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Licensed Unlicensed Requires Authentication Published by De Gruyter July 5, 2017

Differentiation of Toxocara canis and Toxocara cati based on PCR-RFLP analyses of rDNA-ITS and mitochondrial cox1 and nad1 regions

Fattaneh Mikaeili, Alexander Mathis, Peter Deplazes, Hossein Mirhendi, Afshin Barazesh, Sepideh Ebrahimi and Eshrat Beigom Kia
From the journal Acta Parasitologica

Abstract

The definitive genetic identification of Toxocara species is currently based on PCR/sequencing. The objectives of the present study were to design and conduct an in silico polymerase chain reaction-restriction fragment length polymorphism method for identification of Toxocara species. In silico analyses using the DNASIS and NEBcutter softwares were performed with rDNA internal transcribed spacers, and mitochondrial cox1 and nad1 sequences obtained in our previous studies along with relevant sequences deposited in GenBank. Consequently, RFLP profiles were designed and all isolates of T. canis and T. cati collected from dogs and cats in different geographical areas of Iran were investigated with the RFLP method using some of the identified suitable enzymes. The findings of in silico analyses predicted that on the cox1 gene only the MboII enzyme is appropriate for PCR-RFLP to reliably distinguish the two species. No suitable enzyme for PCR-RFLP on the nad1 gene was identified that yields the same pattern for all isolates of a species. DNASIS software showed that there are 241 suitable restriction enzymes for the differentiation of T. canis from T. cati based on ITS sequences. RsaI, MvaI and SalI enzymes were selected to evaluate the reliability of the in silico PCR-RFLP. The sizes of restriction fragments obtained by PCR-RFLP of all samples consistently matched the expected RFLP patterns. The ITS sequences are usually conserved and the PCR-RFLP approach targeting the ITS sequence is recommended for the molecular differentiation of Toxocara species and can provide a reliable tool for identification purposes particularly at the larval and egg stages.

Acknowledgements

The authors would like to thank Jeannine Hauri, laboratory technician of the vector entomology unit at the institute of parasitology, University of Zurich for kind assistance. This research has been partly supported by the Tehran University of Medical Sciences (Project No. 90-02-27-14163) and Shiraz University of Medical Sciences (Project No. 93-7422). The authors declare that there is no conflict of interests.

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Received: 2016-7-26
Revised: 2017-3-2
Accepted: 2017-4-7
Published Online: 2017-7-5
Published in Print: 2017-9-26

© 2017 W. Stefański Institute of Parasitology, PAS