Parasitology Research

, Volume 118, Issue 12, pp 3469–3478 | Cite as

Development and evaluation of high-resolution melting curve analysis for rapid detection and subtyping of Blastocystis and comparison the results with sequencing

  • Hanieh Mohammad Rahimi
  • Hamed MirjalaliEmail author
  • Maryam NiyyatiEmail author
  • Ali Haghighi
  • Hamid Asadzadeh Aghdaei
  • Mohammad Reza Zali
Protozoology - Original Paper


Blastocystis is a prevalent parasite that has a wide distribution. In order to design HRM real-time PCR, primers were selected from SSU rRNA gene to amplify specific fragment with different melting temperatures for each subtype of Blastocystis. Subsequently, HRM real-time PCR was performed and melting curve analysis was done by Rotor-Gene Q software. The results of HRM real-time PCR was then compared with sequence results of “barcoding region” of SSU rRNA gene of Blastocystis. To evaluate sensitivity of test, 10-fold serial dilutions of the parasite were prepared from ~ 106 to 1 parasite per mL of stool sample and were investigated by HRM real-time PCR. In order to determine specificity of method, HRM real-time PCR was done for some microorganisms and Blastocystis-negative stool samples. In silico analysis showed that all seventeen subtypes of Blastocystis were distinguish. In vitro analysis revealed that the test discriminated subtypes with specific melting temperatures.


Blastocystis Subtypes 18S ribosomal RNA gene High-resolution melting curve analysis 



High-resolution melting curve




Ribosomal RNA



This paper has been extracted from the thesis written by Mrs. Hanieh Mohammad Rahimi who is studying in Shahid Beheshti University of Medical Science. The authors thank all members of Foodborne and Waterborne Diseases Research Center for their collaborations.

Availability of data and materials

The data associated with this manuscript consisted of normalized melting curves are included in the article. Furthermore, we used from available isolates in our laboratory that were previously sequenced and subtyped.

Authors’ contributions

HM conceived and designed the experiments. HMR performed the experiments. HM and HMR analyzed the data. MN, AH, HAA, and MRZ contributed reagents/materials/analysis tools/positive samples.HM, HMR, and MN wrote the paper. All authors read and approved the final version of the manuscript.

Funding information

This study was financially supported by Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran (grant no. 970).

Compliance with ethical standards

Ethics approval and consent to participate

All procedures performed in this study were in accordance with the ethical standards (IR.SBMU.RIGLD.REC.1396.163) released by Ethical Review Committee of the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Consent is not applicable.

Informed consent

Not applicable.

Competing interest

The authors declare that they have no conflict of interest.


  1. Alfellani MA, Jacob AS, Perea NO, Krecek RC, Taner-Mulla D, Verweij JJ, Levecke B, Tannich E, Clark CG, Stensvold CR (2013a) Diversity and distribution of Blastocystis sp. subtypes in non-human primates. Parasitology 140(8):966–971PubMedGoogle Scholar
  2. Alfellani MA, Stensvold CR, Vidal-Lapiedra A, Onuoha ES, Fagbenro-Beyioku AF, Clark CG (2013b) Variable geographic distribution of Blastocystis subtypes and its potential implications. Acta Trop 126(1):11–18PubMedGoogle Scholar
  3. Alfellani MA, Taner-Mulla D, Jacob AS, Imeede CA, Yoshikawa H, Stensvold CR, Clark CG (2013c) Genetic diversity of Blastocystis in livestock and zoo animals. Protist 164(4):497–509PubMedGoogle Scholar
  4. Alinaghizade A, Mirjalali H, Mohebali M, Stensvold CR, Rezaeian M (2017) Inter- and intra-subtype variation of Blastocystis subtypes isolated from diarrheic and non-diarrheic patients in Iran. Infect Genet Evol 50:77–82PubMedGoogle Scholar
  5. Andersen LO, Stensvold CR (2016) Blastocystis in health and disease: are we moving from a clinical to a public health perspective? J Clin Microbiol 54(3):524–528PubMedPubMedCentralGoogle Scholar
  6. Bart A, Wentink-Bonnema EM, Gilis H, Verhaar N, Wassenaar CJ, van Vugt M et al (2013) Diagnosis and subtype analysis of Blastocystis sp. in 442 patients in a hospital setting in the Netherlands. BMC Infect Dis 13:389PubMedPubMedCentralGoogle Scholar
  7. Basak S, Rajurkar MN, Mallick SK (2014) Detection of Blastocystis hominis: a controversial human pathogen. Parasitol Res 113(1):261–265PubMedGoogle Scholar
  8. Bustin S, Huggett J (2017) qPCR primer design revisited. Biomolecular Detection and Quantification 14:19–28PubMedPubMedCentralGoogle Scholar
  9. Clark CG (1997) Extensive genetic diversity in Blastocystis hominis. Mol Biochem Parasitol 87(1):79–83PubMedGoogle Scholar
  10. Clark CG, van der Giezen M, Alfellani MA, Stensvold CR (2013) Recent developments in Blastocystis research. Adv Parasitol 82:1–32PubMedGoogle Scholar
  11. Coyle CM, Varughese J, Weiss LM, Tanowitz HB (2012) Blastocystis: to treat or not to treat. Clin Infect Dis 54(1):105–110PubMedGoogle Scholar
  12. Debode F, Marien A, Janssen É, Bragard C, Berben G (2017) Influence of the amplicon length on real-time PCR results. Biotechnol Agron Soc Environ 21(1):3–11Google Scholar
  13. El Safadi D, Meloni D, Poirier P, Osman M, Cian A, Gaayeb L et al (2013) Molecular epidemiology of Blastocystis in Lebanon and correlation between subtype 1 and gastrointestinal symptoms. Am J Trop Med Hyg 88(6):1203–1206PubMedPubMedCentralGoogle Scholar
  14. Eymael D, Schuh GM, Tavares RG (2010) Standardization of Blastocystis hominis diagnosis using different staining techniques. Rev Soc Bras Med Trop 43(3):309–312PubMedGoogle Scholar
  15. Hoevers J, Holman P, Logan K, Hommel M, Ashford R, Snowden K (2000) Restriction-fragment-length polymorphism analysis of small-subunit rRNA genes of Blastocystis hominis isolates from geographically diverse human hosts. Parasitol Res 86(1):57–61PubMedGoogle Scholar
  16. Jalallou N, Iravani S, Rezaeian M, Alinaghizade A, Mirjalali H (2017) Subtypes distribution and frequency of Blastocystis sp. isolated from diarrheic and non-diarrheic patients. Iran J Parasitol 12(1):63–68PubMedPubMedCentralGoogle Scholar
  17. Jones MS 2nd, Ganac RD, Hiser G, Hudson NR, Le A, Whipps CM (2008) Detection of Blastocystis from stool samples using real-time PCR. Parasitol Res 103(3):551–557PubMedGoogle Scholar
  18. Katsarou-Katsari A, Vassalos CM, Tzanetou K, Spanakos G, Papadopoulou C, Vakalis N (2008) Acute urticaria associated with amoeboid forms of Blastocystis sp. subtype 3. Acta Derm Venereol 88(1):80–81PubMedGoogle Scholar
  19. Kurt O, Dogruman Al F, Tanyuksel M (2016) Eradication of Blastocystis in humans: really necessary for all? Parasitol Int 65(6 Pt B):797–801PubMedGoogle Scholar
  20. Mirjalali H (2017) Abbasi, MR, Naderi N, Hasani Z, Mirsamadi ES, Stensvold CR, et al. Distribution and phylogenetic analysis of Blastocystis sp. Subtypes isolated from IBD patients and healthy individuals in Iran. Eur J Clin Microbiol Infect Dis 36(12):2335–2342Google Scholar
  21. Moosavi A, Haghighi A, Mojarad EN, Zayeri F, Alebouyeh M, Khazan H, Kazemi B, Zali MR (2012) Genetic variability of Blastocystis sp. isolated from symptomatic and asymptomatic individuals in Iran. Parasitol Res 111(6):2311–2315PubMedGoogle Scholar
  22. Parkar U, Traub RJ, Kumar S, Mungthin M, Vitali S, Leelayoova S, Morris K, Thompson RC (2007) Direct characterization of Blastocystis from faeces by PCR and evidence of zoonotic potential. Parasitology 134(Pt 3):359–367PubMedGoogle Scholar
  23. Poirier P, Wawrzyniak I, Albert A, El Alaoui H, Delbac F, Livrelli V (2011) Development and evaluation of a real-time PCR assay for detection and quantification of Blastocystis parasites in human stool samples: prospective study of patients with hematological malignancies. J Clin Microbiol 49(3):975–983PubMedPubMedCentralGoogle Scholar
  24. Rezaei Riabi T, Mirjalali H, Haghighi A, Rostami Nejad M, Pourhoseingholi MA, Poirier P et al (2018) Genetic diversity analysis of Blastocystis subtypes from both symptomatic and asymptomatic subjects using a barcoding region from the 18S rRNA gene. Infect Genet Evol 61:119–126PubMedGoogle Scholar
  25. Salehi R, Haghighi A, Stensvold CR, Kheirandish F, Azargashb E, Raeghi S, Kohansal C, Bahrami F (2017) Prevalence and subtype identification of Blastocystis isolated from humans in Ahvaz, Southwestern Iran. Gastroenterol Hepatol Bed Bench 10(3):235–241PubMedPubMedCentralGoogle Scholar
  26. Scicluna SM, Tawari B, Clark CG (2006) DNA barcoding of Blastocystis. Protist 157(1):77–85PubMedGoogle Scholar
  27. Sekar U, Shanthi M (2013) Blastocystis: consensus of treatment and controversies. Trop Parasitol 3(1):35–39PubMedPubMedCentralGoogle Scholar
  28. Stensvold CR (2013a) Blastocystis: Genetic diversity and molecular methods for diagnosis and epidemiology. Trop Parasitol 3(1):26–34PubMedPubMedCentralGoogle Scholar
  29. Stensvold CR (2013b) Comparison of sequencing (barcode region) and sequence-tagged-site PCR for Blastocystis subtyping. J Clin Microbiol 51(1):190–194PubMedPubMedCentralGoogle Scholar
  30. Stensvold CR (2015) Laboratory diagnosis of Blastocystis spp. Trop Parasitol 5(1):3–5PubMedPubMedCentralGoogle Scholar
  31. Stensvold CR, Clark CG (2016) Current status of Blastocystis: a personal view. Parasitol Int 65(6 Pt B):763–771PubMedGoogle Scholar
  32. Stensvold CR, Arendrup MC, Jespersgaard C, Molbak K, Nielsen HV (2007a) Detecting Blastocystis using parasitologic and DNA-based methods: a comparative study. Diagn Microbiol Infect Dis 59(3):303–307PubMedGoogle Scholar
  33. Stensvold CR, Suresh GK, Tan KS, Thompson RC, Traub RJ, Viscogliosi E et al (2007b) Terminology for Blastocystis subtypes--a consensus. Trends Parasitol 23(3):93–96PubMedGoogle Scholar
  34. Stensvold CR, Nielsen HV, Molbak K, Smith HV (2009) Pursuing the clinical significance of Blastocystis--diagnostic limitations. Trends Parasitol 25(1):23–29PubMedGoogle Scholar
  35. Stensvold CR, Ahmed UN, Andersen LO, Nielsen HV (2012a) Development and evaluation of a genus-specific, probe-based, internal-process-controlled real-time PCR assay for sensitive and specific detection of Blastocystis spp. J Clin Microbiol 50(6):1847–1851PubMedPubMedCentralGoogle Scholar
  36. Stensvold CR, Alfellani M, Clark CG (2012b) Levels of genetic diversity vary dramatically between Blastocystis subtypes. Infect Genet Evol 12(2):263–273PubMedGoogle Scholar
  37. Suresh K, Smith H (2004) Comparison of methods for detecting Blastocystis hominis. Eur J Clin Microbiol Infect Dis 23(6):509–511PubMedGoogle Scholar
  38. Taghipour A, Javanmard E, Mirjalali H, Haghighi A, Tabarsi P, Sohrabi MR, Zali MR (2019) Blastocystis subtype 1 (allele 4); predominant subtype among tuberculosis patients in Iran. Comp Immunol Microbiol Infect Dis 65:201–206PubMedGoogle Scholar
  39. Tan KS (2008) New insights on classification, identification, and clinical relevance of Blastocystis spp. Clin Microbiol Rev 21(4):639–665PubMedPubMedCentralGoogle Scholar
  40. Tan KS, Mirza H, Teo JD, Wu B, Macary PA (2010) Current views on the clinical relevance of Blastocystis spp. Curr Infect Dis Rep 12(1):28–35PubMedGoogle Scholar
  41. Toro Monjaraz EM, Vichido Luna MA, Montijo Barrios E, Cervantes Bustamante R, Zarate Mondragon F, Huante Anaya A et al (2017) Blastocystis hominis and chronic abdominal pain in children: is there an association between them? J Trop Pediatr 64(4):279–283Google Scholar
  42. Vogelberg C, Stensvold CR, Monecke S, Ditzen A, Stopsack K, Heinrich-Grafe U et al (2010) Blastocystis sp. subtype 2 detection during recurrence of gastrointestinal and urticarial symptoms. Parasitol Int 59(3):469–471PubMedGoogle Scholar
  43. Yoshikawa H, Nagono I, Yap EH, Singh M, Takahashi Y (1996) DNA polymorphism revealed by arbitrary primers polymerase chain reaction among Blastocystis strains isolated from humans, a chicken, and a reptile. J Eukaryot Microbiol 43(2):127–130PubMedGoogle Scholar
  44. Yoshikawa H, Nagano I, Wu Z, Yap EH, Singh M, Takahashi Y (1998) Genomic polymorphism among Blastocystis hominis strains and development of subtype-specific diagnostic primers. Mol Cell Probes 12(3):153–159PubMedGoogle Scholar
  45. Yoshikawa H, Abe N, Iwasawa M, Kitano S, Nagano I, Wu Z, Takahashi Y (2000) Genomic analysis of Blastocystis hominis strains isolated from two long-term health care facilities. J Clin Microbiol 38(4):1324–1330PubMedPubMedCentralGoogle Scholar
  46. Yoshikawa H, Wu Z, Kimata I, Iseki M, Ali IK, Hossain MB, Zaman V, Haque R, Takahashi Y (2004) Polymerase chain reaction-based genotype classification among human Blastocystis hominis populations isolated from different countries. Parasitol Res 92(1):22–29PubMedGoogle Scholar
  47. Yoshikawa H, Koyama Y, Tsuchiya E, Takami K (2016) Blastocystis phylogeny among various isolates from humans to insects. Parasitol Int 65(6 Pt B):750–759PubMedGoogle Scholar
  48. Zhang X, Qiao JY, Zhou XJ, Yao FR, Wei ZC (2007) Morphology and reproductive mode of Blastocystis hominis in diarrhea and in vitro. Parasitol Res 101(1):43–51PubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Medical Parasitology and Mycology, Faculty of MedicineShahid Beheshti University of Medical SciencesTehranIran
  2. 2.Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver DiseasesShahid Beheshti University of Medical SciencesTehranIran
  3. 3.Basic and Molecular Epidemiology of Gastrointestinal Disorders Research Center, Research Institute for Gastroenterology and Liver DiseasesShahid Beheshti University of Medical SciencesTehranIran
  4. 4.Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver DiseasesShahid Beheshti University of Medical SciencesTehranIran

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