JRC: digital RT-PCR tells highly pathogenic avian influenza H5N1 (clade 2.3.4.4b) apart from seasonal flu in wastewater — a method based on two duplex assays, described in Emerging Infectious Diseases
On 21 October 2026 the European Commission’s Joint Research Centre (JRC) announced a laboratory method that distinguishes seasonal influenza viruses from highly pathogenic avian influenza H5N1 of clade 2.3.4.4b in wastewater samples. The method combines two duplex assays in digital RT-PCR: the influenza A and B assays of the CDC Flu SC2 panel and the JRC-HA/JRC-MP duplex developed by the JRC in 2025.
- The JRC-HA assay targets a conserved region of the haemagglutinin gene of H5N1 viruses of clade 2.3.4.4b, and JRC-MP a conserved region of the matrix gene, confirming the presence of influenza A virus; the two assays of the JRC duplex are run simultaneously. According to the JRC, this makes it possible to detect seasonal flu and identify the avian virus even at very low levels in the same sample.
- The method was published in Emerging Infectious Diseases (vol. 32, no. 11, November 2026, DOI 10.3201/eid3211.261234). The study used 10 samples of H5N1 clade 2.3.4.4b viruses from wild birds and poultry in northern Italy (October–December 2025) and RNA extracts from influenza-negative wastewater spiked with defined amounts of RNA. The limit of detection was 5 copies per reaction.
- With 10 or 20 copies/µL of H5N1 RNA on a background of 35 copies/µL of seasonal influenza A RNA, the JRC-HA and JRC-MP assays selectively detected H5N1, while the CDC influenza A assay detected both viruses without distinction. On a background of 3,500 copies/µL of seasonal influenza, JRC-HA still detected H5N1 without cross-reactivity. The authors propose a two-step use: the CDC duplex for routine influenza A and B surveillance and the JRC duplex to identify clade 2.3.4.4b and give early warning — and note that field evaluation under natural conditions and in different wastewater matrices is still needed.
- The JRC says the method is already being applied in wastewater surveillance in Lombardy; the partners were the EU Reference Laboratory for Avian Influenza and Newcastle disease, the Istituto Superiore di Sanità and the University of Milan Bicocca. The recast Urban Wastewater Treatment Directive (EU) 2024/3019 requires Member States to monitor selected pathogens, including influenza viruses, through their national surveillance systems.