RESUMO
In response to a recent outbreak in China, detection assays for a novel avian influenza A(H7N9) virus need to be implemented in a large number of public health laboratories. Here we present real-time reverse-transcription polymerase chain reaction (RT-PCR) assays for specific detection of this virus, along with clinical validation data and biologically-safe positive controls.
Assuntos
Vírus da Influenza A/genética , Influenza Aviária/virologia , Influenza Humana/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Aves/virologia , China , Humanos , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/transmissão , Influenza Humana/diagnósticoRESUMO
We present a rigorously validated and highly sensitive confirmatory real-time RT-PCR assay (1A assay) that can be used in combination with the previously reported upE assay. Two additional RT-PCR assays for sequencing are described, targeting the RdRp gene (RdRpSeq assay) and N gene (NSeq assay), where an insertion/deletion polymorphism might exist among different hCoV-EMC strains. Finally, a simplified and biologically safe protocol for detection of antibody response by immunofluorescence microscopy was developed using convalescent patient serum.
Assuntos
Infecções por Coronavirus/diagnóstico , Coronavirus/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Coronavirus/classificação , Coronavirus/genética , Infecções por Coronavirus/virologia , Imunofluorescência , Alemanha , Humanos , Laboratórios/normas , Polimorfismo de Fragmento de Restrição , RNA Viral/sangue , RNA Viral/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA , Virologia/métodosRESUMO
We present two real-time reverse-transcription polymerase chain reaction assays for a novel human coronavirus (CoV), targeting regions upstream of the E gene (upE) or within open reading frame (ORF)1b, respectively. Sensitivity for upE is 3.4 copies per reaction (95% confidence interval (CI): 2.56.9 copies) or 291 copies/mL of sample. No cross-reactivity was observed with coronaviruses OC43, NL63, 229E, SARS-CoV, nor with 92 clinical specimens containing common human respiratory viruses. We recommend using upE for screening and ORF1b for confirmation.
Assuntos
Infecções por Coronavirus/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/isolamento & purificação , Coronavirus Humano 229E/genética , Coronavirus Humano 229E/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/genética , Coronavirus Humano NL63/genética , Coronavirus Humano NL63/isolamento & purificação , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/isolamento & purificação , Humanos , Fases de Leitura Aberta , Arábia Saudita , Sensibilidade e Especificidade , Viagem , Proteínas do Envelope Viral , Proteínas ViroporinasRESUMO
Influenza A(H1N1)v virus was first identified in April 2009. A novel real-time RT-PCR for influenza A(H1N1)v virus was set up ad hoc and validated following industry-standard criteria. The lower limit of detection of the assay was 384 copies of viral RNA per ml of viral transport medium (95% confidence interval: 273-876 RNA copies/ml). Specificity was 100% as assessed on a panel of reference samples including seasonal human influenza A virus H1N1 and H3N2, highly pathogenic avian influenza A virus H5N1 and porcine influenza A virus H1N1, H1N2 and H3N2 samples. The real-time RT-PCR assay for the influenza A matrix gene recommended in 2007 by the World Health Organization was modified to work under the same reaction conditions as the influenza A(H1N1)v virus-specific test. Both assays were equally sensitive. Clinical applicability of both assays was demonstrated by screening of almost 2,000 suspected influenza (H1N1)v specimens, which included samples from the first cases of pandemic H1N1 influenza imported to Germany. Measuring influenza A(H1N1)v virus concentrations in 144 laboratory-confirmed samples yielded a median of 4.6 log RNA copies/ml. The new methodology proved its principle and might assist public health laboratories in the upcoming influenza pandemic.
Assuntos
Surtos de Doenças/estatística & dados numéricos , Vírus da Influenza A Subtipo H1N2/genética , Vírus da Influenza A Subtipo H1N2/isolamento & purificação , Influenza Humana/epidemiologia , Influenza Humana/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Alemanha/epidemiologia , Humanos , Influenza Humana/diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeAssuntos
Técnicas Bacteriológicas/métodos , Coxiella burnetii/isolamento & purificação , Análise em Microsséries/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Febre Q/diagnóstico , Febre Q/veterinária , Animais , Bovinos , Coxiella burnetii/genética , Cabras , Humanos , Nova Zelândia , Sensibilidade e Especificidade , OvinosRESUMO
This paper reports significant improvements in the efficacy of sequence-independent amplification and quality of sequencing of viruses with segmented double-stranded RNA (dsRNA) genomes. We demonstrate that most remaining bottlenecks in dsRNA virus genome characterization have now been eliminated. Both the amplification and sequencing technologies used require no previous sequence knowledge of the viral dsRNA, there is no longer a need to separate genome segments or amplicons and the sequence-determined bias observed in cloning has been overcome. Combining very efficient genome amplification with pyrophosphate-based 454 (GS20/FLX) sequencing enabled sequencing of complete segmented dsRNA genomes and accelerated the sequence analysis of the amplified viral genomes. We report the complete consensus sequence of seven viruses from four different dsRNA virus groups, which include the first complete sequence of the genome of equine encephalosis virus (EEV), the first complete sequence of an African horsesickness virus (AHSV) genome determined directly from a blood sample and a complete human rotavirus genome determined from faeces. We also present the first comparison between the complete consensus sequence of a virulent and an attenuated strain of AHSV1. Ultra-deep sequencing (>400-fold coverage) of the AHSV1 reference and attenuated strains revealed different ratios of reassortants in the reference strain and allowed quasispecies detection in the plaque-purified attenuated strain of AHSV1. This approach amounts to a paradigm shift in dsRNA virus research, since it is sensitive and specific enough for comprehensive investigations of the evolution and genetic diversity in dsRNA virus populations.
Assuntos
Genoma Viral , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA de Cadeia Dupla/genética , RNA Viral/genética , Análise de Sequência de DNA/métodos , Vírus da Doença Equina Africana/genética , Sequência de Bases , Humanos , Dados de Sequência Molecular , Orbivirus/genética , Orbivirus/isolamento & purificação , Vírus Reordenados , Rotavirus/genética , Rotavirus/isolamento & purificaçãoRESUMO
Polymorphisms in cytokine genes can influence immune responses and inflammation and thereby affecting the outcome of hematopoietic stem-cell transplantation. We analyzed a single-nucleotide polymorphism in the gene for the interleukin-23 receptor (IL-23R) (1142G>A) in a cohort of 221 transplant recipients and their human leukocyte antigen (HLA)-identical sibling donors and in a second cohort of 186 transplant recipients and their HLA-identical unrelated donors. Genotypes were tested for an association with graft-versus-host disease (GVHD) by multivariate analysis. The donor's IL-23R genotype was significantly associated with a reduced risk of acute GVHD in both cohorts for patients after transplant. Analysis of all 407 transplant recipients showed that IL-23R (1142G>A, Arg381Gln) genotype of the donor was associated with a decreased risk of grades 2-4 acute GVHD (31.6 compared to 51.0%, P=0.02) and grades 3-4 severe acute GVHD (3.9 compared to 23.4%, P=0.003). Death in remission was significantly lower in patients transplanted from donors with variant IL23-R (11.7 versus 27.7%, P=0.028), whereas overall survival or relapse rates were not influenced significantly by the IL-23R genotype. Among recipients of hematopoietic cells from HLA-identical donors, the IL-23R (Arg381Gln) gene variant on the donor side has a protective effect on the occurrence of acute GVHD in recipients after transplantation.
Assuntos
Doença Enxerto-Hospedeiro/genética , Transplante de Células-Tronco Hematopoéticas , Doadores Vivos , Polimorfismo de Nucleotídeo Único , Receptores de Interleucina/genética , Doença Aguda , Adolescente , Adulto , Idoso , Criança , Intervalo Livre de Doença , Feminino , Genótipo , Doença Enxerto-Hospedeiro/mortalidade , Antígenos HLA/genética , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Irmãos , Taxa de SobrevidaRESUMO
UDP-glucuronosyltransferases (UGT) catalyze the glucuronidation of various compounds and thus inactivate toxic substrates. Genetic variations reducing the activity of UGT1A7 have been associated with various gastrointestinal cancers. Most recently, the UGT1A7*3 allele has been reported as a significant risk factor for pancreatic disorders, but we could not confirm these data. This study focused on the possible causes for the noted discrepancy. UGT1A7 genotypes were assessed in 37 samples, which were previously analyzed for UGT1A7 polymorphisms by others. We determined genotypes by melting curve analysis and by DNA sequencing. Additionally, we produced UGT1A7*1 and *3 constructs with or without a mutation at position - 57 of UGT1A7 and analyzed various combinations of these constructs. In 14/37 samples UGT1A7 genotyping results differed. The discrepancy could be explained by polymerase chain reaction bias owing to an unbalanced allelic amplification which was caused by a -57T>G variant located within the sequence of the chosen primer template in previous studies. Our findings indicate that most of the previously reported genetic associations between UGT1A7 and gastrointestinal cancers are based on primer-dependent genotyping errors.
Assuntos
Glucuronosiltransferase/genética , Pancreatite Crônica/enzimologia , Pancreatite Crônica/genética , Polimorfismo Genético/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Clonagem Molecular , Códon , DNA/genética , Primers do DNA , Transferência Ressonante de Energia de Fluorescência , Genótipo , Humanos , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , TemperaturaRESUMO
BACKGROUND: A recent study reported that a non-synonymous single nucleotide polymorphism (rs11209026, p.Arg381Gln) located in the IL23R gene is a protective marker for inflammatory bowel disease. AIM: To analyse the frequency of p.Arg381Gln in three independent European inflammatory bowel disease cohorts and to evaluate how this variant influences disease behaviour. METHODS: We assessed a European cohort of 919 inflammatory bowel disease patients and compared the IL23R p.Arg381Gln genotype frequency with 845 healthy controls. Inflammatory bowel disease patients originated from Germany [Crohn's disease (CD): n = 318; ulcerative colitis (UC): n = 178], Hungary (CD: n = 148; UC: n = 118) and the Netherlands (CD: n = 157). Ethnically matched controls were included. We performed subtyping analysis in respect to CARD15 alterations and clinical characteristics. RESULTS: The frequency of the glutamine allele of p.Arg381Gln was significantly lower in inflammatory bowel disease patients compared with controls in a pooled analysis of all three cohorts (P < 0.000001) as well as in the individual cohorts (Germany: P = 0.001, Hungary: P = 0.02 and the Netherlands: P = 0.0002). The p.Arg381Gln genotype distribution was similar between CD and UC. We did not observe either statistical interactions between p.Arg381Gln and CARD15 variants or any significant associations between p.Arg381Gln genotype and subphenotypes. CONCLUSIONS: The p.Arg381Gln IL23R variant confers a protective effect against both CD and UC, but does not determine disease phenotype.
Assuntos
Colite Ulcerativa/genética , Neoplasias do Colo/prevenção & controle , Doença de Crohn/genética , Proteína Adaptadora de Sinalização NOD2/genética , Polimorfismo de Nucleotídeo Único/genética , Receptores de Interleucina/genética , Adulto , Estudos de Coortes , Feminino , Triagem de Portadores Genéticos/métodos , Genótipo , Humanos , Masculino , Fenótipo , Receptores de Interleucina/análiseRESUMO
The P-glycoprotein (P-gp) plays an important role in carcinogen distribution and is connected with cell differentiation and apoptotic processes leading to carcinogenesis. Interindividual differences in P-gp activity could modulate susceptibility to cancer development. The MDR1 gene, coding for P-gp, is highly polymorphic and some mutations modulate P-gp activity. Recently, association between the MDR1 C3435T polymorphism and the cancer susceptibility was shown. We have hypothesized that MDR1 polymorphism could influence endometrial cancer susceptibility. We have matched 198 women with endometrial cancer and 198 controls. An additional group of 488 healthy volunteers was investigated. The MDR1 C3435T polymorphism was tested by LightCycler assay. The distribution of MDR1 3435 genotypes was significantly different between cases and controls (P = 0.006). Genotypes containing at least one 3435T allele were statistically significant more frequent in the endometrial cancer group (86.8% vs 75.2%, OR 2.18, P = 0.004). Our observation suggests that MDR1 C3435T polymorphism is correlated with endometrial cancer susceptibility.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Carcinoma/genética , Neoplasias do Endométrio/genética , Mutação Puntual , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Nucleophosmin mutations of exon 12 (NPM1 mutations) represent the most frequent molecular aberration that can be found in patients with acute myeloid leukaemia (AML) and can be detected in about 35% of AML patients. NPM1 mutations are characterised by four basepair insertions within the region corresponding to the C-terminus of the protein leading to its translocation out of the nucleus. Until now, more than 40 different subsets of mutations have been identified and about 90% of NPM1 mutations are represented by subtype A and B (78% versus 12%, respectively). So far, standard screening of NPM1 mutations using conventional polymerase chain reaction (PCR) followed by capillary electrophoresis is rather time consuming. We established a new method for rapid screening of NPM1 mutations using the fluorescence resonance energy transfer (FRET) principle. Furthermore, based on individual NPM1 mutations type A and B, we designed mutation specific primers to perform a highly sensitive PCR assay that can be applied for the detection of minimal residual disease (MRD). In summary, we demonstrate new methodological approaches for rapid screening of NPM1 mutations as well as for MRD analyses based on the most frequent NPM1 mutations.
Assuntos
Éxons/genética , Testes Genéticos , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/genética , Mutação/genética , Proteínas Nucleares/genética , Doença Aguda , Primers do DNA , Humanos , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Nucleofosmina , Fosfoproteínas/genética , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Xenobiotic mediated cellular injury is thought to play a major role in the pathogenesis of pancreatic diseases. Genetic variations that reduce the expression or activity of detoxifying phase II biotransformation enzymes such as the UDP-glucuronosyltransferases might be important in this respect. Recently, a UGT1A7 low detoxification activity allele, UGT1A7*3, has been linked to pancreatic cancer and alcoholic chronic pancreatitis. OBJECTIVE: To investigate whether UGT1A7 polymorphisms contribute to the risk of pancreatitis and pancreatic cancer. METHODS: Genetic polymorphisms in the UGT1A7 gene were assessed in a large cohort of patients with different types of pancreatitis and pancreatic cancer originating from the Czech Republic (n = 93), Germany (n = 638), Netherlands (n = 136), and Switzerland (n = 106), and in healthy (n = 1409) and alcoholic (n = 123) controls from the same populations. Polymorphisms were determined by melting curve analysis using fluorescence resonance energy transfer probes. In addition, 229 Dutch subjects were analysed by restriction fragment length polymorphism. RESULTS: The frequencies of UGT1A7 genotypes did not differ between patients with acute or chronic pancreatitis or pancreatic adenocarcinoma and alcoholic and healthy controls. CONCLUSIONS: The data suggest that, in contrast to earlier studies, UGT1A7 polymorphisms do not predispose patients to the development of pancreatic cancer and pancreatitis.
Assuntos
Predisposição Genética para Doença , Glucuronosiltransferase/genética , Pancreatopatias/enzimologia , Polimorfismo Genético , Adenocarcinoma/metabolismo , Adulto , Estudos de Coortes , Feminino , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Pancreatite/enzimologia , XenobióticosAssuntos
Ácidos Nucleicos/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Alelos , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Genótipo , Humanos , Polimorfismo Genético/genética , Receptor de Pregnano X , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TemperaturaRESUMO
The authors investigated whether different types of aggression relate to the A779C tryptophan hydroxylase (TPH) polymorphism and to serotonergic activity in volunteers. A factor analysis of the Buss-Durkee Hostility Inventory yielded 2 factors representing Neurotic Hostility (NH) and Aggressive Hostility (AH). The authors used a neuroendocrine challenge with Citalopram in 48 volunteers and increased cortisol concentrations only in those with high levels of AH. Finally, an association study with 58 volunteers revealed that the A779C TPH polymorphism significantly relates to AH, with the highest aggression levels for the genotype AA and the lowest aggression levels for the genotype CC, but not to NH. Results are discussed with respect to inconsistent findings in the literature, which may be explained by this distinction of types of aggression.
Assuntos
Agressão/efeitos dos fármacos , Citalopram/farmacologia , Polimorfismo Genético , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Serotonina/farmacologia , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/farmacologia , Adulto , Citalopram/administração & dosagem , Genótipo , Humanos , Hidrocortisona/sangue , Masculino , Escalas de Graduação Psiquiátrica , Inibidores Seletivos de Recaptação de Serotonina/administração & dosagemAssuntos
Citosina/metabolismo , Guanina/metabolismo , Histidina/genética , Queratinas/genética , Hepatopatias/genética , Mutação de Sentido Incorreto/genética , Tirosina/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Doença Crônica , Hepatite Autoimune/genética , Hepatite Viral Humana/genética , Humanos , Queratina-8 , Cirrose Hepática/genética , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Keratin 8 is a major component of intermediate filaments in single-layered epithelia of the gastrointestinal tract. Keratin 8 deficient mice display signs of colitis and diarrhoea characteristic for inflammatory bowel disease. Very recently, two keratin 8 mutations, Y54H and G62C, were identified. AIMS: We investigated if these keratin 8 missense mutations were associated with inflammatory bowel disease. PATIENTS: In total, 217 German patients with Crohn' s disease, 131 German patients with ulcerative colitis, and 560 German control subjects were enrolled in this study. METHODS: Samples were analysed by PCR amplification and subsequent melting curve analysis using fluorescence resonance energy transfer probes. RESULTS: The G62C mutation was detected in five (2.3%) patients presenting with Crohn's disease and in three (2.3%) with ulcerative colitis. In comparison, 9 (1.6%) out of 560 controls were heterozygous for this mutation. No patient or control was homozygous for this mutation. Patients carrying one mutant allele did not show any noticeable characteristics in their corresponding phenotype. In contrast, the Y54H mutation was observed in neither any of the 348 patients with inflammatory bowel disease nor in any control subject. CONCLUSIONS: Our data indicate that both keratin 8 mutations, G62C and Y54H, do not play a relevant pathogenic role in inflammatory bowel disease.
Assuntos
Doenças Inflamatórias Intestinais/genética , Queratinas/genética , Mutação de Sentido Incorreto , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Análise Mutacional de DNA , Feminino , Heterozigoto , Humanos , Queratina-8 , Masculino , Pessoa de Meia-IdadeAssuntos
Proteínas de Transporte/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Mutação/genética , Diabetes Mellitus Tipo 1/epidemiologia , Diabetes Mellitus Tipo 2/epidemiologia , Triagem de Portadores Genéticos , Alemanha/epidemiologia , Humanos , Índia/epidemiologia , Razão de Chances , Inibidor da Tripsina Pancreática de KazalRESUMO
Recently, various mutations within the Abl sequence have been described that negatively affect imatinib binding to Bcr/Abl resulting in cellular resistance of chronic myeloid leukemia (CML) cells. So far, little is known as to whether these mutations are preexisting or develop under imatinib therapy as current mutation analyses are limited by a low sensitivity of approximately 1:2 (50%) to 1:5 (20%). By combining peptide nucleic acid (PNA)-based DNA clamping with a fluorescence hybridization probe assay, we developed a new and highly sensitive technique for the detection of known mutations within the Bcr/Abl kinase domain. With this approach we investigated 19 cases of CML refractory to imatinib treatment before and during therapy. By clamping of wild-type Abl through PNA we could effectively enhance the detection sensitivity for the Bcr/Abl mutations Thr315Ile, Glu255Lys, and Tyr253His such that 1 mutant cDNA molecule could be detected in 500 negatives (0.2%). We observed in one case that a Gly255Lys mutation was detectable before treatment. By DNA analysis of buccal swaps, a genetic polymorphism could be excluded. In two cases clonal evolution of known mutations developed gradually under treatment. In another case an initially detectable Tyr253His mutation disappeared after therapy onset but was again observed after 6 weeks of imatinib treatment. Preexisting and evolving Bcr/Abl mutations associated with an unfavorable prognosis could be safely detected by the presented technique. This may facilitate risk stratification in CML and may serve as a model for individualized molecular monitoring and therapeutic strategies in other malignant diseases.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Ácidos Nucleicos Peptídicos , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Benzamidas , Ligação Competitiva , Células Clonais , Sondas de DNA , Feminino , Proteínas de Fusão bcr-abl/análise , Proteínas de Fusão bcr-abl/genética , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Mutação , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
Most cases of dopa-responsive dystonia (DRD) are thought to be caused by mutations in the GCHI gene; however, by sequencing, mutations are found in only 40% to 60%. Recently, a single report identified, via Southern blot analysis, a large genomic GCHI deletion in a "mutation-negative" case. This report describes four families with DRD, two of which carry large deletions, thus confirming that deletions are an important subtype of GCHI mutations. These deletions were detected by quantitative duplex PCR that is amenable to DNA diagnostics.