Development In Detection Of Enterohemorrhagic Escherichia Coli O157 And H5 Subtype Avian Influenza Virus Using Real-time Fluorescence PCR

Recently, real-time polymerase chain reaction (RT-PCR) has been widely used in the research of molecular biology, especially in clinic diagnosis, because of its high specificity, sensitivity and without any further processing of the samples and opening the test tubes.This dissertation consists of two parts. The first part is the development of a new fluorescence probe - equal-length double-stranded fluorescence probe , also named"Y-shape"fluorescence probe. Based on the fluorescence probe technique and real-time PCR, we establish a diagnostic assay system, and use it in the detection of E. coli O157. The second part, we use the fluorescence probe technique to detect H5 subtype avian influenza virus.In the first part, we found the best equal-length double-stranded fluorescence probe combination from several different probe combinations after selecting and optimizing. The probe consists of two partially complementary equal-length oligonucleotides, one of which is labeled with a fluorophore and the other is labeled with a quencher. The strand labeled with the fluorophore (fluorescence strand) matches perfectly to the target sequence while the strand labeled with the quencher (quencher strand) is only partly complementary to the strand labeled with the fluorophore. Without the target sequence, both strands will anneal to form a duplex of"Y-shape"structure, which keeps the fluorophore and quencher in close proximity. Therefore, the fluorophore is quenched. After amplification of PCR reaction, the"fluorescent strand"hybridizes perfectly to the target sequence and the...