Establishment of Fast Identification Method for Lactic Cervi Molecule ID and Development of Detection Kit
WANG Yan-shuang1a,2, JIANG Hai-ying1b, ZHANG Zhi-jie1a, SUN Li-yuan1b, AI Jin-xia1b, GAO Li-jun1b, LI Ming-cheng1b,2, YUAN Guang-xin1c*, ZHANG Li-hua3
1a. School of Medical Faculty; 1b. School of Laboratory Medicine; 1c. School of Pharmacy, Beihua University, Jilin 132013, China; 2. Chinese Medicine DNA Fingerprint Detection Technology Technology Innovation Center, Beihua University, Jilin 132013, China; 3. Jilin Leining Food and Drug Testing Technology Service Co., Ltd., Jilin 132013, China
Abstract：OBJECTIVE To establish a fast identification method of Lactic Cervi molecule ID and develop a fast detection kit for Lactic Cervi molecule ID. METHODS With the Lactic Cervi mtDNA Cytb as the target gene, the Lactic Cervi specific primer was designed with an amplified fragment of 46 bp in length, and the Lactic Cervi molecule ID was determined. Molecular cloning and sequencing technology were used to clone Lactic Cervi DNA detection standards and verify the correctness of the sequence of the molecular ID. Then, the fast detection kit of Lactic Cervi molecule ID was developed, and the specificity, reproducibility, stability and sensitivity of the reagent were investigated. RESULTS The designed small fragment primers by using mtDNA Cytb gene of Lactic Cervi showed the strongest specificity at 56 ℃. A specific amplification band appeared at 46 bp for both the control drug and the authentic product of Lactic Cervi, while no amplification band appeared in the negative and blank control, which confirmed the molecule ID of Lactic Cervi. After cloning, the DNA sequence of Lactic Cervi was 100% homologous with mtDNA specific fingerprint segment sequence of Lactic Cervi, which verified the correctness of the sequence of Lactic Cervi molecular ID. The self-developed reagent had good specificity, reproducibility and stability, and the minimum detection amount of template DNA was 5 pg·μL-1. CONCLUSION The fast identification method for Lactic Cervi molecule ID established in this study is specific, accurate and reliable. The detection kit is easy to operate and stable, which is suitable for materials with serious DNA degradation.
王艳双, 姜海瀛, 张志杰, 孙丽媛, 艾金霞, 高丽君, 李明成, 苑广信, 张丽华. 乳鹿分子身份证快速鉴定方法的建立及检测试剂盒的研制[J]. 中国药学杂志, 2020, 55(21): 1787-1794.
WANG Yan-shuang, JIANG Hai-ying, ZHANG Zhi-jie, SUN Li-yuan, AI Jin-xia, GAO Li-jun, LI Ming-cheng, YUAN Guang-xin, ZHANG Li-hua. Establishment of Fast Identification Method for Lactic Cervi Molecule ID and Development of Detection Kit. Chinese Pharmaceutical Journal, 2020, 55(21): 1787-1794.
GU Y J, ZHANG L H, FU G L. Identification of velvet by mitochondrial DNA fingerprint[J]. Chin Pharm J (中国药学杂志), 2013, 48(3):170-173.
[2]
GAO L J, HE S Y, LI Y N, et al. Charateristics and identification of DNA fingerprint of velvet antler and its counterfeits based on duplex PCR technique [J]. J Jilin Univ(Med Ed)(吉林大学学报:医学版), 2018, 44(4):839-843.
[3]
AI J X, LI M C, XIA W, et al. Development and evaluation of DNA detection kit for Fetus Cervi [J]. Chin Pharm J (中国药学杂志), 2018, 53(4):306-301.
[4]
WANG Y S, YUAN G X, ZHANG L H, et al. Establishment of mink heart identification method based on mitochondrial cytochrome b gene and development of its detection kit[J]. Mitochondrial NA Part A, 2019, 30(2):325-331.
[5]
WANG S, WANG H, ZHANG L H, et al. Identification of Panax ginsheng C. A. Meyer Cv. Sylvatica and cultivated Panax ginseng by DALP fingerprint[J]. Chin Pharm J (中国药学杂志), 2013, 48(9):677-680.
[6]
LIU T H, WANG J, ZHANG L H. Identification of cytochrome b gene of Chinese Herb Testudinis Carapax et Plastom [J]. Chin Pharm J (中国药学杂志), 2012, 47(3):182-185.
[7]
ZHOU T T, YU W J, LI M C, et al. Development and evaluation of Fritillaria cirrhosa D. Don DNA detection kit [J]. Chin Pharm J(中国药学杂志), 2014, 49(6):501-504.
[8]
YU W J, ZHOU T T, ZHANG L H, et al. Evaluation of Agkistrodon acutus(Güenther) DNA test kit and its application in quality inspection of commercial products [J]. Chin Pharm J (中国药学杂志), 2014, 49(11):999-1003.
[9]
ZHANG G J, WANG J J. Science connotation on identification of Chinese materia medica origin[J]. Drugs Clinic (现代药物与临床), 2011, 26(1):1-3.
[10]
LONG F, LI H J, LI P. Application of new technologies and methods in morphologica and microscopic identification of Chinese materia medica[J]. China J Chin Mater Med (中国中药杂志), 2012, 37(8):1076-1080.
[11]
CHEN S L, HAN J P, WANG X Y, et al. Panax notoginseng molecule ID and identification method[P]. China Patent:CN104830969A, 2015-08-12.
[12]
HAN J P, SONG J Y, WANG L L, et al. American ginseng molecule ID and identification method[P]. China Patent:CN104830971A, 2015-08-12.
[13]
HAN J P, XIANG L, LIAN B S, et al. Cordyceps sinensis molecule ID and identification method[P]. China Patent:CN104830970A, 2015-08-12.
[14]
LIU Y, WANG L L, WANG X Y, et al. A nucleotide singnature for the identification of american ginseng and its products[J]. Front Plant Sci, 2016, 7:319.
[15]
LIAO B S, WANG L L, WANG X Y, et al. A fast identification method of Notoginseng Radix Etrhizoma based on molecular signature[J]. Chin Pharm J (中国药学杂志), 2015, 50(22):1954-1959.
[16]
CRIMI M, RIGOLIO R. The mitochondrial genome, a growing interest inside an organelle [J]. Int J Nanomed, 2008, 3(1):51-57.
[17]
PETROVSKAIA A V. Genetic structure of the sable Martes zibellina L. populations from magadan oblast as inferred from mitochondrial DNA variation [J]. Genetika, 2007, 43(4):530-536.
2020, Vol. 55 
