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1.
Euro Surveill ; 25(6)2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32046815

RESUMO

Timely detection of novel coronavirus (2019-nCoV) infection cases is crucial to interrupt the spread of this virus. We assessed the required expertise and capacity for molecular detection of 2019-nCoV in specialised laboratories in 30 European Union/European Economic Area (EU/EEA) countries. Thirty-eight laboratories in 24 EU/EEA countries had diagnostic tests available by 29 January 2020. A coverage of all EU/EEA countries was expected by mid-February. Availability of primers/probes, positive controls and personnel were main implementation barriers.


Assuntos
Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/diagnóstico , Coronavirus/genética , Coronavirus/isolamento & purificação , Laboratórios/normas , Pneumonia Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Técnicas de Laboratório Clínico/métodos , Coronavirus/classificação , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Surtos de Doenças , União Europeia , Humanos , RNA Viral/genética , Padrões de Referência , Sensibilidade e Especificidade , Vigilância de Evento Sentinela , Análise de Sequência
2.
PLoS Negl Trop Dis ; 13(12): e0007884, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31877142

RESUMO

Hantavirus Pulmonary Syndrome is an, often fatal, emerging zoonotic disease in the Americas caused by hantaviruses (family: Hantaviridae). In Brazil, hantavirus routine diagnosis is based on serology (IgM-ELISA) while RT-PCR is often used to confirm acute infection. A Semi-nested RT-PCR and an internally controlled RT-qPCR assays were developed for detection and quantification of four hantaviruses strains circulating in the Brazilian Amazon: Anajatuba (ANAJV) and Castelo dos Sonhos (CASV) strains of Andes virus (ANDV) species; and Rio Mamoré (RIOMV) and Laguna Negra (LNV) strains of LNV species. A consensus region in the N gene of these hantaviruses was used to design the primer sets and a hydrolysis probe. In vitro transcribed RNA was diluted in standards with known concentration. MS2 bacteriophage RNA was detected together with hantavirus RNA as an exogenous control in a duplex reaction. RT-qPCR efficiency was around 100% and the limit of detection was 0.9 copies/µL of RNA for RT-qPCR and 10 copies/µL of RNA for Semi-nested RT-PCR. There was no amplification of either negative samples or samples positive to other pathogens. To assess the protocol for clinical sensitivity, specificity and general accuracy values, both assays were used to test two groups of samples: one comprising patients with disease (n = 50) and other containing samples from healthy individuals (n = 50), according to IgM-ELISA results. A third group of samples (n = 27) infected with other pathogens were tested for specificity analysis. RT-qPCR was more sensitive than semi-nested RT-PCR, being able to detect three samples undetected by conventional RT-PCR. RT-qPCR clinical sensitivity, specificity and general accuracy values were 92.5%, 100% and 97.63%, respectively. Thus, the assays developed in this study were able to detect the four Brazilian Amazon hantaviruses with good specificity and sensitivity, and may become powerful tools in diagnostic, surveillance and research applications of these and possibly other hantaviruses.


Assuntos
Testes Diagnósticos de Rotina/métodos , Síndrome Pulmonar por Hantavirus/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Adulto , Brasil , Testes Diagnósticos de Rotina/normas , Hantavirus/classificação , Hantavirus/isolamento & purificação , Humanos , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Sensibilidade e Especificidade , Adulto Jovem
3.
J Vet Diagn Invest ; 31(6): 909-912, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31650901

RESUMO

Swine diarrhea can be caused by multiple agents, including porcine epidemic diarrhea virus (PEDV), porcine sapelovirus (PSV), and porcine sapovirus (SaV). We designed a one-step triplex reverse-transcription PCR (RT-PCR) detection method including 3 pairs of primers that focused on the S1 gene of PEDV, a conserved gene of PSV, and the VP1 gene of SaV. The optimal concentrations of upstream and downstream primers in the triplex RT-PCR were 0.24 µM for PEDV, 0.15 µM for PSV, and 0.2 µM for SaV, and the optimal annealing temperature was 55.5°C. Triplex RT-PCR assessment of 402 piglet diarrhea samples was compared with conventional individual RT-PCR. Concordance rates in both tests for individual viruses were 100%, 97.6%, and 94.4% for PEDV, PSV, and SaV, respectively. PEDV, PSV, and SaV were detected in 57.2%, 10.4%, and 9.0% of the samples, respectively. The high sensitivity and specificity of this triplex RT-PCR-based detection method for PEDV, PSV, and SaV could allow rapid detection and analysis of mixed infections by these 3 viruses.


Assuntos
Infecções por Caliciviridae/veterinária , Infecções por Coronavirus/veterinária , Diarreia/veterinária , Reação em Cadeia da Polimerase Multiplex/veterinária , Infecções por Picornaviridae/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Doenças dos Suínos/diagnóstico , Animais , Infecções por Caliciviridae/diagnóstico , Infecções por Coronavirus/diagnóstico , Diarreia/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Picornaviridae/isolamento & purificação , Infecções por Picornaviridae/diagnóstico , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sapovirus/isolamento & purificação , Suínos , Doenças dos Suínos/virologia
4.
Lett Appl Microbiol ; 69(5): 318-324, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31529706

RESUMO

Peach is a major crop in China, and like any other stone fruit, virus and virus-like diseases can reduce the yield and quality of the fruit. Herein, we developed a multiplex RT-PCR (mRT-PCR) assay for simultaneously detecting three viruses known to infect peach: peach-associated luteovirus (PaLV), peach virus D (PeVD) and nectarine stem-pitting-associated virus (NSPaV). Plant nad5 mRNA was used as the internal control. Field samples that were co-infected with PaLV, PeVD and NSPaV were used; we identified three primer pairs to be the most specific for detecting these viruses, followed by determining the ideal concentration of each primer pair and optimizing the annealing temperature for mRT-PCR. We also assessed the detection limit using serial dilutions of RNA and cDNA. The newly developed mRT-PCR assay could simultaneously detect PaLV, PeVD and NSPaV. To validate the reliability of mRT-PCR for virus detection, mRT-PCR was used to detect viruses in the leaves of 21 peach plants collected in Liaoning Province, China. The obtained results revealed the presence of single and co-infections. To conclude, the mRT-PCR assay developed herein is sensitive, reliable and economical, and we believe that it can thus be used for large-scale surveys of PaLV, PeVD and NSPaV. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we developed a multiplex reverse transcriptase PCR (mRT-PCR) assay for simultaneously detecting three viruses that infect peach: peach-associated luteovirus (PaLV), peach virus D (PeVD) and nectarine stem-pitting-associated virus (NSPaV). This assay is simple, easy to perform, reliable and cost-effective, and can thus be applied for large-scale surveys of PaLV, PeVD and NSPaV.


Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Doenças das Plantas/virologia , Prunus persica/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Vírus/isolamento & purificação , China , Primers do DNA/genética , Folhas de Planta/virologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Vírus/classificação , Vírus/genética
5.
Clin Lab ; 65(9)2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31532093

RESUMO

BACKGROUND: Recently molecular chimerism analysis after allogeneic hematopoietic stem cell transplantation (AHSCT) has become more important. The use of quantitative chimerism methods aims to assess the kinetics of engraftment to determine graft rejection and failure or relapse of the underlying disease after AHSCT. An accurate and sensitive determination of chimerism status is mandatory after AHSCT. This study aimed to compare two chimerism methods: Multiplex Short Tandem Repeat-Polymerase Chain Reaction (STR-PCR) and quantitative Real Time-PCR (qRT-PCR). METHODS: Thirty-nine blood samples at +28 day were used to extract DNA. Most patients had been diagnosed with acute leukemia (74.3 %) and other hematological diseases. For Multiplex STR-PCR method, PCR products were separated on an ABI 3130 Genetic Analyzer (Applied Biosystem, USA) and for qRT-PCR, an ABI 7500 (Applied Biosystem, USA) Plate System Real Time Analyzer was used to determine the quantification of chimerism per-centage. RESULTS: Of the 39 analyzed samples, 82% concordant chimerism results were detected for both STR-PCR and qRT-PCR methods. Ten mixed chimerisms (MC) were found by qRT-PCR whereas of only 3 MC cases were detected by STR-PCR. In the discordant group of 7 by qRT-PCR, we observed Acute Graft versus Host Disease (aGVHD). Three MC cases that were detected by both STR- and qRT-PCR methods died because of relapse. CONCLUSIONS: The quantitative chimerism method along with multiplex STR-PCR method is important for early detection of MC. qRT-PCR methods can be valuable options in the prevention of graft failure and assisting with fast and early treatment strategies for patients undergoing AHSCT.


Assuntos
Quimerismo , Transplante de Células-Tronco Hematopoéticas/métodos , Leucemia/terapia , Repetições de Microssatélites/genética , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Quimeras de Transplante/genética , Transplante Homólogo , Adulto Jovem
6.
Clin Lab ; 65(9)2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31532107

RESUMO

BACKGROUND: Although expression of WWOX was extensively studied in different tumors, WWOX gene expression in acute myeloid leukemia is not well-known or recognized. METHODS: Fifty control donors and fifty acute myeloid leukemia patients were enrolled in this research. The levels of WWOX gene in all participants were detected by RT-qPCR. RESULTS: Levels of WWOX gene in the AML group were significantly lower as compared to the control group (0.27205 ± 1.19812 vs. 10.501 ± 9.0338, respectively, p < 0.001**). CONCLUSIONS: Decreased expression of WWOX gene ran parallel with acute myeloid leukemia regression.


Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteínas Supressoras de Tumor/genética , Oxidorredutase com Domínios WW/genética , Adulto , Intervalo Livre de Doença , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Proteínas Supressoras de Tumor/metabolismo , Oxidorredutase com Domínios WW/metabolismo
7.
Intervirology ; 62(3-4): 164-168, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31487720

RESUMO

Despite the significant medical advances which have taken place in the last decades, acute diarrhoea cases remain a public health issue of major significance, with gastroenteritis agents being associated with severe symptoms in adults and high morbidity in infants and children. Regarding rotaviruses, while children are the predominant victims of rotavirus infection, adults (often caretakers or parents of these children) may experience the same symptoms of fever, vomiting, and non-bloody diarrhoea. Three different routine schemes for the detection of rotaviruses in archived stool samples were evaluated in terms of diagnostic performance. A total of 640 archived stool samples were included in the study. The samples were screened with three different techniques: a commercial rapid immunochromatographic test, a modified in-house conventional one-step reverse-transcription polymerase chain reaction (PCR) screen protocol, and a com-mer-cial one-step real-time PCR kit. Technical aspects and considerations are discussed.


Assuntos
Testes Diagnósticos de Rotina/métodos , Imunoensaio/métodos , Infecções por Rotavirus/diagnóstico , Rotavirus/isolamento & purificação , Diarreia/diagnóstico , Diarreia/virologia , Fezes/virologia , Gastroenterite/diagnóstico , Gastroenterite/virologia , Grécia , Humanos , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rotavirus/imunologia , Sensibilidade e Especificidade , Centros de Atenção Terciária
8.
J Vet Med Sci ; 81(10): 1533-1539, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31406032

RESUMO

Duck hepatitis A virus (DHAV) infection is characterized by severe hepatitis. In recent years, DHAV-A has become widespread in Asia and has led to economic losses. Conventional methods of DHAV-A detection must often be performed in the laboratory with inconvenience equipment. We have developed a rapid reverse transcription insulated isothermal (RT-iiPCR) technique for the on-site detection of DHAV-A based on the POCKITTM system in a convenient minitype device. We optimized the PCR primers and probes for the amplification of the DHAV-A 3C/3D genes, and successfully amplified a specific fragment of DHAV-A, but no fragment from 18 other duck pathogens. The limit of detection for viral RNA was 49 copies per reaction, and the sensitivity and specificity were each 100% in the analysis of 60 liver samples. By comparison, the sensitivities of RT-iiPCR was comparable in sensitivity to existing rRT-PCR. Furthermore, the RT-iiPCR results were 98.3% in agreement with those of the rRT-PCR, with a kappa value of 0.938. In conclusion, this new method not only offers a higher sensitivity and specificity than existing techniques, but also time-saving and better suited to field diagnoses because device is portable.


Assuntos
Patos , Hepatite A/veterinária , Vírus da Hepatite do Pato/isolamento & purificação , Fígado/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Animais , China/epidemiologia , Hepatite A/diagnóstico , Vírus da Hepatite A/genética , Vírus da Hepatite do Pato/genética , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
9.
J Appl Oral Sci ; 27: e20180256, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31365706

RESUMO

OBJECTIVE: The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. METHODOLOGY: Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). RESULTS: Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). CONCLUSIONS: The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.


Assuntos
DNA Ribossômico/isolamento & purificação , Cavidade Pulpar/microbiologia , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Streptococcus/isolamento & purificação , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Ribossômico/genética , Humanos , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Ribossômico/genética , Reprodutibilidade dos Testes , Tratamento do Canal Radicular/métodos , Streptococcus/genética
10.
Arch Virol ; 164(11): 2683-2690, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31428915

RESUMO

Current antiviral therapies against hepatitis B virus (HBV) infections, such as treatment with nucleos(t)ide analogs (NAs) and interferon alpha, can significantly lower HBV DNA titers, eventually to undetectable levels. However, it is still difficult to completely eliminate the stable template of HBV, the covalently closed circular DNA (cccDNA), and this contributes to viral rebound when treatment is discontinued. HBV pregenomic RNA (pgRNA), which was recently found to be present in the enveloped mature HBV viral particle in blood, is tentatively regarded, with still accumulating clinical evidence, as a novel bona fide virological marker reflecting the amount and status of cccDNA when serum HBV DNA becomes undetectable. HBV pgRNA and DNA share almost identical sequences, and it is therefore difficult to differentiate pgRNA from viral DNA using normal PCR methods. To exclude interference from viral DNA, methods for measuring pgRNA usually require a selective DNA degradation step, which is complicated and time-consuming and also compromises the accuracy of detection. In this study, we developed a simplified quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay with improved accuracy achieved by probing the polyA tail of pgRNA. Using clinical serum samples, we observed that not all patients share the same 3' sequence, suggesting slight differences between HBV strains in the way they end transcription. We then designed and evaluated a universal primer and probe set for distinguishing HBV pgRNA from HBV DNA. Our results demonstrated that a one-step qRT-PCR assay could selectively amplify HBV pgRNA from a mixture of HBV RNA and DNA, which is valuable for clinical applications.


Assuntos
Vírus da Hepatite B/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , DNA Viral/análise , DNA Viral/genética , Hepatite B/virologia , Humanos , RNA Viral/análise , RNA Viral/genética
11.
Int J Mol Sci ; 20(16)2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426285

RESUMO

High-throughput RNA sequencing and novel bioinformatic pipelines have identified thousands of circular (circ)RNAs containing backsplice junction sequences. However, circRNAs generated from multiple exons may contain different combinations of exons and/or introns arising from alternative splicing, while the backsplice junction sequence is the same. To be able to identify circRNA splice variants, we developed a method termed circRNA-Rolling Circle Amplification (circRNA-RCA). This method detects full-length circRNA sequences by performing reverse transcription (RT) in the absence of RNase H activity, followed by polymerase chain reaction (PCR) amplification of full-length circRNAs using a forward primer spanning the backsplice junction sequence and a reverse primer exactly upstream of the forward primer. By sequencing the PCR products, circRNA splice variants bearing the same backsplice junctions, which were otherwise only predicted computationally, could be experimentally validated. The splice variants were further predicted to associate with different subsets of target RNA-binding proteins and microRNAs, supporting the notion that different circRNA splice variants can have different biological impacts. In sum, the circRNA-RCA method allows the accurate identification of full-length circRNA sequences, offering unique insight into their individual function.


Assuntos
DNA Complementar/genética , Processamento de RNA , /genética , Processamento Alternativo , Sequência de Bases , Células HeLa , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de RNA/métodos
12.
PLoS One ; 14(7): e0219292, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31276479

RESUMO

Rabies is a neglected disease mostly affecting the developing world. Accurate and reliable diagnostic and surveillance data forms the foundation for the formulation and monitoring of control strategies. Although various sensitive and specific tests are available for detection of rabies virus, implementation of these tests in low-resource settings are challenging and remains limited. In this study, we describe the developed of a reverse transcription recombinase polymerase amplification assay for the detection of rabies virus. The analytical sensitivity of this assay was determined to be 562 RNA copies and was performed in 20 minutes. The diagnostic sensitivity of the RT-RPA was 100% for detection of rabies virus in field samples. In conclusion, the RT-RPA assay allowed for very quick and sensitive detection of rabies virus and could be adapted for use in low-source settings.


Assuntos
Vírus da Raiva/genética , Raiva/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , África/epidemiologia , Animais , Bioensaio , Cães/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Raiva/veterinária , Vírus da Raiva/patogenicidade , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade
13.
Zoonoses Public Health ; 66(7): 729-738, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31259469

RESUMO

Hepatitis E virus (HEV) is currently considered as a global health concern due to the recognition of its zoonotic transmission to humans, mainly from swine, and its association with the development of severe cases of hepatitis in human risk populations. The lack of updated data on HEV state of infection in swineherds of Argentina, and the necessity of robust technologies for its detection in complex biological samples, positions HEV as an emerging issue in public health. Here, we have optimized a RT-qPCR with internal control for a more precise and accurate HEV RNA detection in swine stool samples. We implemented this optimized molecular tool to analyse the current epidemiological scenario of HEV infection in swine from the core region of commercial activity of Argentina. A total of 135 stool samples were collected from 16 different farms and tested for HEV presence, resulting in 11 positive cases (8.1%). Phylogenetic analysis demonstrated that all of them correspond to HEV genotype 3 and that different subtypes circulate in the region. Moreover, two of the detected strains presented a high nucleotide similarity with a previously identified isolate from human sewage discharges, suggesting the zoonotic transmission of HEV to humans. Collectively, this work provides a better understanding of HEV epidemiology in Argentina while contributes to the improvement of HEV detection technologies.


Assuntos
Vírus da Hepatite E/isolamento & purificação , Hepatite E/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Doenças dos Suínos/virologia , Animais , Argentina/epidemiologia , Genótipo , Vírus da Hepatite E/genética , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Suínos , Doenças dos Suínos/epidemiologia , Zoonoses
14.
PLoS One ; 14(6): e0218717, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31233538

RESUMO

The diversity of antibody variable regions makes cDNA sequencing challenging, and conventional monoclonal antibody cDNA amplification requires the use of degenerate primers. Here, we describe a simplified workflow for amplification of IgG antibody variable regions from hybridoma RNA by a specialized RT-PCR followed by Sanger sequencing. We perform three separate reactions for each hybridoma: one each for kappa, lambda, and heavy chain transcripts. We prime reverse transcription with a primer specific to the respective constant region and use a template-switch oligonucleotide, which creates a custom sequence at the 5' end of the antibody cDNA. This template-switching circumvents the issue of low sequence homology and the need for degenerate primers. Instead, subsequent PCR amplification of the antibody cDNA molecules requires only two primers: one primer specific for the template-switch oligonucleotide sequence and a nested primer to the respective constant region. We successfully sequenced the variable regions of five mouse monoclonal IgG antibodies using this method, which enabled us to design chimeric mouse/human antibody expression plasmids for recombinant antibody production in mammalian cell culture expression systems. All five recombinant antibodies bind their respective antigens with high affinity, confirming that the amino acid sequences determined by our method are correct and demonstrating the high success rate of our method. Furthermore, we also designed RT-PCR primers and amplified the variable regions from RNA of cells transfected with chimeric mouse/human antibody expression plasmids, showing that our approach is also applicable to IgG antibodies of human origin. Our monoclonal antibody sequencing method is highly accurate, user-friendly, and very cost-effective.


Assuntos
Anticorpos Monoclonais/genética , Região Variável de Imunoglobulina/genética , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Reações Antígeno-Anticorpo , Primers do DNA/genética , DNA Complementar/genética , Células HEK293 , Humanos , Hibridomas/imunologia , Imunoglobulina G/genética , Camundongos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fluxo de Trabalho
15.
Transbound Emerg Dis ; 66(6): 2271-2278, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31233656

RESUMO

Porcine reproductive and respiratory syndrome virus 1 (PRRSV1) and 2 (PRRSV2) (including 3 major subtypes: classical (CA-PRRSV2), highly pathogenic (HP-PRRSV2) and NADC30-like (NL-PRRSV2)) are currently coexisting in Chinese swine herds but with distinct virulence. Reliable detection and differentiation assays are crucial to monitor the prevalence of PRRSV and to adopt effective control strategies. However, current diagnostic methods cannot simultaneously differentiate the four major groups of PRRSV in China. In this study, universal and quadruplex real-time RT-PCR assays using TaqMan-MGB probes were developed for simultaneous detection and differentiation of Chinese PRRSV isolates. The newly developed real-time RT-PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. In addition, the newly developed real-time RT-PCR assays were further validated by comparing with a universal PRRSV conventional RT-PCR assay on the detection of 664 clinical samples collected from 2016 to 2019 in China. Based on the clinical performance, the agreements between the universal and quadruplex real-time RT-PCR assays and the conventional RT-PCR assay were 99.55% and 99.40%, respectively. Totally 90 samples were detected as PRRSV-positive, including 2 samples that were determined to be co-infected with NL-PRRSV2 and HP-PRRSV2 isolates by the quadruplex real-time RT-PCR assay. ORF5 sequencing confirmed the real-time RT-PCR results that 2, 6, 27 and 57 of the 92 sequences were PRRSV1, CA-PRRSV2, NL-PRRSV2 and HP-PRRSV2, respectively. This study provides promising alternative tools for simultaneous detection and differentiation of PRRSV circulating in Chinese swine herds.


Assuntos
Síndrome Respiratória e Reprodutiva Suína/diagnóstico , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , China , Reprodutibilidade dos Testes , Suínos
16.
Methods Mol Biol ; 1982: 191-229, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172474

RESUMO

The identification of NADPH oxidase (NOX) isoforms in tissues is essential for interpreting experiments and for next step decisions regarding cell lines, animal models, and targeted drug design. Two basic methods, immunoblotting and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), are important to monitor NOX protein and messenger RNA (mRNA) levels, respectively, for a range of investigations from understanding cell signaling events to judging NOX inhibitor efficacies. For many other genes that are expressed in high abundance, these methods may seem rather simple. However, detecting the low expression levels of endogenous NOX/DUOX is difficult and can be frustrating, so some guidelines would be helpful to those who are facing difficulties. One reason why detection is so difficult is the limited availability of vetted NOX/DUOX antibodies. Many of the commercial antibodies do not perform well in our hands, and dependable antibodies, often generated by academic laboratories, are in limited supply. Another problem is the growing trend in the NOX literature to omit end-user validation of antibodies by not providing appropriate positive and negative controls. With regard to NOX mRNA levels, knockdown of NOX/DUOX has been reported in cell lines with very low endogenous expression (C q values ≥30) or in cell lines devoid of the targeted NOX isoform (e.g., NOX4 expression in NCI-60 cancer cell panel cell line 786-0). These publications propagate misinformation and hinder progress in understanding NOX/DUOX function. This chapter provides overdue guidelines on how to validate a NOX antibody and provides general methodologies to prepare samples for optimal detection. It also includes validated methodology to perform RT-qPCR for the measurement of NOX mRNA levels, and we suggest that RT-qPCR should be performed prior to embarking on NOX protein detection.


Assuntos
Immunoblotting , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Animais , Linhagem Celular , Ativação Enzimática , Guias como Assunto , Humanos , Immunoblotting/métodos , Isoenzimas , Cinética , NADPH Oxidases/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
17.
J Neurooncol ; 143(3): 393-403, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31104222

RESUMO

INTRODUCTION: Molecular classification of medulloblastomas (MB) is prognostically and therapeutically relevant and helps in better risk-stratification. Translation of this subgrouping to routine practice still remains a challenge. The most pathologist accessible techniques for molecular subgrouping include immunohistochemistry (IHC), fluorescent in-situ hybridization (FISH) and NanoString. OBJECTIVES: (1) Molecular subgrouping of MBs by IHC and FISH, and NanoString assay (2) To compare their efficacy and cost for applicability in resource constrained centers. METHODS: Ninety-five cases of MB with adequate tissue were included. Molecular subgrouping was performed by IHC for ß-catenin, GAB1 and YAP1; FISH for MYC amplification, and sequencing for CTNNB1, and by NanoString Assay on the same set of MBs. A subset of cases was subjected to 850k DNA methylation array. RESULTS: IHC + FISH classified MBs into 15.8% WNT, 16.8% SHH, and 67.4% non-WNT/non-SHH subgroups; with MYC amplification identified in 20.3% cases of non-WNT/non-SHH. NanoString successfully classified 91.6% MBs into 25.3% WNT, 17.2% SHH, 23% Group 3 and 34.5% Group 4. However, NanoString assay failure was seen in eight cases, all of which were > 8-years-old formalin-fixed paraffin-embedded tissue blocks. Concordant subgroup assignment was noted in 88.5% cases, while subgroup switching was seen in 11.5% cases. Both methods showed prognostic correlation. Methylation profiling performed on discordant cases revealed 1 out of 4 extra WNT identified by NanoString to be WNT, others aligned with IHC subgroups; extra SHH by NanoString turned out to be SHH by methylation. CONCLUSIONS: Both IHC supplemented by FISH and NanoString are robust methods for molecular subgrouping, albeit with few disadvantages. IHC cannot differentiate between Groups 3 and 4, while NanoString cannot classify older-archived tumors, and is not available at most centres. Thus, both the methods complement each other and can be used in concert for high confidence allotment of molecular subgroups in clinical practice.


Assuntos
Neoplasias Cerebelares/classificação , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Meduloblastoma/classificação , Nanotecnologia/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Criança , Pré-Escolar , Estudos de Coortes , Terapia Combinada , Metilação de DNA , Feminino , Seguimentos , Recursos em Saúde , Humanos , Lactente , Masculino , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Adulto Jovem
18.
Western Pac Surveill Response J ; 10(1): 32-38, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31110840

RESUMO

Introduction: There are two methods of reverse transcription polymerase chain reaction (RT-PCR) that have been the common methods to detect influenza infections: conventional and real-time RT-PCR. From December 2017 to March 2018, several missed diagnoses of influenza A(H1)pdm09 using real-time RT-PCR were reported in northern Viet Nam. This study investigated how these missed detections occurred to determine their effect on the surveillance of influenza. Methods: The haemagglutinin (HA) segments of A(H1N1)pdm09 from both real-time RT-PCR positive and negative samples were isolated and sequenced. The primer and probe sets in the HA gene were checked for mismatches, and phylogenetic analyses were performed to determine the molecular epidemiology of these viruses. Results: There were 86 positive influenza A samples; 32 were A(H1)pdm09 positive by conventional RT-PCR but were negative by real-time RT-PCR. Sequencing was conducted on 23 influenza (H1N1)pdm09 isolates that were recovered from positive samples. Eight of these were negative for A(H1)pdm09 by real-time RT-PCR. There were two different mismatches in the probe target sites of the HA gene sequences of all isolates (n = 23) with additional mismatches only at position 7 (template binding site) identified for all eight negative real-time RT-PCR isolates. The prime target sites had no mismatches. Phylogenetic analysis of the HA gene showed that both the positive and negative real-time RT-PCR isolates were grouped in clade 6B.1; however, the real-time RT-PCR negative viruses were located in a subgroup that referred to substitution I295V. Conclusion: Constant monitoring of genetic changes in the circulating influenza A(H1N1)pdm09 viruses is important for maintaining the sensitivity of molecular detection assays.


Assuntos
Diagnóstico Tardio/tendências , Influenza Humana/diagnóstico , Análise de Sequência de DNA/normas , Testes de Hemaglutinação/métodos , Hemaglutininas/análise , Hemaglutininas/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/epidemiologia , Influenza Humana/mortalidade , Mutação/genética , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/tendências , Vietnã
19.
BMC Infect Dis ; 19(1): 418, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088375

RESUMO

BACKGROUND: The global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health. Because these arboviruses are transmitted by the same mosquito species and co-circulate in the same area, a sensitive diagnostic assay that detects them together, with discrimination, is needed. METHODS: We present here a diagnostics panel based on reverse transcription-PCR amplification of viral RNA and an xMap Luminex architecture involving direct hybridization of PCRamplicons and virus-specific probes. Two DNA innovations ("artificially expanded genetic information systems", AEGIS, and "self-avoiding molecular recognition systems", SAMRS) increase the hybridization sensitivity on Luminex microspheres and PCR specificity of the multiplex assay compared to the standard approach (standard nucleotides). RESULTS: The diagnostics panel detects, if they are present, these viruses with a resolution of 20 genome equivalents (DENV1), or 10 (DENV3-4, CHIKV) and 80 (DENV2, ZIKV) genome equivalents per assay. It identifies ZIKV, CHIKV and DENV RNAs in a single infected mosquito, in mosquito pools comprised of 5 to 50 individuals, and mosquito saliva (ZIKV, CHIKV, and DENV2). Infected mosquitoes and saliva were also collected on a cationic surface (Q-paper), which binds mosquito and viral nucleic acids electrostatically. All samples from infected mosquitoes displayed only target-specific signals; signals from non-infected samples were at background levels. CONCLUSIONS: Our results provide an efficient and multiplex tool that may be used for surveillance of emerging mosquito-borne pathogens which aids targeted mosquito control in areas at high risk for transmission.


Assuntos
Vírus Chikungunya/genética , Culicidae/virologia , Vírus da Dengue/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Zika virus/genética , Animais , Febre de Chikungunya/diagnóstico , Febre de Chikungunya/virologia , Vírus Chikungunya/isolamento & purificação , Dengue/diagnóstico , Dengue/virologia , Vírus da Dengue/isolamento & purificação , Humanos , Hibridização de Ácido Nucleico , RNA Viral/genética , RNA Viral/metabolismo , Kit de Reagentes para Diagnóstico , Saliva/virologia , Zika virus/isolamento & purificação , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/virologia
20.
BMC Vet Res ; 15(1): 168, 2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31126297

RESUMO

BACKGROUND: Seneca Valley virus (SVV) has emerged in multiple countries in recent years. SVV infection can cause vesicular lesions clinically indistinguishable from those caused by other vesicular disease viruses, such as foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV), and vesicular exanthema of swine virus (VESV). Sensitive and specific RT-PCR assays for the SVV detection is necessary for differential diagnosis. Real-time RT-PCR (rRT-PCR) has been used for the detection of many RNA viruses. The insulated isothermal PCR (iiPCR) on a portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, SVV rRT-PCR and RT-iiPCR were developed and validated. RESULTS: Neither the SVV rRT-PCR nor the RT-iiPCR cross-reacted with any of the vesicular disease viruses (20 FMDV, two SVDV, six VSV, and two VESV strains), classical swine fever virus (four strains), and 15 other common swine viruses. Analytical sensitivities of the SVV rRT-PCR and RT-iiPCR were determined using serial dilutions of in vitro transcribed RNA as well as viral RNA extracted from a historical SVV isolate and a contemporary SVV isolate. Diagnostic performances were further evaluated using 125 swine samples by two approaches. First, nucleic acids were extracted from the 125 samples using the MagMAX™ kit and then tested by both RT-PCR methods. One sample was negative by the rRT-PCR but positive by the RT-iiPCR, resulting in a 99.20% agreement (124/125; 95% CI: 96.59-100%, κ = 0.98). Second, the 125 samples were tested by the taco™ mini extraction/RT-iiPCR and by the MagMAX™ extraction/rRT-PCR system in parallel. Two samples were positive by the MagMAX™/rRT-PCR system but negative by the taco™ mini/RT-iiPCR system, resulting in a 98.40% agreement (123/125; 95% CI: 95.39-100%, κ = 0.97). The two samples with discrepant results had relatively high CT values. CONCLUSIONS: The SVV rRT-PCR and RT-iiPCR developed in this study are very sensitive and specific and have comparable diagnostic performances for SVV RNA detection. The SVV rRT-PCR can be adopted for SVV detection in laboratories. The SVV RT-iiPCR in a simple field-deployable system could serve as a tool to help diagnose vesicular diseases in swine at points of need.


Assuntos
Picornaviridae/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Doenças dos Suínos/virologia , Animais , Variação Genética , Picornaviridae/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/diagnóstico
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