SpringerLink
Log in

Specific PCR method for detection of species origin in biochemical drugs via primers for the ATPase 8 gene by electrophoresis

  • Original Paper
  • Published:
Microchimica Acta Aims and scope Submit manuscript

Abstract

A PCR method is described to identify the species origin of various animal and human tissue-derived biochemical drugs. Four commercialized drugs, including spermary tablets, compound embryonic bovine liver extract tablets, spleen aminopeptide solution, and placenta polypeptide injection, were used as a proof-of-principle in this study. Primers were designed to amplify conservative regions of mitochondrial cytochrome b and ATPase 8 genes from beef, pork, lamb and human DNA, respectively. The specificity of primers for ATPase 8 gene is found to be higher than those for cytochrome b under the given experimental conditions. The amplicon sizes of ATPase 8 were 212, 271, 293 and 405 bp for pork, beef, lamb and human tissue, respectively. The minimum detectable concentration of DNA sample for species identification is 0.05–0.5 pg·μL−1. The species origin can be distinguished by this method in extremely low concentrations of template DNAs extracted. Conceivably, this PCR method for meat authentication may be extended to quality control of other biochemical drugs and raw materials.

A specific PCR method was developed for the detection of species origin in biochemical drugs via species-specific primers targeting mitochondrial ATPase 8 genes. The PCR products were separated by gel electrophoresis and species origins were indicated by comparison to references.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Germany)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Ren L, Fan H (2016) Quality status and future perspectives of national biochemical drugs. Chin J Biochem Pharm 36(10):1–4

    Google Scholar 

  2. Goeddel DV, Kleid DG, Bolivar F, Heyneker HL, Yansura DG, Crea R, Hirose T, Kraszewski A, Itakura K, Riggs AD (1979) Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc Natl Acad Sci U S A 76(1):106–110

    Article  CAS  Google Scholar 

  3. Gurramkonda C, Polez S, Skoko N, Adnan A, Gabel T, Chugh D, Swaminathan S, Khanna N, Tisminetzky S, Rinas U (2010) Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin. Microb Cell Factories 9:31. https://doi.org/10.1186/1475-2859-9-31

    Article  CAS  Google Scholar 

  4. Thim L, Hansen MT, Norris K, Hoegh I, Boel E, Forstrom J, Ammerer G, Fiil NP (1986) Secretion and processing of insulin precursors in yeast. Proc Natl Acad Sci U S A 83(18):6766–6770

    Article  CAS  Google Scholar 

  5. Chang CH, Lico LS, Huang TY, Lin SY, Chang CL, Arco SD, Hung SC (2014) Synthesis of the heparin-based anticoagulant drug fondaparinux. Angew Chem Int Ed Eng 53(37):9876–9879. https://doi.org/10.1002/anie.201404154

    Article  CAS  Google Scholar 

  6. Xu Y, Masuko S, Takieddin M, Xu H, Liu R, **g J, Mousa SA, Linhardt RJ, Liu J (2011) Chemoenzymatic synthesis of homogeneous ultralow molecular weight heparins. Science 334(6055):498–501. https://doi.org/10.1126/science.1207478

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kumar A, Kumar RR, Sharma BD, Gokulakrishnan P, Mendiratta SK, Sharma D (2015) Identification of species origin of meat and meat products on the DNA basis: a review. Crit Rev Food Sci Nutr 55(10):1340–1351. https://doi.org/10.1080/10408398.2012.693978

    Article  CAS  PubMed  Google Scholar 

  8. Safdar M, Junejo Y (2016) The development of a hexaplex-conventional PCR for identification of six animal and plant species in foodstuffs. Food Chem 192:745–749. https://doi.org/10.1016/j.foodchem.2015.07.082

    Article  CAS  PubMed  Google Scholar 

  9. Jung M-J, Yang D-H, Sung Y-K, Kim M-J, Kang S-J, Kwon D-Y, Kwon Y (2008) Rapid determination of trace methylmercury in natural crude medicine of animal origin. Microchim Acta 164(3–4):345–349. https://doi.org/10.1007/s00604-008-0063-9

    Article  CAS  Google Scholar 

  10. Ali ME, Razzak MA, Hamid SB, Rahman MM, Amin MA, Rashid NR, Asing (2015) Multiplex PCR assay for the detection of five meat species forbidden in Islamic foods. Food Chem 177:214–224. https://doi.org/10.1016/j.foodchem.2014.12.098

    Article  CAS  PubMed  Google Scholar 

  11. Zhu P, Fu W, Wang C, Du Z, Huang K, Zhu S, Xu W (2016) Development and application of absolute quantitative detection by duplex chamber-based digital PCR of genetically modified maize events without pretreatment steps. Anal Chim Acta 916:60–66. https://doi.org/10.1016/j.aca.2016.02.020

    Article  CAS  PubMed  Google Scholar 

  12. Wang Q, Cai Y, He Y, Yang L, Pan L (2018) Collaborative ring trial of two real-time PCR assays for the detection of porcine- and chicken-derived material in meat products. PLoS One 13(10):e0206609. https://doi.org/10.1371/journal.pone.0206609

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. DuVall JA, Le Roux D, Thompson BL, Birch C, Nelson DA, Li J, Mills DL, Tsuei AC, Ensenberger MG, Sprecher C, Storts DR, Root BE, Landers JP (2017) Rapid multiplex DNA amplification on an inexpensive microdevice for human identification via short tandem repeat analysis. Anal Chim Acta 980:41–49. https://doi.org/10.1016/j.aca.2017.04.051

    Article  CAS  PubMed  Google Scholar 

  14. Thanakiatkrai P, Dechnakarin J, Ngasaman R, Kitpipit T (2019) Direct pentaplex PCR assay: an adjunct panel for meat species identification in Asian food products. Food Chem 271:767–772. https://doi.org/10.1016/j.foodchem.2018.07.143

    Article  CAS  PubMed  Google Scholar 

  15. Di Pinto A, Bottaro M, Bonerba E, Bozzo G, Ceci E, Marchetti P, Mottola A, Tantillo G (2015) Occurrence of mislabeling in meat products using DNA-based assay. J Food Sci Technol 52(4):2479–2484. https://doi.org/10.1007/s13197-014-1552-y

    Article  CAS  PubMed  Google Scholar 

  16. Yang L, Tan Z, Wang D, Xue L, Guan MX, Huang T, Li R (2014) Species identification through mitochondrial rRNA genetic analysis. Sci Rep 4:4089. https://doi.org/10.1038/srep04089

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Roda A, Mirasoli M, Roda B, Bonvicini F, Colliva C, Reschiglian P (2012) Recent developments in rapid multiplexed bioanalytical methods for foodborne pathogenic bacteria detection. Microchim Acta 178(1–2):7–28. https://doi.org/10.1007/s00604-012-0824-3

    Article  CAS  Google Scholar 

  18. Thanakiatkrai P, Kitpipit T (2017) Meat species identification by two direct-triplex real-time PCR assays using low resolution melting. Food Chem 233:144–150. https://doi.org/10.1016/j.foodchem.2017.04.090

    Article  CAS  PubMed  Google Scholar 

  19. Kaltenbrunner M, Hochegger R, Cichna-Markl M (2018) Tetraplex real-time PCR assay for the simultaneous identification and quantification of roe deer, red deer, fallow deer and sika deer for deer meat authentication. Food Chem 269:486–494. https://doi.org/10.1016/j.foodchem.2018.07.023

    Article  CAS  PubMed  Google Scholar 

  20. Guan F, ** YT, Zhao J, Xu AC, Luo YY (2018) A PCR method that can be further developed into PCR-RFLP assay for eight animal species identification. J Anal Methods Chem 2018:5890140:1–6. https://doi.org/10.1155/2018/5890140

    Article  CAS  Google Scholar 

  21. Song KY, Hwang HJ, Kim JH (2018) Data for the optimization of conditions for meat species identification using ultra-fast multiplex direct-convection PCR. Data Brief 16:15–18. https://doi.org/10.1016/j.dib.2017.11.004

    Article  PubMed  Google Scholar 

  22. Matsunaga T, Chikuni K, Tanabe R, Muroya S, Shibata K, Yamada J, Shinmura Y (1999) A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Sci 51(2):143–148

    Article  CAS  Google Scholar 

  23. Song KY, Hwang HJ, Kim JH (2017) Ultra-fast DNA-based multiplex convection PCR method for meat species identification with possible on-site applications. Food Chem 229:341–346. https://doi.org/10.1016/j.foodchem.2017.02.085

    Article  CAS  PubMed  Google Scholar 

  24. Giusti A, Tinacci L, Sotelo CG, Marchetti M, Guidi A, Zheng W, Armani A (2017) Seafood identification in multispecies products: assessment of 16SrRNA, cytb, and COI universal primers' efficiency as a preliminary analytical step for setting up Metabarcoding next-generation sequencing techniques. J Agric Food Chem 65(13):2902–2912. https://doi.org/10.1021/acs.jafc.6b05802

    Article  CAS  PubMed  Google Scholar 

  25. Chen A, Wei C, Chen G, Zhao Y, Yang S (2015) Duplex PCR approach for the detection and quantification of donkey, horse and mule in raw and heat-processed meat products. Int J Food Sci Technol 50(3):834–839. https://doi.org/10.1111/ijfs.12720

    Article  CAS  Google Scholar 

  26. Kesmen Z, Gulluce A, Sahin F, Yetim H (2009) Identification of meat species by TaqMan-based real-time PCR assay. Meat Sci 82(4):444–449. https://doi.org/10.1016/j.meatsci.2009.02.019

    Article  CAS  PubMed  Google Scholar 

  27. Prusakova OV, Glukhova XA, Afanas'eva GV, Trizna YA, Nazarova LF, Beletsky IP (2018) A simple and sensitive two-tube multiplex PCR assay for simultaneous detection of ten meat species. Meat Sci 137:34–40. https://doi.org/10.1016/j.meatsci.2017.10.017

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This research was supported by grants from CAMS Innovation Fund for Medical Sciences (2017-I2M-1-012), National Natural Science Foundation of China (31870059 & 81760778), Shandong Natural Science Foundation (CR2014CL010) and The Drug Innovation Major Project (2018ZX09711001-007-003 & 2017ZX09101002-003-003). We specially thank Dr. Mengxin Geng in Texas A&M University for critical reading of the manuscript.

Author information

Authors and Affiliations

  1. NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Bei**g, 100050, China

    Limei Ai, Juanjuan Liu, Yu Jiang, Weiwei Guo & Li** Bai

  2. Department of medical immunology, School of Basic Medical Sciences, Taishan Medical University, Taian, 271000, China

    ** Wei

  3. CAMS Key Laboratory of Synthetic Biology for Drug Innovation, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tian Tan ** Bai

Authors
  1. Limei Ai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Juanjuan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yu Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Weiwei Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. ** Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Li** Bai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to ** Wei or Li** Bai.

Ethics declarations

The author(s) declare that they have no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(DOCX 595 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ai, L., Liu, J., Jiang, Y. et al. Specific PCR method for detection of species origin in biochemical drugs via primers for the ATPase 8 gene by electrophoresis. Microchim Acta 186, 634 (2019). https://doi.org/10.1007/s00604-019-3738-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00604-019-3738-5

Keywords

Search

Navigation