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A detection kit for the expression of AR-V7

A detection kit, AR-V7 technology, applied in the field of molecular biology, can solve the problems of inability to distinguish the difference of several bases at both ends, reduce the signal-to-noise ratio and sensitivity, and have no fluorescent signal amplification system, so as to avoid non-specific Heterohybridization, improved specificity, and high signal-to-noise ratio

Active Publication Date: 2018-10-26
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, detection methods for AR spliceosomes mainly include PCR, PCR-SSCP, Northern blot, and immunohistochemical methods. None of these methods have a fluorescent signal amplification system, which limits the sensitivity and accuracy of detection to a certain extent.
However, in the current in situ hybridization method using probes to directly detect AR-V7, the probes selected are all linear oligonucleotide probes, which cannot distinguish the difference of several bases at both ends, and there is non-specific hybridization; During the needle hybridization process, it is difficult to avoid the non-specific binding of the probe molecule to the matrix itself (such as a filter membrane), resulting in a certain fluorescence background, reducing the signal-to-noise ratio and sensitivity

Method used

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  • A detection kit for the expression of AR-V7
  • A detection kit for the expression of AR-V7
  • A detection kit for the expression of AR-V7

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The AR-V7 detection kit described in this embodiment mainly includes:

[0035] 1. Capture probe

[0036] The capture probe consists of four parts, from the 5' end to the 3' end are the stem structure sequence, the specific sequence P1 that can bind to the target mRNA to be detected, the spacer sequence, and the P2 that can bind to the sequence of the amplification probe P3 The sequence of P2 in the capture probe of the same target mRNA is 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 hybridization reaction . The spacer arm of the capture probe of the present invention is preferably 5-10 T, preferably 5 T in this embodiment. 10 capture probes are designed for each mRNA to improve the specificity of detection while ensuring the stability of the...

Embodiment 2

[0054] Example 2 Using the kit in Example 1 to detect the sample

[0055] The formula of described various solutions is as follows:

[0056]

[0057]

[0058] In this embodiment, blood samples from patients with prostate cancer are preferred, and the expression level of circulating tumor cells (circulating tumor cells, CTCs) AR-V7 in the samples is detected, wherein the capture mixture, amplification mixture, and color development mixture are all used in Example 1. All probes in the corresponding list of AR-V7 detection kit.

[0059] 1. Sample pretreatment, filter the cells to be tested onto the filter membrane

[0060] 1. Collect the cell suspension to be tested, centrifuge horizontally at 600×g for 5 minutes, and discard the supernatant.

[0061] 2. Add 4mL PBS and 1mL fixative, vortex to mix, and let stand at room temperature for 8min.

[0062] 3. Sample filtration: Transfer the liquid in the sample storage tube to the filter, turn on the vacuum filtration pump to ...

Embodiment 3

[0107] Example 3 The impact of capture probe structure on the detection effect of the kit

[0108] 1. Design of kit preparation (capture probe structure design)

[0109] The present invention aims at the detection kit of AR-V7 and designs a stem-loop capture probe for the detection of AR-V7. Compared with traditional linear oligonucleotide probes, the stem-loop capture probe can more effectively avoid Non-specific hybridization, while reducing the binding of the probe to the matrix, has higher specificity and accuracy.

[0110] In order to evaluate the detection effect of the kits composed of capture probes with different structures, design experimental groups 1-2, wherein experimental group 1 selects stem-loop capture probes in the kit of the present invention, and experimental group 2 selects linear capture probes for use. The experimental groups were the same except for the different capture probes. The composition and structure of the linear capture probe is as follows: ...

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Abstract

The invention discloses a detection kit for the expression of AR-V7. The kit includes a capture probe for detecting AR-V7 mRNA and a signal amplification system, and the signal amplification system includes an amplification probe and a labeled probe the tail end of which is modified by a fluorophore; wherein the capture probe is in a stem-loop shape and is used for connecting the target mRNA and the amplification probe; the amplification probe is used for connecting the capture probe and the labeled probe, the labeled probe is used for connecting the amplification probe and the fluorophore, each labeled probe has a P5 sequence which is complementary to and paired with the corresponding amplification probe P4 sequence, and the end is modified by the fluorophore. The detection kit has the advantages of high specificity, high signal to noise ratio, and high accuracy.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to an AR-V7 expression detection kit. Background technique [0002] Prostate cancer is an epithelial malignant tumor that occurs in the prostate gland and is one of the most common malignant tumors in the male genitourinary system. Its occurrence is affected by many factors, including age, race, living habits, obesity, and family medical history. According to statistics, in 2012, the incidence rate of prostate cancer in my country's tumor registration areas was 9.92 / 100,000, ranking sixth in the incidence rate of male malignant tumors. Prostate cancer is an androgen-dependent tumor. Androgen can promote the proliferation of prostate cancer cells. Reducing the level of androgen in the body or antagonizing its effect through endocrine therapy can inhibit the growth of tumor cells and promote their apoptosis. Therefore, endocrine therapy is a clin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/118C12Q2600/158C12Q2600/166
Inventor 刘苏燕吴诗扬董艳
Owner SUREXAM BIO TECH
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