Comparação da taxa de detecção do DNA do EBV em amostras de saliva total e células exfoliadas de pacientes com leucoplasia bucal através da nPCR

Abstract

The aim of this essay was to compare the detection rate of Epstein-Barr virus (EBV) DNA in total saliva samples with samples of exfoliated cells, obtained through the use of cytobrush of patients with oral leukoplakia, through n-PCR. In the laboratory phase, the DNA was extracted from the samples according to the instructions of the DNA extraction Kit QIAamp® DNA mini kit and all samples were submitted to spectrophotometry to verify presence and integrity of the DNA. Subsequently the PCRs were performed for β-globin, with primers PCO3 and PCO4 (260pb). To verify the presence of human DNA, an analysis was performed using 2% agarose gel electrophoresis. n-PCR was performed with initiator oligonucleotides for the EBV's DNA. All samples were tested in duplicate and the n-PCR products were subjected to 8% polyacrylamide gel electrophoresis. It was verified the existence of association between variables of both groups studied by applying statistical tests (Chi-square or Fisher's exact Test) with a significance level of 5%. Saliva and exfoliated cells samples collected in the laboratory phase for DNA extraction and PCR for β-globin, which was positive for all samples, showing a good quality DNA. EBV's reaction standardization revealed that 80% of the samples (24 patients) of cytobrush were positive for the presence of the virus. In this analysis the prevalence was in males who presented positivity in 84% of cases (16 patients). As for the analysis of the saliva samples, the positivity was 77% (23 patients) also maintaining the prevalence of male with 69.5% of cases (16 patients). However the research showed no statistical significance in the comparison of the detection rate of Epstein-Barr virus (EBV) DNA in samples evaluated of patients with oral leukoplakia

O objetivo deste trabalho foi comparar a taxa de detecção do DNA do Epstein Barr vírus (EBV) em amostras de saliva total e células exfoliadas, em 30 pacientes com leucoplasia bucal, através da nPCR. Na fase laboratorial realizou-se a extração de DNA das amostras segundo as instruções do Kit de extração de DNA QIAamp® DNA mini kit e todas as amostras foram submetidas à espectrofotometria para verificação da presença e integridade do DNA. Posteriormente utilizou-se as PCRs para β-globina, com os primers PCO3 e PCO4 (260pb), seguindo com a análise através da eletroforese em gel de agarose 2% para a verificação da presença do DNA humano. Foi executada nPCR com os ologonucleotídeos iniciadores para o DNA do EBV. Todas as amostras foram testadas em duplicata e os produtos da nPCR submetidos à eletroforese em gel de poliacrilamida 8%. Utilizou-se os testes estatísticos (Qui-quadrado ou Teste exato de Fisher) com nível de significância de 5%. Obteve-se como resultado que 80% das amostras (24 pacientes) de citobrush foram positivas para a presença do EBV. Das amostras positivas, 84% eram homens, com média de idade de 59 anos. Já na análise quanto as amostras de saliva, a positividade foi de 77% (23 pacientes) mantendo também a prevalência do gênero masculino com 69,5% dos casos (16 pacientes). No entanto a pesquisa não apresentou significância estatística na comparação da taxa de detecção do DNA do Epstein-Barr vírus nas amostras avaliadas de pacientes com leucoplasia bucal