AimTo improve the efficacy of intercalating dyes to distinguishing between infectious and inactivated hepatitis A virus (HAV) in food. Methods and ResultsDifferent intercalating dyes were evaluated for the discrimination between infectious and thermally inactivated HAV suspensions combining with the RT-qPCR proposed in the ISO 15216. Among them, PMAxx was the best dye in removing the RT-qPCR signal from inactivated HAV. Applied to lettuce and spinach, PMAxx-Triton pretreatment resulted in complete removal of the RT-qPCR signal from inactivated HAV. Likewise, this study demonstrates that this pretreatment is suitable for the discrimination of inactivated HAV in shellfish without further sample dilution. In mussels and oysters, the developed viability RT-qPCR method reduced the signal of inactivated HAV between 17 and 22 logs at high inoculation level, and signal was completely removed at low inoculation level. ConclusionsThis study showed that the use of PMAxx is an important improvement to assess HAV infectivity by RT-qPCR. It was shown that PMAxx-Triton pretreatment is suitable for the analysis of infectious HAV in complex food samples such as vegetables and shellfish. Significance and Impact of the StudyThe PMAxx-Triton pretreatment can be easily incorporated to the ISO norm for infectious virus detection.