这项工作得到了中国国家自然科学基金 （82060341,81560304） 和海南省院士创新平台科学研究项目 （YSPTZX202134） 的支持。

注意：本方案共使用了 5 个 C. megacephala 样品 - 3 个新鲜样品（新鲜 1、新鲜 2 和新鲜 3），1 个样品在酒精中储存 2 年（Old 1），1 个样品在干燥密封储存中保存 2 年仅避光（Old 2）。样品必须根据实验要求进行标记。
1. 一般样品储存和制备
<ol><li>样品选择和制备 注意：使用乙酸乙酯处决坏死蝇时，请务必在通风橱中工作。称重前，从果蝇表面去除液体。<ol><li>新鲜样品：用乙酸乙酯杀死后，将收集的三个苍蝇样品放入气瓶中。称量样品并将其命名为 Fresh 1、Fresh 2 和 Fresh 3，并立即在实验中使用。 注：该方案中的新鲜果蝇样品质量为 56.8 mg、49.3 mg 和 52.1 mg。</li>
		<li>旧样品 1：取出被乙酸乙酯杀死的果蝇样品，并在充满酒精的离心管中密闭保存 2 年，在室温下避光保存。用滤纸吸附表面液体后称取样品;称之为 Old 1。 注意：在该方案中，Old 1 的质量为 42.3 mg。</li>
		<li>旧样品 2：取出被乙酸乙酯杀死的果蝇样品，并在离心管中密闭保存 2 年，在室温下避光保存。称重并称其为 Old 2。</li>
	</ol></li>
	<li>提取前的准备<ol><li>加入少量液氮冷却不锈钢容器;等到所有液氮都蒸发完毕。</li>
		<li>将苍蝇样本放入容器中;缓慢倒入液氮，以防止液氮飞溅并影响昆虫样品。</li>
		<li>将样品冷冻在液氮中。液氮蒸发后取出样品。</li>
		<li>单独分离胸部，放入 2 mL 离心管中，切成尽可能小的切片。 注：将样品切成小块，使其可以完全裂解，并且可以大量释放 DNA。</li>
		<li>对每只果蝇重复上述步骤，单独冷冻，然后转移到新的离心管中，以防止 DNA 交叉污染。</li>
	</ol></li>
</ol>2. DNA 提取
注：所有离心步骤必须在室温 （15-25 °C） 下在微量离心机中进行。所有步骤都必须严格遵守无菌原则并避免交叉污染。
<ol><li>组织裂解<ol><li>将 180 μL 组织裂解缓冲液添加到含有夹住样品的 2 mL 离心管中。</li>
		<li>加入 20 μL 蛋白酶 K。以 3 000 rpm 涡旋 10 秒充分混合，并在 56 °C 下孵育直至昆虫组织完全裂解。 注意：如果组织未完全消化，则应添加更多蛋白酶 K。裂解通常在 1-3 小时内完成，也可延长裂解时间过夜。</li>
	</ol></li>
	<li>DNA 沉淀<ol><li>以 3,000 rpm 的速度涡旋 15 秒。向样品中加入 200 μL 裂解缓冲液，并以 3,000 rpm 涡旋 15 秒充分混合。</li>
		<li>加入 200 μL 乙醇 （96-100%），并以 3,000 rpm 涡旋 15 秒，再次充分混合。 注：裂解缓冲液和乙醇可以一步预添加在一起，以节省时间。加入裂解缓冲液和乙醇后可能会形成白色沉淀。</li>
	</ol></li>
	<li>将混合物移液到放置在 2 mL 收集管中的 DNA 吸附柱中。以 6,000 × g 离心 1 分钟。丢弃流通管和收集管。</li>
	<li>将上一步的 DNA 吸附柱放入新的 2 mL 收集管中，加入 500 μL 蛋白质去除缓冲液，并以 6,000 × g 离心 1 分钟。丢弃流通管和收集管。</li>
	<li>将 DNA 吸附柱转移到新的 2 mL 收集管中，加入 500 μL 脱盐缓冲液，并以 20,000 × g 离心 3 分钟以干燥 DNA 吸附柱膜。丢弃流通管和收集管。 注：此离心步骤可确保在下一次洗脱之前去除任何残留的乙醇。如果有任何乙醇残留，再次以 20,000 × g 离心 1 分钟。</li>
	<li>将 DNA 吸附柱置于干净的 2 mL 微量离心管中，直接将 100 μL DNA 洗脱缓冲液吸至柱膜上，室温孵育 1 分钟，然后以 6,000 × g 离心 1 分钟洗脱。 注：用 100 μL（而不是 200 μL）洗脱会增加洗脱液中的最终 DNA 浓度，但也会降低 DNA 总产量。为了最大限度地提高 DNA 产量，请按照步骤 2.8 中的说明重复洗脱一次。</li>
	<li>将 DNA 样品储存在 4 °C（长达 1 周）或 -80 °C 下进行长期储存。</li>
</ol>3. DNA 浓度检测
<ol><li>通过酶标仪确定 OD 260 和 OD 280 的值，以比较 DNA 纯度和核酸浓度。对每个样品重复 3 次。</li>
	<li>使用其配套试剂盒通过荧光计测定提取的 DNA 的浓度，每个样品重复 3 次。<ol><li>取 190 μL 工作溶液放入两个 0.5 mL 离心管中，取标准 #1 和标准 #2 各 10 μL，加入工作溶液中，避光放置不少于 15 分钟。 注：标准品 #1 和标准品 #2 针对一定浓度的 DNA 进行了精确校准。应准确配置标准溶液，以确保形成准确的标准曲线。标准 #1 是 0.2 ng/μL 双链 DNA （dsDNA） 样品;标准 #2 为 2,000 ng/μL。工作溶液是一种与 dsDNA 特异性结合的染料。</li>
		<li>取 2 μL 检测 DNA，在 0.5 mL 离心管中与 198 μL 工作溶液混合，避光不少于 15 分钟。 注：如果 DNA 浓度低，则添加 5 μL 测试 DNA 和 195 μL 工作溶液。</li>
		<li>15 分钟后，打开仪器并选择 dsDNA 浓度测量 |长距离。首先，测试标准 #1，然后测试标准 #2 以获得标准曲线。 注意：标准溶液必须在配置后的同一天使用;放置时间过长会导致标准曲线出现较大的误差。</li>
		<li>将待测样品逐一放入检测孔中，将 DNA 加标体积调节至 2 μL，重复测量 3 次，取平均值，记录。</li>
	</ol></li>
</ol>4. PCR 和电泳检测
注：取长度为 278 bp 的线粒体 CO <img alt="Equation 1" src="/files/ftp_upload/66737/66737eq01v2.jpg" style="margin: auto;"> 基因序列，用正向引物 C1-J-2495：CAGCTACTTTATGAGCTTTAGG，反向引物 C1-N-2800：CATTTCAAGCTGTGTAAGCATC8 进行 PCR 扩增，然后进行 2% 琼脂糖凝胶电泳，验证扩增效果和扩增产物的长度。
<ol><li>通过添加 10 μL 2x Taq 混合物、1 μL 每种引物、5 μL ddH2O 和 3 μL 模板 DNA，构建 20 μL PCR 扩增系统;在离心机中充分混合 10 秒。</li>
	<li>使用以下PCR循环参数：预变性95°C，10分钟;变性 95 °C 持续 30 秒，退火 48 °C 持续 30 秒，延伸 72 °C 持续 45 秒，共 40 个循环;最终延伸 72 °C 10 分钟。</li>
	<li>将 PCR 后的产物进行 2% 琼脂糖凝胶电泳，电泳后，将它们放在凝胶成像系统上进行光分析。</li>
</ol>

评估新鲜和老嗜坏死果蝇样品中的 DNA 纯度和浓度

<ol>
	<li>Amendt, J., Richards, C. S., Campobasso, C. P., Zehner, R., Hall, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forensic+entomology:+applications+and+limitations.">Forensic entomology: applications and limitations.</a> Forensic Sci Med Pathol. 7 (4), 379-392 (2011).</li><li>Wydra, J., Matuszewski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+optimal+post-eclosion+interval+while+estimating+the+post-mortem+interval+based+on+an+empty+puparium.">The optimal post-eclosion interval while estimating the post-mortem interval based on an empty puparium.</a> Forensic Sci Med Pathol. 17 (2), 192-198 (2021).</li><li>Mazzanti, M., Alessandrini, F., Tagliabracci, A., Wells, J. D., Campobasso, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+degradation+and+genetic+analysis+of+empty+puparia:+genetic+identification+limits+in+forensic+entomology.">DNA degradation and genetic analysis of empty puparia: genetic identification limits in forensic entomology.</a> Forensic Sci Int. 195 (1-3), 99-102 (2010).</li><li>Odeniran, P. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=molecular+identification+and+distribution+of+Trypanosome-transmitting+Dipterans+from+cattle+settlements+in+Southwest+Nigeria.">molecular identification and distribution of Trypanosome-transmitting Dipterans from cattle settlements in Southwest Nigeria.</a> Acta Parasitol. 66 (1), 116-128 (2021).</li><li>Wells, J. D., Stevens, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+DNA-based+methods+in+forensic+entomology.">Application of DNA-based methods in forensic entomology.</a> Annu Rev Entomol. 53, 103-120 (2008).</li><li>Kotrba, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+barcoding+in+forensic+entomology-establishing+a+DNA+reference+library+of+potentially+forensic+relevant+arthropod+species.">DNA barcoding in forensic entomology-establishing a DNA reference library of potentially forensic relevant arthropod species.</a> J Forensic Sci. 64 (2), 593-601 (2019).</li><li>Weedon, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+SNP+chips+to+detect+rare+pathogenic+variants:+Retrospective,+population+based+diagnostic+evaluation.">Use of SNP chips to detect rare pathogenic variants: Retrospective, population based diagnostic evaluation.</a> BMJ. 372, 214 (2021).</li><li>Cai, J. -. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+availability+of+mitochondrial+DNA+cytochrome+oxidasegene+for+the+distinction+of+forensically+important+flies+in+China.">The availability of mitochondrial DNA cytochrome oxidasegene for the distinction of forensically important flies in China.</a> Acta Entomologica Sinica. 48 (3), 380-385 (2005).</li><li>Khayrova, A., Lopatin, S., Varlamov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtaining+chitin,+chitosan+and+their+melanin+complexes+from+insects.">Obtaining chitin, chitosan and their melanin complexes from insects.</a> Int J Biol Macromol. 167, 1319-1328 (2021).</li><li>Guo, Y. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+three+DNA+extracting+methods+from+different+parts+of+common+necrophagous+flies.">Comparison of three DNA extracting methods from different parts of common necrophagous flies.</a> J Henan Univ Sci Tech (Med Sci). 36 (02), 130-133 (2018).</li><li>Du, H., Xie, H., Ma, M., Igarashi, Y., Luo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+methods+obtain+high-quality+DNA+and+RNA+from+anaerobic+activated+sludge+at+a+wide+range+of+temperatures.">Modified methods obtain high-quality DNA and RNA from anaerobic activated sludge at a wide range of temperatures.</a> J Microbiol Methods. 199, 106532 (2022).</li><li>Sukumaran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+determination+of+nucleic+acids+and+proteins+using+the+micro-volume+BioSpec-nano-spectrophotometer.">Concentration determination of nucleic acids and proteins using the micro-volume BioSpec-nano-spectrophotometer.</a> J Vis Exp. (48), e2699 (2011).</li><li>Masago, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+between+fluorimetry+(Qubit)+and+spectrophotometry+(NanoDrop)+in+the+quantification+of+DNA+and+RNA+extracted+from+frozen+and+FFPE+tissues+from+lung+cancer+patients:+A+real-world+use+of+genomic+tests.">Comparison between fluorimetry (Qubit) and spectrophotometry (NanoDrop) in the quantification of DNA and RNA extracted from frozen and FFPE tissues from lung cancer patients: A real-world use of genomic tests.</a> Medicina (Kaunas). 57 (12), 1375 (2021).</li><li>Ponti, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+value+of+fluorimetry+(Qubit)+and+spectrophotometry+(NanoDrop)+in+the+quantification+of+cell-free+DNA+(cfDNA)+in+malignant+melanoma+and+prostate+cancer+patients.">The value of fluorimetry (Qubit) and spectrophotometry (NanoDrop) in the quantification of cell-free DNA (cfDNA) in malignant melanoma and prostate cancer patients.</a> Clin Chim Acta. 479, 14-19 (2018).</li><li>Adams, Z. J., Hall, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+used+for+the+killing+and+preservation+of+blowfly+larvae,+and+their+effect+on+post-mortem+larval+length.">Methods used for the killing and preservation of blowfly larvae, and their effect on post-mortem larval length.</a> Forensic Sci Int. 138 (1-3), 50-61 (2003).</li><li>Fliegerova, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+DNA+extraction+and+sample+preservation+method+on+rumen+bacterial+population.">Effect of DNA extraction and sample preservation method on rumen bacterial population.</a> Anaerobe. 29, 80-84 (2014).</li><li>Rodrigues, B. L., Galati, E. A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+taxonomy+of+phlebotomine+sand+flies+(Diptera,+Psychodidae)+with+emphasis+on+DNA+barcoding:+A+review.">Molecular taxonomy of phlebotomine sand flies (Diptera, Psychodidae) with emphasis on DNA barcoding: A review.</a> Acta Trop. 238, 106778 (2023).</li></ol>

作者声明他们没有相互竞争的经济利益。

DNA 提取是嗜坏死果蝇分子鉴定中最关键的环节，其提取方法和提取的 DNA 质量直接影响后续检测。在本实验中，我们使用普通的血液试剂盒成功地从嗜坏死的果蝇中提取了 DNA，而无需购买特殊的昆虫 DNA 提取试剂盒，这使得昆虫 DNA 提取更容易完成。
果蝇表面富含几丁质，尤其是在幼虫和蛹期，表皮几丁质可达 60-70%，这使得嗜坏死果蝇的 DNA 提取变得困难9?...

<a href="https://app.jove.com/t/6604">This corrects</a> the article <a href="https://dx.doi.org/10.3791/66737">10.3791/66737</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/66737">DNA Extraction and Comparison Between Old and Fresh Necrophilic Fly Samples</a>. The Authors section was updated.

Erratum: DNA Extraction and Comparison Between Old and Fresh Necrophilic Fly Samples

死亡时间的推论一直是司法实践中需要解决的关键和难点问题之一。在法医学实践中，对于刑事案件，确定验尸间隔对于推断犯罪时间、锁定嫌疑人、缩小侦查范围起着至关重要的作用。传统的推断死亡时间的方法主要基于早期的死后现象，通常只能用于推断 24 小时内的死亡时间。然而，死亡时间的长短不能由死后现象来确定。现在，法医科学界普遍认为，法医昆虫学有可能成为推断较长死亡时间的有效方法。
Necrophagous 昆虫以其以尸体为食的幼虫而得名。其中，每当有尸体出现时，成年嗜尸蝇就会第一个到达尸体并产卵或幼虫。幼虫以尸体组织为食，成熟为蛹，蛹长成成虫。嗜尸蝇由于其有规律的生命周期和地理分布，在法医学实践中发挥着巨大的作用，例如，扣除死亡时间并确定死亡地点 1,2。然而，在法医实践中使用嗜尸蝇的障碍是物种鉴定。形态学方法仍被用作嗜坏死果蝇物种鉴定的权威方法。然而，形态学物种鉴定需要高度的昆虫完整性。如果果蝇处于不同的发育阶段，或者由于环境选择引起的形态变化，这些都会使形态学检查更加困难。特别是，最常见于犯罪现场的蛹和蛹壳的形态几乎无法辨认。这一点，再加上形态学专家的稀缺，使物种的形态学鉴定变得非常困难。因此，出现了分子生物学方法对果蝇物种鉴定的应用，它降低了形态学鉴定的难度，并且独立于发育阶段 3,4,5,6,7。
基于分子生物学方法对嗜坏死果蝇进行物种鉴定的第一步是高效提取 DNA，而目前不常用用于昆虫 DNA 提取的专用试剂盒。如何使用普通血液试剂盒提取嗜坏死果蝇的 DNA 变得更具法医意义。在犯罪现场发现的嗜尸蝇通常被肢解或经历腐烂和 DNA 降解。果蝇样品在采集后不能立即用于 DNA 提取，或者由于证据保存要求，如果可能，需要保留完整的样品。基于上述要求，本研究采用基于蛋白酶K消化的血液DNA试剂盒，选取3个新鲜 的大头金丝菌（双翅目、鳞翅目） 样本（图1），1个大 头念珠 菌老样本在酒精中放置两年，1个巨 头念珠 菌样本在避光保存中放置两年，对5个样本各称重， 选择了昆虫胸部组织进行 DNA 提取。比较从新旧样品中提取的 DNA 的质量和纯度，使用位于线粒体 CO <img alt="Equation 1" src="/files/ftp_upload/66737/66737eq01v2.jpg" style="margin: auto;"> 基因序列中的嗜坏苍蝇特异性引物对提取的 DNA 进行 PCR 扩增和电泳。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer AE</td>
			<td>Qiagen</td>
			<td>172026832</td>
			<td>DNA elution buffer</td>
		</tr>
		<tr>
			<td>Buffer AL</td>
			<td>Qiagen</td>
			<td>172028374</td>
			<td>lysis buffer</td>
		</tr>
		<tr>
			<td>Buffer ATL</td>
			<td>Qiagen</td>
			<td>172028162</td>
			<td>tissue lysis buffer</td>
		</tr>
		<tr>
			<td>Buffer AW1</td>
			<td>Qiagen</td>
			<td>172028760</td>
			<td>protein removal buffer</td>
		</tr>
		<tr>
			<td>Buffer AW2</td>
			<td>Qiagen</td>
			<td>57203108</td>
			<td>desalination buffer</td>
		</tr>
		<tr>
			<td>C1-J-2495</td>
			<td>Taihe Biotechnology</td>
			<td>TW21109216</td>
			<td>forword primer</td>
		</tr>
		<tr>
			<td>C1-N-2800</td>
			<td>Taihe Biotechnology</td>
			<td>TW21109217</td>
			<td>reverse primer</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>DNeasy Mini spin column</td>
			<td>Qiagen</td>
			<td>166050343</td>
			<td>DNA adsorption column</td>
		</tr>
		<tr>
			<td>Dry Bath Incubator</td>
			<td>Miulab</td>
			<td>DKT200-2D</td>
			<td>used for heating</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>Qiagen</td>
			<td>172026218</td>
			<td>Inactivation of intracellular nucleases and other proteins</td>
		</tr>
		<tr>
			<td>Qubit 3.0 Fluorometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>2321611188</td>
			<td></td>
		</tr>
		<tr>
			<td>Speed Micro-Centrifuge</td>
			<td>Scilogex</td>
			<td>9013001121</td>
			<td>centrifuge</td>
		</tr>
		<tr>
			<td>Standard#1</td>
			<td>Thermo Fisher Scientific</td>
			<td>2342797</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard#2</td>
			<td>Thermo Fisher Scientific</td>
			<td>2342797</td>
			<td></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>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>Working Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>2342797</td>
			<td></td>
		</tr>
	</tbody>
</table>

dna extraction and comparison between old and fresh necrophilic fly samples

总共选择了 5 个大 头金鸡 孢菌样品 - 3 个新鲜样品，1 个样品在酒精中储存 2 年，1 个样品在干燥密封储存中避光保存 2 年 - 以研究血液 DNA 提取试剂盒是否可以从嗜坏蝇中提取 DNA，并确定酒精是否可以延长嗜坏蝇 DNA 的保存时间。首先，使用血液 DNA 提取试剂盒从他们的胸部组织中提取 DNA。然后，使用酶标仪和荧光计检查 DNA 纯度和浓度。最后，使用位于线粒体 CO I 基因序列中的嗜坏苍蝇特异性引物对提取的 DNA 进行 PCR 扩增和电泳。结果表明，所有样品的 DNA 纯度均大于 2.0。观察到 DNA 浓度按以下顺序排列：新鲜样品>酒精保存的旧样品>未经处理的旧样品。PCR 扩增后所有样品均具有特异性电泳条带。综上所述，血液 DNA 提取试剂盒可用于成功提取嗜坏死果蝇的 DNA，且鲜果蝇样本的 DNA 浓度大于老果蝇样本。苍蝇可以长时间储存在酒精中。

我们使用酶标仪测量提取的 DNA 溶液的 OD260 和 OD280 值，然后获得 OD260/OD280 值以评估 DNA 的纯度。所有新鲜样品和旧样品 1 的 OD260/OD280 均大于 2。旧样品 2 的 OD260/OD280 的最大值为 2.187，尽管平均值为 1.753，并且由于其 OD280 和 OD280 的值都较小 （≤0.01），其三次测量波动很大（表 1）。根据获得的 OD260 值，我们估计了 DNA 的浓度。三种样品的 DNA 浓度依次为：新鲜样品>旧样品 1 >旧样品 2...

Read this Scientific Article Protocol about DNA Extraction and Comparison between Old and Fresh Necrophilic Fly Samples at JoVE.com

作者聚焦：使用简单技术推进嗜坏死果蝇样品的 DNA 提取

本文介绍了使用改良的常用 DNA 提取试剂盒提取新鲜和旧嗜坏死果蝇 DNA 的方案。

新旧嗜坏死果蝇样品的 DNA 提取和比较

A total of five samples of Chrysomya megacephala samples - three fresh samples, one sample stored in alcohol for 2 years, and one sample stored in dry ...

dna-extraction-comparison-between-old-fresh-necrophilic-fly

Research

JoVE Journal

Medicine

DNA Extraction and Comparison Between Old and Fresh Necrophilic Fly Samples

331 次观看.  Hainan Medical University, Hainan Provincial Academician Workstation (tropical forensic medicine). 我们的颗粒旨在简单方便地提取果蝇 DNA，并比较嗜坏死果蝇新旧样本之间的 DNA 浓度。我们正在尝试研究血液 DNA 提取试剂盒是否能有效地从果蝇中提取 DNA，以及嗜坏死果蝇的新旧样本的 DNA 浓度是否有变化？我们提供了一种使用普通血液检测试剂盒提取果蝇 DNA 的方法，解决了昆虫提取试剂盒昂贵和稀缺的问题，为这些试剂盒的分子生物学分析铺平...

视频: 作者聚焦：使用简单技术推进嗜坏死果蝇样品的 DNA 提取

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

科研

医学

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

收集和唾液DNA提取用于新一代测序

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

收集，分离，和人类宫颈样品的流式细胞仪分析

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

猪生物医学模型生物库组织样品的取样策略与处理

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, we describe the protocol for using&#160;an absorbent microneedle-based device, absorbent microbiopsy, for minimally invasive sampling of skin and blood mixture. Our goal is to help facilitate rapid progress in clinical research, the establishment of biomarkers for skin disease and reducing the risk for clinical research participants. In contrast to conventional skin biopsy techniques, the absorbent microbiopsy can be performed within seconds and does not require intensive training due to its simple design. In this report, we describe the use of absorbent microbiopsy, including loading and application, on a&#160;volunteer. Then, we show how to isolate RNA from the absorbed sample. Finally, we demonstrate the use of quantitative reverse transcription PCR (RT-qPCR) to quantify mRNA expression levels of both blood (CD3E and CD19) and skin (KRT14 and TYR). The methods that we describe utilize off the shelf kits and reagents. This protocol offers a minimally invasive approach for simultaneous sampling of skin and blood within the same absorbent microbiopsy matrix. We have found human ethics committees, clinicians and volunteers to be supportive of this approach to dermatological research.

In this article, we demonstrate how the absorbent microbiopsy technique is performed and how the sample can be used for RNA extraction for simple and simultaneous sampling of skin and blood in a minimally invasive manner.

吸收性微活检取样和 rna 提取用于微创同时进行血液和皮肤分析

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

使用hiPSC衍生心肌细胞的缺血性心脏病模型和基于视频的心肌细胞收缩比较

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

代谢组学和利皮多米分析脑肿瘤的现场采样与提取

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation of the extrahepatic bile duct (EBD) in mouse neonates without damaging the bile duct during preparation. Because of its exceptionally close preparation compared to the cholangiocytes cell line and harvesting of the entire extrahepatic bile duct system (EBDS), the described approach is extremely useful in researching animal models of newborn bile duct disorders, such as biliary atresia. After euthanasia, the peritoneal cavity was accessed, and the bile duct system, duodenum, and liver were extracted with the unique En-bloc-Resection (EbR). The extracted sample is placed on a foam mat, and the EBD is dissected from contaminating cells atraumatically without necessary touch. The dissection of the entire EBDS is a significant advantage of this method. Caution must be taken due to the small size and amount of bile duct tissue. Using the described technique, there is no damage to the cholangiocytes. Further, the purity of the technique is reproducible (n = 10). Therefore, optimally comparable samples can be harvested. Furthermore, no bile duct tissue is harmed, because any contact with the bile duct system can be avoided during preparation, leaving the bile fluid inside the gall bladder. Most importantly, while performing the final gall bladder and bile duct dissection, atraumatic microinstruments were used only slightly lateral of the bile duct without squeezing it. This is the key to a clean and intact sample, and essential for further histological investigation or the isolation of cholangiocytes. To summarize, the described innovative dissection technique enables especially inexperienced operators with the necessary equipment to isolate the EBDS as cleanly as possible.

For the observation of murine neonatal bile duct disorders, an intact bile duct and efficient preparation are required. Therefore, a new approach for isolating the entire extrahepatic bile duct system in murine neonates was successfully developed while maintaining the integrity of the bile duct.

9日龄小鼠新生儿肝外胆管和胆囊清扫术

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation ...

Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature

Lumican is a small leucine-rich proteoglycan in the human amniotic membrane (AM) that promotes corneal epithelialization and the organization of collagen fibers, maintaining corneal transparency. In the present work, a method for protein extraction from AM to obtain lumican is proposed. Additionally, the stability of lumican in the AM extract (AME) stored at different temperatures and time periods is evaluated. 100 mg of AM were thawed and mechanical de-epithelialized. The de-epithelialized AM was frozen and crushed until a fine powder was obtained, which was solubilized with 2.5 mL of saline buffer with protease inhibitors and centrifuged for protein extraction. The supernatant was collected and stored at -20 &#176;C, 4 &#176;C, and room temperature (RT) for 6, 12, 20, and 32 days. Afterward, lumican was quantified in each AME. This technique allows an accessible and acquirable protocol for lumican extraction from AM. Lumican concentration was affected by storage time and temperature conditions. Lumican in the AME of 12 days stored at -20 &#176;C and 4 &#176;C was significantly higher than other AME. This lumican extraction could be useful for developing treatments and pharmaceutical solutions. Further studies are needed to determine the uses of AME lumican in re-epithelialization and wound healing process.

The present protocol describes the extraction of lumican from the amniotic membrane (AM) and their storing conditions as AM extract (AME) at -20 &#176;C, 4 &#176;C, and room temperature (RT) for 6, 12, 20, and 32 days to quantify its proteins and lumican concentration.

从羊膜中提取Lumican及其储存温度的测定

Lumican is a small leucine-rich proteoglycan in the human amniotic membrane (AM) that promotes corneal epithelialization and the organization of collagen ...

使用基于 Web 的放射计划助手增强放射治疗

Radiation Planning Assistant - A Web-based Tool to Support High-quality Radiotherapy in Clinics with Limited Resources

Access to radiotherapy worldwide is limited. The Radiation Planning Assistant (RPA) is a fully automated, web-based tool that is being developed to offer fully automated radiotherapy treatment planning tools to clinics with limited resources. The goal is to help clinical teams scale their efforts, thus reaching more patients with cancer. The user connects to the RPA via a webpage, completes a Service Request (prescription and information about the radiotherapy targets), and uploads the patient&#39;s CT image set. The RPA offers two approaches to automated planning. In one-step planning, the system uses the Service Request and CT scan to automatically generate the necessary contours and treatment plan. In two-step planning, the user reviews and edits the automatically generated contours before the RPA continues to generate a volume-modulated arc therapy plan. The final plan is downloaded from the RPA website and imported into the user&#39;s local treatment planning system, where the dose is recalculated for the locally commissioned linac; if necessary, the plan is edited prior to approval for clinical use.

作者聚焦:使用 Radiation Planning Assistant 改善放射治疗可及性

This protocol describes a series of automated tools designed for high-quality radiotherapy autocontouring and autoplanning that are being packaged into a web-based service to maximize robustness and scalability while minimizing operational costs.

放射计划助手 - 一个基于网络的工具，以支持资源有限的诊所进行高质量的放射治疗

Access to radiotherapy worldwide is limited. The Radiation Planning Assistant (RPA) is a fully automated, web-based tool that is being developed to offer ...