DNA purity over yield: optimizing extraction and PCR in degraded Asian elephant (Elephas maximus) fecal samples.

IF 2 4区生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY

Genes & genomics Pub Date : 2026-05-19 DOI:10.1007/s13258-026-01772-w

Supansa Rerkdee, Worapong Singchat, Thitipong Panthum, Trifan Budi, Warong Suksavate, Aingorn Chaiyes, Wichanon Saenphala, Kyudong Han, Prateep Duengkae, Kornsorn Srikulnath

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引用次数: 0

Abstract

Background: Non-invasive genetic monitoring is essential for conserving the endangered Asian elephant (Elephas maximus). However, fecal DNA is highly susceptible to degradation and polymerase chain reaction (PCR) inhibition.

Objective: Various DNA extraction techniques and optimized downstream PCR parameters were compared to establish a workflow that balances DNA quantity and quality.

Methods: A modified phenol-chloroform-isoamyl alcohol (PCI) protocol was compared with a commercial extraction kit using 35 unpreserved, degraded fecal specimens to evaluate the trade-offs between DNA yield and purity.

Results: The PCI method produced significantly higher concentrations (> 500 ng/µL in some samples) but poor purity ratios (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀), resulting in complete PCR failure. In contrast, the kit yielded lower DNA amounts but superior purity (A₂₆₀/A₂₃₀ = 2.0-2.2), enabling a 57.1% (20/35) amplification success rate for the mitochondrial D-loop when combined with high-processivity KOD polymerase and optimized Mg²⁺ concentrations. Sanger sequencing confirmed E. maximus mitochondrial DNA in 15 specimens, whereas environmental eDNA was detected in two samples.

Conclusion: These findings demonstrate that DNA purity, rather than yield, is the critical determinant of PCR success in degraded wildlife samples, underscoring the importance of inhibitor-removal technologies for the reliable and effective management of endangered megafauna.

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DNA纯度高于产量：优化降解亚洲象粪便样品的提取和PCR。

背景：非侵入性基因监测是保护濒危亚洲象的必要条件。然而，粪便DNA极易被降解和聚合酶链反应（PCR）抑制。目的：比较各种DNA提取技术和优化的下游PCR参数，建立平衡DNA数量和质量的工作流程。方法：将改良的苯酚-氯仿-异戊醇（PCI）方案与商业提取试剂盒进行比较，使用35份未保存的降解粪便标本来评估DNA产量和纯度之间的权衡。结果：PCI法产生的浓度明显较高（部分样品的浓度为500ng /µL），但纯度比较差（A260/A280和A260/A230），导致PCR完全失败。相比之下，该试剂盒产生的DNA量较低，但纯度较高（A260/A230 = 2.0-2.2），当与高处理率的KOD聚合酶和优化的Mg2⁺浓度结合时，线粒体D-loop的扩增成功率为57.1%（20/35）。Sanger测序在15个标本中证实了E. maximus线粒体DNA，而在2个样本中检测到环境eDNA。结论：这些发现表明，DNA纯度，而不是产量，是在退化野生动物样本中PCR成功的关键决定因素，强调了抑制剂去除技术对濒危巨型动物可靠和有效管理的重要性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Genes & genomics 生物-生化与分子生物学

CiteScore

3.70

自引率

4.80%

发文量

131

审稿时长

6-12 weeks

期刊介绍： Genes & Genomics is an official journal of the Korean Genetics Society (http://kgenetics.or.kr/). Although it is an official publication of the Genetics Society of Korea, membership of the Society is not required for contributors. It is a peer-reviewed international journal publishing print (ISSN 1976-9571) and online version (E-ISSN 2092-9293). It covers all disciplines of genetics and genomics from prokaryotes to eukaryotes from fundamental heredity to molecular aspects. The articles can be reviews, research articles, and short communications.