Department of Medical Parasitology and Mycology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Parasitol Res. 2019 Dec;118(12):3469-3478. doi: 10.1007/s00436-019-06486-5. Epub 2019 Nov 5.
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 ~ 10 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.
芽囊原虫是一种分布广泛的寄生虫。为了设计 HRM 实时 PCR，我们从 SSU rRNA 基因中选择引物，以扩增每个芽囊原虫亚型具有不同熔点的特异性片段。随后，我们进行了 HRM 实时 PCR，并通过 Rotor-Gene Q 软件进行了熔解曲线分析。然后将 HRM 实时 PCR 的结果与芽囊原虫 SSU rRNA 基因“条形码区”的序列结果进行比较。为了评估试验的灵敏度，我们从约 10 到 1 个寄生虫/毫升粪便样本中制备了寄生虫的 10 倍系列稀释液，并通过 HRM 实时 PCR 进行了研究。为了确定方法的特异性，我们对一些微生物和芽囊原虫阴性粪便样本进行了 HRM 实时 PCR。计算机分析表明，所有 17 种芽囊原虫亚型均可区分。体外分析表明，该检测可区分具有特定熔点的亚型。
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