A lateral flow strip combined with Cas9 nickase-triggered amplification reaction for dual food-borne pathogen detection
BIOSENSORS & BIOELECTRONICS
Authors: Wang, Luying; Shen, Xingying; Wang, Ting; Chen, Pinru; Qi, Nan; Yin, Bin-Cheng; Ye, Bang-Ce
Nucleic acid-based detection methods are accurate and rapid, which are widely-used in food-borne pathogen detection. However, traditional nucleic acid-based detection methods usually rely on special instruments, weakening their practicality for on-site tests in resource-limited locations. In this work, we developed a convenient and affordable method for food-borne pathogen detection based on a lateral flow strip combined with Cas9 nickase-triggered isothermal DNA amplification, which allows instrument-free and dual target detection. The genomic DNAs of two most common foodborne pathogens, Salmonella typhimurium and Escherichia coli, were simultaneously amplified in a one-pot reaction using specific sgRNAs and primers. The amplicons of genomic DNAs were double-labelled by digoxin/biotin and FITC/biotin tags, respectively, and directly visualized on a simple lateral flow strip. Our method exhibited a high specificity and sensitivity with a detection limit of 100 copies for genomic DNAs and 100 CFU/mL for bacteria. We believe that this method has potential to provide a convenient and low-cost point-of-care test for pathogen detection in the food quality surveillance.
ppGpp Coordinates Nucleotide and Amino-Acid Synthesis in E. coli During Starvation
Authors: Wang, Boyuan; Grant, Robert A.; Laub, Michael T.
(p)ppGpp is a nucleotide messenger universally produced in bacteria following nutrient starvation. In E. coli, ppGpp inhibits purine nucleotide synthesis by targeting several different enzymes, but the physiological significance of their inhibition is unknown. Here, we report the structural basis of inhibition for one target, Gsk, the inosine-guanosine kinase. Gsk creates an unprecedented, allosteric binding pocket for ppGpp by restructuring terminal sequences, which restrains conformational dynamics necessary for catalysis. Guided by this structure, we generated a chromosomal mutation that abolishes Gsk regulation by ppGpp. This mutant strain accumulates abnormally high levels of purine nucleotides following amino-acid starvation, compromising cellular fitness. We demonstrate that this unrestricted increase in purine nucleotides is detrimental because it severely depletes pRpp and essential, pRpp-derived metabolites, including UTP, histidine, and tryptophan. Thus, our results reveal the significance of ppGpp's regulation of purine nucleotide synthesis and a critical mechanism by which E. coli coordinates biosynthetic processes during starvation.