Limited sequence information and assay development

The issues concerning sequence variation can be further complicated by the fact that sequence information on public databases may comprise only a limited number of viral strains usually from discrete geographic locations. Therefore, the development of PCR assays based on such limited information may affect the sensitivity of assay performance. This can be most pronounced for newly detected viruses. Following the initial characterisation of human metapneumovirus (van den Hoogen et al., 2001), our laboratory developed a PCR assay to detect this virus in clinical specimens. This assay used a 5' nuclease probe targeting the nucleocapsid protein gene (hMPV-N-5N; Mackay et al., 2003), and was developed using the limited sequence data present on Genbank at the time. However, subsequent studies revealed that human metapneumovirus comprised at least four genetic lineages (A1, A2, B1 and B2) with considerable sequence variation between these lineages (van den Hoogen et al., 2004). As a result we found that the hMPV-N-5N assay failed to detect some human metapneumovirus strains of the B lineage. Sequencing revealed that mismatches were present between the primers and probe and the nucleocapsid gene sequences of some human metapneumovirus B viral strains, and that these were responsible for the limitations of the hMPV-N-5N assay. Subsequently another hMPV 5' nucle-ase assay targeting the human metapneumovirus nucleocapsid protein gene was described by Maertzdorf et al., (2004), and reportedly was capable of detecting human metapneumovirus from all known genetic lineages. We have since used this assay for the detection of human metapneumovirus lineage B strains in our local patient population. Overall, the initial limitations of our hMPV-N-5N assay was entirely due to the fact that the assay was designed using limited sequence data available on Genbank at the time, and predominantly comprised human metapneumovirus type A sequences.

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