Multiplex PMA–qPCR Assay with Internal Amplification Control for Simultaneous Detection of Viable Legionella pneumophila, Salmonella typhimurium, and Staphylococcus aureus in Environmental Waters

Pathogenic microorganisms are responsible for many infectious diseases, and pathogen monitoring is important and necessary for water quality control. This study for the first time explored a multiplex quantitative real-time PCR (qPCR) technique combined with propidium monoazide (PMA) to simultaneously detect viable Legionella pneumophila, Salmonella typhimurium, and Staphylococcus aureus in one reaction from water samples. Sodium lauroyl sarcosinate (sarkosyl) was applied to enhance the dead bacterial permeability of PMA. The sensitivity of the multiplex PMA–qPCR assay achieved two colony-forming units (CFU) per reaction for L. pneumophila and three CFU per reaction for S. typhimurium and S. aureus. No PCR products were amplified from all nontarget control samples. Significantly, with comparable specificity and sensitivity, this newly invented multiplex PMA–qPCR assay took a much shorter time than did conventional culture assays when testing water samples with spiked bacteria and simulated environmental water treatment. The viable multiplex PMA–qPCR assay was further successfully applied to pathogen detection from rivers, canals, and tap water samples after simple water pretreatment

Identification of Edible Bird’s Nest with Amino Acid and Monosaccharide Analysis

This study describes the approach of amino acid and monosaccharide combined with Hotelling T2 range plot to identify edible bird nests (EBN) and non-EBN. Prior to the approach, an analytical method was developed and validated to quantify monosaccharides in EBN. Hotelling T2 range plots of both compounds were successful in predicting the different types of EBN and differentiating EBN and non-EBN. This outcome suggests EBN contains a group of glycoproteins which is not affected by the EBN’s coloration, country of origin, and/or the processing method of the food item. In addition, the glycoproteins were shown to be unique to EBN. EBN were revealed to be rich in protein and essential amino acids as well as contain a wider variety of monosaccharides than most food items. The overall findings suggest that amino acid and monosaccharide provide information not only on the detected compounds and also insights into the glycoproteins of EBN

Intact NIST monoclonal antibody characterization—Proteoforms, glycoforms—Using CE-MS and CE-LIF

<p>Determining and linking the structural heterogeneity of recombinant antibodies to function is critical in the biopharmaceutical industry. We introduce a new microfluidic capillary electrophoresis—mass spectrometry (μCE-MS) approach to characterize intact monoclonal antibody (mAb) and simultaneously quantifying distinct variants. Our MS analysis of intact NIST mAb (RM8671) shows 18 variants identified amongst proteolytic and glycolytic modifications with a range of relative abundances between 0.1% and 100%. In order to verify our quantitative MS results, we used an established system based on capillary electrophoresis—with laser induced fluorescence (CE-LIF) for profiling the N-glycans. All major glycans were identified and further substantiated by exoglycosidase digestion. Interestingly, the µCE-MS analysis of intact NIST mAb consistently yielded higher amounts of G2FG2F-Hex glycoform (~3.4%), whereas the CE-LIF analysis indicates that only 1.4% of G2F-Gal is present. Therefore, the additional hexose adduct observed in µCE-MS may have been the glycation product of the mAb. Further analysis of deglycosylated mAb with µCE-MS made it possible to reveal the glycation with 10.5% of one hexose product and 4.9% of two hexose product in the intact deglycosylated antibody. An integrated solution using two orthogonal and complementary techniques for characterizing antibody glycosylation is provided here.</p