Papers by Lorraine Lillis
Molecular and cellular probes, Jan 4, 2016
Recombinase Polymerase Amplification (RPA) can be used to detect pathogen-specific DNA or RNA in ... more Recombinase Polymerase Amplification (RPA) can be used to detect pathogen-specific DNA or RNA in under 20 min without the need for complex instrumentation. These properties enable its potential use in resource limited settings. However, there are concerns that deviations from the manufacturer's protocol and/or storage conditions could influence its performance in low resource settings. RPA amplification relies upon viscous crowding agents for optimal nucleic acid amplification, and thus an interval mixing step after 3-6 min of incubation is recommended to distribute amplicons and improve performance. In this study we used a HIV-1 RPA assay to evaluate the effects of this mixing step on assay performance. A lack of mixing led to a longer time to amplification and inferior detection signal, compromising the sensitivity of the assay. However lowering the assay volume from 50 μL to 5 μL showed similar sensitivity with or without mixing. We present the first peer-reviewed study that ...
Journal of Virological Methods, 2016
A low complexity diagnostic test that rapidly and reliably detects HIV infection in infants at th... more A low complexity diagnostic test that rapidly and reliably detects HIV infection in infants at the point of care could facilitate early treatment, improving outcomes. However, many infant HIV diagnostics can only be performed in laboratory settings. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that can rapidly amplify proviral DNA from multiple subtypes of HIV-1 in under twenty minutes without complex equipment. In this study we added reverse transcription (RT) to RPA to allow detection of both HIV-1 RNA and DNA. We show that this RT-RPA HIV-1 assay has a limit of detection of 10-30 copies of an exact sequence matched DNA or RNA, respectively. In addition, at 100 copies of RNA or DNA, the assay detected 171 of 175 (97.7%) sequence variants that represent all the major subtypes and recombinant forms of HIV-1 Groups M and O. This data suggests that the application of RT-RPA for the combined detection of HIV-1 viral RNA and proviral DNA may prove a highly sensitive tool for rapid and accurate diagnosis of infant HIV.
Retrovirology, 2006
The Tax proteins encoded by human T lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) are tr... more The Tax proteins encoded by human T lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) are transcriptional activators of both the viral long terminal repeat (LTR) and cellular promoters via the CREB and NFkB pathways. In contrast to HTLV-1, HTLV-2 has been classified into four distinct genetic subtypes A, B, C and D defined by phylogenetic analysis of their nucleotide sequences and the size and amino acid sequence of their Tax proteins. In the present study we have analysed and compared the transactivating activities of three Tax 2A and one Tax 2B proteins using LTR and NFkB reporter assays. We found that with the exception of the prototype Tax 2A Mo protein, the other two Tax 2A proteins failed to transactivate either the viral LTR or NFkB promoter in Jurkat and 293T cells. Loss of activity was not associated with either expression levels or an alteration in subcellular distribution as all Tax 2 proteins were predominantly located in the cytoplasm of transfected cells. Analysis...
PloS one, 2014
Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification techno... more Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25-43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was anal...
PloS one, 2014
In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse a... more In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phas...
Veterinary Immunology and Immunopathology, 2007
Epithelia play important immunological roles at a variety of mucosal sites. We examined NFkB acti... more Epithelia play important immunological roles at a variety of mucosal sites. We examined NFkB activity in control and TNF-a treated bovine mammary epithelial monolayers (BME-UV cells). A region of the bovine IL-8 (bIL-8) promoter was sequenced and a putative kB consensus sequence was identified bioinformatically. We used this sequence to analyse nuclear extracts for IL-8 specific NFkB activity. As a surrogate marker of NFkB activation, we investigated IL-8 release in two models. Firstly in BME-UV monolayers, IL-8 release in the presence of pro-and anti-inflammatory agents was determined by enzyme-linked immunosorbent assay (ELISA). Secondly, we measured IL-8 secretion from a novel model of intact mucosal sheets of bovine teat sinus. IL-8 release into bathing solutions was assessed following treatment with pro-and anti-inflammatory agents.
Journal of Applied Microbiology, 2009
Aims: To examine the effect of the pollutant 2,4-dichlorophenol on DNA-and RNA-based bacterial co... more Aims: To examine the effect of the pollutant 2,4-dichlorophenol on DNA-and RNA-based bacterial communities in soil. Methods and Results: Soil was exposed to 100 mg kg )1 of 2,4-dichlorophenol (2,4-DCP), and degradation was monitored over 35 days. DNA and RNA were coextracted, and terminal restriction fragment length polymorphism (T-RFLP) was used to report changes in bacterial communities in response to the presence of the chlorophenol. The phylogenetic composition of the soil during degradation was determined by creating a clone library of amplified 16S rRNA sequences from both DNA and reverse-transcribed RNA from exposed soil. Resulting clones were sequenced, and putative identities were assigned. Conclusions: A significant difference between active (RNA-based) and total (DNA-based) bacterial community structure was observed for both T-RFLP and phylogenetic analyses in response to 2,4-DCP, with more pronounced changes seen in RNA-based communities. Phylogenetic analysis indicated the dominance of Proteobacteria in both profiles. Significance and Impact of the Study: This study describes the response of soil bacterial communities to the addition of the xenobiotic compound 2,4-DCP, and highlights the importance of including RNA-based 16S rRNA analysis to complement any molecular study in a perturbed soil.
Journal of Applied Microbiology, 2013
Anaerobic rumen fungi (Neocallimastigales) play important roles in the breakdown of complex, cell... more Anaerobic rumen fungi (Neocallimastigales) play important roles in the breakdown of complex, cellulose-rich material. Subsequent decomposition products are utilized by other microbes, including methanogens. The aim of this study was to determine the effects of dietary changes on anaerobic rumen fungi diversity. Altered diets through increasing concentrate/forage (50 : 50 vs 90 : 10) ratios and/or the addition of 6% soya oil were offered to steers and the Neocallimastigales community was assessed by PCR-based fingerprinting with specific primers within the barcode region. Both a decrease in fibre content and the addition of 6% soya oil affected Neocallimastigales diversity within solid and liquid rumen phases. The addition of 6% soya oil decreased species richness. Assemblages were strongly affected by the addition of 6% soya oil, whereas unexpectedly, the fibre decrease had less effect. Differences in volatile fatty acid contents (acetate, propionate and butyrate) were significantly associated with changes in Neocallimastigales assemblages between the treatments. Diet clearly influences Neocallimastigales assemblages. The data are interpreted in terms of interactions with other microbial groups involved in fermentation processes within the rumen. Knowledge on the influence of diet on anaerobic fungi is necessary to understand changes in microbial processes occurring within the rumen as this may impact on other rumen processes such as methane production.
Journal of Applied Microbiology, 2011
Methane emissions from ruminants are a significant contributor to global greenhouse gas productio... more Methane emissions from ruminants are a significant contributor to global greenhouse gas production. The aim of this study was to examine the effect of diet on microbial communities in the rumen of steers. The effects of dietary alteration (50 : 50 vs 90 : 10 concentrate-forage ratio, and inclusion of soya oil) on methanogenic and bacterial communities in the rumen of steers were examined using molecular fingerprinting techniques (T-RFLP and automated ribosomal intergenic spacer analysis) and real-time PCR. Bacterial diversity was greatly affected by diet, whereas methanogen diversity was not. However, methanogen abundance was significantly reduced (P = 0.009) in high concentrate-forage diets and in the presence of soya oil (6%). In a parallel study, reduced methane emissions were observed with these diets. The greater effect of dietary alteration on bacterial community in the rumen compared with the methanogen community may reflect the impact of substrate availability on the rumen bacterial community. This resulted in altered rumen volatile fatty acid profiles and had a downstream effect on methanogen abundance, but not diversity. Understanding how rumen microbial communities contribute to methane production and how these microbes are influenced by diet is essential for the rational design of methane mitigation strategies from livestock.
Environmental Science & Technology, 2013
The leaching of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) from particulates deposited in live... more The leaching of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) from particulates deposited in live-fire military training range soils contributes to significant pollution of groundwater. In situ microbial degradation has been proposed as a viable method for onsite containment of RDX. However, there is only a single report of RDX degradation in training range soils and the soil microbial communities involved in RDX degradation were not identified. Here we demonstrate aerobic RDX degradation in soils taken from a target area of an Eglin Air Force Base bombing range, C52N Cat's Eye, (Eglin, Florida U.S.A.). RDX-degradation activity was spatially heterogeneous (found in less than 30% of initial target area field samples) and dependent upon the addition of exogenous carbon sources to the soils. Therefore, biostimulation (with exogenous carbon sources) and bioaugmentation may be necessary to sustain timely and effective in situ microbial biodegradation of RDX. High sensitivity stable isotope probing analysis of extracted soils incubated with fully labeled (15)N-RDX revealed several organisms with (15)N-labeled DNA during RDX-degradation, including xplA-bearing organisms. Rhodococcus was the most prominent genus in the RDX-degrading soil slurries and was completely labeled with (15)N-nitrogen from the RDX. Rhodococcus and Williamsia species isolated from these soils were capable of using RDX as a sole nitrogen source and possessed the genes xplB and xplA associated with RDX-degradation, indicating these genes may be suitable genetic biomarkers for assessing RDX degradation potential in soils. Other highly labeled species were primarily Proteobacteria, including: Mesorhizobium sp., Variovorax sp., and Rhizobium sp.
mBio, 2013
Early diagnosis and treatment of human immunodeficiency virus type 1 (HIV-1) infection in infants... more Early diagnosis and treatment of human immunodeficiency virus type 1 (HIV-1) infection in infants can greatly reduce mortality rates. However, current infant HIV-1 diagnostics cannot reliably be performed at the point of care, often delaying treatment and compromising its efficacy. Recombinase polymerase amplification (RPA) is a novel technology that is ideal for an HIV-1 diagnostic, as it amplifies target DNA in <20 min at a constant temperature, without the need for complex thermocycling equipment. Here we tested 63 HIV-1-specific primer and probe combinations and identified two RPA assays that target distinct regions of the HIV-1 genome (long terminal repeat [LTR] and pol) and can reliably detect 3 copies of proviral DNA by the use of fluorescence detection and lateral-flow strip detection. These pol and LTR primers amplified 98.6% and 93%, respectively, of the diverse HIV-1 variants tested. This is the first example of an isothermal assay that consistently detects all of the major HIV-1 global subtypes.
…, Jan 1, 2006
Background: The Tax proteins encoded by human T lymphotropic virus type 1 (HTLV-1) and type 2 (HT... more Background: The Tax proteins encoded by human T lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) are transcriptional activators of both the viral long terminal repeat (LTR) and cellular promoters via the CREB and NFkB pathways. In contrast to HTLV-1, HTLV-2 has been classified into four distinct genetic subtypes A, B, C and D defined by phylogenetic analysis of their nucleotide sequences and the size and amino acid sequence of their Tax proteins. In the present study we have analysed and compared the transactivating activities of three Tax 2A and one Tax 2B proteins using LTR and NFkB reporter assays.
FEMS microbiology ecology, Jan 1, 2010
The tfdC and C23O genes encode two catechol dioxygenases that catalyse ortho and meta cleavage of... more The tfdC and C23O genes encode two catechol dioxygenases that catalyse ortho and meta cleavage of a key metabolite (chlorocatechol) of 2,4-dichlorophenol (2, 4-DCP) metabolism, respectively. Primers were designed and a real-time PCR assay was developed to assess the abundance and expression of both tfdC and C23O genes in a soil amended with 2,4-DCP over a 21-day period. tfdC, the gene encoding the ortho cleaving dioxygenase, was significantly more abundant than the meta cleaving dioxygenase gene (C23O) throughout the experiment. The highest levels of tfdC were observed 2 days after amendment of soil with 2,4-DCP, at which stage the rate of 2,4-DCP degradation was at its maximum. In contrast, C230 copy numbers declined initially and peaked when degradation had slowed considerably. mRNA of the two chlorocatechol dioxygenase genes was not detected on day 0, but both genes were expressed after this time point. tfdC was expressed at a significantly higher level than C23O in 2,4-DCP-amended soil throughout the course of the microcosm, indicating the dominance of the ortho metabolic pathway. Phylogenetic analysis revealed a wide diversity of chlorocatechol dioxygenase genes in the 2,4-DCP-exposed soil examined.
Veterinary …, Jan 1, 2007
... Title: TNF-α increases NFB activity in and IL-8 release from bovine mammary epithelial cells... more ... Title: TNF-α increases NFB activity in and IL-8 release from bovine mammary epithelial cells. Authors: Fitzgerald D, Kieran Meade, Alice McEvoy, Lorraine Lillis, Evelyn Murphy, David MacHugh, Alan Baird. Issue Date: 2007. ...
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Papers by Lorraine Lillis