Hydrogel affinity particles to enable high throughput screening of soft fruits for intact HAV virions

Current food safety testing for human viruses makes use of molecular methods (i.e., polymerase chain reaction, PCR) to detect virus genetic material on fruit. One important limitation of PCR-based methods is that they detect environmental or “free” genetic materials that are not contained within intact virus particles and therefore pose no risk of causing an infection. This limitation can cause large economic losses for soft fruit growers, buyers, and sellers associated with product recalls and introduces large uncertainty into food safety management decisions. Here we propose the use of Nanotrap particles (hydrogel nanoparticles that are design to capture intact viruses) to capture only intact viruses during soft fruit testing. If successful, these Nanotraps would improve the accuracy of food safety testing by excluding free RNA from the PCR-based analysis. The proposed method can be performed using automated equipment which would greatly increase the number of fruit samples that could be tested while simultaneously decreasing the time and costs required to produce the results. The novel method proposed would deliver benefits to consumers, regulators, growers, buyers, and sellers by improving the reliability of food safety testing to detect infectious viruses and inform risk management decisions.

Currently screening of soft fruits for hepatitis A virus (HAV) is performed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). While RT-qPCR can detect extremely low amounts of RNA, it cannot discriminate between residual HAV genetic material (RNA), which is harmless, and infectious HAV virions due to the long persistence of RNA compared to infectious virions. This exposes those in the produce supply chain to ongoing risks of large economic losses associated with product recalls premised on RT-qPCR screening. In alignment with the 2024 CPS Research Priority 13a “Screening assay for HAV”, herein, we propose the development of a novel workflow using functionalized hydrogel nanoparticles (Nanotrap Microbiome A particles), automated RNA isolation, and RT-digital PCR to concentrate intact virus capsids from fruit wash eluate, extract HAV RNA from the resulting concentrate, and detect and quantify the RNA template. The method will be developed and validated in a step-wise approach from the RT-dPCR analytical endpoint upstream to the Nanotrap concentration using Armored HAV RNA (RNA within capsid) and in vitro transcribed RNA (free RNA) certified quantitative control materials. Quantitative and qualitative performance of the whole process workflow will be assessed by determining the method’s 95% limit of detection, limit of quantification, and recovery efficiency for Armored HAV RNA and the exclusion efficiency for free HAV RNA. If successful, the developed method would be compatible with automated platforms for concentration and extraction greatly increasing screening throughput while simultaneously decreasing the times to results and analytical costs and increasing specificity for potentially infectious HAV virions. The workflow would deliver benefits throughout the soft fruit supply chain including decreased economic losses to growers, buyers, and sellers due to false positive screening results, improved risk management data and decision making for regulators, and improved product safety for consumers.

Objective 1: Adapt existing RT-qPCR HAV assays to multiplex RT-dPCR format and assess performance for quantification and detection of HAV RNA. 

Objective 2: Assess the HAV RNA extraction efficiencies of two RNA extraction kits compatible with an automated workflow. 

Objective 3: Compare Nanotrap concentration to azo-dye pretreatment for the detection and quantification of HAV RNA within intact virus capsids. 

Objective 4: Assess the efficiency and performance characteristics of the complete workflow for the recovery and quantification of HAV RNA within intact virus capsids.

This project is ongoing. A final report will be provided when the project is finished.