366 research outputs found
Characterizing the Impact of Primer-Template Mismatches on Recombinase Polymerase Amplification
Recombinase polymerase amplification (RPA) is an isothermal amplification assay that has been ubiquitously utilized in the detection of infectious agents. Like any nucleic acid amplification technology, primer-template complementarity is critical to RPA reaction success. Mismatches arising in the primer-template complex are known to impact reaction kinetics, invalidate downstream analysis, such as nucleic acid quantification, and result in false negatives if used in a diagnostic capacity. Although the impact of specific primer-template mismatches has been well characterized for techniques such as PCR, characterization remains limited for RPA. Through our study, we systematically characterize the impact of mismatches on the RPA reaction, when located in the 3'-anchor region of the primer-template complex. Our investigation identified that the nucleotides involved, as well as position of each mismatch, influence the size of the impact, with terminal cytosine-thymine and guanine-adenine mismatches being the most detrimental. The presence of some mismatch combinations, such as a penultimate cytosine-cytosine and a terminal cytosine-adenine mismatch pairing, led to complete RPA reaction inhibition. Through the successful characterization of 315 mismatch combinations, researchers can optimize their RPA assay accordingly and seek to implement RPA technology for rapid, in-field genotyping
Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.
Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n = 71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n = 90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays could be of use for integration into a point-of-care test for use in resource constrained settings
High-throughput analysis of candidate imprinted genes and allele-specific gene expression in the human term placenta.
BACKGROUND: Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE) is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue. RESULTS: Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA) and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes) remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs) were found in the vicinity of ZNF331. None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2, a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more individuals in 39 candidate genes (18%). CONCLUSIONS: Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes in the human term placenta. ZNF331 is imprinted in human term placenta and might be a new ubiquitously imprinted gene, part of a primate-specific locus. Demonstration of partial imprinting of PHACTR2 calls for re-evaluation of the allelic pattern of expression for the PHACTR2-PLAGL1 locus. ASE was common in human term placenta.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are
Discovery of an intermediate-luminosity red transient in M51 and its likely dust-obscured, infrared-variable progenitor
We present the discovery of an optical transient (OT) in Messier 51,
designated M51 OT2019-1 (also ZTF19aadyppr, AT 2019abn, ATLAS19bzl), by the
Zwicky Transient Facility (ZTF). The OT rose over 15 days to an observed
luminosity of (), in the
luminosity gap between novae and typical supernovae (SNe). Spectra during the
outburst show a red continuum, Balmer emission with a velocity width of
km s, Ca II and [Ca II] emission, and absorption features
characteristic of an F-type supergiant. The spectra and multiband light curves
are similar to the so-called "SN impostors" and intermediate-luminosity red
transients (ILRTs). We directly identify the likely progenitor in archival
Spitzer Space Telescope imaging with a m luminosity of
and a color redder than 0.74 mag, similar
to those of the prototype ILRTs SN 2008S and NGC 300 OT2008-1. Intensive
monitoring of M51 with Spitzer further reveals evidence for variability of the
progenitor candidate at [4.5] in the years before the OT. The progenitor is not
detected in pre-outburst Hubble Space Telescope optical and near-IR images. The
optical colors during outburst combined with spectroscopic temperature
constraints imply a higher reddening of mag and higher
intrinsic luminosity of
() near peak than seen in previous ILRT
candidates. Moreover, the extinction estimate is higher on the rise than on the
plateau, suggestive of an extended phase of circumstellar dust destruction.
These results, enabled by the early discovery of M51 OT2019-1 and extensive
pre-outburst archival coverage, offer new clues about the debated origins of
ILRTs and may challenge the hypothesis that they arise from the
electron-capture induced collapse of extreme asymptotic giant branch stars.Comment: 21 pages, 5 figures, published in ApJ
PCORnet Antibiotics and Childhood Growth Study: Process for Cohort Creation and Cohort Description
OBJECTIVES: The National Patient-Centered Clinical Research Network (PCORnet) supports observational and clinical research using health care data. The PCORnet Antibiotics and Childhood Growth Study is one of PCORnet’s inaugural observational studies. We sought to describe the processes used to integrate and analyze data from children across 35 participating institutions, the cohort characteristics, and prevalence of antibiotic use.
METHODS:We included children in the cohort if they had at least one same-day height and weight measured in each of 3 age periods: 1) before 12 months, 2) 12 to 30 months, and 3) after 24 months. We distributed statistical queries that each institution ran on its local version of the PCORnet Common Data Model, with aggregate data returned for analysis. We defined overweight or obesity as age- and sex-specific body mass index ≥85th percentile, obesity ≥95th percentile, and severe obesity ≥120% of the 95th percentile.
RESULTS: A total of 681,739 children met the cohort inclusion criteria, and participants were racially/ethnically diverse (24.9% black, 17.5% Hispanic). Before 24 months of age, 55.2% of children received at least one antibiotic prescription; 21.3% received a single antibiotic prescription; 14.3% received 4 or more; and 33.3% received a broad-spectrum antibiotic. Overweight and obesity prevalence was 27.6% at age 4 to(n = 362,044) and 36.2% at 9 to(n = 58,344).
CONCLUSIONS: The PCORnet Antibiotics and Childhood Growth Study is a large national longitudinal observational study in a diverse population that will examine the relationship between early antibiotic use and subsequent growth patterns in children
Recommended from our members
Global biome patterns of the Middle and Late Pleistocene
Our primary aim was to assess the hypothesis that distinctive features of the patterns of vegetation change during successive Quaternary glacial–interglacial cycles reflect climatic differences arising from forcing differences. We addressed this hypothesis using 207 half-degree resolution global biome pattern simulations, for time slices between 800 and 2 ka, made using the LPJ-GUESS dynamic global vegetation model. Simulations were driven using ice-core atmospheric CO2 concentrations, Earth's obliquity, and outputs from a pre-industrial and 206 palaeoclimate experiments; four additional simulations were driven using projected future CO2 concentrations. Climate experiments were run using HadCM3. Using a rule-based approach, above-ground biomass and leaf area index of LPJ-GUESS plant functional types were used to infer each grid cell's biome. The hypothesis is supported by the palaeobiome simulations. To enable comparisons with the climatic forcing, multivariate analyses were performed of global vegetation pattern dissimilarities between simulations. Results showed generally similar responses to glacial–interglacial climatic variations during each cycle, although no two interglacials or glacials had identical biome patterns. Atmospheric CO2 concentration was the strongest driver of the dissimilarity patterns. Dissimilarities relative to the time slice with the lowest atmospheric CO2 concentration show the log-linear relationship to atmospheric CO2 concentration expected of an index of ecocarbon sensitivity. For each simulation, extent and total above-ground biomass of each biome were calculated globally and for three longitudinal segments corresponding to the major continental regions. Mean and minimum past extents of forest biomes, notably Temperate Summergreen Forest, in the three major continental regions strongly parallel relative tree diversities, hence supporting the hypothesis that past biome extents played an important role in determining present diversity. Albeit that they reflect the climatic consequences only of the faster Earth system components, simulated potential future biome patterns are unlike any during the past 800 ky, and likely will continue to change markedly for millennia if projected CO2 concentrations are realised
Developing an intervention to facilitate family communication about inherited genetic conditions, and training genetic counsellors in its delivery.
Many families experience difficulty in talking about an inherited genetic condition that affects one or more of them. There have now been a number of studies identifying the issues in detail, however few have developed interventions to assist families. The SPRinG collaborative have used the UK Medical Research Council's guidance on Developing and Evaluating Complex Interventions, to work with families and genetic counsellors (GCs) to co-design a psycho-educational intervention to facilitate family communication and promote better coping and adaptation to living with an inherited genetic condition for parents and their children (<18 years). The intervention is modelled on multi-family discussion groups (MFDGs) used in psychiatric settings. The MFDG was developed and tested over three phases. First focus groups with parents, young people, children and health professionals discussed whether MFDG was acceptable and proposed a suitable design. Using evidence and focus group data, the intervention and a training manual were developed and three GCs were trained in its delivery. Finally, a prototype MFDG was led by a family therapist and co-facilitated by the three GCs. Data analysis showed that families attending the focus groups and intervention thought MFDG highly beneficial, and the pilot sessions had a significant impact on their family' functioning. We also demonstrated that it is possible to train GCs to deliver the MFDG intervention. Further studies are now required to test the feasibility of undertaking a definitive randomised controlled trial to evaluate its effectiveness in improving family outcomes before implementing into genetic counselling practice.The National Institute of Health Research funded the study but any views expressed do not necessarily reflect those of the Authority. Funded by NIHR reference number: RP-DG-1211-10015
- …