Barazesh Afshin, Sarkari Bahador, Ebrahimi Sepideh, Hami Mehdi
Student Research Committee, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
The Persian Gulf Tropical Medicine Research Centre, Bushehr University of Medical Sciences, Bushehr, Iran.
Vet World. 2018 Feb;11(2):231-234. doi: 10.14202/vetworld.2018.231-234. Epub 2018 Feb 22.
The current study aimed to find out a simple, practical and high throughput DNA isolation method for extraction of DNA from hydatid cyst samples.
Cattle and sheep isolate of hydatid cysts were obtained from the slaughterhouse, and hydatid fluid and protoscolices were collected in a sterile condition. Protoscolices were washed, 3 times with phosphate buffered saline, and DNA was extracted by different methods including manual extraction with freeze/thawing and phenol-chloroform, Triton X-100 extraction, and by a commercial kit (YTA, Yekta Tajhiz Azma, Iran) with three different modifications in the kit's manufacturer instructions. The obtained DNA from the different methods was evaluated by Nanodrop in terms of the yield of DNA and carbohydrates or protein contaminations. To compare the quality of the extracted DNA, two pieces of the mitochondrial genome of , cox1, and nad1, were polymerase chain reaction (PCR)-amplified, using each of the DNA prepared by different methods. Electrophoresis of PCR products was carried out on the agarose gel.
The DNA extracted by manual method, using phenol/chloroform, had the highest yield, yet with the highest level of protein and carbohydrate contamination. The DNA extracted using two-step incubations, initially at 60°C for 2 h and then overnight at 37°C, was the most purified DNA with the lowest rate of contamination.
Findings of the study demonstrated that modification in the currently available commercially DNA extraction kit resulted in the development of a high throughput DNA isolation method. This method can be recommended for the extraction of DNA from hydatid cysts, especially the cattle isolate where the extraction of DNA in these samples are usually problematic.
本研究旨在找到一种简单、实用且高通量的DNA提取方法，用于从包虫囊肿样本中提取DNA。
从屠宰场获取牛和羊的包虫囊肿分离株，在无菌条件下收集包虫液和原头节。原头节用磷酸盐缓冲盐水洗涤3次，采用不同方法提取DNA，包括冻融法结合苯酚 - 氯仿手动提取、Triton X - 100提取，以及使用商业试剂盒（YTA，伊朗Yekta Tajhiz Azma公司）并按照试剂盒制造商说明进行三种不同的改进。通过Nanodrop评估不同方法获得的DNA的产量以及碳水化合物或蛋白质污染情况。为比较提取的DNA质量，使用不同方法制备的每种DNA对细胞色素c氧化酶亚基1（cox1）和烟酰胺腺嘌呤二核苷酸脱氢酶亚基1（nad1）的两段线粒体基因组进行聚合酶链反应（PCR）扩增。PCR产物在琼脂糖凝胶上进行电泳。
采用苯酚/氯仿手动方法提取的DNA产量最高，但蛋白质和碳水化合物污染水平也最高。采用两步孵育法（最初在60°C孵育2小时，然后在37°C过夜）提取的DNA是最纯净的，污染率最低。
该研究结果表明，对现有的商业DNA提取试剂盒进行改进可开发出一种高通量DNA提取方法。这种方法可推荐用于从包虫囊肿中提取DNA，特别是对于牛分离株，这类样本中DNA的提取通常存在问题。
