Maroju Pranay Amruth, Yadav Sonu, Kolipakam Vishnupriya, Singh Shweta, Qureshi Qamar, Jhala Yadvendradev
Wildlife Institute of India, Chandrabani, Dehradun, 248001, India.
BMC Genet. 2016 Feb 9;17:37. doi: 10.1186/s12863-016-0344-y.
Non-invasive sampling has opened avenues for the genetic study of elusive species, which has contributed significantly to their conservation. Where field based identity of non-invasive sample is ambiguous (e.g. carnivore scats), it is essential to establish identity of the species through molecular approaches. A cost effective procedure to ascertain species identity is to use species specific primers (SSP) for PCR amplification and subsequent resolution through agarose gel electrophoresis. However, SSPs if ill designed can often cross amplify non-target sympatric species. Herein we report the problem of cross amplification with currently published SSPs, which have been used in several recent scientific articles on tigers (Panthera tigris) and leopards (Panthera pardus) in India. Since these papers form pioneering research on which future work will be based, an early rectification is required so as to not propagate this error further.
We conclusively show cross amplification of three of the four SSPs, in sympatric non-target species like tiger SSP amplifying leopard and striped hyena (Hyaena hyaena), and leopard SSP amplifying tiger, lion (Panthera leo persica) and clouded leopard (Neofelis nebulosa), with the same product size. We develop and test a non-cross-amplifying leopard specific primer pair within the mitochondrial cytochrome b region. We also standardize a duplex PCR method to screen tiger and leopard samples simultaneously in one PCR reaction to reduce cost and time.
These findings suggest the importance of an often overlooked preliminary protocol of conclusive identification of species from non-invasive samples. The cross amplification of published primers in conspecifics suggests the need to revisit inferences drawn by earlier work.
非侵入性采样为难以捉摸的物种的遗传学研究开辟了道路，这对它们的保护做出了重大贡献。当基于野外的非侵入性样本的身份不明确时（例如食肉动物的粪便），通过分子方法确定物种身份至关重要。一种经济有效的确定物种身份的方法是使用物种特异性引物（SSP）进行PCR扩增，随后通过琼脂糖凝胶电泳进行分析。然而，如果设计不当，SSP常常会交叉扩增非目标同域物种。在此，我们报告了当前已发表的SSP存在交叉扩增的问题，这些引物已被用于印度最近几篇关于老虎（Panthera tigris）和豹（Panthera pardus）的科学文章中。由于这些论文构成了未来研究的开创性工作基础，因此需要尽早纠正，以免进一步传播这一错误。
我们确凿地表明，四个SSP中的三个存在交叉扩增现象，在同域非目标物种中，例如老虎的SSP会扩增豹和条纹鬣狗（Hyaena hyaena），豹的SSP会扩增老虎、狮子（Panthera leo persica）和云豹（Neofelis nebulosa），且产物大小相同。我们在细胞色素b线粒体区域内开发并测试了一对不交叉扩增的豹特异性引物。我们还标准化了一种双重PCR方法，以便在一个PCR反应中同时筛选老虎和豹的样本，从而降低成本和时间。
这些发现表明了一个常常被忽视的初步方案的重要性，即从非侵入性样本中确凿鉴定物种。已发表引物在同物种中的交叉扩增表明有必要重新审视早期研究得出结论。
