这项工作是由海军研究办公室通过美国海军研究实验室的核心资金支持。

1。 LNA的探测和实验设计<ol><li>设计的LNA探针，是您的转录斯尔纳/ mRNA的反向补充或他们的自定义<a href="http://www.exiqon.com">设计</a> www.exiqon.com 。如果使用自定义的设计选项，你会知道的顺序，但不低噪声放大器（LNA）扣球模式。此外，在增加的T 之间米到每个LNA残留的DNA或RNA寡核苷酸结果两个10 ° C的低噪声放大器- RNA杂交双工 14 。理想的情况下，设计低噪声放大器（LNA）探头应20-25个核苷酸（较长的探头更难以合成） 之间的85-90之间的Tm ° C的RNA的杂交。 </li><li>设计序列后，利用高炉<a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a>或探针序列比较本地数据库，以确保探头是只对特定的斯尔纳/ mRNA的利益。 </li><li>订货时的LNA探针，LNA的寡核苷酸的5&#39;端添加生物素甘醇修改。生物素是杂交后用链霉亲和染料共轭染色所需。它可以添加此修改的3&#39;端，如果有必要，但另外的5&#39;端更便宜，并应导致更高的收益率。 </li><li>应包括三个阴性对照的方法：（一）“无LNA的控制，在其中LNA探头没有添加在杂交步骤，（二）”无染“控制的LNA探针杂交目标斯尔纳但杂交事件是没有检测到荧光染色的情况下，（三）“非表示斯尔纳/，表达”的控制，采用了低噪声放大器探头的目标是为了不存在或不表达斯尔纳监控非特异性杂交​​。 </li><li>具体LNA探针这里是相辅相成的斯尔纳CsrB和序列5&#39; -生物素- TEG - gTccAttTccCgtCctTagCagC - 3&#39;（LNA的单体大写字母）。 </li><li>在收到冻干低噪声放大器，在10-20微升等分无核酸酶水悬浮在-20 ° C至50微克/毫升和存储</li></ol>解决方案<ul><li> paraformaldehye 8％，10％的乙酸在PBS（PFA）：混合1 mL 32％的煤灰，400μL乙酸，400μL10X PBS，pH = 7.4的，2.2毫升无核酸酶水。对于冻结等分，冻结在-20 ° C情况下使用醋酸等分单。 </li><li> 60％的硫酸葡聚糖溶液：添加6.0克硫酸葡聚糖50 mL锥形管，并加水至终体积10毫升。这种混合物加热至65 ° C水浴中溶解，直到硫酸葡聚糖（可能需要1-2小时）。的增溶过程中保持10毫升量增加，可能需要更多的水。分装在60％硫酸葡聚糖解决方案，并在-20 ° C储存，直到使用。 </li></ul> 2。固定<ol><li>收获1 × 10 8个细胞的生物发光<em弧菌campbellii或您感兴趣的细菌和他们转移到1.5 ml离心管。这种细胞的数量，提供足够的起始原料，四流的鱼类样品（一个特定的LNA探针样品和3个阴性对照）。应收集额外的1.5 ml离心管分装，如果需要进行测试，其他斯尔纳/ mRNA的物种。 </li><li>球离心五分钟在2300 XG的细菌细胞。倒掉上清，吹打在400μL1 × PBS重悬细胞。 </li><li>这种混合物，添加400μLparaformaldehye 8％（PFA）和1X磷酸10％的醋酸缓冲液（1X PBS）的解决方案，拌匀，并孵育10分钟，在25 °彗星搅拌一次五分钟后。在加热管的持有人，除非另有说明，所有孵化执行。的煤灰的解决方案应准备新鲜或冷冻分装后用醋酸此外。多聚甲醛是一种交联固色剂的H电子学习产品，以维持细胞的形态和保留细胞内的RNA。 </li><li>离心沉淀细胞从这种混合物在4500 XG两分钟，吹打除去上清液。所有需要离心未来步骤应该使用相同的设置（7K，2分钟）。重要的是要注意以下的离心上清应吹打，而不是调迁。 </li><li>两次用400μL1 × PBS清洗细胞和悬浮在400μL1 × PBS最后的细胞沉淀。现在，这些细胞是固定的，并可以保存在4 ° C在400μL1 × PBS为七天。 </li></ol> 3。焦碳酸二乙酯处理<ol><li>准备400μL1X PBS（400μL该解决方案需要为每个样品进行测试，以便规模相应）0.1％的焦碳酸二乙酯（DEPC）解决方案。从残留的DEPC股票应准备新鲜的DEPC处理的解决方案。 DEPC处理可灭活carbethoxylation核糖核酸酶和减少背景信号。 </li><li>加入400μL0.1％DEPC解决每一个固定的细菌细胞样本，并拌匀吹打。这种混合物在25 ° C孵育12分钟。清洗细胞两次与400μL1 × PBS，颗粒细胞（7K，2分钟），去除上清。 </li></ol> 4。通透<ol><li>溶菌酶添加1.0毫克，1.0 mg / mL的溶液1毫升的Tris - EDTA缓冲液（10毫米的Tris，1 mM的EDTA，pH值8.0）。该解决方案应准备新鲜。添加800μL1 mg / mL的溶菌酶溶液的细胞，调匀吹打，并培育25 ° C时30分钟，偶尔搅拌。沉淀细胞（7K，2分钟），去除上清。溶菌酶行为水解细菌细胞壁的肽聚糖目前作为一个通透试剂。 </li><li>准备在TE缓冲液中的蛋白酶K 3.0微克/ mL的溶液。从20 mg / mL的蛋白酶K的股票是储存在-20 ° C，这一解决方案应准备新鲜蛋白酶K是一种丝氨酸蛋白酶，消化各种蛋白质，并有助于的RNA的探针和试剂的可访问性。 </li><li>加入800μL的3.0微克/ mL蛋白酶K溶液细胞沉淀，调匀吹打。在25 ° C孵育15分钟，中途通过孵化与涡旋。 </li><li>颗粒细胞（7K，2分钟），取出上清液，用400μL1 × PBS洗细胞一次。取出后洗净上清，400μL1 × PBS重悬细胞，并添加到四个新的1.5 ml离心管100μL的再悬浮。沉淀细胞（7K，2分钟），去除上清。 </li></ol> 5。杂交<ol><li>准备每个样品100μL杂交缓冲液（HB）。高亮度的50％甲酰胺，按量，按重量的10％右旋糖酐硫酸（添加16.7μL的60％硫酸葡聚糖溶液，每100μL，），1X登哈特的解决方案，50 mM磷酸钠pH值7.0，2X柠檬酸钠（SSC），20微克剪切鲑鱼精子DNA（SSSD）和20微克酵母tRNA。 </li><li>高亮度的组成部分，每个人都有不同的目的。甲酰胺降低核酸，从而帮助双链RNA的变性和减少细胞损伤的熔融温度。硫酸葡聚糖是作为一个体积不包括聚合物集中探头和提高杂交率。 Denhardts解决方案，酵母tRNA和SSSD作为一个阻断剂，以减少非特异性结合。 </li><li>再悬浮含1.5毫升离心管，在每个细胞中添加95μLHB和5.0斯尔纳/μL，表达特定的LNA或水（没有LNA的阴性对照）[20 pmol具体LNA终浓度]。例如： <ol class="lalpha"><li>管1 - 95μL，HB + 5.0μL，斯尔纳/ mRNA的特异性探针</li><li>管2 - 95μLHB + 5.0μL，斯尔纳/ mRNA的特异性探针（“不染”阴性对照） </li><li>管3 - 95μL，HB + 5.0μL，斯尔纳/基因探针（“非表示斯尔纳/ mRNA的阴性对照） </li><li>管4 - 95μL，HB + 5.0μL水（“没有LNA的”阴性对照） </li></ol></li></ol><ol start="4"><li>所有四个样品在60℃孵育60分钟。杂交温度取决于LNA的探针（S） 的 Tm，并应设置约30 ° C以下，预测的RNA退火米T为。高架杂交温度，并在HB甲酰胺确保斯尔纳或利益的mRNA的二级结构变性。 </li></ol> 6。后杂交洗<ol><li>杂交后，添加0.1％吐温20（SSCT）1.0毫升0.1X SSC杂交混合物。这是必要的，允许粘性杂交液中取出的细胞从。沉淀细胞（7K，2分钟），去除上清。 </li><li>加入200μL50％甲酰胺，2X SSC，0.1％Tween - 20的细胞沉淀，调匀和incub30分钟吃65 ° C的加热块。 </li><li>加入1 ml 0.1X SSCT的混合物，颗粒细胞（7K，2分钟），去除上清。 </li><li>加入500μl的0.1X SSCT悬浮细胞和一个加热块40分钟，65℃孵育。沉淀细胞（7K，2分钟），去除上清。 </li></ol> 7。阻塞和染色<ol><li>准备封闭液（BB）和链霉菌染色解决方案。从5倍的股票（市售，见试剂），准备一个1X BB的解决方案。对于每四个管子，准备1.2毫升1X BB。 </li><li>加入200μl1X的BB，重悬细胞沉淀，孵育30分钟，25 ° C。 </li><li>一个链霉亲和共轭染料是用来检测的生物素标记的低噪声放大器（LNA）探针。同时阻止，准备2微克/毫升DyLight 488链霉亲和素或1X为30分钟（三个样品，使300μL）BB共轭染料 - 亲你想要的解决方案。 </li><li>后incubATION与1X BB，颗粒细胞（7K，2分钟），去除上清。加入50μL的链霉亲和染液细胞沉淀，调匀，孵育12分钟，25 ° C恒定混合旋涡，或在thermomixer。 “不染”的控制，只添加50μL的1X封闭液。对于其余的协议，保护使用铝箔的光管。 </li><li>用500μl的0.1X SSCT缓冲洗细胞一次，一次400μL1X PBS 0.1％吐温20（PBST）。沉淀细胞（7K，2分钟），去除上清。 </li><li>继最初的链霉亲和共轭染色，信号被放大，引入生物素标记的抗链霉亲和抗体的链霉亲和染料的共轭，然后染色与链霉亲和染料从而增加每LNA的探针杂交信号输出（图2）第二次。 </li><li>加入100μL1微克/毫升的生物素化的抗 - 亲和抗体1X PBS，resuspend的细胞，孵育在25℃30分钟，偶尔搅拌彗星。颗粒细胞（7K，2分钟），取出上清液400μL1X PBST洗两次。 </li><li>加入50μL的2微克/毫升链霉菌细胞染液，调匀，孵育12分钟，25 °恒定混合旋涡，或在thermomixer C。 “不染”的控制，只添加50μL的1X封闭液。 </li><li>清洗细胞一次用500μl的0.1X SSCT缓冲区，一旦与400μL1X PBST。 </li><li>细胞重悬于200μL1X PBST和储存于4 ° C，直到准备流式细胞仪分析。对于规模较小的细菌细胞集流速缓慢下降的核心大小。此外，与前向散射阈值截断流式细胞仪，截止必须尽可能低，以确保小细菌细胞检测。 </li><li> DyLight 488检测，流式细胞仪应配备一个488 nm激光和标准排放过滤为FITC标记。应收集事件至少2X10 4。与流式细胞仪配备不同的激光器兼容，其他的链霉亲和共轭荧光团可用于本议定书。 </li></ol> 8。代表性的成果例如，有效固定和通透的细胞在流式细胞仪散点图图 3A所示。使用通透描述了上述条件时，细胞的人口是小和同质表明一些细胞聚集。当溶菌酶浓度较高（5毫克/毫升）的使用，细胞更容易聚集并显示增加向前的分散值，表明较大的颗粒（ 图3B） 。流式细胞仪从一个成功的LNA流FISH实验数据的一个例子是如图4所示。直方图表明，随着三个阴性对照的具体目标斯尔纳检测。“不染”阴性对照，产生最少的荧光，“没有低噪声放大器”和“非表示斯尔纳阴性对照。最后，特定SRNA - LNA探针与样品产生最大的荧光。一旦以这种方式进行验证，方法和LNA探针，可以进行更多的试验旨在监视斯尔纳信号的变化，随着时间的推移，在应对突变或不同的文化条件等<img alt="图1。" src="/files/ftp_upload/3655/3655fig1.jpg" /> 图1描绘了一个LNA流鱼的细菌斯尔纳检测试验总体方案。 <img alt="图2。" src="/files/ftp_upload/3655/3655fig2.jpg" /> 图2。染色和LNA的流动FISH信号放大。 A）生物素化的LNA探针杂交。 B）染色与荧光DyLight 488链霉亲和共轭。 C）的生物素化抗- S绑定treptavidin抗体DyLight 488链霉。抗体可以通过其抗原结合位点结合的链亲和素或可通过其生物素残留链霉亲和约束。 D）进一步荧光DyLight 488链霉亲和共轭染色的信号放大。 <img alt="图3。" src="/files/ftp_upload/3655/3655fig3.jpg" /> 图3流式细胞仪检测点图显示侧散射结果与A）1 mg / mL的溶菌酶，3微克/ mL的蛋白酶K通透和B）5毫克/毫升溶菌酶，3μg/ ml的蛋白酶K通透。 <img alt="图4。" src="/files/ftp_upload/3655/3655fig4.jpg" /> 图4。直方图分析LNA流FISH结果。从每个样品产生的荧光信号是由四个痕迹所示：黑色 - 具体SRNA - LNA探针;红色 - “非表示斯尔纳”阴性对照;蓝色 - “没有LNA的”阴性对照;紫色 -“不染阴性对照。

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+Situ+Hybridization+Analysis+of+Transcript+Dynamics+in+Cells.">Fluorescence in Situ Hybridization Analysis of Transcript Dynamics in Cells.</a> Methods. 25, 111-118 (2001).</li><li>Silveira, A. C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+non-coding+RNAs+in+environmental+vibrios.">Identification of non-coding RNAs in environmental vibrios.</a> Microbiology. 156, 2452-2458 (2010).</li></ol>

作者什么都没有透露。

LNA流鱼这里介绍的方法是用来检测革兰氏阴性的海洋弧菌campbellii的表达先前已确认通过芯片为基础的表达谱和逆转录聚合酶链反应16 SRNA的表达。到目前为止，我们已经利用这种方法来监视多种RNA分子（如跨编码斯尔纳，斯尔纳调节蛋白质的活性，核糖开关，和mRNA）的表达。因此，我们有信心，该方法的适应性和可用于检测任何斯尔纳或mRNA的目标，在任何细菌的物种或细胞类型，可使用修改。如果本协议的修改是必要的其他类型的细胞中，最重要的变量要考虑操纵的通透性一步。例如，当修改这里描述的通透条件，溶菌酶和蛋白酶K的浓度变化，应进行测试。我们发现，加倍或三倍量的溶菌酶和蛋白酶K在LNA流FISH结果导致了重大变化。此外，在测试时附加的通透条件，细胞应为聚集，细胞的损失，并获得最好的信号背景荧光比分析。细胞聚集程度可以通过显微镜和/或流式细胞仪进行测试。流式细胞仪，细胞团块，目前正向与侧向散射散点图和正确的通透性方法的尾巴，将最大限度地减少这种拖尾效果。可以添加额外的LNA与其他半抗原，如地高辛标记的探针在杂交步骤，然后与抗地高辛抗体和二级抗体荧光基团共轭检测，这种方法也适合复所描述的协议没有重大修改，斯尔纳检测。目前，唯一的限制，我们用这种方法遇到的是它无法产生足够的信号从弱前按下斯尔纳物种和正在努力，以确定检测阈值（每个单元格的绝对拷贝数）。 总体而言，低噪声放大器（LNA）的流FISH方法提供了机会，测量单细胞高通量地为客户提供一个斯尔纳物种的存在和相对丰度和其表达变化在何种程度上的见解级细菌斯尔纳或mRNA表达在人群中。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 10X磷酸盐缓冲液（PBS），pH值7.4 </td><td>应用生物系统公司/ Ambion公司</td><td> AM9625 </td><td></td></tr><tr><td>无核酸酶水</td><td>应用生物系统公司/ Ambion公司</td><td> AM9932 </td><td> （不DEPC处理） </td></tr><tr><td> 32％多聚甲醛</td><td>电子显微镜科学</td><td> RT 15714 </td><td>乙醇免费</td></tr><tr><td>乙酸</td><td> Acros Organics公司</td><td> 327290010 </td><td></td></tr><tr><td>焦碳酸二乙酯（DEPC） </td><td>西格玛爱秩序</td><td> D5758 </td><tD&gt;保持干燥</td></tr><tr><td>溶菌酶</td><td>西格玛爱秩序</td><td> L2879 </td><td></td></tr><tr><td>蛋白酶K（20毫克/毫升） </td><td>应用生物系统公司/ Ambion公司</td><td> AM2546 </td><td></td></tr><tr><td>甲酰胺</td><td>应用生物系统公司/ Ambion公司</td><td> AM9342 </td><td>去离子水，分装和冻结</td></tr><tr><td>硫酸葡聚糖分子量&gt; 500,000 </td><td>西格玛爱秩序</td><td> D8906 </td><td>分装60％的解决方案，并冻结</td></tr><tr><td>登哈特的解决方案50X集中</td><td>西格玛爱秩序</td><td> D2532 </td><td>分装和冻结</td></tr><tr><td>柠檬酸钠缓冲液20X </td><td>应用生物系统公司/ Ambion公司</td><td> AM9770 </td><td></td></tr><tr> &lt;TD&gt;鲑鱼精子DNA（剪切）10毫克/毫升</td><td>应用生物系统公司/ Ambion公司</td><td> AM9680 </td><td>分装和冻结</td></tr><tr><td>酵母tRNA（10毫克/毫升） </td><td>生命科技/ Invitrogen公司</td><td> 15401-011 </td><td>分装和冻结</td></tr><tr><td>吐温20 </td><td>西格玛爱秩序</td><td> P9416 </td><td></td></tr><tr><td> 5X 在原位杂交封闭液</td><td>媒介实验室</td><td> MB - 1220 </td><td></td></tr><tr><td> DyLight 488链霉亲和素</td><td>媒介实验室</td><td> SA - 5488 - 1 </td><td></td></tr><tr><td>生物素化抗 - 亲</td><td>媒介实验室</td><td> BA - 0500 </td><td>分装和冻结</td></tr></tbody></table>

locked nucleic acid flow cytometry-fluorescence in situ hybridization (lna flow-fish): a method for bacterial small rna detection

荧光原位杂交（FISH）是一个强大的技术，用于检测和定位在蜂窝环境特定的核酸序列。为了增加吞吐量，鱼可以用流式细胞仪（流FISH）相结合，使数以千计的单个细胞的有针对性的核酸序列检测。因此，流鱼超过裂解/合奏基于核酸检测方法提供了一个独特的优势，因为每一个细胞都被视为一个独立的观察，从而允许强大的统计分析和方差分析。这些属性有提示的鱼和流量鱼的方法，在不同的应用程序的使用和端粒长度的测定1,2，细胞识别和基因表达的3,4已经成功地展示了这些方法的效用，监测病毒的乘法感染的细胞和细菌群落分析和枚举6。 传统上，鱼和流FISH方法的特异性DNA寡核苷酸探针传授。然而，最近增加鱼和流FISH探针的核酸类似物的DNA寡核苷酸探针更换由于每种技术的灵敏度和特异性较高的熔融温度，这些类似物 （T米）7,8自然核酸。锁核酸（LNA）探针是一个类型的核酸类似物，含有低噪声放大器（LNA）核苷酸添加整个DNA或RNA序列9,10。当与流动FISH相结合的LNA探针先前已被证明优于传统的DNA探针7,11，并已成功地用于检测5哺乳动物细胞真核生物mRNA的12和病毒RNA。 在这里，我们扩大这种能力和描述一个LNA流鱼方法，该方法允许在细菌细胞中的RNA特异性检测S（ 图1）。具体来说，我们有兴趣在检测小的非编码调控RNA（SRNA）在过去几年获得相当大的兴趣，因为他们已经发现，作为在许多关键的13细胞过程中的关键调控元件。然而，有限的工具来研究sRNAs和检测在细菌细胞斯尔纳的挑战，部分是由于规模相对较小（通常为50-300个核苷酸）和低丰度斯尔纳分子以及一般的工作难度较小的生物细胞与不同的细胞膜。在此方法中，我们描述的固定和permeabilzation条件，保护细菌细胞的结构和允许的LNA探针的渗透以及信号放大的步骤，使低丰度斯尔纳的具体检测（图2）。

Watch this Scientific Journal Video about Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection at JoVE.com

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

一种新型的高通量方法使单个细菌细胞，使用锁定的核酸探针和流式细胞仪荧光检测和小分子RNA和mRNA表达的相对定量描述<em&gt;原位</em&gt;杂交。

锁核酸流式细胞仪，荧光原位杂交（LNA流FISH）：细菌小RNA检测方法

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

locked-nucleic-acid-flow-cytometry-fluorescence-situ-hybridization

Research

JoVE Journal

Immunology and Infection

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

25.2K 次观看.  Naval Research Laboratory. 一种新型的高通量方法使单个细菌细胞，使用锁定的核酸探针和流式细胞仪荧光检测和小分子RNA和mRNA表达的相对定量描述原位杂交。

视频: Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new infections each year. Despite decades of research, there is still no vaccine against malaria, highlighting the need for novel control strategies. One innovative approach is the use of genetically modified mosquitoes to effectively control malaria parasite transmission. Deliberate alterations of cell signaling pathways in the mosquito, via targeted mutagenesis, have been found to regulate parasite development 1. From these studies, we can begin to identify potential gene targets for transformation. Targeted mutagenesis has traditionally relied upon the homologous recombination between a target gene and a large DNA molecule. However, the construction and use of such complex DNA molecules for generation of stably transformed cell lines is costly, time consuming and often inefficient. Therefore, a strategy using locked nucleic acid-modified oligonucleotides (LNA-ONs) provides a useful alternative for introducing artificial single nucleotide substitutions into episomal and chromosomal DNA gene targets (reviewed in 2). LNA-ON-mediated targeted mutagenesis has been used to introduce point mutations into genes of interest in cultured cells of both yeast and mice 3,4. We show here that LNA-ONs can be used to introduce a single nucleotide change in a transfected episomal target that results in a switch from blue fluorescent protein (BFP) expression to green fluorescent protein (GFP) expression in both Anopheles gambiae and Anopheles stephensi cells. This conversion demonstrates for the first time that effective mutagenesis of target genes in mosquito cells can be mediated by LNA-ONs and suggests that this technique may be applicable to mutagenesis of chromosomal targets in vitro and in vivo.

Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.

酸修饰的寡核苷酸锁定核酸转染和蚊虫细胞的靶基因突变

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

“发光法”的分类检测细菌病原体的记者噬菌体

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

杂交<em&gt;原位</em&gt;和蚊虫唾液腺，卵巢，胚胎

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

荧<em在原位</em&gt;蚊虫的有丝分裂染色体上的杂交

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

细菌人工染色体：对于正链RNA病毒研究的功能基因组学工具

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

KSHV感染细胞中特定基因产物的定量原位荧光杂交（FISH）和免疫荧光（IF）

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T&#160;overexpressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.&#160;HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.&#160;&#160;

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

非肝细胞293T-NE-3NRS细胞中乙型肝炎病毒感染的建模

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV ...

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia coli and Salmonella Typhimurium, could repress the expression of outer membrane proteins. To further investigate the regulation function of micC in Salmonella Enteritidis, we cloned the micC gene in the Salmonella Enteritidis strain 50336, and then constructed the mutant 50336&#916;micC by the &#955; Red-based recombination system and the complemented mutant 50336&#916;micC/pmicC carrying recombinant plasmid pBR322 expressing micC. qRT-PCR results demonstrated that transcription of ompD in 50336&#916;micC was 1.3-fold higher than that in the wild type strain, while the transcription of ompA and ompC in 50336&#916;micC were 2.2-fold and 3-fold higher than those in the wild type strain. These indicated that micC represses the expression of ompA and ompC. In the following study, the pathogenicity of 50336&#916;micC was detected by both infecting 6-week-old Balb/c mice and 1-day-old chickens. The result showed that the LD50 of the wild type strain 50336, the mutants 50336&#916;micC and 50336&#916;micC/pmicC for 6-week-old Balb/c mice were 12.59 CFU, 5.01 CFU, and 19.95 CFU, respectively. The LD50 of the strains for 1-day-old chickens were 1.13 x 109 CFU, 1.55 x 108 CFU, and 2.54 x 108 CFU, respectively. It indicated that deletion of micC enhanced virulence of S. Enteritidis in mice and chickens by regulating expression of outer membrane proteins.

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

An &#955;-Red-mediated recombination system was used to create a deletion mutant of a small non-coding RNA micC.

非编码小RNA MicC有助于肠炎 沙门氏菌 外膜蛋白的毒力

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia ...