CN104928363A

CN104928363A - Kit for quickly detecting circulating tumor cells of peripheral blood

Info

Publication number: CN104928363A
Application number: CN201510190133.4A
Authority: CN
Inventors: 冯文峰; 黄鹏; 昌芒
Original assignee: Guangzhou Stemove Biotechnology Co Ltd
Current assignee: Guangzhou Stemove Biotechnology Co Ltd
Priority date: 2015-04-21
Filing date: 2015-04-21
Publication date: 2015-09-23

Abstract

The invention belongs to the field of in-vitro diagnosis and discloses a kit for quickly detecting circulating tumor cells of peripheral blood. By the design of three types of probes, circulating or scattered living cells in body fluid such as blood or tissue samples mixed with liquid are detected. A method comprises the following steps: (a) acquiring the peripheral blood, and extracting exosome nucleic acids of the circulating tumor cells; (b) detecting the exosome nucleic acids; (c) interpreting a result. By detecting the circulating tumor cells of the peripheral blood, a highly-valuable scientific basis can be provided for clinic development, treatment effect judgment, prognosis evaluation and the like of tumors.

Description

A kind of test kit for rapid detection Peripheral Circulation tumour cell

Technical field

The invention belongs to in-vitro diagnosis field, relate to a kind of test kit for rapid detection Peripheral Circulation tumour cell, be specifically related to a kind of method for rapid detection Peripheral Circulation tumour cell born of the same parents ectosome nucleic acid.

Background technology

The tumour cell of peripheral blood is entered when circulating tumor cell in peripheral blood (Circulating Tumor Cell is called for short CTC) refers to spontaneous or operation of diagnosis and treatment.The CTC with height vigor and height metastatic potential can be survived in the recycle system, and breeds in suitable environment, leads oncogenic recurrence and transfer.Valuable scientific basis can be provided for the clinical progress of tumour, Outcome measure and prognosis evaluation etc. to the monitoring of CTC.

Born of the same parents' ectosome (exosome, EO) a kind of small vesica with double membrane structure that to be eukaryotic cell secrete under normal and pathological state, includes albumen, lipid, RNA and multiple other biological macromole.Because its signal communication between cell, the effect in the adjustment of tumour immunity microenvironment are significantly subject to extensive concern.Research shows, the composition of tumor cell secretion EO and quantity and normal cell there are differences, and therefore its function in tumour is formed and progress, immunological status regulate also has diversity.And EO may predict in tumour, by stages, have great potential value in Diagnosis and Treat.

In prior art, the method that traditional CT C detects is filtration method, Graded Density method and immunomagnetic beads method, but these methods exist that susceptibility is low, purity difference and the problem such as cytotoxicity is large.

Summary of the invention

In order to solve above-mentioned prior art Problems existing, the invention provides a kind of test kit for rapid detection Peripheral Circulation tumour cell, be intended to provide a kind of technology for rapid detection Peripheral Circulation tumour cell, be specifically related to a kind of method for rapid detection Peripheral Circulation tumour cell born of the same parents ectosome nucleic acid.

For a test kit for rapid detection Peripheral Circulation tumour cell, described test kit comprises capture probe Capture Probe(SEQ ID NO.1) assist probes Signal Probe 1 (SEQ ID NO.2) and Signal Probe2(SEQ ID NO.3).

A kind of test kit for rapid detection Peripheral Circulation tumour cell of the present invention, that detect the circulation in body fluid (such as blood) or spread viable cell, or being mixed with the tissue samples of liquid, detection method comprises draws together following steps: a) acquisition of peripheral blood and the extraction of circulating tumor cell born of the same parents ectosome nucleic acid; B) detection of born of the same parents' ectosome nucleic acid; C) interpretation of result.

Further, the method for test kit preparation of the present invention and detection application is:

1) incomplete DNAzyme(SEQ ID NO.4 is designed) and complete DNAzyme(SEQ ID NO.5), the substrate of DNAzyme, the capture probe Capture Probe(SEQ ID NO.1 for separating of single stranded product), assist probes Signal Probe 1 (SEQ ID NO.2) and Signal Probe2(SEQ ID NO.3);

Described Capture Probe can catch 5 ' end of object fragment (miR-105) (SEQ ID NO.6) single stranded product, Signal Probe 1 can hold complementation to be double-strand with 3 ' of single stranded product, Signal Probe2 can hold complementation to be double-strand with 3 ' of Signal Probe 1 again, the final duplex structure forming length;

2) add in reaction solution by incomplete DNAzyme, cupric ion, xitix and sample, mixing, deposits in case at sample MiRNA, and T4 connects acid becomes DNAzyme completely by incomplete DNAzyme repairing;

3) in reaction solution, add the substrate of DNAzyme, it is two single stranded DNAs that substrate is cracked into;

4) adopt the magnetic bead (SA-MBs) being connected with Streptavidin (SA) to be separated by product single stranded DNA, other dissociative DNA chains are removed in cleaning;

5) by single stranded DNA, assist probes Signal Probe 1 and Signal Probe2 that the band after cleaning is separated, add reaction solution, react completely to form self-assembly double-strand;

6) add Sybr Green I, detect fluorescent signal.

The extraction of described born of the same parents' ectosome nucleic acid; it is characterized in that being completed by differential centrifugation method; described differential centrifugation verifies that top condition is by experiment: 2000rpm/min centrifugal segregation cell debris; add RNA protective material after 8000rpm/min collected by centrifugation born of the same parents ectosome, born of the same parents' ectosome nucleic acid of purifying can be obtained.

Beneficial effect:

Test kit of the present invention, have accurately, repeatability and specificity is high, feature flexibly, effectively solve the present invention and can provide valuable scientific basis for the clinical progress of tumour, Outcome measure and prognosis evaluation etc. by the monitoring of human peripheral blood circulating tumor cell.

Accompanying drawing explanation

A kind of detection of born of the same parents' ectosome nucleic acid for rapid detection Peripheral Circulation tumour cell technology of Fig. 1 and the interpretation schematic diagram of result.

A:A figure is that incomplete DNAzyme is assembled into complete DNAzyme when object fragment to be checked exists

B:B figure is that complete DNAzyme DNAzyme when interpolation bivalent cupric ion and xitix reacts substrate, cuts off substrate and forms capture probe

C:C figure is result interpretation schematic diagram, forms continuous print duplex structure when signal probe 1 exists with signal probe 2 with capture probe, carries out fluorescent dye when interpolation SYBR Green I.

Embodiment

A kind of Peripheral Circulation tumor cell gene detection chip, described chip comprises capture probe Capture Probe assist probes Signal Probe 1 and Signal Probe2.

The concrete construction process of described chip and detection application:

1) substrate of incomplete DNAzyme and complete DNAzyme, DNAzyme is designed, for separating of capture probe Capture Probe, the assist probes Signal Probe 1 and Signal Probe2 of single stranded product;

Described Capture Probe can catch 5 ' end of single stranded product, and Signal Probe 1 can hold complementation to be double-strand with 3 ' of single stranded product, and Signal Probe2 can hold complementation to be double-strand with 3 ' of Signal Probe 1 again, the final duplex structure forming length;

6) add Sybr Green I, detect fluorescent signal.

Si Danfu bio tech ltd, <110> Guangzhou

<120> Peripheral Circulation tumor cell gene detection chip

<130> 2014

<160> 23

<170> PatentIn version 3.3

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<212> DNA

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<212> DNA

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Claims

1. a Peripheral Circulation tumor cell gene detection chip, described chip comprises capture probe Capture Probe(SEQ ID NO.1) assist probes Signal Probe 1 (SEQ ID NO.2) and Signal Probe2(SEQ ID NO.3).

2. gene chip as claimed in claim 1, its method detecting application is:

The acquisition of peripheral blood and the extraction of circulating tumor cell born of the same parents ectosome nucleic acid; B) detection of born of the same parents' ectosome nucleic acid; C) interpretation of result.

3. gene chip as claimed in claim 2; it is characterized in that: the extracting method that described tumour cell born of the same parents ectosome is adjusted is differential centrifugation method; described differential centrifugation 2000rpm/min centrifugal segregation cell debris; add RNA protective material after 8000rpm/min collected by centrifugation born of the same parents ectosome, born of the same parents' ectosome nucleic acid of purifying can be obtained.