这项工作得到了国家自然科学基金（82060341,81560304）和海南省院士创新平台科研项目（YSPTZX202134）的支持。

该研究已获得海南医科大学伦理委员会的批准。本研究中使用的组织样本不被视为涉及人类受试者的研究。这些标本是出于法医病理诊断的目的而获得的，其余的用于本实验中硅藻DNA的提取。研究人员无法轻易识别个人以获得相关利益相关者的知情同意。
注：为保证本实验中报告的研究方法的普遍适用性，本实验基本遵循所用试剂盒的操作说明，仅修改了部分步骤。本实验中使用的水样是从实验室附近的池塘中随机采集的（补充图1A）。在该实验中，确认溺水尸体的肺组织为研究组织，以证明提取方案（补充图1B）。在法医实践中，有时还需要使用溺水尸体的其他器官和组织（如肝、脾、肾、骨髓等）来提取硅藻DNA，这需要对这种实验方法进行微小的相应改进，这将在实验的相应章节中解释。该实验中使用的肺组织样本来自法医案件中明显淹死的尸体。已经进行了形态学测试，以证明肺组织含有硅藻（补充图2）。
1.样品的预处理
<ol><li>水样的预处理<ol><li>将 10 mL 水样放入离心管中，并以 13,400 x g 离心 5 分钟。用移液管小心弃去 9.8 mL 上清液，并将剩余在底部的约 200 μL 富集硅藻水样品转移到 2 mL 离心管中。 注意：可以先进行预实验，如果 10 mL 水样不足以富集，可以增加初始量。</li>
	</ol></li>
	<li>组织样本的预处理<ol><li>从溺水的尸体中取出0.5克肺边缘组织，将肺组织完全切碎或研磨，直到它变得浑浊。防止外源硅藻污染是这一步的核心。用剪刀反复将组织切成小块。</li>
	</ol></li>
</ol>2. DNA提取
注意：所有离心步骤均在室温下完成。使用离心力为14,500 x g的台式离心机;在实验开始之前，需要准备预热到70°C的水浴（或金属浴）。所有步骤必须严格遵循无菌操作原则。
<ol><li>水样和组织的组装 注意：水样和组织硅藻DNA提取方法相同。<ol><li>将玻璃珠加入含有预处理水样的 2 mL 离心管中。倒置 10 倍至 15 倍以充分混合。添加的玻璃珠由大小玻璃珠以1：1的质量比混合而成。大玻璃珠的直径为 1.5-2.0 毫米，小玻璃珠的直径为 0.4-0.6 毫米。</li>
		<li>取 0.5 g 切碎的组织，如上所述将玻璃珠加入含有组织的 2 mL 离心管中。倒置 10 倍至 15 倍混合。</li>
	</ol></li>
	<li>向试管中加入 40 μL 蛋白酶 K （20 mg/mL）。在室温下放置 15 分钟，在此期间，倒置，每 3 分钟混合 10 次。 注意：如果组织没有完全消化，可以适当增加蛋白酶K的量，直到溶液变得透明。</li>
	<li>向离心管中加入200μL结合缓冲液，立即涡旋，混合4分钟（振荡混合频率：3000rpm）。 注意：在此步骤中，应严格控制振荡强度和时间，以确保足够的混合强度和时间。</li>
	<li>将离心管置于70°C水浴中10分钟，溶液变得澄清。</li>
	<li>向离心管中加入100μL异丙醇，涡旋并混合15秒，此时可能出现絮状沉淀。 注意：在上述操作步骤中，以适当的强度充分混合非常重要，但应避免剧烈摇晃，以防止DNA剪切。</li>
	<li>将上一步得到的溶液与絮凝剂沉淀物一起放入吸附柱中（吸附柱置于收集管中）。吸附柱长度为3.0厘米，直径为1.0厘米，吸附基体为硅基膜。</li>
	<li>以8,000× g 离心30秒，弃去收集管中的废液，并将吸附柱放回收集管中。</li>
	<li>向吸附柱中加入 500 μL 抑制剂去除缓冲液。以 13,400 x g 离心 30 秒，然后丢弃收集管中的废液。</li>
	<li>向吸附柱中加入700μL洗涤缓冲液。以 13,400 x g 离心 30 秒，然后丢弃收集管中的废液。 注意： 首次使用前，将指定量的无水乙醇加入洗涤缓冲液瓶中。</li>
	<li>向吸附柱中加入 500 μL 洗涤缓冲液。以 13,400 x g 离心 30 秒，然后丢弃收集管中的废液。</li>
	<li>将吸附柱放回空的收集管中。以14,500× g 离心2分钟，并尽可能除去洗涤缓冲液，以避免洗涤缓冲液中残留的乙醇抑制下游反应。</li>
	<li>取出吸附柱，放入干净的离心管中。向吸附膜的中间部分加入 100 μL 洗脱缓冲液。</li>
	<li>将吸附柱置于室温下3-5分钟，并以13,400× g 离心1分钟。</li>
	<li>将上一步得到的溶液再次加入离心吸附柱中。将离心吸附柱置于室温下2分钟，并以13,400× g 离心1分钟。 注：洗脱缓冲液应在70°C水浴中预热约10分钟。洗脱体积应不小于50μL;否则，DNA产量将降低。</li>
	<li>将提取的硅藻DNA储存在2-8°C以备将来使用。如果要长时间储存DNA溶液，请将其储存在-20°C。</li>
</ol>3. PCR检测
注意：由于法医样本中硅藻的含量通常很低，溺水尸体的组织样本提取物也可能含有不同程度的组织和器官（例如本实验中的肺）及其自身的DNA。因此，直接检测DNA提取物中的总DNA并不能反映硅藻DNA提取的情况。本实验选择硅藻特异性引物，采用PCR产物对提取物中的硅藻DNA提取情况进行评价。产物可采用琼脂糖凝胶电泳进行观察分析，也可采用实时荧光定量PCR熔解曲线进行分析，灵敏度更高。
<ol><li>加入 2 μL 从水样和组织中提取的 DNA 作为模板。选择以下两种方法之一进行检查。</li>
	<li>常规PCR检测<ol><li>使用可特异性扩增硅藻 18S rDNA 片段的引物22 。具体请参见 表1。</li>
		<li>根据引物的特性建立PCR反应体系和扩增条件。具体请参见 表2。</li>
		<li>在2%琼脂糖凝胶上运行PCR扩增产物。使用凝胶成像仪观察和分析成像。</li>
	</ol></li>
	<li>荧光定量PCR检测<ol><li>使用上述可特异性扩增硅藻18S rDNA片段的引物，制备实时荧光定量PCR反应体系。具体请参见 表3。</li>
		<li>进行PCR扩增并分析获得的扩增曲线和Ct值。同时，设置程序，通过荧光定量PCR熔融曲线技术，将扩增产物的双链逐渐熔融成单链，然后对得到的熔解曲线进行分析。</li>
	</ol></li>
</ol>

使用改良试剂盒进行经济的硅藻 DNA 提取

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硅藻细胞受到坚硬的硅质细胞壁17的保护，必须破坏这种结构才能提取硅藻DNA。普通试剂盒不易破坏硅藻的硅质外壳;因此很难成功提取硅藻DNA21。我们的实验室改进了最常用的血液DNA提取试剂盒，在硅藻提取过程中添加了不同直径和不同质量比的玻璃珠。同时进行涡旋振荡，可充分打破硅藻壳，提高硅藻DNA提取效果。如上一份报告21所示，玻璃...

在法医实践中，确定尸体是在水中溺水还是死后被扔进水里，对于案件的妥善解决至关重要1.这也是法医学实践中亟待解决的难题之一2.硅藻在自然环境中（尤其是在水中）含量丰富3,4。在溺水过程中，由于缺氧和应激反应，患者会出现剧烈的呼吸运动，吸入大量的溺水液。因此，水中的硅藻随溺水液进入肺部，一些硅藻可以通过肺泡-毛细血管屏障进入血液循环，并随着血流扩散到内脏器官5,6。在肺、肝和骨髓等内部组织和器官中检测到硅藻是死前溺水的有力证据 7,8。目前，法医硅藻测试主要基于形态学测试方法。在对组织进行一系列预消化后，在显微镜下对未消化的硅藻进行形态定性和定量估计。在此期间，需要使用危险且对环境不友好的试剂，例如硝酸。这个过程非常耗时，需要研究人员具有扎实的分类学专业知识和丰富的经验。这些都给法医人员带来了一定的挑战9.硅藻DNA检测技术是近年来发展起来的一种用于硅藻检测的新技术10,11,12。该技术通过分析硅藻的特定DNA序列组成来实现硅藻的物种鉴定13,14。PCR技术和测序技术是常用的技术方法，但它们的基础是从硅藻中成功提取DNA。然而，硅藻具有不同于其他生物的特殊结构，使其DNA提取技术也不同。
硅藻的细胞壁具有高度的硅化程度，其主要成分是二氧化硅15,16,17。硅质细胞壁非常坚硬，在提取DNA之前必须将其破坏。普通的DNA提取试剂盒通常难以直接用于硅藻DNA的提取，因为它们不能破坏硅藻的硅质外壳18。因此，破坏硅藻的硅质外壳是提取硅藻DNA需要解决的关键技术问题之一。
同时，由于法医研究样本中所含的硅藻数量，无论是水样还是溺水尸体的器官和组织，往往都是有限的，因此有必要富集硅藻。富集的本质是物质的分离。在尝试将硅藻聚集在一起时，尽量减少其他材料成分（干扰成分）的含量。在法医工作中，实验室经常使用离心或膜过滤富集方法来分离硅藻细胞19。然而，由于真空泵设备应用不广，膜富集法在普通的初级法医实验室中并不常用。因此，离心法仍然是法医实验室20中常用的硅藻富集法。
从硅藻中提取DNA目前主要用于法医实践，其应用存在很大的局限性。目前，市场上用于法医学的硅藻DNA提取试剂盒很少，而且价格普遍昂贵21。本文提供了一种改进的硅藻DNA提取方法，使硅藻DNA提取变得简单、方便且具有成本效益。这增加了后续硅藻分子生物学检测的应用，可以通过硅藻检测更好地解决法医溺水相关问题。该方法通过添加玻璃珠并设置适当的涡旋时间来破坏硅藻的硅质细胞壁。通过这种方式，蛋白酶 K 和结合溶液迅速裂解细胞并使细胞中的各种酶失活。基因组DNA在吸附柱的基质膜中被吸收，最后被洗脱缓冲液洗脱。这种改进的全血基因提取试剂盒提高了血液试剂盒在法医检验材料中的硅藻DNA提取效果，降低了法医实践中硅藻DNA提取的成本，可以更好地应用于基层法医研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Binding Buffer</td>
			<td>BioTeke</td>
			<td>B010006022</td>
			<td>rapidly lysing cells</td>
		</tr>
		<tr>
			<td>ChemoHS qPCR Mix</td>
			<td>Monad</td>
			<td>00007547-120506</td>
			<td>qPCR Mix</td>
		</tr>
		<tr>
			<td>D2000 DNA ladder</td>
			<td>Real-Times(Beijing) Biotechnology</td>
			<td>RTM415</td>
			<td>Measure the position of electrophoretic bands</td>
		</tr>
		<tr>
			<td>D512</td>
			<td>Taihe Biotechnology</td>
			<td>TW21109196</td>
			<td>forword primer</td>
		</tr>
		<tr>
			<td>D978</td>
			<td>Taihe Biotechnology</td>
			<td>TW21109197</td>
			<td>reverse primer</td>
		</tr>
		<tr>
			<td>Elution buffer</td>
			<td>BioTeke</td>
			<td>B010006022</td>
			<td>A low-salt elution buffer washes off the DNA</td>
		</tr>
		<tr>
			<td>Glass bead</td>
			<td>Yingxu Chemical Machinery(Shanghai)&#160;</td>
			<td>70181000</td>
			<td>Special glass beads for dispersing and grinding</td>
		</tr>
		<tr>
			<td>Import adsorption column</td>
			<td>BioTeke</td>
			<td>B2008006022</td>
			<td>Adsorption column with silica matrix membrane</td>
		</tr>
		<tr>
			<td>Inhibitor Removal Buffer</td>
			<td>BioTeke</td>
			<td>B010006022</td>
			<td>Removal of Inhibitors in DNA Extraction</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>BioTeke</td>
			<td>B010006022</td>
			<td>Precipitate or isolate DNA</td>
		</tr>
		<tr>
			<td>MIX-30S Mini Mixer</td>
			<td>Miulab</td>
			<td>MUC881206</td>
			<td>oscillatory action</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>BioTeke</td>
			<td>B010006022</td>
			<td>Inactivation of intracellular nucleases and other proteins</td>
		</tr>
		<tr>
			<td>Rotor-Gene Q 5plex HRM</td>
			<td>Qiagen</td>
			<td>R1116175</td>
			<td>real-time fluorescence quantification PCR</td>
		</tr>
		<tr>
			<td>Speed Micro-Centrifuge</td>
			<td>Scilogex</td>
			<td>9013001121</td>
			<td>centrifuge</td>
		</tr>
		<tr>
			<td>Tanon 3500R Gel Imager</td>
			<td>Tanon</td>
			<td>16T5553R-455</td>
			<td>gel imaging</td>
		</tr>
		<tr>
			<td>Taq Mix Pro</td>
			<td>Monad</td>
			<td>00007808-140534</td>
			<td>PCR Mix</td>
		</tr>
		<tr>
			<td>Thermo Cycler</td>
			<td>Zhuhai Hema</td>
			<td>VRB020A</td>
			<td>ordinary PCR</td>
		</tr>
		<tr>
			<td>Wash Buffer</td>
			<td>BioTeke</td>
			<td>B010006022</td>
			<td>Remove impurities such as cell metabolites</td>
		</tr>
	</tbody>
</table>

extraction of diatom dna from water samples and tissues

硅藻测试是法医实践中确定尸体是否溺水、推断溺水地点的重要辅助手段。硅藻试验也是环境和浮游生物领域的重要研究内容。以硅藻DNA为主要研究对象的硅藻分子生物学检测技术是一种新的硅藻检测方法。硅藻DNA提取是硅藻分子检测的基础。目前，常用用于硅藻DNA提取的试剂盒价格昂贵，增加了开展相关研究的成本。本实验室对通用全血基因组DNA快速提取试剂盒进行了改进，获得了满意的硅藻DNA提取效果，从而为相关研究提供了一种经济实惠的基于玻璃珠的DNA提取解决方案。使用该方案提取的硅藻DNA可以满足许多下游应用，例如PCR和测序。

In forensic practice, determining whether a corpse found in the water drowning or was thrown into the water after death is essential for the proper resolution of the case1. It is also one of the difficult issues that need to be solved urgently in forensic practice2. Diatoms are abundant in the natural environment (especially in water)3,4. In the process of drowning, due to hypoxia and stress response, people will have intense breathing movements and inhale a large amount of drowning liquid. Therefore, the diatoms in the water enter the lung with the drowning liquid, and some diatoms can enter the blood circulation through the alveolar-capillary barrier and spread to internal organs with the blood flow5,6. The detection of diatoms in internal tissues and organs such as lung, liver, and bone marrow is a strong evidence of drowning before death7,8. Currently, forensic diatom testing is mainly based on morphological testing methods. After a series of pre-digestion of the tissue, the morphological qualitative and quantitative estimations of undigested diatoms are carried out under the microscope. During this period, dangerous and environmentally unfriendly reagents such as nitric acid need to be used. This process is time-consuming and requires researchers to have solid taxonomic expertise and extensive experience. These all bring certain challenges to the forensic staff9. Diatom DNA testing technology is a new technology for diatom testing developed in recent years10,11,12. This technology realizes the species identification of diatoms by analyzing the specific DNA sequence composition of diatoms13,14. PCR technology and sequencing technology are commonly used technical methods, but their basis is the successful extraction of DNA from diatoms. However, diatoms have a special structure different from other organisms, making their DNA extraction techniques also different.
The cell wall of diatom has a high degree of silicification, and its main component is silicon dioxide15,16,17. The siliceous cell wall is very hard, and it must be destroyed before extract the DNA. Ordinary DNA extraction kits are often difficult to use directly for the extraction of diatom DNA because they cannot destroy the siliceous shell of diatoms18. Therefore, destroying the siliceous shell of diatoms is one of the key technical problems to be solved in extracting diatom DNA.
At the same time, since the number of diatoms contained in forensic research samples, whether water samples or organs and tissues of drowned bodies, is often limited, it is necessary to enrich diatoms. The essence of enrichment is the separation of substances. While trying to gather diatoms together, minimize the content of other material components (interfering components). In forensic work, laboratories often use centrifugation or membrane filtration enrichment methods to separate diatom cells19. However, since vacuum pumping equipment is not widely used, the membrane enrichment method is not often used in ordinary primary forensic laboratories. So, the centrifugation method is still commonly diatom enrichment in forensic laboratories20.
DNA extraction from diatoms is currently used primarily in forensic practice, and there are significant limitations to its application. At present, there are few diatom DNA extraction kits used in forensic science on the market and they are generally expensive21. This article provides an improved diatom DNA extraction method, making diatom DNA extraction simple, convenient and cost effective. This increases the application of subsequent molecular biology testing of diatoms and can better solve problems related to drowning in forensic medicine through diatom testing. This method breaks the siliceous cell walls of diatoms by adding glass beads and setting an appropriate time for vortex. In this way, the proteinase K and the binding solution rapidly lyse the cells and inactivate various enzymes in the cells. The genomic DNA is absorbed in the matrix membrane in the adsorption column and finally eluted by the elution buffer. Such an improved whole blood gene extraction kit improves the diatom DNA extraction effect of the blood kit in forensic examination materials, reduces the cost of diatom DNA extraction in forensic practice, and can be better applied to grassroot forensic research.

作者聚焦： 用于法医溺水检查的硅藻检测 

从水样和组织中提取硅藻DNA

Our research mainly focus on the topic of drowning. We wanted to answer the question of whether the body found in the water died from drowning, as well to answer another question of where the person fell into the water. That means to know the location of the drowning.Compared with other technologies, we provide a simple, convenient, and economical diatom DNA extraction protocol that we can use better in forensic research, especially in basic forensic practice. Our laboratory will further focus on the drowning related problems based on the pattern in the water, such as reason of death, post mortem interval and so on. What's more, we also carry on the research through the forensic entomology and a some special phenomenons in the tropical forensic medicines.

由于目前使用的DNA提取方法提取的DNA溶液中含有来自样品中不同来源的所有DNA成分，因此通过该协议获得的DNA也不例外。因此，DNA溶液不仅仅是硅藻基因组DNA的溶液。通过查阅文献22,23,24选择可特异性扩增硅藻18S rDNA片段的引物。采用NCBI-Blast Primer对引物进行验证，结果表明，18S rDNA的正向引物D512和反向引物D978是藻类特异性引?...

Watch this Scientific Journal Video about Diatom Testing for Forensic Drowning Examination at JoVE.com

本文介绍了使用改良的通用 DNA 提取试剂盒提取硅藻 DNA 的方案。

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. ...

我们的研究主要集中在溺水的话题上。我们想回答在水中发现的尸体是否死于溺水的问题，以及回答另一个人落入水中的另一个问题。这意味着要知道溺水的位置。与其他技术相比，我们提供了一种简单、方便、经济的硅藻DNA提取方案，我们可以更好地用于法医研究，特别是在基本的法医实践中。我们的实验室将根据水中的模式，如死亡原因、死后间隔等，进一步关注溺水相关问题。此外，我们还通过法医昆虫学和热带法医中的一些特殊现象进行研究。

extraction-of-diatom-dna-from-water-samples-and-tissues

Research

JoVE Journal

Medicine

Extraction of Diatom DNA from Water Samples and Tissues

1.3K 次观看.  Hainan Medical University, Hainan Provincial Academician Workstation (tropical forensic medicine). 我们的研究主要集中在溺水的话题上。我们想回答在水中发现的尸体是否死于溺水的问题，以及回答另一个人落入水中的另一个问题。这意味着要知道溺水的位置。与其他技术相比，我们提供了一种简单、方便、经济的硅藻DNA提取方案，我们可以更好地用于法医研究，特别是在基本的法医实践中。我们的实验室将根据水中的模式，如死亡原因、死后间隔等，进一步关注?...

作者聚焦： 用于法医溺水检查的硅藻检测

作者聚焦：用于法医溺水检查的硅藻检测