Loop-mediated isothermal amplification (LAMP) assay for specific and rapid detection of Dickeya fangzhongdai targeting a unique genomic region.

Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae) causes soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai. Multiple genomes of D. fangzhongdai, and other species of Dickeya, Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with tenfold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18-20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies.

Getting your leadership game on in virtual environments.

This chapter explores strategies for using virtual games in leadership education. Pedagogical practices, examples, and connections to leadership learning frameworks, learning goals, and competencies are also included. Implications for practice such as intentional design, pairing with other instructional strategies, and debriefing are also discussed with an emphasis on purposeful utilization.

Chemical stabilization of dispersed Escherichia coli for enhanced recovery with a handheld electroflotation system and detection by Loop-mediated Isothermal AMPlification.

Constraints related to sample preparation are some of the primary obstacles to widespread deployment of molecular diagnostics for rapid detection of trace quantities (≤103 CFU/mL) of food-borne pathogens. In this research, we report a sample preparation method using a novel handheld electroflotation system to concentrate and recover dilute quantities (102-103 CFU/mL) of Escherichia coli (E. coli) 25922 in artificially contaminated samples for reliable, rapid detection by loop-mediated isothermal amplification (LAMP). To protect suspended cells from shear stresses at bubble surfaces, a non-ionic surfactant (Pluronic-F68) and flocculant (chitosan oligosaccharide) were used to aggregate cells and reduce their surface hydrophobicity. Effective conditions for recovery were determined through multifactorial experiments including various concentrations of Pluronic-F68 (0.001, 0.01, 0.1, 1 g L-1), chitosan oligosaccharide (0.01, 0.1, 1, 10 g L-1), bacteria (102, 103, 104 CFU/mL E. coli 25922), recovery times (10, 15 and 20 minutes), and degrees of turbulent gas flux ("high" and "low"). The automated electroflotation system was capable of concentrating effectively all of the bacteria from a large sample (380 mL 0.1 M potassium phosphate buffer containing 102 CFU/mL E. coli) into a 1 mL recovered fraction in less than 30 minutes. This enabled detection of bacterial contaminants within 2 hours of collecting the sample, without a specialized laboratory facility or traditional enrichment methods, with at least a 2-3 order of magnitude improvement in detection limit compared to direct assay with LAMP.

Real-time optical analysis of a colorimetric LAMP assay for SARS-CoV-2 in saliva with a handheld instrument improves accuracy compared with endpoint assessment.

Controlling the course of the Coronavirus Disease 2019 (COVID-19) pandemic will require widespread deployment of consistent and accurate diagnostic testing of the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Ideally, tests should detect a minimum viral load, be minimally invasive, and provide a rapid and simple readout. Current Food and Drug Administration (FDA)-approved RT-qPCR-based standard diagnostic approaches require invasive nasopharyngeal swabs and involve laboratory-based analyses that can delay results. Recently, a loop-mediated isothermal nucleic acid amplification (LAMP) test that utilizes colorimetric readout received FDA approval. This approach utilizes a pH indicator dye to detect drop in pH from nucleotide hydrolysis during nucleic acid amplification. This method has only been approved for use with RNA extracted from clinical specimens collected via nasopharyngeal swabs. In this study, we developed a quantitative LAMP-based strategy to detect SARS-CoV-2 RNA in saliva. Our detection system distinguished positive from negative sample types using a handheld instrument that monitors optical changes throughout the LAMP reaction. We used this system in a streamlined LAMP testing protocol that could be completed in less than 2 h to directly detect inactivated SARS-CoV-2 in minimally processed saliva that bypassed RNA extraction, with a limit of detection (LOD) of 50 genomes/reaction. The quantitative method correctly detected virus in 100% of contrived clinical samples spiked with inactivated SARS-CoV-2 at either 1× (50 genomes/reaction) or 2× (100 genomes/reaction) of the LOD. Importantly, the quantitative method was based on dynamic optical changes during the reaction and was able to correctly classify samples that were misclassified by endpoint observation of color.

What Do We Know About Formal Leadership Courses and Their Effects?

In this chapter, the authors review and critique the existing evidence supporting current leadership education pedagogical practices and their effects on student leadership development. They conclude with a series of suggestions for increasing deliberate practices in formal courses designed to improve leadership education.

Followership Education for Postsecondary Students.

Many universities offer one or more courses on leadership that include followership as a topic. A few have offered a complete course on followership. However, there is little information on postsecondary followership training. This chapter explores the unique issues of introducing followership to students, many of whom have been told their entire lives that leadership is everything. This chapter highlights followership courses and programs at institutions that offer them, and explores the delivery of current practices, with suggestions for increasing efficacy.

SNAPS: Sensor Analytics Point Solutions for Detection and Decision Support Systems.

In this review, we discuss the role of sensor analytics point solutions (SNAPS), a reduced complexity machine-assisted decision support tool. We summarize the approaches used for mobile phone-based chemical/biological sensors, including general hardware and software requirements for signal transduction and acquisition. We introduce SNAPS, part of a platform approach to converge sensor data and analytics. The platform is designed to consist of a portfolio of modular tools which may lend itself to dynamic composability by enabling context-specific selection of relevant units, resulting in case-based working modules. SNAPS is an element of this platform where data analytics, statistical characterization and algorithms may be delivered to the data either via embedded systems in devices, or sourced, in near real-time, from mist, fog or cloud computing resources. Convergence of the physical systems with the cyber components paves the path for SNAPS to progress to higher levels of artificial reasoning tools (ART) and emerge as data-informed decision support, as a service for general societal needs. Proof of concept examples of SNAPS are demonstrated both for quantitative data and qualitative data, each operated using a mobile device (smartphone or tablet) for data acquisition and analytics. We discuss the challenges and opportunities for SNAPS, centered around the value to users/stakeholders and the key performance indicators users may find helpful, for these types of machine-assisted tools.

ABE-VIEW: Android Interface for Wireless Data Acquisition and Control.

Advances in scientific knowledge are increasingly supported by a growing community of developers freely sharing new hardware and software tools. In this spirit we have developed a free Android app, ABE-VIEW, that provides a flexible graphical user interface (GUI) populated entirely from a remote instrument by ascii-coded instructions communicated wirelessly over Bluetooth. Options include an interactive chart for plotting data in real time, up to 16 data fields, and virtual controls including buttons, numerical controls with user-defined range and resolution, and radio buttons which the user can use to send coded instructions back to the instrument. Data can be recorded into comma delimited files interactively at the user's discretion. Our original objective of the project was to make data acquisition and control for undergraduate engineering labs more modular and affordable, but we have also found that the tool is highly useful for rapidly testing novel sensor systems for iterative improvement. Here we document the operation of the app and syntax for communicating with it. We also illustrate its application in undergraduate engineering labs on dynamic systems modeling, as well as for identifying the source of harmonic distortion affecting electrochemical impedance measurements at certain frequencies in a novel wireless potentiostat.

Aligning Instructional Strategies with Learning Outcomes and Leadership Competencies.

This chapter provides recommendations for aligning instructional strategies with learning outcomes and leadership competencies to foster intentional student leadership development.

Real-time duplex applications of loop-mediated AMPlification (LAMP) by assimilating probes.

Isothermal nucleic-acid amplification methods such as Loop-Mediated isothermal AMPlification (LAMP) are increasingly appealing alternatives to PCR for use in portable diagnostic system due to the low cost, weight, and power requirements of the instrumentation. As such, interest in developing new probes and other functionality based on the LAMP reaction has been intense. Here, we report on the development of duplexed LAMP assays for pathogen detection using spectrally unique Assimilating Probes. As proof of principle, we used a reaction for Salmonella enterica as a model coupled with a reaction for λ-phage DNA as an internal control, as well as a duplexed assay to sub-type specific quarantine strains of the bacterial wilt pathogen Ralstonia solanacearum. Detection limits for bacterial DNA analyzed in individual reactions was less than 100 genomic equivalents in all cases, and increased by one to two orders of magnitude when reactions were coupled in duplexed formats. Even so, due to the more robust activity of newly available strand-displacing polymerases, the duplexed assays reported here were more powerful than analogous individual reactions reported only a few years ago, and represent a significant advance for incorporation of internal controls to validate assay results in the field.

Phylogeny and classification of Dickeya based on multilocus sequence analysis.

Bacterial heart rot of pineapple reported in Hawaii in 2003 and reoccurring in 2006 was caused by an undetermined species of Dickeya. Classification of the bacterial strains isolated from infected pineapple to one of the recognized Dickeya species and their phylogenetic relationships with Dickeya were determined by a multilocus sequence analysis (MLSA), based on the partial gene sequences of dnaA, dnaJ, dnaX, gyrB and recN. Individual and concatenated gene phylogenies revealed that the strains form a clade with reference Dickeya sp. isolated from pineapple in Malaysia and are closely related to D. zeae; however, previous DNA-DNA reassociation values suggest that these strains do not meet the genomic threshold for consideration in D. zeae, and require further taxonomic analysis. An analysis of the markers used in this MLSA determined that recN was the best overall marker for resolution of species within Dickeya. Differential intraspecies resolution was observed with the other markers, suggesting that marker selection is important for defining relationships within a clade. Phylogenies produced with gene sequences from the sequenced genomes of strains D. dadantii Ech586, D. dadantii Ech703 and D. zeae Ech1591 did not place the sequenced strains with members of other well-characterized members of their respective species. The average nucleotide identity (ANI) and tetranucleotide frequencies determined for the sequenced strains corroborated the results of the MLSA that D. dadantii Ech586 and D. dadantii Ech703 should be reclassified as Dickeya zeae Ech586 and Dickeya paradisiaca Ech703, respectively, whereas D. zeae Ech1591 should be reclassified as Dickeya chrysanthemi Ech1591.

Loop-mediated amplification of the Clavibacter michiganensis subsp. michiganensis micA gene is highly specific.

Loop-mediated amplification (LAMP) was used to specifically identify Clavibacter michiganensis subsp. michiganensis, causal agent of bacterial canker of tomato. LAMP primers were developed to detect micA, a chromosomally stable gene that encodes a type II lantibiotic, michiganin A, which inhibits growth of other C. michiganensis subspecies. In all, 409 bacterial strains (351 C. michiganensis subsp. michiganensis and 58 non-C. michiganensis subsp. michiganensis) from a worldwide collection were tested with LAMP to determine its specificity. LAMP results were compared with genetic profiles established using polymerase chain reaction (PCR) amplification of seven genes (dnaA, ppaJ, pat-1, chpC, tomA, ppaA, and ppaC). C. michiganensis subsp. michiganensis strains produced eight distinct profiles. The LAMP reaction identified all C. michiganensis subsp. michiganensis strains and discriminated them from other C. michiganensis subspecies and non-Clavibacter bacteria. LAMP has advantages over immunodiagnostic and other molecular detection methods because of its specificity and isothermal nature, which allows for easy field application. The LAMP reaction is also not affected by as many inhibitors as PCR. This diagnostic tool has potential to provide an easy, one-step test for rapid identification of C. michiganensis subsp. michiganensis.

Molecular diagnostics in a teacup: Non-Instrumented Nucleic Acid Amplification (NINA) for rapid, low cost detection of Salmonella enterica.

We report on the use of a novel non-instrumented platform to enable a Loop Mediated isothermal Amplification (LAMP) based assay for Salmonella enterica. Heat energy is provided by addition of a small amount (<150 g) of boiling water, and the reaction temperature is regulated by storing latent energy at the melting temperature of a lipid-based engineered phase change material. Endpoint classification of the reaction is achieved without opening the reaction tube by observing the fluorescence of sequence-specific FRET-based assimilating probes with a simple handheld fluorometer. At or above 22°C ambient temperature the non-instrumented devices could maintain reactions above a threshold temperature of 61°C for over 90 min-significantly longer than the 60 min reaction time. Using the simple format, detection limits were less than 20 genome copies for reactions run at ambient temperatures ranging from 8 to 36°C. When used with a pre-enrichment step and non-instrumented DNA extraction device, trace contaminations of Salmonella in milk close to 1 CFU/mL could be reliably detected. These findings illustrate that the non- instrumented amplification approach is a simple, viable, low-cost alternative for field-based food and agricultural diagnostics or clinical applications in developing countries.

Handheld device for real-time, quantitative, LAMP-based detection of Salmonella enterica using assimilating probes.

A simple handheld instrument was designed to enable real-time detection of the LAMP reaction in a standard PCR tube using newly described assimilating probes as sequence-specific reporter molecules. The system was validated using DNA isolated from Salmonella enterica, demonstrating accurate temperature control with little power and little overshoot of setpoint temperatures, with rapid and accurate detection often in less than 30 min and within 20 min for reactions with high (>10(5)) genome copy numbers. The system could be used for quantitative determination of pathogen DNA, with a limit of detection of about 15 genome copies in purified DNA or 25 cells in DNA extracts from chicken rinsate--comparable to values obtained when running the same reaction on a commercial benchtop real-time PCR instrument. Positive classification of standards nominally containing a single genome equivalent was demonstrated, and no false positives were reported. Detection of S. enterica in rinsate from a contaminated chicken sample required 48 h enrichment prior to the LAMP reaction or plating on semi-selective media. The new system demonstrates a major compelling advantage of the LAMP reaction, in that it may be enabled in simple, low-power, handheld devices without sophisticated custom miniaturized disposables. This new diagnostic system is especially promising for on-site diagnostics in the food and agricultural industries where laboratory space is often primitive if it is available at all.

Rapid concentration of bacteria using submicron magnetic anion exchangers for improving PCR-based multiplex pathogen detection.

Rapid concentration of bacterial targets from dilute solutions to improve subsequent PCR detection is investigated in this study. Submicron (average size 500nm) superparamagnetic anion-exchangers (SiMAG-DEAE) were used successfully to concentrate target bacteria from very dilute solutions. A mass-balance model predicted that for Escherichia coli, the extent of cell concentrating increases almost linearly with increasing sample/SiMAG volume ratio up to about 2000, accompanied by only a slight decrease in the capture efficiency (<10%). Our experimental data generally support this analysis in that the SiMAG beads concentrated bacterial targets by two to three orders of magnitude using a sample/bead volume ratio of about 1000, and lowered the PCR detection limit to a level of 10(2)CFU/mL, from 10(4) to 10(5)CFU/mL without concentrating. Several target bacteria can be concentrated concurrently and detected via multiplex PCR, as illustrated using E. coli and Agrobacterium tumefaciens as model bacteria. Finally, concentration and detection of bacteria in fresh produce samples were demonstrated. The integration of submicron magnetic ion exchangers and PCR detection provides an appealing alternative to immunomagnetic separation/PCR in improving pathogen detection.

Non-Instrumented Nucleic Acid Amplification (NINA) for Rapid Detection of Ralstonia solanacearum Race 3 Biovar 2.

We report on the use of a non-instrumented device for the implementation of a loop-mediated amplification (LAMP) based assay for the select-agent bacterial-wilt pathogen Ralstonia solanacearum race 3 biovar 2. Heat energy is generated within the device by the exothermic hydration of calcium oxide, and the reaction temperature is regulated by storing latent energy at the melting temperature of a renewable lipid-based engineered phase-change material. Endpoint detection of the LAMP reaction is achieved without opening the reaction tube by observing the fluorescence of an innovative FRET-based hybridization probe with a simple custom fluorometer. Non-instrumented devices could maintain reactions near the design temperature of 63°C for at least an hour. Using this approach DNA extracted from the pathogen could be detected at fewer than ten copies within a 25 µL reaction mix, illustrating the potential of these technologies for simple, powerful agricultural diagnostics in the field. Furthermore, the assay was just as reliable when implemented in a tropical environment at 31°C as it was when implemented in an air-conditioned lab maintained at 22°C, illustrating the potential value of the technology for field conditions in the tropics and subtropics.

Detection of Ralstonia solanacearum in natural substrates using phage amplification integrated with real-time PCR assay.

A sensitive, selective, and rapid protocol for detecting Ralstonia solanacearum from soil and plant tissues was developed based on the integration of the rapid self-replicating ability of bacteriophages with quantitative PCR (q-PCR). Six bacteriophages were isolated and selected for their ability to specifically infect and lyse R. solanacearum. Sixty-three strains of R. solanacearum and 72 isolates of other bacterial species were tested for their susceptibility to the bacteriophages. Based on the large host range and observed replication speed and reproductive burst sizes in ginger infecting R. solanacearum strain GW-1, phage M_DS1 was selected for the development of the phage-based indirect assay. With primers based on the phage genome, the protocol was used to detect R. solanacearum from a number of substrates. In pure R. solanacearum cultures, the protocol consistently detected approximately 3.3 CFU/ml after an hour's incubation with 5.3x10(2) PFU/ml M_DS1. We used the protocol to confirm the presence of the pathogen in infected potted ginger plants, detecting levels near 10(2) CFU/g in 0.1 g of leaf tissue and levels near 10(3) CFU/ml in drainage water from the pots. In soils emended with the bacteria, we observed detection limits down to approximately 10(2) CFU/g.

Simultaneous determination of hydrolysis and mutarotation rates during the enzymatic hydrolysis of lactose.

An experiment is described in which a custom-made glucose electrode is used to directly monitor the enzymatic hydrolysis of lactose to glucose. The transient profile of beta- d-glucose can be used to simultaneously determine the rate constants for mutarotation and for enzymatic hydrolysis by applying a dynamic nonlinear regression routine. Due to differences in the mutarotation rate constants between lactose and glucose, the beta- d-glucose concentration "overshoots" equilibrium under certain conditions, which can be modeled mathematically. This overshoot can be observed reliably and used to quantify the differences in mutarotational equilibria between glucose and lactose. These observations may be important for the analysis of dairy products and commercial lactase preparations and illustrate an unusual kinetic phenomenon caused by intramolecular forces. This approach may also be important for the accurate determination of a variety of oligosaccharides such as glycogen, which tend to be composed primarily of one stereoisomer.

Hybridization probe for femtomolar quantification of selected nucleic acid sequences on a disposable electrode.

Mixed monolayers of electroactive hybridization probes on gold surfaces of a disposable electrode were investigated as a technology for simple, sensitive, selective, and rapid gene identification. Hybridization to the ferrocene-labeled hairpin probes reproducibly diminished cyclic redox currents, presumably due to a displacement of the label from the electrode. Observed peak current densities were roughly 1000x greater than those observed in previous studies, such that results could easily be interpreted without the use of algorithms to correct for background polarization currents. Probes were sensitive to hybridization with a number of oligonucleotide sequences with varying homology, but target oligonucleotides could be distinguished from competing nontarget sequences based on unique "melting" profiles from the probe. Detection limits were demonstrated down to nearly 100 fM, which may be low enough to identify certain genetic conditions or infections without amplification. This technology has rich potential for use in field devices for gene identification as well as in gene microarrays.

An immersible manometric sensor for measurement of humidity and enzyme mediated changes in dissolved gas.

An immersible manometric sensor was made by covering the gaseous cavity of a pressure transducer with a 1 microm controlled pore membrane. Transfer of gas across the membrane allowed the pressure transducer to record changes in humidity or dissolved gas when immersed in solution. By immersing the sensor in distilled water, atmospheric humidity could be estimated by the deficit of atmospheric vapor pressure from saturation. In another application of the sensor, CO(2) was monitored continuously. This was not possible in previous closed-reactor type manometric sensors, and may allow the new technology to be used in applications requiring continuous monitoring of a process or stream. By coupling the sensor with enzymes liberating or consuming dissolved gas, different chemicals could be estimated. Urea was estimated by first hydrolyzing it with urease and then measuring the resulting CO(2) gas in solution. Glucose was measured through its enzymatic oxidation by glucose oxidase. The sensitivity to urea over the range 0-2.5 mM was about 1.02 kPa/mM, and the standard error was 0.086 mM. Due to the lower solubility of oxygen, the sensitivity to glucose in a range from 0 to 10 microM was over 100 kPa/mM, with a standard error of only 0.76 microM. This sensitivity was not possible in closed-reactor type manometric sensors due to constraints of dimensioning the head space gas volume for reproducibility and effective mass transfer. The 90% rise times for the sensor ranged from about 1-60 min for the different applications. The dynamic characteristics of the device may be improved by using a membrane with greater porosity, higher rigidity and lower thickness, and by reducing the dimensions of the cavity volume in the sensor through integrated microfabrication of the membrane onto the transducer.