作者感谢比尔和梅林达·盖茨基金会的支持技术开发和培训。

注意：此协议包括体积和浓度为96孔板，8联管和毛细管为基础的系统（10微升反应体积）;然而，HRM，也可以在384孔平板的系统通过缩放所有卷相应进行了500微升反应体积。检测对于大多数系统的范围是10至第10纳克模板DNA。 1.准备模板<ol><li>直接从参与野外现场研究中的患者获得与全血储存血液，沉淀的红细胞，或在滤纸上获得的恶性疟原虫的样品。 注：样品也可以培养适应体外生长;一些标准实验室菌株进行分析可以从疟疾研究和参考试剂资源中心（MR4排序，样品可以直接从沉淀红血细胞或培养使用或从全血，红细胞，或滤纸萃取。 </li><li>从提取的DNA滤纸或培养适应株 注：人力资源管理是盐浓度敏感;由于可变提取方法浓度差异可能显著影响熔化温度。虽然具体最终缓冲区并不成功的显著的因素，使用标准的共同洗脱或悬浮缓冲液（ 即水 ，1个特[&#39;小特“或”DNA悬挂缓冲“：10毫摩尔Tris-CL，0.10毫摩尔EDTA]或1×TE [10mM的三Cl，1mM EDTA中]）对于所有样品，以避免异常的或不一致的熔融曲线。 <ol><li>准备模板稀释10微克-10纳克每微升的标准特，TE，或水，每10微升反应。 </li></ol></li><li>从沉淀的红血细胞的直接放大<ol><li>确定红细胞原虫使用超薄涂片显微镜 注：可从美国疾病控制和预防中心（CDC）的方法来确定受感染的红血细胞的原虫：HTTP：<html> //www.cdc.gov/dpdx/diagnosticProcedures/blood/microexam.html </li><li>在PCR级水，TE，或碲400的稀释：parasitemias 0.5％以上1。使用3微升为每5〜10微升反应。 </li></ol></li><li>控制 注意：HRM可用于基因扫描，以检测在一个人口12序列变体;但是，准确的基因分型要求的标准序列验证，以包含SNP被审问。质粒构建或疟疾菌株与已知的基因型都可以使用。对于大多数应用，3D7（MR4 MRA-151）用作野生型对照。由于运行之间的变化，总是包括阳性和阴性对照。 <ol><li>阳性对照：准备10微克 - 10纳克每质粒1μl的稀释或标准将序列经过证明的SNP基因型。 </li><li>阴性对照：用PCR级水作为一种无模板对照（NTC）的扩增反应。 </li></ol></li></ol>ve_title“&gt; 2。PCR扩增<ol><li>准备反应混合物<ol><li> （可选）矿物油覆盖。注意：一些人力资源管理系统是独立熔化平台，并没有热盖，以防止扩增过程中凝聚和产品的蒸发。 <ol><li>加入20微升的轻质矿物油，每孔装版的反应混合物之前。 </li></ol></li><li>准备好所有的化验工作10倍的股票<ol><li>请参阅表1为推荐的10倍的股票工作浓度以及终浓度为阻止探针为基础的人力资源管理的基因分型。 </li></ol></li><li>准备反应混合物<ol><li>向每个孔中板或管中，加入1微升10X正向工作原液和反向引物和探针，4.0 - 5.0微升2.5倍或2.0倍的人力资源管理主混合物，1μl的模板，PCR级水为10微升的总反应体积。 </li></ol></li><li>盖板或试管<ol><li>使用光学薄板Seals的板为基础的放大，光纤帽带管为基础的系统，及塑料瓶盖毛细管为基础的系统。要确保覆盖已经完成，板和盖完全密封，并固定，防止扩增过程中蒸发。 </li></ol></li><li>旋转样品<ol><li>旋板3分钟，在1800×g离心以除去气泡，如果矿物油用于分离油相和水相。 </li></ol></li></ol></li><li>扩增<ol><li>将反应板或管的热循环仪。运行标准的阻塞的探针或MAAB扩增方案（ 表2和3，分别）。注意在这些方法之间的退火温度的差异。 </li></ol></li></ol> 3.人力资源管理分析<ol><li>熔化<ol><li> PCR扩增后，前往熔炼分析。对于不具有内置放大的独立熔炼系统 （如，BioFire防御LightScanner系统），再从热循环仪，并装在人力资源管理工具移动放大板。从系统软件接口，熔化板40 - 80℃下同时生产探针和扩增子的熔融峰。 注意：为了节省熔化和分析时间，熔化窗口可被缩短以覆盖温度范围只是探针或扩增子熔化区域。熔化后，将板和管可以再熔融如有必要并保存在-20℃或4℃。商店玻璃管仅在4℃，以防止管从开裂。 </li></ol></li><li>熔融分析<ol><li>删除负面样本<ol><li>在分析软件，从进一步的分析样本未扩增或有锯齿熔融峰删除。注意：选择分析模式针对单独运行文件后，仪器自动组的正和负样本。前基因型每个子集，用户可以手动改变呼叫。 <ol><li>无论是从融曲线或熔融峰认为，在曲线上右键单击要删除，选择它们。选择错误分类样本后，进入“新呼叫”，选择相应的组，然后单击“应用更改”。 </li></ol></li></ol></li><li>规范化<ol><li>从标准化荧光的负导数相对于温度（-dF​​ / dT）的（&#39;差分曲线“）视图，移动正常化棒以包围探针熔化区域。 注意：探针熔融区域是两个熔融区域的较低的温度。正常化杆应该在的熔融峰的碱。 </li></ol></li><li>计算组<ol><li>标准化后，选择“计算组的自动分配样本组。 如果需要的话，手动重新分配样品到特定组。 注：有些分析软件已经不能改变高灵敏度阈值;在这些情况下，作者oftware可以分配样本，以在视觉上属于另一个不同的组。 </li></ol></li><li>指定基因型<ol><li>基于标准（阳性对照），每个组分配的基因型。 <ol><li>在用户确认所计算的群体是正确的（如果不是，那正确的修改过的分组选项卡下选择错误分类样本，并从“新呼叫”旁边的下拉框中选择正确的组），选择“编辑组名“下的”分组“选项卡。 </li><li>输入的标准基因型为相应的彩色框（例如，如果标准3D7有一个已知的A基因型，是红豆集团，红豆集团拥有基因型A）。按“确定”按钮，保存结果。 </li></ol></li></ol></li><li>导出结果<ol><li>出口型称作为一个电子表格做进一步样本人群的分析。 </li></ol></li></ol></li></ol>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World Health Organization report 2013. , 1-286 (2013).</li><li>Hayton, K., Su, X. -. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+resistance+and+genetic+mapping+in+Plasmodium+falciparum.">Drug resistance and genetic mapping in Plasmodium falciparum.</a> Current genetics. 54 (5), (2008).</li><li>Neafsey, D. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+genomics+and+genome+biology+to+understand+malaria+biology.">Harnessing genomics and genome biology to understand malaria biology.</a> Nature Reviews Genetics. 13 (5), 315-328 (2012).</li><li>Manske, 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=Analysis+of+Plasmodium+falciparum+diversity+in+natural+infections+by+deep+sequencing.">Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing.</a> Nature. 487 (7407), 375-379 (2012).</li><li>Auburn, S., 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=Characterization+of+within-host+Plasmodium+falciparum+diversity+using+next-generation+sequence+data.">Characterization of within-host Plasmodium falciparum diversity using next-generation sequence data.</a> PLoS ONE. 7 (2), e32891 (2012).</li><li>Daniels, R., 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=A+general+SNP-based+molecular+barcode+for+Plasmodium+falciparum+identification+and+tracking.">A general SNP-based molecular barcode for Plasmodium falciparum identification and tracking.</a> Malaria Journal. 7, 223 (2008).</li><li>Daniels, R., 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=Genetic+surveillance+detects+both+clonal+and+epidemic+transmission+of+malaria+following+enhanced+intervention+in+Senegal.">Genetic surveillance detects both clonal and epidemic transmission of malaria following enhanced intervention in Senegal.</a> PLoS ONE. 8 (4), e60780 (2013).</li><li>Daniels, R. R., 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=field-deployable+method+for+genotyping+and+discovery+of+single-nucleotide+polymorphisms+associated+with+drug+resistance+in+Plasmodium+falciparum.">field-deployable method for genotyping and discovery of single-nucleotide polymorphisms associated with drug resistance in Plasmodium falciparum.</a> Antimicrobial agents and chemotherapy. 56 (6), 2976-2986 (2012).</li><li>Olbadia, 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=Clonal+outbreak+of+Plasmodium+falciparum+in+eastern+Panama.">Clonal outbreak of Plasmodium falciparum in eastern Panama.</a> The Journal of infectious diseases. , (2014).</li><li>Montgomery, J., Wittwer, C. 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作者什么都没有透露。

连续人力资源管理是一个后-PCR分析步骤;因此，样品制备和分析的设置是类似于标准PCR方案，以用于跟踪从过渡的荧光嵌入染料的额外掺入双链期间高分辨率熔化步骤到单链DNA。对于成功的人力资源管理数据的一个最重要的因素是一个强大的PCR产物。分析设计是关键，可用于人力资源管理的分析设计进行优化的多种工具，商业和在线13。的80-150个碱基对伴随〜20个碱基对的扩增子的长度阻止探针集中于与SNP或感兴趣的工作最好的SNP区分SNP位点以及多个SNP的单元型的探针区域内。探针可以使用一个3&#39;C3垫片或2个碱基对错配的3&#39;端，这防止扩增期间延伸被阻止。探针可被设计为匹配沃森或克里克模板链;选择要看实践的检验，以确定哪些探头设计，可以接受的熔融峰。过量正向或反向引物被用于产生单链扩增产物退火到阻止探头。如果探针包含正向链序列，然后反向引物过量使用，典型地以1：5的比例，反之亦然为匹配反向链是探针。 同样，人力资源管理效果最好有足够的和强大的PCR产物。 PCR反应优化需要梯度PCR和琼脂糖凝胶或生物分析仪分析，以确定最佳退火温度。模板浓度可使用的方法，例如预扩增，使用低引物浓度和循环数，以增加模板浓度13的所有目标偏置多路复用扩增来增加。 只有极少数的仪器都与MAAB兼容。玻璃毛细管管的热性能用我ñ这些系统有助于此方法。毛细管的小内径以及通过玻璃的传热迅速提供更严格的温度控制比标准PCR的塑料制品，其具有由于塑料的绝热性能退火和变性循环之间较慢转移速率。用这种方法，检测灵敏度可以从2-5％减少到一个突变等位基因的小于1％的野生型和突变等位基因10的混合物。 HRM为SNP基因分型一个轻便，高效且经济的工具中的各种设置和许多应用，从传染病监测到扫描癌基因的变体。其他一些工具，如罗氏Nano和的LightCycler系统（480和96），以及生态实时荧光定量PCR系统提供HRM除了标准扩增，实时，和拷贝数的功能。使用高通量的机器，人力资源管理条码费莱总比$ 0.50元法在我们手中，包括所有的试剂和耗材。 在这里描述的背景下，基于现场的应用程序允许实时监测人口与疟疾防治工作，包括降低种群的多样性，检测进口寄生虫种类，并降低药物的敏感性相关的SNP的出现和传播相关的变化。改进的方法提供额外的灵敏度，以便向该疟疾消灭和根除的进展非常有用。

尽管疟疾控制和消除新的兴趣，疟疾仍然是一个世界性的负担，有可能感染全球近一半的人口，每年超过55万的死亡，在撒哈拉以南非洲1特别是儿童。 这些新的控制和根除方案已经支持的基因组文艺复兴时期，有大量测序和分析以减少的药物敏感性相关的基因突变疟原虫，毒力增强，以及人口2,3特性。单核苷酸多态性（SNP）是最常见的确定遗传变异体4-7之间。 便携式SNP基因分型方法提供现场实时监测人口与跟踪8。除了指纹个体寄生虫中，“分子条形码&#39;也被用来检测等位基因频率戏剧性颞移以及方差有效群体大小和感染9的复杂性。 虽然这组信息的SNP很容易适应多种基因分型平台，高分辨率熔解（HRM）分析，特别适合基于场的研究，其中灵敏，简便的操作和检测的低成本新的突变相比，排序等方法是在资源贫乏地区的吸引力。 HRM开始用纳入荧光染料标准聚合酶链式反应（PCR）。 PCR后熔炼分析确定峰值扩增子的熔化温度;在很短的扩增子的单个SNP差可能会导致大量峰值熔融温度（Tm）的差异。 一些改进这种方法提供更好的基因分型的分辨率来区分单核苷酸多态性，包括IV级（AT）的SNP，并检测轻微的突变等位基因与目前多个等位基因的样品（polygenomic感染）。首先，将测定法整合集中于与SNP区域的短探针除了向前和反向引物中存在不同浓度。这些封端的探针在PCR过程中没有放大，而是结合在过量产生铸坯由于不对称的引物浓度是增加生产探针 - 模板产物。这些独立的双链扩增子由探头势必多余的模板链是〜20-30基点;相比整个扩增子（80-150碱基）导致具有单个或多个碱基探针-模板关联错配10大得多的Tm差异其显著降低长度。 第二，突变等位基因扩增偏压（MAAB）降低反应的退火温度，以偏向存在于在polygenomic感染低比率的突变等位基因的反应。退火温度设为完全匹配（野生型）的Tm值和错配（突变体）的探针之间秒。在此温度下，野生型等位基因的结合是足够稳定相比，它的失配等效的扩增受到阻碍，从而偏压朝当两者都存在于单一样品 10的突变等位基因的扩增。 有了这些人力资源管理的改进，这种技术允许起源跟踪，同时在南美11疫情的感染和检测的新的基因突变，位于序列彼此紧邻多个SNP位点的差异，和突变有关寄生虫和身份出现在不到1 ％polygenomic样品10的等位基因。 增加的敏感性是地区接近预消除状态和那些在危险中为新兴的耐药性显得尤为重要。快速，轻松地找出与现场的敏感性降低有关进口的寄生虫和SNP的能力通知有关监测工作和控制方案它们的实现和识别热点增加根除疟疾和消除工作的成效。本协议描述的方法来阻止探针为基础的MAAB以​​提高人力资源管理的基因分型的敏感性。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>LightScanner Master Mix</td>
			<td>BioFire Defense</td>
			<td>HRLS-ASY-0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Light mineral oil</td>
			<td>Sigma</td>
			<td>M5904</td>
			<td>for BioFire Defense plate-based HRM systems</td>
		</tr>
		<tr>
			<td>TE</td>
			<td>Teknova</td>
			<td>T0226</td>
			<td></td>
		</tr>
		<tr>
			<td>Te</td>
			<td>Teknova</td>
			<td>T0221</td>
			<td>TE buffer with reduced EDTA</td>
		</tr>
		<tr>
			<td>PCR grade water</td>
			<td>Teknova</td>
			<td>W3330</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmodium falciparum standards</td>
			<td>MR4</td>
			<td>MRA-151, 156, 205, 330</td>
			<td>MR4.org offers free genomic DNA</td>
		</tr>
		<tr>
			<td>Light Scanner Primer Design Software</td>
			<td>BioFire Defense</td>
			<td></td>
			<td>commercial sofware for HRM assay design</td>
		</tr>
		<tr>
			<td>LightCycler 480 High Resolution Melting Master Mix</td>
			<td>Roche</td>
			<td>4909631001</td>
			<td>For use in LightCycler-480, -96, and nano. 2x concentration</td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent</td>
			<td>G2938A</td>
			<td>Agilent 2100 bioanalyzer</td>
		</tr>
		<tr>
			<td>LightCycler 2.0</td>
			<td>Roche</td>
			<td>3531414001</td>
			<td>Capillary-based system for MAAB</td>
		</tr>
		<tr>
			<td>LightCycler Nano</td>
			<td>Roche</td>
			<td>6407773001</td>
			<td>Strip tube-based HRM and amplification</td>
		</tr>
		<tr>
			<td>LightCycler 96</td>
			<td>Roche</td>
			<td>5815916001</td>
			<td>Strip-tube and plate-based HRM and amplification</td>
		</tr>
		<tr>
			<td>LightCycler 480</td>
			<td>Roche</td>
			<td>5015278001</td>
			<td>plate-based HRM (96 and 384-well plates) and amplification</td>
		</tr>
		<tr>
			<td>LightScanner-96</td>
			<td>BioFire Defense</td>
			<td>LSCN-ASY-0011</td>
			<td>plate-based HRM (96-well)&#160;</td>
		</tr>
		<tr>
			<td>LightScanner-384</td>
			<td>BioFire Defense</td>
			<td>LSCN-ASY-0001</td>
			<td>plate-based HRM (96-well)</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Roche</td>
			<td>4729692001</td>
			<td>96-well plates for HRM (LightCycler</td>
		</tr>
		<tr>
			<td>384-well plates</td>
			<td>Roche</td>
			<td>4729749001</td>
			<td>384-well plates for HRM (LightCycler)</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Bio-Rad</td>
			<td>HSP-9665&#160;</td>
			<td>96-well plates for HRM (LightScanner)</td>
		</tr>
		<tr>
			<td>384-well plates</td>
			<td>Bio-Rad</td>
			<td>HSP-3865&#160;</td>
			<td>384-well plates for HRM (LightScanner)</td>
		</tr>
		<tr>
			<td>LightCycler 8-Tube Strips (clear)</td>
			<td>Roche</td>
			<td>6327672001</td>
			<td>strip tubes for HRM (Light Cycler 96 and Light Cycler Nano)</td>
		</tr>
		<tr>
			<td>LightCycler Capillaries (20 &#956;l)</td>
			<td>Roche</td>
			<td>4929292001</td>
			<td>capillaries for HRM</td>
		</tr>
		<tr>
			<td>Optical plate seals</td>
			<td>Roche</td>
			<td>4729757001</td>
			<td>other brands also work</td>
		</tr>
	</tbody>
</table>

methods to increase the sensitivity of high resolution melting single nucleotide polymorphism genotyping in malaria

尽管几十年来根除努力，疟疾仍是一个全球性的负担。近期在区域消灭和根除全球新的兴趣一直陪伴左右疟原虫寄生物种造成疟疾，包括地理种群的敏感性降低有关的抗疟疾药物的特征以及变化增加了基因组信息。 一个常见的​​遗传变异，单核苷酸多态性（SNP），提供有吸引力的目标寄生虫的基因分型。这些标记不仅为跟踪药物抗性标记也用于跟踪使用不根据药物或其他选择压力标记寄生虫种群是有用的。 SNP基因分型方法提供了跟踪耐药性以及指纹个别寄生虫人口监测，特别是在应对疟疾防治工作的地区接近eliminat的能力离子状态。 虽然单核苷酸多态性信息已经确定是不可知的特定基因分型技术，高分辨率熔解（HRM）分析，尤其适合到外地为基础的研究。相比，需要在一个单一的标记探针个体的SNP，并提供最好的10％的灵敏度检测的SNP在包含多个基因组（polygenomic）样品的标准荧光探针为基础的方法，人力资源管理，提供2-5％的敏感性。修改HRM，如阻止探针和不对称引物浓度以及扩增的退火温度的优化偏压PCR朝向次要等位基因的扩增，进一步增加HRM的灵敏度。而改善灵敏度取决于具体的实验中，我们已经增加检测灵敏度的次要等位基因的不到1％。 在地区接近根除疟疾，尽早发现新出现的或进口的耐药性是至关重要的公关的OmpT反应。同样，检测polygenomic感染和区分神秘的地方水塘进口寄生虫类型的能力可以通知控制程序。 这份手稿描述修改高分辨率熔解技术，进一步提高其灵敏度，以确定患者样品中polygenomic感染。

数据可以被可视几种不同的方式，根据分析软件和仪器。通常情况下，标准化荧光的负导数的相对于温度（-dF​​ / dT）的对温度的结果的曲线图是最直接的可视化熔融峰以及确定基因型。 全熔化窗口（40℃-80℃）下，将导致在两种扩增子和探针熔解区域。 图1示出了归一化的熔融峰为两个区域的一个例子。分析窗口设置为只是在探头区域中的结果对应于特定的SNP峰明确分化（ 图2）。 基于探测的分析清楚地显示了两个等位基因与纯合匹配的SNP的峰（ 图3），该熔融温度的单个峰的存在。 图4表明 progressively减少期间扩增（MAAB）的结果，退火温度在朝向突变型等位基因（左侧峰）（图4A）的偏置，结果在混合等位基因（图4B）的群体突变等位基因的敏感性增加。 <img alt="图1" src="/files/ftp_upload/52839/52839fig1.jpg" /> 图1.探针和扩增子熔化的区域。绘制归一化的荧光的负导数相对于温度在宽的熔化窗口的结果在这两个探针（较低的温度）和扩增子的熔解峰可以被分析。 （改编自丹尼尔斯等人DOI：10.1128 / AAC.05737-11） <a href="https://www.jove.com/files/ftp_upload/52839/52839fig1large.jpg" target="_blank">点击此处查看该图的放大版本。</a> 图2.探针熔解峰。完美匹配（通常是野生型等位基因）带来更高的熔融峰（红色），而SNP错配具有较低的熔融温度（灰色）。 <a href="https://www.jove.com/files/ftp_upload/52839/52839fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52839/52839fig3.jpg" /> 图3. Polygenomic熔化峰。当两个等位基因是存在于样品中，基于探针的分析HRM表示两个等位基因作为两个峰曲线（橙色）与匹配的单等位基因样品（红色和灰色），该峰。 （改编自丹尼尔斯等人DOI：10.1128 / AAC.05737-11） <a href="https://www.jove.com/files/ftp_upload/52839/52839fig3large.jpg" target="_blank">请点击这里查看大v</a>版为这一数字的。 <img alt="图4" src="/files/ftp_upload/52839/52839fig4.jpg" /> 图4.突变等位基因扩增偏差（MAAB）。A.渐进地降低扩增退火温度偏压朝向对应于突变体（较低的温度）等位基因在一个或polygenomic polyallelic样品峰的熔解峰反应。 B. MAAB导致人力资源管理灵敏度以检测polygenomic或polyallelic样品中存在低于1％的次要等位基因。 （B部分改编自丹尼尔斯等人DOI：10.1128 / AAC.05737-11） <a href="https://www.jove.com/files/ftp_upload/52839/52839fig4large.jpg" target="_blank">点击此处查看该图的放大版本。</a> 表1.建立了基于探针的高分辨率熔解扩增反应。 <table><tbody><tr><td>零件</td><td>每重卷动作（96孔板和毛细管） </td><td>每个反应体积（384孔板） </td><td>最终浓度</td></tr><tr><td> HRM预混</td><td> 4-5微升</td><td> 2-2.5微升</td><td> 1个</td></tr><tr><td> 10×过量引物</td><td> 1微升</td><td> 0.5微升</td><td> 0.50μM </td></tr><tr><td> 10 x其它底漆</td><td> 1微升</td><td> 0.5μL </td><td> 0.1μM </td></tr><tr><td> 10X探头</td><td> 1微升</td><td> 0.5微升</td><td> 0.40μM </td></tr><tr><td> PCR级水</td><td> 1-2微升</td><td> 0.5-1微升</td><td></td></tr><tr><td>模板DNA </td><td> 1微升</td><td> 0.5微升</td><td> 0.01-10纳克/微升</td></tr><tr><td></td><td> 10微升</td><td> 5微升</td><td></td></tr></tbody></table> 表2. STANDARD高高分辨率熔解扩增条件。 <table><tbody><tr><td>温度</td><td>时间</td></tr><tr><td>保持</td><td> 95℃ </td><td> 120秒</td></tr><tr><td rowspan="2"> 45个循环</td><td> 95℃ </td><td> 30秒</td></tr><tr><td> 68°C * </td><td> 30秒</td></tr><tr><td rowspan="2"> 1周期</td><td> 95℃ </td><td> 30秒</td></tr><tr><td> 28°C </td><td> 30秒</td></tr></tbody></table> *该温度是扩增子依赖性 表3.扩增条件突变等位基因扩增偏见。 <table><tbody><tr><td>温度</td><td>时间</td><td>周期</td><td>斜坡率</td><td>采集方式</td><td>分析模式</td></tr><tr><td>变性</td><td> 95℃ </td><td> 30秒</td><td> 1 </td><td> 20 </td><td>无</TD&gt; <td>无</td></tr><tr><td rowspan="2">周期</td><td> 95℃ </td><td> 2秒</td><td rowspan="2"> 55 </td><td> 20 </td><td>无</td><td rowspan="2">定量</td></tr><tr><td> 56°C * </td><td> 15秒</td><td> 20 </td><td>单</td></tr><tr><td rowspan="3">熔化</td><td> 40℃ </td><td> 0秒</td><td rowspan="3"></td><td rowspan="3"> 0.3 </td><td>无</td><td>无</td></tr><tr><td> 45°C * </td><td> 0秒</td><td rowspan="2">连续</td><td rowspan="2">熔化</td></tr><tr><td> 90°C * </td><td> 0秒</td></tr></tbody></table> *该温度是扩增子依赖性和表演MAAB时将减少

Watch this Scientific Journal Video about Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria at JoVE.com

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

虽然高分辨率熔解分析提供了一个异质群体的单核苷酸多态性区分的能力，突变等位基因扩增偏置可提高其检测存在于样品中相对较低的百分比等位基因的能力。本协议描述的改进，提高了高分辨率熔解分析的灵敏度。

方法来提高高分辨率的敏感性疟疾熔单核苷酸多态性基因分型

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

methods-to-increase-sensitivity-high-resolution-melting-single

Research

JoVE Journal

Immunology and Infection

11.4K 次观看.  Harvard University. 虽然高分辨率熔解分析提供了一个异质群体的单核苷酸多态性区分的能力，突变等位基因扩增偏置可提高其检测存在于样品中相对较低的百分比等位基因的能力。本协议描述的改进，提高了高分辨率熔解分析的灵敏度。 

视频: Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

科研

免疫与感染

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

两种异核体的形成方法，发现丙型肝炎病毒的制约因素

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

疟疾蚊子中的二维和三维染色体绘画

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

建立和高通量安装程序研究优化<em&gt;表皮葡萄球菌</em&gt;和<em&gt;分枝杆菌</em&gt;感染作为模型药物发现

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

使用复分析定性测量细菌生产和敏感性，以肺炎球菌细菌素

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the &#8220;pilus&#8221;5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

的高分辨电子显微<em&gt;幽门螺杆菌</em&gt;恰IV型分泌系统的霹雳产于铁可用性的条件变

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

一个<em&gt;体外</em&gt;模型在HIV合并感染的背景下测量免疫应答疟疾

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

方法来抑制细菌Pyomelanin生产和确定敏感性氧化应激的影响相应增加

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

对于血液处理快速的方法，以提高血浆肽水平的人血产量

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...