XÂY DỰNG KỸ THUẬT QPCR DÙNG CHẤT HUỲNH QUANG SYBR GREEN ĐỊNH LƯỢNG DNA EBV

Abstract

Introduction: Quantification of high levels of EBV DNA in plasma can be considered an important marker of primary disease at different times during treatment. Currently, qPCR technique has a high level of sensitivity for quantitatively targeting EBV DNA and can be commonly applied in clinical practice. Research has been conducted to develop a qPCR technical process using SYBR DNA fluorescence to quantify EBV, which would increase options for clinicians willing to apply it appropriately to the target application and economic conditions. Objective: Establishment of SYBR GREEN-BASED qPCR to help quantitative Epstein – Barr Viruss. Method: Construction of a plasmid containing the BamHI-W gene fragment of EBV by recombinant DNA technique. Optimization of qPCR reaction using SYBR GREEN fluorescence quantification of EBV DNA. Reaction conditions related to reaction volume, primer, concentrations were reduced compared to the recommended. Applying the qPCR technical process just built on the laboratory simulation sample, recording the technical criteria of the qPCR reaction, accuracy, sensitivity, and specificity. Result: Successfully generated a plasmid containing the BamHI-W gene fragment of EBV identified by Sanger decoding. Successfully built a qPCR technique using SYBR GREEN fluorescent agent, including a qPCR reaction at a reaction volume of 10 µL with primer concentrations 500nM which was contrary to the technical specifications of the reaction. The PCR reaction had high accuracy with one-day and three-day CVs of less than 11%, an E% response efficiency of 95,67%, a value slope of -3.432, and a correlation coefficient R² of 0,999. The limit of detection (LOD) of qPCR was 2.10⁴ copies/ µL. In a simulated test on a definite precision recorded plasmid sample, the sensitivity and specificity were 100%. Conclusion: Successfully built a qPCR technical process using SYBR Green fluorescence to quantify EBV DNA.