By Erica Spackman MS, PhD (auth.), Erica Spackman MS, PhD (eds.)
With the growing to be worldwide worry of a massive pandemic, avian influenza virus examine has elevated enormously in value in this younger century. In Avian Influenza Virus, knowledgeable crew of researchers and diagnosticians study the elemental but crucial virological equipment for AI virus study and diagnostics in addition to the various most recent molecular approaches presently used for easy and utilized study. intriguing, state-of-the-art new tools specialise in learning the virus itself and paintings with avian hosts, a space vastly missing in study. Following the layout of the hugely winning Methods in Molecular Biology™ sequence, each one bankruptcy offers conveniently reproducible laboratory protocols supplying step by step guide and lists of the required gear for the task.
Comprehensive and well timed, Avian Influenza Virus equips diagnosticians and researchers with the present instruments and data they should research extra approximately this excessive impression disease.
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Reagent-grade H2O (nuclease-free). 3. TE buffer (for probe reconstitution). 4. Qiagen OneStep RT-PCR Kit (reverse transcriptase, taq polymerase, dNTPs, reaction buffer) or equivalent reagents (see Note 2). 5. Dual-labeled probes (see Note 3) (Table 1). 6. Primers (see Note 4) (Table 1). 7. MgCl2 (25 mM stock). 8. RNase inhibitor (13 units/µL stock). 9. Positive control RNA. 10. 25-µL Smart Cycler tubes or appropriate reaction vessel for the real-time PCR instrument being used. 3. 1. Primer and Probe Handling and Dilution 1.
The HA assay is not an identification assay. , paramyxoviruses and adenovirus) and certain bacteria also have hemagglutinating properties [3–5]. The HA assay should be followed by a hemagglutinationinhibition assay (see Chapter 8) to determine the type and/or subtype of virus From: Methods in Molecular Biology, Vol. 436: Avian Influenza Virus Edited by: Erica Spackman © Humana Press, Totowa, NJ 47 48 M. L. Killian present. The HA assay does not necessarily indicate the presence of a viable virus .
L. Suarez The general principle of amplification of viral RNA to detectable levels of PCR DNA is the same for real-time PCR as it is for standard PCR. The difference between the two methods is in how the PCR product is detected. , during each PCR cycle). Numerous chemistries are available for real-time PCR. The type A influenza test reported here uses hydrolysis probes, also called dual-labeled probes or Taqman probes. In the hydrolysis/dual-labeled probe system, in addition to the target-specific PCR primers, a DNA probe with a sequence complementary to the amplification target is labeled with a fluorogenic reporter dye on one end (usually the 5′ end) and a quencher dye on the other end (usually the 3′ end).
Avian Influenza Virus by Erica Spackman MS, PhD (auth.), Erica Spackman MS, PhD (eds.)