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. 2025 Aug 21;30(33):2500183. doi: 10.2807/1560-7917.ES.2025.30.33.2500183

Figure 2.

Performance evaluation of two digital PCR assays for the detection of influenza A(H5Nx) clade 2.3.4.4b

CDC: United States Centers for Disease Control and Prevention; Cp: copies; dd: droplet digital; HA: JRC-HA assay; HPAI: highly pathogenic avian influenza; Hum inf: human influenza; JRC: Joint Research Centre; LOD: limit of detection; MP: JRC-MP assay.

Panel A: LOD were determined by ddRT-PCR analysing a HPAI A(H5N1) 2.3.4.4b positive RNA isolated from infected Vulpes vulpes. Panel C: Synthetic control RNAs for human influenza A, human influenza B (Twist Bioscience) or HPAI A(H5N1) 2.3.4.4b RNA were used as a template. The performance of JRC-HA and JRC-MP assays was compared with the CDC human influenza duplex assay. Both JRC and CDC assays were run in a duplex format.

Performance of two digital RT-PCR assays - JRC-HA and JRC-MP - targeting the HA and MP genes of HPAI A(H5Nx) clade 2.3.4.4b. The figure includes three panels: detection limits, amplification linearity, and specificity. Panel A shows detection sensitivity using RNA from an HPAI-infected red fox. The HA assay achieved a limit of detection of 6.1 RNA copies per microliter, with strong linearity (R² = 0.95). The MP assay showed slightly better sensitivity at 4.7 copies per microliter (R² = 0.84). Panel B demonstrates that both assays amplified consistently across RNA concentrations from 20 to 10,000 copies per reaction. Amplification was linear, with an R-squared value of 1.0, confirming their suitability for quantification. Panel C compares the JRC assays with the CDC’s duplex assay using synthetic RNAs. The HA assay showed no amplification for human influenza A or B RNAs, confirming specificity. The MP assay did cross-react with human influenza A, though with much lower sensitivity than the CDC assay. All assays detected HPAI A(H5N1) RNA effectively. Overall, the figure confirms that both assays are sensitive and quantitative, with the HA assay providing higher specificity.