Navegando por Palavras-chave "Polymerase Chain Reaction"
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- ItemAcesso aberto (Open Access)Análise da frequência, viabilidade e caracterização genética do Toxoplasma gondii em diversos tipos de amostras provenientes de diferentes regiões do Brasil(Universidade Federal de São Paulo (UNIFESP), 2020-11-26) Costa, Deise Fialho Da [UNIFESP]; Mattos Junior, Rubens Belfort [UNIFESP]; Universidade Federal de São PauloObjective: The goal of this study was to determine the frequency of Toxoplasma gondii (T. gondii) DNA in retinas from eye banks, fresh sausage, cured salami, and pork heart samples. The viability and genetic characteristics of T. gondii strains in pork heart samples was also analyzed. Methods: A total of 162 eyes were collected from eye banks of Manaus (n=60), São Paulo (n=60), Chapecó (n=26), and Joinville (n=16). The samples of 118 sausages (n=59) and salami (n=59) were collected from 8 different producers from Rio Grande do Sul; and 35 fresh pork heart samples were collected at a slaughterhouse in Erechim city. The retinas were analyzed macroscopically, collected, and DNA was extracted using the QIAamp DNA Blood Mini kit (Qiagen, Valencia, CA, USA), and the quantitative polymerase chain reaction (qPCR) was performed to identify T. gondii using B1 marker. After collection, sausage and salami samples were cut into small pieces, DNA was extracted using the DNeasy Mericon Food kit (Qiagen, Valencia, CA, USA), and the qPCR was performed using B1 marker to detect T. gondii. Parasite quantification for each sample was performed. In regard to pork hearts, DNA was extracted using the DNeasy Mericon Food kit (Qiagen, Valencia, CA, USA), and the qPCR was performed using B1 marker. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for genotyping the strain of T. gondii. Positive pork heart samples were digested and inoculated in mice for viability analysis. Results: Our study has shown a higher frequency of T. gondii DNA in the retinas from Joinville (25%) when compared to Manaus (5%). The retinas from São Paulo and Chapecó were qPCR negative for T. gondii infection. Macroscopic analysis showed retinal lesions compatible with toxoplasmosis in the following frequencies: Joinville (62.5%), Manaus (10%), São Paulo (6.7%), and Chapecó (15.4%). The frequency of T. gondii DNA among the total number of sausage and salami samples was 39% (46/118). Among these, we observed a higher frequency of positivity in the sausage samples (47.5%) when compared to the salami samples (17%). However, the mean parasite concentration was significantly higher in the salami samples (p=0.006). The results related to pork hearts showed that T. gondii DNA was detected in 25.7% (9/35) of the samples. The genotyping analysis revealed a new atypical T. gondii strain, assigned as TgPkErBra, in a sample of a pork heart. Out of 9 samples which were qPCR positive, 5 of them were intraperitoneally inoculated in mice and we observed that 4 of 10 mice (40%) presented clinical signs of T. gondii infection. Among 4 mice, 3 were qPCR positive for T. gondii in the lung, 1 in the liver, and 2 in the brain. Also, the histopathology analysis showed retinal disorganization, retinal detachment, inflammatory cell infiltration and fibrosis in 4 of 5 eyes analyzed. Conclusions: These studies confirmed the high frequency of toxoplasmic DNA presented in the retinas from eye banks, and in fresh and processed pork meats from Southern Brazil. Besides, the presence of T. gondii DNA in pork meat can contain live organisms and can be an important source of infections and a public health risk to be considered. Our results also demonstrate that pork meat can contain live T. gondii, and this result can be associated with the high frequency of toxoplasmosis in Southern Brazil. Furthermore, a new strain of T. gondii was found circulating in the same region.
- ItemSomente MetadadadosAplicação da reação em cadeia da polimerase em estudos epidemiológicos da infecção pelo citomegalovirus humano em diferentes populações pediátricas(Universidade Federal de São Paulo (UNIFESP), 1999) Souza, Inara Espinelli Lemes de [UNIFESP]; Pignatari, Antonio Carlos Campos [UNIFESP]A implementacao de medidas adequadas de prevencao e controle de disseminacao de doencas infecciosas exige um conhecimento amplo de diversos aspectos epidemiologicos do microrganismo responsavel pelo processo infeccioso. Essa caracterizacao epidemiologica, por outro lado, exige a utilizacao de tecnicas e metodologias, muitas vezes especificas para cada microrganismo ou situacao epidemiologica, que sejam capazes de agrupar isolados relacionados epidemiologicamente e diferenciar aqueles nao relacionados. Este trabalho teve como objetivo avaliar o desempenho de uma metodologia, baseada na tecnica da reacao em cadeia da polimerase (PCR), em estudos epidemiologicos da infeccao pelo citomegalovirus (CMV). Para isso, a tecnica foi desafiada em populacoes pediatricas e situacoes distintas, e continuamente aperfeicoada para responder as questoes epidemiologicas necessarias para uma melhor abordagem da transmissao do CNW em criancas. No primeiro estudo a metodologia foi utilizada para caracterizacao de cepas de CMV isoladas de pacientes pediatricos submetidos a transplante de medula ossea. Os isolados de um mesmo paciente apresentaram padroes identicos enquanto isolados de diferentes pacientes apresentaram padroes distintos. Alem de mostrar que essa populacao geralmente excreta a mesma cepa por longos periodos e que a transmissao paciente-paciente e baixa, a tecnica mostrou reprodutibilidade e poder discriminatorio ao agrupar isolados relacionados epidemiologicamente e diferenciar os nao relacionados, nessa populacao. No segundo estudo a metodologia foi aperfeicoada para ser desafiada em uma situacao epidemologica mais complexa. A regiao genomica do CMV estudada foi ampliada para se aumentar o poder discriminatorio do metodo. Este estudo mostrou que membros de uma mesma familia excretaram cepas identicas, enquanto que cepas de diferentes familias eram distintas. No terceiro estudo uma situacao ainda mais complexa foi avaliada: transmissao de uma crianca para outra (horizontal) em creches. Foram avaliadas amostras seriadas de 37 criancas (dois a seis isolados por crianca). O maior aperfeicoamento da tecnica permitiu demonstrar a reinfeccao por cepas distintas e a transmissao horizontal nessa populacao. O quarto estudo envolveu uma populacao bem maior e avaliou a diversidade geneticas de cepas excretadas por recem-nascidos (RNs) com infeccao congenita de uma determinada regiao. Os resultados obtidos sugeriram a existencia de uma relacao entre 91...(au)
- ItemAcesso aberto (Open Access)Avaliação da expressão tecidual do gene de reparo MLH1 e dos níveis de dano oxidativo ao DNA em doentes com câncer colorretal(Cidade Editora Científica Ltda, 2009-09-01) Martinez, Carlos Augusto Real; Cordeiro, Adriana Teixeira; Priolli, Denise Gonçalves; Miranda, Daniel Duarte Da Conceição; Bartchewsky Júnior, Waldemar [UNIFESP]; Margarido, Nelson Fontana [UNIFESP]; Ribeiro, Marcelo Lima; Universidade São Francisco; Universidade São Francisco Hospital Universitário São Francisco Serviço de Cirurgia Geral; Universidade Federal de São Paulo (UNIFESP)The oxidative DNA damage caused by oxygen free radicals is one of the most important mechanisms responsible for the initial steps of colorectal carcinogenesis. The oxidative stress can cause errors in the pairing of nitrogenous bases that form the DNA, allowing mutations in controlling genes of the cell cycle. The cells have a defense system represented by the DNA mismatch repair genes that correct the errors of matching prevent the development of DNA mutations. Few studies have evaluated the relationship between oxidative DNA damage and the tissue expression of mismatch repair genes. AIM: The aim of the present study was evaluate the levels of oxidative DNA and the tissue expression of MLH1 mismatch repair gene in the cells of normal and neoplastic colonic mucosa of patients with colorectal cancer. MATERIAL AND METHODS: Were studied 44 patients with diagnosis of colorectal adenocarcinoma. Were excluded patients with hereditary colorectal cancer, with colorectal cancer associate with inflammatory bowel diseases and those undergoing neoadjuvant radioquimiotherapy. To evaluate the levels of oxidative DNA damage was used the single cell gel electrophoresis (comet assay) evaluating 100 cells obtained from normal and neoplastic tissues. For the evaluation of the tissue expression of MLH1 gene was employed the technique of polymerase chain reaction in real time (RT-PCR) with primer specifically designed for MLH1 gene. The comparison among the levels of DNA oxidative stress and expression of MLH1 mismatch repair gene in normal and neoplastic tissues was done by Student t test adopting a significance level of 5% (p< 0.05). RESULTS: The levels of oxidative DNA damage in tumor tissue were significantly higher when compared to the level of the normal tissue (p = 0.0001). The tissue expression of MLH1 mismatch repair gene in tumor tissue was significantly lower when compared to normal tissue (p=0.02). CONCLUSION: The mismatch repair gene MLH1 are less expressed in tumor tissue and inversely related to levels of oxidative DNA damage.
- ItemSomente MetadadadosDetecção do citomegalovirus humano em leucócitos de pacientes imunocomprometidos pela técnica de reação em cadeia da polimerase(Universidade Federal de São Paulo (UNIFESP), 1995) Souza, Inara Espinelli Lemes de [UNIFESP]; Granato, Celso Francisco Hernandes [UNIFESP]
- ItemSomente MetadadadosIdentificação de região correspondente ao gene determinante do sexo no cromossomo Y (SRY) em pacientes com síndrome de Turner através de reação em cadeia por polimerase(Universidade Federal de São Paulo (UNIFESP), 1996) Strufaldi, Maria Wany Louzada [UNIFESP]; Verreschi, Ieda Therezinha do Nascimento [UNIFESP]
- ItemSomente MetadadadosMarcadores moleculares do cromossomo Y em DNA de mucosa oral e sangue periferico de pacientes com Síndrome de Turner(Universidade Federal de São Paulo (UNIFESP), 2020-08-05) Barbosa, Lene Garcia [UNIFESP]; Castro, Angela Maria Spinola E [UNIFESP]; Universidade Federal de São PauloConsidering the high percentage of mosaicism and the possible clinical impact of the presence of fragments of the Y chromosome in patients with Turner Syndrome, we proposed in this study to develop a multiplex polymerase chain reaction (mPCR) to evaluate the presence of these fragments in two tissues with origin different embryonic cells, such as peripheral blood lymphocytes (mesoderm) and oral mucosa cells (ectoderm). DNA samples were collected from 109 patients. Primers for mPCR were built with the help of the MPprimer program to assess possible interactions between them. Two genes present on the Y chromosome and one on the X were used. The amplified samples were evaluated by conventional electrophoresis on 1.8% agarose gel and subsequently by capillary electrophoresis. We found 14 patients (12.8%) with positive molecular markers for Y chromosome, both in the blood and in the oral mucosa swab. The karyotype showed a sensitivity of 95.8%, but a specificity of 28.6% in relation to the molecular test. In conclusion, the mPCR protocol established in this study, combined with capillary electrophoresis, proved to be efficient for identifying Y fragments and must be used in the outpatient routine. The study of tissues of different embryological origin, such as blood and oral tissue obtained the same degree of agreement, as well as the same sensitivity and specificity.
- ItemAcesso aberto (Open Access)Otimização de técnica de PCR em tempo real para detecção das regiões pol e env dos subtipos B e F de HIV-1 e triagem de seus recombinantes(Universidade Federal de São Paulo (UNIFESP), 2009-01-28) Teixeira, Daniela [UNIFESP]; Diaz, Ricardo Sobhie [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Background: The discovery of 42 HIV-1 circulating recombinant forms (CRF) together with the innumerous unique HIV recombinants forms, makes clear the role of genetic recombination for the epidemic. In Brazil, clades B, F, and C co-circulate, with 5 recently described CRFs. Real Time PCR is a rapid and reliable tool capable of detecting different HIV-1 subtypes and recombinant profiles. Objective: The aim of this study was to develop real time PCR systems in order to detect the Brazilian CRF_28 and CRF_29, which are B/F recombinants, as well as detect B/F recombinants generated by in vitro competition assays. In future, these systems should be able to discriminate subtypes in clinical surveys. Methodology: MT-4 cells were separately infected with the viral strains BZ167 (subtype B) and BR020 (subtype F), and supernatant was collected in order to optimizing the real time PCR systems (TaqMan®) developed to detect the subtype profile of different genomic regions, including pol gene (protease, reverse transcriptase, integrase) and env gene (gp120 and gp41). The designed primers should be able to equally amplify the subtype B and F, which should be discriminated by subtype-specific probes. For future validation of these PCR systems, 157 clinical samples from the city of Santos were sequenced and phylogeneticaly analyzed in order to perform the clade assignment with Neighbor-Joining algorithm (Phylip software package v3.5). Results: The designed systems were able to differentiate the utilized viral strains. The estimated efficiencies for each system, for each probe, subtypes B and F separately, were respectively: 80,97 and 85,16% for protease region; 89,80 and 75,09% for reverse transcriptase region; 80,90% and 83,83% for integrase region; 93,49 and 98,93% for gp120 region; and 88,45 and 80,19% for gp41 region. For the co-infected cell culture, the detection of each subtype was performed in the first and fifth passages. Generally, the initial concentration of subtype B appeared to have decreased, some of them becoming undetectable, whereas subtype F seemed to increase with the passages, for protease, reverse transcriptase, gp120 and gp41 regions. The integrase region was an exception, since only the subtype B was detected, with increasing Cycle threshold (Ct) values over time. Sequencing results revealed that 65 out of 157 samples had the subtype profile defined for all regions. 43 out of 71 were defined as B, whereas 3 were F in all regions. 12 samples presented the CRF_28 profile, and 9 samples presented the CRF_29 profile. There were no subtype C samples in any genomic regions analyzed. Conclusion: The use of real time PCR technique for identification of fragments’ subtypes in cell culture and for evaluation of replicative dynamics of recombination in co-infected cultures warrants its potential use in future in vivo surveys. This methodology proved to be efficient, fast, less cumbersome and less expensive than DNA sequencing. The newly designed systems performed for supernatant of competition assays had suggested a divergent distribution of subtypes for the different regions, which reflects the possibility of genetic recombination. Results from clinical samples revealed a high prevalence of CRF_28/29 in this geographic region, thus reflecting the resulting consequence of different co-circulating strains and pointing to the need for a careful surveillance.