Parasitology Research

, Volume 104, Issue 1, pp 79–84 | Cite as

Specific PCR assays for the identification of common anisakid nematodes with zoonotic potential

  • Q. Chen
  • H. Q. Yu
  • Z. R. Lun
  • X. G. Chen
  • H. Q. Song
  • R. Q. Lin
  • X. Q. Zhu
Original Paper


Based on the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) for six taxa of anisakids, namely, Anisakis simplex (s.s.), Anisakis typica, Anisakis pegreffii, Hysterothylacium aduncum, Hysterothylacium sp, and Contracaccum osculatum C, specific primers were designed in the ITS-1 and/or ITS-2 for each of the six anisakid taxa. These specific primers were used to develop polymerase chain reaction (PCR) tools for the identification of these anisakid taxa of sea fish by amplifying partial ITS-1 and/or ITS-2 of rDNA from anisakid nematodes. This approach allowed their specific identification, with no amplicons being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the DNA fragments amplified. The minimum amounts of DNA detectable using the PCR assays were 0.5–1 ng. These PCR tools were then applied to ascertain the specific identity of 143 anisakid larval samples collected from fish in China, Canada, Thailand, and Indonesia, and these anisakid samples were identified to represent one of the six anisakid taxa. These PCR assays based on ITS sequences should provide useful molecular tools for the accurate identification and molecular epidemiological investigations of anisakid infections in humans and fish.


Polymerase Chain Reaction Assay Specific Polymerase Chain Reaction Anisakiasis Anisakid Nematode Specific Polymerase Chain Reaction Product 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Project support was provided by a grant from the Program for Changjiang Scholars and Innovative Research Team in University (Garnt No. IRT0723) to XQZ. The authors are grateful to Dr. M. Podolska of Sea Fisheries Institute, Poland for providing some anisakid samples from Poland. The experiments comply with the current laws of the countries in which the experiments were performed.


  1. Abollo E, Gestal C, Pascual S (2001) Anisakis infestation in marine fish and cephalopods from Galician waters: an updated perspective. Parasitol Res 87:492–499PubMedCrossRefGoogle Scholar
  2. Adams AM, Murrell KD, Cross JH (1997) Parasites of fish and risks to public health. Rev Sci Tech Off Int Epizoot 16:652–660Google Scholar
  3. Chai JY, Murrell KD, Lymbery AJ (2005) Fish-borne parasitic zoonoses: status and issues. Int J Parasitol 35:1233–1254PubMedCrossRefGoogle Scholar
  4. Costa G, Pontes T, Mattiucci S (2003) The occurrence and infection dynamics of anisakis larvae in the black-scabbard fish, Aphanopus carbo, chub mackerel, Scomber japonicus, and oceanic horse mackerel, Trachurus from Madeira, Portugal. J Helminthol 77:163–166PubMedCrossRefGoogle Scholar
  5. Couture C, Measures L, Gagnon J, Desbiens C (2003) Human intestinal anisakiosis due to consumption of raw salmon. Am J Surg Pathol 27:1167–1172PubMedCrossRefGoogle Scholar
  6. D’Amelio S, Mathiopoulos KD, Santos CP, Pugachev ON, Webb SC, Picanço M, Paggi L (2000) Genetic markers in ribosomal DNA for the identification of members of the genus Anisakis (Nematoda: ascaridoidea) defined by polymerase-chain-reaction-based restriction fragment length polymorphism. Int J Parasitol 30:223–226PubMedCrossRefGoogle Scholar
  7. Dick TA, Dixon BR, Choudhury A (1991) Diphyllobothrium, Anisakis and other fish-borne parasitic zoonoses. Southeast Asian J Trop Med Public Health 22(suppl):150–152PubMedGoogle Scholar
  8. Doupe RG, Lymbery AJ, Wong S, Hobbs RP (2003) Larval anisakid infections of some tropical fish species from north-west Australia. J Helminthol 77:363–365PubMedCrossRefGoogle Scholar
  9. Fagerholm HP (1988) Incubation in rats of a nematodal larva from cod to establish its specific identity: Contracaecum osculatum (Rudolphi). Parasitol Res 75:57–63PubMedCrossRefGoogle Scholar
  10. Gasser RB (2006) Molecular tools—advances, opportunities and prospects. Vet Parasitol 136:69–89PubMedCrossRefGoogle Scholar
  11. Hartwich G (1974) Keys to genera of the ascaridoidea. In: Anderson RC, Chabaud AG, Willmott S (eds) CIH keys to the nematode parasites of vertebrates. No. 2. Commonwealth Agricultural Bureaux. Farnham Royal, Bucks, UK, pp 1–15Google Scholar
  12. Li MW, Lin RQ, Chen HH, Sani RA, Song HQ, Zhu XQ (2007) PCR tools for the verification of the specific identity of ascaridoid nematodes from dogs and cats. Mol Cell Probes 21:349–354PubMedCrossRefGoogle Scholar
  13. Lin RQ, Dong SJ, Nie K, Wang CR, Li AX, Song HQ, Huang WY, Zhu XQ (2007) Sequence analysis of the first internal transcribed spacer of rDNA supports the existence of an intermediate Fasciola between F. hepatica and F. gigantica in mainland China. Parasitol Res 101:813–817PubMedCrossRefGoogle Scholar
  14. Maggi P, Caputi-Iambrenghi O, Scardigno A, Scoppetta L, Saracino A, Valente M, Pastore G, Angarano G (2000) Gastrointestinal infection due to Anisakis simplex in southern Italy. Eur J Epidemiol 16:75–78PubMedCrossRefGoogle Scholar
  15. Masiga DK, Tait A, Turner CM (2000) Amplified restriction fragment length polymorphism in parasite genetics. Parasitol Today 16:350–353PubMedCrossRefGoogle Scholar
  16. Mattiucci S, Nascetti G, Cianchi R, Paggi L, Arduino P, Margolis L, Brattey J, Webb S, D’Amelio S, Orecchia P, Bullini L (1997) Genetic and ecological data on the Anisakis simplex complex, with evidence for a new species (Nematoda, Ascaridoidea, Anisakidae). J Parasitol 83:401–416PubMedCrossRefGoogle Scholar
  17. McCarthy J, Moore TA (2000) Emerging helminth zoonoses. Int J Parasitol 30:1351–1360PubMedCrossRefGoogle Scholar
  18. Nascetti G, Cianchi R, Mattiucci S, D’Amelio S, Orecchia P, Paggi L, Brattey J, Berland B, Smith JW, Bullini L (1993) Three sibling species within Contracaecum osculatum (Nematoda, Ascaridida, Ascaridoidea) from the Atlantic Arctic-Boreal region: reproductive isolation and host preferences. Int J Parasitol 23:105–120PubMedCrossRefGoogle Scholar
  19. Olson AC, Lewis MD, Hauser ML (1983) Proper identification of Anisakine worms. Am J Med Technol 49:111–114PubMedGoogle Scholar
  20. Pellegrini M, Occhini R, Tordini G, Vindigni C, Russo S, Marzocca G (2005) Acute abdomen due to small bowel anisakiasis. Dig Liver Dis 37:65–67PubMedCrossRefGoogle Scholar
  21. Prichard R, Tait A (2001) The role of molecular biology in veterinary parasitology. Vet Parasitol 98:169–194PubMedCrossRefGoogle Scholar
  22. Rohlwing T, Palm HW, Rosenthal H (1998) Parasitation with Pseudoterranova decipiens (Nematoda) influences the survival rate of the European smelt Osmerus eperlanus retained by a screen wall of a nuclear power plant. Dis Aquat Organ 32:233–236PubMedCrossRefGoogle Scholar
  23. Ruan TQ, Zhang HM (2007) Review on prevalence of anisakid infection in sea fish in China. Chin J Zoon 23:948–949, (in Chinese)Google Scholar
  24. Szostakowska B, Myjak P, Kur J (2002) Identification of anisakid nematodes from the Southern Baltic Sea using PCR-based methods. Mol Cell Probes 16:111–118PubMedCrossRefGoogle Scholar
  25. Tang LQ, Zhang XL, Guo XA, Zhang MS, Liao YM (2001) Investigation of larval anisakids in sea fish sold in Shenzhen market. Chin J Zoon 17:103–104, (in Chinese)Google Scholar
  26. Umehara A, Kawakami Y, Araki J, Uchida A (2008) Multiplex PCR for the identification of Anisakis simplex sensu stricto, Anisakis pegreffii and the other anisakid nematodes. Parasitol Int 57:49–53PubMedCrossRefGoogle Scholar
  27. Zhang L, Hu M, Shamsi S, Beveridge I, Li H, Xu Z, Li L, Cantacessi C, Gasser RB (2007) The specific identification of anisakid larvae from fishes from the Yellow Sea, China, using mutation scanning-coupled sequence analysis of nuclear ribosomal DNA. Mol Cell Probes 21:386–390PubMedCrossRefGoogle Scholar
  28. Zhou P, Chen N, Zhang RL, Lin RQ, Zhu XQ (2008) Food-borne parasitic zoonoses in China: perspective for control. Trends Parasitol 24:190–196PubMedCrossRefGoogle Scholar
  29. Zhu XQ, Gasser RB, Podolska M, Chilton NB (1998) Characterisation of anisakid nematodes with zoonotic potential by nuclear ribosomal DNA sequences. Int J Parasitol 28:1911–1921PubMedCrossRefGoogle Scholar
  30. Zhu XQ, D’Amelio S, Hu M, Paggi L, Gasser RB (2001) Electrophoretic detection of population variation within Contracaecum ogmorhini (Nematoda: Ascaridoidea: Anisakidae). Electrophoresis 22:1930–1934PubMedCrossRefGoogle Scholar
  31. Zhu XQ, Podolska M, Liu JS, Yu HQ, Chen HH, Lin ZX, Luo CB, Song HQ, Lin RQ (2007a) Identification of anisakid nematodes with zoonotic potential from Europe and China by single-strand conformation polymorphism analysis of nuclear ribosomal DNA. Parasitol Res 101:1703–1707PubMedCrossRefGoogle Scholar
  32. Zhu XQ, D’Amelio S, Gasser RB, Yang TB, Paggi L, He F, Lin RQ, Song HQ, Ai L, Li AX (2007b) Practical PCR tools for the delineation of Contracaecum rudolphii A and Contracaecum rudolphii B (Ascaridoidea: Anisakidae) using genetic markers in nuclear ribosomal DNA. Mol Cell Probes 21:97–102PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Q. Chen
    • 1
  • H. Q. Yu
    • 2
  • Z. R. Lun
    • 3
  • X. G. Chen
    • 4
  • H. Q. Song
    • 1
  • R. Q. Lin
    • 1
  • X. Q. Zhu
    • 1
  1. 1.College of Veterinary MedicineSouth China Agricultural UniversityGuangzhouPeople’s Republic of China
  2. 2.Laboratory of Animal QuarantineGuangzhou Entry–Exit Inspection and QuarantineZhujiang New City, GuangzhouPeople’s Republic of China
  3. 3.Center for Parasitic Organisms, State Key Laboratory of Biocontrol, School of Life SciencesSun Yat-Sen UniversityGuangzhouPeople’s Republic of China
  4. 4.Department of ParasitologySchool of Public Health and Tropical Medicine, Southern Medical UniversityGuangzhouPeople’s Republic of China

Personalised recommendations