Webster Bonnie L
Biomedical Parasitology, Wolfson Wellcome Laboratories, Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK.
Parasit Vectors. 2009 Oct 23;2(1):50. doi: 10.1186/1756-3305-2-50.
Although field-sampling procedures to capture gDNA from individual schistosome larval stages directly from their natural hosts exist, they do pose some technical and logistical challenges hampering certain epidemiological studies. The aim of this study was to develop, refine and evaluate an alternative methodology, which enables better preservation of large numbers of individual schistosome larval stages and eggs collected in low resource endemic areas, to provide PCR-quality DNA for multi-locus genetic analysis. The techniques reported here present simple and effective short-term field and long-term laboratory preservation and storage systems for individually sampled schistosome eggs and larval stages using a commercially available aqueous stabilisation reagent, RNAlater(R) eliminating the need for more cumbersome resources such as refrigerators, heaters and centrifuge equipment for immediate specimen processing. Adaptations to a general gDNA extraction method are described, that enables the acquisition of a gDNA extract (~50 mul), facilitating multiple molecular analyses of each sampled schistosome. The methodology provided PCR-quality mitochondrial and nuclear DNA from laboratory cercariae, miracidia and eggs that had been stored at up to 37 degrees C for 2 weeks and at 4 degrees C for 6 months and also from field collected samples. This present protocol provides significant epidemiological, ethical and practical advantages over existing sampling methods and has the potential to be transferred to studies on other organisms, especially where specimens are unable to be seen by the naked eye, are difficult to handle and need to be obtained from a field environment.
尽管存在从自然宿主中直接捕获各个血吸虫幼虫阶段的基因组DNA（gDNA）的现场采样程序，但这些程序确实带来了一些技术和后勤方面的挑战，阻碍了某些流行病学研究。本研究的目的是开发、完善和评估一种替代方法，该方法能够更好地保存低资源流行地区收集的大量单个血吸虫幼虫阶段和虫卵，以提供用于多位点基因分析的PCR级DNA。本文报道的技术提供了简单有效的短期现场和长期实验室保存及存储系统，用于使用市售水性稳定试剂RNAlater®对单个采样的血吸虫虫卵和幼虫阶段进行保存，无需使用冰箱、加热器和离心机设备等更繁琐的资源进行即时标本处理。文中描述了对一般gDNA提取方法的改进，该方法能够获取gDNA提取物（约50微升），便于对每个采样的血吸虫进行多次分子分析。该方法从在37摄氏度下储存2周和在4摄氏度下储存6个月的实验室尾蚴、毛蚴和虫卵以及现场采集的样本中提供了PCR级的线粒体和核DNA。本方案相对于现有采样方法具有显著的流行病学、伦理和实际优势，并且有可能应用于其他生物的研究，特别是在标本肉眼不可见、难以处理且需要从野外环境获取的情况下。
