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African swine fever virus (ASFV) causes a highly contagious and fatal disease affecting both domesticated and wild pigs. Substandard therapies and inadequate vaccinations cause severe economic damages from pig culling and removal of infected carcasses. Therefore, there is an urgent need to develop a rapid point-of-use approach that assists in avoiding the spread of ASFV and reducing economic loss. In this study, we developed a colorimetric sensing platform based on dual enzymatic amplification that combined the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system and the enzyme urease for accurate and sensitive detection of ASFV. The mechanism of the sensing platform involves a magnetic bead-anchored urease-conjugated single-stranded oligodeoxynucleotide (MB@urODN), which in the presence of ASFV dsDNA is cleaved by activated CRISPR/Cas12a. After magnetically separating the free urease, the presence of virus can be confirmed by measuring the colorimetric change in the solution. The advantage of this method is that it can detect the presence of virus without undergoing a complex target gene duplication process. The established method detected ASFV from three clinical specimens collected from porcine clinical tissue samples. The proposed platform is designed to provide an adequate, simple, robust, highly sensitive and selective analytical technique for rapid zoonotic disease diagnosis while eliminating the need for vast or specialized tools.

Viruses have been a continuous threat to human beings. The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a pandemic that is still ongoing worldwide. Previous pandemic influenza A virus (pH1N1) might be re-emerging through a drug-resistant mutation. We report a colorimetric viral detection method based on the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 endonuclease dead (dCas9) system. In this method, RNA in the viral lysate was directly recognized by the CRISPR/dCas9 system with biotin-protospacer adjacent motif (PAM)-presenting oligonucleotide (PAMmer). Streptavidin-horseradish peroxidase then bound to biotin-PAMmer, inducing a color change through the oxidation of 3,3',5,5'-tetramethylbenzidine. Using the developed method, we successfully identified SARS-CoV-2, pH1N1, and pH1N1/H275Y viruses by the naked eye. Moreover, the detection of viruses in human nasopharyngeal aspirates and sputum was demonstrated. Finally, clinical samples from COVID-19 patients led to a successful diagnosis. We anticipate that the current method can be employed for simple and accurate diagnosis of viruses.

Biological metamaterials with a specific size and spacing are necessary for developing highly sensitive and selective sensing systems to detect hazardous bacteria in complex solutions. Herein, the construction of peptidoglycan-binding protein (PGBP)-based metamaterials to selectively capture Gram-positive cells with high efficacy is reported. Nanoimprint lithography was used to generate a nanohole pattern as a template, the inside of which was modified with nickel(II)-nitrilotriacetic acid (Ni-NTA). Then, PGBP metamaterials were fabricated by immobilizing PGBP via chelation between Ni-NTA and six histidines on PGBP. Compared to the flat and spread PGBP-covered bare substrates, the PGBP-based metamaterials enabled selective capturing of Gram-positive bacteria with high efficacy, owing to enhanced interactions between the metamaterials and bacterial surface not shown in bulk materials. Thereafter, the specific strain and quantitative information of the captured bacteria was obtained by surface-enhanced Raman scattering mapping analysis in the 1 to 1 × 106 cfu/mL range within 30 min. It should be noted that no additional signal amplification process was required for lowly abundant bacteria, even at the single-bacterium level. The PGBP-based metamaterials could be regenerated multiple times with preserved sensing efficiency. Finally, this assay can detect specific Gram-positive bacteria, such as Staphylococcus aureus, in human plasma.

Influenza viruses cause respiratory infection, spread through respiratory secretions, and are shed into the nasal secretion and saliva specimens. Therefore, nasal fluid and saliva are effective clinical samples for the diagnosis of influenza virus-infected patients. Although several methods have been developed to detect various types of influenza viruses, approaches for detecting mutant influenza viruses in clinical samples are rarely reported. Herein, we report for the first time a surface-enhanced Raman scattering (SERS)-based sensing platform for oseltamivir-resistant pandemic H1N1 (pH1N1) virus detection in human nasal fluid and saliva. By combining SERS-active urchin Au nanoparticles and oseltamivir hexylthiol, an excellent receptor for the pH1N1/H275Y mutant virus, we detected the pH1N1/H275Y virus specifically and sensitively in human saliva and nasal fluid samples. Considering that the current influenza virus infection testing methods do not provide information on the antiviral drug resistance of the virus, the proposed SERS-based diagnostic test for the oseltamivir-resistant virus will inform clinical decisions about the treatment of influenza virus infections, avoiding the unnecessary prescription of ineffective drugs and greatly improving therapy.