Hepatitis A is an acute self-limiting disease caused by the HAV, primarily affecting young children(Gholizadeh et al., 2023). HAV is mainly transmitted through the fecal-oral route among humans, with approximately 1.5 million individuals suffering from hepatitis A globally each year(Lemon, Ott et al. 2017). Freeze-dried live attenuated hepatitis A (HepA-L-fd) vaccine has played a positive role in the prevention and control of hepatitis A(Ma et al., 2022). The infectious titer of the HepA-L-fd vaccine is a pivotal quality control parameter for the evaluation of vaccine production processes and potency. It is evident that HAV infection of cells does not generally result in the induction of cytopathic effects (CPE). Consequently, the viral titer cannot be ascertained based on the presence or extent of CPE.(Brack et al., 1998, Konduru et al., 2008).

The Cell-based enzyme-linked immunosorbent assay (ELISA), as the legal method included in the Chinese Pharmacopoeia (2020 edition), is currently used to measure the infectious titer of the HepA-L-fd vaccine. However, this method requires approximately one month to complete a single experimental run (McKnight and Lemon, 2018, Gholizadeh et al., 2023), and numerous interfering factors often lead to experimental failure, thereby impacting the production efficiency of the HepA-L-fd vaccine (de Paula et al., 2009). Therefore, this study aims to develop a rapid method for quantifying the vaccine titer.

Propidium monoazide (PMA) is a photo-reactive dye that can penetrate inactive RNA virus particles and bind to the RNA within or released from them. However, it has no effect on infectious virus particles.(Parshionikar et al., 2010, Sberna et al., 2025). Under UV light, the photosensitive azide group on the dye undergoes photolysis to form a highly reactive nitrene free radical. This radical readily forms a nitrogen-carbon covalent bond with the hydrocarbons of nucleic acids at the binding site. The PMA-RNA conjugate selectively inhibits the qPCR procedure, allowing only infectious virus particles to be detected.(Golpayegani et al., 2019). PMAxx is an upgraded version of PMA with higher sensitivity, enabling better differentiation between dead and live viruses(Randazzo et al., 2018, Liu et al., 2022). Currently, several studies have attempted to apply PMA-qPCR and PMAxx-qPCR to distinguish between infectious and non-infectious viruses (Karim et al., 2015, Hong et al., 2021, Razafimahefa et al., 2021, Li et al., 2023). Additionally, some studies have applied the combination of PMA and RT-qPCR technology to the detection of HAV. For example, Lian-lian et al. (2020) used PMA-PCR to analyse the copy numbers of HAV, but this did not include the infectious titer. Sánchez et al. (2012) developed the PMA-RT-qPCR to measure the HAV infectious titer. However, this approach can only analyse samples under 4 log10CCID50/mL, which is insufficient for vaccine requirements. At present, the application of PMA-qPCR technology for the detection of infectious titers of HAV vaccines has not been successful.

This study details the development of a PMAxx-RT-qPCR assay specifically for quantifying infectious titers in HAV vaccine. Firstly, we determined the optimal PMAxx concentration and optimized the coating conditions for samples using PMAxx, as well as the photolysis conditions. Subsequently, we found out the optimal dilution fold to avoid the impact of the stabilizer. Eventually, we validated the developed PMAxx-RT-qPCR in terms of accuracy, precision, and specificity. The established PMAxx-RT-PCR protocol provides a robust analytical platform for quality control of HAV vaccine titer. This method enables rapid virus titer detection within a day. When measuring the same batches of HAV vaccine samples, the results from the PMAxx-RT-qPCR show no significant difference compared with those from the Cell-based ELISA.