The work carried out in this study was supported in part by the Royal Veterinary College through the student research projects fund, as well as the Biotechnology and Biological Sciences Research Council and the Department for International Development (grant number BB/H009337/2). This manuscript has been assigned the reference PPB_00795 by the RVC.

1. Şablon hazırlama Not: tavuk enfekte yedi Eımerıa türü birinden türetilen DNA ihtiva ettiğinden şüphelenilen herhangi bir genomik DNA şablon lamba tabanlı Eimeria türleri tanımlanması için bir şablon olarak kullanılabilir. Burada açıklandığı gibi alan teşhis analizi için bağırsak doku örnekleri rutin otopsi sırasında alınmalıdır. <ol><li> Bağırsak bölümünde (veya bölümler) seçin test edilecek. Örnek site seçimi için bir rehber ve örnekleme siteleri dağıtımı ve konumu türe özgü aralığı için 18, mevcut ve Şekil 1 olması en muhtemel Eimeria türleri için Tablo 1&#39;e bakınız. NOT: Eimeria özellikle ev sahibi sitesi özeldir çünkü bu anahtar öneme sahiptir. Tavuk bozar Her türün bu 19 hedef bağırsak bölgeye göre tanımlanır. Karar bir veya mo önceki çiftlik deneyimi, ilgi etkilenebilirBelirli Eimeria türlerinin ya da diğer tanısal göstergelere 6 yeniden. </li><li> Tüketim 5 cm veya daha Eimeria steril makas veya bir bisturi bıçağını kullanarak test etmek için seçilen bağırsak bölüm (ler) in daha uzunlukları. İsteğe bağlı olarak, örneğin RNAlater 18 ya da% 95 etanol içinde, örneğin bir sabitleyici olarak bir sonraki analiz için numune depolamak. Not: etanol numune depolamak steril Tris-etilendiamintetraasetik asit iyice yıkanmalıdır (TE), kullanımdan önce tampon. </li><li> Uzunlamasına açık örnek kesin ücretsiz steril cam mikroskop lamı kenarına ya da bir etanol / alev sterilize makas bıçak kullanarak mukoza tabakasının hücreleri en bağırsak içeriği kaldırmak (varsa) ve kazıyın. İsteğe bağlı olarak, bir araya getirilmiş numune için tek bir tüp içerisinde türe özgü bağırsak tüm siteleri dört gelen hücreler bulunmaktadır. </li><li> 1 de dahil olmak üzere 100 ul steril TE tamponu ihtiva eden bir steril 1.5 ml&#39;lik vidalı kapaklı mikrosantrifüj tüpü içine kazınmış malzemenin koyun0% (ağırlık / hacim) Chelex® 100 reçinesi elde edildi. </li><li> 1 dakika boyunca kuvvetli bir şekilde her bir örnek çalkalayın. Vida üst sıkıca kapalı olduğundan emin olun ve daha sonra 10 dakika boyunca bir kaynar su banyosunda inkübe. </li><li> Kaynatıldıktan sonra her bir numune 1-2 dakika süre ile oda sıcaklığına soğumaya bırakın. </li><li> Santrifüj 1 dakika en yüksek hızda (örneğin, ~ 10.000 xg) mikrosantrifüj kullanarak her örnek. </li><li> Her Lamba tayininde şablon gerçekleştirilecek olmak Elde edilen üstte 2 ul toplayın. İsteğe bağlı olarak, çok-taraflı bir deney temin etmek üzere tek bir tüp içerisinde birden fazla bağırsak sitesi havuz. </li></ol> 2. Eimeria LAMBA Astar Hazırlama (Ön-deneyi) <ol><li> 100 tahliller için yeterli Eimeria LAMBA astar stokları hazırlayın: <ol><li> (Üretici tarafından belirtildiği gibi), 100 uM bir konsantrasyona kadar moleküler sınıf su ilave edilerek her bir liyofilize Eimeria lamba primeri sulandırın. Belirtilen değilse, moleküler sınıf su gerekli usin hacmini hesaplamakg her bir primerden fiziksel ve moleküler ağırlıkları. </li><li> Her Eimeria türleri için ayrı 0.5 ml flip-top mikrosantrifüj tüp içine Pipet 60 ul moleküler sınıf su tahlil edilecek. </li><li> Ekleme primerleri FIP BIP F3, B3, yedi türe özgü primer karışımları, bir dizi oluşturma Tablo 2&#39;de gösterilen miktarlar kullanılarak su hedef Eımerıa türü özel LF ve LB. </li><li> Kısaca vorteks sonra, astar çözüm karıştırmak mikrofuge&#39;de darbe ve gerekli kadar dondurma. </li></ol></li><li> Her Eimeria türleri tahlil edilmesi için bir lamba reaksiyon MasterMix hazırlayın. Örneklerin sayısına göre Tablo 3&#39;te gösterilen miktarlar çarpın ve bir pozitif kontrol, bir negatif kontrol ve pipetleme yedek için üç ekleyin. 0.5 veya 1.5 ml pipet flip-top mikrosantrifüj tüp. </li></ol> 3. Eimeria LAMBA Testi <ol><li> Pipet 23 ul Eimeria türe özgü Bst DNA polimeraz / LAMP bir 0&#39;a içine MASTERMIX.5 ml mikrosantrifüj tüpü. </li><li> 25 ul bir son reaksiyon hacmini hale (Bölüm 1 &#39;de hazırlandı) 2 ul DNA şablonu ekleyin. </li><li> Bir reaksiyonda (pozitif kontrol) 2 ul Eimeria türe özel genomik DNA ekleyin. Reaksiyonun (negatif kontrol) 2 ul moleküler sınıf su ilave edilir. NOT: Bu tür özel genomik DNA, mevcut değilse, bir önceki pozitif lamba ya da standart PCR ürünü, bunun yerine kullanılabilir. </li><li> 30 dakika boyunca 62 C &#39;de bir su banyosunda ya da ısıtma bloğu içinde inkübe edin. Reaksiyon hemen okumak olacak değilse İsteğe bağlı, 10 dakika boyunca 80 o C ısıtılarak Bst DNA polimeraz de-aktive. </li></ol> 4. LAMP Testi Okuma-out <ol><li> Inkübasyon bitiminde kapalı ışık altında gözle her reaksiyonun rengini değerlendirmek. Negatif sonuçlar, olumlu sonuçlar gökyüzü 20 mavi görünür renkli menekşe pembe görünür. </li><li> İsteğe bağlı olarak, bir Laborat içinde lamba tahlil sonucunu teyit1 x Trıs / borat / EDTA (TBE) tampon maddesi içinde bir% 2 agaroz jel kullanılarak Agaroz jel elektroforez için 1 ul DNA, jel yükleme tampon maddesi ile 5 ul LAMP reaksiyonu ürünü karıştırarak Ory (5 ul başına bir nükleik asit leke kullanılarak önceden boyanmış 50 mi agaroz). Fragman boyutu hesaplama izin jel 1 şeritli bir 1Kb moleküler boyutta DNA merdiveni 5 ul ekleyin. </li></ol>

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The authors declare that they have no competing financial interests.

The Eimeria species-specific LAMP assays described in this paper offer a new diagnostic tool kit in support of effective control of coccidia and the disease coccidiosis. The outcomes of eimerian infection can include severe economic loss as well as seriously compromised bird welfare and increased susceptibility to colonisation by zoonotic pathogens21. Opportunities to monitor flocks for the occurrence of some, or all Eimeria species can provide early warning of a breakdown in anticoccidial control efficacy. Key advantages of LAMP include robust target specificity, resulting from the requirement for six different DNA sequence targets, as well as high sensitivity, boosted by the inclusion of loop primers13, although the qualitative, not quantitative nature of LAMP may be considered a limitation. It is not currently possible to discriminate low level parasite escape from routine chemoprophylaxis or live vaccine replication from unchecked eimerian replication. Nonetheless, the technical ease of the protocol and definitive readout offers considerable improvement over the existing specialist and frequently subjective pathology- and morphology-led approaches6,7. Each assay may be completed at a cost of ~&#163;0.75 per sample, independent of labour and equipment set up expenses. Thus, LAMP assays are also more cost effective than other molecular diagnostics such as PCR, since they require an isothermal incubation with no need for specialist equipment. 
For many years access to Eimeria genomic DNA as template has limited the development and application of molecular field diagnostics. The oocyst is the most readily accessible phase of the eimerian lifecycle, but routine DNA extraction requires laboratory facilities22. Other, more labile intestinal lifecycle stages require purification prior to DNA preparation to prevent PCR inhibition and a consequent loss of sensitivity23,24. The ability to extract eimerian DNA of a quality suitable for LAMP using equipment no more specialised than a microcentrifuge and a water bath, supplemented by inhibitor adsorption using chelex resin, now promotes the wider use of molecular biology in eimerian diagnostics. Intriguingly, the reported detection of quantitative PCR-measurable Eimeria DNA in intestinal tissue 20 days after the initiation of parasite infection, 11 days after the last detectable oocyst output, raises the suggestion that LAMP may be used to detect resolved parasite exposure as well as ongoing infection, even after any visible lesions may have been resolved25. 
The relatively low cost and low technical requirements of LAMP Eimeria diagnostics can promote their application in the developing world where other more established approaches may not be available or appropriate. For this to be applicable each assay must be capable of detecting all strains which may be circulating within each region. While understanding of the genetic diversity prevailing among Eimeria species is limited26, the use of target sequences previously validated for use in quantitative PCR with strains from Africa, Asia, Europe and South America provides some evidence of conservation, supporting the utility of these LAMP assays around the world10.

Küresel tavuk üretimi gelişmekte olan dünyada gelişmiş ülkelerde tanık yaklaşık dört kat genişleme barındırma on kat üzerinde son 50 yıldır arttı <a href="http://www.faostat.org">(www.faostat.org)</a> . Dünya gıda güvenliği tavuk üretiminin önemi büyüdü kadar çok tavuklarda ciddi hastalığa neden olabilir patojenlerin profiline sahiptir. Bir örnektir enterik hastalık koksidiyoz 1 neden olabilir Eimeria türleri, her yerde protozoon parazitler vardır. Tavuk yetiştirilen her yerde, bir ya da daha fazla Eimeria türleri 2-4 yaygın olması muhtemeldir. Gelişmiş dünyada Eimeria öncelikle direnç 5 etkisini en aza indirmek için servis veya rotasyon programları kullanılarak, kemoprofilaksi tarafından kontrol edilir. Canlı aşılar da kuş değeri elde etme maliyeti haklı göstermek için yeterli olduğu sistemlerde kullanılır (örneğin, stok, katmanları ve bazı piliç 5 damızlık). Bir resu olarakKlinik coccidiosis genellikle iyi kontrol edilir, bu önlemlerin lt, sub-klinik enfeksiyon 5 yaygın olmasına rağmen. Gelişen dünya aşılama nadirdir ve ilaç uygulaması sık sık az iyi bilgilendirilmiş. Sonuç olarak-sub klinik ve klinik coccidiosis daha yaygındır ve önemli bir ekonomik etkisi 3 uygular. En yaygın olarak kullanılan skorlama sistemi bile yazarlar, bazı türler için &quot;böyle bir prosedür herhangi fakat orta şiddetli enfeksiyonlarda denenmelidir şüphelidir görünüyor&quot; yorumunu rağmen eimerian enfeksiyon tanısı geleneksel, otopsiyi puanlama lezyon dayandığı 6. Morfolojisi örtüşen uzman 6,7 ama bulandırabilir rağmen Ek delil, dışkı veya çöp örneklerinde çevre dirençli Ookist yaşam döngüsü aşamasında mikroskobik algılama yoluyla elde edilebilir. Polimeraz zincir reaksiyonu kullanılarak moleküler alternatifler (PCR) kullanılarak, rasgele amplificatiopolimorfik DNA PCR (RAPD-PCR) ve kantitatif PCR teknolojileri n 20 yıla kadar 8-10 mevcut olmuştur, ancak bugüne kadar popüler hale başarısız olmuştur. Bağıl gider ve uzman laboratuar ekipmanları veya işleme gereksinimi genellikle subjektif ve teknik olarak zor yaşlı pathology- doğası ve mikroskopi tabanlı 10,11 yaklaşımlar rağmen, kendi alımını sınırlıdır. Bu tür sınırlamalar yoksulluk üzerindeki koksidiyoz etkisi 12 orantılı büyük olabilir gibi Güney Doğu Asya gibi dünyanın yoksul bölgelerinde, birçok abartılı olabilir. Buna karşılık, Eimeria türe özgü teşhis deneyleri, yeni basit ve hassas, ancak maliyeti etkin bir açık bir talep var. Döngü aracılı izotermal amplifikasyon (LAMP), büyük bir DNA miktarlarda büyütme yeteneğine sahip bir DNA polimerazı güdümlü bir yöntem hazırlamak çok kolaydır. En önemlisi, LAMP bir Bst DNA polymera kullanırse yerine termal çevrim 13,14 için gerek olmaksızın, tek bir sabit sıcaklıkta, DNA amplifikasyonu kolaylaştıran genel olarak PCR&#39;de kullanılan Taq DNA polimerazı. LAMBA bile en ilkel laboratuvarda veya sahada uygulama için uygun olabilir. Birçok PCR inhibitörleri, yüksek duyarlılık ve özgünlüğü göre direnç ile karakterize, LAMP deneyleri enfeksiyöz bursa hastalığı virüsü, Clostridium perfringens ve Cryptosporidium 15-17 olmak üzere patojenlerin geniş bir yelpazede geliştirilmiştir. Buna karşılık tavuk enfekte yedi Eimeria türlerinin her özel LAMP tahliller panel 18 geliştirilmiştir yeni maliyet-etkin Eimeria türe özgü teşhis için talep. Yeni tahliller için başvurular böyle kötü ekonomik pe ile Eimeria maxima veya Eimeria necatrix gibi türlerin derneği verilen belirli değerin izlenmesi parazit oluşumunu içerir3,4 rformance. Diğer uygulamalar çiftlik antikoksidiyal stratejisi, sub-klinik enfeksiyon veya klinik hastalık ve bir çiftlik Eimeria yarattığı riskin değerlendirilmesi tanı etkinliğini değerlendirmek içerir.

<table border="0" cellpadding="0" cellspacing="0" width="889">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Name</td>
			<td style="width:161px;">Company</td>
			<td style="width:139px;">Catalogue number</td>
			<td style="width:295px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">RNAlater</td>
			<td style="width:161px;">Ambion</td>
			<td style="width:139px;">AM7024</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Ethanol</td>
			<td style="width:161px;">VWR Chemicals</td>
			<td style="width:139px;">20821.321</td>
			<td style="width:295px;">Caution, highly flammable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">100 x Tris-EDTA (TE) buffer concentrate</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">T9285</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Chelex 100 resin</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:139px;">142-1253</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Molecular grade water</td>
			<td style="width:161px;">Invitrogen</td>
			<td style="width:139px;">10977035</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. acervulina F3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CCTAACATTTCGCTTCACGGAC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. acervulina B3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*ATGAGCAAGTGGAACACCTTG</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. acervulina FIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*AGAGCACAGTGGCAGTGC-AGCAGACAGCATGGCTTACCT</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. acervulina BIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GAAGACCCTCTGAAGAACGGA-CCTTCTCACCGCTTACCGG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. acervulina LB</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*TAAGGTTACACCCGTGGAGG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. acervulina LF</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GCCATGCACAAAGCGACTT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. brunetti F3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GGCCATCAAGTTCCATGAGC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. brunetti B3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*TCAACCTCCTGAGTGTGGTT</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. brunetti FIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GAAAATGCCTTCGTAGCTGCT-GCTGGGTACGGAGCGTCTT</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. brunetti BIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*TACTTCCTAGGATCCATCCTCGC-AGTTTCGCTGCCGCCTC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. brunetti LB</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GAAACGCTCGAACATGGC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. brunetti LF</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CTTCTCCACAGACCCAGAGGT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. maxima F3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*ACTACGGAAAAGTGCGTAGCT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. maxima B3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CCTTCCTCCCTTCTGAAAACTG</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. maxima FIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GAGTCACTGCTGATGTACCAAA 
			AG-GAACTATGCCGCTTTCCCCTG</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. maxima BIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*AGAATGCGGATTTGTTAGCAGC-AGCAAGTCCAAGGTGTGTGTA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. maxima LB</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CAAGCCTACGCGGACATC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. maxima LF</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*TTATGCAGCTGGGTCAACG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. mitis F3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*ACGATAGCCAAGACACGTAAGG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. mitis B3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CCCCGTGATAAGAGTAGGAACA</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. mitis FIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CGCGGGTCGTGAGATTTAAATT 
			AT-GGAAGATCAGGACGGGCACT</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. mitis BIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GTTTCAGTTGATGAACAAGCGA 
			GA-TGCGCCTCTAGAATCAAGACG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. mitis LB</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*TCCATGCATCCCCTTGTT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. mitis LF</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CGTGGGCACAGATTGATTC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. necatrix F3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*TGGCTTTCCCGCGTACC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. necatrix B3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CGGCCCAACACAAAGACTG</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. necatrix FIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CGCTTGAGTTTTAAGCTATGCA 
			CA-GACCCAAGCAGCTCACCAA</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. necatrix BIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CGCCATGCCATTCAATGAACG-*GAGGCATACCGGCGTTGTC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. necatrix LB</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GTCTGTAACTTGGGACGTTGT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. necatrix LF</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GAACAGCCGGAGCCTCTC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. praecox F3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GCCCTTGTATGTTGCTGTTTCT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. praecox B3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GCGCACGAATCTGAATCACAC</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. praecox FIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*ATCTCCTCAAAGACTTTCGCGT 
			A-GCGCTTGGCTATATCCATAGG</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. praecox BIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GCTCTCGTGGCATACTTGC-GCCAGGAGCCACTGATTGT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. praecox LB</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GAATAGCATTGCCAGGTGG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. praecox LF</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GTCCACTGTCATTAATATTGC 
			TGC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. tenella F3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GCTTGTGAAGGTCAGCGTG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. tenella B3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GCTGAGTCCATACGTACTTCCT</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. tenella FIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GCCACTGCTATGGAAAGTCAC 
			AC-CATAACTGGCATGCAGGGGT</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. tenella BIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GTTTGGCCCGAAAGTTGTGAA 
			GA-CGTCAGAAATTGCTGCCCAAT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. tenella LB</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CGCATGTGCAGTTGAAGACA</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. tenella LF</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CCAAATGTATCTGCTAGTTATA 
			TTAACAAG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">10 x ThermoPol reaction buffer</td>
			<td style="width:161px;">New England Biolabs</td>
			<td style="width:139px;">B9004S</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:295px;">MgSO4</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">M7506</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">dNTPs</td>
			<td style="width:161px;">Promega</td>
			<td style="width:139px;">U1330</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Betaine solution (5 M)</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">B0300</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Bst polymerase</td>
			<td style="width:161px;">New England Biolabs</td>
			<td style="width:139px;">M0275S</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Hydroxynaphthol blue</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">33936</td>
			<td style="width:295px;">Dissolved in molecular grade water.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">UltraPure agarose</td>
			<td style="width:161px;">Invitrogen</td>
			<td style="width:139px;">16500-500</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">10 x Tris/Borate/EDTA (TBE) buffer</td>
			<td style="width:161px;">Invitrogen</td>
			<td style="width:139px;">AM9863</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Blue/Orange DNA loading dye (x6)</td>
			<td style="width:161px;">Promega</td>
			<td style="width:139px;">G1881</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">GeneRuler 1Kb DNA ladder</td>
			<td style="width:161px;">Thermo Scientific</td>
			<td style="width:139px;">SM0313</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">SafeView nucleic acid stain</td>
			<td style="width:161px;">NBS Biologicals</td>
			<td style="width:139px;">NBS-SV</td>
			<td style="width:295px;"></td>
		</tr>
	</tbody>
</table>

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.

Tahlil doğrulama Doğrulama esnasında, her Eimeria türe özgü lamba deney, bir ana kontrol olarak tavuk, hem de tavuk genomik DNA enfekte yedi Eimeria türü temsil saf DNA örnekleri oluşan bir panel kullanılarak test edilir. Agaroz jel elektroforezi her deney çözmek için kullanılan ve herhangi bir ana çapraz reaksiyon 18 mutlak türlere göre gösterilmiştir. Daha sonra, bir on misli seri seyreltme serileri genomik DNA, bir ila on genom kopyalarının 18&#39;in bir deney hassasiyet limiti ortaya saflaştırılmış Eimeria tenella kullanılarak hazırlandı. Üst sınır yok, en yüksek konsantrasyon (100.000 genom kopya) 18 olmak üzere pozitif kadar elde edilen sonuçlara ve tespit edilmiştir. Alan örnekleri ile Uygulama Eimeria testleri için alınan örnekler p bir sonucu olarak itlaf ölü bulundu tavuklardan elde edilmesi olasıdıroor sağlık ya da bir ila üç olası örneklem büyüklüğü gösteren, sentinel sağlık gözetimi için itlaf zaman rutin bir parçası. Bir gözetim programının bir parçası olarak ABD broyler çiftliği toplanan üç kuş Test bağırsak örneklerinin üç set vermiştir. Her Eimeria türleri (Tablo 1) için tercih edilen bir bağırsak sitesi öncelik, türe özgü lamba deneyleri uygulanmasını hedefleyen bir göstergesi olarak mavi hydroxynaphthol (Şekil 2A) ile test edilen tüm kuşlarda eimerian enfeksiyonun görsel tespitine olanak tanıdı. hydroxynaphthol mavi kullanırken olumsuz LAMP reaksiyonu ile elde rengi pembe mor arasında değişir, ama her zaman pozitif bir sonuç elde mavi ayrıdır yapabilirsiniz. Agaroz jel elektroforezi ile teyit benzer sonuçlar (Şekil 2B) elde edilmiştir. Alan uygulaması sırasında kullanıcı yedi türlere karşı tam ekran uygulamak için tercih, ya da öncelik yalnızca türlerin hedefleyebilirÖnemli veya bilinen çiftlikte dolaşan veya çevredeki edilecek. Eimeria oluşumu herhangi bir yeni test basitlik ihtiyacını vurgular için PCR-temelli yaklaşımların yetersizliği teşhisi olarak benimsenmesi. LAMP PCR daha basit hazırlık ve işleme sunarken, kuş başına birden fazla bağırsak sitelerini test etmek şartı kırıcı kalır. Bir veya daha fazla lamba tahlilleri ile test edilebilir kuş başına tek havuza DNA örneği, üretimi, daha çekici olması muhtemeldir. Her bağırsak Alanı ayrı ayrı işlendi olduğu gibi aynı sonucu (Şekil 2, yedi lamba deneyleri ile test edilmesi için, Tablo 1 &#39;de tarif edilen DNA hazırlanmadan önce bir araya getirilmiş özel bağırsak sitelerinin yolunun her birinden toplanan malzemesini temsil eden, kuş başına bir araya getirilmiş numune Resim işleme ) Şekil 3 ile karşılaştırıldığında. <img alt="Şekil 1," src=&quot;/files/ftp_upload/52552/52552fig1.jpg&quot; /&gt; Tavuk bulaştırmak Eimeria tür parazitlerin LAMP tespiti için Şekil 1. Bağırsak örnekleme siteleri. Her Eimeria türlerinin hedeflenen bağırsak bölgeleri E. (noktalı siyah çizgiler arasındaki sayısına göre belirtilen örnekleme tercih siteleri ile, renkli çizgilerle vurgulanır acervulina: Sarı / 1, E. brunetti: pembe / 2, E. maxima: mavi / 3, E. mitis: kırmızı / 5, E. praecox: turuncu / 4 E. necatrix yeşil / 6 ve E. tenella: gri / 7). <a href="https://www.jove.com/files/ftp_upload/52552/52552fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 2," src="/files/ftp_upload/52552/52552fig2.jpg" /> Eimerian enfeksiyon Şekil 2. LAMBA tanı froBir gök mavisi tepki olumlu oldu ve pembe reaksiyona mor negatif oldu, ve (B) agaroz jel elektroforezi burada m, üç ticari etlik tavuk. LAMBA reaksiyonları, (A) kullanarak mavi hydroxynaphthol çözüldü. örneklenmiş bağırsak siteler gibi her parazit türleri için Tablo 1&#39;de gösterilmiştir. A = E acervulina, B = E. brunetti, Ma = E. maksimum Mi = E. mitis, N = E. necatrix, p = E. praecox ve T = E. tenella. Şerit 1 GeneRuler 1Kb DNA merdiveni içeriyordu. <a href="https://www.jove.com/files/ftp_upload/52552/52552fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 3," src="/files/ftp_upload/52552/52552fig3.jpg" /> Eimerian enfeksiyon Şekil 3. LAMP tanı üç ayrı ticari etlik tavuk örnekleri toplanmış kullanarak. LAMBA REACları bir gök mavisi tepki olumlu oldu ve pembe reaksiyona menekşe negatif hydroxynaphthol mavi kullanılarak çözüldü. A = E acervulina, B = E. brunetti, Ma = E. maksimum Mi = E. mitis, N = E. necatrix, p = E. praecox ve T = E. tenella. <a href="https://www.jove.com/files/ftp_upload/52552/52552fig3large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Örnek sitesi </td><td> Eimeria türleri tahlil (büyük olasılıkla) </td></tr><tr><td> Duodenal (D) </td><td> E acervulina, E. praecox </td></tr><tr><td> Jejunum / ileum * (J / I) </td><td> E maxima, E. necatrix </td></tr><tr><td> Çekumu (C) </td><td> E. necatrix, E. tenella </td></tr><tr><td> Terminal ileum (TI) </td><td> E. brunetti, E mitis </td></tr&gt; <tr><td> Bir araya toplanan numune (P) </td><td> E acervulina, E. brunetti, E maxima, E mitis, E. necatrix, E. praecox, E tenella </td></tr></tbody></table> Aday Eimeria türleri deneyleri Tablo 1. Bağırsak bölgeye özgü bir seçim. Bölgelerin seçimi, Şekil 1 &#39;de gösterildiği gibi her bir Eımerıa turu için değişir numune için. Toplanmış örnek daha sonra DNA hazırlanması için bir araya getirilmiş dört özel sitelerde toplanan malzeme içerir. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Astar * </td><td> Hazır konsantrasyonu (uM) </td><td> Hacim (ul) </td></tr><tr><td> Su </td><td> - </td><td> 60 </td></tr><tr><td> İleri İç Astar (FIP) </td><td> 100 </td><td> 40 </td></tr><tr><td> Geriye İç Primer (BIP) </td><td> 100 </td><td> 40 </td></tr><tr><td> İleri Dış Astar (F3) </td><td> 100 </td><td> 10 </td></tr><tr><td> Geriye Dış Astar (B3) </td><td> 100 </td><td> 10 </td></tr><tr><td> Döngü İleri (LF) </td><td> 100 </td><td> 20 </td></tr><tr><td> Geriye Döngü (LB) </td><td> 100 </td><td> 20 </td></tr><tr><td> Toplam </td><td></td><td> 200 </td></tr></tbody></table> LAMP astar ön karışımın Tablo 2. Hazırlık. LAMP için bir astar ön karışım hazırlamak için gerekli bileşenleri ve oranlar. Gösterilen Ciltler 100 LAMBA reaksiyonlar içindir. * Malzeme gösterildiği gibi Astar kimlikleri Barkway ark (2011) 18. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> Stok kons n </td><td> Nihai reaksiyon kons n </td><td> Reaksiyon başına Hacim (ul) </td></tr><tr><td> DDW </td><td> - </td><td> - </td><td> 10.1 </td></tr><tr><td> ThermoPol tampon </td><td> 10 x </td><td> 1 x </td><td> 2.5 </td></tr><tr><td> MgSO 4 </td><td> 100 mM </td><td> 2 mM </td><td> 0.5 </td></tr><tr><td> Primer karışımı * </td><td colspan="2"> Tablo 2 </td><td> 2.5 </td></tr><tr><td> dNTP </td><td> 25 mM </td><td> 400 um </td><td> 0,4 </td></tr><tr><td> Betain </td><td> 5M </td><td> 1M </td><td> 5 </td></tr><tr><td> Hydroxynaphthol mavi </td><td> 3mM </td><td> 120 uM </td><td> 1 </td></tr><tr><td> Bst DNA polimerazı </td><td> 8000 U / ml </td><td> 8 U </td><td> 1 </td></tr><tr><td> Toplam </td><td></td><td></td><td> 23 </td></tr></tbody></table> Bir LAM Tablo 3. HazırlıkP reaksiyon masterkarışım. * Eimeria türe özgü.

Watch this Scientific Journal Video about Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens at JoVE.com

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 ...

loop-mediated-isothermal-amplification-lamp-assays-for-species

Research

JoVE Journal

Immunology and Infection

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

26.8K Görüntüleme.  Royal Veterinary College, London. 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.

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

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

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Standart Klinik Tahliller Algılama Sınırı Aşağıda Viral Yükler HIV enfekte kişilerde kuvvetlendirme ve HIV-1 RNA miktarının

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

Bluetongue Virüs karşı Roman Antiviraller belirlenmesi için Tahliller

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

Bulaştırmak için bir protokol<em&gt; Caenorhabditis elegans</em&gt; Ile<em&gt; Salmonella typhimurium</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Erken Viral Giriş Tespiti için Tahliller ve Antiviral Bileşiklerin Değerlendirilmesi

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

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 ...

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

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

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

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

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

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

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Vitronectin bakteriyel yapışma ve Serum direnç rolü eğitim için deneyleri

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug&#39;s pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples.
In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-na&#239;ve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

Here we present a cell-based flow cytometry method to detect neutralizing antibodies or other factors that interfere with the cellular uptake of enzyme replacement therapies in a human matrix, such as cerebral spinal fluid (CSF) or human serum.

Enzim replasman terapileri ile hücre tabanlı tahlil hücresel alımını nötralize antikorların tespiti

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The ...

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 ...