Difference between revisions of "Team:JNFLS/Description"

 
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<h1 style="text-align:center;color:#FFFFFF;background-color:#A9A9A9">Development of a biosensor for detecting HCV antigen (C) by the nucleic acid aptamer.
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<h3 style="text-align:center;color:#FFFFFF;background-color:#A9A9A9">Development of a biosensor for detecting HCV Core antigen by the nucleic acid aptamer.
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<h3 style="text-align;center;color:#FFFFFF;background-color:#A9A9A9">Window period：</h3>
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<h3 style="text-align;center;color:#FFFFFF;background-color:#A9A9A9">window period：</h3>
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<p>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.  
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<p>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.  
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<h3 style="text-align;center;color:#FFFFFF;background-color:#A9A9A9">Our Motivation：</h3>
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<h3 style="text-align;center;color:#FFFFFF;background-color:#A9A9A9">motivation：</h3>
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<p>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.
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<p>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.
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<h3 style="text-align;center;color:#FFFFFF;background-color:#A9A9A9">How does our device work：</h3>
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<p>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. </p>
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<img src="https://static.igem.org/mediawiki/2018/b/b3/T--JNFLS--ban.jpg"style="width:70%">
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<p>Reference:</p>
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<p>[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.
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[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.
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[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.
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[4]Zhou hui. Studies on competitive mechanism triggered signal amplification based aptasensors [D]. Hunan University,2009.
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[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.
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[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.
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[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.<p>
 
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<h3 style="text-align;center;color:#FFFFFF;background-color:#A9A9A9">how did we do it：</h3>
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<p>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.</p>
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Latest revision as of 03:58, 18 October 2018





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.