This work is supported by the National Natural Science Foundation of China (82060341,81560304) and by the Academician Innovation Platform Scientific Research Project of Hainan Province (YSPTZX202134).

This study was approved by the Ethics Committee of Hainan Medical University. The tissue samples used in this study are not considered to be studies involving human subjects. These specimens were obtained for the purpose of forensic pathological diagnosis, and the rest were used for the extraction of diatom DNA in this experiment. Researchers cannot readily identify individuals to obtain informed consent from relevant stakeholders.
NOTE: To ensure the general applicability of the research method reported in this experiment, this experiment basically followed the operating instructions of the kit used, and only some steps were modified. The water samples used in this experiment were randomly taken from ponds near the laboratory (Supplementary Figure 1A). In this experiment, the lung tissue of the drowned body was confirmed as the research tissue to demonstrate the extraction protocol (Supplementary Figure 1B). In forensic practice, it is also sometimes necessary to use other organs and tissues of drowned bodies (such as liver, spleen, kidney, bone marrow, etc.) to extract diatoms DNA, which requires minor corresponding improvements to this experimental method, which will be explained in the corresponding section of the experiment. The lung tissue samples used in this experiment came from corpses that were clearly drowned in forensic cases. Morphological tests have been conducted to prove that the lung tissue contains diatoms (Supplementary&#160;Figure 2).
1. Pretreatment of samples
<ol>
	<li>Pretreatment of water samples
	<ol>
		<li>Take 10 mL of water sample into the centrifuge tube and centrifuge at 13,400 x g for 5 min. Carefully discard 9.8 mL of supernatant with a pipette gun, and transfer about 200 &#181;L of enriched diatom water sample remaining at the bottom into a 2 mL centrifuge tube. 
		NOTE: Pre-experiment can be carried out first, and the initial amount can be increased if a 10 mL water sample is not enough for enrichment.</li>
	</ol>
	</li>
	<li>Pretreatment of tissue samples
	<ol>
		<li>Take 0.5 g of the lung margin tissue from the drowned body, fully chop or grind the lung tissue until it becomes muddy. Preventing contamination of exogenous diatoms is the core of this step. Cut the tissue into pieces repeatedly using scissors.</li>
	</ol>
	</li>
</ol>
2. DNA extraction
NOTE: All centrifugation steps are completed at room temperature. Using a desktop centrifuge with a centrifugal force of 14,500 x g; a water bath (or metal bath) preheated to 70 &#176;C needs to be prepared before the experiment starts. All steps must strictly follow the principles of aseptic operation.
<ol>
	<li>Assembly of water samples and tissues 
	NOTE: Water sample and tissue diatom DNA extraction method are the same.
	<ol>
		<li>Add glass beads to a 2 mL centrifuge tube containing the pretreated water sample. Invert 10x-15x to mix well. The added glass beads are composed of large and small glass beads mixed in a mass ratio of 1:1. The diameter of large glass beads is 1.5-2.0 mm and of the small glass beads is 0.4-0.6 mm.</li>
		<li>Take 0.5 g of finely chopped tissue, add glass beads to a 2 mL centrifuge tube containing the tissue as described above. Invert 10x-15x to mix.</li>
	</ol>
	</li>
	<li>Add 40 &#181;L of proteinase K (20 mg/mL) to the tubes. Place at room temperature for 15 min, during this period, invert, and mix 10x every 3 min. 
	NOTE: If the tissue is not fully digested, the amount of proteinase K can be appropriately increased until the solution becomes clear.</li>
	<li>Add 200 &#181;L of binding buffer to the centrifuge tubes, immediately vortex, and mix for 4 min (oscillation mixing frequency: 3000 rpm). 
	NOTE: In this step, the shaking intensity and time should be strictly controlled to ensure sufficient mixing intensity and time.</li>
	<li>Put the centrifuge tubes in a 70 &#176;C water bath for 10 min, and the solution becomes clear.</li>
	<li>Add 100 &#181;L of isopropanol to the centrifuge tubes, vortex and mix for 15 s, flocculent precipitation may appear at this time. 
	NOTE: It is very important to mix well with appropriate strength in the above operation steps, but vigorous shaking should be avoided to prevent DNA shearing.</li>
	<li>Put the solution obtained in the previous step together with the flocculent precipitate into the adsorption column (the adsorption column is placed in the collection tube). The adsorption column length is 3.0 cm, the diameter is 1.0 cm, and the adsorption matrix is silicon matrix membrane.</li>
	<li>Centrifuge at 8,000 x g for 30 s, discard the waste liquid in the collection tube and put the adsorption column back into the collection tube.</li>
	<li>Add 500 &#181;L of inhibitor removal buffer to the adsorption column. Centrifuge at 13,400 x g for 30 s and discard the waste liquid in the collection tube.</li>
	<li>Add 700 &#181;L of washing buffer to the adsorption column. Centrifuge at 13,400 x g for 30 s and discard the waste liquid in the collection tube. 
	NOTE: Add the specified amount of absolute ethanol to the wash buffer bottle before first use.</li>
	<li>Add 500 &#181;L of wash buffer to the adsorption column. Centrifuge at 13,400 x g for 30 s and discard the waste liquid in the collection tube.</li>
	<li>Put the adsorption column back into the empty collection tube. Centrifuge at 14,500 x g for 2 min, and remove the washing buffer as much as possible, to avoid the residual ethanol in the washing buffer from inhibiting downstream reactions.</li>
	<li>Take out the adsorption column and put it into a clean centrifuge tube. Add 100 &#181;L of elution buffer to the middle part of the adsorption membrane.</li>
	<li>Place the adsorption column at room temperature for 3-5 min, and centrifuge at 13,400 x g for 1 min.</li>
	<li>Add the solution obtained in the previous step to the centrifugal adsorption column again. Place the centrifugal adsorption column at room temperature for 2 min, and centrifuge at 13,400 x g for 1 min. 
	NOTE: The elution buffer should be preheated in a 70 &#176;C water bath for about 10 min. The elution volume should not be less than 50 &#181;L; otherwise, the DNA yield will be reduced.</li>
	<li>Store the extracted diatom DNA at 2-8 &#176;C for future use. If the DNA solution is to be stored for a long time, store it at -20 &#176;C.</li>
</ol>
3. PCR test
NOTE: Since the content of diatoms in forensic samples is often low, the tissue sample extracts of the drowned bodies may also contain varying degrees of tissue and organs (such as the lungs in this experiment) with their own DNA. Therefore, direct detection of total DNA in DNA extracts does not reflect the situation of diatom DNA extraction. In this experiment, diatom-specific primers were selected, and PCR products were used to evaluate the diatom DNA extraction in the extract. The products can be observed and analyzed by agarose gel electrophoresis and can also be analyzed by real-time fluorescent quantitative PCR-melting curve, which has higher sensitivity.
<ol>
	<li>Add 2 &#181;L of the extracted DNA from water samples and tissues as the template. Choose one of the following two methods for inspection.</li>
	<li>Conventional PCR test
	<ol>
		<li>Use primers22 that can specifically amplify diatom 18S rDNA fragments. For details, see Table 1.</li>
		<li>Establish the PCR reaction system and amplification conditions according to the characteristics of the primers. For details, see Table 2.</li>
		<li>Run the products of PCR amplification on 2% agarose gel. Observe and analyze the imaging with a gel imager.</li>
	</ol>
	</li>
	<li>Fluorescent quantitative PCR test
	<ol>
		<li>Use the above primers that can specifically amplify diatom 18S rDNA fragments to prepare a real-time fluorescent quantitative PCR reaction system. For details, see Table 3.</li>
		<li>Perform PCR amplification and analyze the obtained amplification curve and Ct values. At the same time, set a program, melt the double-strands of the amplified products into single-strands gradually through fluorescent quantitative PCR-melting curve technology, and then analyze the obtained melting curves.</li>
	</ol>
	</li>
</ol>

Economical Diatom DNA Extraction Using a Modified Kit

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The authors declare that they have no competing financial interests.

Diatom cells are protected by hard siliceous cell walls17, and this structure must be destroyed to extract diatom DNA. Ordinary kits do not easily destroy the siliceous shell of diatoms; thus it is difficult to successfully extract diatom DNA21. Our laboratory improved the most commonly used blood DNA extraction kit, adding glass beads of different diameters and different mass ratio in the process of diatom extraction. Vortex oscillation is carried out at the same time, whi...

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.

<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

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. Diatom testing is also an important research content in the field of the environment and plankton. The diatom molecular biology testing technology, which focuses on diatom DNA as the primary research object, is a new method of diatom testing. Diatom DNA extraction is the basis of diatom molecular testing. At present, the kits commonly used for diatom DNA extraction are expensive, which increases the cost of carrying out related research. Our laboratory improved the general whole blood genomic DNA rapid extraction kit and obtained a satisfactory diatom DNA extraction effect, thus providing an alternative economical and affordable DNA extraction solution based on glass beads for related research. The diatom DNA extracted using this protocol could satisfy many downstream applications, such as PCR and sequencing.

Author Spotlight: Diatom Testing for Forensic Drowning Examination 

Extraction of Diatom DNA from Water Samples and Tissues

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.

Since the DNA solution extracted by the currently used DNA extraction method contains all DNA components from different sources in the sample, the DNA obtained by this protocol was no exception. So, the DNA solution was not just a solution of diatom genomic DNA. The primers that can specifically amplify diatom 18S rDNA fragments were selected by consulting the literature22,23,24. The primers were verified by NCBI-Blast Primer, a...

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

This article describes a protocol for diatom DNA extraction using a modified common DNA extraction kit.

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

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

Research

JoVE Journal

Medicine

Diatom DNA Extraction from Water Sample and Human Lung Tissue Using a Modified DNA Extraction Kit

To begin, take 10 milliliters of the diatom water sample in a centrifuge tube. Centrifuge the tube at 13, 400 G for five minutes. With a pipette gun, carefully discard 9.8 milliliters of the supernatant.Then transfer 200 microliters of the enriched diatom water sample into a two-milliliter centrifuge tube. Next, take 0.5 grams of lung margin tissue from a drowned human body. With scissors, cut the lung tissue until it becomes muddy.For DNA extraction off both diatom and lung samples, first add glass beads to both samples. After mixing the samples well, add 40 microliters of proteinase K to the tubes. After a 15-minute incubation at room temperature, add 200 microliters of binding buffer to both tubes.Immediately mix the samples with a vortex mixer for four minutes. Then place the tubes in a water bath at 70 degrees Celsius for 10 minutes. until the solution becomes clear.Next, transfer the clear solution with flocculent precipitate. into a three-centimeter long adsorption column packed with silicon matrix. Centrifuge the column at 8, 000 G for 30 seconds.Then discard the waste liquid in the collection tube and put the column back into the collection tube. Now add 500 microliters of the inhibitor removal buffer into the column and centrifuge at 13, 400 G for 30 seconds. After discarding the waste liquid, add 700 microliters of the washing buffer to the column and centrifuge again.After discarding the waste liquid, place the adsorption column back into the empty collection tube. Centrifuge the column at 14, 500 G for two minutes to remove as much wash buffer as possible. Now take the column out and place it in a clean centrifuge tube.Add 100 microliters of elution buffer to the middle part of the adsorption membrane. Centrifuge the column at 13, 400 G for one minute after a five-minute incubation at room temperature. Transfer the solution obtained in the collection tube back into the adsorption column before centrifuging again.Store the eluted diatom DNA at two to eight degrees Celsius for future use. Agarose gel electrophoresis showed diatom DNA bands between 250 to 500 BP in both water samples and tissues after PCR amplification.

1.3K Views.  Hainan Medical University, Hainan Provincial Academician Workstation (tropical forensic medicine).  This article describes a protocol for diatom DNA extraction using a modified common DNA extraction kit.

Economical Diatom DNA Extraction Using a Modified Kit

概要