A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for Salmonella Detection.

BACKGROUND: Foodborne pathogenic bacteria threaten worldwide public health, and simple bacterial detection methods are in urgent need. Here, we established a lab-on-a-tube biosensor for simple, rapid, sensitive, and specific detection of foodborne bacteria. METHODS: A rotatable Halbach cylinder magnet and an iron wire netting with magnetic silica beads (MSBs) were used for simple and effective extraction and purification of DNA from the target bacteria, and recombinase-aided amplification (RAA) was combined with clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins12a(CRISPR-Cas12a) to amplify DNA and generate fluorescent signal. First, 15 mL of the bacterial sample was centrifuged, and the bacterial pellet was lysed by protease to release target DNA. Then, DNA-MSB complexes were formed as the tube was intermittently rotated and distributed uniformly onto the iron wire netting inside the Halbach cylinder magnet. Finally, the purified DNA was amplified using RAA and quantitatively detected by the CRISPR-Cas12a assay. RESULTS: This biosensor could quantitatively detect Salmonella in spiked milk samples in 75 min, with a lower detection limit of 6 CFU/mL. The fluorescent signal of 102 CFU/mL Salmonella Typhimurium was over 2000 RFU, while 104 CFU/mL Listeria monocytogenes, Bacillus cereus, and E. coli O157:H7 were selected as non-target bacteria and had signals less than 500 RFU (same as the negative control). CONCLUSIONS: This lab-on-a-tube biosensor integrates cell lysis, DNA extraction, and RAA amplification in one 15 mL tube to simplify the operation and avoid contamination, making it suitable for low-concentration Salmonella detection.

An automatic centrifugal system for rapid detection of bacteria based on immunomagnetic separation and recombinase aided amplification.

This study reported an automatic centrifugal system for rapid quantification of foodborne pathogenic bacteria based on immunomagnetic separation (IMS) for target bacteria enrichment and recombinase aided amplification (RAA) for nucleic acid detection. First, target bacteria were captured by immune magnetic nanoparticles (MNPs) to form magnetic bacteria, which were purified and enriched by magnetic separation. Then, nucleic acid extraction buffer was used to extract genomic DNA of magnetic bacteria and dissolve lyophilized RAA reagent. Finally, isothermal amplification and fluorescent detection were conducted for bacteria quantification. Bacteria magnetic separation, nucleic acid extraction and fluorescent RAA detection were elaborately achieved in a centrifugal disc with unique functional chambers and multistage siphon channels. A supporting device was developed to automatically and successively perform the programmed centrifugal protocol, including temperature control for isothermal amplification and fluorescence detection for real-time RAA analysis. Under optimal conditions, this centrifugal system enabled Salmonella detection as low as 10 CFU mL-1 in spiked chicken samples in 1 h with average recovery of 105.6% and average standard deviation of 8.4%. It has been demonstrated as an alternative for rapid detection of Salmonella.

A Salmonella Microfluidic Chip Combining Non-Contact Eddy Heater and 3D Fan-Shaped Mixer with Recombinase Aided Amplification.

Foodborne pathogenic bacteria have become a worldwide threat to human health, and rapid and sensitive bacterial detection methods are urgently needed. In this study, a facile microfluidic chip was developed and combined with recombinase-aided amplification (RAA) for rapid and sensitive detection of Salmonella typhimurium using a non-contact eddy heater for dynamic lysis of bacterial cells and a 3D-printed fan-shaped active mixer for continuous-flow mixing. First, the bacterial sample was injected into the chip to flow through the spiral channel coiling around an iron rod under an alternating electromagnetic field, resulting in the dynamic lysis of bacterial cells by this non-contact eddy heater to release their nucleic acids. After cooling to ~75 °C, these nucleic acids were continuous-flow mixed with magnetic silica beads using the fan-shaped mixer and captured in the separation chamber using a magnet. Finally, the captured nucleic acids were eluted by the eluent from the beads to flow into the detection chamber, followed by RAA detection of nucleic acids to determine the bacterial amount. Under the optimal conditions, this microfluidic chip was able to quantitatively detect Salmonella typhimurium from 1.1 × 102 to 1.1 × 105 CFU/mL in 40 min with a detection limit of 89 CFU/mL and might be prospective to offer a simple, low-cost, fast and specific bacterial detection technique for ensuring food safety.

The Beneficial Effects of Edible Kynurenic Acid from Marine Horseshoe Crab (Tachypleus tridentatus) on Obesity, Hyperlipidemia, and Gut Microbiota in High-Fat Diet-Fed Mice.

The marine horseshoe crab (Tachypleus tridentatus) has been considered as food and traditional medicine for many years. Kynurenic acid (KA) was isolated from horseshoe crab in this study for the first time in the world. A previous study in 2018 reported that intraperitoneal administration of KA prevented high-fat diet- (HFD-) induced body weight gain. Now, we investigated the effects of intragastric gavage of KA on HFD mice and found that KA (5 mg/kg/day) inhibited both the body weight gain and the increase of average daily energy intake. KA reduced serum triglyceride and increased serum high-density lipoprotein cholesterol. KA inhibited HFD-induced the increases of serum low-density lipoprotein cholesterol, coronary artery risk index, and atherosclerosis index. KA also suppressed HFD-induced the increase of the ratio of Firmicutes to Bacteroidetes (two dominant gut microbial phyla). KA partially reversed HFD-induced the changes in the composition of gut microbial genera. These overall effects of KA on HFD mice were similar to that of simvastatin (positive control). But the effects of 1.25 mg/kg/day KA on HFD-caused hyperlipidemia were similar to the effects of 5 mg/kg/day simvastatin. The pattern of relative abundance in 40 key genera of gut microbiota from KA group was closer to that from the normal group than that from the simvastatin group. In addition, our in vitro results showed the potential antioxidant activity of KA, which suggests that the improvement effects of KA on HFD mice may be partially associated with antioxidant activity of KA. Our findings demonstrate the potential role of KA as a functional food ingredient for the treatment of obesity and hyperlipidemia as well as the modulation of gut microbiota.

Characterization and comparison of milk fat globule membrane N-glycoproteomes from human and bovine colostrum and mature milk.

Human and bovine milk fat globule membrane (MFGM) proteins have been identified and characterized; however, their glycosylation during lactation remains unclear. We adopted a glycoproteomics approach to profile and compare MFGM N-glycoproteomes in human and bovine milk during lactation. A total of 843, 718, 614, and 273 N-glycosite peptides corresponding to 465, 423, 334, and 176 glycoproteins were identified in human colostrum, human mature milk, bovine colostrum, and bovine mature milk, respectively. The biological functions of these MFGM N-glycoproteins were revealed through bioinformatics. Substantial differences were observed between human and bovine milk, and immune-related MFGM N-glycoproteins varied between colostrum and mature milk from both species. Our results expand current knowledge of MFGM N-glycoproteomes, and further demonstrate the complexity and biological functions of MFGM N-glycosylation. These data can provide references for the application of bovine MFGM N-glycoproteins in infant formula to resemble human milk and in functional foods.

Quantitative N-glycoproteomics of milk fat globule membrane in human colostrum and mature milk reveals changes in protein glycosylation during lactation.

Milk fat globule membrane (MFGM) proteins have recently gained increasing attention, due to their significant biological function. However, the glycosylation of proteins in human MFGM during lactation has not been studied in detail. In this study, through mass spectroscopy-based N-glycoproteomics, we analyzed protein glycosylation of human MFGM. A total of 912 N-glycosylation sites on 506 N-glycoproteins were identified in human colostrum and mature milk MFGM. Among them, 220 N-glycoproteins with 304 N-glycosylation sites were differentially expressed in colostrum and mature milk MFGM. Gene Ontology (GO) analysis revealed various biological processes, cellular components, and molecular functions of the differentially expressed N-glycoproteins. Specifically, these glycoproteins were involved in biological processes such as single-organism processes, biological regulation, regulation of biological processes, response to stimulus and localization; were cellular components in organelles, membranes, and the extracellular region; and had different molecular functions such as protein binding, receptor activity, and hydrolase activity. KEGG pathway analysis suggested that the majority of the differentially expressed N-glycoproteins were associated with phagosome, cell adhesion molecule and some disease-related pathways. Our results provide an in-depth understanding of the quantitative changes in N-glycosylation of proteins in human colostrum and mature MFGM, and extend our knowledge of the N-glycoproteome and of the distribution of N-glycosylation sites in human MFGM during lactation, providing insight into the biological functions of the highlighted glycoproteins.

Chaotic extension neural network theory-based XXY stage collision fault detection using a single accelerometer sensor.

The collision fault detection of a XXY stage is proposed for the first time in this paper. The stage characteristic signals are extracted and imported into the master and slave chaos error systems by signal filtering from the vibratory magnitude of the stage. The trajectory diagram is made from the chaos synchronization dynamic error signals E1 and E2. The distance between characteristic positive and negative centers of gravity, as well as the maximum and minimum distances of trajectory diagram, are captured as the characteristics of fault recognition by observing the variation in various signal trajectory diagrams. The matter-element model of normal status and collision status is built by an extension neural network. The correlation grade of various fault statuses of the XXY stage was calculated for diagnosis. The dSPACE is used for real-time analysis of stage fault status with an accelerometer sensor. Three stage fault statuses are detected in this study, including normal status, Y collision fault and X collision fault. It is shown that the scheme can have at least 75% diagnosis rate for collision faults of the XXY stage. As a result, the fault diagnosis system can be implemented using just one sensor, and consequently the hardware cost is significantly reduced.