Skip to main content

Advertisement

SpringerLink
Log in

High resolution melting technique for molecular epidemiological studies of cystic echinococcosis: differentiating G1, G3, and G6 genotypes of Echinococcus granulosus sensu lato

  • Original Paper
  • Published:
Parasitology Research Aims and scope Submit manuscript

Abstract

Reliable and rapid genotyping of large number of Echinococcus granulosus sensu lato isolates is crucial for understanding the epidemiology and transmission of cystic echinococcosis. We have developed a method for distinguishing and discriminating common genotypes of E. granulosus s.l. (G1, G3, and G6) in Iran. This method is based on polymerase chain reaction coupled with high resolution melting curve (HRM), ramping from 70 to 86 °C with fluorescence data acquisition set at 0.1 °C increments and continuous fluorescence monitoring. Consistency of this technique was assessed by inter- and intra-assays. Assessment of intra- and inter-assay variability showed low and acceptable coefficient of variations ranging from 0.09 to 0.17 %. Two hundred and eighty E. granulosus s.l. isolates from sheep, cattle, and camel were used to evaluate the applicability and accuracy of the method. The isolates were categorized as G1 (93, 94, and 25 %), G3 (7, 4, and 4 %), and G6 (0, 2, and 71 %) for sheep, cattle, and camel, respectively. HRM results were completely compatible with those obtained from sequencing and rostellar hook measurement. This method proved to be a valuable screening tool for large-scale molecular epidemiological studies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Addy F, Alakonya A, Wamae N et al (2012) Prevalence and diversity of cystic echinococcosis in livestock in Maasailand, Kenya. Parasitol Res 111(6):2289–2294

    Article  PubMed  Google Scholar 

  • Andriantsoanirina V, Lascombes V, Ratsimbasoa A et al (2009) Rapid detection of point mutations in Plasmodium falciparum genes associated with antimalarial drugs resistance by using high-resolution melting analysis. J Microbiol Methods 78(2):165–170

    Article  PubMed  CAS  Google Scholar 

  • Bagheri R, Haghi SZ, Amini M et al (2011) Pulmonary hydatid cyst: analysis of 1,024 cases. Gen Thorac Cardiovasc Surg 59(2):105–109

    Article  PubMed  Google Scholar 

  • Bastien R, Lewis TB, Hawkes JE et al (2008) High throughput amplicon scanning of the TP53 gene in breast cancer using high resolution fluorescent melting curve analyses and automatic mutation calling. Hum Mutat 29(5):757–764

    Article  PubMed  CAS  Google Scholar 

  • Bowles J, Blair D, McManus DP (1992) Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing. Mol Biochem Parasitol 54(2):165–173

    Article  PubMed  CAS  Google Scholar 

  • Bowles J, McManus DP (1993) NADH dehydrogenase 1 gene sequences compared for species and strains of the genus Echinococcus. Int J Parasitol 23(7):969–972

    Article  PubMed  CAS  Google Scholar 

  • Breyer I, Georgieva D, Kurdova R et al (2004) Echinococcus granulosus strain typing in Bulgaria: the G1 genotype is predominant in intermediate and definitive wild hosts. Parasitol Res 93(2):127–130

    Article  PubMed  Google Scholar 

  • Budke CM, Deplazes P, Torgerson PR (2006) Global socioeconomic impact of cystic echinococcosis. Emerg Infect Diseases 12(2):296

    Article  Google Scholar 

  • Cardona GA, Carmena DA (2013) Review of the global prevalence, molecular epidemiology and economics of cystic echinococcosis in production animals. Vet Parasitol 192(1–3):10–32

    Article  PubMed  Google Scholar 

  • Costa JM, Cabaret O, Moukoury S et al (2011) Genotyping of the protozoan pathogen Toxoplasma gondii using high-resolution melting analysis of the repeated B1 gene. J Microbiol Meth 86(3):357–363

    Article  CAS  Google Scholar 

  • Gasser RB (2006) Molecular tool advances, opportunities and prospects. Vet Parasitol 136(2):69–89

    Article  PubMed  CAS  Google Scholar 

  • Hajialilo E, Harandi MF, Sharbatkhori M et al (2012) Genetic characterization of Echinococcus granulosus in camels, cattle and sheep from the south-east of Iran indicates the presence of the G3 genotype. J Helminthol 86(3):263

    Article  PubMed  CAS  Google Scholar 

  • Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  • Harandi MF, Budke CM, Rostami S (2012) The monetary burden of cystic echinococcosis in Iran. PLoS Negl Trop Dis 6(11):e1915

    Article  Google Scholar 

  • Harandi MF, Hobbs RP, Adams PJ et al (2002) Molecular and morphological characterization of Echinococcus granulosus of human and animal origin in Iran. Parasitology 125(Pt 4):367–373

    PubMed  CAS  Google Scholar 

  • Kia EB, Rahimi H, Sharbatkhori M et al (2010) Genotype identification of human cystic echinococcosis in Isfahan, central Iran. Parasitol Res 107(3):757–760

    Article  PubMed  Google Scholar 

  • Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16(2):111–120

    Article  PubMed  CAS  Google Scholar 

  • Krypuy M, Ahmed A, Etemadmoghadam D et al (2007) High resolution melting for mutation scanning of TP53 exons 5–8. BMC Cancer 7(1):168

    Article  PubMed  Google Scholar 

  • Kumar S, Nei M, Dudley J et al (2008) MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinform 9(4):299–306

    Article  PubMed  CAS  Google Scholar 

  • Lymbery AJ, Thompson RCA (2012) The molecular epidemiology of parasite infections: tools and applications. Mol Biochem Parasitol 181(2):102–116

    Article  PubMed  CAS  Google Scholar 

  • Ma J, Wang H, Lin G et al (2012) Molecular identification of Echinococcus species from eastern and southern Qinghai, China, based on the mitochondrial cox1 gene. Parasitol Res 111(1):179–184

    Article  PubMed  Google Scholar 

  • Maurelli MP, Rinaldi L, Capuano F et al (2009) Development of a real-time PCR for the differentiation of the G1 and G2/G3 genotypes of Echinococcus granulosus. Parasitol Res 105(1):255–259

    Article  PubMed  Google Scholar 

  • Millat G, Chanavat V, Rodriguez-Lafrasse C et al (2009) Rapid, sensitive and inexpensive detection of SCN5A genetic variations by high resolution melting analysis. Clin Biochem 42(6):491–499

    Article  PubMed  CAS  Google Scholar 

  • Maillard S, Benchikh-Elfegoun MC, Knapp J et al (2007) Taxonomic position and geographical distribution of the common sheep G1 and camel G6 strains of Echinococcus granulosus in three African countries. Parasitol Res 100(3):495–503

    Article  PubMed  CAS  Google Scholar 

  • Murray CJL, Vos T, Lozano R et al (2012) Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990–2010: a systematic analysis for the Global burden of disease study 2010. Lancet 380(9859):2197–2223

    Article  PubMed  Google Scholar 

  • Nakao M, Yanagida T, Okamoto M et al (2010) State-of-the-art Echinococcus and Taenia phylogenetic taxonomy of human-pathogenic tapeworms and its application to molecular diagnosis. Infect Genet Evol 10(4):444–452

    Article  PubMed  CAS  Google Scholar 

  • Nasereddin A, Jaffe CL (2010) Rapid diagnosis of Old World Leishmaniasis by high-resolution melting analysis of the 7SL RNA gene. J Clin Microbiol 48(6):2240–2242

    Article  PubMed  CAS  Google Scholar 

  • Ngui R, Lim YAL, Chua KH (2012) Rapid detection and identification of human hookworm infections through high resolution melting (HRM) analysis. PLoS One 7(7):e41996

    Article  PubMed  CAS  Google Scholar 

  • Pangasa A, Jex AR, Campbell BE et al (2009) High resolution melting-curve (HRM) analysis for the diagnosis of cryptosporidiosis in humans. Mol Cell Probes 23(1):10–15

    Article  PubMed  CAS  Google Scholar 

  • Sharbatkhori M, Fasihi Harandi M, Mirhendi H et al (2011) Sequence analysis of cox1 and nad1 genes in Echinococcus granulosus G3 genotype in camels (Camelus dromedarius) from central Iran. Parasitol Res 1–7

  • Sharbatkhori M, Mirhendi H, Jex AR et al (2009) Genetic categorization of Echinococcus granulosus from humans and herbivorous hosts in Iran using an integrated mutation scanningphylogenetic approach. Electrophoresis 30(15):2648–2655

    Article  PubMed  CAS  Google Scholar 

  • Tamura K, Peterson D, Peterson N et al (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739

    Article  PubMed  CAS  Google Scholar 

  • Thompson JD, Gibson TJ, Plewniak F et al (1997) The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25(24):4876–4882

    Article  PubMed  CAS  Google Scholar 

  • Thompson RCA (2008) The taxonomy, phylogeny and transmission of Echinococcus. Exp Parasitol 119(4):439–446

    Article  PubMed  CAS  Google Scholar 

  • Thompson RCA, McManus DP (2002) Towards a taxonomic revision of the genus Echinococcus. Trends Parasitol 18(10):452–457

    Article  PubMed  Google Scholar 

  • Zhang P, Liu Y, Alsarakibi M et al (2011) Application of HRM assays with EvaGreen dye for genotyping Giardia duodenalis zoonotic assemblages. Parasitol Res 1–7

Download references

Acknowledgments

The authors wish to thank all veterinary staff of different abattoirs that help collecting parasite specimen for this study. This work is carried out as a part of a Ph.D. thesis done by Sima Rostami and was financially supported by the vice-chancellor for Research, Kerman University of Medical Sciences, grant no. 90-107.

Author information

Authors and Affiliations

  1. Research Center of Tropical and Infectious Diseases, Kerman University of Medical Sciences, Kerman, Iran

    Sima Rostami, Zahra Babaei & Naser Ziaali

  2. Department of Medical Parasitology, School of Medicine, Kerman University of Medical Sciences, Kerman, 76169-14111, Iran

    Sima Rostami, Zahra Babaei & Naser Ziaali

  3. Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran

    Saeed Talebi

  4. Infectious Diseases Research Center, Department of Medical Parasitology and Mycology, Golestan University of Medical Sciences, Gorgan, Iran

    Mitra Sharbatkhori

  5. Shariati Hospital, Karaj, Alborz, Iran

    Habib Rostami

  6. Research Center for Hydatid Disease in Iran, Kerman University of Medical Sciences, Kerman, 76169-14111, Iran

    Majid Fasihi Harandi

Authors
  1. Sima Rostami
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Saeed Talebi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zahra Babaei
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mitra Sharbatkhori
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Naser Ziaali
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Habib Rostami
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Majid Fasihi Harandi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Majid Fasihi Harandi.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rostami, S., Talebi, S., Babaei, Z. et al. High resolution melting technique for molecular epidemiological studies of cystic echinococcosis: differentiating G1, G3, and G6 genotypes of Echinococcus granulosus sensu lato. Parasitol Res 112, 3441–3447 (2013). https://doi.org/10.1007/s00436-013-3523-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00436-013-3523-z

Keywords

Search

Navigation