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1.
J Clin Microbiol ; 58(3)2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-31915287

RESUMO

Antibiotic resistance in Mycoplasma genitalium is rising globally, and resistance-guided diagnostics can facilitate targeted and timely treatment. The ResistancePlus MG FleXible (RPMG Flex) assay for the detection of M. genitalium and macrolide resistance-mediating mutations (MRMM) was evaluated for analytical sensitivity, specificity, reproducibility, and inhibition in the presence of interfering substances by simulating M. genitalium-negative pooled urine and swab matrices with M. genitalium cultures. Furthermore, the clinical sensitivity of the assay was evaluated and compared with a reference real-time PCR assay. The analytical sensitivity of the RPMG Flex assay was 157 genomes/ml for wild-type (WT) and 387 genomes/ml for MRMM strains in both matrices. For clinical specimens, the RPMG assay had an overall sensitivity of 96.1% (95% urine: 10/10 WT, 9/10 MRMM; 96.5% swab: 25/26 WT, 26/29 MRMM) compared to 85.7% for the MgPa/MagNAPure24 assay (95% urine: 19/20; 87% swab: 48/57). Clinical specificity was 100% for urine and 98.5% for swab specimens, respectively. No inhibition due to the presence of any of the tested interfering substances was observed. The RPMG Flex assay was more sensitive than the reference MgPa assay, in particular, for swab specimens. The implementation of this assay may increase ease of use and considerably decrease hands-on time for sample preparation compared to a standard block-based assay. The RPMG Flex assay for the GeneXpert Dx system provides a much-needed platform for the simultaneous detection of MG and MRMM and may thereby facilitate resistance-guided therapy for M. genitalium infections.

2.
J Clin Microbiol ; 57(11)2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31434719

RESUMO

Mycoplasma genitalium causes a common sexually transmitted infection with a marked propensity to develop antimicrobial resistance. As few treatment options exist, this poses significant challenges to clinicians. Recent diagnostic advances have resulted in tests that report the simultaneous detection of M. genitalium and any resistance to macrolides, the first-line treatment. This allows for therapy to be tailored to the individual, thereby optimizing treatment outcomes. However, resistance to fluoroquinolones, the second-line treatment, is increasing in M. genitalium In this study, we describe a new assay, MG+parC (beta), which simultaneously reports the detection of M. genitalium and five parC mutations that have been associated with resistance to fluoroquinolones. These mutations affect the amino acid sequence of ParC at residues S83R (A247C), S83I (G248T), D87N (G259A), D87Y (G259T), and D87H (G259C). The study tested the MG+parC (beta) assay with 202 M. genitalium-positive clinical samples from Australia (n = 141) and Spain (n = 61). Compared to Sanger sequencing, the assay performed with a kappa value of 0.985 (95% confidence interval [CI], 0.955 to 1.000), with a mutation detection sensitivity of 97.6% (95% CI, 87.4 to 99.9), and specificity of 100.0% (95% CI, 97.7 to 100.0). Fluoroquinolone resistance-associated mutations in parC targeted by the assay were more prevalent among the Australian cohort (23.4% [95% CI,16.3 to 31.8]) compared to the Spanish population (8.8% [95% CI, 2.9% to 19.3%]) (P = 0.019). The MG+parC (beta) kit is a simple and reliable method for simultaneous detection of M. genitalium and fluoroquinolone resistance-associated mutations in clinical settings. This novel diagnostic tool may extend the utility of the second line of antimicrobial therapies in M. genitalium infection.

3.
J Antimicrob Chemother ; 74(10): 2913-2915, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31340021

RESUMO

BACKGROUND: The emergence of drug-resistant Neisseria gonorrhoeae has prompted the development of rapid molecular assays designed to determine antimicrobial susceptibility. One common assay uses high-resolution melt analysis to target codon 91 of the gyrase A gene (gyrA) to predict N. gonorrhoeae susceptibility to ciprofloxacin. METHODS: We extracted DNA from remnant clinical specimens that had previously tested positive for N. gonorrhoeae using the Aptima Combo 2 for CT/NG assay (Hologic, San Diego, CA, USA). We selected DNA extracts from specimens with indeterminate, WT and mutant gyrA genotype results from a previous study using high-resolution melt analysis to detect the gyrA codon 91 mutation. We re-tested those specimens using the recently CE-marked ResistancePlus GC (beta) assay (SpeeDx, Sydney, Australia). RESULTS: Of 86 specimens with indeterminate gyrA genotypes on high-resolution melt analysis, the ResistancePlus GC (beta) assay (SpeeDx) identified 30 (35%) WT, 22 (26%) mutant and 34 (40%) indeterminate gyrA genotypes. CONCLUSIONS: The ResistancePlus GC (beta) assay showed improved N. gonorrhoeae gyrA genotype determination compared with a prior gyrA genotypic high-resolution melt assay.

4.
BMC Infect Dis ; 19(1): 571, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266450

RESUMO

BACKGROUND: Carbapenemase-producing organisms (CPOs) have emerged as antibiotic-resistant bacteria of global concern. Here we assessed the performance of the Carba (beta) assay, a multiplex real-time PCR assay developed by SpeeDx for the detection of key carbapenemase-encoding genes: KPC, NDM, OXA-48-like, IMP-4-like, and VIM. METHODS: DNA extracts of 180 isolates were tested with the Carba (beta) assay, using previously validated in-house TaqMan probe assays for the relevant carbapenemase genes as the reference standard. The Carba (beta) assay was then directly used to screen 460 DNA extracts of faecal specimens, with positive results subjected to the aforementioned in-house assays plus Sanger sequencing. RESULTS: The Carba (beta) assay correctly identified the presence of the respective carbapenemase genes in 154 of 156 isolates and provided negative results for all 24 non-CPO isolates. Two isolates provided positive results for OXA-48-like carbapenemase by the Carba (beta) assay only. The Carba (beta) assay had sensitivities of 100% for all targets, and specificities of 100% for KPC, NDM, IMP-4-like, and VIM targets, and 98.5% for OXA-48-like targets. When applied directly to faecal specimens, eight samples were positive by the Carba (beta) assay, two of which were confirmed by in-house TaqMan probe PCR or DNA sequencing. CONCLUSIONS: The Carba (beta) assay is highly sensitive and specific for detecting key carbapenemase genes in isolates. Further testing is required to assess this assay's suitability for direct screening of clinical specimens.


Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , beta-Lactamases/genética , Antibacterianos , Bactérias/efeitos dos fármacos , Técnicas Bacteriológicas/métodos , Proteínas de Escherichia coli/genética , Fezes/microbiologia , Humanos , Sensibilidade e Especificidade
5.
J Antimicrob Chemother ; 74(7): 1820-1824, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30897201

RESUMO

OBJECTIVES: To evaluate the performance of the ResistancePlus GC (beta) assay for the simultaneous detection of Neisseria gonorrhoeae and gyrA S91 markers of resistance (S91F) and susceptibility (WT) to ciprofloxacin, from both clinical specimens and isolates. METHODS: Performance was assessed on several sample banks, including N. gonorrhoeae isolates (n = 822), non-gonococcal isolates (n = 110), N. gonorrhoeae-positive clinical specimens (n = 402) and N. gonorrhoeae-negative specimens (n = 290). Results were compared with previous testing data, including S91 genotyping and phenotypic resistance profiles. RESULTS: Overall, the assay demonstrated 100% sensitivity for N. gonorrhoeae detection in clinical isolates. For gyrA S91 mutation detection in clinical isolates, the assay showed 100% sensitivity/specificity compared with the genotype, and >99%/>97% sensitivity/specificity when compared with phenotype. For positive clinical specimens, the assay demonstrated >96% sensitivity for N. gonorrhoeae detection and 100% sensitivity/specificity for gyrA S91 mutation detection. The assay demonstrated >99% specificity for N. gonorrhoeae detection against non-gonococcal isolates and 100% specificity for negative clinical specimens. CONCLUSIONS: The ResistancePlus GC (beta) assay is suitable for the detection of N. gonorrhoeae and gyrA markers associated with resistance/susceptibility to ciprofloxacin directly in clinical samples. This assay could be implemented for the individualized treatment of gonorrhoea infections as well as to enhance current antimicrobial resistance surveillance methods.

6.
Diagnostics (Basel) ; 8(4)2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30501041

RESUMO

BACKGROUND: PIK3CA pathways are the most frequently mutated oncogenic pathway in head and neck squamous cell carcinoma (HNSCC), including virally driven HNCs. PIK3CA is involved in the PI3K-PTEN-mTOR signalling pathway. PIK3CA has been implicated in HNSCC progression and PIK3CA mutations may serve as predictive biomarkers for therapy selection. Circulating tumour DNA (ctDNA) derived from necrotic and apoptotic tumour cells are thought to harbour tumour-specific genetic alterations. As such, the detection of PIK3CA alterations detected by ctDNA holds promise as a potential biomarker in HNSCC. METHODS: Blood samples from treatment naïve HNSCC patients (n = 29) were interrogated for a commonly mutated PIK3CA hotspot mutation using low cost allele-specific Plex-PCRTM technology. RESULTS: In this pilot, cross sectional study, PIK3CA E545K mutation was detected in the plasma samples of 9/29 HNSCC patients using the Plex-PCRTM technology. CONCLUSION: The results of this pilot study support the notion of using allele-specific technologies for cost-effective testing of ctDNA, and further assert the potential utility of ctDNA in HNSCC.

7.
PLoS One ; 12(1): e0170087, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28114309

RESUMO

BACKGROUND: Whilst qPCR provides an extremely powerful tool for genetic analysis, some applications such as multiplexing variant alleles (eg SNPs, point mutations or deletions), remain challenging using current primer/probe systems. The novel design features of PlexPrimers allow sensitive, multiplexed analysis of variant alleles even when these are tightly clustered. METHOD: PlexPrimers were combined with PlexZymes in qPCR assays for the detection of SNPs in human absorption, distribution, metabolism, and excretion (ADME) genes; clustered mutations in the 23S rRNA gene which confer antibiotic resistance to Mycoplasma genitalium; and deletions within the human epidermal growth factor receptor (EGFR) gene. RESULTS: The combination of PlexPrimers and PlexZymes allowed robust multiplexing of targets which resulted in 100% concordance with results obtained using hydrolysis probe kits for 14 SNPs in the ADME genes. A 7-plex qPCR assay targeting M. genitalium, 5 clustered mutations associated with macrolide resistance and an internal control, allowed efficient amplification of all targets, with all 5 mutations detected in a single channel. Finally, the strategy was employed to analyse common EGFR mutants with high sensitivity, detecting deletions present at only 0.01%. CONCLUSION: PlexPrime is a novel technology for the detection of genetic variants. Unlike previous strategies, the combination of PlexPrimers with PlexZymes enables both allele-specific detection and allele-specific amplification in qPCR. The study demonstrated highly sensitive and specific detection of mutations and SNPs, and superior multiplexing capacity. The ability to multiplex clustered genetic variants reduces the time to result providing more actionable information.


Assuntos
Mutação , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Primers do DNA , Humanos , Sensibilidade e Especificidade
8.
Nucleic Acids Res ; 43(11): 5586-600, 2015 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-25940628

RESUMO

Polypyrimidine tract binding protein (PTBP1) is a widely expressed RNA binding protein that acts as a regulator of alternative splicing and of cytoplasmic mRNA functions. Vertebrates contain two closely-related paralogs with >75% amino acid sequence identity. Early replacement of PTBP1 by PTBP2 during neuronal differentiation causes a concerted set of splicing changes. By comparison, very little is known about the molecular functions or physiological roles of PTBP3, although its expression and conservation throughout the vertebrates suggest a role in haematopoietic cells. To begin to understand its functions we have characterized the mRNA and protein isoform repertoire of PTBP3. Combinatorial alternative splicing events at the 5' end of the gene allow for the generation of eight mRNA and three major protein isoforms. Individual mRNAs generate up to three protein isoforms via alternative translation initiation by re-initiation and leaky scanning using downstream AUG codons. The N-terminally truncated PTBP3 isoforms lack nuclear localization signals and/or most of the RRM1 domain and vary in their RNA binding properties and nuclear/cytoplasmic distribution, suggesting that PTBP3 may have major post-transcriptional cytoplasmic roles. Our findings set the stage for understanding the non-redundant physiological roles of PTBP3.


Assuntos
Processamento Alternativo , Iniciação Traducional da Cadeia Peptídica , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Animais , Células Cultivadas , Códon de Iniciação , Humanos , Células K562 , Camundongos Endogâmicos C57BL , Proteína de Ligação a Regiões Ricas em Polipirimidinas/biossíntese , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA/metabolismo
9.
Nucleic Acids Res ; 42(1): 276-89, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24106088

RESUMO

Transcription factors (TFs) are often regarded as being composed of a DNA-binding domain (DBD) and a functional domain. The two domains are considered separable and autonomous, with the DBD directing the factor to its target genes and the functional domain imparting transcriptional regulation. We examined an archetypal zinc finger (ZF) TF, Krüppel-like factor 3 with an N-terminal domain that binds the corepressor CtBP and a DBD composed of three ZFs at its C-terminus. We established a system to compare the genomic occupancy profile of wild-type Krüppel-like factor 3 with two mutants affecting the N-terminal functional domain: a mutant unable to contact the cofactor CtBP and a mutant lacking the entire N-terminal domain, but retaining the ZFs intact. Chromatin immunoprecipitation followed by sequencing was used to assess binding across the genome in murine embryonic fibroblasts. Unexpectedly, we observe that mutations in the N-terminal domain generally reduced binding, but there were also instances where binding was retained or even increased. These results provide a clear demonstration that the correct localization of TFs to their target genes is not solely dependent on their DNA-contact domains. This informs our understanding of how TFs operate and is of relevance to the design of artificial ZF proteins.


Assuntos
DNA/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Sequência Consenso , Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/química , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína
10.
BMC Genomics ; 11: 565, 2010 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-20946641

RESUMO

BACKGROUND: In addition to acting as an RNA quality control pathway, nonsense-mediated mRNA decay (NMD) plays roles in regulating normal gene expression. In particular, the extent to which alternative splicing is coupled to NMD and the roles of NMD in regulating uORF containing transcripts have been a matter of debate. RESULTS: In order to achieve a greater understanding of NMD regulated gene expression we used 2D-DiGE proteomics technology to examine the changes in protein expression induced in HeLa cells by UPF1 knockdown. QPCR based validation of the corresponding mRNAs, in response to both UPF1 knockdown and cycloheximide treatment, identified 17 bona fide NMD targets. Most of these were associated with bioinformatically predicted NMD activating features, predominantly upstream open reading frames (uORFs). Strikingly, however, the majority of transcripts up-regulated by UPF1 knockdown were either insensitive to, or even down-regulated by, cycloheximide treatment. Furthermore, the mRNA abundance of several down-regulated proteins failed to change upon UPF1 knockdown, indicating that UPF1's role in regulating mRNA and protein abundance is more complex than previously appreciated. Among the bona fide NMD targets, we identified a highly conserved AS-NMD event within the 3' UTR of the HNRNPA2B1 gene. Overexpression of GFP tagged hnRNP A2 resulted in a decrease in endogenous hnRNP A2 and B1 mRNA with a concurrent increase in the NMD sensitive isoforms. CONCLUSIONS: Despite the large number of changes in protein expression upon UPF1 knockdown, a relatively small fraction of them can be directly attributed to the action of NMD on the corresponding mRNA. From amongst these we have identified a conserved AS-NMD event within HNRNPA2B1 that appears to mediate autoregulation of HNRNPA2B1 expression levels.


Assuntos
Processamento Alternativo/genética , Códon sem Sentido/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Homeostase/genética , Proteômica/métodos , Estabilidade de RNA/genética , Transativadores/metabolismo , Regiões 3' não Traduzidas/genética , Proteínas de Transporte/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Ribonucleoproteína Nuclear Heterogênea A1 , Humanos , Espectrometria de Massas , Fases de Leitura Aberta/genética , Fosfatidilinositol 3-Quinases/metabolismo , Biossíntese de Proteínas/genética , RNA Helicases , RNA Antissenso/metabolismo , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo , Eletroforese em Gel Diferencial Bidimensional
11.
Nat Struct Mol Biol ; 17(9): 1114-23, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20711188

RESUMO

To gain global insights into the role of the well-known repressive splicing regulator PTB, we analyzed the consequences of PTB knockdown in HeLa cells using high-density oligonucleotide splice-sensitive microarrays. The major class of identified PTB-regulated splicing event was PTB-repressed cassette exons, but there was also a substantial number of PTB-activated splicing events. PTB-repressed and PTB-activated exons showed a distinct arrangement of motifs with pyrimidine-rich motif enrichment within and upstream of repressed exons but downstream of activated exons. The N-terminal half of PTB was sufficient to activate splicing when recruited downstream of a PTB-activated exon. Moreover, insertion of an upstream pyrimidine tract was sufficient to convert a PTB-activated exon to a PTB-repressed exon. Our results show that PTB, an archetypal splicing repressor, has variable splicing activity that predictably depends upon its binding location with respect to target exons.


Assuntos
Processamento Alternativo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Sequência de Bases , Éxons , Células HeLa , Humanos , Íntrons , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Elementos Silenciadores Transcricionais
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