Polony Detection and Genotyping of Hepatitis A Virus from Frozen Raspberries Possibly Implicated in an Acute Case of Hepatitis A

Introduction: Hepatitis A virus (HAV) is one of the leading causes of foodborne infections. HAV can be transmitted by ingestion of contaminated food or water as well as direct contact with HAV-infected individuals through the fecal-oral route. It is highly critical to develop rapid and sensitive detection methods for HAV in food samples in order to prevent and control HAV-related foodborne diseases.

Purpose: In this study, we described a polymerase colony (polony) method for simultaneous detection and genotyping of HAV from frozen raspberries that possibly implicated in an acute case of hepatitis A.

Methods: RNA extraction was performed on frozen raspberries (collected in New Hampshire outbreak, July 2013) by ultracentrifugation concentration and QiaAMP viral RNA mini kit. Polony assay was used to detect and genotype the virus. Additional analysis using nested reverse transcription PCR (RT-PCR) was completed to confirm the presence of HAV, and obtain the amplicon sequence information for phylogenetic analysis and confirmation of virus genotype.

Results: HAV was detected and identified as sub-genotype IB in the frozen raspberries using polony approach. This approach amplifies multiple individual cDNA molecules reverse transcribed from viral RNA to produce immobilized colonies within a thin acrylamide gel on a microscope slide. The resulting polonies were then genotyped by single base extensions with fluorescent dye-labeled nucleotides. This sample was also tested positive for HAV by nested RT-PCR. Phylogenetic sequence analysis of the nested RT-PCR amplicon from the sub-genomic region of VP1/P2A confirmed that the virus belonged to sub-genotype IB.

Significance: The polony method was designed to be an effective tool with the ability to simultaneously detect and genotype HAV embedded in the food matrix in a single assay.