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Circulating tumor cell identification kits and methods

A tumor cell and kit technology, applied in the field of molecular biology, can solve problems such as high requirements, false positives, contaminated blood samples, etc., and achieve the effects of improving sensitivity, increasing fluorescence signal intensity, and improving detection sensitivity.

Active Publication Date: 2016-06-29
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Because there may be a certain number of non-neoplastic epithelial cells in peripheral blood, and blood sampling may cause normal epithelial cells to contaminate blood samples, and some CTCs cannot be detected due to the loss of epithelial antigens due to EMT, resulting in false positives and false negatives. In addition, immunomagnetic sorting (MACS) technology combined with reverse transcription-polymerase chain reaction (RT-PCR) is also a commonly used technology for the separation and identification of CTCs, but the RT-PCR process has high requirements for the environment and operation. Moreover, mRNA is easily degraded, and CTC cell typing cannot be performed. Therefore, a technology that can accurately detect CTCs is urgently needed to realize the identification and accurate typing of CTCs.

Method used

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  • Circulating tumor cell identification kits and methods
  • Circulating tumor cell identification kits and methods
  • Circulating tumor cell identification kits and methods

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] There are two types of circulating tumor cell identification kits described in this example, those with labeled probes and those without labeled probes.

[0048] Among them, the circulating tumor cell identification kit A with labeled probes mainly includes:

[0049] 1. Capture probe

[0050] The capture probe is composed of three parts, the 5' end to the 3' sequence is the sequence P1 complementary to the mRNA of the corresponding marker gene, the spacer sequence, and the P2 sequence complementary to the corresponding amplification probe specific sequence P3, Marker genes of the same category have the same P2 sequence in their capture probes. 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 hybridization reaction . The spacer arm of the capture probe of ...

Embodiment 2

[0077] Example 2 Using the kit A in Example 1 to detect circulating tumor cells in human peripheral blood

[0078] The formula of described various solutions is as follows:

[0079]

[0080]

[0081] All the probes in the corresponding gene list in Example 1 were used in the probe mixture, amplification mixture, and chromogenic mixture in this example.

[0082] 1. Sample pretreatment, filter CTCs onto the filter membrane

[0083] 1. Preserve the blood sample in RI preservation solution in the sample preservation tube, centrifuge at 600×g for 5 minutes, discard the supernatant, and remove the red blood cells.

[0084] 2. Add 4 mL of LPBS and 1 mL of RI fixative, vortex and mix well, and let stand at room temperature for 8 minutes.

[0085] 3. Sample filtration: Transfer the liquid in the sample storage tube to the filter, turn on the vacuum filtration pump to pump out the liquid; add 4mL PBS to the storage tube, wash the tube wall and filter the liquid.

[0086] 4. Tra...

Embodiment 3

[0146] Example 3 Selection of the target detection marker gene of the kit

[0147] 1. Design of kit preparation (selection of the number and types of target detection marker genes)

[0148] The epithelial cell marker gene of the kit of the present invention is selected from: EPCAM, KRT16, KRT8, KRT18, KRT19, and the mesenchymal cell marker gene is selected from: CDH2, VIMENTIN, FN1, AKT2. When the epithelial cell marker gene and the mesenchymal cell marker gene are selected differently When the number and type of genes were tested, the detection results were consistent.

[0149] The selection of epithelial cell marker genes as an example, refer to the implementation group 1-3, select one, three and five marker genes respectively, and compare their detection results, while mesenchymal cell marker genes use all 4 genes and The specific design of leukocyte marker gene CD45 is shown in Table 10.

[0150]Taking leukocyte marker genes as an example, experimental group 4 used all e...

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Abstract

The invention discloses a circulating tumor cell identification kit and a circulating tumor cell identification method. The circulating tumor cell identification kit comprises a detection probe for detecting mRNA of an epithelial cell marking gene and / or mRNA of a mesenchymal cell marking gene, wherein the epithelial cell marking gene is selected from one or more of CDH2, VIMENTIN, FN1 and AKT2. Multiple RNA probes are adopted in the identification method, specific genes of a plurality of circulating tumor cells (CTCs) can be marked at the same time and are divided into genes I (epithelial genes), genes II (epithelial-mesenchymal genes) and genes III (mesenchymal genes), and false positive results caused by lack of part of the specific genes of the CTCs in a process that the CTCs enter peripheral blood for circulation are reduced; the method can be finished within eight hours, and a single copied mRNA hybridization probe is combined with a corresponding fluorescent probe by virtue of a signal amplification system, so that the detection sensitivity of in-situ RNA (Ribonucleic Acid) hybridization is remarkably improved.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a circulating tumor cell identification kit and method. Background technique [0002] Circulating tumor cells (circulating tumor cells, CTCs) are tumor cells that enter the peripheral blood of the human body, and may be a sign of distant metastasis of tumors. Studies in recent years have shown that epithelial-mesenchymal transition (ETM, Epithelial-mesenchymal Transition) may occur when tumor cells enter the peripheral blood circulation. The expression of interstitial molecular markers and their transcription factors also changed, and the adhesion between tumor cells undergoing EMT changed, and their migration and invasion abilities were enhanced. After detaching from the primary tumor, they entered the blood to form circulating tumor cells. At present, according to the difference of CTCs antigen markers, CTCs can be divided into epithelial...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6841C12Q1/6881C12Q2600/158C12Q2543/10C12Q2563/107
Inventor 许嘉森吴诗扬刘苏燕刘志明胡文晖
Owner SUREXAM BIO TECH
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