Identification of Cervi Cornu Pantotrichum and Cervi Cornu by Allele-specific PCR

Run QIAN; Na TIAN; Xue-yan ZHANG; Chao JIANG; Yuan YUAN

doi:10.13422/j.cnki.syfjx.20191712

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Identification of Cervi Cornu Pantotrichum and Cervi Cornu by Allele-specific PCR

Topic on DNA Molecular Identification of Animal Medicinal Materials | 更新时间：2021-02-09

- Identification of Cervi Cornu Pantotrichum and Cervi Cornu by Allele-specific PCR
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 25, Issue 17, Pages: 118-123(2019)
- 作者机构：
  
  1.中国中医科学院　中药资源中心，道地药材国家重点实验室培育基地，北京　 100700；
  2.安徽中医药大学　药学院，合肥　 230012
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20191712
  CLC： R284.2; R285; R22; R2-031
- Received：04 January 2019，
  
  Published Online：17 May 2019，
  
  Published：05 September 2019
- 稿件说明：
移动端阅览
Run QIAN, Na TIAN, Xue-yan ZHANG, et al. Identification of Cervi Cornu Pantotrichum and Cervi Cornu by Allele-specific PCR[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(17): 118-123.
DOI：

Run QIAN, Na TIAN, Xue-yan ZHANG, et al. Identification of Cervi Cornu Pantotrichum and Cervi Cornu by Allele-specific PCR[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(17): 118-123. DOI： 10.13422/j.cnki.syfjx.20191712.

摘要

目的：

建立一种快速、高效、简便的鹿茸、鹿角位点特异性聚合酶链式反应(PCR)鉴别方法。

方法：

通过比较梅花鹿、马鹿及其混伪品的COI，Cytb基因序列差异，根据Cytb片段上的3个SNP (single nucleotide polymorphism)变异位点设计出一对鹿茸的特异性鉴别引物LR-238上游和LR-238下游，并对影响PCR结果的主要因素退火温度，PCR循环次数，模板DNA浓度，

Taq

酶用量和及影响重复性的

Taq

酶种类和PCR仪型号等进行方法学考察和优化。

结果：

进行方法学考察确定最优鉴别条件的基础上，当PCR退火温度为56 ℃，循环次数为35次的条件下，鹿茸、鹿角正品均能扩增出约250 bp片段，其他9种混伪品麋鹿、白唇鹿、水鹿、坡鹿、黇鹿、骡鹿、驯鹿、驼鹿、狍及阴性对照无条带。不同来源的药用的马鹿亚种东北马鹿、塔河马鹿、天山马鹿、阿拉善马鹿、阿勒泰马鹿、新西兰马鹿，梅花鹿亚种东北梅花鹿、华南梅花鹿样品使用建立的位点特异性PCR鉴别方法均产生约250 bp特异性鉴别条带。

结论：

该文设计的鉴别引物具有高度的专属性，所建立的位点特异性PCR鉴别方法可用于鹿茸、鹿角的准确鉴别。

Abstract

Objective:

To obtain a rapid

efficiency and convenient polymerase Chain reaction(PCR) identification method for medicinal Cervi Cornu Pantotrichum

Cervi Cornu and its common adulterates.

Method:

Based on three single nucleotide polymorphisms (SNP) of Cytb gene DNA sequences among

Cervus nippon

elaphus

and its adulterants

a pair of species-specific primers (LR-238.F and LR-238.R) was designed

the reaction conditions were optimized

and the PCR method for identification was explored and verified in terms of tolerance and feasibility.

Result:

Through the established allele-specific PCR method

under the annealing temperature of 56 ℃ and cycle number of 35

250 bp of fragments were amplified from DNA templates of Cervi Cornu Pantotrichum

Cervi Cornu and its subspecies in origin animal samples as well as herbal medicines. All of the adulterants species of

Przewalskium albirostris

Cervus eldi

Odocoileus hemionus

Dama dama

Alces alces

Elaphurus davidianus

Capreolus pygargus

Rusa unicolor

and

Rangifer tarandus

were negative by the PCR assay.

Conclusion:

The identification primer is highly specific

and the allele-specific PCR identification method established in this paper can accurately identify the medicinal Cervi Cornu Pantotrichum and Cervi Cornu.

关键词

Keywords

references

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