The funding for this research project was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) via a Discovery Grant to YL.

1.试剂和缓冲液的制备<ol><li>的0.5M乙二胺四乙酸（EDTA）的<ol><li>在2L烧杯中，186.1克EDTA添加到800毫升去离子蒸馏水的（DDH 2 O的）。调节溶液至8.0用NaOH粒料的pH值。添加DDH 2 O的以1.0升的最终体积，并在4℃下将溶液转移到一个高压灭菌玻璃瓶高压灭菌和储存。 </li></ol></li><li> 10×Tris-硼酸盐-EDTA（10×TBE） <ol><li>在一个4升的塑料烧杯中，使用搅棒，直到所有的组分432克Tris-碱将200克硼酸中，加入80ml的0.5M EDTA（pH 8.0）中和DDH 2 O的的添加4 L.混合的最终体积溶解。在4℃下将溶液转移至1升的玻璃瓶高压灭菌和储存。 </li></ol></li><li> 10％的变性聚丙烯酰胺股票<ol><li>在4升的塑料烧杯中，加入1681.7克尿素，加入400ml 10X TBE，1升40％的丙烯酰胺/双丙烯酰胺（29:1）解决方案和DDH 2 O的，直到在FINAL量为4 L.混合使用搅拌棒直到尿素溶解。在4℃下将溶液转移至1L的琥珀色玻璃瓶和存储。 </li></ol></li><li> 2X凝胶缓冲液（2X GLB） <ol><li>在200毫升玻璃烧杯中，加入44克尿素8克蔗糖，10毫克溴酚蓝，10毫克二甲苯青FF，400微升10％十二烷基硫酸钠的，和4毫升10×TBE的。添加DDH 2 O的以40 ml的终体积，并在50℃下溶解有轻度加热的部件，同时用磁力搅拌棒混合。传输1毫升一份到1.5 ml离心管和储存在4℃。 注意:使用前需要在90℃加热简要重新溶解固体的任何。 </li></ol></li><li>的1M Tris盐酸（Tris-盐酸）（pH7.5）中<ol><li>在玻璃瓶中，添加12.1克Tris-碱和70ml DDH 2 O的的并混合直到固体溶解。调整使用1M盐酸（HCl）将pH值至7.5。添加DDH 2 O的至100ml，高压釜和STO终体积再在4℃ </li></ol></li><li>的5M氯化钠（NaCl）中<ol><li>在玻璃瓶中，溶解58.4克NaCl的150毫升的DDH 2 O的调节音量至200毫升的DDH 2 O保存在4℃。 </li></ol></li><li> DNA洗脱缓冲液<ol><li>在玻璃瓶中，混合2.0毫升的1M的Tris-HCl（pH为7.5），8.0毫升5摩尔NaCl和0.4ml的0.5M的EDTA（pH8.0）中的。调节音量到200毫升的DDH 2 O，高压灭菌器和储存在4℃。 </li></ol></li><li> 1M的4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）（pH 7.4）中<ol><li>在玻璃瓶中，溶解2.38克HEPES的80ml的DDH 2 O的调整用5N氢氧化钠将pH至7.4，并添加DDH 2 O的至100ml的最终体积。 </li></ol></li><li> 1 1M氯化镁（ 氯化镁 ） <ol><li>在玻璃瓶中，添加2.03克的MgCl 2 -6H 2 O和使体积为100毫升的DDH 2 O </li></ol></li><li>反应缓冲液（RB） <ol><li&gt;在50ml锥形管中，加入50微升的1M HEPES（pH7.4）中，将1.5ml 5M的NaCl，0.75毫升的1摩尔的 MgCl 2，和5μl吐温-20组成。添加的DDH 2 O至50ml终体积。混合溶液中，并使用注射器驱动的滤器（0.22μm），并储存在4℃直至使用过滤到另一个锥形管中。 </li></ol></li><li>结合缓冲液（BB） <ol><li>在一个50毫升锥形管，加入500微升的1M的Tris-HCl（pH7.5）中的，8.8克NaCl，50微升的1M 的 MgCl 2，和5μl吐温20的添加DDH 2 O的至最终体积为50毫升。混合溶液中，并使用注射器驱动的滤器（0.22μm），并储存在4℃直至使用过滤到另一个锥形管中。 </li></ol></li><li>底物溶液（SS） <ol><li>在50ml锥形管中，加入5.8克NaCl的3毫升的1M MgCl 2的，1.5克尿素，和40毫升的DDH 2 O的调节溶液至5.0用10mM的HCl的pH值。由于该解决方案不进行缓冲，使用盐酸调节pH小心通过添加盐酸小等分。添加的DDH 2 O至50ml终体积。 </li><li>混合溶液中，并使用注射器驱动的滤器（0.22μm），并储存在4℃直至使用过滤到另一个锥形管中。 </li></ol></li><li>卢里亚BERTANI（LB）肉汤<ol><li>在烧杯中，溶解20.0克LB粉1升的DDH 2 O的转移到玻璃瓶中，高压釜中，并储存在4℃。 </li></ol></li><li> 1.5％LB琼脂<ol><li>在250毫升烧瓶中，添加1.5克琼脂和100ml LB肉汤中。高压灭菌器和储存在4℃。 </li></ol></li><li>琼脂平板<ol><li>再溶解在微波的LB琼脂并冷却溶液至〜50℃。将溶液倒入皮氏培养皿，使得〜5块板，并允许它们固化。 </li></ol></li></ol> 2.合成和E的纯化coli-响应核酶EC1 <ol><li>通过模板介导的酶连接EC1的合成<ol><li>净化COMMercially合成的寡核苷酸BS1，DE1，和T1 10％，根据标准方法变性聚丙烯酰胺凝胶电泳（dPAGE）（ 表1中提供了序列）。 </li><li>准备BS1，DE1，和T1的100微米的股票。储存在-20℃直到使用。 </li><li>在1.5ml微量离心管中，添加38.5微升DDH 2 O的，5微升DE1和5微升10×T4多核苷酸激酶反应缓冲液中由酶的供应商（500毫摩尔Tris盐酸（pH值7.6），100mM的MgCl 2的设置，的50mM二硫苏糖醇（DTT），1.0mM的亚精胺）。 </li><li>加入1μl三磷酸腺苷（ATP）（100毫米）的。加入5单位T4多核苷酸激酶（10U /微升）。通过仔细吹打反应混合物混合。 </li><li>孵育在37℃下进行30分钟的反应。 </li><li>淬火加热5分钟，将反应至90℃。 </li><li>加入118微升的DDH 2 O，5微升BS1和5微升T1的。加热反应至90℃2英里n和然后在10分钟内冷却至室温。 </li><li>加入20μl的酶的供应商提供的10×T4 DNA连接酶反应缓冲液（400毫摩尔Tris盐酸（pH值7.8），100mM的MgCl 2的，100毫摩尔DTT，5毫摩尔ATP）。加10单位T4 DNA连接酶（5U /微升）和通过吸液仔细混合。 </li><li>在室温下孵育2小时。 </li><li>加入20μl的3M乙酸钠（NaOAc）（pH为5.2），500微升冷的100％乙醇。混合通过涡旋该溶液，然后将管在-20℃下冷冻30分钟。 </li><li>离心机离心在20000 XG在4℃下20分钟。小心通过移液除去上清液。 </li><li>在4℃下以20,000 xg离心再次洗用冷70％乙醇，离心沉淀10分钟。再次去除上清液并干燥在真空下将沉淀10分钟。 </li><li>重悬DNA与15微升的DDH 2 O，然后加入15微升2×GLB的。 </li><li>涡大力并加热至90℃下2分钟。如下所述通过10％dPAGE纯化全长DNA。 </li></ol></li><li>设置10％dPAGE <ol><li>清洁两块玻璃板（一个完整的板和一个切口），两个0.75毫米厚间隔物，和一个12孔的梳子。放置在平坦的表面上的一个玻璃板上每侧的两个间隔件和在顶部的切口板。剪辑在一起使用由供应商提供的四个片段的两大板块。 </li><li>在150毫升的塑料烧杯中，倒入40含10％dPAGE的，四甲基乙二胺的40微升（TEMED）和400微升10％过硫酸铵（APS）的。混合组分和倾慢慢板之间的溶液。 </li><li>插入梳子，然后使凝胶聚合在10分钟内。一旦凝胶聚合，慢慢取出梳子，用的DDH 2 O冲洗井</li><li>安装在板上的凝胶电泳装置。使用金​​属板来消散所产生，以防止过热的热量。 </li><li>倾1×TBE上的顶部和底部腔室并确保井在缓冲器井淹没。冲洗使用吸液管或注射器用1×TBE的孔中。 </li><li>设置装载样品前15分钟在35马润的设备和预运行。 </li></ol></li><li>从10％dPAGE结扎EC1的溶出<ol><li>以下步骤2.2.6，运行在35毫安直到底部染料（溴酚蓝）凝胶到达玻璃板的底部。这大约需要1.5小时。关闭电源，并从该装置中删除所述板。 </li><li>躺在纸巾几片板并小心地从玻璃板取下隔离物。小心取出顶部玻璃板并用保鲜膜包住凝胶。 </li><li>翻转凝胶上以除去第二玻璃板，并再次用保鲜膜覆盖。请注意，以避免保鲜膜的皱纹。 </li><li>通过使用UV遮蔽（260纳米），这将产生4个不同的DNA条带（完全连接的EC1，DE1，BS1和T1）可视化的连接产物。 </li><li&gt;消费用无菌刀片并转移到1.5ml的微离心管的顶部带（EC1）。粉碎用无菌枪头（200微升针尖大小），凝胶，直到它变成一个精膏。加入550微升DNA洗脱缓冲液，覆盖铝箔管，以保护嵌入荧光团，振摇15分钟。 </li><li>离心以20,000 xg离心凝胶溶液5分钟，并小心地加入400μl的上清液转移至另外的150 ml离心管。避免在移液吸胶块。 </li><li>为了与转移的上清液的离心管中添加40微升的3M醋酸钠（pH值5.2）和1.0冷100％乙醇的溶液中。混合通过涡旋该溶液，然后将管在-20℃下冷冻30分钟。 </li><li>离心机离心在20000 XG在4℃下20分钟。小心通过移液除去上清液。 </li><li>在4℃下以20,000 xg离心再次洗用冷70％乙醇，离心沉淀10分钟。再次，删除上清液并干燥在真空下将沉淀10分钟。 </li><li>通过在260nm处测量UV吸光度确定EC1的浓度。使10μM的库存和样品储存在-20℃直到使用。 </li></ol></li></ol> 3.尿素酶的DNA的共轭<ol><li>琥珀酰亚胺4-（N-马来酰亚胺甲基）环己烷-1-羧酸酯（SMCC）股票的制备<ol><li>溶于676微升DMSO的1毫克SMCC。涡置于冰上直至使用。 </li></ol></li><li>股票脲酶的制备<ol><li>在1毫升1×PBS（无镁离子和钙离子 ）的溶解1毫克尿素酶。在冰上放置直至使用。 注:结晶尿素酶溶解缓慢且需要温和的混合，以避免变性。 </li></ol></li><li>脲酶DNA的合成（UrDNA） <ol><li>加10μl100μMLD1至2.5 ml离心管。加入140微升的DDH 2 O，40微升10X的PBS，旋涡。 </li><li>添加80.5微升SMCC的S滴答，DMSO，旋涡，短暂离心的159.5微升使用台式离心机。 </li><li>孵育在37℃下60分钟。避免离心帽下冷凝。 </li><li>加入200μl1×PBS中的60微升的3M醋酸钠（pH值5.2）和1.5冷100％乙醇的溶液中。混合通过涡旋该溶液并在-20℃下孵育30分钟。 </li><li>离心在4℃下以20,000 xg离心该溶液20分钟。除去上清液，并在真空下干燥沉淀。避免过度干燥。 </li><li>向干燥的沉淀中添加400μl的脲酶的库存中，并在室温下孵育5小时。 </li><li>转移200μl的粗共轭物的到预先洗涤100k的MWCO离心过滤柱。在14000 xg离心离心柱5分钟。 </li><li>传输剩余200μl的粗共轭到柱和离心机在14,000rpm xg离心5分钟。删除列，把它倒挂在一个新的2.0 ml离心管（"收集管"）。 </li><li> CentrifUGE的收集管（与倒置列）在1000 xg离心2分钟。取下收集管，并添加30微升1×PBS中的离心柱来洗膜对偶联物的额外的恢复。 </li><li>再次反转柱放回收集管。在1000 xg离心离心收集管（与倒置列）2分钟。移走和处置列。 </li><li>存储UrDNA在4℃直至使用。 </li></ol></li></ol> 4. EC1和UrDNA装配到磁珠<ol><li>混合磁珠（MB）的股票以及100微升MB悬挂转移到1.5 ml离心管中。放置离心管上用于隔离所述MB的磁性架子保持部。 </li><li>通过移液除去上清液，并添加150微升的结合缓冲液（BB）的管中。从保持器中取出管，并仔细挖掘管悬浮MB到均匀的溶液。避免飞溅悬挂到T管或盖的运算。如果发生这种情况，可使用台式离心机，以旋转简要残余物中回悬浮液中。 </li><li>重复步骤4.2两次。 </li><li>在此悬浮液加入10微升10μMEC1的。小心地敲打管子混用。 </li><li>孵育温和振摇30分钟的溶液中。挖掘管每隔2-3分钟，以避免对MB的沉淀。 </li><li>放置离心管背面上的磁性机架隔离MB和通过移液除去上清液。用的BB 150微升（如在步骤4.2中所述）洗MB的三倍。 </li><li>一旦在洗涤完成后，暂停所述MB在总共150微升的BB。向此溶液中，添加15微升UrDNA和加热该溶液至45℃持续2分钟。冷却该溶液至室温，并孵育2小时。 </li><li>放置离心管背面上的磁性机架隔离MB和通过移液除去上清液。 </li><li>加入100微升反应缓冲液（RB）的。除去叔他从磁架离心管，小心地悬浮MB。 </li><li>通过重复步骤4.9洗MB三次。 注意:洗涤上清液可以快速测试，以确定未杂交UrDNA仍然存在，这可能会导致假阳性信号。测试可以通过加入50mM的尿素的10微升和10微升0.04％酚红的洗涤溶液来完成。继续洗MB直到洗涤液中不引起颜色变化。在100微升的悬浮液贮存于4℃直至使用。 </li></ol> 5.制备细菌细胞20的<ol><li> E.培养大肠杆菌从股票<ol><li> E.板大肠杆菌 K12（MG1655）上从一个甘油下火焰或在生物安全柜的LB琼脂平板上。 </li><li>使用无菌移液管尖端，轻轻触摸甘油和轻轻条纹的琼脂平板的表面上，以避免刺穿LB琼脂。 </li><li>反转挑染板孵育在37℃下14小时。 </li><li>密封在4℃下用封口膜板和存储最多3周。 </li></ol></li><li> E.培养大肠杆菌的细胞计数<ol><li>在无菌14毫升培养管，取2毫升LB肉汤。 </li><li>使用无菌移液管尖端，从在步骤5.1中制备的划线琼脂平板挑取单菌落，并将其转移到培养管中。 </li><li>孵育培养在37℃和以230rpm 14小时摇动。 </li><li>连续稀释细菌培养的10倍区间。 </li><li>对于每个稀释样品，均匀地镀上分开的LB琼脂平板个100微升等分试样。倒置平板，并且在37°C孵育14小时。 </li><li>算每个样品的细胞，以获得每个稀释的平均细胞浓度。 注:10 7个细胞经常被用来设置为E.基准石蕊试大肠杆菌作为这个级别的E.大肠杆菌可引发快速的陈色GE。然而，正确执行试金石可检测低至50​​0个细胞，如结果部分讨论。 </li></ol></li><li> E.准备大肠杆菌细胞试验<ol><li>对于所需的细胞悬液，转移1毫升培养股票到1.5 ml离心管。 </li><li>离心细胞以6000 xg离心在4℃下10分钟。小心取出上清液，而不会干扰细胞沉淀。 </li><li>添加10微升反应缓冲液中的细胞沉淀重悬细胞。超声处理的细胞悬浮液5分钟。细胞悬液转移到冰盒5分钟。 </li><li>声波处理另一个5分钟的细胞悬浮液。 </li><li>离心细胞悬浮液以13,000rpm xg离心在4℃下10分钟。用于测试的上清液（10微升）。 </li></ol></li></ol> 6.试金石<ol><li>在1.5 ml离心管中，通过在离心管中添加和涡旋100微升反应缓冲液（RB）的的预洗管弃去缓冲液。 </li><li>转移15微升组装EC1（协议4）向洗涤微量离心管。 </li><li>通过将离心管上的磁性机架洗涤磁珠。通过移液除去上清液。从机架上卸下的离心管中，添加的RB的100微升，并小心地重悬磁珠。 </li><li>通过重复步骤6.3洗MB两次以上。 </li><li>放置离心管放回磁架，除去上清液，并添加10微升大肠杆菌从步骤5.3编写的大肠杆菌样本。 </li><li>通过在离心管轻轻拍打仔细混合样品和磁性珠粒。 </li><li>在室温下孵育1小时的反应。 </li><li>向反应中，添加90微升DDH 2 O的和离心管放置到一个磁架。 </li><li>后大约3分钟磁选，仔细转移85微升上清液至0.5 ml离心管。慢慢拔出上清避免收集任何磁珠。 </li><li>向上述离心管加入15微升的0.04％酚红，和100微升的底物溶液。 </li><li>采取以特定时间间隔的照片以记录颜色变化。 注:在pH值的变化也可以使用pH计用微电极监测。起始pH值应为大约5.2-5.5（溶液是黄色）。如果不是，则溶液可以通过添加1mM醋酸缓冲液pH 5.0）进行调整。 </li></ol>

<ol>
	<li>Daar, A. 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=Top+ten+biotechnologies+for+improving+health+in+developing+countries.">Top ten biotechnologies for improving health in developing countries.</a> Nat. Genet. 32, 229-232 (2002).</li><li>Newman, J. D., Turner, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Home+blood+glucose+biosensors:+a+commercial+perspective.">Home blood glucose biosensors: a commercial perspective.</a> Biosens. Bioelectron. 20, 2435-2453 (2005).</li><li>Turner, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensors:+sense+and+sensibility.">Biosensors: sense and sensibility.</a> Chem. Soc. Rev. 42, 3184-3196 (2013).</li><li>Tram, K., Kanda, P., Salena, B. J., Huan, S. Y., Li, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+Bacterial+Detection+by+DNAzymes+into+a+Litmus+Test.">Translating Bacterial Detection by DNAzymes into a Litmus Test.</a> Angew. Chem. Int. Ed. 53, 12799-12802 (2014).</li><li>Ali, M. M., Aguirre, S. D., Lazim, H., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorogenic+DNAzyme+probes+as+bacterial+indicators.">Fluorogenic DNAzyme probes as bacterial indicators.</a> Angew. Chem. Int. Ed. 50, 3751-3754 (2011).</li><li>Schlosser, K., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologically+inspired+synthetic+enzymes+made+from+DNA.">Biologically inspired synthetic enzymes made from DNA.</a> Chem. Biol. 16, 311-322 (2009).</li><li>Tuerk, C., Gold, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+evolution+of+ligand+by+exponential+enrichment:+RNA+ligands+to+bacteriophage+T4+DNA+polymerase.">Systematic evolution of ligand by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.</a> Science. 249, 505-510 (1990).</li><li>Ellington, A. D., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+selection+of+RNA+molecules+that+bind+specific+ligands.">In vitro selection of RNA molecules that bind specific ligands.</a> Nature. 346, 818-822 (1990).</li><li>Breaker, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+catalytic+DNAs.">Making catalytic DNAs.</a> Science. 290, 2095-2096 (2000).</li><li>Liu, J., Cao, Z., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+nucleic+acid+sensors.">Functional nucleic acid sensors.</a> Chem. Rev. 109, 1948-1998 (2009).</li><li>Navani, N. K., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+aptamers+and+enzymes+as+sensors.">Nucleic acid aptamers and enzymes as sensors.</a> Curr. Opin. Chem. Biol. 10, 272-281 (2006).</li><li>Li, J., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+sensitive+and+selective+catalytic+DNA+biosensor+for+lead+ions.">A highly sensitive and selective catalytic DNA biosensor for lead ions.</a> J. Am. Chem. Soc. 122, 10466-10467 (2000).</li><li>Liu, J., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+colorimetric+lead+biosensor+using+DNAzyme-directed+assembly+of+gold+nanoparticles.">A colorimetric lead biosensor using DNAzyme-directed assembly of gold nanoparticles.</a> J. Am. Chem. Soc. 125, 6642-6643 (2003).</li><li>Liu, Z., Mei, S. H. J., Brennan, J. D., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assemblage+of+signaling+DNA+enzymes+with+intriguing+metal+specificity+and+pH+dependence.">Assemblage of signaling DNA enzymes with intriguing metal specificity and pH dependence.</a> J. Am. Chem. Soc. 125, 7539-7545 (2003).</li><li>Liu, J., 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+catalytic+beacon+sensor+for+uranium+with+parts-per-trillion+sensitivity+and+millionfold+selectivity.">A catalytic beacon sensor for uranium with parts-per-trillion sensitivity and millionfold selectivity.</a> Proc. Natl. Acad. Sci. USA. 104, 2056-2061 (2007).</li><li>Huang, P. J., Vazin, M., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+selection+of+a+new+lanthanide-dependent+DNAzyme+for+ratiometric+sensing+lanthanides.">In vitro selection of a new lanthanide-dependent DNAzyme for ratiometric sensing lanthanides.</a> Anal. Chem. 86, 9993-9999 (2014).</li><li>Chiuman, W., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+fluorescent+sensors+engineered+with+catalytic+DNA+'MgZ'+based+on+a+non-classic+allosteric+design.">Simple fluorescent sensors engineered with catalytic DNA 'MgZ' based on a non-classic allosteric design.</a> PLoS One. 2, e1224 (2007).</li><li>Xiang, Y., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+personal+glucose+meters+and+functional+DNA+sensors+to+quantify+a+variety+of+analytical+targets.">Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets.</a> Nat. Chem. 3, 697-703 (2011).</li><li>Aguirre, S. D., Ali, M. M., Salena, B. J., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+DNA+enzyme-based+fluorescent+assay+for+bacterial+detection.">A sensitive DNA enzyme-based fluorescent assay for bacterial detection.</a> Biomolecules. 3, 563-577 (2013).</li><li>Aguirre, S. D., Ali, M. M., Kanda, P., Li, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Bacteria+Using+Fluorogenic+DNAzymes.">Detection of Bacteria Using Fluorogenic DNAzymes.</a> J. Vis. Exp. (63), e3961 (2012).</li><li>Shen, Z., 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+catalytic+DNA+activated+by+a+specific+strain+of+bacterial+pathogen.">A catalytic DNA activated by a specific strain of bacterial pathogen.</a> Angew. Chem. Int. Ed. 54, (2015).</li><li>He, 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=Highly+specific+recognition+of+breast+tumors+by+an+RNA-cleaving+fluorogenic+DNAzyme+probe.">Highly specific recognition of breast tumors by an RNA-cleaving fluorogenic DNAzyme probe.</a> Anal. Chem. 87, 569-577 (2015).</li><li>Sumner, J. B., Hand, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoelectric+point+of+crystalline+urease.">Isoelectric point of crystalline urease.</a> J. Am. Chem. Soc. 51, 1255-1260 (1929).</li><li>Karplus, P. A., Pearson, M., Hausinger, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=70+Years+of+crystalline+urease:+What+have+we+learned.">70 Years of crystalline urease: What have we learned.</a> Acc. Chem. Res. 30, 330-337 (1997).</li><li>Omiccioli, E., Amagliani, G., Brandi, G., Magnani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+platform+for+Real-Time+PCR+detection+of+Salmonella+spp.,+Listeria+monocytogenes+and+Escherichia+coli+O157+in+milk.">A new platform for Real-Time PCR detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 in milk.</a> Food Microbiol. 26, 615-622 (2009).</li><li>Cui, S., Schroeder, C. M., Zhang, D. Y., Meng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+sample+preparation+method+for+PCR-based+detection+of+Escherichia+coli+O157:H7+in+ground+beef.">Rapid sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef.</a> J. Appl. Microbiol. 95, 129-134 (2003).</li><li>Ibekwe, A. M., Watt, P. M., Grieve, C. M., Sharma, V. K., Lyons, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+fluorogenic+real-time+PCR+for+detection+and+quantification+of+Escherichia+coli+O157:H7+in+dairy+wastewater+wetlands.">Multiplex fluorogenic real-time PCR for detection and quantification of Escherichia coli O157:H7 in dairy wastewater wetlands.</a> Appl. Environ. Microbiol. 68, 4853-4862 (2002).</li><li>Strachan, N. J., Ogden, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+microsphere+coagulation+ELISA+for+Escherichia+coli+O157:H7+using+Russell's+viper+venom.">A sensitive microsphere coagulation ELISA for Escherichia coli O157:H7 using Russell's viper venom.</a> FEMS Microbiol Lett. 186, 79-84 (2000).</li><li>de Boer, E., Beumer, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+detection+and+typing+of+foodborne+microorganisms.">Methodology for detection and typing of foodborne microorganisms.</a> Int. J. Food Microbiol. 50, 119-130 (1999).</li><li>Gracias, K. S., McKillip, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+conventional+detection+and+enumeration+methods+for+pathogenic+bacteria+in+food.">A review of conventional detection and enumeration methods for pathogenic bacteria in food.</a> Can. J. Microbiol. 50, 883-890 (2004).</li></ol>

The authors have nothing to disclose.

的细菌响应核酶到石蕊试验的RNA裂解活性的作用的翻译是通过使用脲酶和磁分离的成为可能，由图1所示。虽然改性石蕊试验的细菌检测示范是与E.完成大肠杆菌依赖性的RNA切割核酶，5,19,20设计通常可延伸为任何的RNA切割核酶。定为不同的分析物，并且各种方法的RNA切割DNA核酶的极大可用性来隔离从新的目标随机序列池新的RNA切割DNA核酶，我们预计，改性石蕊测试平台可以扩展到检测的感兴趣的多样目标。 为E.的试金石大肠杆菌的检测可以检测5000和500个细胞时，报告的反应时间被设定为1和2小时，分别。流行的聚合酶链反应（PCR）和夹心酶联免疫吸附测定（ELISA）方法可达到约10 4 -10 5 大肠杆菌的检测限大肠杆菌细胞在类似的测试时间。25,26因此，细菌试金石提供可比的检测灵敏度。 虽然细菌石蕊测试是容易进行，并且能够产生鲜明的色彩的变化，有几个因素可以显著影响测试结果。首先，脲酶的质量是非常重要的。我们已经从不同的源中使用的脲酶，并发现这些检测结果可以显著变化。我们建议使用尿素酶从材料部分指定的来源。 核酶的/脲酶/磁珠大会需要特别注意。磁珠彻底清洗以除去未杂交的UrDNA必须防止假阳性结果。护理也需要采取避免升的内表面上的残留磁性珠的堆积微量离心管中，这可能是难以看到的标识。一旦出现，使磁珠不再进行磁分离，因此，可能会携带一些未杂交UrDNA，可导致在记者反应假阳性信号。同样重要的是，以避免在磁分离步骤留下的磁性机架离心管长于10分钟。珠粒可聚集或粘到离心管，这可以减少洗涤效率并引入批与批不一致性。在洗涤溶液0.01％吐温-20的夹杂物可以改善批与批之间的一致性，并应来实现。 磁珠涂覆有链霉亲和，将其用作锚组装核酶脲酶偶联物上的磁性珠粒。两个链霉和脲酶是可以储存期间变性的蛋白质分子。我们通常在4℃下保存组装的DNA酶脲酶磁珠长达4周并定期作出新的批次，以获得更一致的结果。 护理也需要注意避免意外服用磁珠在以下核酶活化的磁性分离步骤（步骤6.9）。从我们的经验，细胞碎片和在溶液中的其它颗粒可降低磁分离效率，并且因此，一些磁性珠可以无意中移液中取出。这将导致假阳性结果。我们建议采取以下措施来缓解这个问题:一个较长的分离时间（如5-10分钟），压力对吸管较慢的释放，使上清温柔的撤离，以及对上清液进行另一轮磁选。 最后，为了避免其中多个样品进行测试的实验过程中，记者原液通过脲酶的意外污染是重要的。由于u的高反应性rease，这种性质的污染可导致假阳性结果。

Bacterial pathogens are one of the major causes of global morbidity and mortality. Outbreaks from hospital-acquired infections, food-borne pathogens, and bacterial contaminants in the environment pose serious and on-going threats to public health and safety. To prevent these outbreaks, effective tools are needed that permit pathogen detection in a timely fashion under a variety of settings. Simple but still effective tests that are portable and cost-effective are greatly coveted, especially in regions that are susceptible to outbreaks but cannot afford expensive testing facilities.1-3 Although there exists a multitude of methods to detect bacteria, many of them are not suitable as screening or on-site testing tools because they require long test times, expensive instruments and complicated testing procedures. 
Colorimetric tests are particularly attractive for point-of-care or field applications as color changes can be easily detected by the naked eye. The litmus test for pH is simple, quick, and effective. Although it is a very old technology, it is still widely used today because of its simplicity and effectiveness. Surprisingly, this simple test had never been modified to achieve the detection of other analytes before we recently developed an approach of modifying this test for E. coli testing.4
The expanded litmus test for E. coli employs three additional components: an E. coli activated RNA-cleaving DNAzyme (EC1), 5 urease, and magnetic beads. DNAzymes refer to synthetic single-stranded DNA molecules with catalytic activity.6 They can be isolated from random-sequence DNA pools using in vitro selection.7,8 They are highly stable and can be produced cost-effectively using high-efficiency automated DNA synthesis.9 For these reasons, DNAzymes, particularly RNA-cleaving DNAzymes, have been widely examined for biosensing applications.6,10,11 RNA-cleaving DNAzyme sensors have been developed to detect metal ions,12-16 small molecules,17,18 bacterial pathogens5,19-21 and cancer cells.22 Given the great availability of target-induced RNA-cleaving DNAzymes, any assay that utilizes a DNAzyme can be potentially expanded to detect a diverse range of analytes.
Urease is chosen for its ability to hydrolyze urea into ammonia,23,24 resulting in a pH increase. Urease is also highly efficient, stable and amenable for conjugation to other biomolecules. Therefore, we postulated that a conjugate of an RNA-cleavage DNAzyme with urease would allow the use of litmus test for the detection of other targets.5
The action of the RNA-cleaving DNAzyme is relayed to urease-mediated increase of pH through the use of magnetic beads that are immobilized with the DNAzyme-urease conjugate. Because the activity of the DNAzyme under investigation is strictly dependent on E. coli, the presence of this bacterium in the test solution will result in the release of urease from the magnetic beads to the solution, which is then taken and used to hydrolyze urea in a reporter solution that contains a pH-sensitive dye. The final outcome of this procedure is a color change that can be conveniently reported by the dye or pH paper.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:275px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:183px;">VWR AMRESCO</td>
			<td style="width:163px;">0105</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Hydroxide (NaOH) pellets</td>
			<td>BIO BASIC CANADA INC.</td>
			<td>SB6789</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tris-base</td>
			<td>VWR AMRESCO</td>
			<td>0497</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Boric acid</td>
			<td>AMRESCO</td>
			<td>0588</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Urea</td>
			<td>VWR AMRESCO</td>
			<td>M123</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">40% acrylamide/bisacrylamide (29:1) solution</td>
			<td>BIO BASIC CANADA INC.</td>
			<td>A0007</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sucrose</td>
			<td>Bioshop Canada inc.</td>
			<td>SUC507</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Bromophenol blue</td>
			<td>Bioshop Canada inc.</td>
			<td>BRO777</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Xylenecyanol FF</td>
			<td>SIGMA-ALDRICH</td>
			<td>X-4126</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10% sodium dodecyl sulfate</td>
			<td>Bioshop Canada inc.</td>
			<td>SDS001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Hydrochloric Acid (HCl)</td>
			<td>CALEDON LABORATORIES LTD</td>
			<td>6026</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Chloride (NaCl)</td>
			<td>Bioshop Canada inc.</td>
			<td>SOD001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Bioshop Canada inc.</td>
			<td>HEP001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Magnesium Chloride (II) hexahydrate</td>
			<td>VWR AMRESCO</td>
			<td>0288</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tween 20</td>
			<td>Bioshop Canada inc.</td>
			<td>TW508</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Adenosine Triphospahte (ATP)</td>
			<td>AMRESCO</td>
			<td>0220</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Acetate trihydrate (NaOAc)</td>
			<td>SIGMA-ALDRICH</td>
			<td>S8625</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Ethanol</td>
			<td>Commercial Alcohols</td>
			<td>P016EAAN</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tetramethyleneethylenediamine (TEMED)</td>
			<td>AMRESCO</td>
			<td>0761</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10% Ammonium persulfate (APS)</td>
			<td>BIO BASIC CANADA INC.</td>
			<td>AB0072</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC)</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>22360</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dimethyl sulfoxide (DMSO)</td>
			<td>CALEDON LABORATORIES</td>
			<td>803540</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Urease</td>
			<td>SIGMA-ALDRICH</td>
			<td>U0251</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1&#215; Phosphate Buffered Saline (PBS)</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>70011-069</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">0.04% Phenol red</td>
			<td>SIGMA-ALDRICH</td>
			<td>P3532</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10&#215;T4 polynucleotide kinase reaction buffer</td>
			<td>Lucigen</td>
			<td>30061-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10&#215; T4 DNA ligase reaction buffer</td>
			<td>Bio Basics Canada</td>
			<td>B1122-B</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">T4 DNA ligase (5 U/uL)</td>
			<td>Thermo Fischer Scientific</td>
			<td>B1122</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Luria Bertani (LB) Broth</td>
			<td>AMRESCO</td>
			<td>J106</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Agar</td>
			<td>AMRESCO</td>
			<td>J637</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">T4 polynucleotide kinase (10 U/uL)</td>
			<td>Lucigen</td>
			<td>30061-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">E. coli K12 (MG1655)</td>
			<td>ATCC</td>
			<td>ATCC700926</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Centrifuge</td>
			<td>Beckman Coulter, Inc.</td>
			<td>392187</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Glass plates</td>
			<td>CBS scientific</td>
			<td>ngp-250nr</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">0.75 mm thick spacers</td>
			<td>CBS scientific</td>
			<td>VGS-0725r</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">12-well comb</td>
			<td>CBS scientific</td>
			<td>VGC-7512</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">UV Lamp</td>
			<td>UVP</td>
			<td>95-0017-09</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Spectrophotometer (NanoVue)</td>
			<td>GE Healthcare</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Metal plate</td>
			<td>CBS scientific</td>
			<td>CPA165-250</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Vortex</td>
			<td>VWR International</td>
			<td>58816-123</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Gel electrophoresis apparatus</td>
			<td>CBS scientific</td>
			<td>ASG-250</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Petri dishes</td>
			<td>VWR International</td>
			<td>25384-342</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">100 kDa MWCO centrifugal filters</td>
			<td>EMD Millipore</td>
			<td>UFC510024</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Magnetic Bead (BioMag)</td>
			<td>Bangs Laboratories Inc</td>
			<td>BM568</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Magnetic Seperation Rack</td>
			<td>New England BioLabs</td>
			<td>S1506S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Microfuge tubes</td>
			<td>Sarstedt</td>
			<td>72.69</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Syringe filter (0.22 um)</td>
			<td>VWR International</td>
			<td>28145-501</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">14 mL culture tube</td>
			<td>VWR International</td>
			<td>60818-725</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Cell culture incubator</td>
			<td>Eppendorf Scientific</td>
			<td>M13520000</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Branson Ultrasonic cleaner</td>
			<td>Branson</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Camera (Canon Powershot G11)</td>
			<td>Canon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">50 mL conical tube</td>
			<td>VWR International</td>
			<td>89004-364</td>
			<td></td>
		</tr>
	</tbody>
</table>

colorimetric detection of bacteria using litmus test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

细菌石蕊试验的原理在图1中说明的测试使用了三个关键材料:由一特定细菌酶，脲酶和磁珠活化的RNA切割核酶。所述核酶被用作分子识别元件，以实现所关注的细菌具有高度特异性的检测。脲酶和磁性珠粒用来实现DNA核酶的RNA的切割活性的信号转导。这涉及含有尿素酶结合物核酶磁珠的创建。在目标细菌的存在，所述核酶裂解其RNA的联动。这个动作引起脲酶从磁珠的解离。被释放的尿素酶可以从磁珠容易分离并用于产生在一个记者溶液，其中包含脲和pH敏感染料的颜色变化。尿素酶水解尿素成氨，伴随着触发℃pH值的增加染料的olor变化。 图2给出细菌试金石EC1的地方，一个E.大肠杆菌响应性的RNA切割核酶，用作核酶和酚红被用作pH值报告染料。 EC1先前由我们的组使用体外选择的技术中一个随机序列的DNA池隔离。5我们先前的研究已经表明，EC1为大肠杆菌高度特异性大肠杆菌和表现出最小的活动对其他细菌。5,19业已发现，EC1由来自大肠杆菌的蛋白质分子活化大肠杆菌。虽然这种蛋白质生物标记的身份还没有被破译，高识别特异性表明，这种蛋白是唯一的大肠杆菌大肠杆菌 。记者溶液被设置为具有5.5的初始pH值。在此pH，酚红呈现黄色。作为脲酶水解尿素成氨，记者溶胶的碱度ution增加。这是通过颜色从黄色到粉红色逐渐变化反映出来。颜色变化的深度依赖于以下两个参数，通过图2所示: 大肠杆菌的数量在核酶活化步骤和所允许的尿素水解步骤时所使用的大肠杆菌细胞。更多E.大肠杆菌细胞产生了更强的色彩变化，通过一个渐进的黄色到粉红色过渡时E.观察反映大肠杆菌细胞被连续从增加的5至5×10 7（10倍，每次增加）。同时，允许用于检测大肠杆菌的人数较少的尿素水解的时间较长大肠杆菌细胞（5,000个细胞在1小时的反应和500个细胞在2小时的反应）。 细菌石蕊测试的pH值的变化，也可以使用手持式pH计监测和代表性的结果在图3中示出。这WA作者发现10 7 E.存在的大肠杆菌细胞产生了由3个单位在10分钟内pH值的逐渐增加。与此相反，不存在E的大肠杆菌细胞没有造成相同的设置下可检测的pH变化。 <img alt="图1" src="/files/ftp_upload/54546/54546fig1.jpg" /> 图1: 细菌试金石的设计原理 （A）由感兴趣的细菌的生物标志物特定的RNA裂解脱氧核酶的激活。在生物标志物的存在下，该RNA切割核酶固定于磁珠裂解RNA的联动，导致从磁性珠溶液中标记的脲酶的释放。 （B）的三步骤检测程序。第1步:激活核酶，如面板A.步骤2中所述:磁选 - 释放的脲酶是从磁珠分离。第3步:尿素水解 - [R12;被释放的尿素酶加入到一个含脲 - 报道溶液。尿素酶水解尿素成氨，导致pH的变化，可以通过pH敏感的染料进行报告。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54546/54546fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54546/54546fig2.jpg" /> 图 2:E. 试金石 使用 大肠杆菌 E.大肠杆菌 响应性脱氧核酶EC1代表性的颜色变化结果与大肠杆菌的不同数量每个试管上方设置的大肠杆菌细胞。酚红被用作pH敏感染料。 E.没有一个测试大肠杆菌用作阴性对照。更多E.预期的大肠杆菌细胞以引起更多的脲酶分子的释放，伴随bŸ较强的颜色变化。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54546/54546fig2large.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/54546/54546fig3.jpg" /> 图3: 使用pH计监测pH上升引起的10 7 大肠杆菌的pH的变化。使用便携式pH计的大肠杆菌细胞进行监控。 E.没有一个测试大肠杆菌用作阴性对照。 10 E. 7的存在大肠杆菌细胞中的测试解决方案可以增加3〜pH单位的碱度在10分钟内。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54546/54546fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>名称</td><td>硒quence（5&#39;-3&#39;） </td><td>注意</td></tr><tr><td> BS1 </td><td> BTTTT TTTTT TTTAC TCTTC CTAGC FRQGG TTCGA TCAAG一个</td><td> A:5&#39;-生物素; R:腺嘌呤核苷酸; F:荧光胸苷;问:DABCYL胸苷</td></tr><tr><td> DE1 </td><td> GATGT GCGTT GTCGA GACCT GCGAC CGGAA CACTA CACTG TGTGG GGATG GATTT CTTTA CAGTT GTGTG TTGAA CGCTG TGTCA AAAAA AAAA </td><td></td></tr><tr><td> T1 </td><td> GACAA CGCAC ATCTC TTGAT CGAACÇ </td><td></td></tr><tr><td> LD1 </td><td> XTTTT TTTTT TTTTT TTGAC ACAGC GTTCA一个</td><td> X:5&#39;-NH 2的</td></tr></tbody></table> 表1: 合成的寡核苷酸的序列。

Watch this Scientific Journal Video about Colorimetric Detection of Bacteria Using Litmus Test at JoVE.com

Colorimetric Detection of Bacteria Using Litmus Test

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

细菌利用试金石比色法检测

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

colorimetric-detection-of-bacteria-using-litmus-test

Nanyang Technological University

Research

JoVE Journal

Biology

9.1K 次观看.  McMaster University. We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads. 

视频: Colorimetric Detection of Bacteria Using Litmus Test

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

蛋白泛素化的检测

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

科研

生物学

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

利用磁调制生物传感系统的生物检测的快速均质检测

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

罕见的基因变异检测从集资使用斯普林特测序

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

使用荧光显微镜可视化中的细菌线虫

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

测量细菌的影响<em&gt; C。线虫</em&gt;行为使用一个鸡蛋保留含量

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

检测miRNA靶在高通量的使用3&#39;LIFE分析

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

对肌红蛋白鲸类检测基于金免疫层析胶体试纸条的研制

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

胞嘧啶修饰形式使用敏感免疫组化检测

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

Detection of Tissue-resident Bacteria in Bladder Biopsies by 16S rRNA Fluorescence In Situ Hybridization

Visualization of the interaction of bacteria with host mucosal surfaces and tissues can provide valuable insight into mechanisms of pathogenesis. While visualization of bacterial pathogens in animal models of infection can rely on bacterial strains engineered to express fluorescent proteins such as GFP, visualization of bacteria within the mucosa of biopsies or tissue obtained from human patients requires an unbiased method. Here, we describe an efficient method for the detection of tissue-associated bacteria in human biopsy sections. This method utilizes fluorescent in situ hybridization (FISH) with a fluorescently labeled universal oligonucleotide probe for 16S rRNA to label tissue-associated bacteria within bladder biopsy sections acquired from patients suffering from recurrent urinary tract infection. Through use of a universal 16S rRNA probe, bacteria can be detected without prior knowledge of species, genera, or biochemical characteristics, such as lipopolysaccharide (LPS), that would be required for detection by immunofluorescence experiments. We describe a complete protocol for 16S rRNA FISH from biopsy fixation to imaging by confocal microscopy. This protocol can be adapted for use in almost any type of tissue and represents a powerful tool for the unbiased visualization of clinically-relevant bacterial-host interactions in patient tissue. Furthermore, using species or genera-specific probes, this protocol can be adapted for the detection of specific bacterial pathogens within patient tissue.

This protocol is for the unbiased detection of tissue-associated bacteria in patient biopsies by 16S rRNA in situ hybridization and confocal microscopy.

16S rRNA 荧光原位杂交检测膀胱活检中的组织驻留细菌

Visualization of the interaction of bacteria with host mucosal surfaces and tissues can provide valuable insight into mechanisms of pathogenesis. While ...