African swine fever (ASF) is one of the most severe diseases of pigs. In this study, a CRISPR-Cas12a (also known as Cpf1) system coupled with nucleic acid amplification was optimized for the detection of ASF virus (ASFV). Two novel single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters were developed to increase the brightness of the fluorescent signal for the visualization of nucleic acid detection. The CRISPR-Cas12a system was used to simultaneously cleave the polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP) amplicons and the newly developed ssDNA-FQ reporter, resulting in fluorescence that could be easily detected in multiple platforms, especially on cheap and portable blue or UV light transilluminators. This specific cleavage with fluorescence reveals the presence of the amplicon and confirms its identity, thereby preventing false-positive test results from nonspecific amplicons. This method is also uninterfered by the presence of large amounts of irrelevant background DNA and displays no cross-reactivity with other porcine DNA or RNA viruses. When coupled with LAMP, the Cas12a platform can detect a plasmid containing p72 with as few as 2 copies/μL reaction. Our results indicate that the CRISPR-Cas12a enhanced fluorescence assay coupled with nucleic acid amplification is robust, convenient, specific, confirmatory, affordable, and potentially adaptable for ASF diagnosis.

Rapid diagnosis based on naked-eye colorimetric detection remains challenging, but it could build new capacities for molecular point-of-care testing (POCT). In this study, we evaluated the performance of 16 types of single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters for use with clusters of regularly spaced short palindrome repeats (CRISPR)/Cas12a-based visual colorimetric assays. Among them, nine ssDNA-FQ reporters were found to be suitable for direct visual colorimetric detection, with especially very strong performance using ROX-labeled reporters. We optimized the reaction concentrations of these ssDNA-FQ reporters for a naked-eye read-out of assay results (no transducing component required for visualization). In particular, we developed a convolutional neural network algorithm to standardize and automate the analytical colorimetric assessment of images and integrated this into the MagicEye mobile phone software. A field-deployable assay platform named RApid VIsual CRISPR (RAVI-CRISPR) based on a ROX-labeled reporter with isothermal amplification and CRISPR/Cas12a targeting was established. We deployed RAVI-CRISPR in a single tube toward an instrument-less colorimetric POCT format that required only a portable rechargeable hand warmer for incubation. The RAVI-CRISPR was successfully used for the high-sensitivity detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and African swine fever virus (ASFV). Our study demonstrates this RAVI-CRISPR/MagicEye system to be suitable for distinguishing different pathogenic nucleic acid targets with high specificity and sensitivity as the simplest-to-date platform for rapid pen- or bed-side testing.

A series of 4-phenyl-acyl-substituted 3-(2,5-dimethylphenyl)-4-hydroxy-1-azaspiro[4.5]dec-3-ene-2,8-dione derivatives were designed and synthesized, and their structures were characterized using (1)H NMR (or (13)C NMR), mass spectrometry, and elemental analysis. The bioactivities of the new compounds were evaluated. These compounds exhibited good inhibition activities against bean aphids (Aphis fabae) and carmine spider mite (Tetranychus cinnabarinus), and 4-phenyl acyl esters showed stronger bioactivity than 4-arylesterases and alkyl esters. The results showed that compound 8-I-e, which contains a para-methoxy group on the phenyl acyl, and compound 8-I-m, which contains a para-trifluoromethyl group on the phenyl acyl, displayed potent insecticidal activity against A. fabae and T. cinnabarinus respectively. The insecticidal activity showed a clear structure-activity relationship, confirming the importance of the flexible bridge. The DFT/B3LYP/6-31(d) level method was used to calculate molecular geometries and electronic descriptors. These factors included total energy, charge distribution, and the linear orbital level of the title compounds. Quantitative structure-activity relationship studies were performed on these compounds using quantum-chemical and physicochemical parameters as independent variables and insecticidal activity as a dependent variable. Insecticidal activity was most closely correlated (r > 0.8) with quantum chemical and physicochemical parameters.