Identification of newly established Spodoptera littoralis cell lines by two DNA barcoding markers



Cell line authentication is crucial in determining the identity of cell lines and detecting any cross-contamination. The identity of three newly established Spodoptera littoralis cell lines (Spli-C, Spli-B, and Spli-S) was confirmed by DNA fingerprinting. In this study, we used two universal primers sets to amplify two DNA fragments in different positions in the mitochondrial cytochrome C oxidase 1 gene (COI). The PCR reaction succeeded in amplifying two target DNA amplicons. The first amplicon had ~650 bp, while the second had ~410 bp. By comparing the obtained informative sequences with those in the GenBank sequence database, the results showed 100% similarity between the S. littoralis cell lines and their host. The same similarity ratio was observed between the Sf21, Tni, and Cp cell lines, which are used widely, and their hosts. The informative sequences were then used for phylogenetic analyses. In addition to the high efficiency of this technique, it showed high reproducibility in two different laboratories. DNA barcoding using the two sets of the universal primers used in this study can be a fast and a reliable method for insect cell line identification.


DNA fingerprinting DNA barcoding Insect cell lines Spodoptera littoralis 



This work was supported by the Institute of Bioprocess Engineering/Erlangen University.


  1. Adams RP, Baker LE, Pandey RN (2008) Optimizing procedures to obtain reliable DNA fingerprinting data for use in systematic, ecological and evolutionary studies. Phytologia 90:83–96Google Scholar
  2. Ahmed I, Huebner H, Buchholz R (2014) Establishment and characterization of three new embryonic Spodoptera littoralis cell lines and testing their susceptibility to SpliMNPV. JSM Biotechnol Bioeng 2:1031Google Scholar
  3. Belloncik S, Petcharawan O, Couillard M, Charpentier G, Larue B, Guardado H, Chareonsak S, Imanishi S (2007) Development and characterization of a continuous cell line, AFKM-On-H, from hemocytes of the European corn borer Ostrinia nubilalis (Hübner) (Lepidoptera, Pyralidae). In Vitro Cell Dev-An 43:245–254CrossRefGoogle Scholar
  4. Capes-Davis A, Theodosopoulos G, Atkin I, Drexler HG, Kohara A, MacLeod RA, Masters JR, Nakamura Y, Reid YA, Reddel RR, Freshney RI (2010) Check your cultures! A list of cross-contaminated or misidentified cell lines. Int J Cancer 127:1–8CrossRefPubMedGoogle Scholar
  5. Cox MM (2012) Recombinant protein vaccines produce in insect cells. Vaccine 30:1759–1766CrossRefPubMedGoogle Scholar
  6. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294–299PubMedGoogle Scholar
  7. Goodman CL, McIntosh AH, Sayed GNE, Grasela J, Stiles B (2001) Production of selected baculoviruses in newly established lepidopteran cell lines. In Vitro Cell Dev-An 37:374–379CrossRefGoogle Scholar
  8. Hebert PDN, Cywinska A, Ball SL, Waard JR (2003) Biological identifications through DNA barcodes. Proc R Soc Lond B 270:313–321CrossRefGoogle Scholar
  9. Hebert PDN, deWaad JR, Landry J (2009) DNA barcodes for 1/1000 of the animal kingdom. Biology letters Evolutionary biology Biol Lett. doi: 10.1098/rsbl.2009.0848 Google Scholar
  10. Ikonomou L, Schneider YJ, Agatho SN (2003) Insect cell culture for industrial production of recombinant proteins. Appl Microbiol Biot 62:1–20CrossRefGoogle Scholar
  11. Jones CJ, Edwards KJ, Castaglione S, Winfield MO, Sala F, Wiel CV, Bredemeijer G, Vosman B, Matthes M, Daly A, Brettschneider R, Bettini P, Buiatti M, Maestri E, Malcevschi A, Marmiroli N, Aert R, Volckaert G, Rueda J, Linacero R, Vazquez A, Karp A (1997) Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories. Mol Breeding 3:381–390CrossRefGoogle Scholar
  12. Kress WJ, Erickson DL (2008) DNA barcodes: genes, genomics, and bioinformatics. PNAS 105:2761–2762CrossRefPubMedPubMedCentralGoogle Scholar
  13. Lery X, LaRue B, Cossette J, Charpentier G (2003) Characterization and authentication of insect cell lines using RAPD markers. Insect Biochem Molec 33:1035–1041CrossRefGoogle Scholar
  14. Markovic O, Markovic N (1998) Cell cross-contamination in cell cultures: the silent and neglected danger. In Vitro Cell Dev-An 34:1–8CrossRefGoogle Scholar
  15. McCarroll L, King LA (1997) Stable insect cell cultures for recombinant protein production. Curr Opin Biotech 8:590–594CrossRefPubMedGoogle Scholar
  16. Mclntosh AH, Grasela JJ, Matted RL (1996) Identification of insect cell lines by DNA amplification fingerprinting (DAF). Insect Mol Biol 5:187–195CrossRefGoogle Scholar
  17. Mitsuhashi J (2002) Invertebrate tissue culture methods. Springer, TokyoCrossRefGoogle Scholar
  18. Nagoshi RN, Brambila J, Meagher RL (2011) Use of DNA barcodes to identify invasive armyworm Spodoptera species in Florida. J Insect Sci 11:1–11CrossRefGoogle Scholar
  19. Nagoshi RN, Silvie P, Meagher RL (2007) Comparison of haplotype frequencies differentiate fall armyworm (Lepidoptera: Noctuidae) corn-strain populations from Florida and Brazil. J Econ Entomol 100:954–961CrossRefPubMedGoogle Scholar
  20. Sass C, Little DP, Stevenson DW, Specht CD (2007) DNA barcoding in the Cycadales: testing the potential of proposed barcoding markers for species identification of cycads. PLoS One 2:e1154. doi: 10.1371/journal.pone.0001154 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Savolainen V, Cowan RS, Vogler AP, Roderick GK, Lane R (2005) Towards writing the encyclopaedia of life: an introduction to DNA barcoding. Phil Trans R Soc B 360:1805–1811CrossRefPubMedPubMedCentralGoogle Scholar
  22. Stacey G, Bolton B, Doyle A, Griffiths B (1992) DNA fingerprinting a valuable new technique for the characterization of cell lines. Cytotechnology 9:211–216CrossRefPubMedGoogle Scholar
  23. Szewczyk B, Rabalski L, Krol E, Sihler W, Lobo de Souza M (2009) Baculovirus biopesticides—a safe alternative to chemical protection of plants. J Biopesticides 2:64–71Google Scholar

Copyright information

© The Society for In Vitro Biology 2017

Authors and Affiliations

  • I. Ahmed
    • 1
    • 2
  • H. Huebner
    • 1
  • Y. I. Mamoori
    • 2
  • R. Buchholz
    • 1
  1. 1.Bioprocess Engineering Institute, Department of Chemical and Biological EngineeringFriedrich Alexander University, Erlangen-NurembergErlangenGermany
  2. 2.College of Applied BiotechnologyAl-Nahrain UniversityBaghdadIraq

Personalised recommendations