In two research projects funded by the Center for Produce Safety (CPS), scientists are investigating novel ways to improve detection methods for hepatitis A virus (HAV) in food, increasing their sensitivity, reducing false positives, and making them faster and more affordable.

At present, the usefulness of standard virus detection methods for indicating the actual food safety risk presented by HAV is limited, because these tests often produce positive results based on the presence of inactivated virus RNA fragments that do not cause illness.

Getting Around Non-Infectious Virus Fragments with Chemical Pre-Treatments

Addressing this limitation in real time qualitative polymerase chain reaction (RT-qPCR) detection methods, a team of researchers led by Lee-Ann Jaykus, Ph.D., Emerita, North Carolina University (NCSU) are exploring chemical sample pre-treatments that will prevent HAV tests from detecting inactivated RNA. Joining Dr. Jaykus as co-investigators are NCSU’s Benjamin Chapman, Ph.D. and Lynette Johnson, Ph.D.

The first technique uses enzymes to digest RNA fragments so that they cannot be detected via RT-qPCR. The second technique deploys chemical compounds that can enter capsids, which is a protein shell protecting virus genetic material, and bind to RNA to prevent PCR amplification, which is when copies of specific genetic material are made, enabling their detection and study.

After these pre-treatments are optimized, Dr. Jaykus and her team will test them against the only laboratory-cultivatable HAV strain, which will be inactivated using chemical and heat treatments, producing virus suspensions with different ratios of infectious to non-infectious RNA fragments. Next, they will compare the results of RT-qPCR coupled with their optimized pre-treatments against the results of cell culture infectivity assays, applied to suspensions of fully infectious, partially inactivated, or fully inactivated HAV. Finally, the researchers will test the efficacy of the top-performing pre-treatments in an experiment using soft fruits inoculated with varying proportions of infectious and non-infectious HAV, which will be processed according to the ISO standard.

Using CRISPR-Cas for Simple Rapid Tests

In a second CPS-funded project, Jose Santos García Alvarado, Ph.D. of Universidad Autónoma de Nuevo León in Mexico is leading work to develop another enzyme pre-treatment that destroys non-infectious fragments while retaining the infectious capsid. Joining Dr. García as Co-Investigators is Universidad Autónoma de Nuevo León’s Norma L. Heredia, Ph.D. Their experiments focus on frozen berries, a significant vehicle of HAV foodborne illness, and irrigation water, an important produce contamination risk factor.

His team is looking at using the same enzyme pre-treatment in two different assays: one that uses the ISO-standardized RT-qPCR method, and one that replaces the RT-qPCR amplification step with CRISPR-Cas technology, similar to what was used to develop rapid at-home COVID tests during the pandemic.

Unlike RT-qPCR, CRISPR-Cas does not require prohibitively expensive laboratory equipment or specialized technicians, Dr. García explained. Additionally, CRISPR-Cas can produce results in as few as 36 minutes—much shorter than the two-hour turnaround time required for RT-qPCR.

Dr. García’s team is currently working with laboratories at the University of Delaware and Emory University—with the help of Kalmia Kniel, Ph.D. and Juan León, Ph.D., respectively—to validate the new techniques. They also intend to develop a user manual for the assays, and they hope to acquire an industry partner to commercialize the HAV rapid test.