Team:JNFLS

Welcome to Team JNFLS' iGEM 2018 Wiki!

Our Motivation

In the past 3 years, there are 3.4 million persons donated their blood in Shandong province. All the donors negative blood tested by ELISA , were tested again by qPCR method. The positive rate of HIV is 0.002%，its about 20 persons per year . However, the positive rate of HCV is 0.which reminds us that the HCV detection methods are deficient. So we designed our project.In the past 3 years, there are 3.4 million persons donated their blood in Shandong province. All the donors negative blood tested by ELISA , were tested again by qPCR method. The positive rate of HIV is 0.002%，its about 20 persons per year . However, the positive rate of HCV is 0.which reminds us that the HCV detection methods are deficient. So we designed our project.

Abstract

Development of a biosensor for detecting HCV C antigen by the nucleic acid aptamer. In the blood test of blood donation without compensation, the antibodies and RNA detection are usually used for HCV, but these detections are insufficient. Nucleic acid aptamer has been widely used for its specificity. In this project, the nucleic acid aptamer is used to detect HCV antigen, and the specific detection of trace HCV antigen could be realized by the signal amplification, which has great significance to shorten the window period in clinic transfusion. HCV C gene is subcloned from pCMV CE1E2, respectively, into pCold II plasmid and transformed into E. Coli. Therefore, C protein can be collected in the supernatant liquid of E.Coli. At the same time, the ssDNA aptamer library is constructed. The length of nucleotide insertion is 40nt, and the storage capacity is about 106. HCV C protein is used to screen the nucleic acid aptamer specifically bound to HCV C protein by SELEX technology. Using the competing reaction of the target antigen, the adapter sequence, padlock probe and complementary sequence of the adapter, a highly sensitive fluorescent adapter sensor is developed based on the rolling circle replication. When there is no target antigen, the complementary sequence binders with aptamer probe instead of the padlock probe, which triggers rolling circle amplification reaction. Whereas when the aptamer-probe binds with the target antigen, the complementary sequence hybridizes with the padlock probe. Under the action of DNA ligase, the padlock probe is further cyclized and a rolling circle amplification occurs under the action of DNA polymerase. By designing different aptamer sequences and related nucleic acid sequences, the sensing system can be used as a general method to detect another targets antigen.