基于内部转录间隔区（ITS1、5.8S rDNA、ITS2），利用PCR-RFLP方法对片形吸虫分离株进行分子鉴定与区分
Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA, ITS2).
作者信息
Mahami-Oskouei M, Dalimi A, Forouzandeh-Moghadam M, Rokni Mb
机构信息
Department of Medical Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
出版信息
Iran J Parasitol. 2011 Aug;6(3):35-42.
BACKGROUND
In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species.
METHODS
The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety trematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and analyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species.
RESULTS
The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp509I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates.
CONCLUSION
The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates.
背景
在本研究中，我们使用内转录间隔区1（ITS1）和内转录间隔区2（ITS2）对片形吸虫属物种进行分子鉴定。
方法
采用核糖体DNA的18S和28S之间的区域，通过聚合酶链反应-限制性片段长度多态性（PCR-RFLP）方法对片形吸虫属物种进行分子鉴定。在屠宰场检查期间，从伊朗霍拉桑省、东阿塞拜疆省和法尔斯省自然感染的绵羊和牛的肝脏中收集了90条片形吸虫。DNA提取后，进行PCR以扩增ITS1区域、5.8S核糖体DNA、ITS2。为选择合适的限制性内切酶，我们对绵羊和牛的肝片吸虫和巨片吸虫样本的PCR产物进行了测序和分析。选择Tsp509I快速消化限制性内切酶用于RFLP方法，该方法可特异性分离片形吸虫属物种。
结果
所有片形吸虫样本中均扩增出约1000bp的片段，然后用Tsp509I限制性内切酶进行消化。在总共90个片形吸虫分离株中，鉴定出70个肝片吸虫和20个巨片吸虫。
结论
使用Tsp509I限制性内切酶的新型PCR-RFLP检测方法为片形吸虫分离株的鉴定和区分提供了一种简单、实用、快速、低成本且可靠的方法。
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