New methods of removing debris and high-throughput counting of cyst nematode eggs extracted from field soil
New methods of removing debris and high-throughput counting of cyst nematode eggs extracted from field soil
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DOI：
10.1371/journal.pone.0223386
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发表时间：
2019-10-15
期刊：
影响因子：
3.7
通讯作者：
Pandey, Santosh
中科院分区：
文献类型：
--
作者：
Kalwa, Upender;Legner, Christopher;Pandey, Santosh
大豆胞囊线虫（SCN），大豆胞囊线虫（Heterodera glycines），是美国大豆最具破坏性的病原体。为了评估田间线虫侵染的严重性，测定SCN卵群体密度。必须从土壤样品中提取线虫的囊（死亡的雌性），然后研磨以提取其中的卵。蔗糖离心通常用于从提取的线虫卵的悬浮液中分离碎片。我们提出了一种使用OptiPrep作为密度梯度介质的方法，与蔗糖离心技术相比，该方法具有改进的提取卵的分离和回收。此外，还开发了计算机化方法来自动识别和计数处理样本中的线虫卵。在一种方法中，使用高分辨率扫描仪拍摄滤纸上提取的鸡蛋和碎片的静态图像，并训练深度学习网络来识别和计数碎片中的鸡蛋。在第二种方法中，使用现成的组件开发了无透镜成像装置，并将处理过的鸡蛋样品通过由双面胶带制成的微流体流动芯片。记录经过的鸡蛋和碎片的全息视频，并通过定制软件程序重建和处理视频以获得鸡蛋计数。鸡蛋计数的软件程序的性能，其特征在于从两个农场收集SCN感染的土壤，并使用这些方法的结果进行了比较，通过手工计数。
The soybean cyst nematode (SCN), Heterodera glycines, is the most damaging pathogen of soybeans in the United States. To assess the severity of nematode infestations in the field, SCN egg population densities are determined. Cysts (dead females) of the nematode must be extracted from soil samples and then ground to extract the eggs within. Sucrose centrifugation commonly is used to separate debris from suspensions of extracted nematode eggs. We present a method using OptiPrep as a density gradient medium with improved separation and recovery of extracted eggs compared to the sucrose centrifugation technique. Also, computerized methods were developed to automate the identification and counting of nematode eggs from the processed samples. In one approach, a high-resolution scanner was used to take static images of extracted eggs and debris on filter papers, and a deep learning network was trained to identify and count the eggs among the debris. In the second approach, a lensless imaging setup was developed using off-the-shelf components, and the processed egg samples were passed through a microfluidic flow chip made from double-sided adhesive tape. Holographic videos were recorded of the passing eggs and debris, and the videos were reconstructed and processed by custom software program to obtain egg counts. The performance of the software programs for egg counting was characterized with SCN-infested soil collected from two farms, and the results using these methods were compared with those obtained through manual counting.