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

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