Typing and identifying kit for circulating tumor cells of lung cancer
A tumor cell, typing and identification technology, used in lung cancer circulating tumor cell typing and identification kits, medical and biological fields, can solve the problems of false positives, contaminated blood samples, false negatives, etc., to solve false negatives, improve accuracy and Confidence, the effect of improving sensitivity
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Embodiment 1
[0043] There are two types of lung cancer circulating tumor cell typing and identification kits described in this example, those with labeled probes and those without labeled probes.
[0044] Among them, the lung cancer circulating tumor cell typing and identification kit A with labeled probes mainly includes:
[0045] 1. Capture probe
[0046] The capture probe consists of three parts, from the 5' end to the 3' sequence is the sequence P1 that is complementary to the mRNA of the corresponding marker gene, the spacer sequence, and the P2 sequence that is complementary to the corresponding amplification probe P3 sequence, the same category The P2 sequences in the capture probes of the marker genes are the same. The spacer is used to space the capture probe P2 sequence from the target mRNA, and by setting a spacer sequence of appropriate length inside the probe, it can reduce steric hindrance, improve the efficiency of the hybridization reaction and the specificity of the hybri...
Embodiment 2
[0077] Example 2 Using kit A in Example 1 to detect circulating tumor cells in peripheral blood of patients with lung cancer
[0078] The formula of described various solutions is as follows:
[0079]
[0080] The probe mixture, amplification mixture, and chromogenic mixture in this example used all the probes in the corresponding gene list of the Lung Cancer Circulating Tumor Cell Typing and Identification Kit A with Labeled Probes in Example 1.
[0081] 1. Sample pretreatment, filter lung cancer circulating tumor cells onto the filter membrane
[0082] 1. Preserve the blood sample in the sample preservation tube with preservation solution, centrifuge at 600×g for 5 minutes, discard the supernatant, and remove the red blood cells.
[0083] 2. Add 4mL PBS and 1ml fixative, vortex to mix, and let stand at room temperature for 8min.
[0084] 3. Sample filtration: transfer the liquid in the sample storage tube to the filter, turn on the vacuum filtration pump to pump out the...
Embodiment 3
[0142] Example 3 Using kit A in Example 1 to detect lung cancer cell lines
[0143] 1. Selection of cell lines
[0144] The lung cancer cell marker gene of the kit of the present invention is selected from: two or more of TTF1, NSE, ASC; the epithelial cell marker gene is selected from: one or more of EpCAM, KRT5, KRT7, KRT18, KRT19, KRT20; The mesenchymal cell marker gene is selected from one or more of: SNAI2, FOXC2, TWIST1, ZEB1, SERPINE1; the leukocyte marker gene is CD45. The various marker genes in the lung cancer cell marker gene, epithelial cell marker gene, mesenchymal cell marker gene and leukocyte marker gene selected in the present invention are specifically expressed on lung cancer CTCs obtained by the inventor through a large number of experiments and statistical analysis. Gene, the detection of lung cancer cells has good specificity and accuracy.
[0145] In this example, the epithelial-mesenchymal mixed lung cancer cell lines NCI-H1975 and HCC827 were selecte...
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