On-site and visual detection of genotype VII Newcastle disease virus based on RT-RPA and CRISPR/Cas13a

Abstract

Newcastle Disease Virus (NDV) is not only a significant and persistent threat to the healthy development of poultry industry, but also an important pathogen endangering food safety and harboring potential zoonotic risks. In this study, a rapid and highly sensitive diagnostic assay was established for genotype VII NDV by integrating reverse transcription recombinase polymerase amplification (RT-RPA) with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system, in combination with a lateral flow dipstick (LFD) for visual signal readout. Specific RT-RPA primers were designed based on the fusion (F) gene of genotype VII NDV, and CRISPR RNAs (crRNAs) were constructed to establish a simplified CRISPR/Cas13a-based diagnostic workflow targeting the fusion (F) gene of genotype VII NDV. The optimized detection platform integrates RT-RPA amplification at 39 °C for 20 min and CRISPR/Cas13a-mediated cleavage at 37 °C for 20 min. The final results can be quantified by fluorescence signals or visualized via LFD band within 5 min, with the total detection procedure completed in less than 45 min. Performance evaluation demonstrated that this assay achieved a limit of detection (LOD) as low as 6 copies per microliter of input and high specificity, with no cross-reactivity against other common avian pathogens. Clinical validation of 154 samples showed 100% concordance between the RT-RPA-CRISPR/Cas13a-(LFD) assay and RT-PCR sequencing for genotype VII NDV detection. This method requires no sophisticated instruments and enables simple operation. It provides a field-deployable tool for rapid on-site detection of genotype VII NDV, which is suitable for preliminary screening in primary laboratories, and holds promising application potential for the early surveillance and food safety monitoring of virulent NDV strains.