Argonaute-integrated isothermal amplification for rapid, portable, multiplex detection of SARS-CoV-2 and influenza viruses.

Isothermal amplification methods are a promising trend in virus detection because of their superiority in rapidity and sensitivity. However, the generation of false positives and limited multiplexity are major bottlenecks that must be addressed. In this study, we developed a multiplex Argonaute (Ago)-based nucleic acid detection system (MULAN) that integrates rapid isothermal amplification with the multiplex inclusiveness of a single Ago for simultaneous detection of multiple targets such as SARS-CoV-2 and influenza viruses. Owing to its high specificity, MULAN can distinguish targets at a single-base resolution for mutant genotyping. Moreover, MULAN also supports portable and visible devices with a limit of detection of five copies per reaction. Validated by SARS-CoV-2 pseudoviruses and clinical samples of influenza viruses, MULAN showed 100% agreement with quantitative reverse-transcription PCR. These results demonstrated that MULAN has great potential to facilitate reliable, easy, and quick point-of-care diagnosis for promoting the control of infectious diseases.

Argonaute integrated single-tube PCR system enables supersensitive detection of rare mutations.

Technological advances in rare DNA mutations detection have revolutionized the diagnosis and monitoring of tumors, but they are still limited by the lack of supersensitive and high-coverage procedures for identifying low-abundance mutations. Here, we describe a single-tube, multiplex PCR-based system, A-Star, that involves a hyperthermophilic Argonaute from Pyrococcus furiosus (PfAgo) for highly efficient detection of rare mutations beneficial from its compatibility with DNA polymerase. This novel technique uses a specific guide design strategy to allow PfAgo selective cleavage with single-nucleotide resolution at 94°C, thus mostly eliminating wild-type DNA in the denaturation step and efficiently amplifying rare mutant DNA during the PCR process. The integrated single-tube system achieved great efficiency for enriching rare mutations compared with a divided system separating the cleavage and amplification. Thus, A-Star enables easy detection and quantification of 0.01% rare mutations with ≥5500-fold increase in efficiency. The feasibility of A-Star was also demonstrated for detecting oncogenic mutations in solid tumor tissues and blood samples. Remarkably, A-Star achieved simultaneous detection of multiple oncogenes through a simple single-tube reaction by orthogonal guide-directed specific cleavage. This study demonstrates a supersensitive and rapid nucleic acid detection system with promising potential for both research and therapeutic applications.

Highly sensitive and selective detection of naproxen via molecularly imprinted carbon dots as a fluorescent sensor.

The overuse and inappropriate discharge of naproxen, a common anti-inflammatory medication and an emerging contaminant in water, is detrimental to human health and bodies of water. Here, we design a fluorescent sensor based on molecularly imprinted carbon dots (CDs) for highly selective detection of trace amounts of naproxen. The CDs were encapsulated into the pores of silica through a sol-gel based method and provide fluorescent signal. After removal of the template molecules, a molecularly imprinted polymer layer was formed and the fluorescence of the CDs sensor was selectively quenched by naproxen. A detection limit of as low as 0.03 µM and a linear range of 0.05-4 µM for detecting naproxen in aqueous solution were obtained. High recoveries of naproxen levels in waste water and urine samples for practical application were also achieved. In addition, the accurate detection performance of sensor was maintained during the UV degradation of naproxen.

Solid-Phase Microwave Reduction of WO3 by GO for Enhanced Synergistic Photo-Fenton Catalytic Degradation of Bisphenol A.

The synergistic photocatalytic Fenton reaction is a powerful advanced oxidation technique for the degradation of persistent organic pollutants. However, microwave-induced thermal effects on the formation of novel structures facilitating the photocatalytic degradation have been rarely reported. Herein, a two-step microwave thermal strategy was developed to synthesize a new hybrid catalyst comprising defective WO3-x nanowires coupled with reduced graphene oxides (rGOs). Conventionally, microwave methods could induce superhot spots on the GO surface, resulting in the site-specific crystallization and oriented growth of WO3. However, in the solid phase, localized microwave thermal effects could reduce the interfacial area between WO3 and rGO and enhance the bonding between them. As for the unique structure and surface properties, the synthesized catalyst enhanced the light absorption, promoted the interfacial charge separation, and increased the carrier density in the photocatalytic processes. In addition, surface formation of W4+ provided a new pathway for Fe3+/Fe2+ cycling which linked the photocatalytic reaction and the Fenton process. The optimized catalyst exhibited a remarkable performance in the degradation of bisphenol A with a ∼83% removal yield via a photo-Fenton route. These microwave-induced functionalities of materials for synergistic reactions could also give a new avenue to other photoelectrocatalytic fields and solar cells.

Characterisation of the Virome of Tonsils from Conventional Pigs and from Specific Pathogen-Free Pigs.

Porcine respiratory disease is a multifactorial disease that can be influenced by a number of different microorganisms, as well as by non-infectious factors such as the management and environment of the animals. It is generally believed that the interaction between different infectious agents plays an important role in regard to respiratory diseases. Therefore, we used high-throughput sequencing combined with viral metagenomics to characterise the viral community of tonsil samples from pigs coming from a conventional herd with lesions in the respiratory tract at slaughter. In parallel, samples from specific pathogen-free pigs were also analysed. This study showed a variable co-infection rate in the different pigs. The differences were not seen at the group level but in individual pigs. Some viruses such as adenoviruses and certain picornaviruses could be found in most pigs, while others such as different parvoviruses and anelloviruses were only identified in a few pigs. In addition, the complete coding region of porcine parvovirus 7 was obtained, as were the complete genomes of two teschoviruses. The results from this study will aid in elucidating which viruses are circulating in both healthy pigs and in pigs associated with respiratory illness. This knowledge is needed for future investigations into the role of viral-viral interactions in relation to disease development.

Detection and genetic characterisation of porcine circovirus 3 from pigs in Sweden.

Porcine circovirus 3 (PCV3) is a newly detected circovirus belonging to the family Circoviridae with a circular ssDNA genome of 2000 bp that encodes two proteins-the replicase protein and the capsid protein. PCV3 was discovered for the first time in the US in 2016. After this initial discovery, PCV3 was detected in other parts of the world such as in China, South Korea, Italy and Poland. In this study, 49 tissue samples from Swedish pig herds were screened for PCV3 using PCR and 10 samples were positive and one was uncertain. The entire PCV3 genome and a mini PCV-like virus (MPCLV) were obtained from one of these samples. These two viruses showed a high sequence identity to PCV3 viruses from other countries as well as to MPCLV from the US. However, the sequence identity to PCV1 and 2 was only 31-48% on amino acid level. This is the first detection and complete genetic characterisation of PCV3 in Swedish pigs. It is also interesting to note that one of the positive samples was collected in 1993, showing that PCV3 has been present for a long time.