Detection and Identification of Common Food-Borne Viruses with a Tiling Microarray

Chen, Haifeng; Mammel, Mark; Kulka, Mike; Patel, Isha; Jackson, Scott; Goswami, Biswendu B.

Validation of Norovirus Microarray Genotyping. To confirm the microarray genotyping result of the norovirus sample #186 (NoV#186), we performed a specific PCR with a published primer set G1SKF (5'-CTGCCCGAATTYGTA AATGA-3') and G1SKR (5'-CCAACCCARCCATTRTAC A-3'); G2SKF (5'-CNTGGGAGGGCGATCGCAA-3') and G2SKR (5'-CCRCCNGCATRHCCRTTRTACAT-3'; Y=C/T; N=A/T/G/C; R=A/G; H=A/T/C). These primer pairs were used for the amplification of genogroup I and group II norovirus. The PCR was carried out as described previously [21] . The amplicon was sequenced and its relationship to known NoV strains was determined by phylogenetics analysis program ClustalX as described by Thompson et al. [22] . A phylogenetic tree from boot-strap analysis was generated by Neighbor-Joining method. Other norovirus samples were genotyped by Dr. William Burkhardt (FDA, Dauphin Island, AL) and by Dr. Jan Vinje (CDC, Atlanta, GA) via RT-PCR. Fig. (1) shows the results of hybridization of the array probes to samples of coxsackievirus, hepatitis A virus, and norovirus samples. Coxsackievirus samples CXKV B2, B3, B4 and A2 demonstrated clear-cut hybridization patterns. In CXKV A5, a minimal degree of cross-hybridization to A16, B3, B5 and A7 as well as B1 were also detected. The reason is the considerable localized sequence identity between different CXKV strains. However, their signal intensities were much lower than that of the probes from the same strain to targets derived from A5 itself (58.2-fold). For HAV, strong hybridization signals are only observed in HAVderived oligoprobes and genotypic-specific probes generate high intensity signals although cross-hybridization among strains is also observed, which reflects the sequence conservation within the HAV genus.