutton and duck, and provides a PCR-RFLP detection technology for identifying mutton and duck. Mutton and duck can be identified by the following methods: extract sample DNA, use universal primers to amplify and detect by gel electrophoresis, and then perform PCR-RFLP analysis with Bsu36I and SpeI restriction endonucleases respectively to distinguish mutton and duck Meat. The invention adopts the PCR-RFLP method, which has the characteristics of simplicity, quickness, low cost and high accuracy.

Description

A kind of PCR-RFLP method of differentiating mutton and duck

Technical field

The invention belongs to the molecular Biological Detection field, relate to the discriminating of mutton and duck.

Technical background

Contain abundant quality protein and a large amount of mineral element etc. in the mutton, have very high nutritive value.Eating mutton severe winter can beneficial gas qi-restoratives, stimulates circulation, and strengthens the resisting cold ability.The traditional Chinese medical science thinks that mutton also has the effect of kidney invigorating and YANG supporting, so mutton is subjected to people's favor deeply.Yet in recent years, some illegal retailers sell the incident of cheating the human consumer with the duck string as mutton cubes roasted on a skewer and occur repeatedly, this behavior of selling with the alternative mutton of duck not only brings loss to the human consumer, causes detrimentally affect also for the sound development of mutton industry.In order to ensure consumers in general's rights and interests,, need differentiate accurately mutton and duck for the law enfrocement official provides technical support.

When fresh meat was carried out the sense organ discriminating, according to the characteristics difference of some species meat at aspects such as color, smell, flavour, fiber thickness, by " see, hear, touch " means, comprehensive three judgements that main points draw were carried out sense organ to meat and are differentiated.There are the following problems in actual applications for this: for the meat product through processing, owing to add materials such as pigment, foodstuff additive, spices, be difficult to accurately differentiate by the sense organ means.For example: the counterfeiter brushes sheep oil with the duck string, adds materials such as seasoning matter, is difficult to check by traditional sense organ method.

Immunology detection is based on the immune response of soluble antigen and antibody, uses the method that interspecies specific antigen carries out design studies.The ELISA method that GiovannacciI etc. detected animal species in 2005 has been carried out comprehensive review, thinks that sample can influence the detection accuracy of ELISA method through the heat treated of differing temps.Particularly after sample process pyroprocessing, sex change has taken place in albumen, has changed the specific antigen determinant, therefore is difficult to differentiate and distinguish animal species.The test kit sensitivity that is used for animal derived detection at present is lower, is unsuitable for promoting the use of.

The PCR-RFLP technology is quick, easy, existing relevant report aspect the kind discriminating of meat product, but do not see the report that mutton and duck discriminating aspect are arranged.

Summary of the invention

The objective of the invention is to solve present mutton and duck discrimination method defects of low accuracy, the PCR-RFLP method that provides a kind of mutton and duck to differentiate replaces the malpractice of mutton sale that technical support is provided for hitting duck.

Mutton and duck can be differentiated by the following method: one, will get the EP pipe (the EP pipe is answered autoclaving) that 50mg sample to be checked is put into 1.5mL, and it be shredded with little scissors.Two, the SDS solution 15 μ L that add

STE extract

600 μ L, ProK (20mg/mL) 20 μ L and 20%, abundant mixing on the earthquake device.Three, the EP pipe is inserted water-bath, 55 ℃ of digestion electrophorograms." the moon " negative control sample among three figure, M are mark in the molecular weight.

Embodiment

Present embodiment one: one, will get the EP pipe (the EP pipe is answered autoclaving) that 50mg sample to be checked is put into 1.5mL, it be shredded with little scissors.Two, the SDS solution 15 μ L that add

STE extract

600 μ L, ProK (20mg/mL) 20 μ L and 20%, abundant mixing on the earthquake device.Three, the EP pipe is inserted water-bath, 55 ℃ of digestion are spent the night (12-16h), can take out during this period and put upside down mixing several times, so that digestion thoroughly.Four, the EP pipe is taken out, every pipe adds the saturated phenol of Tris of 600 μ L, shakes mixing 15min gently, then at the centrifugal 10min of 12000rpm.Five, shift supernatant liquor to new EP pipe, add isopyknic phenol chloroform isoamyl alcohol mixed solution (phenol: chloroform: the primary isoamyl alcohol volume ratio is 25: 24: 1), put upside down mixing 10min, then the centrifugal 10min of 12000rpm.Six, shift supernatant liquor to new EP pipe, add isopyknic chloroform isoamyl alcohol mixed solution (chloroform: the primary isoamyl alcohol volume ratio is 24: 1), mixing 10min gently is then at the centrifugal 10min of 12000rpm.Seven, shift supernatant liquor to new EP pipe, add 1mL ice ethanol earlier, add the NaAc (3M) of 60 μ L again, level is shaken gently, and as seen cotton-shaped, white DNA occurs.The EP pipe is put into-20 ℃ of freezing 30min of refrigerator, and then at the centrifugal 10min of 12000rpm, visible DNA is sunken to the pipe end.Abandon dehydrated alcohol, use 75% washing with alcohol DNA twice again, then at the centrifugal 10min of 12000rpm; After the seasoning, add the dissolving of 50 μ L TE solution.Eight, pcr amplification and detection: primer general upstream primer the F:5 '-TAC CAT GAG GAC AAA TAT CAT TCT G-3 ' and general downstream primer the R:5 '-CCT CCT AGT TTG TTA GGG ATT GAT CG-3 ' of all vertebrates Cytb sequences that can increase.PCR reaction cumulative volume is 25 μ L, 10 * PCR Buffer, 2.5 μ L wherein, Taq archaeal dna polymerase 0.2 μ L (2.5U/ μ L), dNTPs (2.5mmol/L) 2 μ L, each 2 μ L of the upstream and downstream primer of 10pmol/ μ L, template DNA 250ng, sterile purified water is supplied cumulative volume.Amplification condition is: 95 ℃ of sex change 10min, and 95 ℃ of sex change 45s, 53 ℃ of annealing 60s, 72 ℃ are extended 60s, continue to extend 7min at 72 ℃ after 35 circulations.The PCR product of getting 4 μ L mixes with 6 * loadding buffer of 1 μ L, adopts the TAE buffering system, and 2% sepharose (adding staining agent Gold view I) about 25min under 5v/cm constant voltage deposition condition observes and record in gel imaging system.Nine, Bsu36I restriction analysis: the PCR product in the step 8 is carried out PCR-RFLP with restriction enzyme Bsu36I analyze; Bsu36I can only be cut into the PCR product of duck the fragment of 95bp and 377bp, and can not cut the PCR product of goat and sheep, and the bar that whether can detect 95bp and 377bp according to the sepharose detected result brings and judges whether to be duck.The enzyme system of cutting is 20 μ L, wherein: DNA (PCR product) 1 μ g, 10 * NEB buffer, 2 μ L, 100 * BSA0.2 μ L, Bsu36I restriction enzyme (10000U/mL) 0.5 μ L, sterile purified water is supplied volume.Mixing and centrifugal back are at 37 ℃ of digestion 16h, handle 20min deactivation endonuclease activity at 65 ℃ then, getting 7 μ LPCR products mixes with 1 μ L, 6 * loadding buffer, adopt the TAE buffering system, 2% sepharose (adding Gold view I staining agent) about 25min under 5v/cm constant voltage deposition condition observes and record in gel imaging system.Ten, SpeI restriction analysis: the PCR product in the step 8 is carried out PCR-RFLP with restriction enzyme SpeI analyze; SpeI can be cut into the PCR product of sheep and

goat

326 and spend the night (12-16h), can take out during this period and put upside down mixing several times, so that digestion thoroughly.Four, the EP pipe is taken out, every pipe adds the saturated phenol of Tris of 600 μ L, shakes mixing 15min gently, then at the centrifugal 10min of 12000rpm.Five, shift supernatant liquor to new EP pipe, add isopyknic phenol chloroform isoamyl alcohol mixed solution (phenol: chloroform: the primary isoamyl alcohol volume ratio is 25: 24: 1), put upside down mixing 10min, then the centrifugal 10min of 12000rpm.Six, shift supernatant liquor to new EP pipe, add isopyknic chloroform isoamyl alcohol mixed solution (chloroform: the primary isoamyl alcohol volume ratio is 24: 1), mixing 10min gently is then at the centrifugal 10min of 12000rpm.Seven, shift supernatant liquor to new EP pipe, add 1mL ice ethanol earlier, add the NaAc (3M) of 60 μ L again, level is shaken gently, and as seen cotton-shaped, white DNA occurs.The EP pipe is put into-20 ℃ of freezing 30min of refrigerator, and then at the centrifugal 10min of 12000rpm, visible DNA is sunken to the pipe end.Abandon dehydrated alcohol, use 75% washing with alcohol DNA twice again, then at the centrifugal 10min of 12000rpm; After the seasoning, add the dissolving of 50 μ L TE solution.Eight, pcr amplification and detection: primer general upstream primer the F:5 '-TAC CAT GAG GAC AAA TAT CATTCT G-3 ' and general downstream primer the R:5 '-CCT CCT AGT TTG TTA GGG ATT GAT CG-3 ' of all vertebrates Cyt b sequences that can increase.PCR reaction cumulative volume is 25 μ L, 10 * PCR Buffer, 2.5 μ L wherein, Taq archaeal dna polymerase 0.2 μ L (2.5U/ μ L), dNTPs (2.5mmol/L) 2 μ L, each 2 μ L of the upstream and downstream primer of 10pmol/ μ L, template DNA 250ng, sterile purified water is supplied cumulative volume.Amplification condition is: 95 ℃ of sex change 10min, and 95 ℃ of sex change 45s, 53 ℃ of annealing 60s, 72 ℃ are extended 60s, continue to extend 7min at 72 ℃ after 35 circulations.The PCR product of getting 4 μ L mixes with 6 * loadding buffer of 1 μ L, adopts the TAE buffering system, and 2% sepharose (adding staining agent Gold view I) about 25min under 5v/cm constant voltage deposition condition observes and record in gel imaging system.Nine, Bsu36I restriction analysis: the PCR product in the step 8 is carried out PCR-RFLP with restriction enzyme Bsu36I analyze; Bsu36I can only be cut into the PCR product of duck the fragment of 95bp and 377bp, and can not cut the PCR product of goat and sheep, and the bar that whether can detect 95bp and 377bp according to the sepharose detected result brings and judges whether to be duck.Ten, SpeI restriction analysis: the PCR product in the step 8 is carried out PCR-RFLP with restriction enzyme SpeI analyze; SpeI can be cut into 326bp and 146bp with the PCR product of sheep and goat, but can not cut the PCR product of duck, and the bar that whether can detect 326bp and 146bp according to the sepharose detected result brings and judges whether to be mutton.

The present invention adopts the PCR-RFLP method in discrimination process, have easy, quick, cheapness and the high characteristics of accuracy.

Description of drawings

Fig. 1 is the gel electrophoresis figure that the sheep that step 8 obtains in the embodiment one (S), goat (G) and duck (D) PCR product detect; Fig. 2 is that the PCR product of step 9 obtains step 8 in the embodiment one with Bsu36I restriction endonuclease digestion in the embodiment one sheep (S), goat (G) and duck (D) carries out the gel electrophoresis figure that PCR-RFLP analyzes the back detection; Fig. 3 is that the PCR product of the sheep that step 10 obtains step 8 in the embodiment one with the SpeI digestion with restriction enzyme in the embodiment one (S), goat (G) and duck (D) carries out gel bp and the 146bp that PCR-RFLP analyzes the back detection, but can not cut the PCR product of duck, the bar that whether can detect 326bp and 146bp according to the sepharose detected result brings and judges whether to be mutton.The enzyme system of cutting is 20 μ L, wherein: DNA (PCR product) 1 μ g, 10 * NEBbuffer, 2 μ L, 100 * BSA, 0.2 μ L, SpeI restriction enzyme (10000U/mL) 0.5 μ L, sterile purified water is supplied volume.Mixing and centrifugal back are at 37 ℃ of digestion 16h, handle 20min deactivation endonuclease activity at 65 ℃ then, getting 7 μ L PCR products mixes with 1 μ L, 6 * loadding buffer, adopt the TAE buffering system, 2% sepharose (adding Gold view I staining agent) about 25min under 5v/cm constant voltage deposition condition observes and record in gel imaging system.

Attached: the restriction enzyme site of differentiating sheep, goat and the used restriction enzyme of duck

Restriction enzyme Bsu36I (CC ↓ TNAGG)

Restriction enzyme SpeI (A ↓ CTAGT)

Attached: solution preparation table

STE extract: 10mL Tris-HCl (1M; PH 8.0), 2mL EDTA (0.5M; PH 8.0), 100mL NaAc (1M), 888mL water, autoclaving.

20%SDS:200g SDS is dissolved in the 900mL water, with hydrochloric acid adjust pH to 7.2, adds water and is settled to 1000mL.

1M Tris-HCl:121.4g Tris is dissolved in the 800mL distilled water, with hydrochloric acid adjust pH to 8.0, is settled to 1000mL, autoclaving.

TE damping fluid (pH 8.0): 1mL Tris-HCl (1M; PH 8.0), 0.1mL EDTA (0.5M; PH 8.0), add distilled water and be settled to 50mL, autoclaving.

20mg/mL ProK:100mg Proteinase K is dissolved in 5mL sterilization distilled water, and-20 ℃ of packing are preserved.

TE damping fluid (pH 8.0): 1mL Tris-HCl (1M; PH 8.0), 0.1mL EDTA (0.5M; PH 8.0), add distilled water and be settled to 50mL, autoclaving.

Claims (5)

1.一种鉴别羊肉和鸭肉的PCR-RFLP方法，其特征在于可将羊肉和鸭肉按以下方法加以鉴别：一、将取50mg待检样品放入1.5mL的EP管(EP管应高压灭菌)中，用小剪刀将其剪碎。二、加入STE抽提液600μL、ProK(20mg/mL)20μL及20％的SDS溶液15μL，在震荡器上充分混匀。三、将EP管置入水浴锅，55℃消化过夜(12-16h)，在此期间可取出颠倒混匀几次，以便消化彻底。四、将EP管取出，每管加入600μL的Tris饱和酚，轻轻摇动混匀15min，然后在12000rpm离心10min。五、转移上清液至新的EP管，加入等体积的酚氯仿异戊醇混合液(酚∶氯仿∶异戊醇体积比为25∶24∶1)，颠倒混匀10min，然后12000rpm离心10min。六、转移上清液至新的EP管，加入等体积的氯仿异戊醇混合液(氯仿∶异戊醇体积比为24∶1)，轻轻混匀10min，然后在12000rpm离心10min。七、转移上清液至新的EP管，先加入1mL冰乙醇，再加入60μL的NaAc(3M)，轻轻水平摇动，可见絮状、白色DNA出现。将EP管放入-20℃冰箱冷冻30min，然后在12000rpm离心10min，可见DNA沉于管底。弃无水乙醇，再用75％的乙醇洗涤DNA两次，然后在12000rpm离心10min；自然干燥后，加入50μL TE溶液溶解。八、PCR扩增及检测：引物用可扩增所有脊椎动物Cyt b序列的通用上游引物F：5’-TAC CAT GAG GAC AAA TAT CAT TCT G-3’和通用下游引物R：5’-CCT CCT AGT TTG TTA GGG ATT GAT CG-3’。PCR反应总体积为25μL，其中10×PCR Buffer 2.5μL，Taq DNA聚合酶0.2μL(2.5U/μL)，dNTPs(2.5mmol/L)2μL，10pmol/μL的上、下游引物各2μL，模板DNA 250ng，灭菌蒸馏水补足总体积。扩增条件为：95℃变性10min，95℃变性45s，53℃退火60s，72℃延伸60s，35个循环后在72℃继续延伸7min。取4μL的PCR产物与1μL的6×loadding buffer混合，采用TAE缓冲系统，2％琼脂糖凝胶(加入染色剂Gold view I)在5v/cm恒压电泳条件下约25min，在凝胶成像系统中观察并记录。九、Bsu36I酶切分析：对步骤八中的PCR产物用限制性内切酶Bsu36I进行PCR-RFLP分析；Bsu36I只能将鸭的PCR产物切割为95bp和377bp的片段，而不能切割山羊和绵羊的PCR产物，根据琼脂糖凝胶检测结果是否可检测到95bp和377bp的条带来判断是否为鸭肉。十、SpeI酶切分析：对步骤八中的PCR产物用限制性内切酶SpeI进行PCR-RFLP分析；SpeI可将绵羊和山羊的PCR产物切割为326bp和146bp，但不能切割鸭的PCR产物，根据琼脂糖凝胶检测结果是否可检测到326bp和146bp的条带来判断是否为羊肉。1. a kind of PCR-RFLP method of discriminating mutton and duck is characterized in that mutton and duck can be differentiated by following method: one, will get the EP tube of 50mg sample to be checked and put into 1.5mL (EP tube should high pressure Sterilization), cut it up with small scissors. 2. Add 600 μL of STE extract, 20 μL of ProK (20 mg/mL) and 15 μL of 20% SDS solution, and mix well on a shaker. 3. Put the EP tube into a water bath and digest overnight at 55°C (12-16h). During this period, it can be taken out and mixed several times by inversion to ensure thorough digestion. 4. Take out the EP tubes, add 600 μL of Tris saturated phenol to each tube, gently shake and mix for 15 minutes, and then centrifuge at 12000 rpm for 10 minutes. 5. Transfer the supernatant to a new EP tube, add an equal volume of phenol-chloroform-isoamyl alcohol mixture (phenol: chloroform: isoamyl alcohol volume ratio: 25:24:1), invert and mix for 10 minutes, then centrifuge at 12000rpm for 10 minutes . 6. Transfer the supernatant to a new EP tube, add an equal volume of chloroform-isoamyl alcohol mixture (chloroform: isoamyl alcohol volume ratio: 24:1), mix gently for 10 min, and then centrifuge at 12000 rpm for 10 min. 7. Transfer the supernatant to a new EP tube, first add 1mL of ice ethanol, then add 60μL of NaAc (3M), shake gently horizontally, flocculent and white DNA can be seen. Place the EP tube in a -20°C refrigerator for 30 minutes, then centrifuge at 12,000 rpm for 10 minutes, and the DNA can be seen to sink to the bottom of the tube. Discard absolute ethanol, wash DNA twice with 75% ethanol, and then centrifuge at 12000rpm for 10min; after natural drying, add 50μL TE solution to dissolve. 8. PCR amplification and detection: primers use the general upstream primer F: 5'-TAC CAT GAG GAC AAA TAT CAT TCT G-3' and the general downstream primer R: 5'-CCT that can amplify all vertebrate Cyt b sequences CCT AGT TTG TTA GGG ATT GAT CG-3'. The total volume of the PCR reaction is 25 μL, including 2.5 μL of 10×PCR Buffer, 0.2 μL of Taq DNA polymerase (2.5 U/μL), 2 μL of dNTPs (2.5 mmol/L), 2 μL of 10 pmol/μL upstream and downstream primers, and 2 μL of template DNA 250ng, make up the total volume with sterile distilled water. The amplification conditions were: denaturation at 95°C for 10 min, denaturation at 95°C for 45 s, annealing at 53°C for 60 s, extension at 72°C for 60 s, and extension at 72°C for 7 min after 35 cycles. Take 4 μL of PCR product and mix it with 1 μL of 6×loading buffer, use TAE buffer system, run 2% agarose gel (with staining agent Gold view I) under 5v/cm constant voltage electrophoresis condition for about 25min, in the gel imaging system observed and recorded. Nine, Bsu36I enzyme digestion analysis: carry out PCR-RFLP analysis to the PCR product in step 8 with the restriction endonuclease Bsu36I; Bsu36I can only cut the PCR product of the duck into 95bp and 377bp fragments, but cannot cut the fragments of goat and sheep As for the PCR product, it can be judged whether it is duck meat according to whether the 95bp and 377bp bands can be detected in the agarose gel test result. 10. SpeI digestion analysis: carry out PCR-RFLP analysis on the PCR product in step 8 with the restriction endonuclease SpeI; SpeI can cut the PCR products of sheep and goats into 326bp and 146bp, but cannot cut the PCR products of ducks, According to whether the 326bp and 146bp bands can be detected in the agarose gel test results, it can be judged whether it is mutton. 2.根据权利要求1所述的一种鉴别羊肉和鸭肉的PCR-RFLP方法，其特征在于步骤二用STE抽提液。2. a kind of PCR-RFLP method for distinguishing mutton and duck according to claim 1, is characterized in that step 2 uses STE extraction solution. 3.根据权利要求1所述的一种鉴别羊肉和鸭肉的PCR-RFLP方法，其特征在于步骤八中扩增引物为用通用上游引物F：5’-TAC CAT GAG GAC AAA TAT CAT TCT G-3’和通用下游引物R：5’-CCT CCT AGT TTG TTA GGG ATT GAT CG-3’。3. A kind of PCR-RFLP method for distinguishing mutton and duck according to claim 1, characterized in that the amplification primer in step 8 is the general upstream primer F: 5'-TAC CAT GAG GAC AAA TAT CAT TCT G -3' and universal downstream primer R: 5'-CCT CCT AGT TTG TTA GGG ATT GAT CG-3'. 4.根据权利要求1所述的一种鉴别羊肉和鸭肉的PCR-RFLP方法，其特征在于步骤九用限制性内切酶Bsu36I进行PCR-RFLP分析。4. a kind of PCR-RFLP method for distinguishing mutton and duck according to claim 1, is characterized in that step 9 carries out PCR-RFLP analysis with restriction endonuclease Bsu36I. 5.根据权利要求1所述的一种鉴别羊肉和鸭肉的PCR-RFLP方法，其特征在于步骤十用限制内切酶SpesI进行PCR-RFLP分析。5. a kind of PCR-RFLP method of distinguishing mutton and duck according to claim 1 is characterized in that step ten carries out PCR-RFLP analysis with restriction endonuclease SpesI.

CN 201010226683 2010-07-15 2010-07-15 A PCR-RFLP method for differentiating mutton and duck Pending CN101875975A (en)

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