Экспресс-диагностики вируса птичьего гриппа у диких птиц: Использование портативных-ПЦР и сублимированные реагенты в поле
Summary
Это исследование описывает диагностики птичьего гриппа у диких птиц с помощью портативного-ПЦР системы. Метод использует лиофилизированной реагентов для скрининга диких птиц в не лабораторных условиях, характерных для вспышки сценарию. Использование молекулярных инструментов обеспечивает точные и чувствительные альтернатив для экспресс-диагностики.
Abstract
Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.
Protocol
Discussion
Метод экспресс-диагностики, представленные здесь облегчает эффективный по времени и точное тестирование образцов диких птиц для наблюдения ВГП. Гораздо менее строгие требования к хранению образцов переносного-ПЦР подходят для удаленных ситуациях, когда поддержание холодовой цепи м?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Мы хотели бы поблагодарить М. Скаллион и Р. Крисп Айдахо технологии для технической поддержки и USGS западных экологических исследований Центра финансирования (С. Шварцбах) и помощь (К. Spragens, Т. Грэм). Это исследование проводилось под эгидой Центра инновационных технологий – Институт по вопросам обороны и Национальная Безопаность (www.idhs.org), в поддержке Министерства обороны и Исследовательская лаборатория ВВС. Животное обработки протоколов следуют утвержденным уходу и использованию животных комитета Геологической службы США Западная экологический центр исследований и разрешения американского Геологического Обзора кольцевание птиц лаборатории. Любое использование торговых, продуктов и фирменных наименований в данной публикации, в описательных целях и не подразумевает одобрения правительства США.
Materials
| Name of the reagent | Company | Catalogue Number | Comments (optional) |
|---|---|---|---|
| RNeasy mini spin column | Qiagen | 74106 | Included in RNeasy Mini Kit |
| Collection tubes (1.5 & 2 mL) | Qiagen | 74106 | Included in RNeasy Mini Kit |
| Buffer RLT | Qiagen | 74106 | Included in RNeasy Mini Kit |
| Buffer RW1 | Qiagen | 74106 | Included in RNeasy Mini Kit |
| Buffer RPE | Qiagen | 74106 | Included in RNeasy Mini Kit |
| RNase-free water | Qiagen | 74106 | Included in RNeasy Mini Kit |
| 14.3 M β-mercaptoethanol solution | Fisher Scientific | BP176100 | |
| 100% ethanol | Fisher Scientific | NC9602322 | |
| Vortex Genie 2, 120V | Scientific Industries | SI-0236 | |
| Taqman Influenza A Target 1 (Hydrolysis Probe) | Idaho Technologies | ASAY-ASY-0109 | |
| Lightcycler 20ml capillary tubes | Roche Applied Science | 04929292001 | |
| Micro-centrifuge with rotator for 2 ml tubes | Idaho Technologies | Included in RAPID kit | |
| Ruggedized Advanced Pathogen Identification Device (RAPID) 7200 | Idaho Technologies | Included in RAPID kit | |
| Pentium-based laptop with Windows XP Professional | Idaho Technologies | Included in RAPID kit | |
| Lightcycler Data Analysis software | Idaho Technologies | Included in RAPID kit |
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