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.

<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.

يتم ترجمة العمل من النشاط النووي الريبي انشقاق من DNAzyme-البكتيريا تستجيب للاختبارا ممكن من خلال استخدام اليورياز والفصل المغناطيسي، كما يتضح من الشكل 1. على الرغم من أن مظاهرة من اختبارا تعديل للكشف عن البكتيريا هي القيام به مع E. القولونية معتمد على DNAzyme الشق RNA، 5،19،20 تصميم ويمكن تمديد عموما لأي DNAzyme الشق الحمض النووي الريبي. ونظرا لتوافر كبير من DNAzymes الشق RNA للالتحاليل المختلفة ومنهجيات مختلفة لعزل DNAzymes جديدة الشق الحمض النووي الريبي من حمامات العشوائي تسلسل للأهداف جديدة، ونحن نتوقع أن محك منصة الاختبار تعديل يمكن أن تمتد إلى الكشف عن أهداف متنوعة من الفائدة . الاختبار الحقيقي لE. كشف القولونية يمكن الكشف عن 5000 و 500 خلايا عندما يتم تعيين وقت رد الفعل التقارير إلى أن يكون 1 و 2 ساعة، على التوالي. رد الفعل الشعبية البلمرة المتسلسل (PCR) وشطيرة انزيم مرتبط المناعي فحص (ELISA) أساليب يمكن أن تحقق حدود الكشف عن ما يقرب من 10 4 -10 5 هاء خلايا القولونية في الأوقات العصيبة مماثلة. 25،26 وهكذا، فإن الاختبار الحقيقي البكتيرية يقدم كشف حساسية قابلة للمقارنة. على الرغم من أن الاختبار الحقيقي للبكتيريا من السهل القيام بها ويمكن أن تنتج تغييرات اللون نابضة بالحياة، يمكن أن العديد من العوامل تؤثر تأثيرا كبيرا على نتائج الاختبار. أولا، نوعية اليورياز مهم جدا. وقد استخدمنا اليورياز من مصادر مختلفة، وجدت نتائج الاختبار يمكن أن تختلف بشكل كبير. نحن نوصي باستخدام اليورياز من المصدر المحدد في قسم المواد. يحتاج تجميع DNAzyme / اليورياز / حبات مغناطيسية اهتماما خاصا. الغسل الكامل من الخرز المغناطيسي لإزالة UrDNA الغير مهجنة ضروري لمنع نتائج إيجابية كاذبة. يحتاج إلى رعاية أيضا التي يجب اتخاذها لتجنب تراكم حبات مغناطيسية المتبقية على السطح الداخلي لللمعرف من microfuge أنبوب، والذي قد يكون من الصعب أن نرى. مرة واحدة هناك، لم تعد تخضع حبات مغناطيسية إلى الفصل المغناطيسي، وبالتالي، يمكن أن تحمل بعض UrDNA الغير مهجنة التي يمكن أن تؤدي إلى إشارات إيجابية كاذبة في رد فعل المراسل. ومن المهم أيضا لتجنب ترك microfuge أنبوب على الرف المغناطيسي لمدة أطول من 10 دقيقة خلال خطوة فصل المغناطيسية. حبات قد تجميع أو التمسك microfuge أنبوب، الأمر الذي قد يقلل من كفاءة الغسيل وإدخال التناقض دفعة إلى دفعة. إدراج 0.01٪ توين 20 في الحل غسل يمكن تحسين الاتساق دفعة إلى دفعة، وينبغي تنفيذه. والمغلفة حبات مغناطيسية مع streptavidin، الذي كان يستخدم كمركز تجمع لتجميع تقارن DNAzyme-اليورياز على حبات مغناطيسية. كلا streptavidin واليورياز هي عبارة عن جزيئات البروتين التي يمكن التشويه والتحريف أثناء التخزين. ونحن عادة تخزين حبات DNAzyme-اليورياز المغناطيسي تجميعها في 4 درجة مئوية لمدة تصل إلى 4 أسابيعوجعل دفعات جديدة بشكل منتظم لتحقيق نتائج أكثر اتساقا. يحتاج إلى عناية أيضا التي يجب اتخاذها لتجنب الخطأ أخذ حبات مغناطيسية في خطوة الفصل المغناطيسي (الخطوة 6.9) بعد تفعيل DNAzyme. من تجربتنا، والحطام الخلوي والجسيمات الأخرى في الحل يمكن أن تقلل من كفاءة الفصل المغناطيسي، وبالتالي، بعض حبات مغناطيسية يمكن أن تؤخذ عن غير قصد من خلال pipetting ل. وهذا يؤدي إلى نتائج إيجابية كاذبة. ونحن نوصي التدابير التالية للتخفيف من حدة المشكلة: وقتا أطول فصل (مثل 5-10 دقيقة)، بيان أبطأ من الضغط على ماصة للسماح للانسحاب لطيف من طاف، وإخضاع طاف لجولة إضافية من الفصل المغناطيسي . وأخيرا، من المهم تجنب التلوث الحادث من محلول المخزون مراسل بواسطة اليورياز خلال التجربة حيث يتم اختبار عينات متعددة. ونظرا لتفاعل عالية من شrease، تلوث من هذا النوع يمكن أن يؤدي إلى نتائج إيجابية كاذبة.

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.

ويفسر مبدأ اختبارا الجرثومي في الشكل 1 يستخدم الاختبار ثلاث مواد رئيسية هي: وDNAzyme الشق الحمض النووي الريبي التي يتم تفعيلها من بكتيريا معينة، اليورياز وحبات مغناطيسية. يتم استخدام DNAzyme باعتبارها العنصر الاعتراف الجزيئي لتحقيق الكشف محددة للغاية للبكتيريا من الفائدة. تستخدم اليورياز وحبات مغناطيسية لتحقيق نقل الإشارة من النشاط الحمض النووي الريبي انشقاق من DNAzyme. وهذا ينطوي على إنشاء حبات مغناطيسية التي تحتوي على تقارن اليورياز-DNAzyme. في وجود البكتيريا المستهدفة، وDNAzyme يشق لها الحمض النووي الريبي الربط. هذا الإجراء يؤدي إلى تفكك اليورياز من حبات مغناطيسية. واليورياز صدر يمكن فصلها بسهولة من حبات مغناطيسية وتستخدم لتوليد تغيير اللون في حل المراسل، الذي يحتوي على اليوريا وصبغة حساسة درجة الحموضة. اليورياز hydrolyzes اليوريا إلى الأمونيا، يرافقه زيادة درجة الحموضة الذي يقوم بتشغيل جتغيير OLOR للصباغة. ويبين الشكل 2 اختبارا البكتيرية حيث EC1، وهو E. القولونية -responsive DNAzyme الشق RNA، كان يستخدم في DNAzyme، وكان يستخدم أحمر الفينول مثل صبغ الإبلاغ عن الرقم الهيدروجيني. وقد EC1 عزل من قبل مجموعتنا من مجموعة الحمض النووي العشوائي تسلسل باستخدام تقنية في المختبر الاختيار. 5 أظهرت دراساتنا السابقة أن EC1 غير محددة للغاية لE. القولونية ويسلك الحد الأدنى من النشاط تجاه البكتيريا الأخرى. 5،19 وقد وجد أن EC1 يتم تفعيلها من خلال جزيء البروتين من E. القولونية. على الرغم من أن هوية هذه العلامات البيولوجية البروتين لم يتم فك شفرتها، تقترح عالية التحديد الاعتراف بأن هذا البروتين فريد من نوعه لE. القولونية. تم تعيين حل مراسل حتى يكون لها درجة الحموضة الأولية من 5.5. وفي هذا الرقم الهيدروجيني، أحمر الفينول يسلك اللون الأصفر. كما اليورياز hydrolyzes اليوريا إلى الأمونيا، وقاعدية سول مراسلزيادات ution. ويتجلى ذلك من خلال التغيير التدريجي من اللون من الأصفر إلى الوردي. عمق تغيير لون يعتمد على المعلمتين التالية، كما يتضح من الشكل 2: عدد E. خلايا القولونية المستخدمة في تفعيل خطوة DNAzyme والوقت المسموح به للخطوة اليوريا التحلل. المزيد E. أسفرت خلايا القولونية في يتغير لون أقوى، التي تعكسها والمراقبة من E. الأصفر إلى الوردي تحول لون عندما التدريجي وزادت خلايا القولونية متسلسل 5-5 × 10 7 (10 أضعاف زيادة في كل مرة). وفي الوقت نفسه، وقتا أطول لالتحلل اليوريا يسمح للكشف عن أعداد أقل من E. خلايا القولونية (5000 الخلايا في رد فعل 1 ساعة و 500 الخلايا في رد فعل 2 ساعة). تغيير الرقم الهيدروجيني للاختبارا البكتيرية يمكن أيضا رصد باستخدام متر المحمولة الرقم الهيدروجيني وأوضحت نتائج ممثلة في الشكل (3). وواالصورة وجدت أن وجود 10 7 E. أسفرت خلايا القولونية في زيادة تدريجية في درجة الحموضة بمقدار 3 وحدات في غضون 10 دقيقة. في المقابل، فإن غياب E. لم خلايا القولونية لا تسبب تغيرات درجة الحموضة للكشف تحت نفس الإعداد. <img alt="شكل 1" src="/files/ftp_upload/54546/54546fig1.jpg" /> الشكل 1: مبدأ تصميم اختبارا البكتيري (A) تفعيل لDNAzyme الشق الحمض النووي الريبي العلامات البيولوجية محددة من بكتيريا من الفائدة. في وجود العلامات البيولوجية، وDNAzyme الشق RNA ثبتوا على يشق حبات مغناطيسية الربط RNA، مما أدى إلى الإفراج عن اليورياز الموسومة من حبات مغناطيسية إلى حل. (ب) ثلاث خطوات إجراء الفحص. الخطوة 1: تفعيل DNAzyme، كما هو موضح في لوحة A. الخطوة 2: الفصل المغناطيسي - يتم فصل اليورياز صدر من حبات مغناطيسية. الخطوة 3: اليوريا التحلل R12؛ وأضاف اليورياز صدر في حل مراسل التي تحتوي على اليوريا. اليورياز hydrolyzes اليوريا إلى الأمونيا، مما أدى إلى تغير في درجة الحموضة الذي يمكن الكشف عنه بواسطة صبغة حساسة درجة الحموضة. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54546/54546fig1large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <img alt="الشكل 2" src="/files/ftp_upload/54546/54546fig2.jpg" /> الشكل 2: اختبار عباد الشمس مع E. القولونية E. باستخدام القولونية -responsive DNAzyme EC1. التمثيلية تغيير لون النتائج مع أعداد متفاوتة من E. خلايا القولونية الواردة أعلاه كل أنبوب اختبار. تم استخدام أحمر الفينول مثل صبغة حساسة درجة الحموضة. اختبار دون E. وقد استخدم القولونية كعنصر تحكم السلبية. المزيد E. ومن المتوقع أن تتسبب في إطلاق المزيد من الجزيئات اليورياز خلايا القولونية، يرافقه بذ يتغير لون أقوى. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54546/54546fig2large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <img alt="الشكل (3)" src="/files/ftp_upload/54546/54546fig3.jpg" /> الشكل (3): رصد زيادة درجة الحموضة باستخدام مقياس درجة الحموضة وتغيير درجة الحموضة الناجمة عن 10 7 هاء. تم رصد خلايا القولونية باستخدام مقياس درجة الحموضة المحمولة. اختبار دون E. وقد استخدم القولونية كعنصر تحكم السلبية. وجود 10 7 E. خلايا القولونية في حل الاختبار يمكن أن تزيد من قاعدية من قبل وحدات ~ 3 درجة الحموضة في 10 دقيقة. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54546/54546fig3large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم </td><td> سيquence (5&#39;-3 &#39;) </td><td> ملاحظة </td></tr><tr><td> BS1 </td><td> BTTTT TTTTT TTTAC TCTTC CTAGC FRQGG TTCGA TCAAG و </td><td> باء: 5&#39;-البيوتين. R: ريبونوكليوتيد الأدينين. F: فلوريسئين من DT. س: dabcyl من DT </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 CGAAC C </td><td></td></tr><tr><td> LD1 </td><td> XTTTT TTTTT TTTTT TTGAC ACAGC GTTCA و </td><td> X: 5&#39;-NH 2 </td></tr></tbody></table> الجدول 1: متواليات من أليغنوكليوتيد الاصطناعية.

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.

كشف اللونية من البكتيريا عن طريق محك الاختبار

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 Views.  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. 

Video: 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.

اكتشاف بروتين Ubiquitination

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

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.

متجانسة الكشف السريع عن طريق فحوصات البيولوجي المغناطيسي نظام Biosensing التحوير

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.

الكشف عن المتغيرات الجينية النادرة من تسلسل المجمعة عن طريق سبلينتر

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.

تصور البكتيريا في نيماتودا باستخدام المجهر الفلورسنت

 
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.

سريعة وفعالة الزرد التنميط الجيني باستخدام PCR عالية الدقة مع تحليل نذوب

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.

قياس آثار البكتيريا على<em&gt; C. ايليجانس</em&gt; السلوك باستخدام مقايسة الاحتفاظ البيض

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.

الكشف عن أهداف ميرنا في إنتاجية عالية باستخدام الفحص 3&#39;LIFE

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.

تطوير المناعي اختبار قطاع تستند الذهب الغروية عن الكشف عن الحيتان الميوجلوبين

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.

الكشف عن أشكال معدلة من السيتوزين عن طريق الحساسة المناعية

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.

الكشف عن البكتيريا المقيمة في الانسجه في المثانة الخزعات من قبل rRNA الفلورية 16S في الموقع التهجين

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