The Establishment Of Genotyping Q-PCR Multiple Detection System For Group A Rotavirus

Rotavirus is the main pathogens that cause human, mammalian and avian viral diarrheaand group A rotavirus is the leading cause of severe diarrhea in infants less than5yearsworldwide. According to the neutralization antigen activity of virus coat protein,P and Gdouble nomenclature is used for naming.Accurate identification of the rotavirus serum type is important in providing the basis fordevelopment and use of vaccine targeted the disease. The unknown template can be throughthe standard curve of real-time fluorescent quantitative PCR for analysis.This method isstrong in specificity, high in sensitivity, great in automation, repeatability, quantitative andaccurate,which become an important technology of molecular biology and moleculardiagnosis platform.This technology is widely used in genetic epidemiology diagnosis ofpathogens and tumors,therefore the Q-PCR technique is the ideal way to detect the presenceof rotavirus and its subordinate serotype.This experiment uses G1,G3and G9subtype RNA of group A RV as sample to set up asystem of multiple fluorescence quantitative PCR through optimizing design. The multiplesystem could complete a detecting reaction concluded the existence of the RV and itssubordinate serotype in a short time even within1to2hours. Fluorescence curve ofFAM,CY5and JOE can be presented in the same coordinate system at the same time throughABI7500. Firstly this experiment successfully build the G1+G9subtypes of doublefluorescence quantitative PCR system. Results show that the detection sensitivity of thedouble system reachs to10~3copy/ul amount of template, and the accuracy is very high withno cross influence in different subtypes in the system and fluorescence curve of FAM andJOE can be presented in the same coordinate system. Then on base of it,G1+G3+G9detectionsystem is built to identify the three subtypes. Fluorescence curve of FAM,CY5and JOE havegood repeatability with no cross influence in different subtypes,it provides experimental basisfor the future development of rotavirus detection kits.