Research On Dengue Virus Identification And Cytokine MRNA Levels In Patients

ObjectiveUsing high-resolution melting (HRM) analysis technique, for rapid and accurate identification of four dengue virus (DEV) and make up for the lack of existing classification methods; using quantitative polymerase chain reaction (PCR) to detect interleukin 12 (IL-12), interleukin 10 (IL-10), interleukin 6 (IL-6), interferon gamma (IFN-γ), tumor necrosis factor a (TNF-a) mRNA expression levels in dengue patients, in order to clarify the body’s innate and adaptive immunity during dengue virus infection.Methods1. Design HRM genotyping primers according to the sequences published by NCBI to identify different types of dengue virus, the sensitivity and specificity experiment was also carried out, at the same time, using sequencing, fluorescence PCR and conventional PCR methods to verify the results identified by HRM.2.21 cases of dengue fever patients and 21 cases of healthy controls venous blood were collected, using the comparative CT relative quantitative methodOto detect IL-12, IL-10, IL-6, IFN-Y and TNF-a mRNA expression levels, analysis related cytokine mRNA changes in patients with dengue fever and the differences between healthy controls and dengue fever patients.Results1. By using HRM analysis technology, we can identify four types of dengue virus accurately with high sensitivity and specificity. HRM genotyping and sequencing, fluorescence PCR and conventional PCR genotyping results is tantamount.2. Compared with the healthy control group, IL-12、IL-10、IFN-γ、TNF-α mRNA expression levels were significantly increased in dengue patients group with a significant difference(P<0.01,0.05,0.01,0.05); while IL-6 mRNA expression level was lower than healthy control group, but there was no significant difference (P>0.05).Conclusions1. This study established the dengue virus PCR-HRM genotyping method, compared with the sequencing, fluorescence PCR and general PCR, this method provides a more rapid, accurate, low-cost detection method for the molecular diagnosis of dengue virus.2. Dengue virus (DEV) infection can lead to a patient’s five kinds of cytokines mRNA expression level changes including IL-12, IL-10, IL-6, IFN-y and TNF-a these cytokines may play a role in the pathogenesis of dengue fever and immune regulation.3. Thl/Th2 cytokine imbalance may play an important role in the process of infection and disease transformation in dengue fever patients. The increase of IL-12 mRNA in dengue fever patients can promote the secretion of Thl cytokines and inhibit the secretion of Th2 cytokines, and play the advantages of Thl immune response.