Bu çalışma, kısmen Avrupa Birliği Araştırma Çerçeve 7 finanse edilen projeler VIROBATHE (Sözleşme No: 513.648), VIROCLIME (Sözleşme No. 243.923 tarafından, İspanyol Hükümeti &quot;Ministerio de educacion y Ciencia&quot; (proje AGL2008-05275-C01/ALI) tarafından desteklenmiştir. ) ve Su Katalan Ajansı, Agencia Catalana de l&#39;Aigua (ACA), Departament de Kontrolü i Millora dels Ecosistemes Aquatics. Gelişmiş çalışma sırasında Marta Rusiñol Katalan Hükümeti &quot;AGAUR&quot; (FI-DGR) bir adam oldu.

1. Viral parçacıkların konsantrasyonu su örneklerinde mevcut <ol><li> Koleksiyon, klima ve su örnekleri <ol><li> En az 2 plastik kaplar içinde bir proses kontrolü gibi düz dipli ve bir ekstra numune örnek başına 10 L çoğaltır toplayın. Bu son örnek, bilinen bir viral parçacıkların miktarı ile çivili ve kontrol olarak kullanılan olacaktır. Not: Bu çivili numuneler için özel ayrı bir malzeme (şişe, tüp, vb.) Olması tavsiye edilir. </li><li> Conductimeter kalibrasyon kontrol edin ve gerekirse yeniden kalibre. Bir ekstra 10 L plastik konteyner yeterli iletkenliği önceden ayarlanmış musluk suyu (1.6 aşağıya bakınız) kullanarak bir negatif kontrol hazırlayın. </li><li> Çivili örnekleri için örnek kontrol virüsü standart hacmi (10 L su başına yaklaşık 10 5 genom kopya) ekleyin. Sıçrama ve aerosoller kaçınarak karıştırarak karıştırın. Pozitif kontroller adenovirüs s nadir bir zorlanma meydanaHAdV-35 veya MS2 gibi bir bakteriyofaj gibi uch. </li><li> Örnek askıya malzeme (kum veya diğer malzeme) yüksek miktarda sunuyor için tortu, 15 dakika izin verin. Yeni bir kap içine su aktarın. </li><li> 1 N HCl ilave edilerek su numunesi pH 3.5 (± 0.1) ayarlayın. Bu adım, virüs konsantrasyonu için çok önemlidir, bu nedenle pH düzgün ayarlanmış emin olun. HCl eklerken kuvvetli karıştırarak suyu iyice karıştırın. (Not: pH 3.5 eklemek 1 M NaOH daha düşük ise). </li><li> Iletkenlik ayarlayın. Örnek, 1500 μS / cm veya daha yüksek bir iletkenlik varsa, bu adım gerekli değildir. Iletkenliği 1500 μS / cm topaklanmalara malzeme formasyonu (flocs) garanti edilmemektedir daha düşükse yapay deniz tuzu (Sigma) eklenmesiyle 1500 μS / cm iletkenlik ayarlayabilirsiniz. Deniz tuzu ekleyerek karıştırmaya kuvvetlice karıştırın. </li><li> Bir de öncesi ve Klima sonra numunelerin pH kaydedinHCl hacmi kullanılır. Iletkenlik, pH ayarladıktan sonra kayıt altına alınmalıdır. Her zaman taze bir HCl çözeltisi ile pH metre ve conductimeter elektrotlar dezenfekte,% 10 sodyum tiosulphate çözüm dechlorinate ve sonunda saf su ile durulayın. </li></ol></li><li> % 1 yağsız süt-topaklanmalara öncesi hazırlanması (PSM) <ol><li> PH metre ve conductimeter kalibrasyon kontrol edin ve gerekirse yeniden kalibre. </li><li> 1 L yapay deniz suyu 10 gr yağsız süt tozu (Difco) eriterek ön topaklanmalara yağsız süt çözeltisi hazırlayın (% 1 PSM, w / v) (dechlorinated musluk suyu ve otoklav 1 L 33.3 g yapay deniz tuzu çözülür) ve dikkatle 1N HCl ile 3.5 pH ayarlanması. Flocs görünür olmalıdır. 24 saat boyunca 4 ya da mağaza olarak hemen önce çözüm ° C hazırlayın Deklorinasyonu için 1 ml, 100 ml su başına% 10 tiosulphate çözüm kullanın. </li></ol></li><li> Viral parçacıkların flokülasyon su örnekleri mevcut<ol><li> 10-L su numunesi için% 1 PSM 100 ml ekleyin. </li><li> Virüsler flocs tutunmasını sağlamak için 8-10 saat süreyle örnekleri karıştırın. 8-10 saat sonra kapanma karıştırma için bir zamanlayıcı kullanın. </li><li> Karıştırarak 8-10 saat süreyle durdurun ve yerçekimi ile flocs tortu izin </li></ol></li><li> Flocs toplanması ve yeniden-çözünür. Santrifüj <ol><li> Peristaltik bir pompa ve bir plastik tüpe bağlı plastik bir pipet kullanarak süpernatantı. Çivili örnekleri için süpernatant bir şişeye toplanır ve iç prosedürlerine göre dezenfekte. Tüm durumlarda DİKKATLİ pelet toplamak için değil. </li><li> Tortu santrifüj şişeye flocs (yaklaşık 500 ml) ile toplayın. </li><li> PSM pH 3,5 ilave edilerek tencere Dengesi. </li><li> 4, 30 dakika boyunca 8000 xg yüksek hızda santrifüj tencere Santrifüj ° C En kısa sürede santrifüj durur, santrifüj santrifüj tencere dikkatlice çıkarın. </li><li> Çok gkardiak defekt dökün ve süpernatantı atın. Bulaşıcı malzeme için uygun önlemleri izleyin. </li><li> Her santrifüj şişe pelet çözmek için 7 ml fosfat tamponu ekleyin. </li><li> Flocs çözünmüş sonra, ölçü ve toplam hacmi 10 ml ulaşmak için fosfat tamponu ekleyin. </li><li> Vorteks viral konsantre homojenize ve -80 dondurulmuş olmalıdır temiz mikrotüpler içine 10 ml dağıtmak ° C daha fazla analize ihtiyaç duyulan kadar. </li></ol></li></ol> 2. Nükleik asit ekstraksiyon <ol><li> Nükleik asit ekstraksiyon üretici talimatları izleyerek QIAamp viral RNA Mini Kit ile gerçekleştirin. Bu kit, otomatik bir platform kullanımı (örneğin Qiacube, Qiagen) sağlar. </li></ol> 3. Insan adenovirüs (HAdV), JC polyomaviruses (JCPyV), domuz adenovirüs (PAdV) ve sığır polyomaviruses Kantitatif PCR (BPyV) <ol><li> Örneklerinde genom kopya Kantitasyonu <ol><li> HazırlamakTaqMan Çevre PCR Master Mix 2x (Biyosistem Uygulamalı) kullanarak temiz bir ayrı alanda qPCR karıştırın. Reaksiyon optik yapışkanlı kapakları ile kaplı bir 96-iyi optik bir reaksiyon plaka yer alır. Master Mix konsantrasyonları, astar ve sondalar Tablo 1&#39;de açıklanmıştır. </li><li> Sonra karışımı kısım 15μl (3.2) kontrolleri de dahil olmak üzere her birine hazırlanan olmuştur. Hedef eklenmesinden sonra bir reaksiyon için toplam hacmi (15μl karışımı + 10μl örnek veya standart) 25μl olacak. </li><li> Ayrı bir alanda örnekleri (10μl) nükleik asit ekstraksiyon ekleyin. Yinelenen her numune saf su doğrudan ve on kat seyreltme çalıştırın. Kapak yapışkan bir kapak parçası örnekleri içeren kuyu kapağının diğer kısmı, sonraki adım için tutun. </li><li> 10 0 10 6 GC/10μl üç nüsha tarafından DNA standart süspansiyonlar (10μl) dilüsyonları ekleyin ve standartları için özel olarak kullanılan bir mikropipet kullanarakDard DNA. Plazmid DNA yok ve her kullanımdan sonra mikropipet temizlemek için UV radyasyonu ile donatılmış bir alanda standartları eklemek için tavsiye edilir. Kapak önceden kesilmiş yapışkan kapaklı standartlarını içeren kuyu. Not: genom kopya miktarının belirlenmesinde kullanılmak üzere standart süspansiyonlar hazırlamak için, hedef DNA bölgesi plazmid ve lineerleştirilmiş klonlanmış olmalıdır. Aşağıdaki adrese standart eğrileri qPCR kullanılan plazmid DNA şablonları ile nasıl oluşturulacağı hakkında ayrıntılı bir prosedür bulacaksınız: <a href="http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf">http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf</a> </li><li> Uygun parametreler (yapışkanlı kapak ve her iyi, vb toplam hacmi dikkate alınarak) seçmek yeterli bir sistem içine qPCR gerçekleştirin. AmpliTaq Gold aşağıdaki aktivasyon az 10 dakika süreyle 95 ° C, 40 amplifikasyon döngüsü şu şekilde yapılır: 95 ° C&#39;de 15 sve HAdV, JCPyV ve BPyV ve 95 15 sn için 60 ° C&#39;de 1 dakika ° C, 55 20s ° C ve 20s, 60 ° C PAdV için. </li><li> Reaksiyonlar tamamlandığında, ekipman kullanım kılavuzu açıklandığı gibi mağaza verileri ve sonuçları kullanılır. DNA miktarı, seyreltme faktörü gerektiğinde düzelttikten sonra elde edilen veriler medyan olarak tanımlanabilir. </li></ol></li><li> Kontrolleri <ol><li> Pozitif ve negatif kontrolleri kullanın. Test karışımı floresan üretmek değil kanıtlamak için daha fazla olmayan bir şablon kontrolü (NTC) içermelidir. Incelenen örneklerde mevcut inhibitörleri nedeniyle potansiyel enzim inhibisyonu değerlendirmek için olumlu bir süreç kontrolü çalıştırmak için tavsiye edilir. </li><li> Kayıt, standart DNA ve proses kontrolü iki farklı dilüsyonlarının qPCR testlerinin sonuçları. Tahlillerin hassasiyeti ve verimliliği ile ilgili kalite kontrol (QC) programları için kontrol şemaları hazırlamak için kullanın. </li></ol></li><li> Onayısonuçları <ol><li> Pozitif sonuçlar daha suşlarının nükleotid dizilimleri üzerinde ek veri üreten amplikonlarının iç içe-PCR ve nükleotid sıralama 1,5,6,7,9,12 tespit kullanarak teyit edilebilir. </li></ol></li></ol> Prosedürü tarif ardından, insan ve hayvan virüsleri tespit edilmiştir ve banyo suları, deniz suyu ve nehir suyu 2,3 niceliği. Temsili bir örnek olarak, yüksek düzeyde nitrat sunan alanlarda yeraltı suyu örneklerinde kirlilik kaynakları tanımlamak için değerlendirildi. İspanya&#39;nın Kuzeydoğu bölgesi kırsal alanlarda 4 farklı kuyulardan On litre su örnekleri toplanmıştır. Beş süreç kontrolü olarak kullanılan insan adenovirüs ile 2 numaralı seribaşı her bir çoğaltmak toplanmıştır çoğaltır. Örnekler Şekil 1&#39;de temsil edilen protokole göre işlendi. Dört (ortalama değeri 7.7 PAdV için olumlu sonuçlar gösterdi incelenen dört site bir analiz çoğaltırÖrnekleme alanını çevreleyen alanlarda domuz çamurlar varlığı ile ilgili olacak ve fekal porcine kirlenme kaynağı olarak yeraltı suyu nitrat (Tablo 2) destek vereceğini 4x10 2 GC / L). 4. Temsilcisi Sonuçlar: <img src="/files/ftp_upload/2820/2820fig1.jpg" alt="Şekil 1" /> Şekil 1 su virüslerin tespiti ve ölçülmesi için Prosedür. <img src="/files/ftp_upload/2820/2820table1.jpg" alt="Tablo 1" /> Tablo 1. Konsantrasyon qPCR deneyleri için astar ve probları. <img src="/files/ftp_upload/2820/2820table3.jpg" alt="Tablo 3" /> Tablo 2. Tespit ve yeraltı suyu örneklerinde hayvan ve insan adenovirüs ve polyomaviruses ölçümü. n sayısını çoğaltır analiz Pozitif% yüzdesini çoğaltır. (-) Olmayan tespit

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İki patent başvuruları, PAdV ve BPyV ölçümü için protokollerin fikri mülkiyet haklarını korumak için 2009 yılında yayınlandı.

<html> Tekrarlanabilir, güvenilir, basit ve maliyet-etkin açıklanan prosedürü rutin çevre ve halk sağlığı laboratuvarları için uygun bir yöntem için gerekli koşulları yerine getirecektir. Protokol basittir; ancak dikkatli bir şekilde takip edilmelidir. Flokülasyon için karıştırma süresi önemli ölçüde azalır (örneğin en az 5 saat) durumunda olacağı gibi, yapay deniz suyu tuzlarının istenen konsantrasyon eklemeden örneklerinde düşük iletkenlik virüs kurtarma önemli ölçüde azaltaca...

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Yüksek hızlı santrifüj (8.000 xg) </td><td> Berckman Coulter </td><td> Avanti J-20XP </td><td></td></tr><tr><td> pH metre, termometre ve conductimeter </td><td> Afora </td><td> LPPC3003 </td><td></td></tr><tr><td> Plastik borular 100-200 cm uzunluğunda </td><td> Deltalab </td><td> 350059 </td><td></td></tr><tr><td> Steril tek kullanımlık pipetler bitirdi </td><td> Labclinics </td><td> PN10E1 </td><td></td></tr><tr><td> Steril plastik tüpler 1.5 ve 10-15 ml (Eppendorf, Şahinleri, vb.) </td><td> Afora </td><td> KA298/00 </td><td></td></tr><tr><td> Santrifüj tencere (500 mL) </td><td> Fisher Scientific </td><td> SE5753512 </td><td></td></tr><tr><td> Manyetik karıştırıcı ve mıknatıslar (başına birörneği) </td><td> Fisher Scientific </td><td> 10510 </td><td></td></tr><tr><td> Cam veya plastik kaplar, manyetik karıştırıcı kullanılmasına izin vermek düz dipleri </td><td> Deltalab </td><td> 191642 </td><td></td></tr><tr><td> Süpernatant (ya da bir su-jeti vakum pompası) kaldırmak için bir peristaltik pompa </td><td> Watson-Marlow </td><td> 323E / D </td><td></td></tr><tr><td> Timer kapama 8-10 saat sonra karıştırma </td><td> Deltalab </td><td> 900400 </td><td></td></tr><tr><td> Hidroklorik asit (1N ve 0.1N) </td><td> Panreac </td><td> 141020.1611 </td><td></td></tr><tr><td> Sodyum hidroksit (1N) </td><td> Panreac </td><td> 131687.1211 </td><td></td></tr><tr><td> Suni deniz suyu deniz tuzu </td><td> Sigma </td><td> S9883 </td><td></td></tr><tr><td> Yağsız süt (SM) </td><td> Difco </td><td> 232100 </td><td></td></tr><tr><td> Fosfat tampon pH 7, 5 </td><td></td><td></td><td> 01:02 v / v steril Na 2 HPO 4 0,2 M ve pH 7.5 NaH 2 PO 4 0,2 M </td></tr><tr><td> Tiyosülfat </td><td> Panreac </td><td> 121879.1209 </td><td> Su içinde% 10&#39;luk bir çözüm olun. </td></tr><tr><td> QIAamp viral RNA Mini Kit </td><td> Qiagen </td><td> 52904 </td><td></td></tr><tr><td> 96-optik tepki plaka (500 adet) </td><td> Applied Biosystems </td><td> 43426659 </td><td></td></tr><tr><td> Optik yapışkanlı kapakları (100 adet) </td><td> Applied Biosystems </td><td> 4311971 </td><td></td></tr><tr><td> TaqMan Çevre PCR Master Mix 2x </td><td> Applied Biosystems </td><td> 4396838 </td><td></td></tr></tbody></table>

cost-effective method for microbial source tracking using specific human and animal viruses

Ortamında mikrobiyal kirlenme önemli bir sağlık riski temsil eder. Klasik bakteri dışkı göstergeler önemli sınırlamalar olduğu gösterilmiştir, virüs bulaşma kaynağı bilgi birçok inaktivasyon işlemleri ve standart fekal göstergeler daha dayanıklıdır. Virüslerin su ve moleküler testlerin konsantrasyon için maliyet-etkin yöntemlerin geliştirilmesi, fekal kontaminasyon göstergeleri ve mikrobiyal kaynağı olarak izleme (MST) araçları gibi virüslerin uygulanabilirliği kolaylaştırır. Adenovirüsler ve polyomaviruses DNA virüsleri insanlarda dahil olmak üzere belirli omurgalı türlerin bulaşmasını ve ısrarla dışkı ve / veya incelenen tüm coğrafi bölgelerde idrar ile atılır. Daha önceki çalışmalarda, biz insan adenovirüs (HAdV) ve insan fekal kontaminasyonun bir göstergesi olarak kantitatif PCR (qPCR) JC polyomaviruses (JCPyV) miktarının önerdi. Son zamanlarda, biz por özel ölçümü için qPCR testleri geliştirdikcine adenovirüs (PAdV) ve büyükbaş hayvan fekal kontaminasyon belirteç olarak test tüpü başına 1-10 genom kopya hassasiyetleri polyomaviruses (BPyV). Bu çalışmada, biz bu araçları kullanarak su örneklerinde kirlilik kaynağını belirlemek için izlenecek prosedür mevcut. Temsili sonuçlar örnek olarak, yüksek düzeyde nitrat sunan yeraltı suyu virüs analizi gösterilmektedir. Düşük veya orta derecede kirli sularda virüslerin tespiti çok daha küçük bir hacim, genellikle iki seri konsantrasyon adımları içeren bir prosedür en azından birkaç litre su virüslerin konsantrasyonu gerektirir. Bu biraz hantal bir prosedür ve viral kurtarma gözlenen değişkenlik su örneklerinin çok sayıda eşzamanlı işlem önemli ölçüde engel teşkil etmektedir. Önceki Studie geliştirilen bir adım protokolü uygulanan iki adım yordamlar kaynaklanan darboğaz ortadan kaldırmak içins ve su matrisler bir çeşitlilik için de geçerlidir. Asitlenme on litre su örneklerinin, yağsız süt ile flokülasyon, topaklanmalara malzemelerin ağırlık sedimantasyon, çökelti ve santrifüj toplama, 10 ml fosfat tamponu çökelti, tabanda: Bu prosedür içerir. Viral konsantre viral nükleik asitler ve özel adenovirüs ve ilgi polyomaviruses qPCR tarafından sayısal çıkarılması için kullanılır. Çok sayıda numune eş zamanlı olarak bu düşük maliyetli konsantrasyon yöntemi kullanılarak analiz edilebilir. Bu prosedür, suları, deniz suyu ve nehir suyu analiz uygulanmış ve bu çalışmada, yeraltı suyu numuneleri analiz sonuçları sunmak olmuştur. Bu yüksek verim nicel yöntem, basit, güvenilir ve maliyet-etkin.

Watch this Scientific Journal Video about Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses at JoVE.com

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Bu çalışma, MST araçları olarak önerilen insan / / sığır domuz DNA virüsleri, adenovirüs ve polyomaviruses, belirli bir miktar tayini için qPCR kullanarak suda nitrat dışkı / idrar kirlenme veya kontaminasyon kaynağının belirlenmesi için maliyet-etkin bir yöntem açıklanır.

Özgül İnsan ve Hayvan Virüsler Mikrobiyal Kaynak Takip için maliyet-etkin Yöntemi

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

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12.1K Görüntüleme.  University of Barcelona. Bu çalışma, MST araçları olarak önerilen insan / / sığır domuz DNA virüsleri, adenovirüs ve polyomaviruses, belirli bir miktar tayini için qPCR kullanarak suda nitrat dışkı / idrar kirlenme veya kontaminasyon kaynağının belirlenmesi için maliyet-etkin bir yöntem açıklanır.

Video: Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

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

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

HIV / AIDS Kapsamlı ve Maliyet Etkin Laboratuvar Kontrolü: Afrikalı bir Rol Modeli

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

Demir Kaynağı Olarak İnsan Hemoglobin kullanarak Staphylococcus aureus Büyüme

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Polarize İnsan Havayolu bir sıvı kaplı Kültürünün Oluşturulması Solunum Patojenler için Host Hücre Yanıtı Okumak Epitel Calu-3 Hücreler<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

İnsan İmmün Yetmezlik Virüs çoğaltma Fitness belirlenmesi için İkili Büyüme Rekabet Analizi

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Kimerik fare modellerinde Rekombinant Grip aşıların burun içinde yoluyla mukozal bağışıklık Okumak

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

Bakteriyel Yapay Kromozom: RNA Virüsler Pozitif iplikli Araştırmaları Fonksiyonel Genomik Aracı

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

MDCK-SIAT1 hücreleri kullanarak antikor yanıt için Microneutralization tarafından insan Sera A(H3N2) virüsleri nötralize grip ölçme deneyi

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

VirWaTest, su örnekleri virüslerin tespiti için bir nokta-of-kullanım yöntemi

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Proliferasyon ınsan antijeni spesifik CD4+ T hücrelerinin Karboksflorcein Succinimidyl Ester kullanarak ölçülmesini

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...