Breeding technique of Selenothrips rubrocinctus (Giard, 1901) (Thysanoptera: Thripidae) on roses leaflets

The development of adequate methods for maintaining populations of arthropod organisms in the laboratory has been a challenge due to the characteristics of each species. This work has aimed to define a method for breeding Selenothrips rubrocinctus (Giard, 1901) in rose leaflets in order to study this species in the laboratory. A condition which could maintain the leaflets turgor for a longer time was sought, in order to guarantee both the survival and multiplication of the insects, and less influence of abiotic factors. Four types of substrates were tested: a) a filter paper disk moistened with distilled water covering the bottom of a Petri dish and; b) a vegetable sponge moistened with distilled water surrounding the base of the leaflet; c) a potato, dextrose and agar (BDA) in a microcentrifuge tube surrounding the base of the leaflet; and d) hydrogel in a microcentrifuge tube surrounding the base of the leaflet. The filter paper moistened with distilled water allowed 65% of the leaflets to remain turgid over a 10-day period and was the most suitable substrate for thrips breeding. With the results at hand, we described S. rubrocinctus breeding in the laboratory. The adopted methodology provided the population density stability of the bred insects, as well as the obtainment of specimens of S. rubrocinctus in quantity and quality throughout the entire period of development of studies on the biology of the species.

PSVII-17 Use of a dimethyl carbonate extraction for rumen fluid volatile fatty acid quantification by gas chromatograph mass spectrometry

Analysis of rumen fluid volatile fatty acids (VFA) is typically conducted by injecting acidified aqueous rumen fluid into a gas chromatograph (GC) with a flame ignition detector (FID). Water in GC samples can lead to poor peak shape and to contamination of inlets, potentially causing sample carryover. Aqueous methods are not well suited for use in mass spectrometer (MS) detector systems. The objective of this project was to validate a dimethyl carbonate (DMC) extraction process and GCMS method for rumen VFA analysis. To perform the extraction, 100 µL of sample, KHSO4 (50 g/L), and 2-ethylbutyrate (internal standard; 0.86 mM) are added to a microcentrifuge tube (in order) followed by 1 mL of DMC. The mixture is thoroughly vortexed and centrifuged. The organic layer (top) is removed and placed in a GC vial. The DMC extract is injected (0.5 µL) into an Agilent 5977B GCMS (8:1 split injection) with a polar DB-FFAP column. The column was held at 105°C for 5 min, increased at 10°C/min to 150°C, then 65°C/min to 240°C, and held constant for 10 min. The assay is linear for acetate from approximately 2 mM to at least 130 mM and covers the expected values of rumen concentrations for the other VFA. Recovery of VFA from spiked rumen fluid was tested at three concentrations in rumen fluid from steers fed a finishing diet or grazing wheat pasture. Recovery was not affected by the diet of the animals (P > 0.19) or the amount of VFA spiked (P > 0.27) and averaged 99.9% for all VFA, with valerate being the lowest (95.9%). Including the 10 min hold at 240°C at the end of each run prevented carryover from sample to sample. This method appears to perform well in a GCMS system and accurately and precisely quantify rumen fluid VFA.

Microcentrifuge Tubes as Disposable Immunoelectrochemical Cells for the On-Site Determination of GFAP, Biomarker of Hemorrhagic Stroke

Stroke is the leading cause of mortality worldwide. Differentiating patients with intracerebral hemorrhage (ICH) or ischemic stroke in the first hours of symptoms onset is of paramount importance to the optimal management of patients. Current diagnosis of acute stroke relies on neuroimaging techniques that provide valuable information but not always are readily available. In this context, the development of analytical tools capable of a rapid and on-site differentiation between the types of stroke is an important challenge with great socio-economic benefits. Glial fibrillary acidic protein (GFAP) is considered one of the ICH biomarkers in patients with symptoms of acute stroke. In this work, a simple electroanalytical device for the analysis of GFAP was developed combining stainless-steel pins and a microcentrifuge tube. The sandwich immunoassay for the determination of GFAP was carried out inside the microcentrifuge tube immobilizing the capture antibody on the bottom of the tube. The three stainless-steel pins acting as electrodes were inserted in the cap in such a way that, when the immunoassay is finished, the tube is turned bottom up allowing the electrochemical detection in the same tube.

Survival of Salmonella on red meat in response to dry heat

Red meat is associated with Salmonella outbreaks resulting in negative impacts for the processing industry. Little work has been reported on the use of dry heat as opposed to moist heat against Salmonella on red meat. We determined the effect of drying at 25°C and dry heat at 70°C with ~10% relative humidity (RH) for 1 h against eleven Salmonella strains of multiple serovars on beef, lamb, goat, and rubber as an inert surface. Each strain at ~108 cfu/ml was inoculated (100ul) onto ±1g (1cm2) of each surface and allowed to attach for 15 min in a microcentrifuge tube. Samples were then exposed to 70°C and 25°C with ~10% RH in a heating block. Surviving Salmonella numbers on surfaces were enumerated on a thin layer medium. If numbers were below the limit of detection (LOD), (2.01 log cfu/cm2), Salmonella cells were enriched before plating to determine the presence of viable cells. Water loss (%) from meat after at 25°C and 70°C was determined. Whole genomes of Salmonella were interrogated to identify the presence/absence of stress response genes (n=30) related to dry heat which may contribute to the survival of Salmonella. The survival of Salmonella at 25°C was significantly higher across all surfaces (~6.09–7.91 log cfu/cm2) as compared to 70°C (~3.66–6.33 log cfu/cm2). On rubber, numbers of Salmonella were <LOD at 70°C. Water loss at 70°C (~17.72–19.89%) was significantly higher as compared to 25°C (~2.98–4.11%). Salmonella was not detected on rubber while survival occurred on all red meat at 70°C, suggesting its protective effect against the effect of heat. All Salmonella strains carried 30 stress response genes which likely contributed to its survival. A multi-antibiotic resistant S. Typhimurium 2470 exhibited an increase in heat resistance at 70°C on beef and lamb as compared to other strains. Our work shows that dry heat at 70°C for 1 h against Salmonella on red meat is not a practical approach for effectively reducing or eliminating them from red meat.

3D Printed Monolithic Microreactors for Real-Time Detection of Klebsiella pneumoniae and the Resistance Gene blaNDM-1 by Recombinase Polymerase Amplification

We investigate the compatibility of three 3D printing materials towards real-time recombinase polymerase amplification (rtRPA). Both the general ability of the rtRPA reaction to occur while in contact with the cured 3D printing materials as well as the residual autofluorescence and fluorescence drift in dependence on post curing of the materials is characterized. We 3D printed monolithic rtRPA microreactors and subjected the devices to different post curing protocols. Residual autofluorescence and drift, as well as rtRPA kinetics, were then measured in a custom-made mobile temperature-controlled fluorescence reader (mTFR). Furthermore, we investigated the effects of storage on the devices over a 30-day period. Finally, we present the single- and duplex rtRPA detection of both the organism-specific Klebsiella haemolysin (khe) gene and the New Delhi metallo-β-lactamase 1 (blaNDM-1) gene from Klebsiella pneumoniae. Results: No combination of 3D printing resin and post curing protocol completely inhibited the rtRPA reaction. The autofluorescence and fluorescence drift measured were found to be highly dependent on printing material and wavelength. Storage had the effect of decreasing the autofluorescence of the investigated materials. Both khe and blaNDM-1 were successfully detected by single- and duplex-rtRPA inside monolithic rtRPA microreactors printed from NextDent Ortho Clear (NXOC). The reaction kinetics were found to be close to those observed for rtRPA performed in a microcentrifuge tube without the need for mixing during amplification. Singleplex assays for both khe and blaNDM-1 achieved a limit of detection of 2.5 × 101 DNA copies while the duplex assay achieved 2.5 × 101 DNA copies for khe and 2.5 × 102 DNA copies for blaNDM-1. Impact: We expand on the state of the art by demonstrating a technology that can manufacture monolithic microfluidic devices that are readily suitable for rtRPA. The devices exhibit very low autofluorescence and fluorescence drift and are compatible with RPA chemistry without the need for any surface pre-treatment such as blocking with, e.g., BSA or PEG.

Field-deployable, rapid diagnostic testing of saliva samples for SARS-CoV-2

Rapid, scalable, point-of-need, COVID-19 diagnostic testing is necessary to safely re-open economies and prevent future outbreaks. We developed an assay that detects single copies of SARS-CoV-2 virus directly from saliva and swab samples in 30 min using a simple, one-step protocol that utilizes only a heat block and microcentrifuge tube prefilled with a mixture containing the necessary reagents and has a sensitivity and specificity of 97% and 100%, respectively.

Simplifying Glycan Profiling through a High-Throughput Micropermethylation Strategy

Glycoproteins play key roles in various molecular and cellular functions and are among the most difficult to analyze biomolecules on account of their microheterogeneity, non-template-driven synthesis, and low abundances. The stability, serum half-life, immunogenicity, and biological activity of therapeutic glycoproteins, including antibodies, vaccines, and biomarkers, are regulated by their glycosylation profile. Thus, there is increasing demand for the qualitative and quantitative characterization and validation of glycosylation on glycoproteins. One of the most important derivatization processes for the structural characterization of released glycans by mass spectrometry (MS) is permethylation. We have recently developed a permethylation strategy in microscale that allows facile permethylation of glycans and permits the processing of large sample sets in nanogram amounts through high-throughput sample handling. Here, we are reporting the wide potential of micropermethylation-based high-throughput structural analysis of glycans from various sources, including human plasma, mammalian cells, and purified glycoproteins, through an automated tandem electrospray ionization–mass spectrometry (ESI-MSn) platform. The glycans released from the plasma, cells, and glycoproteins are permethylated in microscale in a 96-well plate or microcentrifuge tube and isolated by a C18 tip-based cleanup through a shorter and simple process. We have developed a workflow to accomplish an in-depth automated structural characterization MS program for permethylated N/O-glycans through an automated high-throughput multistage tandem MS acquisition. We have demonstrated the utility of this workflow using the examples of sialic acid linkages and bisecting GlcNAc ( N-acetylglucosamine) on the glycans. This approach can automate the high-throughput screening of glycosylation on large sample sets of glycoproteins, including clinical glycan biomarkers and glycoprotein therapeutics.

Detection of Particulate Matter of Size 2.5 μm with a Surface-Acoustic-Wave Sensor Combined with a Cyclone Separator

A device to monitor particulate matter of size 2.5 μm (PM2.5) that has been designed and developed includes a surface-acoustic-wave sensor operating in a shear horizontal mode (SH-SAW) combined with a cyclone separator. In our tests, aerosols generated as incense smoke were first separated and sampled inside a designed cyclone separator; the sampled PM2.5 was then introduced into the sensing area of an SH-SAW sensor for detection. The use of microcentrifuge tubes as a cyclone separator effectively decreases the size and power consumption of the device; the SAW sensor in a well design and operating at 122 MHz was fabricated with MEMS techniques. After an explanation of the design of the cyclone separator, a simulation of the efficiency and the SAW sensor detection are discussed. A microcentrifuge tube (volume 0.2 mL, inlet and outlet diameters 0.5 mm) as a separator has separation cutoff diameters 50% (d50) at 2.5 μm; the required rate of volumetric flow at the inlet is 0.125 LPM, according to simulation with computational fluid dynamics (CFD) software; the surface-acoustic-wave (SAW) sensor exhibits sensitivity approximately 9 Hz/ng; an experiment for PM2.5 detection conducted with the combined device shows a strong positive linear correlation with a commercial aerosol monitor. The limit of detection (LOD) is 11 μg/m3 with sample time 160 s and total detection duration about 5 min.