Ultraspecific One-Pot CRISPR-Based "Green-Yellow-Red" Multiplex Detection Strategy Integrated with Portable Cartridge for Point-of-Care Diagnosis.

Versatile, informative, sensitive, and specific nucleic acid detection plays a crucial role in point-of-care pathogen testing, genotyping, and disease monitoring. In this study, we present a novel one-pot Cas12b-based method coupled with the "Green-Yellow-Red" strategy for multiplex detection. By integrating RT-LAMP amplification and Cas12b cleavage in a single tube, the entire detection process can be completed within 1 h. Our proposed method exhibits high specificity, enabling the discrimination of single-base mutations with detection sensitivity approaching single molecule levels. Additionally, the fluorescent results can be directly observed by the naked eye or automatically analyzed using our custom-designed software Result Analyzer. To realize point-of-care detection, we developed a portable cartridge capable of both heating and fluorescence excitation. In a clinical evaluation involving 20 potentially SARS-CoV-2-infected samples, our method achieved a 100% positive detection rate when compared to standard RT-PCR. Furthermore, the identification of SARS-CoV-2 variants using our method yielded results that were consistent with the sequencing results. Notably, our proposed method demonstrates excellent transferability, allowing for the simultaneous detection of various pathogens and the identification of mutations as low as 0.5% amidst a high background interference. These findings highlight the tremendous potential of our developed method for molecular diagnostics.

utPCR: A Strategy for the Highly Specific and Absolutely Quantitative Detection of Single Molecules within Only Minutes.

Bloodstream infection is a major health problem worldwide, with extremely high mortality. Detecting infection in the early stage is challenging due to the extremely low concentration of bacteria in the blood. Digital PCR provides unparalleled sensitivity and can achieve absolute quantification, but it is time-consuming. Moreover, the presence of unavoidable background signals in negative controls poses a significant challenge for single-molecule detection. Here, we propose a novel strategy called "Ultrafast flexible thin tube-based droplet digital PCR (utPCR)" that can shorten the digital PCR process from 2 h to only 5 min, with primer annealing/extension time reduced from minutes to only 5 s. Importantly, the ultrafast PCR eliminates nonspecific amplification and thus enables single-molecule detection. The utPCR enabled the sensitive detection and digital quantification of E. coli O157 in the high background of a 106-fold excess of E. coli K12 cells. Moreover, this method also displayed the potential to detect rare pathogens in blood samples, and the limit of detection (LOD) could be as low as 10 CFU per mL of blood without false positive results. Considered ultrafast (<5 min) and highly sensitive (single-molecule detection), the utPCR holds excellent prospects in the next generation of molecular diagnosis.

Widely targeted metabolomics analysis reveals the mechanism of quality improvement of flue-cured tobacco.

Flue-curing of top leaves with stems is a widely applied curing technology. Owing to the presence of stems, the quality of flue-cured leaves was significantly improved. However, the contribution of stems to flue-cured leaves is still unknown. In this study, the differences in physicochemical properties and metabolomics data between separated leaves (stem(-)) and leaves with stems (stem(+)) were investigated. The metabolic profiling of stem(+) was significantly different from that of stem(-), with phytohormone indole-3-acetic acid (IAA) being one of the most differential metabolites. The presence of stems reduced the rate of water loss in leaves, which led to less ROS accumulation, higher antioxidant enzyme activities and a lower level of membrane lipid peroxidation in stem(+) than in stem(-). The presence of stems also helped maintain the cellular membrane integrity of leaf cells by preventing the accumulation of IAA in leaf cells. Better cellular membrane integrity during flue-curing means a lower risk of leaf browning. In addition, stem(+) had a lower starch content than stem(-) because of a higher level of amylase activity. In summary, these results indicated that the presence of stems caused metabolism changes in leaves, prevented flue-cured leaves from browning and enhanced starch degradation in leaves during flue-curing.

Speciation of Main Nutrients (N/P/K) in Hydrochars Produced from the Hydrothermal Carbonization of Swine Manure under Different Reaction Temperatures.

Hydrothermal carbonization (HTC) has been proved to be a promising technology for swine manure (SM) treatment. Currently, there is a lack of systematic understanding of the transformation characteristics of nutrient speciation in the HTC of SM. In this study, the speciation of the main nutrients (N/P/K) in SM-derived hydrochar produced at different reaction temperatures (200-280 °C) was investigated. The recovery of P (61.0-67.1%) in hydrochars was significantly higher than that of N (23.0-39.8%) and K (25.5-30.0%), and the increase in reaction temperature promoted the recovery of P and reduced the recovery of N. After the HTC treatment, the percentage of soluble/available P was reduced from 61.6% in raw SM to 4.0-23.9% in hydrochars, while that of moderately labile/slow-release P was improved from 29.2% in raw SM feedstock to 65.5-82.7%. An obvious reduction was also found in the amounts of available N (from 51.3% in raw SM feedstock to 33.0-40.5% in hydrochars). The percentages of slow-release N and residual N in hydrochars produced at 240 °C reached the maximum and minimum values (46.4% and 18.9%), respectively. A total of 49.5-58.3% of K retained in hydrochars was residual (invalid) potassium. From the perspective of the mobility and availability of N, P and K only, it was suggested that the HTC of SM should be carried out at 220-240 °C. Compared with the original SM, it is safer and more effective to use the SM-derived hydrochar as an organic fertilizer.