Sensitive Genotyping of Foodborne-Associated Human Noroviruses and Hepatitis A Virus Using an Array-Based Platform

Abstract

Human noroviruses (NoV) are the leading cause of human gastroenteritis in populations of all ages and are linked to most of the foodborne outbreaks worldwide. Hepatitis A virus (HAV) is another important foodborne enteric virus and is considered the most common agent causing acute liver disease worldwide. In the present study, a focused, low-density DNA microarray was developed and validated for the simultaneous identification of foodborne-associated genotypes of NoV and HAV. By employing a novel algorithm, capture probes were designed to target variable genomic regions commonly used for typing these foodborne viruses. Validation results showed that probe signals, specific for the tested NoV or HAV genotypes, were on average 200-times or 38-times higher than those detected for non-targeted genotypes, respectively. To improve the analytical sensitivity of this method, a 12-mer oligonucleotide spacer sequence was added to the capture probes and resulted in a detection threshold of less than 10 cRNA transcripts. These findings have indicated that this array-based typing sensor has the accuracy and sensitivity for identifying NoV and HAV genotypic profiles predominantly linked to food poisoning. The implementation of this typing sensor would thus provide highly relevant and valuable information for use in surveillance and outbreak attribution.

Keywords: food safety; foodborne pathogen; genotyping; hepatitis A virus; microarray; norovirus; pathogen detection; viruses.

Figure 1

Schematic diagram of the genomic regions in (a) NoV and (b) HAV targeted by the capture probes in the array-based typing assay.

Figure 2

Steps of the VGM algorithm for the design of capture probes, detecting distinct genotypes of NoV or HAV.

Figure 3

Steps of the array-based method for detecting distinct genotypes of NoV or HAV. The starting material was an RNA sample subjected to RT-PCR, purified, and enzymatically digested to remove the non-complementary strand. The hybridization steps was followed by the microarray labeling and signal amplification and quantification steps. Sample-to-result time is below 8 h.

Figure 4

Hybridization signals of the tested NoV and HAV genotypes at 10² cRNA transcript copies in the absence (grey bars) or presence (blue bars) of a 12-mer oligonucleotide spacer sequence attached to the capture probe. The sensitivity detection threshold of the array-based typing method was set at 4000 fluorescent counts and is indicated by the dashed line.

Figure 5

Hybridization signals of various amounts of in vitro cRNA transcripts from (a) NoV GI genotypes; (b) NoV GII genotypes; and (c) HAV genotypes were tested using the array-based typing method. The sensitivity detection threshold of the typing method is indicated by the dashed line.