It's in the chips: Development of a microarray GeneChip
approach to detect and type waterborne viruses

Nichole Brinkman and Shay Fout • Office of Research and Development, National Exposure Research
Laboratory, Microbiological and Chemical Exposure Assessment Research Division, Cincinnati, Ohio

genetically

Figure 2. Affymetrix
GenFlex GeneChip

Figure 1.

Transmission electron
micrograph of Norwalk
virus, the type strain for the
genus Norovirus. Bar = 50
nanometers. Photo Credit:
F. P. Williams, U.S. EPA

Figure 4.

Flowchart of probe labeling and hybridization to the chip. A) RT-PCR amplifies norovirus templates ( ——' ~ —• - ) for use with the tag-

probes ( 		" 1	) in a labeling reaction. B) At a specific annealing temperature, the tag-probes can anneal to the viral templates. C)

If the tag-probe matches the viral template perfectly, the tag probe will be extended by a labeled nucleotide (•). D) At the end of the labeling reaction,
the tag-probes are pooled then added to the chip. E) The tag-probes hybridize to their respective capture-probes on the chip for detection.

Preliminary Data

Table 2 shows the mean hybridization intensity values for two tag-probes

designed to have a perfect match (5 and 225) and two tag-probes

designed to have mismatches (214 and 226) to the sequence of our

Norwalk virus reference strain.

•	The perfect match tag-probes, 5 and 225, show a higher mean signal
intensity value for the labeled tag-probes as compared to the unlabeled
tag-probes.

•	The mismatched tag-probes, 214 and 226, show no difference in mean
signal intensity value between the labeled and unlabeled tag-probes.

Figure 3.

Design of tag-probes for detection of norovirus
strains. The probe sections in blue represent the
complementary sequences to the capture-probes
on the chip. The black regions represent a 4 base
pair hinge to allow flexibility. The regions of other
colors represent the sequences that match the
individual norovirus strains.

5'-

Chip sequence 1

Norovirus strain 1



Chip sequence 2

Norovirus strain 2



Chip sequence 3

Norovirus strain 3

Figure 5. Results of scanned chip.

siimbsh'.

Table 2.

Hybridization Intensity Values (Mean ± standard deviation)



Control - unlabeled tag-probes

Experimental - labeled tag-probes

Tag-probe 5

5.5 ± 2.65

199.00 ±29.51

Tag-probe 214

4.0 ±0.82

4.6 ±2.19

Tag-probe 225

5.0 ± 1.41

222.00 ±26.51

Tag-probe 226

2.67 ±2.08

5.0 ±2.0

Potential Applications

•	The ability to subtype noroviruses can provide useful information for:

•	Identifying the source of contamination in outbreak situations by comparing waterborne and
clinical isolates.

•	Occurrence data regarding the genetic diversity of noroviruses circulating in the environment.

•	Assessing risk posed by these viruses (after virulence properties are determined).

•	Since the genetic code is universal, this approach can be adapted for detection and identification of
other viruses, bacteria, parasites, algae - all the microorganisms on the CCL - in a single analysis.

Disclaimer: Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official Agency policy.

Science and Innovation to Protect Health and the Environment

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