|
| Status |
Public on Apr 01, 2020 |
| Title |
Celery_NoV_HAV |
| Sample type |
RNA |
| |
|
| Source name |
NoV/HAV-spiked celery
|
| Organism |
Apium graveolens |
| Characteristics |
treatment(s): celery + NoV + HAV
infection: human norovirus, hepatitis A virus
virus strain: Minerva, HM175/18f (ATCC VR1402)
virus genotype: GII.4, IB
|
| Treatment protocol |
HAV/18f culture supernatant was collected and used for inoculation. NoV GII.4 Minerva stool sample was suspended in 10% phosphate-buffered saline (PBS), centrifuged at 9000 ×g for 3 min, and the supernatant was collected and used for inoculation.
|
| Growth protocol |
Confluent culture of FRhK-4 cells in 75 cm2 flasks (5 x 106 cells) were infected with 5-10 pfu/cell of HAV/18f in 1.5 ml of MEM containing 1% heat inactivated FBS. After 1 hr of adsorption, 13.5 ml of the same medium was added to each flask and Incubation continued for 5-6 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
HAV/18f was spiked onto tomatoes, and the samples were shaked in TGBE buffer and homogenized. The eluate was collected and clarified by centrifugation to remove food particulates. The supernatant was recovered, and RNA was precipitated with 70% ice cold isopropanol. The RNA pellet was collected by centrifugation and re-suspended in the lysis buffer following the instructions of RNAqueous Kit. Potassium acetate was added to precipitate any remaining polysaccharides. The extracted RNA samples were further treated with DNase and Dynabeads mRNA Purification Kit. For virus-spiked green onions and celery, material was eluted using either Glycine Buffer or TGBE buffer supplemented with pectinase, respectively, then virus particles were concentrated by ultracentrifugation and RNA was extracted with commercial kits.
|
| Label |
biotin
|
| Label protocol |
Samples were prepared and hybridized to FDA-EVIR GeneChip Tiling Array according to Affymetrix specifications.
|
| |
|
| Hybridization protocol |
Samples were prepared and hybridized to FDA-EVIR GeneChip Tiling Array according to Affymetrix specifications.
|
| Scan protocol |
The tiling arrays were scanned using an Affymetrix GeneChip scanner with a scanning resolution of 1.56 µm pixelsize according to manufacturer's instruction.
|
| Description |
ultracentrifugation: viral RNA
|
| Data processing |
The probe intensities were extracted with Affymetrix PowerTools software (APT 2.10.2.2), and then exported to Excel for further processing.
|
| |
|
| Submission date |
Nov 13, 2019 |
| Last update date |
Apr 03, 2020 |
| Contact name |
Christine Fang Yu |
| E-mail(s) |
Christine.Yu@fda.hhs.gov
|
| Phone |
1-301-796-0643
|
| Organization name |
US Food and Drug Administration
|
| Department |
Center for Food Safety and Applied Nutrition
|
| Lab |
Office of Applied Research and Safety Assessment
|
| Street address |
8301 Muirkirk Rd.
|
| City |
Laurel |
| State/province |
MD |
| ZIP/Postal code |
20708 |
| Country |
USA |
| |
|
| Platform ID |
GPL27751 |
| Series (1) |
| GSE140351 |
Capability of FDA-EVIR Microarray for Detection of Norovirus and Hepatitis A Virus in Inoculated Tomatoes, Green Onions, and Celery |
|
