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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

Abstract

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.

Keywords

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

Abbreviation

HRM

High-resolution melting curve

ST

Subtype

rRNA

Ribosomal RNA

Notes

Acknowledgements

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.

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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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