Karasartova Djursun, Gureser Ayse Semra, Ruh Emrah, Turegun-Atasoy Buse, Calgin Mustafa Kerem, Tasci Leyla, Taylan-Ozkan Aysegul
Department of Medical Microbiology, Faculty of Medicine, Hitit University, Corum, Turkey.
Department of Medical Microbiology, Faculty of Medicine, Hitit University, Corum, Turkey.
Exp Parasitol. 2018 Mar;186:36-41. doi: 10.1016/j.exppara.2018.01.019. Epub 2018 Feb 10.
Polymerase chain reaction (PCR) is an effective technique for diagnosis of Blastocystis infection. Notably, DNA isolation procedure is extremely critical for the PCR step. In the present study, a recently described extraction procedure, named as the "sand method" was modified and adapted for isolation of Blastocystis DNA. To evaluate its efficacy, the current method and QIAamp DNA Stool Mini Kit (Qiagen) were applied to fresh human stool samples. Our results indicated that, the mean DNA concentrations obtained by the sand method and the commercial kit were 48 and 55 ng/μl, respectively. Also, no DNA inhibitors were detected in two methods. The sand method was capable of detecting 16 parasites per 50 mg feces. DNA samples extracted by both methods were subjected to PCR. Blastocystis spp. were detected in 11 (31.4%) of 35 samples, and perfect agreement (κ: 1.000) was found between the PCR-sand method and PCR-commercial kit method. The samples that were detected positive by PCR-sand method were successfully sequenced, and Blastocystis subtypes (STs) were identified as ST3, ST2 and ST1. In conclusion, the present study indicates that the sand method provides a simple, rapid and inexpensive procedure for reliable extraction of Blastocystis DNA from stool samples.
聚合酶链反应（PCR）是诊断芽囊原虫感染的有效技术。值得注意的是，DNA提取程序对PCR步骤极为关键。在本研究中，对最近描述的一种名为“沙法”的提取程序进行了修改，并适用于芽囊原虫DNA的分离。为评估其效果，将当前方法和QIAamp DNA粪便迷你试剂盒（Qiagen）应用于新鲜人类粪便样本。我们的结果表明，沙法和商业试剂盒获得的平均DNA浓度分别为48和55 ng/μl。此外，两种方法均未检测到DNA抑制剂。沙法能够检测出每50 mg粪便中有16个寄生虫。两种方法提取的DNA样本均进行了PCR。在35个样本中的11个（31.4%）中检测到芽囊原虫属，PCR-沙法和PCR-商业试剂盒法之间发现完全一致（κ：1.000）。通过PCR-沙法检测为阳性的样本成功进行了测序，并鉴定出芽囊原虫亚型（STs）为ST3、ST2和ST1。总之，本研究表明，沙法为从粪便样本中可靠提取芽囊原虫DNA提供了一种简单、快速且廉价的程序。
