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Probe and kit for detecting HER-2 (human epidermal growth factor receptor-2) gene amplification

A technology of HER-2 and gene amplification, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as dependence, achieve high accuracy, reduce medical costs and expenses, The result is a stable effect

Active Publication Date: 2015-11-25
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But FISH relies on the quality of the probes being made

Method used

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  • Probe and kit for detecting HER-2 (human epidermal growth factor receptor-2) gene amplification
  • Probe and kit for detecting HER-2 (human epidermal growth factor receptor-2) gene amplification
  • Probe and kit for detecting HER-2 (human epidermal growth factor receptor-2) gene amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Preparation of embodiment 1 HER-2 / CSP17 probe

[0025] Use the InvitrogenBioPrimeDNALabelingSystem (Invitrogen18094-011) kit to prepare HER-2 / CSP17 probes by random primer labeling, including the following process:

[0026] 1. Label the probe in a light-proof environment: take out the kit and related reagents from -20°C, thaw on ice, centrifuge briefly after thawing, and then put it back on ice. The probe labeling reference system is shown in Table 2.

[0027] Table 2 Probe Labeling Reference System

[0028] system

10 μL

20 μL

25 μL

dna

N (40-80ng)

N (80-160ng)

N (100-200ng)

[0029] 2.5×Buffer

4μL

8μL

10 μL

dH 2 o

1-N

2-N

2.5-N

Lack of C / T dNTP

1.6μL

3.2 μL

4μL

Cy3-dCTP / FITC-dUTP

3.2 μL

6.4μL

8μL

Klenow Fragment (40U / μL)

0.2 μL

0.4μL

0.5μL

[0030] The HER-2 BACDNA used in the probe labeling is the BAC clone with th...

Embodiment 2

[0042] Embodiment 2 reagent preparation

[0043] (1) Preparation of pretreatment solution (pH7.0): weigh 22.6gNa 2 EDTA-2H 2 O and 6.34g Tris were dissolved in 800mL deionized water, adjusted to pH 7.0 after dissolving, and adjusted to 1L. Store in airtight at 2-8°C for 12 months. If the reagent becomes turbid or contaminated, please discard it immediately.

[0044] (2) Preparation of 20×SSC solution (pH7.0): weigh 88g of sodium citrate and 176g of NaCl in 800mL of deionized water, adjust the pH to 7.0 after dissolving, and dilute to 1L. Store in airtight at 2-8°C for 12 months. If the reagent becomes turbid or contaminated, please discard it immediately.

[0045] (3) Preparation of 2×SSC solution (pH7.0): Dilute 20×SSC solution 10 times with deionized water and adjust the pH to 7.0. Store in a sealed container at 2-8°C for 12 months. If the reagent is turbid or contaminated, please Discard immediately.

[0046] (4) Preparation of 20mg / mL proteinase K mother solution: Wei...

Embodiment 3

[0051] Application of a FISH kit for detection of HER-2 gene amplification in FFPE samples for detection of clinical samples

[0052] From February 2014 to August 2014, 80 paraffin-embedded tissue sections of clinical breast cancer were sent to our company for FISH detection of HER-2 gene amplification. Detection consists of the following steps:

[0053] 1. Pretreatment of FFPE samples, including the following steps:

[0054] (1) baked slices

[0055] (2) Xylene dewaxing

[0056] (3) Gradient ethanol hydration

[0057] (4) Pretreatment solution for 20 minutes

[0058] (5) Proteinase K digestion for 5-15min.

[0059] (6) Gradient ethanol dehydration step by step

[0060] 2. Under dark conditions, denature and hybridize the probe with the tissue to be tested on the glass slide, specifically including the following steps:

[0061] (1) Turn on the hybridization instrument, set or select the saved program: denaturation at 85°C for 5 minutes, hybridization at 37°C overnight (...

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Abstract

The invention discloses a fluorescence in situ hybridization probe and kit for detecting HER-2 (human epidermal growth factor receptor-2) gene amplification and relates to an FISH (fluorescence in situ hybridization) kit. The fluorescence in situ hybridization kit for detecting HER-2 gene amplification comprises an LSP HER-2 / CSP 17 probe and DAPI redyeing liquid; and the probe is marked by adopting a random primer method and is a fluorescence in situ hybridization double-color probe, an HER-2 gene is indicated by a red signal, and the internal control CSP17 is indicated by a green signal. An FFPE tissue slice is used as a detection sample, the FISH method is adopted for detecting the HER-2 gene amplification state, and the probe and the kit can be used for diagnosis of molecular markers for invasive breast cancer and stomach cancer and assisting in clinical medication selection.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a FISH kit for detecting HER-2 gene amplification. Background technique [0002] The human epidermal growth factor receptor-2 (HER-2) gene is located on chromosome 17q12-q21 and is a member of the epidermal growth factor receptor (EGFR) family. Its encoded protein has tyrosine protein kinase activity and is highly identical to EGFR. source. Studies have confirmed that HER-2-mediated signal transduction is related to the formation, proliferation, metastasis, invasion and resistance to radiotherapy and chemotherapy of various tumors. In the past, most of its related research focused on breast cancer and ovarian cancer. Recent studies have shown that HER-2 is also highly expressed in gastric cancer and is expected to become a new gene therapy target. [0003] In vitro cell culture studies have shown that in breast cancer cells, the copy number of HER-2 gene is significantly increased...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 卢皇彬林清华林煌艺曾伶俐阮力宋庆涛
Owner AMOY DIAGNOSTICS CO LTD
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