Field-Deployable RPA-CRISPR/Cas12a Detection of Decapod Iridescent Virus 1 in Shrimp.

Identification of MDV and REV Using RPA-CRISPR/Cas12a Combined with Fluorescence and Lateral Flow Assays.

Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) are two major immunosuppressive oncogenic viruses that severely threaten the global poultry industry. Field co-infection of these two pathogens is prevalent, accompanied by highly similar pathological lesions, making conventional diagnostic technologies incapable of rapid on-site differential identification. Recombinase polymerase amplification (RPA) coupled with the CRISPR/Cas12a system represents an innovative platform for rapid nucleic acid detection. In this study, target-specific crRNAs were designed against the conserved unique genes of MDV and REV, respectively, to establish a CRISPR-based diagnostic system capable of differentiating the two viruses for the first time. Comprehensive optimization of RPA reaction composition, buffer formulation and incubation temperature improved the overall detection performance. Three independent visual signal readout modalities were supported by this assay, including real-time fluorescence quantification, naked-eye fluorescence visualization under blue light, and lateral flow assay (LFA). Sensitivity evaluation demonstrated that the limit of detection (LOD) reached 1×100 copies/μL for MDV and 1×101 copies/μL for REV. Specificity assays verified that the system only generated specific positive signals in response to target viruses without cross-reactivity against other common avian oncogenic viruses and their subgroups. Blind testing of 30 clinical chick fecal specimens revealed that the assay achieved a 100% positive detection rate for both viruses when referenced to qPCR and RT-qPCR, with outstanding diagnostic efficacy and perfect detection consistency. Collectively, the developed RPA-CRISPR/Cas12a assay overcomes the limitations of traditional diagnostic methods and holds great promise for on-site routine screening in poultry farms and epidemiological surveillance of MDV and REV.

Development of an RPA-CRISPR/Cas12a-Based Assay for Sensitive and Visual Detection of Channel Catfish Calicivirus (ChCV).

Channel catfish calicivirus (ChCV) has emerged as a novel viral pathogen threatening the sustainability of channel catfish (Ictalurus punctatus) aquaculture. The absence of rapid, on-site detection methods has hindered effective disease prevention and control. In this study, a visual RPA-CRISPR/Cas12a detection platform targeting the conserved non-structural protein (NS) gene of ChCV was established, and the key reaction parameters were systematically optimized. The optimal combination was identified as the F3/R3 primer pair with crRNA3-2. The optimal reaction conditions included RPA amplification at 37°C for 30 min, followed by CRISPR/Cas12a detection at 37°C for 30 min using 250 nM ssDNA reporter, 50 nM crRNA3-2 and 200 nM Cas12a. Specificity analysis showed that the established system specifically recognizes ChCV without cross-reactivity with other common aquatic pathogens affecting channel catfish, including channel catfish virus (CCV, also known as Ictalurid herpesvirus 1, IcHV1), channel catfish reovirus (CCRV), Edwardsiella tarda, Aeromonas hydrophila and Aeromonas veronii. Sensitivity evaluation demonstrated that the fluorescence visualization mode of the system achieved a limit of detection (LOD) of 2 copies/μL, while the lateral flow dipstick (LFD) mode exhibited an LOD of 2 × 102 copies/μL. Both detection modes were significantly more sensitive than conventional PCR. When applied to 20 clinical samples, the RPA-CRISPR/Cas12a system yielded a positive detection rate of 60%, substantially higher than that of conventional PCR (25%). In conclusion, the RPA-CRISPR/Cas12a system established in this study exhibits high specificity, ultra-sensitivity and operational simplicity, making it a promising tool for rapid and field-deplorable detection of ChCV.

A streamlined visual RT-RAA-CRISPR/Cas12a assay for rapid detection of Schmallenberg virus

Schmallenberg virus (SBV), an emerging Culicoides‑borne arbovirus, is responsible for febrile illness and reduced milk production in adult ruminants and can induce congenital malformations in fetuses, representing a substantial concern for livestock health worldwide. Consequently, rapid, field‑adaptable diagnostic approaches are urgently needed. In this study, we developed a visual nucleic acid detection assay for SBV that combines reverse transcription recombinase‑aided amplification (RT‑RAA) with the CRISPR/Cas12a system, targeting conserved regions of the SBV S gene. Following systematic optimization of the reaction conditions, the assay was completed within 50 min, with a sensitivity of 8.6 copies/μL for the SBV-S plasmid and 8.6 × 101 copies/μL for the RNA transcripts. The assay exhibited high specificity, with no cross‑reactivity observed against a panel of relevant pathogens, including Seoul orthohantavirus (SEOV), infectious bovine rhinotracheitis virus (IBRV), Rift Valley fever virus (RVFV), Crimean‑Congo hemorrhagic fever virus (CCHFV), and bovine viral diarrhea virus (BVDV). When evaluated using simulated clinical samples, the sensitivity of the method was superior to that of conventional real-time fluorescent reverse transcription‒polymerase chain reaction (RT‑qPCR). Importantly, the entire reaction is performed in a single closed-tube format, significantly reducing the risk of cross-contamination and false-positive results. In summary, this method demonstrates favorable analytical performance for the detection of SBV-S plasmid-spiked nasal swab samples and holds promise for further clinical validation, although its diagnostic utility in authentic clinical specimens remains to be confirmed in future investigations.

A dual-readout RAA-CRISPR/Cas13a diagnostic platform for rapid and sensitive detection of Eggerthella lenta

On-site and visual detection of genotype VII Newcastle disease virus based on RT-RPA and CRISPR/Cas13a

Newcastle Disease Virus (NDV) is not only a significant and persistent threat to the healthy development of poultry industry, but also an important pathogen endangering food safety and harboring potential zoonotic risks. In this study, a rapid and highly sensitive diagnostic assay was established for genotype VII NDV by integrating reverse transcription recombinase polymerase amplification (RT-RPA) with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system, in combination with a lateral flow dipstick (LFD) for visual signal readout. Specific RT-RPA primers were designed based on the fusion (F) gene of genotype VII NDV, and CRISPR RNAs (crRNAs) were constructed to establish a simplified CRISPR/Cas13a-based diagnostic workflow targeting the fusion (F) gene of genotype VII NDV. The optimized detection platform integrates RT-RPA amplification at 39 °C for 20 min and CRISPR/Cas13a-mediated cleavage at 37 °C for 20 min. The final results can be quantified by fluorescence signals or visualized via LFD band within 5 min, with the total detection procedure completed in less than 45 min. Performance evaluation demonstrated that this assay achieved a limit of detection (LOD) as low as 6 copies per microliter of input and high specificity, with no cross-reactivity against other common avian pathogens. Clinical validation of 154 samples showed 100% concordance between the RT-RPA-CRISPR/Cas13a-(LFD) assay and RT-PCR sequencing for genotype VII NDV detection. This method requires no sophisticated instruments and enables simple operation. It provides a field-deployable tool for rapid on-site detection of genotype VII NDV, which is suitable for preliminary screening in primary laboratories, and holds promising application potential for the early surveillance and food safety monitoring of virulent NDV strains.

CRISPR-Cas12a assay for rapid and specific detection of Shigella flexneri 2a in clinical samples

ABSTRACT Shigella flexneri 2a is the most common cause of shigellosis, a major public health concern in developing countries. Rapid and reliable diagnostic tools are critical for timely outbreak detection and management. Leveraging clustered regularly interspaced short palindromic repeats (CRISPR) technology, we developed a CRISPR-Cas12a-based assay for the rapid and specific detection of S. flexneri 2a and validated its performance using stool specimens from patients. Two guide RNAs targeting the gtrII and gtrX genes, unique markers of the S. flexneri 2a serotype, were designed to ensure specificity. Recombinase polymerase amplification (RPA) was coupled with Cas12a-mediated collateral cleavage for signal amplification, with detection by fluorescence or lateral flow. Analytical sensitivity, specificity, and clinical accuracy were compared with conventional PCR using purified DNA and 588 clinical stool specimens. The CRISPR-Cas12a assay achieved a detection limit of 10 copies/µL, comparable to PCR, and showed 100% analytical specificity without cross-reactivity to other bacteria. The isothermal reaction operated at room temperature and was completed within 1 h. Both readouts allowed visual interpretation without specialized equipment. Clinical validation of the CRISPR-Cas12a assay demonstrated a diagnostic sensitivity of 95% and specificity of 98%, comparable to PCR when evaluated using the same clinical specimens. This study provides two key advances: it establishes a CRISPR-Cas12a assay specifically targeting S. flexneri 2a, the predominant serotype, and validates it using a large clinical cohort. The assay’s simplicity, speed, and high diagnostic accuracy make it a valuable tool for clinical diagnostics and field-based surveillance in resource-limited settings. IMPORTANCE Rapid and accessible diagnostics are essential for effective management of infectious diseases such as shigellosis. We developed a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based assay that specifically detects Shigella flexneri 2a, the predominant serotype responsible for the global disease burden. This assay integrates isothermal amplification with CRISPR-mediated detection to achieve low-copy detection (10 copies/µL) within 1 h, eliminating the need for complex instrumentation. Dual fluorescence and lateral-flow readouts enable flexible use in both clinical laboratories and low-resource settings. The method’s simplicity, accuracy, and adaptability demonstrate the practical potential of CRISPR diagnostics for point-of-care applications. By enabling rapid, on-site identification of S. flexneri 2a, this approach can significantly improve clinical diagnosis and strengthen public health responses to enteric pathogen outbreaks. Rapid and accessible diagnostics are essential for effective management of infectious diseases such as shigellosis. We developed a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based assay that specifically detects Shigella flexneri 2a, the predominant serotype responsible for the global disease burden. This assay integrates isothermal amplification with CRISPR-mediated detection to achieve low-copy detection (10 copies/µL) within 1 h, eliminating the need for complex instrumentation. Dual fluorescence and lateral-flow readouts enable flexible use in both clinical laboratories and low-resource settings. The method’s simplicity, accuracy, and adaptability demonstrate the practical potential of CRISPR diagnostics for point-of-care applications. By enabling rapid, on-site identification of S. flexneri 2a, this approach can significantly improve clinical diagnosis and strengthen public health responses to enteric pathogen outbreaks.