The Establishment Of The Norovirus Fluorescence Quantitative Detection Method And The Exploratory Researches On Infectious Clones Of Two Viruses

Objective:The object of this study is to develop a duplex fluorescent quantitative one-step RT-PCR assay for detection and quantitation of GI and GIl norovirus,so that we can complete quantitative and typing at the same time.And to develop a fluorescent quantitative one-step RT-PCR assay for detection and quantitation of GIV norovirus. In addition, we obtained the cloning of the complete genome of a strain of norovirus by use of the application of molecular biology techniques, especially the research method of noroviruses belonging to other members. The transfection effect of the full genomic clone transcripts was evaluated by the real-time detection method and relative quantitative method. We hope to provid some theoretical and technical information for the study of human norovirus.We obtained the cloning of the complete genome of a strain of rhinovirus C by use of the application of molecular biology techniques. The transfection effect of the full genomic clone transcripts was evaluated by the real-time detection method and relative quantitative method. We hope to provid some theoretical and technical information for the study of rhinovirus C.Methods:1. Established a duplex fluorescent quantitative one-step RT-PCR assay for detection and quantitation of norovirus genogroup GI and GllWe develop a duplex fluorescent quantitative one-step RT-PCR assay for detection and quantitation of GI and Gll norovirus by optimizing the reaction system and conditions using the specific primers for norovirus and taqman probe. We constructed the RNA standard of this assay. The sensitivity, specificity, repeatability of the method was assessed. The traditional RT-PCR method was used to evaluate the quantitative fluorescence method through the detection to the same fecal specimens by the traditional RT-PCR method and the real-time RT-PCR.2. Established a fluorescent quantitative one-step RT-PCR assay for detection and quantitation of norovirus genogroup GIVWe develop a duplex fluorescent quantitative one-step RT-PCR assay for detection and quantitation of GIV norovirus by optimizing the reaction system and conditions using the specific primers for norovirus and taqman probe. We also constructed the RNA standard of this assay. The specificity, repeatability of the quantitative one-step RT-PCR assay for detection and quantitation of Norovirus Gl and Gll and the fluorescent quantitative one-step RT-PCR assay for detection and quantitation of Norovirus GIV are well. The methods have high sensitivity and good specificity, can be applied to detecting gastroenteritis patients.This study conducted a preliminary exploration of two virus that difficult to cultivate. Finally we found the increase of viral nucleic acid of the norovirus and rhinovirus C. We also found the cytopathic effect of the transfection cells. We hope to provid some theoretical and technical information for the future study of norovirus and rhinovirus C.