It must be recognized that the success of any real-time PCR assay is highly dependent on the nature of the primer and probe target sequences utilized in the assay. These sequences need to be specific for the target virus and must be well conserved across different strains or types of the virus, otherwise the sensitivity of the assay may be compromised. However, for many viruses it can be difficult to identify such sequences. There are several reasons for this, including sequence polymorphism of the viral genome, a lack of sequence information as well as perceived changes in the clinical relevance of certain viral subtypes. Parainfluenza virus type 3, respiratory syncytial virus, human metapneumovirus, and influenza virus A provide good examples of this phenomenon.
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