基于双重信号放大技术的非洲猪瘟病毒快速现场核酸检测方法的开发与应用OA北大核心CSTPCD

This study introduces a novel nucleic acid detection method based on enzymes mediated duplex exponential amplification,designed for rapid,highly sensitive,and specific on-site testing,which has been scaled for broader application.The method employs specific primers and a recombinase complex to target ASFV genetic sequences,followed by rapid RNA amplification at a constant temperature using T7 RNA polymerase.The amplified RNA hybridizes with a fluorescently labeled DNA probe,and the DSN enzyme-mediated cleavage releases a fluorescent signal,amplifying the probe's detection by more than 1 000 times.By assessing various primer,probe,and reagent combinations,we evaluated sensitivi-ty,specificity,and contamination control on standard plasmids and mock samples,while also streamlin-ing sample processing steps.The results demonstrate that this method can stably and specifically de-tect 5-10 copies of the target gene within 25 minutes,reducing contamination risks compared to qPCR.In a test of 393 clinical samples,all 155 positive and 238 negative samples were correctly identified,achieving a 100％accuracy rate.This research successfully established a nucleic acid detection method based on dual signal amplification,offering reliable technical support for the clinical moni-toring and diagnosis of ASFV.