RNase Hybridization-Assisted Amplification (RHAM) Technology: A High-Sensitivity, Field-Deployable Alternative to Quantitative Polymerase Chain Reaction for the Rapid Detection of African Swine Fever Virus

African Swine Fever (ASF) control is severely limited by a diagnostic gap, as laboratory-based quantitative polymerase chain reaction (qPCR) is highly sensitive but slow, whereas field-deployable immunochromatographic assays (ICAs) are rapid but unreliable. To address this limitation, this study evaluated a novel, rapid isothermal assay, RNase hybridization-assisted amplification (RHAM), as a high-sensitivity, point-of-need diagnostic solution. This study compared the performance of RHAM and a conventional p72-based ICA against the qPCR reference standard using 106 diverse clinical field samples, including oral swabs, blood, serum, and organs, collected from suspected ASF cases in Thailand. The ICA exhibited markedly low diagnostic performance, achieving only 56.76% sensitivity and showing moderate agreement (κ = 0.421) with qPCR, highlighting the need for a more reliable alternative. In contrast, the RHAM assay achieved 94.59% sensitivity and 96.88% specificity, providing results rapidly within 35 min. This statistically superior performance (McNemar's test, <i>p</i> < 0.0001) demonstrated almost perfect agreement (κ = 0.891) with the qPCR reference standard, missing only four samples with very high Ct values (>30). In conclusion, RHAM is a powerful, accurate, and field-deployable diagnostic tool that effectively bridges the diagnostic gap, offering qPCR-like sensitivity for the rapid containment of ASF outbreaks.