A nucleic acid aptamer for detecting human bladder transitional cell carcinoma cells and its application in the preparation of detection preparations
A nucleic acid aptamer, transitional cell technology, applied in measurement devices, biochemical equipment and methods, instruments, etc., can solve the problem of atypical or degenerative cells with a small number of cancer cells, cannot rule out low-grade urothelial cancer, Low sensitivity of bladder cancer, etc., to achieve the effect of short cycle, easy labeling and good reproducibility
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Embodiment 1
[0027] Example 1: High-throughput detection and data analysis
[0028] After repeated verification, it was determined that the library with the strongest binding ability screened for another cancer cell line had a strong specific binding to the T24 cell line. We sent the library to the Sangon Sequencing Department, using Illumina Miseq or Hiseq2000 The platform was sequenced and generated more than 150,000 raw data. We selected the top 100 sequences with the most occurrences by analyzing the original data and combining the number of occurrences of each sequence, and compared the homology of these 100 sequences to classify the sequences with close homology into a family. Finally, select representative sequences from each family and select 10 sequences for further verification based on the sequence of occurrence (see Table 1).
[0029] Table 1
[0030]
Embodiment 2
[0031] Example 2: Determining the sequence with the strongest specific binding ability to the T24 cell line
[0032]First, digest the adherent T24 cells from the culture dish with 0.2% EDTA and 2% EDTA, collect the cells into centrifuge tubes, and centrifuge with washing buffer (PBS, containing 0.45% glucose, 5mM magnesium chloride) Wash several times; secondly, add the 10 sequences and the random library with a final concentration of 250nM to the binding buffer (D-PBS, containing 0.45% glucose, 5mM magnesium chloride, 100mg / L tRNA, 1g / L BSA) soaked In T24 cells; then place it on a shaker at 4°C and incubate for 40min; after incubation, centrifuge and wash twice with washing buffer (PBS, containing 0.45% glucose, 5mM magnesium chloride), and perform fluorescence detection by flow cytometry (results see figure 2 , only a few representative sequences are shown in the figure). The test results showed that D7 had the strongest binding ability with T24 cells.
Embodiment 3
[0033] Example 3: D7 specifically recognizes human bladder transitional cell carcinoma cells
[0034] This experiment has similarities with certain processing steps in cell-selection technology (CELL-SELEX). First, digest adherent T24 and SV-HUC-1 from the culture dish with 0.2% EDTA and 2% EDTA, respectively, collect the cells into centrifuge tubes, and wash with washing buffer (PBS, containing 0.45% Glucose, 5mM magnesium chloride) centrifugation and washing several times; Add equal amount of binding buffer (D-PBS, containing 0.45% glucose, 5mM magnesium chloride, 100mg / L tRNA, 1g / L BSA to T24 and SV-HUC-1 respectively ), and put into D7 and random library with a final concentration of 250nM; then placed on a shaker at 4°C and incubated for 40min; Fluorescent detection by flow cytometry (results in image 3 ). The results showed that D7 had high affinity and specifically combined with T24 cells, but not with normal SV-HUC-1 cells.
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