Team:JNFLS/Description

Development of a biosensor for detecting HCV Core antigen 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.

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.

How does our device work：

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. The aptamer is originally bound with its partially complementary sequence; thus, it remains double-stranded before the detection. If target antigen, or say, HCV Core protein, exists, since the affinity between the aptamer and HCV Core protein is stronger than that between the aptamer and its complementary sequence, the aptamer will bind to the antigen and leave the complementary sequence in the reaction system. The complementary sequence will bind to the padlock probe which contains the complementary sequence on both sides of its gap, and “sew” the gap. Then, after the treatment of DNA ligase, the complementary-sequence-padlock-probe complex will be encircled. Next, under the function of DNA polymerase Phi29, the rolling circle amplification can initiate. After adding the quenching fluorescence group, the signal is able to be detected.

Reference:

[1]Li Shengtao. The expression and purification of the truncated HCV core protein (HCV Core125) and its antibody preparation [D]. Kunming University of technology,2012. [2]Wu Xianbo. Cloning, expression of a gene fragment encoding HCV core antigen and purification, antigenicity analysis of the recombinant protein [D]. First military medical university of the people's liberation army,2003. [3]Zhang Songbai, Zheng Liying, Hu Xia, Shen Guangyu, Liu Xuewen, Shen Guoli, Yu Ruqin. Highly sensitive fluorescent aptasensor for thrombin detection based on competition triggered rolling circle amplification [J]. Chinese Journal of Analytical Chemistry,2015,43(11):1688-1694. [4]Zhou hui. Studies on competitive mechanism triggered signal amplification based aptasensors [D]. Hunan University,2009. [5]Shi S, Yu X, Gao Y, et al. Inhibition of hepatitis C virus production by aptamers against the core protein[J]. Journal of Virology, 2014, 88(4): 1990-1999. [6]Dean F B, Nelson J R, Giesler T L, et al. Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multiply-primed rolling circle amplification[J]. Genome research, 2001, 11(6): 1095-1099. [7]Banér J, Nilsson M, Mendel-Hartvig M, et al. Signal amplification of padlock probes by rolling circle replication[J]. Nucleic acids research, 1998, 26(22): 5073-5078.