Mollet Kayla A, Tembrock Luke R, Zink Frida A, Timm Alicia E, Gilligan Todd M
Department of Agricultural Biology, Colorado State University, Fort Collins, CO 80523-1177, USA.
Insects. 2024 Aug 1;15(8):585. doi: 10.3390/insects15080585.
is among the most problematic agricultural pests worldwide due to its polyphagy and ability to evolve pesticide resistance. Molecular detection methods for have been developed to track its spread, as such methods allow for rapid and accurate differentiation from the native sibling species . Droplet digital PCR (ddPCR) is a preferred method for bulk screening due to its accuracy and tolerance to PCR inhibitors; however, real-time PCR is less expensive and more widely available in molecular labs. Improvements to DNA extraction yield, purity, and throughput are crucial for real-time PCR assay optimization. Bulk DNA extractions have recently been improved to where real-time PCR sensitivity can equal that of ddPCR, but these new methods require significant time and specialized equipment. In this study, we improve upon previously published bulk DNA extraction methods by reducing bench time and materials. Our results indicate that the addition of caffeine and RNase A improves DNA extraction, resulting in lower Cq values during real-time PCR while reducing the processing time and cost per specimen. Such improvements will enable the use of high throughput screening methods across multiple platforms to improve the probability of detection of .
由于其食性广泛且具有进化出抗药性的能力，它是全球最具问题的农业害虫之一。已经开发出针对它的分子检测方法来追踪其传播，因为这些方法能够快速、准确地将其与本地近缘物种区分开来。液滴数字PCR（ddPCR）因其准确性和对PCR抑制剂的耐受性而成为批量筛选的首选方法；然而，实时PCR成本更低，在分子实验室中更广泛可用。提高DNA提取的产量、纯度和通量对于优化实时PCR检测至关重要。批量DNA提取最近已经得到改进，使得实时PCR的灵敏度能够与ddPCR相当，但这些新方法需要大量时间和专门设备。在本研究中，我们通过减少实验台操作时间和材料用量，对先前发表的批量DNA提取方法进行了改进。我们的结果表明，添加咖啡因和核糖核酸酶A可改善DNA提取，在实时PCR过程中产生更低的Cq值，同时减少每个样本的处理时间和成本。这些改进将使高通量筛选方法能够在多个平台上使用，以提高检测到它的概率。
