Bu araştırma Deniz Araştırma Merkezi, araştırma iş birimi 6000.RAD1.J.A0310 tarafından desteklenmiştir. Bu makalede ifade edilen görüşler yazarlara aittir ve ille de Deniz Kuvvetleri, Savunma Bakanlığı, ya da ABD Hükümeti Bölümü resmi politikasını ya da yansıtmaz. Wei-Mei Ching ABD Hükümetinin bir çalışanıdır. Bu eser onun resmi görevlerinin bir parçası olarak hazırlanmıştır. Başlık 17 USC §105 &#39;bu başlık altında Telif hakkı koruması Birleşik Devletler Hükümeti&#39;nin herhangi bir iş için mevcut değildir.&#39; Öngörmektedir Başlık 17 USC §101 o kişinin resmi görevlerinin bir parçası olarak askerlik üyesi veya ABD Hükümeti çalışanı tarafından hazırlanan bir eser olarak bir ABD Hükümeti işi tanımlar.

1. LAMBA Reaksiyon Standardı olarak plazmid DNA dilüsyonları hazırlayın <ol><li> DNA konsantrasyonu plazma (C burnetii RSA 493 ihtiva eden hedef gen IS1111a) belirlenmesi için, 260 nm&#39;de optik yoğunluk (OD) ölçmek için bir spektrofotometre kullanarak. 1 OD 260 DNA 50 ug / ul eşittir. </li><li> kopya sayısında plazmit DNA miktarını dönüştürün. Plazmid büyüklüğü 6330 bp olduğu için, bu plazmid moleküler ağırlığı 4.1 x 10 6 g (6.330 bp&#39;lik bir X 649 g / BP) &#39;dir. (Adım 1.1 belirlenir) plazmidinin DNA konsantrasyonu 34.2 ng / ml. Bu DNA, örnek 1 ul DNA, 5 x 10 9 kopya (34.2 x 10 -9 g / 4.1 x 10 6 gx 6.02 x 10 23) denk, plasmid DNA 34.2 ng içerir. </li><li> 5 x 10 8 kopya DNA örneği yapmak için bir 10-kat seyreltme için su 90 ul plasmid DNA (5 x 10 9 kopya / ml) 10 ul ekle / ml. </li><li> Tekrar adım 1.3, 5 x 10 7 DNA numuneleri hazırlamak için 5 x 10 6, 5 x 10 5, 5 x 10 4, 5 x 10 3, 5 x 10 2, 5 x 10 1 ve 5 x 10 0 kopya / ul. </li></ol> 2. LAMBA Reaksiyon için Örneklerden DNA şablonu hazırlayın <ol><li> imalatçının protokolüne göre, ticari bir DNA mini kiti kullanılarak insan plazma örneklerinden DNA izolasyonu. çıkarılması için 200 ul örnek bir toplam kullanım ve 20 ul hacim içinde ekstre DNA Zehir. </li></ol> 3. 2x LAMP reaksiyonu tamponu hazırlayın <ol><li> 10x Pol tampon 200 ul, 5 M Trimethylglycine 320 ul, E MgSO 4, dNTP karışımı 280 ul (10 mm) ve suyun 188 ul 2x LAMBASI 1 ml tampon olmak için 1 12 ul karıştırın. 10 saniye boyunca darbe vorteks ile karıştırın. </li></ol> 4. Standart LAMBA Reaksiyonu gerçekleştirin <ol><li> 2x LA 12.5 ul karıştırınAşama 3&#39;te hazırlanan MP tamponu (40 mM Tris-HCI (pH 8.8), 20 mM KCI, 16 mM MgSO 4, 20 mM (NH4) 2 SO4,% 0.2 Triton X-100, 1.6 M Trimethylglycine, 1.4 mM dNTP karışımı), primer karışımının 1.2 ul, Bst DNA polimerazı (8 U / ul) 1 ul ve 0.2 ml PCR tüpü içinde, reaksiyon karışımı (20 ul) yapmak için su 5.3 ul. </li><li> 20 ul reaksiyon karışımına 5 (aşama 1 ya da 2) DNA ul ekleyin ve iyice karıştırın. </li><li> Yakın boru ve bir su banyosu ya da ısıtma bloğu içinde 60 dakika süre ile, 60 ° C&#39;de inkübe edin. Bu önceden belirlenmiş optimal reaksiyon sıcaklığıdır. </li><li> Tepkimeyi sonlandırmak için 10x, jel yükleme tampon maddesi 5 ul ekle. </li><li> nükleik asit, leke interkalasyon ile boyanmış% 2 agaroz jeli reaksiyon ürünlerin yük 5 ul. </li><li> 1x TBE tamponu içinde 35 dakika boyunca 100 V&#39;ta jel üzerinde çalıştırıldı. </li><li> UV ışığı ile sonuçları görselleştirmek. </li></ol> 5. sulandırılmış ile LAMBA Reaksiyonu gerçekleştirinReaktifler <ol><li> 10x Pol Tamponu 125 ul 5 M Trimethylglycine 200 ul, 1 M MgSO 4 7.5 ul karıştırın, su 667.5 ul sulandırma tamponu 1 ml yapmak için. 10 saniye boyunca darbe vorteks ile karıştırın. </li><li> mühürlü alüminyum folyo çanta liyofilize reaktif ihtiva tüpleri çıkarın. alüminyum folyo çanta mühürlü değilse tüpleri kullanmayın veya hasarlı olup olmadığını. </li><li> Liyofilize reaktifler yeniden askıya almak için her bir tüp halinde yeniden tamponu 20 ul ekle. </li><li> Tampon yukarı ve aşağı 5 kez pipetleme karıştırın. Liyofilize reaktifler tamamen yeniden askıya alınır emin olmak için bir göz atın. beyaz toz tüp içinde kaybolur. </li><li> yeniden reaktifler (aşama 1 ya da 2) DNA şablonu 5 ul ilave edin ve iyice karıştırın. </li><li> Yakın boru ve bir su banyosu ya da ısıtma bloğu içinde 60 dakika süre ile, 60 ° C&#39;de inkübe edin. </li><li> Tepkimeyi sonlandırmak için 10x, jel yükleme tampon maddesi 5 ul ekle. </li><li> Yük reaksiyon 5 ulnükleik asit, leke interkalasyon ile boyanmış% 2 agaroz jel ürünleri. </li><li> 1x TBE tamponu içinde 35 dakika boyunca 100 V&#39;ta jel üzerinde çalıştırıldı. </li><li> UV ışığı ile sonuçları görselleştirmek. </li></ol> 6. Sulandırma Tampon HNB veya Floresan araya eklenen boyanın İçeren ile LAMBA Reaksiyonu gerçekleştirin <ol><li> 10x Pol Tamponu 125 ul 5 M Trimethylglycine 200 ul, 1 M, 7.5 ul MgSO 4, 20 mM HNB (ya da 100X flüoresan araya eklenen boyanın 12.5 ul) ve 660 ul (veya 655 ul) su için 7.5 ul karıştırın sulandırma tamponu 1 ml yapmak. 10 saniye boyunca darbe vorteks ile karıştırın. </li><li> 5.6 - adımlarını 5.2 tekrarlamak için 6. bölümde hazırlanan sulandırılmış tampon kullanın. </li><li> çıplak gözle sonuçları (HNB içeren reaksiyon) ya da bir UV ışığı (floresan interkalasyon boya içeren reaksiyon) gözünüzde canlandırın. </li></ol> 7. Tüp Tarayıcı ile gerçek zamanlı LAMBASI Reaksiyonu gerçekleştirin <ol><li> reconstitut DNA ul 5 ekleyinfloresan interkalasyon boya, yakın tüp içeren ve tüp tarayıcıya yerleştirin ed reaktifleri. </li><li> 60 ° C&#39;ye ayarlanmış inkübasyon sıcaklığı. 60 dakika boyunca 520 nm de floresan ölçülür. </li></ol>

<ol>
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Yazarlar ifşa hiçbir şey yok.

Daha önce, ekleme elemanı IS1111a 14 hedefleyen duyarlı ve özgül LAMBA testi geliştirdi. Derece C arasında, farklı türleri arasında korunduğu için IS1111 elemanı seçilmiştir burnetti ve bakteri kopya yüksek numarası (110 7) (Klee 2006). Bizim sonuçlarımız LAMP Coxiella DNA gibi kısa bir sürede kromozomal kopyasını ilişkili olabilir IS1111 elemanı, yaklaşık 25 kopya tespit edebilen gösterdi. Bu çalışmada, Bst DNA pol...

Küçük Gram-negatif bakteri Coxiella burnetii dünya çapında bir zoonoz olan Q humması etkeni olduğunu. Nedeniyle Q Ateşi dünya çapında dağıtım ve C yüksek bulaşıcılıklarına burnetii, denizaşırı dağıtılan ABD askeri ve sivil personel endemik bölgelerde enfekte olma riski vardır. akut ve kronik enfeksiyonların: Q humması iki şekilde tezahür eder. Akut Q-humması grip benzeri semptomlar, hepatit, pnömoni veya ile kendini göstermektedir ve genellikle düşük mortalite oranı kendini sınırlayan bir hastalıktır. Kronik Q-humması, daha az yaygın olmakla birlikte, genellikle 1,2 tedavi edilmediği takdirde çok daha yüksek bir ölüm oranına sahip endokardit ile sonuçlanır. Bu nedenle erken tanı uygun bir tedavi hasta bakımı için önemlidir rehberlik. Polimeraz zincir reaksiyonu (PCR) ve kantitatif gerçek zamanlı PCR (qPCR) analizleri C saptanması için geliştirilmiştir hücre kültürleri ve klinik örneklerde 3-5 &lt;de burnetii DNA/ Sup&gt;. PCR ve qPCR Hem masraflı ve rutin işleri için kaynak kısıtlı alanlarda genellikle hazır değildir. Başlangıçta Notomi 6 tarafından açıklanan döngü aracılı izotermal amplifikasyon (LAMP) alternatif bir DNA çoğaltma yöntemi sunuyor. LAMBA hedef genin en az altı bağımsız bölgeleri tanıyan özel olarak tasarlanmış primer kümeleri ile birlikte sarmal yer değiştirme DNA sentezi için Bst DNA polimerazı kullanır. LAMP en önemli avantajı, amplifikasyon izotermal şartlar altında gerçekleşmesidir. Bu nedenle, yalnızca su banyosu ısıtma bloğu ya da bir inkübatör gereklidir. Bu yöntem, birkaç riketsiyal patojenleri 7-9 algılamak için kullanılır olmuştur. jel elektroforezi ile yükseltilmiş DNA ürünleri Görselleştirme nedeniyle nonspesifik amplifikasyon yanlış pozitif gelen gerçek pozitifler ayırt edebilirsiniz en doğru yöntemdir. Ancak jel elektroforezinde ilgili prosedürler kaynakları sınırlı ar için pratik değildirEAS. Birkaç alternatif yöntemler tepkime ürünlerini tespit etmek için geliştirilmiştir. Magnezyum pirofosfat oluşumu 10 veya floresan interkalasyon boya kullanılarak elde edilen bulanıklık UV ışığı 11,12 altında görüntülendi edilecek gibi bu alternatif, bir jel çalışan daha elverişli. Q humması endemik 13 olduğu tropikal ve alt tropikal ülkelerde ortam sıcaklığı olan 25 ° C ve 37 ° C&#39;de saklandığında LAMP reaktifleri bir ay boyunca stabil idi. Bir lamba deneyi C varlığını tespit etmek için laboratuvarda geliştirilmiştir Plazma örneklerinde 14 burnetti. Burada liyofilize reaktifler lamba Reaksiyon karışımının hazırlanması için bir protokol açıklamaktadır. Oda sıcaklığında muhafaza edildiğinde liyofilize reaktif bir ay boyunca stabildir. Kolay bir görselleştirme yöntemi ile kombine edildiğinde, bu C tespit için kullanılacak ideal bir yöntemdir Bir kaynak sınırlı bir ortamda burnetti.

<table border="0" cellpadding="0" cellspacing="0" width="773">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">LAMP primers</td>
			<td style="width:188px;">Eurofins MWG operon</td>
			<td style="width:131px;">10 nmol, salt free</td>
			<td style="width:240px;">Sequence designed by customer</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Bst DNA polymerase</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:131px;">M0275L</td>
			<td style="width:240px;">8 units/ml</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">10X Thermo pol buffer</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:131px;">B9004S</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">dNTP mixture</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:131px;">N0447L</td>
			<td style="width:240px;">10 mM each</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Betaine</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:131px;">B0300</td>
			<td style="width:240px;">5 M, Trimethylglycine</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Magnesium Sulfate</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:131px;">M3409-1ML</td>
			<td style="width:240px;">1 M</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">10X Bluejuice</td>
			<td style="width:188px;">Invitrogen</td>
			<td style="width:131px;">10816-015</td>
			<td style="width:240px;">gel loading buffer</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">SYBR green</td>
			<td style="width:188px;">Invitrogen</td>
			<td style="width:131px;">S7585</td>
			<td style="width:240px;">10,000X, visualize products in tubes</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">GelRed</td>
			<td style="width:188px;">Phenix Research Products</td>
			<td style="width:131px;">RGB-4103</td>
			<td style="width:240px;">10,000X, visualize products in gels</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Lyophilized reagents</td>
			<td style="width:188px;">Gene Reach</td>
			<td style="width:131px;"></td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Hydroxynaphthol blue</td>
			<td style="width:188px;">Fluka</td>
			<td style="width:131px;">33936-10G</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">ESEQuant tube scanner</td>
			<td style="width:188px;">Qiagen</td>
			<td style="width:131px;"></td>
			<td style="width:240px;">real-time detection</td>
		</tr>
	</tbody>
</table>

the development of lyophilized loop-mediated isothermal amplification reagents for the detection of coxiella burnetii

Coxiella burnetii, Q ateşi etkenidir, zorunlu hücre içi bakteridir. PCR bazlı teşhis deneyleri C saptanması için geliştirilmiştir Hücre kültürleri ve klinik örneklerde burnetii&#39;nin DNA. PCR özel ekipman ve kapsamlı son kullanıcı eğitimi gerektirir ve bu nedenle, özellikle kaynak kısıtlı bir alanda rutin işleri için uygun değildir. Bunun C varlığını tespit etmek için bir döngü aracılı izotermal amplifikasyon (LAMP) deneyi geliştirdik hasta numunelerinde burnetti. Bu yöntem, bir su banyosu ya da ısıtma bloğu 60 ° C civarında tek bir sıcaklıkta gerçekleştirilir. Bu lamba tahlilin duyarlılığı reaksiyon başına yaklaşık 25 kopya, algılama sınırı ile PCR için çok benzer. Bu rapor, liyofilize reaktifler ve hydroxynaphthol mavisi (HNB) veya reaksiyon floresan interkalasyon boya ile UV lambası kullanılarak sonuçlar görselleştirmelerine reaksiyon hazırlanmasını tarif etmektedir. LAMP reaktifler lyophili edildized ve algılama hassasiyeti kaybı olmadan bir ay süreyle oda sıcaklığında (RT) saklandı. Reaksiyon karışımı hazırlama karmaşık adımlar içermez, çünkü bu lamba deneyi özellikle sağlamdır. Bu yöntem, Q humması endemik kaynak sınırlı ortamlarda kullanım için idealdir.

0.2 ml tüp içinde liyofilize LAMP reaktifler Bst DNA polimeraz, astar ve dNTP içerir. Sulandırma tamponu 20 ul yeniden askıya liyofilize reaktifler kullanılır. 1 taze hazırlanmış reaktifler ve liyofilize reaktiflerle agaroz jelleri üzerinde LAMP reaksiyonu sonucu göstermektedir. Liyofilize reaktif aktivitesini azaltmaz. DNA şablonu. Şekil 2&#39;nin 25 kopya tespit her iki reaktiflerle hazırlanan LAMP reaksiyonlar?...

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The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

tespiti için Liyofilize Döngü aracılı İzotermal Amplifikasyon Reaktifler Gelişimi<em&gt; Coxiella burnetii</em

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

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Immunology and Infection

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

10.4K Görüntüleme.  Naval Medical Research Center. We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Video: The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Vero Hücre Kültür Testi ile Alan toplanan Sivrisinekler Enfeksiyon Virüs Algılama

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

İmmünoloji ve Enfeksiyon

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

FDA onaylı Vero hücrelerinde Aşı Geliştirme için bir araya getiren Arenavirus üretimi

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

Hematopoetik progenitörlerin Evlatlık Transferi kullanarak Peter Parçacıklannda Murin plazmasitoid dendritik hücreleri Gelişimini değerlendirilmesi

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

Batı Nil Virüsü Mutant Soy Enfeksiyon MyD88-aracılı Hücresel Bağışıklık Tepki Vitro Analizi

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Bir Geliştirilmesi<em&gt; In vitro</emEtkileşimini incelemek için&gt; bir model sistem<em&gt; Equus</em&gt;<em&gt; Caballus</emYüksek afiniteli reseptör FcεRI&gt; ile IgE

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

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 ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Üretim ve Çok fenotipik Yüksek içerik Eleme<em&gt; Coxiella burnetii</em&gt; Transposon Mutantlar

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Nitel ve Nicel Saptanması için Tahliller ve Protein Antimikrobiyal karakterizasyonu

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Uygulanması Floresan Rezonans Enerji Transferi (FRET) tarafından Efektör Translokasyon Verimliliği incelemek için<em&gt; Coxiella burnetii</em&gt; SiRNA susturma sırasında

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Tespiti için daha duyarlı ve özgül Chromogenic Agar Orta Gelişimi<em&gt; Vibrio parahaemolyticus</em&gt; Ve Diğer<em&gt; Vibrio</em&gt; Türler

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

tespiti için Liyofilize Döngü aracılı İzotermal Amplifikasyon Reaktifler Gelişimi

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