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Artur Rze_zutka and Nigel Cook
enhance the sensitivity and specificity of the assay (Loisy et al., 2005). The constant need for the development of more sensitive methods results in new protocols utilizing non-traditional PCR nucleic acid detection techniques. For example, the method developed by Schwab et al. (2001) for detection of NoV and HAV in shellfish join together RTPCR and ELISA, where RTPCR-generated amplicons were detected by virus-specific oligoprobes in an enzyme-linked immunosorbent assay. The most promising methods for virus detection are real-time PCR (Jothikumar et al., 2005; Butot et al., 2007) or NASBA (Jean et al., 2004). These methods are fast, highly specific, and have the potential to detect single virus particles. VI.A.1. Molecular typing of noroviruses Most human caliciviruses belong to the genus Norovirus, which is further divided into genogroups and genotypes. All of the isolated NoV strains were classified to one from over 15 currently recognized genotypes. The identification of each virus genotype detected in clinical or food samples is difficult, because it requires the application of advanced and expensive methods. The present classification of noroviruses, as well as their division into particular genotypes, is based on molecular differences and homology across the capsid protein or RNA dependent RNA polymerase viral genes (Vennema et al., 2002; Vinje et al., 2004). These genes were shown to be the most conserved among all up-to-date characterized NoV genotypes (Duizer and Koopmans, 2006). However, not all currently available PCR-based assays for NoV detection allow typing of viruses. The variability of the NoV genome causes difficulties in the development of the single generic detection test, which is able to identify the NoV genogroup or strain (Koopmans and Duizer, 2004). Therefore some methods utilize additional procedures such as probe hybridization (Rosenfield and Jaykus, 1999; Schwab et al., 2000; Schwab et al., 2001; Sair et al., 2002; Le Guyader et al., 2004) or sequence analysis of the amplified genome fragment (Schwab et al., 2000; Le Guyader et al., 2004) to identify the NoV genotype. Another example is multiplex NASBA for NoV detection and genogroup identification in meat samples (Jean et al., 2004). Nowadays, there is a new perspective based on DNA microarray technology, which undoubtedly will become an alternative method to sequence analysis in the near future. Both methods (sequencing and microarray) consist of several steps, where RTPCR amplification of chosen gene fragments takes place, followed by sequencing or hybridization of the specific oligonucleotide probes to the immobilized DNA. Oligonucleotide microarrays are able to detect single-point mutations in the analyzed sequence, and are useful in epidemiological or phylogenetic studies (Kostrzynska and Bachand, 2006). A distinct advantage of microarray technology is a simultaneous detection