We would like to thank Dr. Nicholas J. Ashbolt and Dr. G. Shay Fout for their review of the manuscript.

Züccaciye ve Filtre gövdeleri 1. Hazırlık <ol style="margin-left: 40px;"><li> Aksi belirtilmediği sürece, 15 dakika için 121 ° C&#39;de tüm cam, filtre gövdesi ve çözümleri sterilize. Sterilite sağlamak için, tüm açıklıkları veya alüminyum folyo ya da sterilizasyon öncesinde bant güvenli kağıt ile ya maruz yüzeyleri kapsar. </li><li> 1 L&#39;lik bir yan kol Erlenmeyer şişesine filtre yuvası (47 mm çapında) takılarak filtreleme aparatı birleştirin. Gerekli filtreleri toplayın: 47 mm çap elektronegatif / elektropozitif disk filtreler. </li><li> 1 L&#39;lik 1.5% sığır eti ekstresi hazırlanması (topaklanması; topaklanma parçacıklarını üreten çözeltinin pH değeri 3,5, ya da olmayan flokülasyon altına düştüğünde, pH 3.5 agrega değildir) eti ekstresi, 15 g içinde çözülerek 0.05 M glisin ile, 1 L deiyonize su ve 3.75 g glisin ilave edilmesi. Not: Non-pıhtılaştırıcı sığır ekstraktlar önce yüksek konsantrasyona kadar selit kullanılmasını gerektirmektedir. </li><li> Elüsyon katkı sodyum polyph ekle% 0.1 konsantrasyonda sığır ekstraktı osphate. </li><li> 1 M hidroklorik asit solüsyonu ekleyin sığır eti ekstresi, istenilen pH pH solüsyonu karıştırma, damla damla. 121 ° C&#39;de, 15-30 dakika süreyle otoklavlanmaktadır sığır özü. </li><li> Ince, orta, ya da (non-flokülan sığır özü yalnızca kullanım için) büyük parçacık celite 0.1 g ölçün. </li><li> Erlenmeyer yan kol şişeye ve vakum prizine uyumlu boru takarak vakum / emme hazırlayın. </li></ol> Çözümler ve Virüs Stokta 2. Hazırlık <ol style="margin-left: 40px;"><li> 1 M HCl 1 L hazırlayın. </li><li> 1 M NaOH ile 1 L hazırlayın. </li><li> 1x PBS çözeltisi hazırlayın (137 mM NaCI, 2.7 mM KCI, 1.47 mM K 2 PO 4 ve 4.3 mM NaH 2 PO 4). <ol><li> 1 M NaOH ile 9.0&#39;a 1x PBS pH ayarlayın. </li></ol></li><li> Hazırlayın ve 9 ml PBS (pH stok virüsü 1 ml karıştırılarak 10 4 -10 5 en muhtemel sayı (EMS) ml -1 stok virüs sulandırmak7), daha önce tarif edildiği 8,13. 1 ml alikotlar içinde -80 ° C&#39;de saklayın. <ol><li> Potansiyel kirleri çıkarmak için şırınga filtreleme (0.22 mikron gözenek boyutu) virüs stok sterilize edin. </li></ol></li><li> Klor Musluk Suyu <ol><li> Ölçü steril kullanılarak steril musluk suyu içinde 1 litre dereceli silindir ve manyetik bir karıştırma çubuğu ihtiva eden bir 2 L&#39;lik bir kap içine dökün. </li><li> Sodyum tiyosülfat 0.7 g eklenir ve granüller eriyene kadar karıştırılır. 1 M HCI veya 1 M NaOH çözeltisi kullanılarak gerekirse 7-7.5&#39;e musluk suyu pH ayarlayın. </li><li> Çözülme virüs başak (1 ml) ve dechlorinated steril musluk suyu 1 L başak tüm hacim ekleyin. 10 dakika boyunca karıştırın. </li><li> Katkılı bir örnekle aynı şekilde, her bir deney ve yöntem ile ilgili bir yöntem boş (adenovirüs olmadan kloru giderilmiş musluk suyu) hazırlayın. </li></ol></li></ol> Millipore Filtre Aparatı 3. Hazırlık <ol style="margin-left: 40px;"><li> Filtre konut üzerinde steril örtü çıkarın. SağlamakFiltre gövdesi üst kase ve alt filtre ekranı arasında uygun uyum filtrasyon sırasında örnek sızıntıyı önlemek için. </li><li> 1 L&#39;lik bir Erlenmeyer şişesi, steril kılıf çıkarılır. İyi bir yalıtım oluşturmak, filtre ünitesinin mantar Erlenmeyer şişesi üst açıklığı içinden rahatça sığar emin olun. </li><li> Alt filtre ekranından filtre ünitesinin üst kase çıkarın ve steril forseps ile ekran üzerinde kare 47 mm elektropozitif filtre diski yerleştirin. Alt ekran filtresi konut kase takın ve saat yönünün tersine çevirerek yerine kilitleyin. </li></ol> 4. dokunun Su Filtrasyon (Birincil Konsantrasyon) <ol style="margin-left: 40px;"><li> Virüsün 100 ml steril dereceli silindir kullanarak musluk suyu çivili ölçün. </li><li> Virüs, 100 mi 47 mm&#39;lik kıvrımlı cam / selüloz filtresi veya bir nano alüminyum oksit / cam elyaf filtre diski ya da ihtiva eden filtre gövdesine musluk suyu çivili dökün. </li><li> Su yavaş yavaş filtre geçmesine izin verin. O kalan artıklar kaldırmak için nazik vakum uygulayınFiltre yüzeyinden f musluk suyu. </li><li> Tripsin tedavisi kullanıldığında, yüzey filtre edilir ve 5 dakika boyunca inkübasyona% 0.25 tripsin 5 ml ilave ediniz. </li><li> Steril deiyonize su, 50 ml kullanılarak filtre yüzeyinden tripsin yıkayın. </li></ol> Filtre 5. Virüs Elüsyon <ol style="margin-left: 40px;"><li> 2 L&#39;lik bir Erlenmeyer şişesi filtre gövdesini kaldırmak ve 250 mi yan kol şişede üzerine yerleştirin. </li><li> Silindir mezun kullanarak sığır ekstraktı 100 ml ölçün. Yavaş yavaş filtre gövdesine sığır ekstraktı 100 ml dökün. </li><li> Sığır özü küçük bir (250 mi) bir Erlenmeyer şişesi içinde sığır eti ekstresi yakalama, üzerine dökülen tipteki filtrenin izin verin. Filtreden kalan sığır özü ile çekmek için vakum uygulayın. </li></ol> 6. Celite İkincil Konsantrasyon <ol style="margin-left: 40px;"><li> Bir heyecan plaka yıkanarak virüs içeren transfer şişeler. Hafif girdap oluşturmak için karıştırma, karıştırma plaka üzerinde steril manyetik karıştırma çubuğu ve yer ekle. </li><li> Selit 0.1 g eklemesığır özü, 100 ml toz ve selit dağılmasını sağlar. Sığır özü içine steril pH probu yerleştirin. </li><li> PH değeri 4.0 ulaşmak için sığır özü akıllıca 1 M HCl damla ekleyin. Yavaş yavaş 10 dakika boyunca karıştırın izin verin. </li><li> 2 L&#39;lik bir Erlenmeyer şişesi kullanarak (daha önce tarif edildiği gibi) filtre yuvası üzerine 47 mm ön filtresi yerleştirin. </li><li> Ön filtre üzerinde 100 ml sığır özü / selit karışımı dökün. Sığır ekstraktı yoluyla dökmek için izin verin. Filtreden kalan sığır özü üzerinden çekin hafif vakum uygulayın. </li><li> Filtre konut ağzının ucunda metal boru klibi yerleştirin. Metal boru klibe steril 15 ml polipropilen toplama tüpü takın. Geri Erlenmeyer şişesi içine filtre muhafazasını yerleştirin. </li><li> Ön filtre üzerinde toplanan selit üzerinde 1x PBS tamponu 5 mi, pH 9.0 ekleyin. </li><li> PBS ön filtre ile damla izin verin. 15 ml toplama tüpüne artık PBS üzerinden çekin hafif vakum uygulayın. </li></ol> 7. Organik Flokülasyonu İkincil Konsantrasyon <ol style="margin-left: 40px;"><li>Bir heyecan plaka yıkanarak virüs içeren beher aktarın. Sığır ekstraktı 100 ml steril manyetik karıştırma çubuğu ekleyin ve hafif vorteks oluşturmak için karıştırın. </li><li> Sığır özü içine steril pH probu yerleştirin. </li><li> Sığır özü akıllıca 1 M HCI damla ekleyin ve 3.5 pH ayarlayın. 30 dakika boyunca karıştırın. </li><li> 4 ° C&#39;de 15 dakika boyunca 2500 x g&#39;de 250 ml&#39;lik santrifüj konik tüplere eluat ve santrifüj dökün. </li><li> Pelet bozmaya değil değil süpernatant alarak bakım kapalı dökün. 1x PBS, pH 9 5 ml pelletini. </li><li> 4 ° C&#39;de 10 dakika boyunca 4.000 xg&#39;de süspansiyon santrifüjleyin. Süpernatantı dökün ve pelet atın. </li><li> 7-7,5 pH ayarlayın, filtre -80 ° C&#39;de 0.22 mikron şırınga filtresi ve dondurma ile sterilize. </li></ol> 8. Viral Nükleik Asit Ekstraksiyon <ol style="margin-left: 40px;"><li> Üreticinin talimatlarına uygun olarak, uygun bir ticari kit kullanılarak viral nükleik asitlerin ekstre edilmesi. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 9. AdV 40/41 QPCR <ol style="margin-left: 40px;"><li> 8,13 yayınlanan primerler ve problar, aynı zamanda reaksiyon karışımı ve termal döngü profili kullanın. NOT: tahlil için algılama Tahmini sınırı reaksiyonu başına yaklaşık 5 qPCR birimdir. </li><li> Aşağıdaki gibi konsantrasyonlarda PCR büyütme katkı maddeleri ekleyin: 10 mM Tris (pH 8.3), 50 mM KCI, 4.5 mM MgCI2, 10 mM dNTP, 10 uM primerleri ve 1 uM probu ve polimeraz 0.5 ul olacak şekilde 45 son hacim ul, 96 oyuklu bir PCR reaksiyonu, plaka içindeki deliklerin uygun sayıda ilave edilebilir. </li><li> Bir 96-yuvalı, hızlı reaksiyon PCR plaka her biri uygun oyuğuna PCR amplifikasyon karışımı yüklenebilir 45 ul. </li><li> DNA 5 ul PCR plaka ihtiva eden uygun oyuklara örnek ekstre ekleyin. </li><li> , Isıya dayanıklı kapak contası ile plaka Kapak PCR plaka kuyuların dibine herhangi damlacıkları taşımak için plaka döndürün. </li><li> Foll olarak ayarla PCR amplifikasyon döngüleriakımları: 15 saniye için 95 ° C 40 döngü, 1 dakika süreyle 60 ° C&#39;de, ardından 10 dakika boyunca 95 ° C olarak ölçülmüştür. </li></ol> 10. Veri Analizleri <ol style="margin-left: 40px;"><li> 5-1: 1 kapsayan örneklerinin seri 5 kat seyreltmeleri üçlü ölçümleri analiz 625 seyreltme aralığı viral parçacıkların en olası sayı (EMS) belirlemek amacıyla, daha önce tarif edildiği gibi 8,13. </li><li> 5-1: 1 kapsayan adenovirüs deney sivri seri 5 kat seyreltmeleri analiz 15.625 seyreltme aralığı viral parçacıkların en olası sayı (EMS) tespit etmek için. </li><li> EPA EMS hesap (http://www.epa.gov/nerlcwww/online.html) kullanılarak, seyreltme tipi için hesap hesap ayarlayın (1: 5 ya da 1:10) dilüsyonlarda adedine ve sayısı seyrelme serisi içindeki örnekleri çoğaltırlar. </li><li> Yapın bir birim hacim vb (örneğin, 1 mi, 100 ml, normalize ardından verileri 10 log transformasyonu </em&gt;). Numunede mevcut viral parçacıkların sayısına bölünmesiyle verilen deney için kurtarma hesaplayın deneysel ani mevcut viral parçacıkların sayısı ile yoğunlaşmaktadır. </li><li> Farklı deneysel değişkenlerin etkisini değerlendirmek (örneğin, farklı celite tipleri, pH aralıkları, farklı filtre türleri ve farklı ikincil konsantrasyon teknikleri) (örn, eşleştirilmiş t-testi, bir ya da iki parametrik ya da parametrik olmayan istatistiksel testler yapılarak AdV verimleri üzerindeki veri dağıtım ve deneysel tasarım bağlı) ANOVA yollu. </li></ol>

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</ol>

No competing financial interests. 
The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

Elektropozitif filtreler su virüsleri konsantre yararlıdır; Ancak bu filtreler sırayla onların etkinliğini değiştirebilir kendi yapısı ve kompozisyon farklı olabilir. Bu sorunun Bileşikler, kapsid yapıları ve ücretleri konsantrasyon teknikleri optimum kurtarma 15 sağlamak için uyarlanabilir gerektiren virüs suşları arasında değişmektedir. (Örneğin, elektropozitif filtreler, dana özü elüsyon), hedef virüs daha etkili konsantrasyon 16,17 elde edilebilir, mevcut k...

İnsan enterik virüsler su kaynaklı hastalıkların 1-3 önemli etken vardır, ama konsantrasyon olmadan kendi algılama zorlaştırır, genellikle kontamine çevre sularında düşük sayılarda bulunurlar. Virüsleri konsantre için kullanılan prosedürler, genellikle filtre yıkama sıvısının bir hava filtresi, yıkama, ardından adım, ve ikinci bir konsantrasyon yer alır. Yaygın bir filtrasyon işlemi (son 4,5 &#39;de gözden geçirilmiştir), elektropozitif filtreler gibi yüklü membran kullanılması ile ilgilidir. Bu filtreler, filtre alanı (pozitif yüklü) ve hedeflenen virüs parçacıkları arasındaki elektrostatik etkileşimleri kullanılarak su içinde süspansiyon haline getirilmiş virüs yakalama dayanır (negatif yüklü). Ticari olarak mevcuttur iki elektropozitif filtreler bu teknoloji güveniyor, cam / selüloz ve nano-alümina / cam elyaf filtreler. Cam / selüloz filtre maliyetleri cam / ce kullanımını sınırlamak kadar 10 kez nano-alümina / cam elyaf ki, vardırRutin virüs kontrolü için llulose filtreler. Son çalışmalar ucuz filtre alternatif kullanımını haklı, farklılıklar ortam su 6,7 den enterovirüslerin kurtarma bu iki filtre arasındaki nominal olarak sonucuna varmışlardır. Örneğin elektronegatif ve cam yün filtre gibi diğer filtre ayarları, ancak, her iki kaynak, su (elektronegatif filtreleri) tabi tutulmasını gerektirir ya da (cam yünü filtreleri), ticari olarak mevcut olmadığında, incelenmiştir. virüs konsantrasyonu prosedürlerinin geliştirilmesi çok sudan virüs verimini artırmak amacıyla, birinci konsantrasyon teknikleri (filtre) optimize odaklanmıştır. Ancak, hacimleri mililitrelik için tipik olarak 1 L yıkama sıvısı hacmini azaltır ikincil konsantrasyon işlemleri, ayrıca virüs geri kazanımlar 8 üzerinde önemli bir etkiye sahip olabilir. Bağırsak virüslerinin ikincil konsantrasyonu tipik olarak sığır özü bazı türleri gibi bir pıhtılaştırıcı madde dayanır (organik floccuyon) veya yağsız süt flokülasyon 9-12 filtre yüzeyleri virüs parçacıkları kaldırmak için. Son zamanlarda, selit (ince parçacık) eklenmesi ile bağlanmış sığır eti ekstresi ile bir ikinci yoğunlaştırma işlemi adenovirüs, enterovirüs ve Norovirus 8,13,14 geri kazanılması için umut verici olduğu gösterilmiştir. Bu virüs parçacıklarının organik flokülasyon yöntem ile aynı prensiplere göre, Celite konsantrasyonu işleri tutunur ve süspansiyon çözeltisinin pH&#39;ını değiştirilmesiyle partiküller (topak veya selit) salınır. Bu iki ikincil konsantrasyon teknikleri arasındaki karşılaştırmalar çivili adenovirüs (AdV) türleri 40 ve 41 8 kurtarma değerlendirilmiştir. Bu çalışma iki ikincil konsantrasyon teknikleri adenovirüslerin kurtarma istatistiksel olarak benzer olduğu sonucuna varıldı. Bununla birlikte, organik çökeltme yöntemi, bir 30 dakika gerektirir. pH 3.5 inkübasyon, selit tekniği pH 4.0 kısa bir inkübasyon (10 dakika) gerektirir ise. Organik flocculatiyonu, tertier konsantrasyon sırasında topaklanma maddeleri toplamak pahalı laboratuar ekipmanları (santrifüj) kullanımını gerektirir, farklı olarak selit tekniği süspansiyondan selit parçacıkları ayırmak için sadece temel laboratuar ekipmanı (vakum filtrasyon) kullanılır. Filtreler ve ikincil yıkama teknikleri belirli kombinasyonları da virüs kurtarma etkileyebilir. Bir çalışma birincil (elektropozitif filtrelerin) belirli kombinasyonları ve ikincil konsantrasyon teknikleri (Celite veya organik flokülasyon) adenovirüs 13 kurtarma önemli bir etkiye sahip olduğu sonucuna varmıştır. Bu bulgular, bu teknikler kullanılarak zaman bu optimizasyon optimum verilen bir su matris hedef virüsü geri kazanmak için gerekli olduğunu düşündürmektedir. Optimizasyon sayısız değişkenler (filtre tipi / marka, pH elüsyon çözeltisi, selit / organik flokülasyon) değerlendirilecektir çünkü, zorlu bir süreç birçok araştırmacı aktif önlemek tüketen bir zaman. Bu s içinHer virüs tipi eşsiz bir kapsid morfolojisi ve özel kapsid şarj görüntüler beri tudy, bir prosedür, Muhtemelen çivili insan adenovirüs 40 suşlar ve 41. kullanarak sudan virüs konsantrasyonu için en uygun koşulları belirlemek için geliştirilen, konsantrasyon protokolleri her virüs için optimize edilmesi gerekebilir Optimal viral kurtarma ulaşmak için hedef. 1) elektropozitif filtre ardından 2 diskler) ikincil konsantrasyon olarak celıte tekniği karşı kurulmuş bir organik flokülasyon yöntemi değerlendirmesini kullanarak musluk suyunda virüs kurtarma değerlendirilmesi, ve 3) değerlendirme: Bu çalışma AdV 40 ve 41 ile konsantrasyon için bir yaklaşım sağlar tertier konsantrasyonu için elüsyon tamponları.

<table><tbody><tr><td>Adenovirus 40 stock</td><td>ATCC</td><td>VR-931</td><td></td></tr><tr><td>Adenovirus 41 stock</td><td>ATCC</td><td>VR-930</td><td></td></tr><tr><td>Sodium Thiosulfate</td><td>Fluka Chemical Co.</td><td>72051</td><td></td></tr><tr><td>Celites #577</td><td>Fluka Chemical Co.</td><td>22142</td><td></td></tr><tr><td>NanoCeram 47 mm</td><td>Argonide</td><td>N/A</td><td></td></tr><tr><td>1MDS 47 mm</td><td>3M</td><td>6408502</td><td></td></tr><tr><td>AP-20 Prefilter 47 mm</td><td>Millipore Corp.</td><td>AP2004700</td><td></td></tr><tr><td>Glycine&amp;nbsp;</td><td>Sigma</td><td>50046-1KG</td><td></td></tr><tr><td>Sodium Polyphosphate</td><td>Acros Organics</td><td>390930010</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200</td><td></td></tr><tr><td>PBS</td><td>Sigma</td><td>P5368</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher</td><td>A481-212</td><td></td></tr><tr><td>BBL Beef Extract</td><td>BD Biosciences</td><td>212303</td><td></td></tr><tr><td>Difco Beef Extract</td><td>BD Biosciences</td><td>211520</td><td></td></tr><tr><td>ABI 7900 Real-time PCR system</td><td>ABI</td><td>N/A</td><td></td></tr><tr><td>Stainless Steel Filter Housing</td><td>Millipore Corp.</td><td>XX2004720</td><td></td></tr><tr><td>Blood DNA Extraction Kit</td><td>Qiagen&amp;nbsp;</td><td>51104</td><td></td></tr><tr><td>EPA MPN Calculator</td><td></td><td></td><td>http://www.epa.gov/nerlcwww/online.html</td></tr></tbody></table>

a small volume procedure for viral concentration from water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

<html> Celite seçimi Celite Üç farklı öncesinde iyi performans varyant seçimi test edildi. Orta ölçekli parçacıklara cezası ile selitler yüksek adenovirüs geri kazanımlarını üretti. Daha büyük selitler kullanımı AdV40 ve 41 (aralık% 32% 100) (Şekil 1) her ikisi için de daha düşük kurtarma ile sonuçlanmıştır. Adenovirüs 40 Ortalama kazanımları 144% ± 52% (ince), 115% ± 28% (orta) ve% 82 ± 53% (büyük) partikül selitler ve AdV41, 132% ± 3...

Watch this Scientific Journal Video about A Small Volume Procedure for Viral Concentration from Water at JoVE.com

A Small Volume Procedure for Viral Concentration from Water

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

Sudan Viral Konsantrasyon için Küçük Bir Ses Prosedürü

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

a-small-volume-procedure-for-viral-concentration-from-water

Universidad San Sebastian

Research

JoVE Journal

Environment

10.1K Görüntüleme.  U.S. EPA. An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation. 

Video: A Small Volume Procedure for Viral Concentration from Water

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.

The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.

Su Sistemleri Virüs Üretim Oranlar tahmin

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

İmmünoloji ve Enfeksiyon

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

Su Kimya ve Deniz Mikrobiyoloji A Field Guide - Okyanusu&#39;nda gaybı Oyuncular çözülüyor

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

Çevre

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

Enterovirus Kültür ve RT-qPCR Su Norovirus Oluşum EPA Metot 1615 ölçülmesi. II. Toplam kültürlenebilir virüs Deneyi

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

Enterovirüs Kültür ve RT-qPCR Su Norovirus Oluşum EPA Method 1615 Ölçümü. Bölüm III. RT-qPCR Virüs Algılama

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

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

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

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 ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Nucleocapsids gelen Viral DNA hazırlanması

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

The key first step in evaluating pathogen levels in suspected contaminated water is concentration. Concentration methods tend to be specific for a particular pathogen group, for example US Environmental Protection Agency Method 1623 for Giardia and Cryptosporidium1, which means multiple methods are required if the sampling program is targeting more than one pathogen group. Another drawback of current methods is the equipment can be complicated and expensive, for example the VIRADEL method with the 1MDS cartridge filter for concentrating viruses2. In this article we describe how to construct glass wool filters for concentrating waterborne pathogens. After filter elution, the concentrate is amenable to a second concentration step, such as centrifugation, followed by pathogen detection and enumeration by cultural or molecular methods. The filters have several advantages. Construction is easy and the filters can be built to any size for meeting specific sampling requirements. The filter parts are inexpensive, making it possible to collect a large number of samples without severely impacting a project budget. Large sample volumes (100s to 1,000s L) can be concentrated depending on the rate of clogging from sample turbidity. The filters are highly portable and with minimal equipment, such as a pump and flow meter, they can be implemented in the field for sampling finished drinking water, surface water, groundwater, and agricultural runoff. Lastly, glass wool filtration is effective for concentrating a variety of pathogen types so only one method is necessary. Here we report on filter effectiveness in concentrating waterborne human enterovirus, Salmonella enterica, Cryptosporidium parvum, and avian influenza virus.

Glass wool filters have been used to concentrate waterborne viruses by a number of research groups around the world. Here we show a simple approach for constructing glass wool filters and demonstrate the filters are also effective in concentrating waterborne viral, bacterial and protozoan pathogens.

Konsantre Su Bazlı Virüsler ve Tarım Zoonotik Patojenler için Cam Yünü Filtreler

The key first step in evaluating pathogen levels in suspected contaminated water is concentration.  Concentration methods tend to be specific for a ...

Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The recovery of microbial fractions, including viruses, bacteria, and microeukaryotes from wastewater and environmental water samples&#160;is one of the challenging steps in these approaches. In this study, we focused on the recovery efficiency of sequential ultrafiltration and skimmed milk flocculation (SMF) methods using Armored RNA as a test virus, which is also used as a control by some other studies. Prefiltration with 0.45 &#181;m and 0.2 &#181;m membrane disc filters were applied to eliminate solid particles before ultrafiltration to prevent the clogging of ultrafiltration devices. Test samples, processed with the sequential ultrafiltration method, were centrifuged at two different speeds. An increased speed resulted in lower recovery and positivity rates of Armored RNA.&#160;On the other hand, SMF resulted in relatively consistent recovery and positivity rates of Armored RNA. Additional tests conducted with environmental water samples demonstrated the utility of SMF to concentrate other microbial fractions. The partitioning of viruses into solid particles might have an impact on the overall recovery rates, considering the prefiltration step applied before the ultrafiltration of wastewater samples. SMF with prefiltration performed better when applied to environmental water samples&#160;due to lower solid concentrations in the samples and thus lower partitioning rates to solids. In the present study, the idea of using a sequential ultrafiltration method arose from the necessity to decrease the final volume of the viral concentrates during the COVID-19 pandemic, when the supply of the commonly used ultrafiltration devices was limited, and there was a need for the development of alternative viral concentration methods.

Virus concentration from environmental water and wastewater samples is a challenging task, carried out primarily for the identification and quantification of viruses. While several virus concentration methods have been developed and tested, we demonstrate here the effectiveness of ultrafiltration and skimmed milk flocculation for RNA viruses with different sample types.

Yağsız Süt Flokülasyonu ve Ultrafiltrasyon Kullanılarak Çevresel Su ve Atık Su Numunelerinden Virüs Partiküllerinin Konsantrasyonu

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The ...

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

Lentivirüs Üretim

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

Biyoloji

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

Kullanımı Çevre Örneklerinde Yeni Faustoviruses İzolasyonunda bir Hızlı Stratejisi<em&gt; Vermamoeba vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Sudan Viral Konsantrasyon için Küçük Bir Ses Prosedürü

Özet

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