Kit for detecting invasiveness of circulating tumor cells

A tumor cell and invasive technology, applied in the field of immunoassay detection, can solve the problem of inability to distinguish invasive and non-invasive CTCs, and achieve the effect of enhancing probe sensitivity, detection sensitivity and high reaction efficiency.

Active Publication Date: 2016-06-15
SHANGHAI MAJORBIO BIO PHARM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, during our research, we found that the current CTC detection methods cannot distinguish between invasive and non-invasive CTCs

Method used

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  • Kit for detecting invasiveness of circulating tumor cells
  • Kit for detecting invasiveness of circulating tumor cells
  • Kit for detecting invasiveness of circulating tumor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1CD133

[0038] Embodiment 1CD133-oligonucleotide probe

[0039] (1) Aldehyde modification of oligonucleotide probes

[0040] 50 nmol of Oligo1 (oligonucleotide) was prepared into a solution with 0.1 M pH 7.4 phosphate buffer. Weigh 500nmol of SFB (N-succinimidyl 4-formylbenzoate), dissolve it with anhydrous DMF (N,N-dimethylformamide), react at room temperature for 2.5h, and purify through a column to obtain Oligo -FB (aldehyde modified oligonucleotide).

[0041] Detection of the concentration of Oligo-FB: detection of A by Nanodrop spectrophotometer 260 value, the calculated concentration of Oligo-FB was 0.73nmol / μL.

[0042]Detection of aldehyde group modification rate: Use quantitative 2-hydrazinopyridine-2-hydrochloride solution to detect the aldehyde group modification rate. Take the above-mentioned Oligo-FB and add it to 2-hydrazinopyridine-2-hydrochloride solution, shake and mix well, and react at 37°C for 1 hour. The absorbance value at 360nm detected by Nanodrop is 1.41, ...

Embodiment 2

[0056] Specific detection of oligo molecules by QPCR amplification

[0057] Oligo1-13 molecules were diluted to three concentrations of 25000 molecules / μl, 83333 molecules / μl and 250000 molecules / μl. Then, taking Oligo1-3 as an example, mix Oligo1-3 at the same concentration to obtain mixed samples A, B, and C, as shown in Table 2.

[0058] Table 2 Example 2 sample formula

[0059]

[0060] The above samples were prepared according to Table 3 for QPCR amplification solution to obtain a QPCR amplification kit. Then carry out QPCR amplification to Oligo1-3 according to the program shown in Table 4, and carry out QPCR amplification respectively to Oligo1-3 in mixed sample A, B, C, take the Oligo1-3 after respectively extending extension as template, amplify The Ct values ​​of the increasing results are shown in Table 5. Among them, the extension primer is RT-P, its 3' end is complementary to the 3' end of the oligonucleotide, the 5' end can form a hairpin structure, and the...

Embodiment 3

[0074] Embodiment 3 makes standard curve

[0075] The same result can be obtained with Oligo1-13. In order to simplify the process, the following examples use Oligo1-3 as an example.

[0076] According to the steps of Example 1, Oligo2 was modified with SFB having a molar equivalent of 5 times to obtain Oligo-FB, and CD24 was hydrazine-modified with SANH having a molar equivalent of 10 times to obtain CD24-SANH, and the molar ratio was 10:1 CD24-Oligo2 was obtained after reacting Oligo-FB with CD24-SANH at room temperature for 16 hours.

[0077] According to the steps of Example 1, modify Oligo3 with SFB with a molar equivalent of 20 times to obtain Oligo-FB, and use SANH with a molar equivalent of 50 times to modify CD44 with a hydrazine group to obtain CD44-SANH, and the molar ratio is 8:1 CD44-Oligo3 was obtained after reacting Oligo-FB with CD44-SANH at room temperature for 24 hours.

[0078] CD133-Oligo1, CD24-Oligo2, and CD44-Oligo3 were amplified by QPCR according to ...

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Abstract

The invention provides a kit for detecting the invasiveness of circulating tumor cells (CTC). The kit is characterized by comprising antibody-oligonucleotide probes obtained by coupling CD133, CD24 and CD44 antibodies with different oligonucleotides, respectively, an amplification primer and a buffering solution which are used for carrying out PCR (Polymerase Chain Reaction) amplification on the antibody-oligonucleotide probes, and a fluorescent probe. According to the kit provided by the invention, the CD133, the CD24 and the CD44 are used as markers of cancer stem cells (CSC) and the content of each antigen in the CTC can be detected in parallel, so that the expression amount of the CSC in the CTC is obtained, and the invasiveness of the CTC is analyzed. The kit provided by the invention is low in detection cost and high in sensitivity, and has very strong practicability.

Description

technical field [0001] The invention relates to the field of immunoassay detection, in particular to a kit for detecting the invasion force of circulating tumor cells. Background technique [0002] Circulating tumor cells (CTCs) refer to tumor cells that are released into the peripheral blood circulation from the primary tumor or metastases of solid tumors spontaneously or due to diagnostic and therapeutic procedures. Metastasis is the main cause of cancer-related death, and CTCs are regarded as the seeds of metastasis. CTC cells are currently considered to be one of the seeds of cancer metastasis. It plays an important role in the early diagnosis and prognosis of cancer. However, during our research, we found that the current CTC detection methods cannot distinguish between invasive and non-invasive CTCs. Due to the different invasiveness of CTCs, when more than 5 CTCs are detected, the number of CTCs does not completely correspond to the survival period of cancer patien...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574
CPCG01N33/57488
Inventor 李静陈昌岳蔡红东邓文斌甘广利张祥林
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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