 |
 |
 |
 |
 |
 |
Sains Malaysiana 48(3)(2019):
555–559 http://dx.doi.org/10.17576/jsm-2019-4803-07
Successful and Efficient DNA Extraction
from Processed Pangolin Scales (Kejayaan dan Kecekapan Pengekstrakan
DNA daripada Sisik
Tenggiling Terproses) XUAN CAI1,
HAO-TING
YE1,
EN-WEI
TIAN1,
FANG
LI1,
CHAN
LI1,
YAO-JUN
YANG2,
CHUN-SHENG
LIU2
& ZHI CHAO1* 1School of Traditional
Chinese Medicine, Southern Medical University, Guangzhou 510515,
China 2School of Chinese
Materia Medica, Beijing University
of Chinese Medicine, Beijing 100029, China Received:
9 March 2018/Accepted: 14 November 2018 ABSTRACT A successful method for total
DNA
extraction from crude and processed pangolin scales
was established. After pretreatment in the soaking solution for
cleansing, the scales were prepared into fine powders and treated
with PBS buffer containing 0.1% collagenase
and 0.1% trypsin
for 24 h, followed by digestion with proteinase
K at 55°C for 120 h. Phenol-chloroform extraction was used to obtain
the total DNA.
PCR
amplification for mitochondrial cytochrome b (cytb) gene was successful using the extracted
DNA
as the template, and sequencing of the amplified fragments
confirmed Manis origin of the scale samples. With an efficiency up to 100%, this
method is expected to provide a powerful tool in molecular identification
of processed as well as crude pangolin scales. Keywords: DNA extraction;
Manis; processed pangolin scales ABSTRAK Kaedah berkesan untuk pengekstrakan DNA total daripada
sisik tenggiling
mentah dan terproses
telah berjaya
dihasilkan. Selepas pra-rawatan dengan cara merendam di dalam larutan untuk
pembersihan, sisik
disediakan dalam bentuk serbuk halus
dan dirawat
dengan penimbal PBS yang
mengandungi 0.1% kolagenase
dan tripsin
0.1% selama 24 jam diikuti penghadaman oleh proteinase K pada 55°C selama 120
jam. Pengekstrakan fenol-kloroform
telah digunakan
untuk mendapatkan DNA total.
Amplifikasi PCR untuk
gen mitokondria sitokrom
b (cytb) telah
berjaya dengan menggunakan ekstrak DNA
sebagai templat dan penjujukan serpihan teramplifikasi membuktikan sampel sisik berasal daripada
Manis. Dengan
kadar kecekapan sehingga 100%, kaedah ini dijangka menjadi
suatu alat berkesan untuk mengenal pasti molekul sisik terproses
serta mentah
tenggiling. Kata kunci: Manis; pengekstrakan
DNA; sisik tenggiling terproses REFERENCES
Chinese Pharmacopoeia
Commission. 2015. Pharmacopoeia of the People’s Republic of China.
Vol. 1: 268. China Medical Science &Technology Press: Beijing,
China. CITES. 2017. Convention
on international trade in endangered species of wild fauna and flora.
https://cites.org/sites/default/ files/eng/app/2017/E-Appendices-2017-10-04.pdf.
Coutinho, M.D.F., Still,
D.W., Lum, M.R. & Hirsch, A.M. 2015.
DNA-based authentication of botanicals and plant-derived dietary
supplements: Where have we been and where are we going? Planta
Medica 81: 687-695. Huang, L.Q., Yuan,
Y., Jiang, C. & Tian, X.X. 2017. Strategy on molecular authentication
of animal medicines. Modern Chinese Medicine 19: 1-10. IUCN. 2017. The
IUCN Red List of Threatened Species. Version 2017-2. http://www.iucnredlist.org.
Accessed on 9 November 2017. Jia, J., Zhang, H.Y.,
Chen, J., Liu, D., Yao, H., Qian, Q.N. & Zhang, H. 2014. Molecular
identification of Manis pentadactyla
using DNA barcoding. China Journal of Chinese Materia
Medica 39: 2212-2215. Lan, H., Wang, W.
& Shi, L.M. 1995. PCR analysis of DNA from fur specimens of
muntjacs (Muntiacus). Zoological
Research 16: 146-152. Liu, C.H., Zhang,
J., Kang, S., Liu, T.S. & Zhao, J. 2014. Identification of the
scales of Manis pentadactyla,
M. tricuspis and their adulterants.
Journal of Chinese Medicinal Materials 37: 1178-1182. Liu, S., Zhang, H.F.,
Liu, X.M., Zhu, Y. & Cui, X.B. 2017. Identification of processed
pangolin scales from 4 different Manis
species. Journal of Chinese Medicinal Materials 40: 585-588.
Liu, X.Q., Peng,
J.J., Gao, S.F., Yu, D.M., Gao, L.F., Wang, L.L., Hu, S.J. &
Fu, M.L. 2011. Smuggling trade overview, species identification
and morphological comparison of pangolin. Practical Forestry
Technology 41: 11-14. Mwale, M., Dalton, D.L.,
Jansen, R., De Bruyn, M., Pietersen,
D., Mokgokong, P.S. & Kotzé,
A. 2017. Forensic application of DNA barcoding for identification
of illegally traded African pangolin scales. Genome 60: 272-284.
Shi, Y., Wu, X.B.,
Yan, P. & Zhao, Z. 2004. A method for DNA extraction from tannage and scales of Chinese alligator Alligator
sinensis. Acta
Zoologica Sinica
50: 297-301. Su, B., Liu, R.Q.,
Wang, Y.X. & Shi, L.M. 1994. Genetic diversity in the Chinese
pangolin (Manis pentadactyla)
inferred from protein electrophoresis. Biochemical Genetics 32:
343-349. Thapa, P. 2013. An overview
of Chinese pangolin (Manis pentadactyla): Its general biology, status, distribution
and conservation threats in Nepal. The Initiation 5: 164-170.
Wu, Z.Y., Chao, J.G.
& Liu, X. 2015. Pharmacognostical
studies on four sources of Manis
spp. Research and Practice on Chinese Medicines 29: 25-28.
Xing, Y.L., Peng,
J.J., Hu, H.J., Yu, D.M., Zhang, L.B. & Yu, B.C. 2013. Extraction
and amplification of DNA from pangolin specimen and scales. Chinese
Journal of Zoology 48: 49-57. Yin, Y., Liu, X.,
Wang, B., Gao, L.H. & Zhang, X.N. 2017. DNA molecular identification
of Manis pentadactyla.
China Journal of Chinese Materia Medica 42: 2078-2084. *Corresponding author; email: chaozhi@smu.edu.cn
|
|||
|
