Sensitive and specific CRISPR-Cas12a assisted nanopore with RPA for Monkeypox detection.

Monkeypox virus (MPXV) poses a global health emergency, necessitating rapid, simple, and accurate detection to manage its spread effectively. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique has emerged as a promising next-generation molecular diagnostic approach. Here, we developed a highly sensitive and specific CRISPR-Cas12a assisted nanopore (SCAN) with isothermal recombinase polymerase amplification (RPA) for MPXV detection. The RPA-SCAN method offers a sensitivity unachievable with unamplified SCAN while also addressing the obstacles of PCR-SCAN for point-of-care applications. We demonstrated that size-counting of single molecules enables analysis of reaction-time dependent distribution of the cleaved reporter. Our MPXV-specific RPA assay achieved a limit of detection (LoD) of 19 copies in a 50 μL reaction system. By integrating 2 μL of RPA amplifications into a 20 μL CRISPR reaction, we attained an overall LoD of 16 copies/μL (26.56 aM) of MPXV at a 95% confidence level using the SCAN sensor. We also verified the specificity of RPA-SCAN in distinguishing MPXV from cowpox virus with 100% accuracy. These findings suggest that the isothermal RPA-SCAN device is well-suited for highly sensitive and specific Monkeypox detection. Given its electronic nature and miniaturization potential, the RPA-SCAN system paves the way for diagnosing a wide array of other infectious pathogens at the point of care.

Ahamed MA ，Khalid MAU ，Dong M ，Politza AJ ，Zhang Z ，Kshirsagar A ，Liu T ，Guan W ... -  《-》

Development of a CRISPR/Cas12a-recombinase polymerase amplification assay for visual and highly specific identification of the Congo Basin and West African strains of mpox virus.

Human mpox is a zoonotic disease, similar to smallpox, caused by the mpox virus, which is further subdivided into Congo Basin and West African clades with different pathogenicity. In this study, a novel diagnostic protocol utilizing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 12a nuclease (CRISPR/Cas12a)-mediated recombinase polymerase amplification (RPA) was developed to identify mpox in the Congo Basin and West Africa (CRISPR-RPA). Specific RPA primers targeting D14L and ATI were designed. CRISPR-RPA assay was performed using various target templates. In the designed CRISPR-RPA reaction system, the exponentially amplified RPA amplification products with a protospacer adjacent motif (PAM) site can locate the Cas12a/crRNA complex to its target regions, which successfully activates the CRISPR/Cas12a effector and achieves ultrafast trans-cleavage of a single-stranded DNA probe. The limit of detection for the CRISPR-RPA assay was 10 copies per reaction for D14L- and ATI-plasmids. No cross-reactivity was observed with non-mpox strains, confirming the high specificity of the CRISPR-RPA assay for distinguishing between the Congo Basin and West African mpox. The CRISPR-RPA assay can be completed within 45 min using real-time fluorescence readout. Moreover, the cleavage results were visualized under UV light or an imaging system, eliminating the need for a specialized apparatus. In summary, the developed CRISPR/RPA assay is a visual, rapid, sensitive, and highly specific detection technique that can be used as an attractive potential identification tool for Congo Basin and West African mpox in resource-limited laboratories.

Yang X ，Zeng X ，Chen X ，Huang J ，Wei X ，Ying X ，Tan Q ，Wang Y ，Li S ... -  《-》

Development of a Copro-RPA-CRISPR/Cas12a assay to detect Echinococcus granulosus nucleic acids isolated from canine feces using NaOH-based DNA extraction method.

Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato (E. granulosus s.l.), remains a significant zoonotic parasitic disease affecting both livestock and humans. It arises from the ingestion of food and water contaminated with canine feces containing E. granulosus eggs. The detection of these eggs in canine feces is essential for guiding effective preventative measures against the disease. Therefore, the development of a novel accurate, rapid, and visually interpretable point-of-care test is crucial for controlling CE. We combined recombinase polymerase amplification (RPA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) with a CRISPR-associated protein 12a (Cas12a) system, forming the RPA-CRISPR/Cas12a assay. This assay targeted the E. granulosus mitochondrial nad2 gene and utilized a lateral flow strip for visual readout. To improve field applicability, we integrated a simple and cost-effective NaOH-Based DNA extraction method. Clinical validation included testing DNA extracted from eighteen canine fecal samples, followed by comparison with quantitative PCR (qPCR) and two commercial enzyme-linked immunosorbent assay (ELISA) kits. The RPA-CRISPR/Cas12a assay showed a detection limit of 1 fg/μL DNA, without any cross-reactivity with related tapeworms such as Echinococcus multilocularis, Dipylidium caninum, Taenia hydatigera, Taenia multiceps, and Taenia pisiformis. When applied to 62 clinical fecal samples from dogs, the RPA-CRISPR/Cas12a assay demonstrated 68% sensitivity, while the developed RPA-CRISPR/Cas12a-NaOH assay exhibited 45% sensitivity. In the field performance comparison of the RPA-CRISPR/Cas12a and the RPA-CRISPR/Cas12a-NaOH assay with qPCR and two ELISA kits, the sensitivity, consistency rate, and Youden's index suggested good or fair agreement with the currently employed detection methods. This study describes the development and validation of the RPA-CRISPR/Cas12a and RPA-CRISPR/Cas12a-NaOH assays for detecting E. granulosus in canine feces. The developed assays surpassed previous detection methods in providing enhanced diagnostic sensitivity and enabling point-of-care testing. Moreover, these assays hold potential for surveilling E. granulosus in low-income countries.

Shao G ，Zhu X ，Hua R ，Chen Y ，Yang G ... -  《-》

Solid-Phase Extraction and Enhanced Amplification-Free Detection of Pathogens Integrated by Multifunctional CRISPR-Cas12a.

Tian Z ，Yan H ，Zeng Y 《-》

The current status and future prospects of CRISPR-based detection of monkeypox virus: A review.

The current pandemic of 2022 global mpox (formerly known as monkeypox), caused by infection with monkeypox virus (MPXV), has now reached over 120 countries. This constitutes a critical public health issue requiring effective disease management and surveillance. Rapid and reliable diagnosis is conducive to the control of infection, early intervention, and timely treatment. Clinical laboratories use various conventional diagnostic methods for detecting MPXV, including PCR, which can be regarded as a gold-standard diagnostic method. However, the application of PCR is limited by its requirements for high-cost equipment, skilled professionals, and a laboratory setting. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic systems have provided promising prospects for the rapid, sensitive, and specific detection of infectious diseases, especially in point-of-care settings. Over the past 2 years, an increasing number of researchers have concentrated on the application of the CRISPR method to mpox diagnosis. In the majority of cases, a two-step method was chosen, with CRISPR/Cas12a and recombinase polymerase amplification (RPA) as pre-amplification methods, followed by a fluorescence readout. Different strategies have been applied to overcome the encountered limitations of CRISPR detection, but no consensus on an integrated solution has been achieved. Thus, the application of the CRISPR/Cas system in mpox detection requires further exploration and improvement. This review discusses contemporary studies on MPXV CRISPR detection systems and the strategies proposed to address the challenges faced by CRISPR diagnosis with the hope of helping the development of CRISPR detection methods and improving pathogen detection technologies.

Chen Y ，Zhao R ，Hu X ，Wang X ... -  《-》