Development and evaluation of a generic tag array to detect and genotype noroviruses in water.
Noroviruses are the leading cause of nonbacterial gastroenteritis outbreaks in the United States, some of which are caused by the ingestion of contaminated water. Detection and genotypic characterization of noroviruses is commonly performed by reverse transcription-polymerase chain reaction (RT-PCR) followed by sequencing. However, sequencing of products amplified from environmental water samples is often hindered by the co-amplification of non-specific cDNA. To overcome this issue, a generic microarray was evaluated to genotype noroviruses by probe hybridization. RT-PCR amplicons are used in a single base extension (SBE) reaction where genotype-specific probes are labeled and then hybridized to an Affymetrix GeneChip GenFlex Tag Array for detection. Using a standardized, multiplex SBE reaction, genotyping of representative strains was accomplished through the identification of genotype-specific patterns of positive hybridization results. Furthermore, the SBE-GenFlex array method was successful in the genotype identification of noroviruses seeded into tap and Ohio River water samples. This study shows the utility of using a microarray to genotype noroviruses in complex environmental matrices.
Brinkman
Nichole E
NE
U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, Biohazard Assessment Research Branch, 26 West Martin Luther King Drive, MS 320, Cincinnati, OH 45268, USA. brinkman.nichole@epa.gov
Fout
G Shay
GS
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D015342:DNA Probes; D004784:Environmental Monitoring / Q000379:methods; D005838:Genotype; D029322:Norovirus / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D010948:Viral Plaque Assay; D014867:Water / Q000032:analysis*
D015342:DNA Probes; D012367:RNA, Viral; D014867:Water