Difference between revisions of "Team:JNFLS/Description"
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Revision as of 11:33, 10 October 2018
Development of a biosensor for detecting HCV antigen (C) by the nucleic acid aptamer.
window period：
window period is the time between first infection and when the test can reliably detect that infection. In antibody-based testing, the window period is dependent on the time that a specific antibody develops and becomes detectable in the blood.
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.
how did we do it：
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 cloned respectively into secretory eukaryotic expression vectors, transfected into eukaryotic cells, and collected secreted HCV virus-like particles (VLP). 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-VLP is used to screen the nucleic acid aptamer specifically bound to HCV-VLP 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.