Palomares F, Godoy J A, Piriz A, O'Brien S J
Department of Applied Biology, Estación Biológica de Doñana, CSIC, Avda. María Luisa s/n, 41013, Sevilla, Spain.
Mol Ecol. 2002 Oct;11(10):2171-82. doi: 10.1046/j.1365-294x.2002.01608.x.
Noninvasive methods using genetic markers have been suggested as ways to overcome difficulties associated with documenting the presence of elusive species. We present and assess a novel, reliable and effective molecular genetic technique for the unequivocal genetic identification of faeces from the endangered Iberian lynx (Lynx pardinus). From mitochondrial DNA (mtDNA) cytochrome b and D-loop region sequences, we designed four species-specific primers (for products 130-161 bp long) that were considered to be likely to amplify degraded DNA. We compared two DNA extraction methods, various DNA amplification conditions and the robustness and specificity of the primer pairs with 87 lynx samples from 5 potentially different lynx populations and with 328 samples of other carnivore species. The utility of the identification technique was tested with faeces of different ages, with faeces from controlled field experiments, and with faeces collected from locales with possible lynx populations from throughout the state of Andalusia, Spain (8052 km2). Faecal mtDNA extraction was more efficient using PBS wash of the faeces instead of a faeces homogenate. Our assay increased from 92.6 to 99% efficiency with a second amplification and a reduction in template concentration to overcome polymerase chain reaction (PCR) inhibition. Our assay never produced false positives, and correctly identified all lynx faeces. Of 252 faeces samples of unknown species collected throughout Andalusia, 26.6% (from three different areas) were classified as Iberian lynx, 1.4% showed evidence of PCR inhibition and 1.2% were of uncertain origin. This method has proven to be a reliable technique that can be incorporated into large-scale surveys of Iberian lynx populations and exemplifies an approach that can easily be extended to other species.
有人提出使用遗传标记的非侵入性方法，作为克服记录 elusive 物种存在相关困难的途径。我们展示并评估了一种新颖、可靠且有效的分子遗传技术，用于明确鉴定濒危伊比利亚猞猁（Lynx pardinus）粪便的遗传特征。从线粒体 DNA（mtDNA）细胞色素 b 和 D 环区域序列，我们设计了四种物种特异性引物（用于扩增长度为 130 - 161 bp 的产物），这些引物被认为有可能扩增降解的 DNA。我们比较了两种 DNA 提取方法、各种 DNA 扩增条件以及引物对与来自 5 个可能不同猞猁种群的 87 个猞猁样本以及 328 个其他食肉动物物种样本的稳健性和特异性。该鉴定技术的实用性通过不同年龄的粪便、来自对照野外实验的粪便以及从西班牙安达卢西亚州（8052 km²）可能存在猞猁种群的地点收集的粪便进行了测试。使用 PBS 冲洗粪便而非粪便匀浆进行粪便 mtDNA 提取效率更高。通过第二次扩增以及降低模板浓度以克服聚合酶链反应（PCR）抑制，我们的检测效率从 92.6%提高到了 99%。我们的检测从未产生假阳性，并正确鉴定了所有猞猁粪便。在整个安达卢西亚收集的 252 个未知物种的粪便样本中，26.6%（来自三个不同区域）被归类为伊比利亚猞猁，1.4%显示出 PCR 抑制的迹象，1.2%来源不确定。该方法已被证明是一种可靠的技术，可纳入伊比利亚猞猁种群的大规模调查中，并且是一种可轻松扩展到其他物种的方法示例。
