Development and evaluation of high-resolution melting curve analysis for rapid detection and subtyping of Blastocystis and comparison the results with sequencing
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.
KeywordsBlastocystis Subtypes 18S ribosomal RNA gene High-resolution melting curve analysis
High-resolution melting curve
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.
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.
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.
The authors declare that they have no conflict of interest.
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