High-Resolution Melting Analysis as an Appropriate Method to Differentiate between Fasciola hepatica and F. gigantica

Background: Fasciolosis is a shared disease between humans and livestock caused by hepatic trematodes; Fasciola hepatica and F. gigantica. Differentiate between the two species of this genus is essential. High-Resolution Melting (HRM) Analysis represents a new approach to this issue. This method can be performed right after termination of Real-Time PCR. This technique has not been used for identification of adult F. hepatica and F. gigantica genotypes. The aim of this study was to determine Fasciola species by using HRM in isolates taken from Iran, respectively. Methods: Ninety-three Fasciola spp. samples were collected from infected slaughtered animals in different regions of Iran, including North West (Ardebil Province) and South East (Zahedan Province) during 2016. Genomic DNA from the samples was extracted using a DNA extraction kit and then after Real-Time PCR amplification, HRM was done. Results: Overall, 59 and 34 isolates were identified as F. hepatica and F. gigantica, respectively. The percentages of each species from animals were as follows: sheep (F. hepatica, 80.39% and F. gigantica, 19.61%), cattle (F. hepatica, 42.85% and F. gigantica, 57.15%). Conclusion: HRM technique developed in the present study is a powerful, rapid and sensitive technique for epidemiological survey and molecular identification between F. hepatica and F. gigantica.

[1]  M. Rokni,et al.  Molecular Evidence of Human Fasciolosis Due to Fasciola gigantica in Iran: A Case Report , 2018, Iranian journal of public health.

[2]  I. Aroch,et al.  Detection and quantification of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocercosis , 2017, Parasites & Vectors.

[3]  M. Rokni,et al.  Molecular and Morphometrical Characterization of Fasciola Species Isolated from Domestic Ruminants in Ardabil Province, Northwestern Iran , 2017, Iranian journal of public health.

[4]  R. Mohammadi,et al.  First report of Tasmanian sheep strain (G2) genotype isolated from Iranian goat using the high resolution melting (HRM) analysis , 2016, Gastroenterology and hepatology from bed to bench.

[5]  H. Rahimi,et al.  Molecular Characterization of Animal Fasciola spp. Isolates from Kermanshah, Western Iran , 2016, Iranian journal of public health.

[6]  Majid Fasihi Harandi,et al.  Rapid Identification of Echinococcus granulosus and E. canadensis Using High-Resolution Melting (HRM) Analysis by Focusing on a Single Nucleotide Polymorphism. , 2016, Japanese journal of infectious diseases.

[7]  R. Mahmud,et al.  Detection of Helminth Eggs and Identification of Hookworm Species in Stray Cats, Dogs and Soil from Klang Valley, Malaysia , 2015, PloS one.

[8]  M. Rokni,et al.  Serological study of Human Fasciolosis in Patients Referring to the School of Public Health, Tehran University of Medical Sciences, Tehran, Iran during 2008–2014 , 2015, Iranian journal of parasitology.

[9]  H. C. Chai,et al.  Development of High Resolution Melting Analysis for the Diagnosis of Human Malaria , 2015, Scientific Reports.

[10]  Guo-Hua Liu,et al.  Rapid Detection and Differentiation of Clonorchis sinensis and Opisthorchis viverrini Using Real-Time PCR and High Resolution Melting Analysis , 2014, TheScientificWorldJournal.

[11]  M. Rokni,et al.  Genotyping and Phylogenetic Analysis of Fasciola Spp. Isolated from Sheep and Cattle Using PCR-RFLP in Ardabil Province, Northwestern Iran , 2014, Iranian journal of public health.

[12]  S. Rafati,et al.  Neglected Tropical Diseases - Middle East and North Africa , 2014, Neglected Tropical Diseases.

[13]  J. Ramírez,et al.  Identification of Trypanosoma cruzi Discrete Typing Units (DTUs) through the implementation of a High-Resolution Melting (HRM) genotyping assay , 2013, Parasites & Vectors.

[14]  Yawen Li,et al.  Application of HRM assays with EvaGreen dye for genotyping Giardia duodenalis zoonotic assemblages , 2012, Parasitology Research.

[15]  K. Chua,et al.  Rapid Detection and Identification of Human Hookworm Infections through High Resolution Melting (HRM) Analysis , 2012, PloS one.

[16]  T. Le,et al.  Development and Evaluation of a Single-Step Duplex PCR for Simultaneous Detection of Fasciola hepatica and Fasciola gigantica (Family Fasciolidae, Class Trematoda, Phylum Platyhelminthes) , 2012, Journal of Clinical Microbiology.

[17]  S. Alasaad,et al.  A TaqMan real-time PCR-based assay for the identification of Fasciola spp. , 2011, Veterinary parasitology.

[18]  S. Şimşek,et al.  Molecular differentiation of Turkey cattle isolates of Fasciola hepatica and Fasciola gigantica , 2011, Helminthologia.

[19]  M. Rokni,et al.  Identification and differentiation of Fasciola hepatica and Fasciola gigantica using a simple PCR-restriction enzyme method. , 2010, Experimental parasitology.

[20]  L. Kádasi,et al.  Development of high-resolution melting (HRM) analysis for population studies of Fascioloides magna (Trematoda: Fasciolidae), the giant liver fluke of ruminants , 2010, Parasitology Research.

[21]  A. Jex,et al.  High resolution melting-curve (HRM) analysis for the diagnosis of cryptosporidiosis in humans. , 2009, Molecular and cellular probes.

[22]  R. Caldeira,et al.  Isolation and detection of Fasciola hepatica DNA in Lymnaea viatrix from formalin-fixed and paraffin-embedded tissues through multiplex-PCR. , 2008, Veterinary parasitology.

[23]  U. Braun,et al.  Prevalence of Fasciola hepatica in the intermediate host Lymnaea truncatula detected by real time TaqMan PCR in populations from 70 Swiss farms with cattle husbandry. , 2007, Veterinary parasitology.

[24]  S. Mas‐Coma,et al.  Fascioliasis and other plant-borne trematode zoonoses. , 2005, International journal for parasitology.

[25]  K. Magalhães,et al.  Detection of Lymnaea columella infection by Fasciola hepatica through Multiplex-PCR. , 2004, Memorias do Instituto Oswaldo Cruz.

[26]  X. Zhu,et al.  Characterisation of Fasciola species from Mainland China by ITS-2 ribosomal DNA sequence. , 2004, Veterinary parasitology.

[27]  A. Marcilla,et al.  A PCR-RFLP assay for the distinction between Fasciola hepatica and Fasciola gigantica. , 2002, Molecular and cellular probes.

[28]  T. Schnieder,et al.  Sequence heterogeneity in a repetitive DNA element of Fasciola. , 1998, International journal for parasitology.

[29]  堤 可厚,et al.  Characterization of Genetic Divergence Among Species within the Genus Fasciola by PCR-SSCP , 1995 .