Gale Stewart

Recombinase Polymerase Amplification for Diagnostic Applications

Abstract BACKGROUND First introduced in 2006, recombinase polymerase amplification (RPA) has stirred great interest, as evidenced by 75 publications as of October 2015, with 56 of them just in the last 2 years. The widespread adoption of this isothermal molecular tool in many diagnostic fields represents an affordable (approximately 4.3 USD per test), simple (few and easy hands-on steps), fast (results within 5–20 min), and sensitive (single target copy number detected) method for the identification of pathogens and the detection of single nucleotide polymorphisms in human cancers and genetically modified organisms. CONTENT This review summarizes the current knowledge on RPA. The molecular diagnostics of various RNA/DNA pathogens is discussed while highlighting recent applications in clinical settings with focus on point-of-care (POC) bioassays and on automated fluidic platforms. The strengths and limitations of this isothermal method are also addressed. SUMMARY RPA is becoming a molecular tool of choice for the rapid, specific, and cost-effective identification of pathogens. Owing to minimal sample-preparation requirements, low operation temperature (25–42 °C), and commercial availability of freeze-dried reagents, this method has been applied outside laboratory settings, in remote areas, and interestingly, onboard automated sample-to-answer microfluidic devices. RPA is undoubtedly a promising isothermal molecular technique for clinical microbiology laboratories and emergence response in clinical settings.

Characterization of the mouse metal-regulatory-element-binding proteins, metal element protein-1 and metal regulatory transcription factor-1

Metal activation of metallothionein gene transcription depends mainly on the presence of regulatory DNA sequences termed metal-regulatory elements (MREs) and involves MRE-binding transcription factor-1 (MTF-1) interacting with the MREs in a Zn(2+)-dependent manner. We previously identified and characterized a nuclear protein, termed metal element protein-1 (MEP-1), specifically binding with high affinity to MRE elements. The precise relationship between MTF-1 and MEP-1 was unclear, and to determine whether MEP-1 and MTF-1 were distinct protein species, we performed DNA binding analyses to characterize the binding properties of both proteins. Electrophoretic mobility-shift assays showed that MTF-1, produced in COS cells, produces a slower-migrating band compared with that obtained with purified MEP-1. Using an anti-MTF-1 antibody, we showed that both the MTF-1-MRE and the MEP-1-MRE complexes are supershifted by an anti-MTF-1 antibody, thus demonstrating that MEP-1 is antigenically related to MTF-1. RNase protection analyses carried out with RNA prepared from different tissues and cell lines failed to reveal the presence of MTF-1 splicing variants. This indicates that MEP-1 may be a proteolytic fragment of MTF-1. MTF-1 DNA-binding activity was rapidly activated in vivo by Zn(2+) ions but not by Cd(2+), UV irradiation or PMA, and occurred on ice as well as at 21 degrees C. In control and Zn(2+)-treated cell extracts, DNA-binding activity was not enhanced in vitro following the addition of exogenous Zn(2+) or a preincubation at 37 degrees C. However, recombinant MTF-1 produced in vitro required Zn(2+) activation for DNA binding. Interestingly, treatment of nuclear extracts with calf intestine phosphatase completely abrogated MTF-1 DNA-binding activity, thus suggesting that phosphorylation is involved in the regulation of MTF-1 activity.

Isothermal Recombinase Polymerase Amplification Assay Applied to the Detection of Group B Streptococci in Vaginal/Anal Samples

BACKGROUND Group B streptococcal infections are the leading cause of sepsis and meningitis in newborns. A rapid and reliable method for the detection of this pathogen at the time of delivery is needed for the early treatment of neonates. Isothermal amplification techniques such as recombinase polymerase amplification have advantages relative to PCR in terms of the speed of reaction and simplicity. METHODS We studied the clinical performance of recombinase polymerase amplification for the screening of group B streptococci in vaginal/anal samples from 50 pregnant women. We also compared the limit of detection and the analytical specificity of this isothermal assay to real-time PCR (RT-PCR). RESULTS Compared to RT-PCR, the recombinase polymerase amplification assay showed a clinical sensitivity of 96% and a clinical specificity of 100%. The limit of detection was 98 genome copies and the analytical specificity was 100% for a panel of 15 bacterial and/or fungal strains naturally found in the vaginal/anal flora. Time-to-result for the recombinase polymerase amplification assay was <20 min compared to 45 min for the RT-PCR assay; a positive sample could be detected as early as 8 min. CONCLUSIONS We demonstrate the potential of isothermal recombinase polymerase amplification assay as a clinically useful molecular diagnostic tool that is simple and faster than PCR/RT-PCR. Recombinase polymerase amplification offers great potential for nucleic acid-based diagnostics at the point of care.

Superparamagnetic Nanoparticle-Polystyrene Bead Conjugates as Pathogen Capture Mimics : A Parametric Study of Factors Affecting Capture Efficiency and Specificity

There is currently significant interest in the miniaturization of disease detection platforms. As detection platforms decrease in size there is a need for the development of sample preparation protocols by which cells or biomarkers of interest can be concentrated from large volumes down to volumes more amenable to analysis within microfluidic devices. To address this issue, we present a series of magnetic confinement assays for polystyrene (PS) beads mediated through their covalent modification with a series of superparamagnetic nanoparticles, where the PS beads have many properties similar to bacteria, but are not pathogenic. The magnetic confinement of the PS beads is investigated as a function of (1) the overall nanoparticle size, (2) the loading of superparamagnetic content within the nanoparticle matrix, and (3) the viscosity and volume of the dispersion medium. We demonstrate that the time required for the magnetic capture of the PS beads by the superparamagnetic nanoparticles (1) decreases as the loading of superparamagnetic material into the nanoparticles increases and (2) increases as the viscosity and volume of the dispersion medium are increased. However, limitations in the magnetic confinement efficiency for the PS beads labeled with nanoparticles comprised of low loadings of superparamagnetic material can be overcome through the use of magnetic columns. These magnetic columns provide a practical and fast mode of sample preparation that should facilitate the magnetic concentration of cells and biomarkers from large volumes to volumes more amenable to incorporation into a microfluidic-based analysis system, where they can be analyzed/detected.

Rapid Filtration Separation-Based Sample Preparation Method for Bacillus Spores in Powdery and Environmental Matrices

ABSTRACT Authorities frequently need to analyze suspicious powders and other samples for biothreat agents in order to assess environmental safety. Numerous nucleic acid detection technologies have been developed to detect and identify biowarfare agents in a timely fashion. The extraction of microbial nucleic acids from a wide variety of powdery and environmental samples to obtain a quality level adequate for these technologies still remains a technical challenge. We aimed to develop a rapid and versatile method of separating bacteria from these samples and then extracting their microbial DNA. Bacillus atrophaeus subsp. globigii was used as a simulant of Bacillus anthracis. We studied the effects of a broad variety of powdery and environmental samples on PCR detection and the steps required to alleviate their interference. With a benchmark DNA extraction procedure, 17 of the 23 samples investigated interfered with bacterial lysis and/or PCR-based detection. Therefore, we developed the dual-filter method for applied recovery of microbial particles from environmental and powdery samples (DARE). The DARE procedure allows the separation of bacteria from contaminating matrices that interfere with PCR detection. This procedure required only 2 min, while the DNA extraction process lasted 7 min, for a total of <10 min. This sample preparation procedure allowed the recovery of cleaned bacterial spores and relieved detection interference caused by a wide variety of samples. Our procedure was easily completed in a laboratory facility and is amenable to field application and automation.

Selected ion monitoring of tert-butyldimethylsilyl cholesterol ethers for determination of total cholesterol content in foods

Abstract A gas chromatography/mass spectrometry (GC/MS) method, using tert-butyldimethylsilyl (tBDMS) derivatives of sterols for detection in the selected ion monitoring (SIM) mode has been applied to the determination of total cholesterol in food (eggs, dairy products). Generally, comparison of results with literature data shows agreement with the amount of cholesterol determined by other chromatographic techniques, and slight underestimation if compared to the amount of cholesterol estimated by colorimetric techniques. The saponification and extraction procedure allowed for 98.6% recovery of spiked cholesterol in milk samples with a coefficient of variation of 2.1%. Amounts as low as 5 ng per 100 g food can be detected using external standards with 95% accuracy.

Double blind study on the efficacy and safety of tetrabamate and chlordiazepoxide in the treatment of the acute alcohol withdrawal syndrome

1. Efficacy and safety of tetrabamate and chlordiazepoxide in the treatment of the acute or Primary Alcohol Withdrawal Syndrome (PAWS) were assessed during a randomized double blind clinical trial, carried out on sixty male alcoholic in-patients. 2. The two drugs were administered four times a day in double dummy conditions, according to a fixed-flexible decreasing dosage schedule (six days basic regimen). 3. Drug efficacy was measured daily throughout the study period using a battery of standard instruments for collecting quantitative clinical, behavioral, psychopathological and laboratory data. Side effects were daily recorded. 4. Tetrabamate was found to be as efficient as chlordiazepoxide in reducing the intensity of the PAWS, improving sleep and vital signs rapidly and alleviating anxiety progressively. 5. Tetrabamate was found particularly beneficial for severe tremor. Psychomotor and mood scores consistently favored tetrabamate, suggesting psychoanaleptic properties of this compound (increased diurnal vigilance). 6. Side effects were minimal with tetrabamate and generally of weak intensity with chlordiazepoxide. 7. The results of this study indicate that tetrabamate may represent a new alternative drug of choice for the therapy of the acute alcohol withdrawal syndrome.

Nosology and diagnosis of alcoholism. Issues and perspectives in subtyping (DSM III) simple alcoholism and alcoholism associated with other psychopathologies

The presence of psychopathological syndromes in alcoholic in-patients was assessed using the NIMH-Diagnosis. The most stricking finding of this study was the high percentage of additional psychopathological syndromes associated to alcoholism. Based on this finding, a tentative classification of alcoholism is proposed. The urgent need for a comprehensive diagnostic scheme for alcoholism is underlined.