| Background: The association of Epstein-Barr virus (EBV) with Nasopharyngeal carcinoma (NPC) has been known for a long time, though the exact role of Epstein-Barr virus in this cancer is not well understood. The result of in situ hybridization has revealed that EBV-DNA is present in almost all of the biopsies of NPC tumors. Further more, recent research suggested that the latency of EBV-DNA will induce the undifferentiation of epithelial cells of nasopharyn and promote the reproduction of the malignant cells. Many current methods for the detection of EBV are based on serum immunology, but serum antibodies against EBV is an indirect sign with low specificity for the diagnosis of NPC. Pathologic examination and in situ hybridzation of EBV-DNA and EBV-RNA in tissue sections allows direct and exact visualization of infected cells by microscop field, however, sample collection for these procedures may cause injury to the focus ,which may add distress to the patients. And that,these procedures are also time consuming. Resent research has showed that there is strong correlation between the quantification of EBV-DNA and the development of undifferentiated cells. Direct and specific method for the detection of EBV in a high-risk population will be an important tool for the screen of NPC.In this research DNA in the cells of nasopharyngeal brush biopsies was used as sample. One set of primers and prob was designed to amplify the target regions in the Epstein-Barr nuclear antigen(EBNA1)and a second set of primers and prob designed to simultaneously amplify DNA segment of human B -globin which served as an internal control. The amplification system was optimized to amplify the two segments in seperated tubes. And then, each sample got a CtE and Ct B, the value of CtE/Ct B is corresponsive with the quantity of EBV-DNA of unit cell and represents the quantitation of Epstein-Barr virus in focus(partial area).Objectives:To establish a Fluorescence quantitative PCR DNA amplification system to quantify EBV-DNA of unit cell in the specimens nasopharyngeal carcinoma brush biopsies.Method:1.Sample collection: Samples were collected by otorhinolaryngologist with aseptic instruments.2.DNA extraction: Brush biopsies samples were collected in standard tubes containing saline. Following centrifugation at 6000 x g, cells samples were collected. The cells samples were transferred to Eppendorf tube, incubated in splitting solution with proteinase K at 56 C for six hours and were then extracted with phenol-chloroform; this was followed by ethanol precipation. The extracted DNA was placed in TE solution and stored at -70 C, which was used for PCR.3.Primers and Probs: One set of primers and prob was designed to amplify the target regions in the Epstein-Barr nuclear antigen(EBNA1) and a second set of primers and prob designed to simultaneously amplify DNA segment of human B -globin which served as an internal control. Target segment (262bp): prmers:5'-GTA GAA GGC CAT TTT TCC AC-3'and 5'-TTT CTA CGT GAC TCC TAG CC-3', prob: 5'-ACC ACC GTG GCC CAG ATGG-3'; Internal control(260bp): primers GH20:5'-GAA GAG CCA AGG ACA GGT AC-3'and PCO4: 5'-CAA CTT CAT CCA CGT TCA CC-3', prob: 5'-ACT GTG AGC AAC CTC AAAC-3',4. Optimization of PCR reaction solution: With equivalent templet in each reaction tube, a strategy was designed to optimize the buffer(ionic strength) and MgCL2concentrations with fixed annealing temperature 55C,which includes two factors with three levels each. Duplicated test was done with a alteration of annealing temperature to 45 C . From the result of above tests, optimal reaction solution was obtained.5. Stability tests for FQ- PCR amplification system: To observe the stability of the FQ-PCR amplification system, five duplicated tests to amplify target EBNA1-DNA segment and internal central (3 -globin-DNA segment simultaneously were performed.6. Using stable FQ-PCR amplification system for the quantificaton of EBV-DNA in 29 samples.Result:1. Optimization of PCR reaction soluti...
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