Gold nanoparticles (GNPs) biosensors can detect low viral loads and differentiate between viruses types, enabling early diagnosis and effective disease management. In the present study, we developed GNPs biosensors with two different capping agent, citrate-GNPs biosensors and polyvinylpyrrolidone (PVP)-GNPs biosensors for detection of EHV-1 and EHV-4 in multiplex real time PCR (rPCR). Citrate-GNPs and PVP-GNPs biosensors can detect dilution 1010 of EHV-1 with mean Cycle threshold (Ct) 11.7 and 9.6, respectively and one copy as limit of detection, while citrate-GNPs and PVP-GNPs biosensors can detect dilution 1010 of EHV-4 with mean Ct 10.5 and 9.2, respectively and one copy as limit of detection. These findings were confirmed by testing 87 different clinical samples, 4 more samples were positive with multiplex GNPs biosensors rPCR than multiplex rPCR. Multiplex citrate-GNPs and PVP-GNPs biosensors for EHV-1 and EHV-4 are a significant breakthrough in the diagnosis of these virus types. These biosensors offer high sensitivity and specificity, allowing for the accurate detection of the target viruses at very low concentrations and improve the early detection of EHV-1 and EHV-4, leading to faster control of infected animals to prevent the spread of these viruses.
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