Food Analytical Methods

, Volume 9, Issue 5, pp 1199–1209 | Cite as

Universal Primers Used for Species Identification of Foodstuff of Animal Origin: Effects of Oligonucleotide Tails on PCR Amplification and Sequencing Performance

  • A. ArmaniEmail author
  • A. Giusti
  • L. Guardone
  • L. Castigliego
  • D. Gianfaldoni
  • A. Guidi


M13 universal non-homologous oligonucleotide tails incorporated into universal primers have been shown to improve amplification and sequencing performance. However, a few protocols use these tails in the field of food inspection. In this study, two types of M13 tails (by Steffens and Messing) were selected to assess their benefits using universal cytochrome oxidase subunit I (COI) and 16S ribosomal RNA gene (16SrRNA) primers in standard procedures. The primer characteristics were tested in silico. Then, using 20 DNA samples of edible species (birds, fishes, and mammals), their performance during PCR amplification (band recovery and intensity) and sequencing (sequence recovery, length, and Phred score) was assessed and compared. While 16SrRNA tailed and non-tailed primers performed similarly, differences were found for COI primers. Messing’s tails negatively affected the reaction outputs, while Steffens’ tails significantly improved the band intensity and the length of the final contigs based on the individual bidirectional read sequence. This different performance could be related to a destabilization effect of certain tails on primers with unfavorable mismatches on the annealing region. Even though our results cannot be generalized because the tail performances are strictly dependent on laboratory conditions, they show that appropriate tails can improve the overall throughput of the analysis, supporting food traceability.


Universal primers M13 oligonucleotide tails Tailed primers Species identification Amplification Sequencing 



The research was performed with funds granted from the University of Pisa.

Conflict of Interest

Armani Andrea declares that he has no conflict of interest. Giusti Alice declares that she has no conflict of interest. Guardone Lisa declares that she has no conflict of interest. Castigliego Lorenzo declares that he has no conflict of interest. Gianfaldoni Daniela declares that she has no conflict of interest. Guidi Alessandra declares that she has no conflict of interest. This article does not contain any studies with human or animal subjects.

Supplementary material

12161_2015_301_MOESM1_ESM.docx (32 kb)
Table 1SM (DOCX 32 kb)
12161_2015_301_MOESM2_ESM.docx (20 kb)
Table 2SM (DOCX 20 kb)
12161_2015_301_MOESM3_ESM.docx (20 kb)
Table 3SM (DOCX 19 kb)
12161_2015_301_MOESM4_ESM.docx (13 kb)
Table 4SM (DOCX 13 kb)


  1. Al-Soud WA, Rådström P (2000) Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces, and meat. J Clin Microbiol 38(12):4463–4470Google Scholar
  2. Armani A, Castigliego L, Tinacci L, Gianfaldoni D, Guidi A (2011) Molecular characterization of icefish, (Salangidae family), using direct sequencing of mitochondrial cytochrome b gene. Food Control 22(6):888–895CrossRefGoogle Scholar
  3. Armani A, Castigliego L, Guidi A (2012) Fish frauds: the DNA challenge. CAB Rev 7(071):1–12Google Scholar
  4. Armani A, Giusti A, Castigliego L, Rossi A, Tinacci L, Gianfaldoni D, Guidi A (2014) Pentaplex PCR as screening assay for jellyfish species identification in food products. J Agric Food Chem 62(50):12134–12143CrossRefGoogle Scholar
  5. Armani A, Guardone L, Castigliego L, D’Amico P, Messina A, Malandra R, Gianfaldoni D, Guidi A (2015a) DNA and mini-DNA barcoding for the identification of Porgies species (family Sparidae) of commercial interest on the international market. Food Control 50:589–596CrossRefGoogle Scholar
  6. Armani A, Tinacci L, Xiong X, Castigliego L, Gianfaldoni D, Guidi A (2015b) Fish species identification in canned pet food by BLAST and forensically informative nucleotide sequencing (FINS) analysis of short fragments of the mitochondrial 16s ribosomal RNA gene (16S rRNA). Food Control 50:821–830CrossRefGoogle Scholar
  7. Armani A, Guardone L, La Castellana R, Gianfaldoni D, Guidi A, Castigliego L (2015c) DNA barcoding reveals commercial and health issues in ethnic seafood sold on the Italian market. Food Control 55:206–214CrossRefGoogle Scholar
  8. Baldwin CC, Mounts JH, Smith DG, Weigt LA (2009) Genetic identification and color descriptions of early life-history stages of Belizean Phaeoptyx and Astrapogon (Teleostei: Apogonidae) with comments on identification of adult Phaeoptyx. Zootaxa 2008:1–22Google Scholar
  9. Barbuto M, Galimberti A, Ferri E, Labra M, Malandra R, Galli P, Casiraghi M (2010) DNA barcoding reveals fraudulent substitutions in shark seafood products: the Italian case of “palombo” (Mustelus spp.). Food Res Int 43(1):376–381CrossRefGoogle Scholar
  10. Bartlett SE, Davidson WS (1992) FINS (forensically informative nucleotide sequencing): a procedure for identifying the animal origin of biological specimens. Biotechniques 12(3):408–411Google Scholar
  11. Binladen J, Gilbert MTP, Campos PF, Willerslev E (2007) 5′-Tailed sequencing primers improve sequencing quality of PCR products. Biotechniques 42(2):174CrossRefGoogle Scholar
  12. Boutin-Ganache I, Raposo M, Raymond M, Deschepper CF (2001) M13-tailed primers improve the readability and usability of microsatellite analyses performed with two different allele-sizing methods. Biotechniques 31:24–28Google Scholar
  13. Carrera E, Garcia T, Cespedes A, Gonzalez I, Fernandez A, Asensio LM, Hernandez PE, Martin R (2000) Identification of smoked Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) using PCR-restriction fragment length polymorphism of the p53 gene. J AOAC Int 83(2):341–346Google Scholar
  14. Casiraghi M, Labra M, Ferri E, Galimberti A, De Mattia F (2010) DNA barcoding: a six-question tour to improve users’ awareness about the method. Brief Bioinform 11:440–453Google Scholar
  15. Cawthorn DM, Harris AS, Witthuhn RC (2012) DNA barcoding reveals a high incidence of fish species misrepresentation and substitution on the South African market. Food Res Int 46(1):30–40CrossRefGoogle Scholar
  16. De Maeseneire SL, Van Bogaert IN, Dauvrin T, Soetaert WK, Vandamme EJ (2007) Rapid isolation of fungal genomic DNA suitable for long distance PCR. Biotechnol Lett 29(12):1845–1855CrossRefGoogle Scholar
  17. Ewing B, Hillier L, Wendl MC, Green P (1998) Base-calling of automated sequencer traces using Phred. I. Accuracy assessment. Genome Res 8(3):175–185CrossRefGoogle Scholar
  18. Folmer O, Black M, Hoen RL, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294–299Google Scholar
  19. Galimberti A, De Mattia F, Losa A, Bruni I, Federici S, Casiraghi M, Labra M (2013) DNA barcoding as a new tool for food traceability. Food Res Int 50(1):55–63CrossRefGoogle Scholar
  20. Griffiths AM, Sotelo CG, Mendes R, Pérez-Martín RI, Schröder U, Shorten M, Mariani S (2014) Current methods for seafood authenticity testing in Europe: is there a need for harmonisation? Food Control 45:95–100CrossRefGoogle Scholar
  21. Gu H, Rajewsky K (2004) B cell protocols, vol 271. Humana Press Inc., TotowaCrossRefGoogle Scholar
  22. Hajibabaei M, Singer GA, Clare EL, Hebert PD (2007) Design and applicability of DNA arrays and DNA barcodes in biodiversity monitoring. BMC Biol 5(1):24CrossRefGoogle Scholar
  23. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  24. Handy SM, Deeds JR, Ivanova NV, Hebert PD, Hanner RH, Ormos A, Yancy HF (2011) A single-laboratory validated method for the generation of DNA barcodes for the identification of fish for regulatory compliance. J AOAC Int 94(1):201–210Google Scholar
  25. Hebert PD, Cywinska A, Ball SL (2003) Biological identifications through DNA barcodes. Proc R Soc Lond B Biol 270(1512):313–321CrossRefGoogle Scholar
  26. Hebert PD, Stoeckle MY, Zemlak TS, Francis CM (2004) Identification of birds through DNA barcodes. PLoS Biol 2(10), e312CrossRefGoogle Scholar
  27. Hillier L, Green P (1991) OSP: a computer program for choosing PCR and DNA sequencing primers. Genome Res 1(2):124–128CrossRefGoogle Scholar
  28. Ivanova NV, Zemlak TS, Hanner RH, Hebert PD (2007) Universal primer cocktails for fish DNA barcoding. Mol Ecol Notes 7(4):544–548CrossRefGoogle Scholar
  29. James SW, Porco D, Decaëns T, Richard B, Rougerie R, Erséus C (2010) DNA barcoding reveals cryptic diversity in Lumbricus terrestris L., 1758 (Clitellata): resurrection of L. herculeus (Savigny, 1826). PLoS One 5(12), e15629CrossRefGoogle Scholar
  30. Kämpke T, Kieninger M, Mecklenburg M (2001) Efficient primer design algorithms. Bioinformatics 17(3):214–225CrossRefGoogle Scholar
  31. Kermekchiev MB, Kirilova LI, Vail EE, Barnes WM (2009) Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples. Nucleic Acids Res. doi: 10.1093/nar/gkn1055 Google Scholar
  32. Kochzius M, Seidel C, Antoniou A, Botla SK, Campo D, Cariani A et al (2010) Identifying fishes through DNA barcodes and microarrays. PLoS One 5(9), e12620CrossRefGoogle Scholar
  33. Kool ET (2001) Hydrogen bonding, base stacking, and steric effects in DNA replication. Annu Rev Biophys Biomol 30(1):1–22CrossRefGoogle Scholar
  34. Kwok S, Chang SY, Sninsky JJ, Wang A (1995) Design and use of mismatched and degenerate primers. PCR primer: a laboratory manual. Cold Spring Harbor Laboratory, New York, pp 143–155Google Scholar
  35. Lang M, Orgogozo V (2011) Identification of homologous gene sequences by PCR with degenerate primers. Methods Mol Biol 772:245–256CrossRefGoogle Scholar
  36. Lindeman R, Hu SP, Volpato F, Trent RJ (1991) PCR mutagenesis enabling rapid non-radioactive detection of common beta-thalassemia mutations in Mediterraneans. Br J Haemaetol 78:100–104CrossRefGoogle Scholar
  37. Linhart C, Shamir R (2005) The degenerate primer design problem: theory and applications. J Comp Biol 12(4):431–456CrossRefGoogle Scholar
  38. Lockley AK, Bardsley RG (2000) DNA-based methods for food authentication. Trends Food Sci Technol 11:67–77CrossRefGoogle Scholar
  39. Lorenz JG, Jackson WE, Beck JC, Hanner R (2005) The problems and promise of DNA barcodes for species diagnosis of primate biomaterials. Philos Trans R Soc B 360(1462):1869–1877CrossRefGoogle Scholar
  40. Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78CrossRefGoogle Scholar
  41. Mikkelsen PM, Bieler R, Kappner I, Rawling TA (2006) Phylogeny of Veneroidea (Mollusca: Bivalvia) based on morphology and molecules. Zool J Linnean Soc 48(3):439–521CrossRefGoogle Scholar
  42. Missiaggia A, Grattapaglia D (2006) Plant microsatellite genotyping with 4-color fluorescent detection using multiple-tailed primers. Genet Mol Res 5:72–78Google Scholar
  43. Model P, Russel M (1988) In: Calendar R (ed) The bacteriophages. Plenum, New YorkGoogle Scholar
  44. Neilan BA, Wilton AN, Jacobs D (1997) A universal procedure for primer labelling of amplicons. Nucleic Acids Res 25(14):2938–2939CrossRefGoogle Scholar
  45. Regulation (EU) No 1379/2013 of the European Parliament and of the Council of 11 December 2013 on the common organisation of the markets in fishery and aquaculture products, amending Council Regulations (EC) No 1184/2006 and (EC) No 1224/2009 and repealing Council Regulation (EC) No 104/2000. Off J Eur Union L 354, p1–21Google Scholar
  46. Oetting WS, Lee HK, Flanders DJ, Wiesner GL, Sellers TA, King RA (1995) Linkage analysis with multiplexed short tandem repeat polymorphisms using infrared fluorescence and M13 tailed primers. Genomics 30(3):450–458CrossRefGoogle Scholar
  47. Palumbi SR (1996) Nucleic acids II: the polymerase chain reaction. Mol Syst 2(1):205–247Google Scholar
  48. Palumbi SR, Martin A, Romano S, McMillan WO, Stice L, Grabowski G (1991) The simple fool’s guide to PCR, version 2.0. Accessed 26 Mar 2015
  49. Park DS, Suh SJ, Oh HW, Hebert PD (2010) Recovery of the mitochondrial COI barcode region in diverse Hexapoda through tRNA-based primers. BMC Genomics 11(1):423CrossRefGoogle Scholar
  50. Porco D, Bedos A, Greenslade P, Janion C, Skarżyński D, Stevens MI, vanVuuren BJ, Deharveng L (2012) Challenging species delimitation in Collembola: cryptic diversity among common springtails unveiled by DNA barcoding. Invertebr Syst 26(6):470–477CrossRefGoogle Scholar
  51. Prosser SW, Velarde-Aguilar MG, León-Règagnon V, Hebert PD (2013) Advancing nematode barcoding: a primer cocktail for the cytochrome c oxidase subunit I gene from vertebrate parasitic nematodes. Mol Ecol Resour 13(6):1108–1115Google Scholar
  52. Ratnasingham S, Hebert P (2007) BOLD: the Barcode of Life Data System (http://www.barcodinglife. org). Mol Ecol Notes 7(3):355–364CrossRefGoogle Scholar
  53. Regier JC, Shi D (2005) Increased yield of PCR product from degenerate primers with nondegenerate, nonhomologous 5′ tails. BioTechniques 38(1):34–38CrossRefGoogle Scholar
  54. Report on the food crisis, fraud in the food chain and the control thereof 2013/2091 (INI), Committee on the Environment, Public Health and Food Safety, 4 December 2013.−//EP//TEXT+REPORT+A7-2013-0434+0+DOC+XML+V0//EN. Accessed 26 Mar 2015
  55. Rougerie R, Decaëns T, Deharveng L, Porco D, James SW, Chang CH, Richard B, Potapov M, Suhardjono Y, Hebert PD (2009) DNA barcodes for soil animal taxonomy. Pesq Agrop Brasileira 44(8):789–802CrossRefGoogle Scholar
  56. Roy R, Steffens DL, Gartside B, Jang GY, Brumbaugh JA (1996) Producing STR locus patterns from bloodstains and other forensic samples using an infrared fluorescent automated DNA sequencer. J Forensic Sci 41(3):418–424CrossRefGoogle Scholar
  57. Rudi K, Rud I, Holck A (2003) A novel multiplex quantitative DNA array based PCR (MQDA‐PCR) for quantification of transgenic maize in food and feed. Nucleic Acids Res 31(11):e62CrossRefGoogle Scholar
  58. Sambrook J, Russell DV (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  59. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74(12):5463–5467CrossRefGoogle Scholar
  60. Schreier PH, Cortese R (1979) A fast and simple method for sequencing DNA cloned in the single-stranded bacteriophage M13. J Mol Biol 129(1):169–172CrossRefGoogle Scholar
  61. Schuelke M (2000) An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol 18(2):233–234CrossRefGoogle Scholar
  62. Singh VK, Govindarajan R, Naik S, Kumar A (2000) The effect of hairpin structure on PCR amplification efficiency. Mol Biol Today 1(3):67–69Google Scholar
  63. Steffens DL, Roy R (1998) Sequence analysis of mitochondrial DNA hypervariable regions using infrared fluorescence detection. BioTechniques 24(6):1044–1047Google Scholar
  64. Steffens DL, Sutter SL, Roemer SC (1993) An alternate universal forward primer for improved automated DNA sequencing of M13. BioTechniques 15(4):580–582Google Scholar
  65. Stoev P, Akkari N, Zapparoli M, Porco D, Enghoff H, Edgecombe GD, Georgiev T, Penev L (2010) The centipede genus Eupolybothrus Verhoeff, 1907 (Chilopoda: Lithobiomorpha: Lithobiidae) in North Africa, a cybertaxonomic revision, with a key to all species in the genus and the first use of DNA barcoding for the group. ZooKeys 50:29–77CrossRefGoogle Scholar
  66. Teletchea F (2009) Molecular identification methods of fish species: reassessment and possible applications. Rev Fish Biol Fish 19(3):265–293CrossRefGoogle Scholar
  67. Van Den Hondel CA, Pennings L, Schoenmakers JG (1976) Restriction-enzyme-cleavage maps of bacteriophage M13. Existence of an intergenic region on the M13 genome. Eur J Biochem 68(1):55–70CrossRefGoogle Scholar
  68. Van Houdt JKJ, Breman FC, Virgilio M, De Meyer M (2010) Recovering full DNA barcodes from natural history collections of Tephritid fruitflies (Tephritidae, Diptera) using mini barcodes. Mol Ecol Resour 10(3):459–465CrossRefGoogle Scholar
  69. van Pelt-Verkuil E, Van Belkum A, Hays JP (2008) Principles and technical aspects of PCR amplification. Springer Science & Business MediaGoogle Scholar
  70. Wells JD, Pape T, Sperling FA (2001) DNA-based identification and molecular systematics of forensically important Sarcophagidae (Diptera). J Forensic Sci 46(5):1098–1102CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • A. Armani
    • 1
    Email author
  • A. Giusti
    • 1
  • L. Guardone
    • 1
  • L. Castigliego
    • 1
  • D. Gianfaldoni
    • 1
  • A. Guidi
    • 1
  1. 1.FishLab, Department of Veterinary SciencesUniversity of PisaPisaItaly

Personalised recommendations