The authors have no acknowledgements.

Alanında 1. Swab Örnekleme <ol><li> eldiven temiz bir çifti giymek. </li><li> bir ölçüm bandı veya cetvel kullanarak yüzeyine dokunmadan örnekleme alanının boyutunu ölçün. Mümkün olduğu kadar doğru bölgeyi tahmin ve bir rapor formu (Ek Tablo 1) doldurmak için deneyin. </li><li> olası sızıntı ve etiket örneği taşıma çantaları ve çubukla kitleri için sürüntü kiti edin. </li><li> şu şekilde numune alanı boyunca çubukla hareket Yatay yönde bir felç, dikey yönde bir vuruş ve bir köşegen yönünde bir vuruş. 700 cm 2 den bir yüzey alanı büyük çubukla etmeyin. </li><li> bir tüp içine her bezi yerleştirin ve güvenli kapağı sıkın. </li></ol> Laboratuvara Swablar 2. Depolama ve Taşıma <ol><li> en fazla 48 saat boyunca 4 ° C &#39;de bezlerden tutun. daha uzun süreler boyunca depolama gerekli ise, -20 ° C &#39;de bezlerden saklamak (ya da -70 ° C). </li><li> 0-4 ° C&#39;de tüpler tutmak (örn. Toplama 24-48 saat içinde laboratuar ve gemiye taşıma sırasında izole edilmiş bir kap içinde) soğuk paketleri kullanır. </li></ol> 3. Virüs Konsantrasyon, Viral RNA Ekstraksiyonu ve Saflaştırılması NOT: Aksi belirtilmedikçe tüm santrifüj adımları, oda sıcaklığında 5 dakika boyunca 5000 x g&#39;de bir masa üstü santrifüjü kullanabilirsiniz. evrensel nükleik asit ekstraksiyon (UNEX) tamponu ile çalışırken ekstra dikkatli olun. gözlük veya yüz siperi takın. <ol><li> bir 15 ml tüp ve her bir numune için bir RNA Midi sütun etiketleyin. Her deneyde negatif çıkarma kontrolü içerir. </li><li> 10 çubuklar için lizis çözeltisinin hazırlanması PBST 25 mL UNEX tamponu 25 ml karışımı (% 0.02 oranında içeren PBS pH 7.2 Tween-80). </li><li> Lizis çözeltisinin 50 mL kolifaj MS2 süspansiyonu (10 6 PFU / ml) 50 ul ekleyin. </li><li> 15 ml bir tüp içinde bir çubukla yerleştirilir ve 5 mi liz çözeltisi ilave edin. Karıştırınız ve oda sıcaklığında 10 dakika inkübe edilir. </li><li>10 sn için, her bir tüp ve vorteks 5 mi% 100 etanol ilave edin. </li><li> Dikkatle 15 ml tüp çubukla kaldırmak aşırı sıvı kaldırmak ve sonra çubukla atmak için tüpün kenarına hafifçe bastırarak (o UNEX tampon içerir). Geri kalan hacim 9-10 ml arasında olmalıdır. </li></ol> 4. Midi Sütun Viral Nükleik Asit Ekstraksiyon <ol><li> Dikkatle bir midi sütun, santrifüj üzerine adım 3.6 4.5 mL UNEX / etanol karışımı aktarın ve süzüntü atın. </li><li> Aynı sütun, santrifüj üzerine aynı karışımdan başka 4.5 mL yükleyin ve süzüntü atın. </li><li> İlk yıkama için, Midi kolonu, santrifüjün üzerine,% 70 etanol 3.5 ml ilave edilir ve filtrat atılır. </li><li> İkinci yıkama için, Midi kolonu üzerine% 70 etanol bir 3,5 ilave edin. Santrifüj süzüntü atın ve. </li><li> kuru spin için, etanol tüm izlerini (olumsuz viral RNA kurtarma etkileyebilir) kaldırmak için Midi sütunları santrifüj. </li><li>yeni bir 15 ml&#39;lik santrifüj tüpüne Midi sütun. Midi kolonuna 250 ul elüsyon tamponu ekleyin; En yüksek Viral RNA iyileşmeyi elde etmek santrifüj edilmeden önce 1 dakika boyunca bekleyin. </li><li> -70 ° C&#39;de çıkarılan nükleik asidi saklayın veya adım 5&#39;e hemen geçin. </li></ol> RNA Temiz ve Konsantratörü Setlerini Kullanma Viral Nükleik Asit 5. Konsantrasyon <ol><li> RNA 10 saniye süreyle yukarıda adım 4.7 ve vorteks yıkandı RNA 250 uL Bağlama Tamponu 500 mcL ekleyin. </li><li> 10 sn için 750 mcL% 100 etanol ve vorteks ekleyin. </li><li> Her bir numune için bir spin kolon etiketleyin. spin kolona 750 uL örnek yükleyin. </li><li> 1 dakika boyunca 12.000 xg&#39;de dönüş sütunları santrifüj. flow-through atın. </li><li> 1 dakika boyunca 12.000 xg&#39;de bir spin kolon ve santrifüj üzerine kalan 750 uL yükleyin. </li><li> ön yıkama için 1 dakika boyunca 12.000 xg&#39;de her spin kolon ve santrifüj 400 uL RNA Hazırlık tampon ekleyin. flow-through atın. </li><li> İlk yıkama için, 1 dakika için 12,000 x g&#39;de her spin kolon ve santrifüj 800 uL RNA yıkama tamponu ilave edin. flow-through atın. </li><li> İkinci yıkama için, 1 dakika için 12,000 x g&#39;de sütun ve santrifüj dönmeye 400 uL RNA yıkama tamponu ilave edin. flow-through atın. </li><li> kuru spin için, 2 dakika süreyle 12,000 xg&#39;de yıkama tamponu tüm izlerini silmek için spin kolon santrifüj. </li><li> Dikkatle temiz bir 1.5 mL mikrosantrifüj tüp spin kolon aktarın. </li><li> Spin kolonu üzerine 25 uL elüsyon tamponu ilave edin ve oda sıcaklığında 1 dakika boyunca inkübe edin. Bu sütundan viral RNA kurtarma artacaktır. </li><li> 1 dakika 10.000 xg&#39;de spin kolon santrifüj edilerek RNA toplayın. </li><li> Her iki durumda da, -70 ° C&#39;de, RT-qPCR (aşama 6) veya mağaza RNA doğrudan devam edin. </li></ol> 6. Genogroup I Mutiplex RT-qPCR Algılama ve II Noroviruses ve Kolifaj MS 2 (Ek Tablo 2) <ol><li> Temiz çalışma yüzeyleri, pipettes, RNaz dekontaminasyon solüsyonu ile ve santrifüjler olası kirlenmeyi azaltmak için. </li><li> buz üzerinde RT-qPCR reaktifler çözülme. (Ayrı bir alan / oda) buz üzerinde Çözülme RNA. </li><li> Reaksiyonların sayısını belirlemek ve bunların en az% 10 daha fazla eklemek (10 reaksiyonlar gerektiği takdirde, örneğin, 11 master karışımı olun). </li><li> 5 saniye için 25x RT-qPCR enzimi haricinde Vortex bireysel master miks bileşenleri. Dikkatli bir parmak ile tüp hafifçe vurarak enzimi karıştırın. </li><li> 5 saniye süreyle kısaca santrifüj master miks bileşenleri. </li><li> Kiti talimatlarına göre Norovirus GI ve GII gerçek zamanlı RT-PCR tespiti için ana karışım hazırlanarak, 1.5 ml mikrosantrifüj tüpü (Ek Tablo 3) norovirus spesifik oligonükleotid primerler ve problar ekleyin. </li><li> yukarı ve aşağı 5-10 kez pipetleme ana karışımı karıştırın (vorteks tavsiye edilmez). Gerçek zamanlı PCR, 96 oyuklu plakanın her bir master karışımı kısım 22 uL. </li><li> Vortex örnek RNA5 saniye için, ve kısa (5 s) santrifüjle toplanır. </li><li> RT-qPCR plaka (Tablo 2 şablonu izleyin) örnek RNA ve GI ve GII pozitif kontroller 3 mcL ekleyin. no-şablon kontrolü (NTC) kuyularda nükleaz Freewater 3 mcL ekleyin. </li><li> Optik yapışkan film ile gerçek zamanlı plaka basım. </li><li> 1 dakika kuyularda mevcut olabilecek herhangi bir hava kabarcığı veya sıvı damlaları kaldırmak için dikkatlice 1,300 xg&#39;de gerçek zamanlı plaka santrifüj. </li><li> 10 dakika 1) RT adım: Bir gerçek zamanlı PCR cihazı ve set-up aşağıdaki thermocycling koşullar ayarlayın. Taq polimeraz, 45 ° C (2) aktivasyonu, 10 dak. 95 ° C ve (3) 95 ° C&#39;de 15 saniye, 45 döngü, 60 ° C&#39;de 60 s. </li></ol> Çubuğu Örneklerinde Norovirus 7. sayısallaştırılması <ol><li> RT-qPCR sonuçları (Ek Tablo 3) doğrulamak için pozitif ve negatif kontrollerin sonuçlarını kontrol edin. </li><li> Her bir standart eğri karşılayıp karşılamadığını belirlemekkabul edilebilir değerler Ek Tablo 2&#39;de verilmiştir ve Eşitlik 1 kullanılarak standart eğri için genel standart sapmayı hesaplar. (Denklem 1)% verim = 100 x 10 (ortalama Ct Değerleri-Intercept) / Eğim. </li><li> PCR termal döngü cihazı yazılımı her standart eğri için eğimi hesaplamak etmezse, eğim ve R istatistik yazılımı kullanılarak regresyon ile 2 değerleri (örneğin Excel, SPSS, ya da SAS) belirler. Buna ek olarak, denklem I aşağıdaki standart eğriler için% verimliliğini hesaplamak </li><li> Ek Tablo 4&#39;te belirtilen kriterlere uygun standart eğrileri dayalı tüm test numuneleri için termal döngü cihazı yazılımı tarafından hesaplanan RNA kopya numarasını kaydedin. Kriterlere uygun ya da yanlış pozitif kontroller yok, standart eğrileri t şapka ile herhangi bir örnekleri yeniden. PCR inhibisyonunu izlemek için bir iç kontrol olarak eklenmiştir kolifajla MS2, Ct değerlerini kontrol edin. </li><li> RNA kopyası p sayısını belirlemekRT-qPCR Reaksiyon için kullanılan RNA (3 uL 5), bu, toplam RNA, yıkama sıvısı hacmi (25 ila 50 uL) oranında adım 7.2 hesaplanan RNA kopya sayısı ile çarpılarak er örneği. Alternatif olarak, analiz nesne yüzey alanı (cm2) toplam RNA kopya sayısına bölünmesi ile RNA yoğunluğunu hesaplamak. </li></ol> Yan yerleştirilmiş geleneksel PCR amplifikasyonu ile gerçek zamanlı RT-PCR pozitif örnek 8. Genotiplendirme <ol><li> RT-PCR testinin her bir primer seti için ana karışımı ilk turda hazırlayın (Ek Tablo 2 ve 5). </li><li> ana karışımı 20 mcL norovirus pozitif RNA 5 mcL ekleyin. </li><li> aşağıdaki koşullar altında, RT-PCR deneyinde (1) içinde oda sıcaklığında 95 ° C&#39;de, Taq polimeraz, 42 ° C (2) aktivasyonu, 15 dakika, 30 dakika boyunca aşama, ve (3) 95 ° C&#39;de 30 saniyelik 40 döngü 50 ° C&#39;de 30 saniye ve 72 ° C&#39;de 60 san. 40 döngü sonrasında, 72 ° C&#39;de ek bir 10 dakika boyunca inkübe edin. </li><li>RT-PCR testinin her bir primer seti (Ek Tablo 2 ve 5) için mater karışımı ikinci tur hazırlayın. </li><li> 23 uL ana karışımı ekleyin ve her tüp (ideal ilk tur ürünü RNaz içermeyen su içinde 1/10 seyreltilmiş olmalıdır) için (ilk turdan) ilk tur RT-PCR ürünlerinin 2 mcL ekleyin. </li><li> Adımı tekrarlayın 8.3. </li><li> (PH 8.3, 40 mM Tris, 20 mM asetik asit ve 1 mM EDTA) 100 mL 1x Tris-asetat EDTA,% 2 agaroz jel hazırlayın. Bir mikrodalga fırında karışımın ısıtılmasıyla agaroz çözüldükten sonra, 60-70 ° C&#39;ye kadar soğutulduktan sonra karışım hazırlanmış jel, 100 ml başına nükleik asit leke 10 mcL jel dökün ve tarak yerleştirin. Jel en az 30 dakika süreyle dinlendiriniz. </li><li> 6x yükleme boyası 3 uL her bir RT-PCR ürünü 15 ul karıştırın. Yük Her numunenin 15 uL ve 1 saat boyunca 100 V&#39;ta agaroz jeli Electrophorese. </li><li> Jel An uygun büyüklükte (GI için 330 bp ve GII 341 bp) tüketim hedefi RT-PCR fragmanlarıticari bir jel özütleme kiti kullanılarak RNA&#39;yı saflaştırmak d. Arıtılmış PCR ürünü, artık Sanger sekanslama için de kullanılabilir. </li></ol>

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<li>Verani, M., Bigazzi, R., Carducci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Viral+contamination+of+aerosol+and+surfaces+through+toilet+use+in+health+care+and+other+settings%5BTitle%5D)+AND+%22Am+J+Infect+Control%22%5BJournal%5D)">Viral contamination of aerosol and surfaces through toilet use in health care and other settings.</a> Am J Infect Control. 42 (7), 758-762 (2014).</li>
</ol>

Authors have no conflicting interest. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Noroviruses 18 ve 10 3 virüs parçacıkları 20 arasında% 50 insan bulaşıcı dozu var. Bu nedenle, yüzeylerin bile düşük seviyeli kirlenme bir halk sağlığı riski doğurabilir. iç çıkarma kontrolü olarak taşıma sırasında 1) farklı sürüntü malzemeleri, 2) depolama koşulu bezlerden, 3) viral RNA konsantrasyonu ve 4) kolifaj MS2: çubukla örnekleme protokolünün çeşitli yönleri de dahil olmak üzere değerlendirildi. Yakın zamana ...

<html> İnsan Noroviruses salgın ve sporadik akut gastroenterit, dünya çapında 1, 2, 3 lider nedenidir. Virüs son derece bulaşıcıdır ve iletim kişi etkileşim ya da dolaylı kirlenmiş yiyecek, su ya da çevresel yüzeylerle temas yoluyla doğrudan kişi aracılığıyla gerçekleşir. Noroviruses uzun süre tutacak ve uzun süreli çevresel yüzeyler üzerinde virüs yaşam 1, 2, 3, belgelenmiştir edilebilir. Pik ağızlık açma işlemi esnasında, virüs partiküllerinin milyarlarca dışkıların gramı başına serbest bırakılır, ve kusmuk da enfeksiyonu 4, 5, 6, 7, 8 neden viral parçacıkların yeterli sayı içerir,ef &quot;&gt; 9, 10. Buna ek olarak, cansız yüzeyler ve insan cilt arasındaki virüsün transferi kolayca oluşabilir 2, 11, 12. Dolayısıyla, çevresel kirlilik izleme salgını soruşturmalarda yardımcı ve temiz-up etkinliğini değerlendirmede olabilir ve dezenfeksiyon işlemleri. Bir çok çevresel numune alma protokolleri rotavirüs saptanması için tarif edilmiştir, kolifaj MS2, kedigiller calicivirüs (FCV) ve bakteriyofaj P22 13, 14, 15, 16. Ancak, hızlı kurumaya (&lt;1 saat) ve küçük yüzey alanları da dahil olmak üzere bu çalışmalarda, açıklanan doğrulama koşulları (25 x 100 cm 2), yeterli alan ayarlarını temsil etmeyebilir. Buna ek olarak, Çevreyi düşük kirlilik seviyeleri tahminnmental yüzeyler çok az virüs partiküllerini tespit edebiliyoruz protokolleri gerektirir. Biz Norovirüs tespiti ve yazmak için bir macrofoam esaslı yüzey örnekleme yöntemi geliştirdi. Bu yöntem, birkaç norovirus salgınlar sırasında valide edilmiştir. protokol içerir 1) (2) nasıl en iyi laboratuvara toplama ve nakliye sırasında numunelerin bütünlüğünü korumak için, ve norovirusa 3) laboratuvar testleri ve yazarak çevresel yüzeylerden sürüntü örnekleri toplamak için nasıl.

<table><tbody><tr><td>&lt;strong&gt;Generic name for kits&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Macrofoam swab&lt;/strong&gt;</td><td>Premoistened EnviroMax Swab kit&amp;nbsp;</td><td>Puritan</td><td>2588060PFUW</td></tr><tr><td>&lt;strong&gt;RNA Lysis buffer&amp;nbsp;&lt;/strong&gt;</td><td>CDC UNEX buffer</td><td>Microbiologics</td><td>Cat No MR0501</td></tr><tr><td>&lt;strong&gt;RNA extraction spin column&lt;/strong&gt;</td><td>Midi column</td><td>Omega Biotek</td><td>Cat No R6664-02</td></tr><tr><td>&lt;strong&gt;RNA purification spin column&lt;/strong&gt;</td><td>Zymol RNA Clean and Concentrator kit&amp;nbsp;</td><td>Zymo Research</td><td>Cat No R1016</td></tr><tr><td>&lt;strong&gt;Real time RT-PCR kit&lt;/strong&gt;</td><td>AgPath kit One-Step RT-PCR Kit</td><td>Life Technologies</td><td>Cat No 4387391</td></tr><tr><td>&lt;strong&gt;Conventional RT-PCR kit&lt;/strong&gt;</td><td>Qiagen one step RT-PCR kit</td><td>Qiagen kit</td><td>Cat No 210212</td></tr><tr><td>&lt;strong&gt;Gel extraction kit&lt;/strong&gt;</td><td>Qiagen QIAquick gel extraction kit</td><td>Qiagen kit</td><td>Cat No 28704 or 28706</td></tr><tr><td></td><td>Coliphage MS2</td><td>ATCC</td><td>Cat No 15597-B1</td></tr><tr><td></td><td>RNA run-off transcripts</td><td></td><td></td></tr><tr><td></td><td>Realtime PCR platform</td><td>Applied Biosystems</td><td>Model ABI 7500</td></tr><tr><td></td><td>Optical 96-well reaction plate</td><td>Thermo Scientific</td><td>Cat No 4316813</td></tr><tr><td></td><td>&lt;a href=&quot;http://www.thermofisher.com/order/catalog/en/US/adirect/%20https:/www.thermofisher.com/order/catalog/en/US/adirect/lt?cmd=catProductDetail&amp;amp;showAddButton=true&amp;amp;productID=4306311&amp;amp;_bcs_=H4sIAAAAAAAAAI1RTWvDMAz9Nb4srDg2yXpNW3rZVyHbuRhHSQx2HWwnJf9%2BUkO6scMYGEmWnqX3%0A5Mec8e0p%2BGbUKWZMlFkNYTIa4h%2F5PqWByYqJI57r9bpJPQTnWxPRb7R3mB4jGrig6b0DdD40WOyT%0As9iBCUmHb1MYge78iYi06ZznPEd0exacF6LkD5h%2BNTr4yg1II0DH5C6rmh6imSA7GuuInsIq5TUy%0AjD6Yf%2FKU1Zav3NBrlZT13cr8s0ajGhNAJ4xsYvKoXcPkAYEHEwer5jrNFuWVdF1ieajNpbPwAhPY%0AvUrQ%2BTAjAt88w4zlm8Ly%2FYRha2yiyeUSUJM35ajHt%2BZiUS0KuWNityrHkLSjuyHUDbkgfuxgbfwx%0AD9Q0vydo9rrs4te675%2FTKhvpd74AhB0C9yUCAAA%3D&amp;amp;returnURL=http%3A%2F%2Fwww.thermofisher.com%3A80%2Forder%2Fcatalog%2Fen%2FUS%2Fadirect%2Flt%3Fcmd%3DcatDisplayStyle%26catKey%3D101%26filterType%3D1%26OP%3Dfilter%26filter%3Dft_1101%252Ff_2005260*&quot;&gt;MicroAmp Clear Adhesive Film&amp;nbsp;&lt;/a&gt;</td><td>Thermo Scientific</td><td>Cat No 4306311</td></tr></tbody></table>

swab sampling method for the detection of human norovirus on surfaces

İnsan Noroviruses salgın ve dünya çapında dağınık gastroenteritin önde gelen nedenidir. En enfeksiyonlar ya çevresel yüzeylerde ya da gıda yoluyla dolaylı olarak kişiden kişiye yolla doğrudan yayılır ya, çünkü kirlenen fomitler ve cansız yüzeyler norovirus salgınları sırasında virüsün yayılması için önemli araçlardır. Biz geliştirdik ve algılama ve sert yüzeylerde insan Noroviruses bir tiplemesi için macrofoam bezlerden kullanarak bir protokol değerlendirildi. Fiber uçlu bezlerden veya antistatik mendil ile karşılaştırıldığında, macrofoam bezlerden 700 cm2 klozet yüzeylerinden virüs kurtarma (aralık 1,2-33,6%) izin verir. Protokol spin sütunları kullanılarak viral RNA&#39;nın bezlerden virüsün çıkarma ve bir daha konsantrasyon yapılması adımlarını içerir. norovirus gastroenterit olmuştu yolcu gemileri ve uzun süreli bakım tesislerinde yüzeylerinden toplanan olmuştu 217 sürüntü örneklerinin toplam olarak, 127 (% 58.5)RT-qPCR GII norovirusa için pozitif test bildirildi. Bu 29 (% 22.8) başarıyla genotip olabilir. Sonuç olarak, klinik örnekler mevcut olmadığında virüsün saptanması yanı sıra salgınlar sırasında çevresel kirlilik düzeyinin belirlenmesinde yardımcı olabilir geliştirilen protokolü kullanılarak çevresel yüzeylerde norovirusa tespiti; aynı zamanda iyileştirme stratejilerinin etkinliğinin izlenmesi kolaylaştırabilir.

Şekil 1 sürüntü örnekleme protokolünün bir akış şemasını göstermektedir. Bu protokol dört ana aşamadan oluşmaktadır; 1) numune toplama, 2) örnek depolama ve nakliye, 3) viral RNA arıtma ve konsantrasyon ve 4) RT-qPCR tahlil ve genotiplendirme. <img alt="Şekil 1" src="/files/ftp_upload/55205/55205fig1.jpg" /> Şekil 1: norovirusa çevresel yüzey örnekleme için nihai protokol?...

Watch this Scientific Journal Video about Swab Sampling Method for the Detection of Human Norovirus on Surfaces at JoVE.com

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Yüzeyler İnsan Norovirus Tespiti sürüntü Örnekleme Yöntemi

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

swab-sampling-method-for-the-detection-of-human-norovirus-on-surfaces

Research

JoVE Journal

Immunology and Infection

19.6K Görüntüleme.  Centers for Disease Control and Prevention. A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces. 

Video: Swab Sampling Method for the Detection of Human Norovirus on Surfaces

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

Enterovirus Kültür ve RT-qPCR Su Norovirüs Oluşum EPA Metot 1615 Ölçüm. Virüs Örneklerinin I. Koleksiyonu

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Çevre

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

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 ...

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

Egzersizle indüklenen Lenfosit Apoptoz belirlenmesi için parmak sopa Kan Örnekleme Metodolojisi

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

Taqman By Çocuk Tabureler gelen Noroviruses tespiti ve Genogrouping Tek aşamalı RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

Tıp

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

Viral kapsid yapısal bütünlük belirlenmesi için alternatif Vitro yöntemleri

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

Akış Sitometrikullanarak Commensal Bakterilere Bağlanan İnsan Norovirüs Benzeri Partiküllerin Ölçülmesi

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

Kentsel Çevrenin Taranması - Çevre Rezervuarlarından SARS-CoV-2'nin Örneklemesi ve Tespiti için Bir Boru Hattı

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

Biyoloji

An Assay to Quantify the Binding of Human Norovirus VLPs to Intestinal Bacteria

After preparing the reagents, antibodies, and bacteria as described in the manuscript, assemble all the materials in a BSL II biosafety cabinet.
Virus-like particles for human norovirus are listed as BSL II pathogens, and all work performed using VLPs should be conducted in a certified biosafety cabinet.
Add 10 micrograms of human norovirus VLPs to each tube of bacteria, and mix thoroughly by pipetting. Incubate the tubes for 1 hour at 37 degrees Celsius with constant rotation. After incubation, centrifuge the tubes at 10,000 times g for 5 minutes.
Aspirate and discard the supernatant and resuspend the bacterial pellet in 1 milliliter of PBS. After repeating the wash steps once, centrifuge the tubes again at 10,000 times g for 5 minutes. Discard the supernatant and resuspend the VLP bacteria pellet in 150 microliters of 5% blocking buffer.
A common error that occurs is that tubes are swapped and the wrong treatment is added. To prevent this, arrange all tubes in lines that correspond to the correct treatment, and add one treatment all at once to the tubes.
For each bacterial sample, prepare 50 microliters of diluted human norovirus G2 antibody. Using 5% blocking buffer, dilute the antibody 1 to 125 for E. coli case samples. Prepare 50 microliters of diluted isotype antibody for each bacterial sample using the same dilution ratios. Divide each attachment assay sample into 350-microliter aliquots by transferring them into clean 1.5-milliliter centrifuge tubes.
To create unstained controls, add 50 microliters of blocking buffer to the first aliquot from each bacterial sample. For the stained samples, add 50 microliters of the G2 antibody dilution to the second set of aliquots. Create the isotype controls by adding 50 microliters of the diluted isotype antibody to the third set of aliquots.
Incubate all the samples on ice and in the dark for 30 minutes. Then, centrifuge all the samples at 10,000 times g for 5 minutes. Discard the supernatants and resuspend each sample in 150 microliters of FCSB. Again, centrifuge all the samples at 10,000 times g for 5 minutes. Again, discard the supernatants and resuspend the samples in 100 microliters of FCSB.
After centrifuging the samples one final time and discarding the supernatant, resuspend the samples in 150 microliters of FCSB. Transfer each sample to a tube containing 400 microliters of FCSB for a total volume of 550 microliters. Store the samples at 4 degrees Celsius until they are analyzed by flow cytometry.

Yüzeyler İnsan Norovirus Tespiti sürüntü Örnekleme Yöntemi

Özet

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