Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Vic. 3030, Australia.
Int J Parasitol. 2009 Sep;39(11):1277-87. doi: 10.1016/j.ijpara.2009.03.002. Epub 2009 Mar 27.
We evaluated a combined microscopic-molecular approach for the diagnosis of key strongylid infections in sheep using panels of well-defined control and test samples. The method established is based on the separation of nematode eggs from faecal samples using a salt flotation procedure, the extraction and column-purification of genomic DNA, followed by real-time PCR and melting-curve analysis. Specific and semi-quantitative amplification from (a minimum of 0.1-2.0pg) genomic DNA of Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus spp., Cooperia oncophora, Oesophagostomum columbianum, Oesophagostomum venulosum or Chabertia ovina is achieved using a specific, forward oligonucleotide primer located in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) together with a conserved reverse primer in the large subunit of rDNA. Using a panel of well-defined genomic DNA samples from eggs from sheep monospecifically infected with H. contortus or Te. circumcincta, there was a correlation between cycle threshold (Ct) values in the PCR and numbers of egg per gram of faeces, thus allowing the semi-quantitation of parasite DNA in faeces. The findings of the present study indicate that a microscopic-molecular approach provides a useful tool for diagnosis, for epidemiological and ecological surveys as well as for integration into parasite monitoring, drug resistance (i.e. 'egg count reduction') testing or control programmes, particularly following semi- or full-automation.
我们使用经过明确定义的对照和测试样本的小组，评估了一种用于诊断绵羊中关键的强旋毛虫感染的组合显微镜-分子方法。所建立的方法基于使用盐浮选程序从粪便样本中分离线虫卵，提取和柱纯化基因组 DNA，然后进行实时 PCR 和熔解曲线分析。使用位于核核糖体 DNA（rDNA）的第二个内部转录间隔区（ITS-2）中的特异性正向寡核苷酸引物与 rDNA 的大亚基中的保守反向引物，从（至少 0.1-2.0pg）基因组 DNA 中特异性和半定量扩增 Haemonchus contortus、Teladorsagia circumcincta、Trichostrongylus spp.、Cooperia oncophora、Oesophagostomum columbianum、Oesophagostomum venulosum 或 Chabertia ovina。使用一组来自绵羊单特异性感染 H. contortus 或 Te. circumcincta 的卵的明确定义的基因组 DNA 样本，PCR 中的循环阈值（Ct）值与粪便中每克卵的数量之间存在相关性，从而允许对粪便中的寄生虫 DNA 进行半定量。本研究的结果表明，显微镜-分子方法为诊断、流行病学和生态调查以及整合到寄生虫监测、药物抗性（即“卵数减少”）测试或控制计划中提供了有用的工具，特别是在半自动或全自动之后。
