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1.
J Infect Dis ; 221(6): 1017-1024, 2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-32031634

RESUMO

BACKGROUND: The basis of fluoroquinolone treatment failure for Mycoplasma genitalium is poorly understood. METHODS: To identify mutations associated with failure we sequenced key regions of the M. genitalium parC and gyrA genes for patients undergoing sequential therapy with doxycycline-moxifloxacin (201 patients, including 21 with failure) or doxycycline-sitafloxacin (126 patients, including 13 with failure). RESULTS: The parC G248T/S83I mutation was more common among patients with failed sequential doxycycline-moxifloxacin (present in 76.2% of failures vs 7.8% cures, P < .001) or doxycycline-sitafloxacin (50% vs 16.8%, respectively; P = .01) treatment. Doxycycline-sitafloxacin was more efficacious than doxycycline-moxifloxacin against infections carrying the parC mutation conferring S83I amino acid change. Treatment was more likely to fail in these infections if they had a concurrent gyrA mutation (M95I or D99N) (P = .07 for doxycycline-moxifloxacin group and P = .009 for doxycycline-sitafloxacin group), suggesting an additive effect. CONCLUSIONS: This study indicates that parC G248T/S83I mutations contribute to failure of moxifloxacin and sitafloxacin, and the findings will inform the development of quinolone resistance assays needed to ensure optimal selection of antimicrobials for M. genitalium.

2.
Clin Infect Dis ; 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31629365

RESUMO

BACKGROUND: Macrolide-resistance in Mycoplasma genitalium (MG) exceeds 50% in many regions and quinolone-resistance is increasing. We recently reported that resistance-guided therapy (RGT) using doxycycline followed by sitafloxacin or 2.5g-azithromycin cured 92% and 95% of macrolide-resistant and macrolide-susceptible infections, respectively. We now present the data on RGT using doxycycline-moxifloxacin, the regimen recommended in international guidelines, and extend the data on the efficacy of doxyxycline-2.5g azithromycin and subsequent de novo macrolide-resistance. METHODS: Patients attending Melbourne Sexual Health Centre between 2017-2018 with STI-related syndromes were treated with doxycycline for 7 days and recalled if positive for MG. Macrolide-susceptible cases then received 2.5g azithromycin (1g, then 500mg daily for 3 days) and resistant cases received moxifloxacin (400 mg daily, 7 days). Test of cure (TOC) was recommended 14-28 days post-completion of antimicrobials. Adherence and adverse effects were recorded. RESULTS: A total of 383 patients (81 females/106 heterosexual males/196 men-who-have-sex-with-men) were included. Microbial cure following doxycycline-azithromycin was 95.4% (95% CI 89.7-98.0) and doxycycline-moxifloxacin was 92.0%(88.1-94.6). De novo macrolide-resistance was detected in 4.6% of cases. Combining doxycycline-azithromycin data with our prior RGT study (n=186) yielded a pooled cure of 95.7% (91.6-97.8). ParC mutations implicated in moxifloxacin failure were present in 15-22% of macrolide-resistant cases at baseline. CONCLUSION: These findings support the inclusion of moxifloxacin in resistance-guided strategies and extend the evidence for use of 2.5g azithromycin, and presumptive use of doxycycline. These data provide an evidence-base for current UK, Australian and European guidelines for the treatment of MG, an STI which is increasingly challenging to cure.

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.
Emerg Infect Dis ; 25(4): 719-727, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30882306

RESUMO

During 2016-2017, we tested asymptomatic men who have sex with men (MSM) in Melbourne, Australia, for Mycoplasma genitalium and macrolide resistance mutations in urine and anorectal swab specimens by using PCR. We compared M. genitalium detection rates for those asymptomatic men to those for MSM with proctitis and nongonococcal urethritis (NGU) over the same period. Of 1,001 asymptomatic MSM, 95 had M. genitalium; 84.2% were macrolide resistant, and 17% were co-infected with Neisseria gonorrhoeae or Chlamydia trachomatis. Rectal positivity for M. genitalium was 7.0% and urine positivity was 2.7%. M. genitalium was not more commonly detected in the rectums of MSM (n = 355, 5.6%) with symptoms of proctitis over the same period but was more commonly detected in MSM (n = 1,019, 8.1%) with NGU. M. genitalium is common and predominantly macrolide-resistant in asymptomatic MSM. M. genitalium is not associated with proctitis in this population.


Assuntos
Homossexualidade Masculina , Infecções por Mycoplasma/diagnóstico , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium , Doenças Bacterianas Sexualmente Transmissíveis/diagnóstico , Doenças Bacterianas Sexualmente Transmissíveis/microbiologia , Antibacterianos/farmacologia , Austrália/epidemiologia , Coinfecção , Estudos Transversais , Farmacorresistência Bacteriana , Humanos , Masculino , Infecções por Mycoplasma/epidemiologia , Infecções por Mycoplasma/transmissão , Mycoplasma genitalium/efeitos dos fármacos , Razão de Chances , Prevalência , Vigilância em Saúde Pública , Doenças Bacterianas Sexualmente Transmissíveis/epidemiologia , Doenças Bacterianas Sexualmente Transmissíveis/transmissão , Avaliação de Sintomas
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.
Clin Infect Dis ; 68(4): 554-560, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29873691

RESUMO

Background: Rising macrolide and quinolone resistance in Mycoplasma genitalium necessitate new treatment approaches. We evaluated outcomes of sequential antimicrobial therapy for M. genitalium guided by a macrolide-resistance assay. Methods: In mid-2016, Melbourne Sexual Health Centre switched from azithromycin to doxycycline (100 mg twice daily for 7 days) for nongonococcal urethritis, cervicitis, and proctitis. Cases were tested for M. genitalium and macrolide-resistance mutations (MRMs) by polymerase chain reaction. Directly after doxycycline, MRM-negative infections received 2.5 g azithromycin (1 g, then 500 mg daily for 3 days), and MRM-positive infections received sitafloxacin (100 mg twice daily for 7 days). Assessment of test of cure and reinfection risk occurred 14-90 days after the second antibiotic. Results: Of 244 evaluable M. genitalium infections (52 women, 68 heterosexual men, 124 men who have sex with men) diagnosed from 20 June 2016 to 15 May 2017, MRMs were detected in 167 (68.4% [95% confidence interval {CI}, 62.2%-74.2%]). Treatment with doxycycline decreased bacterial load by a mean 2.60 log10 (n = 56; P < .0001). Microbiologic cure occurred in 73 of 77 MRM-negative infections (94.8% [95% CI, 87.2%-98.6%]) and in 154 of 167 MRM-positive infections (92.2% [95% CI, 87.1%-95.8%]). Selection of macrolide resistance occurred in only 2 of 76 (2.6% [95% CI, .3%-9.2%]) macrolide-susceptible infections. Conclusions: In the context of high levels of antimicrobial resistance, switching from azithromycin to doxycycline for presumptive treatment of M. genitalium, followed by resistance-guided therapy, cured ≥92% of infections, with infrequent selection of macrolide resistance.


Assuntos
Antibacterianos/uso terapêutico , Monitoramento de Medicamentos/métodos , Farmacorresistência Bacteriana , Macrolídeos/uso terapêutico , Infecções por Mycoplasma/tratamento farmacológico , Mycoplasma genitalium/efeitos dos fármacos , Mycoplasma genitalium/isolamento & purificação , Adulto , Antibacterianos/farmacologia , Feminino , Humanos , Macrolídeos/farmacologia , Masculino , Infecções por Mycoplasma/microbiologia , Estudos Prospectivos , Resultado do Tratamento , Adulto Jovem
7.
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.

8.
Sex Transm Dis ; 45(9): 632-635, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29509566

RESUMO

BACKGROUND: Mycoplasma genitalium is an important cause of bacterial sexually transmitted diseases. Diagnosis and susceptibility testing of M. genitalium are limited by the fastidious nature of the organism. Therefore, the prevalence of infection and azithromycin resistance are poorly studied. METHODS: We conducted an exploratory study on remnant clinical specimens. We collected remnant DNA from consecutive urine samples and clinical swabs (cervical/vaginal, rectal, and pharyngeal) previously tested for Neisseria gonorrhoeae and Chlamydia trachomatis using the Cobas 4800 CT/NG assay (Roche Molecular Systems, Pleasanton, CA) between March-April 2017 from across the University of California, Los Angeles Health System. We then retrospectively tested all specimens with the ResistancePlus MG (550) kit, a molecular assay for the detection of M. genitalium and genetic mutations associated with azithromycin resistance. RESULTS: Among 500 specimens, the prevalence of M. genitalium was 1.1% (95% confidence interval [CI], 0.04%-3.0%) in urine samples (n = 362), 17.4% (95% CI, 5.7%-39.6%) in rectal swabs (n = 23), and 1.9% (95% CI, 0.3%-7.3%) in cervical/vaginal swabs (n = 106). The prevalence of N. gonorrhoeae was 0.6% in urine samples and 4.3% in rectal swabs, whereas the prevalence of C. trachomatis was 2.2% in urine samples, 4.3% in rectal swabs and 3.8% in cervical/vaginal swabs. Of the 10 M. genitalium positive specimens, 8 (80.0%) had a mutation associated with azithromycin resistance. CONCLUSIONS: The prevalence of M. genitalium infection in our population varied by anatomic site of infection. Most M. genitalium infections had at least 1 mutation associated with azithromycin resistance.


Assuntos
Antibacterianos/farmacologia , Azitromicina/farmacologia , Farmacorresistência Bacteriana/genética , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/genética , Doenças Bacterianas Sexualmente Transmissíveis/microbiologia , Colo do Útero/microbiologia , DNA Bacteriano/urina , Feminino , Humanos , Los Angeles/epidemiologia , Mutação , Infecções por Mycoplasma/epidemiologia , Mycoplasma genitalium/efeitos dos fármacos , Faringe/microbiologia , Prevalência , Reto/microbiologia , Estudos Retrospectivos , Doenças Bacterianas Sexualmente Transmissíveis/epidemiologia
10.
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
12.
PLoS One ; 11(6): e0156740, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27271704

RESUMO

Mycoplasma genitalium is a cause of non-gonoccocal urethritis (NGU) in men and cervicitis and pelvic inflammatory disease in women. Recent international data also indicated that the first line treatment, 1 gram stat azithromycin therapy, for M. genitalium is becoming less effective, with the corresponding emergence of macrolide resistant strains. Increasing failure rates of azithromycin for M. genitalium has significant implications for the presumptive treatment of NGU and international clinical treatment guidelines. Assays able to predict macrolide resistance along with detection of M. genitalium will be useful to enable appropriate selection of antimicrobials to which the organism is susceptible and facilitate high levels of rapid cure. One such assay recently developed is the MG 23S assay, which employs novel PlexZyme™ and PlexPrime™ technology. It is a multiplex assay for detection of M. genitalium and 5 mutations associated with macrolide resistance. The assay was evaluated in 400 samples from 254 (186 males and 68 females) consecutively infected participants, undergoing tests of cure. Using the MG 23S assay, 83% (331/440) of samples were positive, with 56% of positives carrying a macrolide resistance mutation. Comparison of the MG 23S assay to a reference qPCR method for M. genitalium detection and high resolution melt analysis (HRMA) and sequencing for detection of macrolide resistance mutations, resulted in a sensitivity and specificity for M. genitalium detection and for macrolide resistance of 99.1/98.5% and 97.4/100%, respectively. The MG 23S assay provides a considerable advantage in clinical settings through combined diagnosis and detection of macrolide resistance.


Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/genética , Macrolídeos/uso terapêutico , Reação em Cadeia da Polimerase Multiplex/métodos , Infecções por Mycoplasma/diagnóstico , Mycoplasma genitalium , Técnicas de Tipagem Bacteriana/métodos , Análise Mutacional de DNA/métodos , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana/métodos , Infecções por Mycoplasma/tratamento farmacológico , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/efeitos dos fármacos , Mycoplasma genitalium/genética , Mycoplasma genitalium/isolamento & purificação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Uretrite/diagnóstico , Uretrite/tratamento farmacológico , Uretrite/microbiologia
13.
Biosens Bioelectron ; 75: 59-66, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26296241

RESUMO

Advancements in molecular biology have improved the ability to characterize disease-related nucleic acids and proteins. Recently, there has been an increasing desire for tests that can be performed outside of centralised laboratories. This study describes a novel isothermal signal amplification cascade called EzyAmp (enzymatic signal amplification) that is being developed for detection of targets at the point of care. EzyAmp exploits the ability of some restriction endonucleases to cleave substrates containing nicks within their recognition sites. EzyAmp uses two oligonucleotide duplexes (partial complexes 1 and 2) which are initially cleavage-resistant as they lack a complete recognition site. The recognition site of partial complex 1 can be completed by hybridization of a triggering oligonucleotide (Driver Fragment 1) that is generated by a target-specific initiation event. Binding of Driver Fragment 1 generates a completed complex 1, which upon cleavage, releases Driver Fragment 2. In turn, binding of Driver Fragment 2 to partial complex 2 creates completed complex 2 which when cleaved releases additional Driver Fragment 1. Each cleavage event separates fluorophore quencher pairs resulting in an increase in fluorescence. At this stage a cascade of signal production becomes independent of further target-specific initiation events. This study demonstrated that the EzyAmp cascade can facilitate detection and quantification of nucleic acid targets with sensitivity down to aM concentration. Further, the same cascade detected VEGF protein with a sensitivity of 20nM showing that this universal method for amplifying signal may be linked to the detection of different types of analytes in an isothermal format.


Assuntos
Técnicas Biossensoriais , Ácidos Nucleicos/isolamento & purificação , Fator A de Crescimento do Endotélio Vascular/isolamento & purificação , Corantes Fluorescentes/química , Humanos , Hibridização de Ácido Nucleico , Ácidos Nucleicos/química , Fator A de Crescimento do Endotélio Vascular/química
14.
Anal Chem ; 86(18): 9106-13, 2014 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-25157928

RESUMO

Diagnostic tests performed in the field or at the site of patient care would benefit from using a combination of inexpensive, stable chemical reagents and simple instrumentation. Here, we have developed a universal "DNA-only Cascade" (DoC) to quantitatively detect target analytes with increased speed. The DoC utilizes quasi-circular structures consisting of temporarily inactivated deoxyribozymes (DNAzymes). The catalytic activity of the DNAzymes is restored in a universal manner in response to a broad range of environmental and biological targets. The present study demonstrates DNAzyme activation in the presence of metal ions (Pb(2+)), small molecules (deoxyadenosine triphosphate) and nucleic acids homologous to genes from Meningitis-causing bacteria. Furthermore, DoC efficiently discriminates nucleic acid targets differing by a single nucleotide. When detection of analytes is orchestrated by functional nucleic acids, the inclusion of DoC reagents substantially decreases time for detection and allows analyte quantification. The detection of nucleic acids using DoC was further characterized for its capability to be multiplexed and retain its functionality following long-term exposure to ambient temperatures and in a background of complex medium (human serum).


Assuntos
DNA Catalítico/metabolismo , DNA/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , DNA/análise , DNA Bacteriano/análise , DNA Bacteriano/metabolismo , Corantes Fluorescentes/química , Humanos , Íons/química , Chumbo/química , Infecções Meningocócicas/diagnóstico , Neisseria meningitidis/genética , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , Streptococcus/genética
15.
Methods Mol Biol ; 1039: 31-49, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24026684

RESUMO

Multicomponent nucleic acid enzymes (MNAzymes) are nucleic acid enzymes composed of multiple oligonucleotide partzymes that only associate to form catalytic complexes in the presence of a target nucleic acid. Once assembled, MNAzymes cleave a separate substrate (probe) between fluorophore and quencher labels to produce a fluorescent signal indicative of the presence of the target. MNAzymes are particularly useful as tools for monitoring the accumulation of amplicons during real-time quantitative PCR (qPCR). The partzyme pairs have sensor domains that are complementary to adjacent regions in the amplicons such that their partial catalytic core domains form a complete active MNAzyme core. The probe-binding domain of the partzymes can be complementary to any one of a series of well-characterized universal probes. Since there is no need to synthesize and optimize new target-specific probes for each new target, MNAzyme qPCR provides a flexible alternative which allows target-specific interrogation with a generic readout. A series of universal probes have been designed that perform with high reliability, yielding consistent and reproducible results for any target, making the development of multiplex qPCR assays faster, cheaper, and simpler. This chapter describes a 5plex MNAzyme RT-qPCR method which simultaneously quantifies five mRNA transcripts with high efficiency and specificity using five unique universal probes, each labeled with a different fluorophore.


Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sequência de Bases , Primers do DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Corantes Fluorescentes/química , Humanos , Células K562 , Complexos Multienzimáticos , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
16.
Clin Chem ; 59(2): 419-26, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23232065

RESUMO

BACKGROUND: MNAzymes (nucleic acid enzymes formed from multiple partial enzymes) can be linked to PCR to provide a highly specific method for target detection and quantification. We investigated the feasibility of multiplexing MNAzyme quantitative PCR (qPCR) methods. METHODS: We combined MNAzyme components with PCR primers and standard qPCR reagents to perform MNAzyme qPCR and reverse-transcription qPCR (RT-qPCR) assays with a set of universal reporter probes. Assays were performed on single targets and in multiplex formats that combined up to 5 different targets in a single reaction. RESULTS: A comparison of 3 targets amplified in single and triplex formats showed no significant differences with respect to detection limit or amplification efficiency. Likewise, we successfully converted single-target assays for 11 transcripts of interest to triplex assays containing 2 reference transcripts without having to optimize or modify the conditions. A quintuplex RT-qPCR that simultaneously quantified 5 transcripts with 5 universal probes produced high amplification efficiencies and r(2) values for all transcripts. Despite the large numbers of oligonucleotides in the reactions, we observed no false-positive signals, owing to the requirement of 4 target-specific binding events to produce a signal. A quadruplex assay that combined MNAzymes with methylation-specific PCR to measure epigenetic biomarkers of prostate cancer was capable of detecting a single methylated DNA allele in a background of 1000-10 000 unmethylated alleles. The MNAzyme qPCR was compatible with a rapid-cycling protocol. CONCLUSIONS: MNAzymes offer a flexible and unique approach to qPCR that is specific, sensitive, and easily multiplexed. The universal nature of MNAzyme reporter probes removes the need for target-specific probes, thereby making the development of new assays easier and cheaper.


Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sondas de DNA/química , Sondas de DNA/genética , Enzimas/química , Genoma Humano , Humanos , Sondas RNA/química , Sondas RNA/genética , Sensibilidade e Especificidade
17.
J Am Chem Soc ; 132(3): 1051-9, 2010 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-20038095

RESUMO

To increase the versatility and utility of nucleic acid enzymes, we developed multicomponent complexes, known as MNAzymes, which produce amplified "output" signals in response to specific "input" signals. Multiple oligonucleotide partzymes assemble into active MNAzymes only in the presence of an input assembly facilitator such as a target nucleic acid. Once formed, MNAzymes catalytically modify a generic substrate, generating an amplified output signal that heralds the presence of the target while leaving the target intact. We demonstrated several applications including sensitive, isothermal target detection; discrimination of polymorphisms; and highly specific monitoring of real-time polymerase chain reaction (PCR). Furthermore, we showed their capacity to function as molecular switches and to work in series to create a molecular cascade. The modular nature of MNAzymes, together with the separation of input and output functionalities, provides potential for their integration into diverse devices such as diagnostic biosensors, molecular computers, and/or nanoscale machines.


Assuntos
Técnicas Biossensoriais , Computadores Moleculares , DNA Catalítico/química , DNA Catalítico/classificação , RNA Catalítico/química , RNA Catalítico/classificação , Nanoestruturas/química , Engenharia de Proteínas
18.
Methods Mol Med ; 125: 127-47, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16502582

RESUMO

The last 15 yr have produced dramatic improvements in the survival rate of patients with acute promyelocytic leukemia (APL). These improvements have been due mainly to the introduction of targeted therapies and improved methods for diagnosing and monitoring this disease. The underlying molecular lesion in APL involves a t(15:17) translocation which leads to the generation of PML-RARalpha fusion transcripts and proteins. The PML-RARalpha fusion transcripts have been shown to be useful markers for establishing the diagnosis and for monitoring the response to treatment. This manuscript describes the application of QZyme reverse-transcription polymerase chain reaction (RT-PCR) to the quantification of PML-RARalpha transcripts as a marker of APL. QZyme is a method for real time detection and quantification of target genes or transcripts. The principle of QZyme analysis is similar to other quantitative PCR systems; however, the mechanism is quite different. QZyme exploits the catalytic activity of DNAzymes (deoxyribozymes), which are oligonucleotides that can bind and cleave nucleic acid substrates. The approach is well suited to monitoring minimal residual disease (MRD) in patients with APL, as a result of its ability to detect low numbers of transcripts and accurately measure differences in concentration over a broad dynamic range. Further, its capacity for duplex analysis has multiple advantages for analysis of clinical specimens. Protocols for duplex, single-tube QZyme RT-PCR assays, which allow simultaneous quantification of PML-RARalpha fusion transcripts (either L-type and V-type, or S-type) and the internal control BCR transcript, are provided. These protocols can be used for analyzing patient RNA specimens and are suitable for clinical trial monitoring. For this type of work, it is recommended that investigators validate the assays to ensure reproducible, accurate, and specific results on the equipment in their own laboratories. Assay validation is critical for real-time quantitative RT-PCR (RQ-PCR) and is often overlooked. A guide to the steps involved in validation and recommendations for acceptance criteria is included in this chapter.


Assuntos
Leucemia Promielocítica Aguda/genética , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcrição Genética , Cromossomos Humanos Par 15 , Cromossomos Humanos Par 17 , Humanos , Monitorização Fisiológica/métodos , RNA Neoplásico/genética , Translocação Genética
19.
J Cancer Res Clin Oncol ; 128(8): 403-11, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12200596

RESUMO

PURPOSE: Identification of germline mutations in mismatch repair genes is increasingly being used to guide clinical practice in hereditary non-polyposis colon cancer. The aim of this study was to retrospectively assess the clinical utility of immunostaining and microsatellite instability testing in a group of individuals in whom germline testing of hMSH2 and hMLH1 had already been performed. METHODS: Individuals were identified from the records of family cancer clinics. A total of thirty-eight tumour blocks were retrieved from 28 kindreds. DNA was extracted and PCR amplification of six microsatellite markers was performed. Immunostaining was used to examine the expression of hMSH2 and hMLH1 protein. RESULTS: Of the 32 assessable tumours, 24 (75%) showed microsatellite instability. Most of the MSI-H cancers (92%) failed to express either hMLH1 or hMSH2. Deleterious germline mutations were identified in the proband in 12 of 28 families. Missense mutations were identified in 11 cases and no mutations in six probands. CONCLUSIONS: The use of germline genetic testing is indicated for a highly selected group of individuals. MSI testing and immunostaining are extremely useful tools which significantly improve the clinical interpretation of germline results. Ambiguity regarding the significance of missense mutations in hereditary bowel cancer suggests that these findings should be interpreted with caution.


Assuntos
Pareamento Incorreto de Bases , Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA , Repetições de Microssatélites/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Transporte , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais Hereditárias sem Polipose/metabolismo , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Análise Mutacional de DNA , Feminino , Genótipo , Mutação em Linhagem Germinativa , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Fenótipo , Prognóstico , Proteínas Proto-Oncogênicas/metabolismo , Estudos Retrospectivos
20.
Clin Chem ; 48(8): 1338-43, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12142392

RESUMO

BACKGROUND: PML/RARalpha fusion transcripts provide a readily accessible marker for diagnosis of acute promyelocytic leukemia (APL) and for monitoring response to therapy. Survival rates are improved by therapies guided by such monitoring. We assessed the potential of DzyNA reverse transcription-PCR (RT-PCR) for measurement of PML/RARalpha fusion transcripts. METHODS: Parallel single-tube DzyNA RT-PCR protocols were developed to allow real-time fluorescent quantification of PML/RARalpha fusion transcripts and a low abundance control transcript, normal BCR. Calibration curves, generated using cell line RNA, allowed estimation of these transcripts in RNA from patients with APL at various stages of the disease. RESULTS: DzyNA RT-PCR calibration curves were linear for both transcripts over a broad range and demonstrated interassay variations of 12% (mean, 658 ng) and 10% (mean, 263 ng), respectively. The protocols detected low concentrations of transcripts and resolved twofold dilutions. PML/RARalpha mRNA was quantified in 10 patients at diagnosis and in 1 patient over a 7-year period. Monitoring of transcript concentrations effectively reflected the disease course in one patient and demonstrated that an increase in PML/RARalpha transcripts can be detected 4-6 months before hematologic relapse, with no false-positive results. CONCLUSION: DzyNA RT-PCR has potential for use in clinical practice as a tool for diagnosis of APL and forsubsequent monitoring of minimal residual disease and detection of molecular relapse.


Assuntos
Leucemia Promielocítica Aguda/diagnóstico , Proteínas de Neoplasias/análise , Proteínas de Fusão Oncogênica/análise , Humanos , Leucemia Promielocítica Aguda/genética , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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