Absolute Quantification of H5-Subtype Avian Influenza Viruses Using Droplet Digital Loop-Mediated Isothermal Amplification.

Human infection with avian influenza A H5N1 viruses can cause severe diseases with high mortality rate and continues to pose a significant threat to global public health. Rapid diagnosis is needed for identifying the types of influenza viruses for making timely treatment decisions. Here, we demonstrate absolute quantification of H5-subtype influenza viruses by digital loop-mediated isothermal amplification (dLAMP) on our recently developed cross-interface emulsification (XiE) method. Our results show that XiE-based dLAMP is highly specific and displays comparable sensitivity to real-time PCR (qPCR) and digital PCR (dPCR). Notably, dLAMP is more tolerant to inhibitory substances than PCR methods and demonstrated similar detection efficiency to qPCR for real H5N1 samples. Therefore, it can serve as a robust and precise alternative to qPCR or dPCR and is especially suitable for environmental and clinical samples with hard-to-remove contaminants. We believe that our dLAMP method offers great potential for rapid and accurate diagnosis of influenza and other infectious diseases.