Quantitative Real-Time PCR Detection of Inactivated H5 Avian Influenza Virus in Raw Milk Samples by Miniaturized Instruments Designed for On-Site Testing

Abstract

Highly Pathogenic Avian Influenza Virus of H5 (HPAI A(H5N1)) subtype has emerged as one of the most important zoonotic pathogens with significant economic consequences. The recent outbreak of H5N1 avian influenza in dairy cattle in the United States highlights the importance of early detection in managing and mitigating HPAI A(H5N1) outbreaks. A new diagnostic platform (the MT platform) consisting of miniaturized instruments for DNA/RNA purification and RT-rtPCR, designed for mobilse, on-site testing, is compared with a platform of benchtop instruments (QIAGEN RNeasy and QuantStudio5) for detecting inactivated HPAI A(H5N1) virus spiked into raw milk samples. Results show that, despite the presence of inhibitors in raw milk, HPAI A(H5N1) virus can be detected in all samples using both platforms. The MT platform shows higher sensitivity than the benchtop platform: the MT Ct values are [~]2 units lower than the benchtop Ct values. Our findings demonstrate the robustness of the MT platform for detecting HPAI A(H5N1) virus in raw milk samples and support its use as an on-site detection and screening for rapid surveillance and response.