Primer probe for detecting amplification of recurrence and metastasis gene WNT2 of neuroblastoma and application of primer probe
A neuroblastoma, recurrence and metastasis technology, applied in the field of primer probes for detecting the amplification of the neuroblastoma recurrence and metastasis gene WNT2, can solve developmental defects, cancer and other problems, and achieve the effect of avoiding false negatives of PCR
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Embodiment 1
[0033] Example 1 Design of Specific Primers and Probes for Detecting the Amplification of Neuroblastoma Recurrent and Metastatic Gene WNT2
[0034] With human WNT2 as the target gene and human EFTUD2 gene as the internal reference gene, specific primers and probes suitable for ddPCR were designed. The sequences of the primers and probes are as follows:
[0035] Target gene upstream primer sequence WNT2-F: 5'-GCGTGGACTTACCACCATGA-3'SEQ ID NO: 1,
[0036]Target gene downstream primer sequence WNT2-R: 5'-CTCTCGGTGGAATCTGGCTC-3'SEQ ID NO: 2,
[0037] Target gene probe sequence WNT2-P: 5'-FAM-TGACCTCGGGGGTGAGCCAGGTCA-BHQ1-3'SEQ IDNO: 3,
[0038] Internal reference gene upstream primer sequence EFTUD2-F: 5'-CTCAAAGTGCGGGGACTGAT-3'SEQ ID NO: 4,
[0039] Internal reference gene downstream primer sequence EFTUD2-R: 5'-GGCATCAGGGTGACTCCAAA-3'SEQ ID NO: 5,
[0040] Internal reference gene probe sequence EFTUD2-P: 5'-HEX-AGCCTGCTTCCTGGGAATGTGCTGCT-BHQ1-3'SEQ ID NO:6.
Embodiment 2
[0041] Example 2 Detection of the Amplification of Neuroblastoma Recurrent and Metastatic Gene WNT2 Using Digital PCR Detection Primers and Probes
[0042] 1. DNA extraction from plasma
[0043] The NucleoSpin Plasma XS kit (product number: 740901.50) produced by MNG Company was used to extract nucleic acid from plasma samples. Take 240 μl of plasma, perform nucleic acid extraction according to the instructions of the extraction kit, and finally resuspend with 20 μl of eluent.
[0044] 2. Digital PCR amplification reaction
[0045] (1) Prepare digital PCR reaction solution; 20 μl of digital PCR reaction solution is formulated as follows: 4 μl of one-step reaction buffer, 2 μl of enzyme mixture, 1.2 μl of 10 μM WNT2-F, WNT2-R, EFTUD2-F and EFTUD2-R primers each, 0.6 μl each of 10 μM WNT2-P and EFTUD2-P, 8 μl of the DNA template of the sample to be tested;
[0046] (2) On the generation chip of droplet PCR, add 20 μ L of droplet PCR reaction system in step 1), then add 40 μ L...
Embodiment 3
[0053] Example 3 Neuroblastoma Sample Digital PCR Primer Probe Sensitivity Experiment
[0054] In this experiment, neuroblastoma-positive samples were used for serial dilution. Detect by the detection method given in Example 2. The results show that this system can detect the copy number ( Figure 3-Figure 4 ).
[0055] Table 2. Detection results of WNT2 gene and EFTUD2 gene of positive samples at the critical concentration.
[0056]
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