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Breast cancer molecular typing real-time fluorescent multiple PCR (Polymerase Chain Reaction) primer and kit and application thereof

A molecular typing and real-time fluorescence technology, applied in the field of nucleic acid detection, can solve problems such as poor prognosis and affect disease diagnosis, and achieve the effects of strong specificity, short detection time and high accuracy

Active Publication Date: 2018-03-06
洛阳科赫医疗器械有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] (4) Basal-like type: the incidence rate is 17.1%, negative for estrogen receptor (ER) and / or progesterone receptor (PR), negative for proto-oncogene human epidermal growth factor receptor 2 (Her2), and ineffective for endocrine , the effect of chemotherapy is good, the prognosis is the worst
③Each set of primers / probes should not have cross-homology to other target and non-target nucleic acid sequences, so as to prevent false positive results and affect the diagnosis of diseases

Method used

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  • Breast cancer molecular typing real-time fluorescent multiple PCR (Polymerase Chain Reaction) primer and kit and application thereof
  • Breast cancer molecular typing real-time fluorescent multiple PCR (Polymerase Chain Reaction) primer and kit and application thereof
  • Breast cancer molecular typing real-time fluorescent multiple PCR (Polymerase Chain Reaction) primer and kit and application thereof

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Experimental program
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Effect test

Embodiment 1

[0050] (1) Design primer / probe combination

[0051] According to the mRNA sequence of each molecular receptor of breast cancer released by GenBank (ER-α66 gene: NM_000125.3; PR gene: NM_000926.4; Her2 gene: NM_001005862.2), the sequences were compared to find conserved regions, using Beacon The software designed primers and Taqman probes for multiplex RT-PCR on mRNA transcribed by these receptor genes, and designed three pairs of primers for each molecule (Table 1). The resulting primer / probe combinations were subjected to BLAST analysis on the NCBI website to ensure no cross-reaction with other genes that may be present in the sample. The different designed primers and probes were tested for crossover, and a total of 81 experiments were carried out. Experimentally validate the primer combination for its best performance. Simultaneously, single-plex RT-PCR primers were designed as controls.

[0052] Table 1 Multiplex RT-PCR Primer / Probe Combinations

[0053]

[0054] (...

Embodiment 2

[0063] (1) Prepare a positive control and detect the minimum detection amount

[0064] ER-α66, PR, and Her2 positive cell lines MFC-7, Bcap-37, and SK-BR-3 (purchased from Shanghai Gefan Biotechnology Co., Ltd.) were cultured, passaged, and amplified, and the cells were collected and extracted. For mRNA, the primers shown in Table 2 were used as amplification primers for RT-PCR, wherein the reaction system (25 μl) was: template 10 μl, 2×RT-PCR Buffer 12.5 μl, 25×RT-PCR Enzyme Mix 1 μl, each primer / Probe combination 1.5 μl, wherein, the final concentration of the template is 5 μM, and the final concentration of each primer / probe is 0.5 μM; then carry out RT-PCR on a real-time fluorescent PCR instrument (ABI 7500, Applied Biosystems) for each reaction tube according to the following procedure Reaction: 50°C for 15min (reverse transcription), 95°C for 10min (hot start); 95°C for 8s (denaturation), 60°C for 34s (annealing / extension, fluorescence signal collection), 40 cycles. T...

Embodiment 3

[0070] RNA was extracted from fresh breast cancer tissue samples or tissue sections, and 2 μl of internal control (final concentration was 5 μM) was added to the samples before extraction, and then RNA was extracted from the samples ( mRNA DIRECT TM Purification (life)). Then use the primer combinations shown in Table 2 as amplification primers for RT-PCR. The reaction system (25 μl) is: RNA template 10 μl, 2×RT-PCR Buffer 12.5 μl, 25×RT-PCR Enzyme Mix 1 μl, each primer / Probe combination 1.5 μl, in which the final concentration of the sample is 5 μM, and the concentration of each primer / probe is 0.5 μM; the amplification program is: 50 ° C for 15 min (reverse transcription), 95 ° C for 10 min (hot start); 95 ° C 8s (denaturation), maintained at 60°C for 34s (annealing / extension, collecting fluorescence signals), 40 cycles.

[0071] Result judgment: if the corresponding fluorescent signal generation index Ct value of a certain receptor gene is less than 33, it is positive,...

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Abstract

The invention belongs to the technical field of nucleic acid detection and in particular relates to a breast cancer molecular typing real-time fluorescent multiple PCR (Polymerase Chain Reaction) primer and kit and application thereof. The breast cancer molecular typing real-time fluorescent multiple PCR primer comprises an estrogen receptor alpha66 gene primer, a progestrone receptor gene primerand a human epidermal growth factor receptor 2 gene primer, and nucleotide sequences of the multiple PCR primer are shown as SEQ ID NO. 1-6. The invention further provides the breast cancer moleculartyping real-time fluorescent multiple PCR rapid detection kit. The kit comprises the primer, 2*RT-PCR Buffer, 25*RT-PCR Enzyme, positive control and negative control. The kit has the characteristics of high specificity, high sensitivity, high accuracy and the like.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a real-time fluorescent multiplex PCR primer for molecular typing of breast cancer, a kit and application thereof. Background technique [0002] Breast cancer is one of the most common malignant tumors in women. According to statistics, the incidence rate accounts for 7-10% of all kinds of malignant tumors in the whole body. It is second only to uterine cancer in women. Its incidence is often related to genetics, and 40-60 It is one of the most common malignant tumors that seriously affects women's physical and mental health and even threatens their lives. [0003] Breast cancer is a highly heterogeneous tumor, and the traditional pathomorphological classification has gradually shown its incompleteness in current clinical practice. With the application of human molecular biology techniques, the concept of molecular typing based on tumor morphology comb...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/112C12Q2600/158C12Q2600/16C12Q2537/143C12Q2561/101C12Q2545/101C12Q2545/113
Inventor 万东山郭樱
Owner 洛阳科赫医疗器械有限公司
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