Application of a rapid and sensitive RPA-CRISPR/Cas12a assay for naked-eye detection of Haemophilus parasuis.

BACKGROUND: Haemophilus parasuis (H. parasuis) is a gram-negative bacterial pathogen that causes severe infections in swine, resulting in substantial economic losses. Currently, the majority of H. parasuis detection methods are impractical for on-site application due to their reliance on large instruments or complex procedures. Thus, there is an urgent need to develop a rapid, visually detectable, and highly sensitive detection method, especially under resource-limited environments and field conditions. RESULTS: In this study, we established a naked eye assay for highly sensitive detection by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Positive samples exhibited a clear red color visible to the naked eye, while negative samples appeared blue. We achieved a remarkable sensitivity, detecting H. parasuis down to a single copy, with no cross-reactivity with other bacteria. In a mouse model, our assay detected H. parasuis infection nearly 8 h earlier than traditional PCR. Compared to qPCR, our detection results were 100 % accurate. To enhance point-of-care applicability and mitigate the risk of aerosol contamination from uncapping, we consolidated RPA and CRISPR/Cas12a cleavage into a single-tube reaction system. This integrated approach was validated with 20 clinical lung samples, yielding results consistent with those obtained from qPCR. The entire procedure, from DNA extraction to detection, was completed in 35 min. SIGNIFICANCE: We present an RPA-CRISPR/Cas12a assay suitable for the early and resource-efficient diagnosis of H. parasuis infections. Its simplicity and visual detection are advantageous for field diagnostics, representing a substantial develpoment in the diagnosis of H. parasuis.

Photonic Crystal-Integrated Optoelectronic Devices with Naked-Eye Visualization and Digital Readout for High-Resolution Detection of Ultratrace Analytes.

The detection of ultratrace analytes is highly desirable for the non-invasive monitoring of human diseases. However, a major challenge is fast, naked-eye, high-resolution ultratrace detection. Herein, a rectangular 3D composite photonic crystal (PC)-based optoelectronic device is first designed that combines the sensitivity-enhancing effects of PCs and optoelectronic devices with fast and real-time digital monitoring. A crack-free, centimeter-scale, mechanically robust ellipsoidal composite PCs with sufficient hardness and modulus, even exceeding most plastics and aluminum alloys, are developed. The high mechanical strength of ellipsoidal composite PCs allows them to be hand-machined into rectangular geometries that can be conformally covered with the centimeter-scale flat light-detection area without interference from ambient light, easily integrating 3D composite PC-based optoelectronic devices. The PC-based device's signal-to-noise ratio increases dramatically from original 30-40 to ≈60-70 dB. Droplets of ultratrace analytes on the device are identified by fast digital readout within seconds, with detection limits down to 5 µL, enabling rapid identification of ultratrace glucose in artificial sweat and diabetes risk. The developed 3D PC-based sensor offers the advantages of small size, low cost, and high reliability, paving the way for wider implementation in other portable optoelectronic devices.

A one-tube rapid visual CRISPR assay for the field detection of Japanese encephalitis virus.

Early and rapid detection of Japanese encephalitis virus (JEV) is necessary for timely preventive and control measures. However, JEV RNA detection remains challenging due to the low level of viremia. In this study, a RApid VIsual CRISPR (RAVI-CRISPR) assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and CRISPR/Cas12a targeting was developed for easy detection of JEV in the field. We showed successful detection of 8.97 or more copies of the C gene sequence of JEV RNA within approximately 60 min. This assay also displayed no cross-reactivity with other porcine pathogens. We applied our one-tube RAVI-CRISPR assay to 18 brain tissue sample for JE diagnosis. The results from both fluorescence intensity measurements and directly naked-eye visualization were consistent with those from real-time PCR analysis. Taken together, our results showed that one-tube RAVI-CRISPR assay is robust, convenient, sensitive, specific, affordable, and potentially adaptable to on-site detection or surveillance of JEV in clinical and vector samples.

A Naked-Eye Visual Reverse Transcription Loop-Mediated Isothermal Amplification with Sharp Color Changes for Potential Pen-Side Test of Foot-and-Mouth Disease Virus.

Visual loop-mediated isothermal amplification (LAMP) is qualified to be applied in the field to detect pathogens due to its simplicity, rapidity and cost saving. However, the color changes in currently reported visual reverse transcription LAMP (RT-LAMP) for foot-and-mouth disease virus (FMDV) detection are not so obvious to the naked eye, so interpretation of results is troublesome. In this study, a new naked-eye visual RT-LAMP to detect all seven distinct serotypes of FMDV was established based on the 3D genes by using pH-sensitive neutral red as the indicator, rendering a sharp contrast of color changes between the negative (light orange) and the positive (pink). Analytical sensitivity tests showed that the detection limit of the visual RT-LAMP was 104 copies/µL while those were 103 and 104 copies/µL for the RT-qPCR and conventional RT-PCR methods, respectively. Specificity tests proved that the established visual RT-LAMP assay had no cross-reactivity with other common livestock viruses. Furthermore, the analysis of 59 clinical samples showed 98.31% and 100% concordance with the RT-qPCR and the RT-PCR, respectively. The pan-serotypic FMD visual RT-LAMP assay could be suitable for a pen-side test of all seven serotypes of FMDV because the results could be easily distinguished by the naked eye without the requirement of complicated instruments and professional technicians. Hence, the novel method may have a promising prospect in field tests which exert an important role in monitoring, preventing, and controlling FMD, especially in regions with no PCR or qPCR instrument available.

Surface Functional Nanofiber Membrane for Ultrasensitive and Naked-Eye Visualization of Bacterial Concentration.

Bacterial contamination in water is a serious health risk to human beings, so it is very important to realize the point-of-care (POC) bacterial detection in water. However, the traditional bacterial detection methods are time-consuming, professional- and equipment-dependent, and do not meet the needs of POC detection. There is a pressing need to develop a platform for POC bacterial detection to defeat the increasing risk of bacterial infections. Herein, a surface functional nanofiber membrane (NFM) is prepared by layer-by-layer (LBL) self-assembly as a platform for POC detection of bacterial concentration; it is naked-eye visualization and ultrasensitive. The platform shows obvious bacterial responsiveness, which allows naked-eye visualization of bacterial concentration (102-106 CFU/mL) within 30 min and can quantitatively detect the bacterial concentration (101-106 CFU/mL) by fluorescence within 5 min. The platform not only exhibits high efficiency but also has a low threshold for bacterial concentration detection. Furthermore, the platform shows good consistency with traditional methods in the detection of bacteria in practical water samples, and has the potential for use in detecting bacterial concentrations in water supplies to protect human beings from health hazards. This work also provides useful reference for research on bacterial detection, taking advantage of the surface characteristics of bacteria and the high sensitivity of NFM.