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
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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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