Bu eser K-INBRE P20GM103418, Kansas soya Komisyonu (KSC/PSU 1663), ACS PRF 56629-UNI7 ve PSU polimer kimya başlangıç Fonu, tüm SS tarafından desteklenmektedir. Biz Üniversitesi videoları çekiyor, Bay Jacob Anselmi, video ile onun en iyi çalışma için teşekkür ederim. Biz de Bay Roger Heckert ve Bayan Katha Heckert cömert destekleri araştırma için teşekkür ederiz.

1. sentez ve çok parametrik Manyeto-floresan Nanosensors (MFnS) Functionalization.
<ol><li>Superparamagnetic demir oksit sentezi nano tanecikleri (IONPs) <ol><li>IONP sentezi için hazırlamak için aşağıdaki 3 çözümleri hazırlamak: çözüm 1: FeCl 3 (0.70 g) ve FeCl 2 H 2 O içinde (2 mL), çözüm 2: NH 4 OH (2.0 mL, 13.4 M) H 2 O (15 mL) ve çözüm 3: polyacrylic asit (0.855 g) H 2 O (5 mL).</li>
		<li>2 M hidroklorik asit (HCl) 90 µL çözüm 1'e ve daha sonra hemen vortexing 875 devirde iken çözüm 2 ile karıştırın. O zaman çözüm 3 ekleyin. Vortexing 60 dakika süreyle devam</li>
		<li>Santrifüj için 1,620 x g., 20 dk santrifüj kapasitesi süpernatant 2.880 x g., 20 dk</li>
		<li>Son süpernatant ile diyaliz arındırmak. Süpernatant bir Diyaliz Ekle (MWCO 6 &#8722; 8 K) ve fosfat tamponlu tuz (PBS) içeren bir ölçek yerleştirin (pH 7.4 =) ve spin bar. Her 2-3 h. ile taze tampon yerine 12 h için emmek izin</li>
	</ol></li> <li>IONPs yüzey için hedef özgü antikorların konjugasyon <ol><li>Aşağıdaki dört çözümleri hazırlamak: çözüm A: 5 mg 1-etil - 3-(3-dimethylaminopropyl) carbodiimide hidroklorid (EDC) 250 µL MES [2-(N- morpholino) ethanesulfonic asit] arabellek (0.1 M, pH 6.8 =), çözüm B: 3 mg, N-Hydroxysuccinimide (NHS) 250 µL MES arabelleği (0.1 M, pH 6.8 =), Igg1, E. coli mAb fosfat tamponlu tuz (PBS) (pH 7,4) 225 µL içinde çözüm C: 5 µg ve IONP çözüm D: 4 mL (5.0 mmol) PBS (pH 7,4) 1 ml.</li>
		<li>Ekleyin 2-(N-Morpholino) ethanesulfonic asit (MES) arabellek eriyik-e doğru A, o zaman hemen çözüm eklemek A çözüm D 15 içinde küçük artışlarla s, çözüm karıştırma için her eklenmesinden sonra ters çevirme.</li>
		<li>Hemen çözüm B çözüm D için küçük aralıklarla üç dakikalık bir süre içinde eklemek için başlayın. Her eklenmesinden sonra çözüm ters çevirme tarafından Mix.</li>
		<li>5 µL artırımlarıyla çözüm çözüm C eklemek çözüm karıştırma için her eklenmesinden sonra ters çevirme.</li>
		<li>Oda sıcaklığında 3 h için devam etmek ve o zaman kuluçka gecede 4 ° C'de devam etmek tepki izin.</li>
		<li>Arındırmak manyetik sütun PBS kullanma yolu ile elde edilen Ab Birleşik IONPs (pH 7.4, final [Fe] = = 3,5 mmol) herhangi bir çekimsiz antikorlar kaldırmak için.
		<ol><li>Yıkama manyetik sütun PBS (1 mL) ile. Sütun için manyetik plaka ekleyin ve IONP çözüm ekleyin.</li>
			<li>Manyetik sütun PBS (1 mL) ile tekrar yıkayın. Manyetik sütun Manyetik plaka kaldırın. PBS sütununu ekleyin ve çözüm toplamak. Mağaza 4 ° C'de</li></ol></li></ol></li> <li>Floresan 1,1 kapsülleme &#39; - Dioctadecyl - 3,3,3 &#39;, 3 &#39;-Tetramethylindocarbocyanine perklorat (dıı) boya kullanarak antikor Birleşik IONPs polyacrylic asit (PAA) kaplama içine bir Solvent Difüzyon yöntemi.
	<ol><li>IONPs 4 mL için dropwise 2.0 ekleyin dıı boya (2 mmol) 100 µL DMSO, sürekli 1100 devir / dakikada karıştırma ile µL.</li>
		<li>[Fe] son demir konsantrasyonu ile son bir MFnS çözüm oluşturma PBS karşı elde edilen çözüm (MWCO 6-8 KDa, 12 h) diyaliz 2 mmol =.</li>
	</ol></li> <li>MFnS karakterizasyonu <ol><li>spektrofotometrik analiz için bir plaka okuyucu ile floresan emisyon 595, kapsüllenmiş dıı boya algılamak için kullanın nM.</li>
		<li>Dinamik ışık saçılma <ol><li>ortalama boyutu ve MFnS yüzey ücret belirlemek için zetasizer MFnS çözüm örneği yerleştirin. 
			Not: Burada, ortalama boyutu ve fonksiyonel MFnS yüzey ücret 77.09 bulundu nm ve-22.3 mV, sırasıyla.</li>
		</ol></li></ol></li></ol> 2. Bakteriyel Culturing ve hisse senedi çözüm hazırlık <ol><li>bakteri kültürü <ol><li>besin suyu 1 ml dondurularak Pelet (E. coli O157: H7) hidrat ve bir ek 5 mL suyu ekleyin.</li>
		<li>Bu çözümün bir agar plaka ve çizgi uygulamak 100 µL kuluçka için 24 h 37 ° C'de ve ardından bir steril aşılama döngü kullanarak</li>
		<li>Sonra 24 h, izole bir koloni agar plaka seçin ve bir 15 mL kültür suyu ekleyin. Süre optik yoğunluk değeri izleme için 4-6 h, 37 ° C'de kuluçkaya (600 Absorbans nm).</li>
		<li>Bir optik yoğunluk değerini 0.1 alındıktan sonra kültür durdurmak ve seri dilutions gerçekleştirmek.</li>
	</ol></li> <li>Seri dilutions <ol><li>ekleyin 900 µL sekiz steril microcentrifuge tüpler için besin suyu.</li>
		<li>Ekle 100 µL bakteriyel süspansiyon ilk tüp (seyreltme 10 -1).</li>
		<li>Al 100 µL ilk tüp ve ikinci 10 -2 seyreltme elde etmek için ekleyin. Bu model 10 -8 son bir seyreltme ile biten kalan tüpler için devam.</li>
		<li>Transfer 100 µL Ağar kaplamalar ayrı ve 37 24 h için kuluçkaya her tüp ° C.</li>
		<li>Ertesi gün, her tabağa birimleri (CFUs) oluşturan colony saymak. ~ 100 CFUs ile kalıbı seçin. Karşılık gelen dilutions daha fazla deneyler için kullanın (100 µL ~ 100 CFUs =). 
		Not: Burada ~ 100 CFUs sonuçlanan 10 -6 seyreltme oldu.</li>
	</ol></li></ol> 3. MFnS kullanarak hızlı algılama E. coli O157: H7 <ol><li>1-100 arasında değişmektedir CFU sonuçlanan 10 -6 bakteriyel hisse senedi, miktarda artan çeşitli PBS Çözümleri (1 X, pH 7.4, 300 µL) Spike. MFnS tutarlı bir miktar eklemek (100 µL) Bu çözümler için.</li>
	<li>Sadece PBS (1 X, pH 7.4, 300 µL) içeren bir temel çözüm oluşturmak ve MFnS (100 µL).</li>
	<li>37 ° C'de 30 dk için çözümler kuluçkaya ve sonra oda sıcaklığında soğumaya izin.</li>
	<li>Manyetik relaxometer (0,47 Tesla) ve kayıt değişiklikleri gevşeme zamanlarda (T2) her çözümde CFUs göre transfer bireysel çözümler.
	<ol>T2 değerlerde değişiklikleri kaydetmek, sadece PBS ve MFnS içeren temel çözüm T2 değerini ölçerek başlamak için <li>.
		<ol><li>Yer manyetik çözümde relaxometer. İlgili yazılım açık, seçin &#34; T2 gevşeme &#34; ayarı ve basın &#34; ölçü birimi. &#34;</li></ol></li> <li>temel T2, topluluğu takip çeşitli ile çivili ek çözümler T2 değerini ölçmek bakteri konsantrasyonları. 
		Not: Taban çizgisi çivili T2 düşülen T2 T2 değişiklik eşdeğerdir.</li>
	</ol></li> <li>Manyetik relaxometer örnekleri kaldırmak ve 10 dakika süreyle 2880 x g de tüpler santrifüj kapasitesi</li>
	<li>Süpernatant dikkatle boşaltmak ve PBS (1 X, pH 7,4) 100 µL içinde bakteri granül resuspend.</li>
	<li>Eklemek 80 µL, her resuspension bir 96-şey plaka ve kayıt Floresans Koyulukları 595 nM. 
	Not: Test sonuçları bölümünde açıklandığı gibi göl su, süt ve diğerleri dahil olmak üzere farklı çözücüler kullanılarak tekrar edilebilir</li></ol>

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Bu nanoteknoloji gösterdiği uygulama henüz FDA tarafından onaylanmış değil.

Bu iletişim kuralı basit tamamen işlevsel MFnS üretmek için tasarlanmış mümkün olduğunca. Ancak, değişiklik Protokolü'nün kullanıcının nihai hedef bağlı olarak yararlı olabilecek çok önemli noktalar vardır. Örneğin, farklı antikor kullanımı birçok diğer patojenleri hedefleme için izin verecek. Ayrıca, bu iletişim kuralını molekülleri hedefleme olarak antikor kullanımı sınırlı değildir. Konak hücre reseptörleri gibi hedef patojenler için özel b...

Bakteriyel kontaminasyon hem de kalıcı geçtiği ticari gıda üretilen ve su kaynaklarının giderek hızlı ve spesifik tanı platformları için bir ihtiyaç yarattı. 1 , 2 , Salmonella, Staphylococcus, Listeria, Vibrio, Shigella, Bacillus ve Escherichia cins daha yaygın bakteriyel kirletici için yiyecek ve su kirlenmesi sorumlu bazılarıdır. 3 , 4 kez bakteriyel kontaminasyon Bu patojenler tarafından ateş, kolera, gastroenterit ve ishal gibi belirtiler olur. 4 su kaynaklarının kirlenme kez yeterince filtrelenmiş su erişimi olmayan topluluklar üzerinde ciddi ve olumsuz etkileri vardır ve gıda kontaminasyonu hastalıklar ve ürün geri çekme çabaları çok sayıda için açmıştır. 5 , 6
Bakteriyel kirlenme tarafından neden olduğu hastalıkların oluşumunu azaltmak için bir dizi hangi su ve yiyecek verimli satış veya tüketim önce taranabilir yöntemleri geliştirmek için çaba olmuştur. PCR,1,7,8,9,10 ELISA,11,12 döngü-aracılı izotermal amplifikasyon () gibi 3 teknikleri LAMBA),13,14 diğerleri arasında15,16,17,18,19,20,21, 22,23,24 son zamanlarda çeşitli patojenler tespiti için kullanılır. Geleneksel bakteriyel kültür yöntemleri ile karşılaştırıldığında, bu tekniklerin özgüllük ve zaman açısından çok daha verimli. Ancak, bu teknikler hala yanlış pozitif ve negatif, karmaşık prosedürleri ve maliyet ile mücadele. 1 , 3 , 25 multiparametric Manyeto-floresan nanosensors (MFnS) bakteriyel algılama için alternatif bir yöntem olarak önerilen bu nedenle içindir.
Bu nanosensors benzersiz olarak birlikte manyetik gevşeme ve floresan yöntemleri, hızlı ve doğru bir çift-algılama platformu için izin eşleyin. E. coli O157: H7 örnek kirletici kullanarak, MFnS dakika içinde olduğu kadar az 1 CFU algılama yeteneğini gösterilmiştir. Patojen özel antikorlar özgüllüğü artırmak için kullanılır ve algılama ve miktar her iki düşük ve yüksek kirlilik aralıklardaki bakteriyel kirletici için manyetik ve floresan modaliteleri kombinasyonu sağlar. 16 bakteriyel kontaminasyon durumunda patojen özgü antikorların hedefleme yetenekleri nedeniyle bakteri çevresinde nanosensors swarm. Manyetik nanosensors ile bakteri arasındaki manyetik demir çekirdekli ve çevresindeki su proton arasındaki etkileşimi sınırlar. Bu artış T2 gevşeme zamanlarda manyetik bir relaxometer tarafından kaydedildiği şekilde neden olur. Çözüm bakterilerde konsantrasyon yükselir gibi bakteri, alt T2 değerleri kaynaklanan artan sayıda nanosensors dağıtmak. Tersine, Floresans emisyon nanosensors patojen için doğrudan bağlı artan sayıda nedeniyle bakteri konsantrasyonu ile orantılı artar. Santrifüjü örnekleri ve bakteriyel Pelet yalıtım sadece doğrudan herhangi bir serbest yüzer nanosensors kaldırılması ve doğrudan Floresans emisyon sayısı ile korele bakteri iliştirilmiş nano tanecikleri tasarruf etmek bakteri içinde çözüm sunuyoruz. Bu mekanizma şematik gösterimi şekil 1' de temsil edilir.
Bu MFnS platform bakım noktası tarama unutmayın, düşük maliyetli ve taşınabilir özellikleri kaynaklanan ile dizayn edilmiştir. MFnS oda sıcaklığında kararlı olduğunu ve sadece doğru algılama bakteriyel kirletici için çok düşük konsantrasyonlarda gerekmektedir. Ayrıca, sentez sonra MFnS kullanımı basittir ve alanında eğitimli uzmanları kullanımı gerektirmez. Son olarak, bu tanı platform, bu tarafından bir platformda her türlü patojenler birçok farklı ortamlarda algılamak için kullanılan bir araç sağlayarak son derece özelleştirilebilir hedefleme için sağlar.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ferrous Chloride Tetrahydrate</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">I90-500</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ferric Chloride Hexahydrate</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td>I88-500</td>
			<td></td>
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			<td height="21" style="height:21px;width:239px;">Ammonium Hydroxide</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">A669S-500</td>
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			<td height="21" style="height:21px;width:239px;">Hydrochloric Acid</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">A144S-500</td>
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			<td height="21" style="height:21px;width:239px;">Polyacryllic Acid</td>
			<td style="width:156px;">Sigma-Aldrich</td>
			<td style="width:197px;">323667-100G</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">EDC</td>
			<td style="width:156px;">Thermofisher Scientific</td>
			<td style="width:197px;">22980</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">NHS</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td>AC157270250</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-E. coli O111 antibody&#160;</td>
			<td style="width:156px;">sera care</td>
			<td>5310-0352</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Anti-E. coli O157:H7 antibody [P3C6]&#160;</td>
			<td style="width:156px;">Abcam</td>
			<td style="width:197px;">ab75244</td>
			<td></td>
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			<td height="21" style="height:21px;width:239px;">DiI Stain</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">D282</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Nutrient Broth</td>
			<td style="width:156px;">Difco</td>
			<td style="width:197px;">233000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freeze-dried E. coli O157:H7 pellet</td>
			<td style="width:156px;">ATCC</td>
			<td style="width:197px;">700728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Magnetic Relaxomteter&#160;</td>
			<td style="width:156px;">Bruker</td>
			<td style="width:197px;">mq20</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Zetasizer</td>
			<td style="width:156px;">Malvern</td>
			<td style="width:197px;">NANO-ZS90</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Plate Reader&#160;</td>
			<td style="width:156px;">Tecan</td>
			<td style="width:197px;">Infinite M200 PRO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Magnetic Column&#160;</td>
			<td style="width:156px;">QuadroMACS</td>
			<td style="width:197px;">130-090-976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Centrifuge</td>
			<td style="width:156px;">Eppendorf</td>
			<td style="width:197px;">5804 Series</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Centrifuge (accuSpin Micro 17)</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">13-100-676</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Floor Model Shaking Incubator</td>
			<td style="width:156px;">SHEL LAB</td>
			<td style="width:197px;">SSI5</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Analytical Balance</td>
			<td style="width:156px;">Metler Toledo</td>
			<td style="width:197px;">ME104E</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Digital Vortex Mixer</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">02-215-370</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Open-Air Rocking Shaker</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">02-217-765</td>
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		</tr>
	</tbody>
</table>

foodborne pathogen screening using magneto-fluorescent nanosensor: rapid detection of e. coli o157:h7

Enterohemorrhagic Escherichia coli O157: H7-var bağlı su bazlı hem de ve gıda kaynaklı hastalıklar ve bir tehdit rağmen gıda ve su filtreleme yöntemleri kullanılan şu anda kalır. Polimeraz zincir tepkimesi (PCR) ve enzim bağlı immunosorbent deneyleri (ELISA) özellikle patojenik kirleticiler, algılayabilir gibi konvansiyonel bakteri algılama yöntemleri, bunlar geniş numune hazırlama ve uzun bekleme süreleri gerektirir. Ayrıca, bu uygulamaları talep gelişmiş Laboratuvar Cihazları ve ayarları ve eğitimli profesyoneller tarafından yürütülmelidir. Burada, bir protokol manyetik ve floresan parametreleri nanoparçacık tabanlı bir platform, eşsiz kombinasyonu özellikleri basit bir tanı tekniği için önerilmiştir. Önerilen multiparametric Manyeto-floresan nanosensors (MFnS) E. coli O157: H7 kirlenme olduğu kadar az 1 adet az 1 h çözümünüzde mevcut koloni oluşturan ile algılayabilir. Ayrıca, MFnS gibi karmaşık medyada son derece işlevsel kalır yeteneği süt ve göl su doğrulanmadı. Ek özgüllük deneyleri de MFnS yalnızca belirli hedef bakteri, hatta huzurunda benzer bakteriyel tür algılama yeteneğini göstermek için kullanılmıştır. Manyetik ve floresan yöntemleri eşleştirme algılama ve patojen bulaşma her iki erken ve geç-aşamalı kirlenme algılama içinde yüksek performans sergileyen konsantrasyonları, geniş bir alanda miktar sağlar. Etkinliği, uygun fiyatta ve MFnS taşınabilirliği için bakım noktası tarama bakteriyel kirletici için ideal bir aday ayarları, geniş bir ticari olarak paketlenmiş gıda su depoları dan onları.

MFnS etki mekanizması şekil 1' de temsil edilir. MFnS bakteriyel kirletici yüzeyi kümeleme MFnS magnetic cores ve çevresindeki hidrojen çekirdekleri arasındaki etkileşimler etkilemektedir. Bu kümeleme, manyetik gevşeme sonucu olarak değerlerini artırmak. Bakteriyel kirletici konsantrasyonu arttıkça, kümeleme azaltır ve T2 değerleri değişikliği azalır. Bu nedenle, bir floresan modality eklenmesi önemlidir. Bakteri konsantrasyonu arttıkça, her iki düşük ve yüksek ko...

Watch this Scientific Journal Video about Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7 at JoVE.com

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Hızlı algılama özellikle patojenik bakterilerin manyetik gevşeme ve floresan emisyon modaliteleri kombinasyonu ile hedef ve bu protokol genel amacı, düşük maliyetli, taşınabilir için fonksiyonel nanosensors sentez etmektir.

Gıda kaynaklı patojen tarama kullanarak Manyeto-floresan Nanosensor: E. Coli O157: H7 hızlı tespiti

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

foodborne-pathogen-screening-using-magneto-fluorescent-nanosensor

Research

JoVE Journal

Immunology and Infection

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

7.6K Görüntüleme.  Pittsburg State University. Hızlı algılama özellikle patojenik bakterilerin manyetik gevşeme ve floresan emisyon modaliteleri kombinasyonu ile hedef ve bu protokol genel amacı, düşük maliyetli, taşınabilir için fonksiyonel nanosensors sentez etmektir.

Video: Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

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

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

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

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

Yapışkan bant tabanlı Örnekleme ve Floresans kombinasyonu In situ</emHızlı Algılama> Hibridizasyon Salmonella Taze üretin

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

İmmünoloji ve Enfeksiyon

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Hücre-kaderi ardından<em&gt; E. coli</em&gt; Faj Lambda tarafından Enfeksiyon Sonrası

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Fungal Patojen Fagositoz Canlı hücre video Mikroskopi

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV ters transkriptaz ve integraz inhibitörleri hızlı taranması

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Türler özgü Algılama Loop-aracılı İzotermal Amplifikasyon (LAMP) Tahliller<em&gt; Eimeria</em&gt; Tavuklar bulaştırmak ki

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Hızlı moleküler algılama ve Nezle Virüsleri A ve B farklılaşması

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Bir yoğun Transposon ekleme kütüphane kullanarak bakteriyel konjugasyon Escherichia Coli Enterobacterial suşları gibi veya Shigella flexneri oluşturulması

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

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

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

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

Tam kan örneklerinde virüs tespiti için hızlı, güvenli ve basit el ile başucu nükleik asit çıkarma

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

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Salmonella spp. testi ile taranması için yüksek üretilen iş platformu /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Gıda kaynaklı patojen tarama kullanarak Manyeto-floresan Nanosensor: E. Coli O157: H7 hızlı tespiti

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