This review summarizes the recent advances and the main strategies to improve the sensitivity of amplification-free CRISPR/Cas-based detection techniques.

Background: During the validation of a highly sensitive panspecies coronavirus (CoV) seminested reverse-transcription polymerase chain reaction (RT-PCR) assay, we found canine CoV (CCoV) RNA in nasopharyngeal swab samples from 8 of 301 patients (2.5%) hospitalized with pneumonia during 2017–2018 in Sarawak, Malaysia. Most patients were children living in rural areas with frequent exposure to domesticated animals and wildlife.Methods: Specimens were further studied with universal and species-specific CoV and CCoV 1-step RT-PCR assays, and viral isolation was performed in A72 canine cells. Complete genome sequencing was conducted using the Sanger method.Results: Two of 8 specimens contained sufficient amounts of CCoVs as confirmed by less-sensitive single-step RT-PCR assays, and 1 specimen demonstrated cytopathic effects in A72 cells. Complete genome sequencing of the virus causing cytopathic effects identified it as a novel canine-feline recombinant alphacoronavirus (genotype II) that we named CCoV–human pneumonia (HuPn)–2018. Most of the CCoV-HuPn-2018 genome is more closely related to a CCoV TN-449, while its S gene shared significantly higher sequence identity with CCoV-UCD-1 (S1 domain) and a feline CoV WSU 79-1683 (S2 domain). CCoV-HuPn-2018 is unique for a 36-nucleotide (12–amino acid) deletion in the N protein and the presence of full-length and truncated 7b nonstructural protein, which may have clinical relevance.Conclusions: This is the first report of a novel canine-feline recombinant alphacoronavirus isolated from a human patient with pneumonia. If confirmed as a pathogen, it may represent the eighth unique coronavirus known to cause disease in humans. Our findings underscore the public health threat of animal CoVs and a need to conduct better surveillance for them.

The cutting‐edge CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas (CRISPR‐associated proteins) system, as an emerging molecular diagnostic technique, is driving revolutionary developments in the detection field due to its high specificity and efficiency. However, the CRISPR‐based assays typically require the combination with an additional pre‐amplification step based on isothermal nucleic acid amplification to meet the requirements of clinical diagnosis, which brings issues including complicated operation and the risk of aerosol contamination. To address these challenges, one‐pot CRISPR platforms are emerging as an attractive solution to streamline workflows, enabling rapid, cost‐effective, and high‐sensitivity diagnostics. This review outlines the current status, challenges, and three key strategies to realize highly efficient one‐pot CRISPR‐based detection. In addition, further perspectives are outlined that will inspire new exploration and promote one‐pot CRISPR/Cas detection as the next generation of diagnostic tools.

Infectious diseases contribute significantly to the global disease burden. Sensitive and accurate screening methods are some of the most effective means of identifying sources of infection and controlling infectivity. Conventional detecting strategies such as quantitative polymerase chain reaction (qPCR), DNA sequencing, and mass spectrometry typically require bulky equipment and well-trained personnel. Therefore, mass screening of a large population using conventional strategies during pandemic periods often requires additional manpower, resources, and time, which cannot be guaranteed in resource-limited settings. Recently, emerging microfluidic technologies have shown the potential to replace conventional methods in performing point-of-care detection because they are automated, miniaturized, and integrated. By exploiting the spatial separation of detection sites, microfluidic platforms can enable the multiplex detection of infectious diseases to reduce the possibility of misdiagnosis and incomplete diagnosis of infectious diseases with similar symptoms. This review presents the recent advances in microfluidic platforms used for multiplex detection of infectious diseases, including microfluidic immunosensors and microfluidic nucleic acid sensors. As representative microfluidic platforms, lateral flow immunoassay (LFIA) platforms, polymer-based chips, paper-based devices, and droplet-based devices will be discussed in detail. In addition, the current challenges, commercialization, and prospects are proposed to promote the application of microfluidic platforms in infectious disease detection.

Mitigating the spread of global infectious diseases requires rapid and accurate diagnostic tools. Conventional diagnostic techniques for infectious diseases typically require sophisticated equipment and are time consuming. Emerging clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated proteins (Cas) detection systems have shown remarkable potential as next‐generation diagnostic tools to achieve rapid, sensitive, specific, and field‐deployable diagnoses of infectious diseases, based on state‐of‐the‐art microfluidic platforms. Therefore, a review of recent advances in CRISPR‐based microfluidic systems for infectious diseases diagnosis is urgently required. This review highlights the mechanisms of CRISPR/Cas biosensing and cutting‐edge microfluidic devices including paper, digital, and integrated wearable platforms. Strategies to simplify sample pretreatment, improve diagnostic performance, and achieve integrated detection are discussed. Current challenges and future perspectives contributing to the development of more effective CRISPR‐based microfluidic diagnostic systems are also proposed.

Ceftriaxone-resistant Neisseria gonorrhoeae FC428-like strains have disseminated across the Asia-Pacific region, with a continuous rise in prevalence during 2015–2022. To mitigate the effect of these strains, we advocate for enhanced molecular diagnostics, expanded surveillance networks, and a regionally coordinated effort to combat the global spread of FC428-like strains.