Development Of Real-time PCR Assays For Three Pediatric Common Viruses And Their Clinical Applications

Children with respiratory infections and intestinal infections are the two most common diseases in pediatric infectious diseases. The over80%of the disease belong to viral infection. In the viral infection, respiratory syncytial virus and group C adenovirus are the most common pathogens during respiratory infection, which can cause severe bronchiolitis and pneumonia;At the same time, Group F adenovirus and rotavirus are the common pathogens distributing and popularing throughout the world, which can cause diarrhea in children. Therefore, there is a great significance in the prevention and control of pediatric communicable diseases to monitor and detect the main pathogens.Since1996, Applied Biosystems company has proposed the real-time PCR technology,which has widely been used in basic research and pathogen detection. Real-time PCR technology can be used to measure the initial template and have good specificity, high sensitivity, easily operated, monitored at any time, making her become a important tool for nucleic acid detectionan which can not be replaced. Complex probe is a new technology which based on real-time PCR.The technology takes advantages of real-time PCR, at the same time, which is efficient, fast, low background interference and low cost to provide a reliable and effective detection methods for the detection of pathogenic microorganisms. This paper is based on the real-time PCR technology to develop the efficient and sensitive real-time PCR detection methods for the pediatric infectious diseases including respiratory syncytial virus, adenovirus and rotavirus. Hope to achieve fast and accurate diagnosis and furtherly expand monitoring for the pediatric infectious diseases and provide reliable and effective method for epidemiological studies。 Based on the TaqMan probe, composite probe real-time PCR primers and probes design principles, specific primers and probes were designed to filter out the best primer probe sets by comparing the experiment; hot start Taq polymerase for PCR amplification system amount, the amount of reverse transcriptase, reverse transcriptase reaction time PCR annealing temperature of the reaction system and the reaction conditions were optimized to establish the best real-time PCR detection method; and establish quantitative PCR sensitivity, specificity and precision was evaluated; simultaneously established method for clinical fever, pneumonia and diarrhea in infants and young children patients sputum, stool specimens, testing, to evaluate the effect of this method of detection.Optimized, established respiratory syncytial virus, adenovirus, rotavirus reaction system and the reaction conditions; respiratory syncytial virus the detection sensitivity RSV A type low as2.0X102copies/mL, RSV B low as5.0×102copies/ml.; There is no cross-reactivity with non-respiratory syncytial virus and other common respiratory viruses; inter-assay coefficient of variation were less than5%; respiratory syncytial virus infection in patients with throat swab specimens test results showed that:The clinical findings coincidence rate was99.23%. The detection sensitivity of the adenovirus type ADV as low as2.0×101PFU/ml the the ADV40type low as5.0×101PFU/ml; There was no cross-reactivity with common respiratory virus, adenovirus, enterovirus; batch intra-The coefficients of variation were less than5%; patients with adenovirus infection throat swab, stool specimens test results showed that:the method and clinical diagnostic accordance rate was99.14%,99.09%. Rotavirus detection sensitivity RV SA-11standard strains minimum up to5.0×101PFU/ml; There was no cross-reactivity with non-rotavirus and other common intestinal virus; between-batch coefficients of variation were less than5%; from The test results showed that three hospitals stool specimens of patients with rotavirus infection: the method and clinical diagnostic accordance rate was98.63%,99.17%and99.14%, respectively.The developed real-time PCR detection method can quickly and accurately, specificity, high sensitivity qualitative analysis of three children viral nucleic acid, providing new and more effective methods for clinical detection.