Development of a DNA microarray for the simultaneous detection and genotyping of noroviruses.
Current methods for detecting and genotyping noroviruses focus on the use of reverse transcriptase (RT)-mediated PCR. A major drawback of this approach is that short target RT-PCR products do not always encompass sequences that can be compared among research laboratories, resulting in difficulties for molecular epidemiology. We describe the use of a microarray-based system for simultaneous detection and molecular characterization of noroviruses. The protocol generates a 917-bp RT-PCR product that encompasses two major regions currently used for detection and analysis of norovirus genomes. The PCR products are then hybridized to an oligonucleotide array (NoroChip) based on 50-mer features, which allows for both confirmation of reaction specificity and molecular characterization of the amplified genome. Parallel sequence analyses of amplicons revealed that our microarray data were robust in separating genogroups I and II, and further subtyping to the cluster level was possible. This approach, combining detection and characterization, overcomes the need for expensive and time-consuming sequence analysis of amplified genome targets for molecular epidemiology.
Pagotto
Franco
F
Microbiology Research Division, Bureau of Microbial Hazards, Food Directorate, Health Products and Food Branch, Health Canada, Sir FG Banting Research Centre, P.L. 2204E, Ottawa, Ontario, Canada. franco_pagotto@hc-sc.gc.ca
Corneau
Nathalie
N
Mattison
Kirsten
K
Bidawid
Sabah
S
D000818:Animals; D016000:Cluster Analysis; D005516:Food Microbiology; D005838:Genotype; D006801:Humans; D008970:Molecular Weight; D029322:Norovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity