RESUMEN
Rapid and sensitive quantification of RNA is critical for detecting infectious diseases and identifying disease biomarkers. Recent direct detection assays based on CRISPR-Cas13a1-4 avoid reverse transcription and DNA amplification required of gold-standard PCR assays5, but these assays have not yet achieved the sensitivity of PCR and are not easily multiplexed to detect multiple viruses or variants. Here we show that Cas13a acting on single target RNAs loaded into droplets exhibits stochastic nuclease activity that can be used to enable sensitive, rapid, and multiplexed virus quantification. Using SARS-CoV-2 RNA as the target and combinations of CRISPR RNA (crRNA) that recognize different parts of the viral genome, we demonstrate that reactions confined to small volumes can rapidly achieve PCR-level sensitivity. By tracking nuclease activity within individual droplets over time, we find that Cas13a exhibits rich kinetic behavior that depends on both the target RNA and crRNA. We demonstrate that these kinetic signatures can be harnessed to differentiate between different human coronavirus species as well as SARS-CoV-2 variants within a single droplet. The combination of high sensitivity, short reaction times, and multiplexing makes this droplet-based Cas13a assay with kinetic barcoding a promising strategy for direct RNA identification and quantification.
RESUMEN
Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided recognition of RNA that triggers cleavage and release of a fluorescent reporter molecule1,2, but long reaction times hamper sensitivity and speed when applied to point-of-care testing. Here we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of [~]30 RNA copies/microliter in 20 minutes. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that detected SARS-CoV-2 RNA from nasopharyngeal samples with PCR-derived Ct values up to 29 in microfluidic chips, using a compact imaging system. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables direct RNA detection in a format amenable to point-of-care infection diagnosis, as well as to a wide range of other diagnostic or research applications.
RESUMEN
Rapid nucleic acid testing is a critical component of a robust infrastructure for increased disease surveillance. Here, we report a microfluidic platform for point-of-care, CRISPR-based molecular diagnostics. We first developed a nucleic acid test which pairs distinct mechanisms of DNA and RNA amplification optimized for high sensitivity and rapid kinetics, linked to Cas13 detection for specificity. We combined this workflow with an extraction-free sample lysis protocol using shelf-stable reagents that are widely available at low cost, and a multiplexed human gene control for calling negative test results. As a proof-of-concept, we demonstrate sensitivity down to 40 copies/L of SARS-CoV-2 in unextracted saliva within 35 minutes, and validated the test on total RNA extracted from patient nasal swabs with a range of qPCR Ct values from 13-35. To enable sample-to-answer testing, we integrated this diagnostic reaction with a single-use, gravity-driven microfluidic cartridge followed by real-time fluorescent detection in a compact companion instrument. We envision this approach for Diagnostics with Coronavirus Enzymatic Reporting (DISCoVER) will incentivize frequent, fast, and easy testing.
RESUMEN
The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic and pre-symptomatic carriers of the virus. CRISPR-based diagnostics that utilize RNA and DNA-targeting enzymes can augment gold-standard PCR-based testing if they can be made rapid, portable and accurate. Here we report the development of an amplification-free CRISPR-Cas13a-based mobile phone assay for direct detection of SARS-CoV-2 from nasal swab RNA extracts. The assay achieved [~]100 copies/L sensitivity in under 30 minutes and accurately detected a set of positive clinical samples in under 5 minutes. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity, and we directly quantified viral load using enzyme kinetics. Combined with mobile phone-based quantification, this assay can provide rapid, low-cost, point-of-care screening to aid in the control of SARS-CoV-2.
RESUMEN
Purpose@#The purpose of this study was to identify the effects of the types of seat surface (static or dynamic seat surface) on the pulmonary functions during prolonged sitting. @*Methods@#Thirty-four participants (20 males and 14 females) were recruited, and distributed randomly into dynamic prolonged sitting (DPS, n=17) and static prolonged sitting (SPS, n=17) groups. The DPS group was seated on a chair with a dynamic air cushion, and the SPS group was seated on a chair without a dynamic air cushion. The pulmonary function was assessed before sitting, and after participants had been seated for one hour. The pulmonary function [forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), and Peak expiratory flow (PEF)] was measured using a spirometer. @*Results@#Statistical analyses revealed significant differences in the time x group interactions of FVC, FEV1, PEF, and FEV1/FVC. The DPS group were significantly different in FVC, FEV1, PEF, and FEV1/FVC after prolonged sitting for one hour, compared to the SPS group (p<0.05). @*Conclusion@#These findings suggest that dynamic sitting can prevent a decrease in the physiological function, such as pulmonary functions, rather than static sitting during prolonged sitting.
RESUMEN
Purpose@#This study examined whether the motivation for rehabilitation influences the functional performance of stroke patients. @*Methods@#To elucidate the same, correlation analysis between the ‘rehabilitation motivation level’ and ‘functional performance improvement’ was performed. Thirty-three stroke patients, who were hospitalized in rehabilitation and nursing hospitals, were enrolled in this study. Functional performance was assessed using the 10MWT, FRT, and the BBS. Statistical analysis was performed to determine the correlation between the reactivation symptoms and rehabilitation therapy at four weeks, eight weeks, and 12 weeks. @*Results@#The results showed a significant correlation between the reactivation symptoms and rehabilitation therapy, as evidenced by the 10MWT, FRT, and BBS (8-12 weeks 10M gait test, Berg balance scale for 4-8 weeks, and 8-12 weeks). The FRT did not reveal any significant correlation between the rehabilitation program and reactivation. @*Conclusion@#The present study confirmed that a higher motivation for rehabilitation in stroke patients resulting in a higher likelihood of it acting as a factor in improving their functional ability. Based on the above-mentioned results, this study suggests that a rehabilitation program could be considered for the motivation of patients to participate in a rehabilitation program.
RESUMEN
Purpose@#The purpose of this study was to identify the effects of the types of seat surface (static or dynamic seat surface) on the pulmonary functions during prolonged sitting. @*Methods@#Thirty-four participants (20 males and 14 females) were recruited, and distributed randomly into dynamic prolonged sitting (DPS, n=17) and static prolonged sitting (SPS, n=17) groups. The DPS group was seated on a chair with a dynamic air cushion, and the SPS group was seated on a chair without a dynamic air cushion. The pulmonary function was assessed before sitting, and after participants had been seated for one hour. The pulmonary function [forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), and Peak expiratory flow (PEF)] was measured using a spirometer. @*Results@#Statistical analyses revealed significant differences in the time x group interactions of FVC, FEV1, PEF, and FEV1/FVC. The DPS group were significantly different in FVC, FEV1, PEF, and FEV1/FVC after prolonged sitting for one hour, compared to the SPS group (p<0.05). @*Conclusion@#These findings suggest that dynamic sitting can prevent a decrease in the physiological function, such as pulmonary functions, rather than static sitting during prolonged sitting.
RESUMEN
Purpose@#This study examined whether the motivation for rehabilitation influences the functional performance of stroke patients. @*Methods@#To elucidate the same, correlation analysis between the ‘rehabilitation motivation level’ and ‘functional performance improvement’ was performed. Thirty-three stroke patients, who were hospitalized in rehabilitation and nursing hospitals, were enrolled in this study. Functional performance was assessed using the 10MWT, FRT, and the BBS. Statistical analysis was performed to determine the correlation between the reactivation symptoms and rehabilitation therapy at four weeks, eight weeks, and 12 weeks. @*Results@#The results showed a significant correlation between the reactivation symptoms and rehabilitation therapy, as evidenced by the 10MWT, FRT, and BBS (8-12 weeks 10M gait test, Berg balance scale for 4-8 weeks, and 8-12 weeks). The FRT did not reveal any significant correlation between the rehabilitation program and reactivation. @*Conclusion@#The present study confirmed that a higher motivation for rehabilitation in stroke patients resulting in a higher likelihood of it acting as a factor in improving their functional ability. Based on the above-mentioned results, this study suggests that a rehabilitation program could be considered for the motivation of patients to participate in a rehabilitation program.