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A real-time fluorescent multiplex PCR primer and kit and application for molecular typing of breast cancer

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

Active Publication Date: 2020-05-19
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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  • A real-time fluorescent multiplex PCR primer and kit and application for molecular typing of breast cancer
  • A real-time fluorescent multiplex PCR primer and kit and application for molecular typing of breast cancer
  • A real-time fluorescent multiplex PCR primer and kit and application for molecular typing of breast cancer

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Experimental program
Comparison scheme
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), sequence alignment was performed to find conserved regions, using Beacon The software performs multiple RT-PCR primer and Taqman probe design for these receptor gene transcription mRNAs, designing three pairs of primers for each molecule (Table 1). Perform BLAST analysis on the NCBI website for each primer / probe combination obtained to ensure that it does not cross-react with other genes that may be present in the sample. In addition, crossover experiments were performed on the designed different primers and probes, and a total of 81 experiments were performed. Experiments to verify the best primer combination. At the same time, single-plex RT-PCR primers were designed as a control.

[0052] Table 1 Multiple RT-PCR primer / probe combinations

[0053]

[0054] (...

Embodiment 2

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

[0064] The ER-α66, PR, Her2 positive cell lines MFC-7, Bcap-37, SK-BR-3 (purchased from Shanghai Gefan Biotechnology Co., Ltd.) were cultured, passaged, expanded and other steps were taken to collect the cells and extract For mRNA, use the primers shown in Table 2 as amplification primers for RT-PCR. The reaction system (25μl) is: template 10μl, 2×RT-PCR Buffer 12.5μl, 25×RT-PCR Enzyme Mix 1μl, each primer / Probe combination 1.5μl, in which the final concentration of the template is 5μM, and the final concentration of each primer / probe is 0.5μM; then each reaction tube is subjected to RT-PCR on a real-time fluorescent PCR machine (ABI 7500, Applied Biosystems) according to the following procedure Reaction: hold at 50°C for 15min (reverse transcription), hold at 95°C for 10min (hot start); hold at 95°C for 8s (denaturation), hold at 60°C for 34s (anneal / extension, collect fluorescence signal), 40 cycle...

Embodiment 3

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

[0071] The result is judged: if the Ct value of the fluorescence signal production index corresponding to a certain receptor gene is less than 33, it is positive, otherwise it is negative.

[0072] Dete...

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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 specifically relates to a real-time fluorescent multiplex PCR primer and kit for molecular typing of breast cancer and its application. 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 various malignant tumors in the whole body. In women, it is second only to uterine cancer. Its incidence is often related to genetics, and 40-60%. The incidence of women before and after menopause is relatively high between ages, and it is one of the most common malignant tumors that seriously affects women’s physical and mental health and even endangers their lives. [0003] Breast cancer is a highly heterogeneous tumor, and the traditional pathomorphological classification has gradually shown its imperfections in current clinical practice. With the application of human molecular biology t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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