Read e-book online SARS- and Other Coronaviruses: Laboratory Protocols PDF

By Leen Vijgen PhD, Elien Moës, Els Keyaerts PhD, Sandra Li, Marc Van Ranst MD, PhD (auth.), Dave Cavanagh (eds.)

ISBN-10: 1588298671

ISBN-13: 9781588298676

ISBN-10: 1597451819

ISBN-13: 9781597451819

In 2003, the observe "coronavirus" unfold around the globe, just a little additional than the virus that sparked the panic. In SARS- and different Coronaviruses: Laboratory Protocols, specialist researchers research those devastating viruses via distinctive laboratory protocols. Chapters take care of such matters as detection and discovery of coronaviruses via nucleic acid and antibody-based methods, virus isolation, propagation and titration, virus purification, constitution research by way of electron cryomicroscopy, expression and crystallization of viral proteins, elevating antibodies opposed to viral proteins, manipulation of the coronavirus genome, and outlines of ways to enquire facets of the mobilephone floor for coronavirus receptors. Composed within the hugely winning Methods in Molecular Biology™ sequence layout, each one bankruptcy encompasses a short creation, step by step equipment, an inventory of important fabrics, and a Notes part which stocks tips about troubleshooting and keeping off identified pitfalls.

Comprehensive and state-of-the-art, SARS- and different Coronaviruses: Laboratory Protocols serves as a great advisor for all virologists and particularly for these operating with coronaviruses.

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1999) Phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay Virus Res. 60, 181–189. 8. Jonassen, C. , Jonassen, T. , Sveen, T. , and Grinde, B. (2003) Complete genomic sequences of astroviruses from sheep and turkey: comparison with related viruses Virus Res. 91, 195–201. 4 RT-PCR Detection of Avian Coronaviruses of Galliform Birds (Chicken, Turkey, Pheasant) and in a Parrot Francesca Anne Culver, Paul Britton, and Dave Cavanagh Abstract Of the many primer combinations that we have investigated for the detection of avian coronaviruses, two have worked better than any of the others: they worked with the largest number of strains/samples of a given coronavirus and the most species of avian coronavirus, and they also produced the most sensitive detection tests.

Agarose Gel Electrophoresis 1. Place a gel-casting tray onto a gel-casting base. Level the base. 2. 75 g of agarose powder. Add it to a 250-ml bottle containing 50 ml 1X TAE buffer. Microwave the bottle with a loosened cap until the gel starts to bubble and become transparent (see Note 10). 2 ␮M 1 U/reaction – – 20 Poon and Peiris Table 3 Conditions for the PCR Reaction Step 1. Heat activation 2. Thermal cycling (45 cycles) Denaturing step Annealing step Extension 3. Final extension 4. Soak Temperature Time ◦ 94 C 10 min 94◦ C 48◦ C 72◦ C 72◦ C 4◦ C 30 sec 30 sec 40 sec 2 min ∞ 3.

Thermocyclers (MJ Research) are used both for RT and PCR. 2. Sequencing and Analysis 1. Agarose (Applied Biosystems) for visualization of PCR products. 2. 3). 3. 68 is used to check for primer hairpin structures and primer dimers and to calculate annealing temperatures for PCR and cycle sequencing. Detection of Coronavirus Genomes Harboring 3’ s2m Sequence 29 4. 1 (Applied Biosystems) is used for sequencing of short (from initial PCR) or long (5 -RACE PCR) PCR products, respectively. 5. Cycle sequencing reaction is performed on an MJ Research thermocycler.

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SARS- and Other Coronaviruses: Laboratory Protocols by Leen Vijgen PhD, Elien Moës, Els Keyaerts PhD, Sandra Li, Marc Van Ranst MD, PhD (auth.), Dave Cavanagh (eds.)

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