ABSTRACT
Introduction Molecular biology procedures to detect, genotype and quantify hepatitis C virus (HCV) RNA in clinical samples have been extensively described. Routine commercial methods for each specific purpose (detection, quantification and genotyping) are also available, all of which are typically based on polymerase chain reaction (PCR) targeting the HCV 5′ untranslated region (5′UTR). This study was performed to develop and validate a complete serial laboratory assay that combines real-time nested reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) techniques for the complete molecular analysis of HCV (detection, genotyping and viral load) in clinical samples. Methods Published HCV sequences were compared to select specific primers, probe and restriction enzyme sites. An original real-time nested RT-PCR-RFLP assay was then developed and validated to detect, genotype and quantify HCV in plasma samples. Results The real-time nested RT-PCR data were linear and reproducible for HCV analysis in clinical samples. High correlations (> 0.97) were observed between samples with different viral loads and the corresponding read cycle (Ct - Cycle threshold), and this part of the assay had a wide dynamic range of analysis. Additionally, HCV genotypes 1, 2 and 3 were successfully distinguished using the RFLP method. Conclusions A complete serial molecular assay was developed and validated for HCV detection, quantification and genotyping. .
Subject(s)
Humans , /genetics , Hepacivirus/genetics , Hepatitis C/diagnosis , RNA, Viral/blood , DNA Primers , Genotype , Hepacivirus/isolation & purification , Polymorphism, Restriction Fragment Length , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Viral LoadABSTRACT
The neuraminidase (NA) genes of A(H1N1)pdm09 influenza virus isolates from 306 infected patients were analysed. The circulation of oseltamivir-resistant viruses in Brazil has not been reported previously. Clinical samples were collected in the state of Rio Grande do Sul (RS) from 2009-2011 and two NA inhibitor-resistant mutants were identified, one in 2009 (H275Y) and the other in 2011 (S247N). This study revealed a low prevalence of resistant viruses (0.8%) with no spread of the resistant mutants throughout RS.
Subject(s)
Humans , Antiviral Agents/pharmacology , Drug Resistance, Viral/genetics , Influenza A Virus, H1N1 Subtype/drug effects , Mutation , Neuraminidase/genetics , Oseltamivir/pharmacology , Brazil , Influenza A Virus, H1N1 Subtype/enzymology , Influenza A Virus, H1N1 Subtype/genetics , Microbial Sensitivity Tests , Reverse Transcriptase Polymerase Chain Reaction , RNA, Viral/geneticsABSTRACT
Mycoplasma gallisepticum (MS) and Mycoplasma synoviae (MS) are important avian pathogens and cause economic losses to the poultry industry. Molecular biology techniques are currently used for a rapid detection of these pathogens and the adoption of control measures of the diseases. The aim of this study was to develop and validate a technique for simultaneous detection of MG and MS by multiplex real time polymerase chain reaction (PCR). The complete assay (Multiplex MGMS) was designed with primers and probes specific for each pathogen and developed to be carried out in a single tube reaction. Vaccines, MG and MS isolates and DNA from other Mycoplasma species were used for the development and validation of the method. Further, 78 pooled clinical samples from different poultry flocks in Brazil were obtained and used to determine the sensitivity and specificity of the technique in comparison to 2 real time PCR assays specific for MG (MG PCR) and MS (MS PCR). The results demonstrated an agreement of 100% (23 positive and 44 negative samples) between Multiplex MGMS and MG PCR in the analysis of 67 samples from MG positive and negative poultry flocks, and an agreement of 96.9% between Multiplex MGMS and MS PCR in the analysis of 64 samples from MS positive and negative poultry flocks. Considering the single amplification tests as the gold standard, the Multiplex MGMS showed 100% of specificity and sensitivity in the MG analysis and 94.7% sensitivity and 100% specificity in the MS analysis. This new assay could be used for rapid analysis of MG and MS in the poultry industry laboratories.
Subject(s)
Animals , Molecular Diagnostic Techniques/methods , Mycoplasma Infections/veterinary , Mycoplasma gallisepticum/isolation & purification , Mycoplasma synoviae/isolation & purification , Poultry Diseases/diagnosis , Real-Time Polymerase Chain Reaction/methods , Veterinary Medicine/methods , Brazil , Bacteriological Techniques/methods , Multiplex Polymerase Chain Reaction/methods , Mycoplasma Infections/diagnosis , Mycoplasma Infections/microbiology , Mycoplasma gallisepticum/genetics , Mycoplasma synoviae/genetics , Poultry , Poultry Diseases/microbiology , Sensitivity and SpecificityABSTRACT
BACKGROUND: It is clinically important to detect and type human papillomavirus (HPV) in a sensitive and specific manner. OBJECTIVES: Development of a nested-polymerase chain reaction-restriction fragment length polymorphism (nested-PCR-RFLP) assay to detect and type HPV based on the analysis of L1 gene. METHODS: Analysis of published DNA sequence of mucosal HPV types to select sequences of new primers. Design of an original nested-PCR assay using the new primers pair selected and classical MY09/11 primers. HPV detection and typing in cervical samples using the nested-PCR-RFLP assay. RESULTS: The nested-PCR-RFLP assay detected and typed HPV in cervical samples. Of the total of 128 clinical samples submitted to simple PCR and nested-PCR for detection of HPV, 37 (28.9 percent) were positive for the virus by both methods and 25 samples were positive only by nested-PCR (67.5 percent increase in detection rate compared with single PCR). All HPV positive samples were effectively typed by RFLP assay. CONCLUSION: The method of nested-PCR proved to be an effective diagnostic tool for HPV detection and typing.
Subject(s)
Female , Humans , Cervix Uteri/virology , Papillomaviridae/genetics , Papillomavirus Infections/diagnosis , Base Sequence , DNA Primers/analysis , DNA, Viral/analysis , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Papillomaviridae/classification , Papillomavirus Infections/virology , Sensitivity and SpecificityABSTRACT
A reaçao em cadeira da polimerase (PCR-Polymerase Chain Reaction) é uma técnica de amplificaçao enzimática de seqüências específicas de ácidos nucleicos. Essa técnica tem sido amplamente descrita para detecçao e tipagem do Papilomavírus Humano (HPV - Human Papilomavirus). Neste trabalho, 460 amostras de colo uterino foran avaliadas para a presença do DNA do HPV pela técnica de PCR. Amostras positivas foram subseqüentemente tipadas por RFLP (Restriction Fragment Lenght Polymorphism). Os resultados de PCR-RFLP foram comparados com os exames colposcópico e citopatológico (Papanicolou). O PCR-RFLP demonstrou ser uma técnica eficaz na detecçao e tipagem virais, apresentando maior sensibilidade do que os exames citopatológico e colposcópico
Subject(s)
Humans , Female , Papillomaviridae/isolation & purification , Papillomavirus Infections/diagnosis , Cervix Uteri/virology , Colposcopy , Polymerase Chain Reaction , Risk Factors , Vaginal SmearsABSTRACT
A Reação em Cadeia da Polimerase (PCR Polymerase Chain Reaction) é uma técnica de amplificação enzimática de sequências específicas de ácidos nucléicos. Essa técnica tem sido amplamente descrita para detecção e tipagem do Papilomavírus Humano (HPV Human Papillomavirus). Neste trabalho, 460 amostras de colo uterino foram avaliadas para a presença do DNA do HPV pela técnica de PCR. Amostras positivas foram subsequentemente tipadas por RFLP (Restriction Fragment Polymorphism). Os resultados de PCR-RFLP foram comparados com os exames colposcópico e citopatológico (Papanicolaou). O PCR-RFLP demonstrou ser uma técnica eficaz na detecção e tipagem virais, apresentando maior sensibilidade do que os exames citopatológico e colposcópico.