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Primer and probe for detecting c-MET gene amplification through digital PCR technology and detection method thereof

A gene amplification and technical detection technology, applied in the field of molecular biology detection of genes in the field of biotechnology, can solve the problems of low accuracy and low sensitivity, achieve simple operation, high sensitivity, and reduce the cost and workload of clinical detection Effect

Inactive Publication Date: 2018-07-24
PRIMBIO GENES BIOTECH WUHAN CO LTD
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Benefits of technology

In this new technology, there were advantages over previous methods but they had weaknesses or limitations during their use due to factors like poor targeting ability caused by hybridization between different sequences within the same sequence. These improvements led to improved accuracy and stability in diagnosing cancer types based on its presence at certain points along genome.

Problems solved by technology

This patents describes how certain type of nucleic acid called catheter endonuclease (CENE1) may cause various diseases such as gastrointestinal stomach adhesion disease or gastritis fibroblasts carcinoma. It also explains why these conditions are associated with increased risk of developing colorectomy related complications like death from colon cancer. Current diagnoses rely heavily upon histology analysis and immunohistochemistry, making them time-consuming processes and subject to errors.

Method used

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  • Primer and probe for detecting c-MET gene amplification through digital PCR technology and detection method thereof
  • Primer and probe for detecting c-MET gene amplification through digital PCR technology and detection method thereof

Examples

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Embodiment 1

[0038] Embodiment 1: ddPCR technology detects the design and synthesis of primers and probes for c-MET gene amplification

[0039] Based on the genome analysis data, analyze the c-MET gene, use Oligo software to analyze the sites of TaqMan primers and probes, and select the best combination as follows:

[0040] c-MET gene upstream and downstream primers:

[0041] c-MET upstream primer SEQ NO1: 5'-AGTTCGCTACGATGCAAGAGT-3'

[0042] c-MET downstream primer SEQ NO2: 5'-AGTTGGGCTTACACTTCGGG-3'

[0043] When using the above primers for PCR amplification, the amplified fragment is 73bp, which is especially suitable for the amplification of small fragment DNA samples such as plasma free DNA.

[0044] c-MET gene detection probe: SEQ NO5: 5'-FAM-TCCTCATTTGGATAGGCTTGTA-MGB-3'

[0045] A gene that can be stably amplified and whose amplification effect is consistent with c-MET after testing is selected as an internal reference gene. The internal reference gene selected in this example i...

Embodiment 2

[0051] Example 2: Detection of c-MET gene amplification in whole blood samples

[0052] 1. The samples to be tested in this embodiment are the whole blood samples of 5 cases of human peripheral blood collected from Wuhan Liangpei Gene Biotechnology Co., Ltd., and the numbers are 1-1, 1-2, 1-3, 1-4, 1-5, where IHC results show that 1-1 and 1-2 are c-MET positive samples, and 1-3, 1-4 and 1-5 are c-MET negative samples.

[0053] 2. Extraction of cfDNA: Use a kit to extract cfDNA from whole blood samples. For specific operations, refer to the instructions of the QIAamp Circulating Nuleacid Kit kit from QIAGEN, and use the extracted cfDNA as a template for PCR detection.

[0054] 3. Prepare a PCR reaction solution in the PCR plate according to the ratio shown in Table 1: the concentrations of the cfDNA template and the internal reference gene are both 1 ng / μL.

[0055] Table 1 PCR reaction solution configuration

[0056] Reagent

[0057] 4. Put the PCR plate containing...

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Abstract

The invention relates to molecular biology detection of genes in the technical field of biology, and particularly relates to a method for detecting c-MET gene amplification by utilizing a digital PCR(Polymerase Chain Reaction) technology. The detection method comprises the following steps: firstly, designing and synthesizing upstream and downstream primers for detecting c-MET gene amplification and the probe for detecting the c-MET gene amplification for c-MET genes; then designing digital PCR detection parameters; judging whether a sample to be detected contains c-MET gene amplification through the digital PCR technology. The primer and the probe designed by the invention have strong specificity, are applied to non-tumorous tissues through the digital PCR detection, the accuracy is high,and the sensitivity can be up to 0.01 percent. The length of an amplification fragment of the primer designed and synthesized by the method disclosed by the invention is less than 100bp, so that thefragment is particularly suitable for amplifying small-fragment DNA samples such as plasma free DNA, and variation of c-MET amplification with extremely low abundance ratio can be detected from cfDNA,tumor DNA is detected from blood circulation of patients in the early stage of tumors; moreover, the technical problem of risk of invasive sampling detection is overcome, and the primer, the probe and the detection method have extremely important effects on guidance of clinical treatment and improvement of prognosis of patients.

Description

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Claims

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Application Information

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Owner PRIMBIO GENES BIOTECH WUHAN CO LTD
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