Application Of In Situ Rolling Circle Amplification Based On Immune Nucleic Acid Probe In Cell Sorting

Objective:Rolling Circle Amplification(RCA)technique which developed in the mid-1990 s is an isothermal enzymatic process that uses a strand displacement polymerase and a circular DNA template to amplify the target DNA sequence with high fidelity and high specificity.The product is a repetitive long single strand complementary to the template sequence.RCA technique has been used to develop sensitive diagnostic methods for various targets,including nucleic acids,proteins and cells,and has also been used in nanotechnology and nanobiotechnology.The detection of some special biomarkers based on RCA technique can achieve the purpose of cell labeling and detection,which can be applied to the diagnosis of major diseases in clinical practice.For example,high-efficiency fluorescent labeling of human immunodeficiency virus(HIV)reservoir cells,and then single-cell sorting and counting of cells by hot-bubble inkjet printing technology,provide detection technology for functional cure of AIDS,for example,the detection of circulating tumor cells(CTCs)can achieve diagnosis of malignant tumors and early screening.This technology does not rely on complex operations,expensive instruments,and both sensitivity and specificity.It is a powerful tool for low-abundance cell detection.Therefore,in this experiment,the primers in the RCA technique were modified to improve the sensitivity and specificity of the RCA technique as the breakthrough point,and the immune nucleic acid probe that can simultaneously recognize and bind proteins and initiate RCA was constructed by using the immune reaction between antigen and antibody,and the high efficiency and high specificity of cell fluorescence labeling technology based on RCA technique was developed to reserve the method for low abundance cell detection.Methods:Firstly,a circular template is prepared by using a padlock probe in RCA technique.The long single strand was cyclized by T4 DNA ligase and purified by exonuclease III.Secondly,an immune nucleic acid probe was constructed.The primers of RCA were ligated with the secondary antibody by the specific binding of biotin and streptavidin.Finally,an immune nucleic acid probe that can trigger RCA and specifically recognize the primary antibody was formed.Then,it is necessary to design and screen molecular beacons that emit fluorescence signals after specifically binding to products that can be amplified by rolling circle.The expression difference of P-g between A2780 and A2780T was verified by RT-PCR and Western Blot.After confirming that P-gp was highly expressed in A2780 T,the membrane protein was verified by immunofluorescence.Finally,the cell climbing film was made,and the above circular template and immune nucleic acid probe were used to trigger the RCA on the surface of A2780 and A2780 T cells.The molecular beacon was combined with the rolling circle amplification product,and the drug-resistant and sensitive cells in the presence or absence of fluorescence signal were detected by laser confocal.Results:1.The ring template constructed by the padlock probe was successfully connected and purified,which can be used as a template for the experiment.2.The positive primers modified with biotin were screened by using the above template,and the connection conditions were optimized to ensure the connection efficiency of biotin-modified nucleic acid and streptavidin-modified secondary antibody.The required immune nucleic acid probe was obtained by ultrafiltration of the connection product,and the probe was verified by polypropylene gel electrophoresis and rolling circle amplification.3.RT-PCR and WB experiments verified that P-gp was highly expressed in A2780 T and hardly expressed in A2780,and the protein was verified as a membrane protein by immunofluorescence.4.A2780 T was successfully labeled by using the above template and probe to trigger the in situ RCA of cells,while A2780 was not labeled,indicating that this experimental method can distinguish A2780 and A2780 T.Conclusions:This experiment successfully constructed a cell surface antigen detection method based on immune nucleic acid probe to initiate RCA,and realized the detection of model cells(A2780 and A2780T),which provided experimental data for the establishment of a new method for HIV reservoir cell detection.