Field-deployable viral diagnostic tools for dengue virus based on Cas13a and Cas12a.

The diagnosis of dengue virus (DENV) has been challenging particularly in areas far from clinical laboratories. Early diagnosis of pathogens is a prerequisite for the timely treatment and pathogen control. An ideal diagnostic for viral infections should possess high sensitivity, specificity, and flexibility. In this study, we implemented dual amplification involving Cas13a and Cas12a, enabling sensitive and visually aided diagnostics for the dengue virus. Cas13a recognized the target RNA by crRNA and formed the assembly of the Cas13a/crRNA/RNA ternary complex, engaged in collateral cleavage of nearby crRNA of Cas12a. The Cas12a/crRNA/dsDNA activator ternary complex could not be assembled due to the absence of crRNA of Cas12a. Moreover, the probe, with 5' and 3' termini labeled with FAM and biotin, could not be separated. The probes labeled with FAM and biotin, combined the Anti-FAM and the Anti-Biotin Ab-coated gold nanoparticle, and conformed sandwich structure on the T-line. The red line on the paper strip caused by clumping of AuNPs on the T-line indicated the detection of dengue virus. This technique, utilizing an activated Cas13a system cleaving the crRNA of Cas12a, triggered a cascade that amplifies the virus signal, achieving a low detection limit of 190 fM with fluorescence. Moreover, even at 1 pM, the red color on the T-line was easily visible by naked eyes. The developed strategy, incorporating cascade enzymatic amplification, exhibited good sensitivity and may serve as a field-deployable diagnostic tool for dengue virus.

A universal CRISPR/Cas12a nucleic acid sensing platform based on proximity extension and transcription-unleashed self-supply crRNA.

The extraordinary genome-editing tool CRISPR/Cas12a has also been utilized as a powerful sensing technology owing to its highly-specificity and isothermal signal amplification. Nevertheless, the widespread application of Cas12a-based sensing methods in nucleic acid detection is limited by the targeting range and high undesired background. Herein, we established a universal Cas12a-based nucleic acid sensing strategy by using proximity extension and transcription-unleashed self-suppling of crRNA. The target was recognized and bound to a pair of adjacent probes, and then triggered the proximity-induced primer extension and transcription amplification to produce numerous crRNAs. The amplified abundant crRNAs assembled with Cas12a and dsDNA activators containing PAM to form a ternary complex, which trans-cleaved ssDNA-FQ reporters continuously to generate a strong fluorescent signal. Thus, the cascade enzymatic amplification was performed and subsequently applied for detecting target DNA down to 41.7 amol with a low nonspecific background. The application of this strategy in RNA detection has also been demonstrated, and it is expected to provide a universal and sensitive sensing platform for molecular diagnosis applications.

A Cascade Signal Amplification Based on Dynamic DNA Nanodevices and CRISPR/Cas12a Trans-cleavage for Highly Sensitive MicroRNA Sensing.

The variations of microRNA (miRNA) expression can be valuable biomarkers in disease diagnosis and prognosis. However, current miRNA detection techniques mainly rely on reverse transcription and template replication, which suffer from slowness, contamination risk, and sample loss. To address these limitations, here we introduce a cascade toehold-mediated strand displacement reaction (CTSDR) and CRISPR/Cas12a trans-cleavage for highly sensitive fluorescent miRNA sensing, namely CTSDR-Cas12a. In this work, the target miRNA hybridizes with the terminal toehold site of a rationally designed probe and subsequently initiates dynamic CTSDR, leading to enzyme-free target recycling and the production of multiple programmable DNA duplexes. The obtained DNA duplex acts as an activator to trigger Cas12a trans-cleavage, generating significantly amplified fluorescence readout for highly sensitive detection of the miRNA target. Under the optimal conditions, the developed sensing method can detect target miRNA down to 70.28 fM with a wide linear range from 100 fM to 100 pM. In particular, by designing a set of probes and crRNAs, we demonstrate its broad applicability for the detection of six kinds of miRNAs with high sequence specificity. Furthermore, the method can be satisfactorily applied to monitor miR-21 in total RNA extracted from cells and clinical serum samples. Considering the high sensitivity, specificity, universality, and ease of handling, this strategy provides a great potential platform for the detection of miRNA biomarkers in molecular diagnostic practice.

[Atmospheric correction of visible-infrared band FY-3A/MERSI data based on 6S model].

Based on the observation data from the meteorological stations in Taiyuan City and its surrounding areas of Shanxi Province, the atmosphere parameters for 6S model were supplied, and the atmospheric correction of visible-infrared band (precision 250 meters) FY-3A/MERSI data was conducted. After atmospheric correction, the range of visible-infrared band FY-3A/MERSI data was widened, reflectivity increased, high peak was higher, and distribution histogram was smoother. In the meantime, the threshold value of NDVI data reflecting vegetation condition increased, and its high peak was higher, more close to the real data. Moreover, the color synthesis image of correction data showed more abundant information, its brightness increased, contrast enhanced, and the information reflected was more close to real.