Name: cost-effective method for microbial source tracking using specific human and animal viruses
Uploaded: 2011-12-03T14:00:00
Description: Watch this Scientific Journal Video about Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses at JoVE.com

Эта работа была частично поддержана правительством Испании &quot;Ministerio де Educación у Ciencia» (проект AGL2008-05275-C01/ALI), Европейским Союзом Рамочной исследовательской 7 проектов, финансируемых VIROBATHE (контракт № 513648), VIROCLIME (контракт № 243923 ) и Агентством каталонского Воды, Agencia Catalana де l&#39;Aigua (ACA), Департамент управления де я Millora ДЕЛЬЗ Ecosistemes водных видов спорта. Во время исследования разработан Мартой Rusiñol был членом правительства каталонской &quot;AGAUR&quot; (FI-DGR).

1. Концентрация вирусной частицы, присутствующие в пробах воды <ol><li> Сбор и кондиционирования проб воды <ol><li> Сбор минимум 2 повторов 10 л на один образец в пластиковых контейнерах с плоским дном и один дополнительный образец в качестве управления процессами. Последний пример будет подс?...

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Microbiol. 68, 4523-4533 (2002).</li><li>Hundesa, A., Bofill-Mas, S., de Motes, M. a. l. u. q. u. e. r., Rodriguez-Manzano, C., Bach, J., Casas, A., M, ., Girones, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+PCR+assay+for+the+quantitation+of+bovine+polyomavirus+as+a+microbial+source-tracking+tool.">Development of a quantitative PCR assay for the quantitation of bovine polyomavirus as a microbial source-tracking tool.</a> J. Virol. 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<html> Процедура, описанная выполнит условия для установки методом для рутинной окружающей среды и лаборатории общественного здравоохранения: воспроизводимый, надежный, простой и экономически эффективной. Протокол прост, однако он должен быть тщательно следили. Низкая проводимость в об...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер по каталогу </td><td> Комментарии </td></tr><tr><td> Высокая скорость центрифуги (8000 мкг) </td><td> Berckman Coulter </td><td> Avanti J-20XP </td><td></td></tr><tr><td> рН-метр, термометр и conductimeter </td><td> Afora </td><td> LPPC3003 </td><td></td></tr><tr><td> Пластиковые трубы длиной 100-200 см </td><td> Deltalab </td><td> 350059 </td><td></td></tr><tr><td> Стерильные окончил одноразовые пипетки </td><td> Labclinics </td><td> PN10E1 </td><td></td></tr><tr><td> Стерильные пластиковые трубы в 1,5 и 10-15 мл (Эппендорф, Соколов и др.) </td><td> Afora </td><td> KA298/00 </td><td></td></tr><tr><td> Центрифуга горшки (500 мл) </td><td> Fisher Scientific </td><td> SE5753512 </td><td></td></tr><tr><td> Магнитные мешалки и магниты (по одному наобразца) </td><td> Fisher Scientific </td><td> 10510 </td><td></td></tr><tr><td> Стеклянные или пластиковые контейнеры с плоским дном, чтобы разрешить использование магнитной мешалки </td><td> Deltalab </td><td> 191642 </td><td></td></tr><tr><td> Перистальтического насоса для удаления надосадочной (или водоструйный вакуумный насос) </td><td> Watson-Marlow </td><td> 323E / D </td><td></td></tr><tr><td> Таймер на отключение шевелясь после 8-10 часов </td><td> Deltalab </td><td> 900400 </td><td></td></tr><tr><td> Соляная кислота (1н и 0,1) </td><td> Panreac </td><td> 141020.1611 </td><td></td></tr><tr><td> Гидроксид натрия (1N) </td><td> Panreac </td><td> 131687.1211 </td><td></td></tr><tr><td> Искусственные морской соли морской воды </td><td> Сигма </td><td> S9883 </td><td></td></tr><tr><td> Обезжиренное молоко (SM) </td><td> Difco </td><td> 232100 </td><td></td></tr><tr><td> Фосфатный буфер рН = 7, 5 </td><td></td><td></td><td> 1:02 об. / стерильных Na 2 HPO 4 0,2 М и NaH 2 PO 4 0,2 М при рН 7,5 </td></tr><tr><td> Тиосульфата </td><td> Panreac </td><td> 121879.1209 </td><td> Сделать 10% раствор в воде </td></tr><tr><td> QIAamp Вирусная РНК Mini Kit </td><td> Qiagen </td><td> 52904 </td><td></td></tr><tr><td> 96-и оптические пластины реакции (500 единиц) </td><td> Applied Biosystems </td><td> 43426659 </td><td></td></tr><tr><td> Оптический клей охватывает (100 единиц) </td><td> Applied Biosystems </td><td> 4311971 </td><td></td></tr><tr><td> TaqMan Экологическая ПЦР 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

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.

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

Исследование описывает эффективный метод для идентификации источника фекальных / мочи заражения или загрязнения нитратами в воде с помощью КПЦР для конкретного количественного человека / свинины / бычьей ДНК-вирусы, аденовирусы и polyomaviruses, предложил в качестве инструментов MST.

Экономически эффективный метод для отслеживания источника микробного Использование Конкретные человека и животных вирусов

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

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